Antibody

Abstract

The present invention provides antibodies or antigen-binding fragments thereof that (a) specifically bind 4R tau protein isoforms in a physiological sample; or (b) specifically bind 3R tau protein isoforms in a physiological sample. The present invention further provides nucleic acids and vectors encoding such antibodies or antigen-binding fragments, and host cells comprising such nucleic acids and vectors. The antibodies and fragments may be used in the treatment and diagnosis of tauopathies.

Description

[Technical field]

[0001] The present invention relates to antibodies specific for certain tau proteins, in particular for the 4R or 3R tau protein isoforms. The antibodies can be used, for example, for detection, diagnosis and therapy. [Background technology]

[0002] The microtubule-associated protein tau (tau) was discovered in 1975. It was initially characterized as a microtubule-binding protein essential for microtubule growth and stabilization within neuronal axons. Binding to microtubules is achieved at the C-terminus of tubulin (encoded via exons 9-12) via the microtubule-binding region of tau (MTBR). Specifically, it has been shown by peptide competition NMR that tau interacts with the interface between α-tubulin and β-tubulin via residues 224-237, 245-253, 275-284, and 300-317.

[0003] The tau gene (MAPT) contains 16 exons located on chromosome 17q21. Alternative splicing from a single tau pre-mRNA results in six splice variant isoforms of tau in the central nervous system ranging from 342 to 441 amino acids in length. Tau isoforms are classified into two categories based on the inclusion or exclusion of exon 10 (within the MTBR), resulting in four MTBR (4-repeat (4R) tau) in which exon 10 is present, and three MTBR (3-repeat (3R) tau) in which exon 10 is excluded. Within each of these categories, there are three N-terminal variants of tau based on the splicing of exons 2 and 3. This results in 0N tau (not including either exon 2 or 3), 1N tau (including only exon 2), and 2N tau (including both exons 2 and 3). Tau nomenclature is based on amino acid numbering from the largest (2N4R) isoform of tau, which itself has four distinct functional domains: the N-terminal protuberance (amino acids 1-165); the proline-rich domain (amino acids 166-242); the MTBR (amino acids 243-367); and the C-terminal domain (amino acids 368-441). The presence or absence of the sequence encoded by exon 10 in the MTBR domain denotes 3R or 4R tau.

[0004] Tau MTBR is a protein repeat region consisting of three (3R tau) or four (4R tau) repeat regions that are identical in sequence across tau isoforms. Each repeat is a different tau exon; repeat 1 (exon 9) (amino acids 242-273); repeat 2 (exon 10) (amino acids 274-304); repeat 3 (exon 11) (amino acids 305-335); repeat 4 (exon 12) (336-367). While there is only 34% complete sequence identity across all four repeat regions, the level of homology between amino acids of similar classes (polar, charged, uncharged) or between at least two of the repeat regions is 90%, with repeat regions 1 and 4 being the most divergent.

[0005] Tau is a largely unstructured protein, but FRET experiments suggest that the N- and C-termini are oriented in close proximity. Further studies using NMR confirmed that the N- and C-termini of tau fold into a "paper clip"-like structure onto the central region of the protein. Tau has also been demonstrated to have a tendency to form more complex transient local secondary structures, specifically, beta strands in the MTBR and polyproline helices in the proline-rich domain.

[0006] Tau is found to be expressed primarily in neurons, with lower levels in oligodendrocytes and astrocytes. Tau was originally identified in association with axonal microtubules, and to a lesser extent with plasma membranes, nuclei, and mitochondria. In healthy adult neurons, the distribution of tau to axons crosslinks tubulin, allowing interconnections with other cytoskeletal components such as neurofilaments and actin. These interactions stabilize tubulin assemblies within microtubules and modulate their dynamically labile nature, allowing cytoskeletal reorganization required for axonal growth. Tau has also been shown to directly regulate the microtubule binding of the motor transport proteins dynein and kinesin, which transport cargo along axons in retrograde and anterograde directions, respectively. This regulatory effect is believed to occur via direct competition with the aforementioned motor proteins for access to microtubules. In tau overexpression systems, a greater impact on kinesin is observed, resulting in net retrograde transport and accumulation of cargo (such as mitochondria) in the cell body rather than diffusion downstream of the axon. However, this regulatory effect is not fully understood and may have complementary mechanisms, as demonstrated in tau knockout mice where axonal transport is unaffected.

[0007] Tau protein and its isoforms are involved in several diseases that are typically characterized by the deposition of tau fibrils, and these conditions are known collectively as tauopathies. Although the majority of tauopathies occur sporadically in the population, there are many tauopathies associated with MAPT mutations that display the same disease phenotype as their sporadic counterparts (Ghetti et al., 2015, Neuropathol Appl Neurobiol, 41(1), 24-46). Tauopathies can be further divided into primary and secondary tauopathies. Primary tauopathies are a subgroup of frontotemporal lobar degeneration (FTLD), which is characterized by neuronal tau inclusions accompanied by cell death predominantly in the frontal and temporal lobes of the brain. Within these lobes, tau inclusions are thought to be the primary driving factor in pathology. A well-characterized example is progressive supranuclear palsy (PSP). In contrast, secondary tauopathies are diseases in which tau pathology is observed in association with other brain pathologies. An example of a well-characterized secondary tauopathy is Alzheimer's disease (AD), whose disease pathology is characterized by both neuronal tau fibrils and extracellular amyloid plaques, as defined by Alois Alzheimer in 1907. It is noteworthy that in AD, there is still an association between tau burden and cognitive decline, indicating that tau still plays a key role in this multifactorial disease.

[0008] The ratio of tau isoforms appears to play an important role in maintaining tau in a non-pathogenic state. Thus, tauopathies can exist as 3R tauopathies or 4R tauopathies, characterized by an excess of 3R or 4R tau, respectively. Pick's disease is a tauopathy primarily associated with an excess of 3R-forming filamentous 3R tau inclusions, known as Pick's globules. The primary pathology associated with Pick's disease is the loss of neurons and glia in the frontal, temporal and parietal lobes of the brain. The by far most common form of tauopathy associated with an imbalance of tau isoforms is 4R tauopathy. Excess 4R tau and 4R tau inclusions have been observed in diseases such as frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17); progressive supranuclear palsy (PSP); and corticobasal degeneration (CBD).

[0009] Given the importance of tau in many pathological conditions, there is a continuing need for new diagnostic and therapeutic approaches that target tau. Summary of the Invention

[0010] The present invention relates to (a) specifically binds a 4R tau protein isoform in a physiological sample; or (b) specifically binds 3R tau protein isoforms in physiological samples; An antibody or antigen-binding fragment thereof is provided.

[0011] If an antibody or antigen-binding fragment thereof specifically binds to 4R tau protein, it: (a) binds to an amino acid region encoded by exon 10 of 4R tau protein, where the 4R tau protein contains a post-translational modification at one or more of amino acid positions 279, 280, 281, 285, and 289 of tau4R; (b) binds to a peptide comprising or consisting of amino acids 294 to 302 of 4R tau protein; (c) binds to a peptide corresponding to a single epitope from the amino acid sequence encoded by exon 10 of tau; or (d) capable of cross-blocking or being cross-blocked by any of the antibodies or fragments of (a)-(c).

[0012] The antibody or antigen-binding fragment thereof may specifically bind the 4R Tau protein isoform in a cell lysate from a cell expressing a physiological 4R Tau protein isoform, preferably in a cell lysate from an iPSC-derived neuronal cell expressing the 4R Tau protein isoform. The antibody or antigen-binding fragment may detect the 4R Tau protein isoform by immunofluorescence on or in a cell expressing the 4R Tau protein isoform.

[0013] The antibody or antigen-binding fragment thereof against 4R tau protein is (a) comprising one or more of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 3, 5 and 7, and one or more of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 11, 13 and 15, preferably comprising the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 3, 5 and 7, and the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 11, 13 and 15, (b) comprising the light chain variable region and the heavy chain variable region of SEQ ID NOs: 1 and 9; (c) an intrabody, preferably comprising the sequence of SEQ ID NO: 17, or a sequence having at least 95% sequence identity thereto, and still capable of specifically binding to the Tau 4R isoform; or (d) capable of cross-blocking or being cross-blocked by any of the antibodies or fragments of (a)-(c).

[0014] If an antibody or antigen-binding fragment thereof specifically binds a 3R tau protein isoform, it: (a) binds to a peptide, e.g., a cyclic peptide, that includes an amino acid sequence encoded by a region spanning exons 9 and 11 of tau; and / or (b) It is capable of binding to a peptide, such as a cyclic peptide, consisting of seven amino acids on either side of the boundary of the sequence encoded by exons 9 and 11 of tau.

[0015] The antibody or antigen-binding fragment thereof against 3R tau protein is (a) comprising one or more of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 20, 22 and 24, and one or more of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 28, 30 and 32, preferably comprising the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 20, 22 and 24, and the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 28, 30 and 32, (b) comprising the light chain variable region and the heavy chain variable region of SEQ ID NOs: 18 and 26; (c) an intrabody, preferably comprising a sequence of SEQ ID NO: 34, or a sequence having at least 95% sequence identity thereto, capable of specifically binding to the Tau 3R isoform; or (d) capable of cross-blocking or being cross-blocked by any of the antibodies or fragments of (a)-(c).

[0016] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds 4R Tau protein, wherein the antibody or antigen-binding fragment thereof binds an epitope of 4R Tau protein that includes amino acids K294, D295, N296, and I297, and optionally the epitope further includes K298 and V300 of 4R Tau protein.

[0017] The antibody or antigen-binding fragment thereof may be an intracellular antibody (intrabody) or a degrabody.

[0018] The present invention also provides one or more nucleic acids encoding the antibodies or antigen-binding fragments of the invention; one or more vectors comprising said nucleic acids; and a host cell comprising said one or more nucleic acids or said one or more vectors.

[0019] The present invention also provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof of the invention, one or more nucleic acids of the invention, or one or more vectors of the invention and a pharmaceutical carrier or excipient.

[0020] The present invention also provides a method for detecting 4R or 3R tau protein isoforms, comprising: (a) contacting a test sample with an antibody or antigen-binding fragment thereof of the present invention; and (b) detecting binding of the antibody or antigen-binding fragment thereof. The present invention also provides a method for determining the levels of 4R and 3R tau protein isoforms. The method may be used to diagnose a tauopathy, preferably PSP, CBD, or Pick's disease.

[0021] The antibody or antigen-binding fragment thereof can be used to treat a tauopathy, for example a tauopathy involving an imbalance between 4R and 3R tau protein isoforms. [Brief description of the drawings]

[0022] [Figure 1] The tau protein sequence exons 9-12 are shown. The TE9 / 11 peptide sequence is outlined in solid boxes. Potentially cross-reactive epitopes at the boundaries of exons 9-10, 10-11, and 11-12 are highlighted in dashed boxes. Amino acid differences between TE9 / 11 are highlighted with asterisks. [Diagram 2] Tau protein sequence exons 9-12 are shown. The TE10 peptide sequence is outlined in a solid box. Potentially cross-reactive epitopes within exons 9, 11 and 12 are highlighted in dashed boxes. Amino acid differences between TE10 are highlighted with asterisks. Documented post-translational modifications at N279, K280 / 281 and S285 / 289 are highlighted in dotted boxes immediately to the left of the solid boxed region. [Diagram 3]A homogeneous fluorescence-based assay for screening primary B cell culture supernatants. Green dots represent those selected for hit picking, red ones were not selected. [Figure 4] ELISA data for TE10 v TE9 / 11 shown as fold change over background, wells selected for 3R specific are marked in red on the top left of the graph and for 4R specific in green on the bottom right. [Diagram 5] ELISA data for 0N3R v 0N4R shown as fold change over background, wells selected for 3R specific are marked in red on the top left and for 4R specific are marked in green on the bottom right. [Figure 6] ELISA of neat TAP IgG expression product binding, one representative TAP expression per focus group. Values ​​are expressed as fold change over background binding. 3R and 4R specific antibodies selected for cloning are highlighted in red at the top left and green at the bottom right, respectively. Black dots represent wells containing antibodies that were not selected for cloning. [Figure 7] A: ELISA optical density (OD) at 630 nm of each cloning transient of rabbit isoform-specific antibodies. This represents the signal at 10 μg / ml of each clone. 4R-selective antibodies are shown in green and 3R-selective antibodies are highlighted in red. Clone 2 is not shown as it could not be repeatedly expressed. Clone 14 (VR7081) and clone 3 (VR7082) are boxed in red and green, respectively, and were selected as the preferred isoform-specific antibodies for further studies. B: Heavy and light chain CDR3 sequencing of cloned rabbit antibodies. Highlighted in green and red boxes are the CDR3 of antibodies selected as 3R and 4R specific by ELSA, respectively. [Figure 8]Exhaustive titration of VR7081 (A) and VR7082 (B) against all tau isoforms coated directly onto ELISA plates. VR7081 is selective for the tau 3 isoform and VR7082 is selective for the tau 4 isoform. [Figure 9] Flow cytometry assay, VR7081 (A) and VR7082 (B) titrated against intracellularly expressed tau isoforms. [Figure 10] Western blots from single isoform-overexpressing cell lysates probed with VR7081 (A) or VR7082 (B). [Figure 11] Western blots on 2N3R or 2N4R tau-containing lysates blotted with non-tau-reactive rabbit (A) or mouse (B) IgG. [Figure 12] Western blots from single isoform-overexpressing cell lysates probed with VR7081 (A) or VR7082 (B) formatted as mouse IgG. [Figure 13] Immunofluorescence staining of CHOK1 cells expressing 0N3R or 0N4R tau co-stained with VR7081 (AF647) and polyclonal anti-total tau antibody (AF488). [Figure 14] Immunofluorescence staining of CHOK1 cells expressing 0N3R or 0N4R tau co-stained with VR7082 (AF647) and polyclonal anti-total tau antibody (AF488). [Figure 15] Immunofluorescence staining of iPSC-derived neurons with VR7081. Immunofluorescence staining of non-mutated (control) iPSC-derived neurons and monoallelic 10+16 MAPT mutant neurons stained with VR7081 (3R-tau) AF647. In the merged images, DAPI is shown in blue and tau staining is shown in red. Green arrows indicate examples of axonal staining with VR7081. [Figure 16]Immunofluorescence staining of iPSC-derived neurons with VR7082. Immunofluorescence staining of non-mutated (control) iPSC-derived neurons and monoallelic 10+16 MAPT mutant neurons stained with VR7082 (4R-tau) AF647. In the merged images, DAPI is shown in blue and tau staining is shown in red. Green arrows indicate examples of axonal staining with VR7082. [Figure 17] Simple Western immunoblots probed with either VR7081 (A) or VR7082 (B). [Figure 18] Western blot of human brain lysates and recombinant taurander probed with VR7081 (A) and VR7082 (B). [Figure 19] scFv mouse Fc conversion confirming VR7082 binding profile. Diagram of rabbit IgG (A) and scFv-Ms-Fc (B) formats. VR7082 rabbit IgG ELISA (C), flow cytometry (E), binding assay and Western blot (G). VR7082 scFv-Ms-Fc ELISA (D), flow cytometry (F), binding assay and Western blot (H). [Figure 20] VR7082 scFv-GFP intrabody study. A Flow cytometry fluorescence of VR7082-scFv-GFP and mock transfected cells. B Western blot from four replicate transfections with VR7082-scFv-GFP. C Western blot of lysate pulldowns from four replicate VR7082-scFv-GFP transfected cells pulled down with either TE10 or control peptide. D Western blot band densitometry from VR7082-scFv-GFP pulldowns. [Figure 21] Degradation screening with potential VR7082 degrabody constructs. A. Representative western blots of total tau and GAPDH following co-transfection of 0N4R tau with degrabody or control intrabody fusions. B. Western blot band densitometry N=3 western blots. [Figure 22] Flow cytometry assay of degradation with VR7082-degrabody. A Representative flow cytometry gating and histogram plots demonstrating levels of tau staining for GFP control, non-degrading intrabody (A-1), degrabody (A-2), and degrabody in the presence of MG132 (A-3). B 0N4R and 0N3R tau staining in the presence and absence of MG132 are expressed as a percentage of the GFP non-degrading intrabody control for all degrabody constructs. [Diagram 23] VR7082-XIAP degradation assay in iPSC-derived neurons. A Gating strategy for GFP-based sorting. A-1 Identification of live cells by ToPro-3 exclusion. A-2 Identification of cells from debris / cellular vesicles by FSC-A v SSC-A. A-3 GFP positive and GFP negative sorting gates. A-4 Overlay plot of sorting gating including VR7082-XIAP IRES GFP AAV treated cells (green) and untreated cells (red). B Peggy Sue simple western of GFP negative sorted cells revealed with polyclonal anti-tau antibody. C Peggy Sue simple western of GFP positive sorted cells revealed with polyclonal anti-tau antibody. [Figure 24]VR7082-XIAP degrabody mitochondrial membrane polarization assay in iPSC-derived neurons. A Representative gating strategy for mitochondrial polarization assay of iPSC-derived neurons. A-1 Identification of nucleated cells by DAPI staining. A-2 Identification of intact cells by FSC-A v SSC-A. A-3 Identification of neurons by βIII tubulin staining. A-4 Mitochondrial membrane polarization assessment. B Representative mitochondrial membrane polarization histograms derived from the ratio of mitochondrial polarized / total mitochondrial content. B-1 WT cells iPSC-derived neurons. B-2 10+16 monoallelic 10+16 MAPT mutant iPSC-derived neurons (10+16 mono). B-3 A12 biallelic MAPT mutant iPSC-derived neurons (10+16 biallelic). C Mitochondrial polarization assay across each of the 3-iPSC-derived neuronal lines and AAV treatment groups. ANOVA is used to determine statistical significance between groups. D Normalized 3R tau staining across each of the 3-iPSC-derived neuronal lines and AAV treatment groups. ANOVA is used to determine statistical significance between groups. [Diagram 25] Comparison of the structures of different tau isoforms. Exon sequences present in different isoforms of tau protein are shown. [Figure 26]VR7082 antibody epitope analysis. The graph shows the relative binding of the VR7082 antibody to various tau mutants, expressed as a percentage, compared to binding to ON4R tau. The results demonstrate that VR7082 binding to tau is dependent on each of K294; D295; N296; and I297. When any of these residues are independently mutated to alanine, the antibody binding to ON4R tau is completely ablated. The results also show that binding is partially dependent on K298 and V300, as binding is reduced by approximately 50% when those positions are mutated to alanine. The results show that H299, P301, and G302 are not involved in VR7082 binding to tau, as there is no difference between the binding levels observed between the alanine mutations and ON4R tau when they are independently mutated to alanine. Finally, the results show that VR7082 is able to bind to both the P301S and P301L forms of tau protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Tau protein The antibodies and antigen-binding fragments thereof provided by the present invention bind tau protein. In a particularly preferred embodiment, the tau protein referred to herein is human tau protein. The amino acid numbering convention based on the largest human isoform of tau protein, namely the 2N4R isoform, is adopted herein. The amino acid sequence of the 2N4R isoform is provided as SEQ ID NO: 35. As further discussed herein, the antibodies and fragments provided are specific for either the 4R or 3R tau protein isoform.

[0024] Antibodies specific to certain tau proteins The present invention provides antibodies and antigen-binding fragments thereof specific for either the 4R or 3R tau protein isoform. As discussed further below, typically, antibodies and antigen-binding fragments thereof specific for the 4R tau protein isoform bind to a region of the 4R tau protein isoform that includes at least a portion of the region encoded by exon 10 of the tau gene (MAPT gene), which region is unique to the 4R tau protein isoform. Conversely, typically, antibodies or antigen-binding fragments of the present invention specific for the 3R tau protein isoform recognize a portion of the protein that includes a bridging sequence encoded by the junction of exons 9 and 11 of the tau gene.

[0025] In one embodiment, a particular advantage of the present invention is that the antibody or antigen-binding fragment thereof binds 4R or 3R tau protein isoforms in physiological conditions, e.g., when the protein is not in a denatured form. For example, the antibody or antigen-binding fragment thereof of the present invention may specifically bind 4R or 3R tau protein isoforms in intact cells, e.g., as determined by immunofluorescence. In one embodiment, the protein can be detected in a flow cytometric analysis of cells. In another embodiment, it may be used to detect the protein in tissue samples, e.g., by immunofluorescence. Alternatively, it may be possible to detect the protein by immunohistochemistry. In a preferred embodiment, it may be possible to detect tau protein via Western blot, in particular by Peggy Sue's Simple Western.

[0026] In one embodiment, "specifically binds" as used herein means that the antibody or antigen-binding fragment thereof binds at least 10 times stronger to both the 4R and 3R tau isoforms for which it is specific. For example, it may bind 50, 100, 200 or 500 times stronger to 4R tau than to the 3R tau protein isoform. In another embodiment, it may bind 50, 100, 200 or 500 times stronger to 3R tau than to the 4R tau protein isoform. In one embodiment, the binding is at least 1000 times stronger to both the 4R and 3R tau protein isoforms for which it is specific. In one embodiment, the K of binding is at least 10 times stronger to both the 4R and 3R tau protein isoforms for which it is specific. D The value is at least 10-fold, 50-fold, 100-fold, 500-fold or 1000-fold lower for either the 3R or 4R tau protein for which it is specific. In another embodiment, the binding may be 5,000-fold, 10,000-fold or 50,000-fold stronger for the form of tau protein for which it is specific. In one embodiment, the antibody or antigen-binding fragment thereof of the invention does not bind at all or does not significantly bind other forms of tau protein. In another embodiment, the level of binding to non-tau proteins is at least as low as the level of binding to forms of tau for which the antibody is not specific. In one embodiment, the antibody or antigen-binding fragment thereof does not bind or does not significantly bind non-tau proteins. The level of binding may be measured by any of the techniques well known in the art, such as surface plasmon resonance or other related techniques disclosed herein. In one embodiment, the level of specificity described above relates to the specificity of the 4R tau protein isoform over the 3R tau protein isoform. In another embodiment, said level of specificity relates to specificity for 3R tau protein isoforms over 4R tau protein isoforms.

[0027] As shown in FIG. 25, 4R tau protein isoforms represent a group that all have the amino acid sequence encoded by exon 10 of the tau gene, whereas 3R tau protein isoforms represent a group of isoforms that all share the common feature of lacking the amino acid sequence encoded by exon 10. These features show how tau proteins are grouped into 4R and 3R tau protein isoforms, but within each of these names, different isoforms exist depending on whether the amino acid sequence encoded by exons 2 and 3 is present. Thus, for example, 4R tau protein isoforms are 2N4R, which has the sequence encoded by exons 2 and 3; 1N4R, which has the sequence encoded by exon 2 but not by exon 3; and 0N4R, which has neither the sequence encoded by exon 2 nor the sequence encoded by exon 3. All three of 2N4R, 1N4R, and 0N4R contain the sequence encoded by exon 10, and are therefore referred to as 4R tau proteins. Thus, when reference is made herein to an antibody or antigen-binding fragment thereof that specifically binds a 4R tau protein isoform, it typically means that the antibody or fragment binds all three of 2N4R, 1N4R, and 0N4R, but does not bind any of the 3R tau protein isoforms 2N3R, 1N3R, and 0N3R. In embodiments where the antibody or antigen-binding fragment specifically binds a 3R tau protein isoform, the converse is also true, so that the antibody will bind 2N3R, 1N3R, and ON3R, but not 2N4R, 1N4R, and 0N4R.

[0028] In one embodiment, the affinity of the antibody or antigen-binding fragment thereof specific for either 4R or 3R tau protein isoform is about 100 nM or more, such as about 50 nM, 20 nM, 10 nM, 1 nM, 500 pM, 250 pM, 200 pM, 100 pM or more. In one embodiment, the binding affinity is 50 pM or more. In one embodiment, the affinity of the antibody specific for either 4R or 3R tau protein can be less than 1 μM, less than 750 nM, less than 500 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 10 pM, less than 1 pM, or less than 0.1 pM. In some embodiments, the Kd is about 0.1 pM to about 1 μM.

[0029] Tau proteins contain repeats with a high level of sequence identity in the MTBR region. In the case of 4R, there are four such repeats, and in the case of 3R, there are three such repeats. In one embodiment, an antibody or antigen-binding fragment thereof of the invention binds only to the repeat containing its epitope and not to other repeats. For example, in one embodiment, an antibody or antigen-binding fragment of the invention binds to the repeat containing the junction between exons 9 and 11, but not to other repeats, and is therefore specific for 3R tau. In another embodiment, an antibody or antibody fragment of the invention binds to a sequence within a repeat present in the sequence encoded by exon 10 of the tau gene, but not to the sequences of the other three repeats.

[0030] Tau4R-specific antibody In a particularly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention is specific for the Tau4R protein isoform. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds the Tau4R protein isoform and not the 3R Tau protein isoform. For example, it may bind to all isoforms of the Tau4R protein of a given species or subject, but not to any isoforms of the Tau3R protein of that species or subject. Thus, in one embodiment, it may bind to all of the 2N4R, 1N4R and 0N4R isoforms of the Tau4R protein, but not to any of the 2N3R, 1N3R and 0N3R isoforms of the Tau3R protein. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to the Tau4R protein isoforms from human, rat and mouse, but not to the 3R Tau protein isoforms from those species.

[0031] A common difference between 4R and 3R tau protein isoforms is that the latter lack the amino acid sequence encoded by exon 10 of the tau gene. Thus, in one embodiment, an antibody or antigen-binding fragment thereof specific for a 4R tau protein isoform binds to a region unique to the 4R tau protein isoform and thus encoded by exon 10. In a preferred embodiment, an antibody or antigen-binding fragment thereof specific for a 4R tau protein isoform binds to an epitope that is entirely or partially within the sequence encoded by exon 10 of the tau gene. In a preferred embodiment, an antibody or antigen-binding fragment of the invention binds to a region within the amino acid sequence encoded by exon 10 of the tau gene and in particular does not bind to the 3R tau protein isoform.

[0032] In one embodiment, the antibody or antigen-binding fragment thereof of the invention specific for the 4R tau protein isoform is capable of binding a peptide comprising at least a portion of the amino acid sequence encoded by exon 10 of the tau gene. In one embodiment, the peptide sequence is entirely within the region encoded by exon 10 of the tau gene. In one embodiment, the peptide is less than 15 amino acids in length. In one embodiment, it is less than 14, 13, 12, 11, or 10 amino acids in length. In one embodiment, the antibody or antigen-binding fragment thereof binds a peptide sequence that is 9 amino acids in length. In a preferred embodiment, it is capable of binding a peptide consisting of amino acids 294 to 302 of the 4R tau protein isoform. In one embodiment, the antibody or antigen-binding fragment thereof is capable of binding such a peptide when the peptide is in linear form. In one embodiment, the antibody or antigen-binding fragment thereof of the invention is capable of binding a peptide corresponding to an amino acid sequence encoded by exon 10 of the tau gene in a length in which only one epitope is present. In one embodiment, the antibody or antigen-binding fragment thereof is capable of binding to such peptides when conjugated to a carrier, such as KLH, ovalbumin, or BSA, particularly when the peptide is in a linear form.

[0033] In a preferred embodiment, the antibody or antigen-binding fragment thereof is capable of specifically binding to 4R Tau protein isoforms, regardless of whether the protein has any post-translational modification at one or more of amino acid positions 279, 280, 281, 285 and 289. In one embodiment, it is capable of specifically binding to Tau4R protein isoforms, regardless of whether the protein is modified at any or all of amino acid positions 279, 280, 281, 285 and 289. Frequent post-translational modifications at these positions include glycosylation, phosphorylation and acetylation. The most frequent post-translational modification at position 279 is glycosylation, the most frequent post-translational modification at positions 281 and 289 is acetylation, and the most frequent post-translational modification at positions 285 and 289 is phosphorylation. In one embodiment, the antibody or antigen-binding fragment thereof of the invention is capable of binding Tau4R protein isoforms, regardless of whether there is glycosylation, acetylation, or phosphorylation at those positions. In one embodiment, an antibody or antigen-binding fragment thereof of the present invention is capable of binding Tau4R protein isoforms regardless of whether the protein is glycosylated at position 279, acetylated at position 281, acetylated at position 289, phosphorylated at position 285, and / or phosphorylated at position 289. In other words, the antibody binds Tau4R protein both when it is glycosylated, acetylated and / or phosphorylated at these positions and when it is not glycosylated, acetylated and / or phosphorylated at these positions.

[0034] In one embodiment, an antibody or antigen-binding fragment specific for a 4R tau isoform binds to repeat 2 within the 4R tau protein sequence, but not to the other three repeats present in the 4R tau protein isoform. Each repeat corresponds to a different tau exon; repeat 1 (exon 9) (amino acids 242-273); repeat 2 (exon 10) (amino acids 274-304); repeat 3 (exon 11) (amino acids 305-335); repeat 4 (exon 12) (336-367). Thus, in one embodiment, the antibody may bind to the sequence encoded by exon 10, but not by the sequence encoded by exons 9, 11 and 12 of the 4R tau protein isoform.

[0035] In one embodiment, the antibody or antigen-binding fragment thereof specific for 4R tau protein is: (a) binds to an amino acid region encoded by exon 10 of Tau, where the Tau4R protein contains a post-translational modification at one or more of amino acid positions 279, 280, 281, 285, and 289 of Tau4R; (b) binds to a peptide comprising or consisting of amino acids 294 to 302 of the Tau4R protein; and / or (c) binds to a peptide corresponding to a single epitope from the amino acid sequence encoded by exon 10 of tau; or (d) cross-blocks or is cross-blocked by any of the antibodies or fragments of (a) to (c).

[0036] In one embodiment, the antibody or antigen-binding fragment thereof of the invention comprises: (a) comprising one or more of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 3, 5 and 7, and one or more of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 11, 13 and 15, preferably comprising the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 3, 5 and 7, and the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 11, 13 and 15, or (b) comprising the light chain variable region and the heavy chain variable region of SEQ ID NOs: 1 and 9; (c) an intrabody, preferably comprising the sequence of SEQ ID NO: 17 or a sequence having at least 95% sequence identity thereto, and still capable of specifically binding to the Tau 4R isoform; or (d) cross-blocks or is cross-blocked by any of the antibodies or fragments of (a) to (c).

[0037] In one embodiment, the antibody or antigen-binding fragment of the invention comprises light chain CDR1, CDR2 and CDR3, or CDRs, corresponding to the sequences of SEQ ID NOs: 3, 5 and 7, where each CDR has no more than two amino acid sequence changes compared to the sequences of SEQ ID NOs: 3, 5 and 7, and the antibody is still able to specifically bind 4R tau protein isoforms. In one embodiment, the sequence changes are conservative amino acid sequence changes. In another embodiment, the antibody or antigen-binding fragment of the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences, or CDRs, corresponding to the sequences of SEQ ID NOs: 11, 13 and 15, where each CDR has no more than two amino acid sequence changes compared to the sequences of SEQ ID NOs: 11, 13 and 15, and the antibody is still able to specifically bind 4R tau protein isoforms. In one embodiment, the antibody or antigen-binding fragment thereof comprises both such light and heavy chain CDRs, and in a preferred embodiment, the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 3, 5, and 7, and the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 11, 13, and 15. In one embodiment, the light chain of the antibody may comprise one or more of the FW1, FW2, FW3, and FW4 framework regions of SEQ ID NOs: 2, 4, 6, and 8. In one embodiment, it may comprise all four of the FW1, FW2, FW3, and FW4 of SEQ ID NOs: 2, 4, 6, and 8. In another embodiment, the heavy chain of the antibody may comprise one or more of the FW1, FW2, FW3, and FW4 framework regions of SEQ ID NOs: 10, 12, 14, and 16. In one embodiment, it may comprise all four of those framework regions. In another embodiment, it may comprise both such heavy and light chain framework regions. In one embodiment, an antibody or antigen-binding fragment thereof comprising the above sequences is humanized, e.g., such that all of the antibody's sequences, except for the CDR sequences, are human sequences, and the antibody or antigen-binding fragment thereof still specifically binds the 4R Tau protein isoform. In another embodiment, all of the antibody's sequences, apart from the variable regions, may be human.

[0038] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO: 1 or a light chain variable region having at least 90% sequence identity thereto, wherein the antibody or antigen-binding fragment is still capable of specifically binding a 4R Tau protein isoform. In one embodiment, the light chain variable region has at least 95%, 98% or 99% sequence identity, or has 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid sequence changes compared to SEQ ID NO: 1, but is still capable of specifically binding a 4R Tau protein isoform. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region of SEQ ID NO: 9 or a heavy chain variable region having at least 90% sequence identity thereto, wherein the antibody or antigen-binding fragment is still capable of specifically binding a 4R Tau protein isoform. In one embodiment, the light chain variable region has at least 95%, 98% or 99% sequence identity or has 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid sequence changes compared to SEQ ID NO: 9 and still specifically binds the 4R tau isoform. In one embodiment, the sequence changes compared to the heavy and light chain sequences are only in the framework regions of the variable regions. In one embodiment, the antibody or antigen-binding fragment thereof comprises both the light chain variable region and the heavy chain variable region as described above. In one embodiment, the antibody or antigen-binding fragment of the invention comprises any of the light and / or heavy chain variable region sequences as described above, but they are humanized such that all of the sequences other than the CDR sequences are human.

[0039] As discussed further below, the antibodies or antigen-binding fragments of the present invention may be provided in a variety of antibody and antibody fragment formats, and those specific for the 4R Tau protein isoform may be provided in any of the antibody formats described herein, and they may also have any of the levels of sequence variation described herein, and any of the properties described herein.

[0040] Tau3R-specific antibody In a further preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention is specific for 3R tau protein isoform. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to 3R tau protein isoform and not to 4R tau protein isoform. For example, it can bind to all isoforms of 3R tau protein of a given species or subject, but not to any isoforms of 4R tau protein of that species or subject. Thus, in one embodiment, it can bind to all 2N3R, 1N3R and 0N3R isoforms of 3R tau protein, but not to any of 2N4R, 1N4R and 0N4R isoforms of 4R tau protein.

[0041] A common difference between 3R tau proteins and those of 4R tau is that the former lack the amino acid sequence encoded by exon 10 of the tau gene. Thus, in one embodiment, an antibody or antigen-binding fragment thereof specific for a 3R tau protein isoform binds to a region unique to the 3R tau protein isoform, in particular to a region comprising an amino acid sequence corresponding to the junction between exons 9 and 11 of the tau gene. In a preferred embodiment, the antibody or antigen-binding fragment of the invention binds to an epitope comprising said junction between exons 9 and 11.

[0042] In one embodiment, an antibody or antigen-binding fragment thereof specific for a 3R Tau protein isoform can bind a peptide comprising a sequence encoded by the junction between exons 9 and 11. In one embodiment, the peptide is less than 15 amino acids long. In one embodiment, it is 14 amino acids long. In a preferred embodiment, it can bind a peptide of 14 amino acids, including 7 amino acids from each side of the junction between exons 9 and 11. In one embodiment, an antibody or antigen-binding fragment thereof specific for a 3R Tau protein isoform can bind a protein comprising amino acids 268-274 and 306-312 of SEQ ID NO: 35, but not including amino acids 275-305 of SEQ ID NO: 35. In one embodiment, an antibody or antigen-binding fragment thereof specific for a 3R Tau protein isoform can bind a peptide comprising the amino acid sequence of SEQ ID NO: 36. In one embodiment, an antibody or antigen-binding fragment thereof can bind such a peptide when the peptide is in cyclic form, particularly when conjugated to a carrier such as KLH, ovalbumin, or BSA.

[0043] In one embodiment, the antibody or antigen-binding fragment thereof specific for the 3R tau protein isoform is (a) comprising one or more of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 20, 22 and 24, and one or more of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 28, 30 and 32, preferably comprising the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 20, 22 and 24, and the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 28, 30 and 32; and / or (b) comprising the light chain variable region and the heavy chain variable region of SEQ ID NOs: 18 and 26; (c) an intrabody, preferably comprising a sequence of SEQ ID NO: 34 or a sequence having at least 95% sequence identity thereto, and still capable of specifically binding to the Tau 3R isoform, (d) cross-blocks or is cross-blocked by any of the antibodies or fragments of (a) to (c).

[0044] In one embodiment, the antibody or antigen-binding fragment of the invention comprises light chain CDR1, CDR2 and CDR3, or CDRs corresponding to the sequences of SEQ ID NOs: 20, 22 and 24, where each CDR has no more than two amino acid sequence changes compared to the sequences of SEQ ID NOs: 20, 22 and 24, and the antibody is still able to specifically bind 3R tau protein isoforms. In one embodiment, the sequence changes are conservative amino acid sequence changes. In another embodiment, the antibody or antigen-binding fragment of the invention comprises heavy chain CDR1, CDR2 and CDR3, or CDRs corresponding to the sequences of SEQ ID NOs: 28, 30 and 32, where each CDR has no more than two amino acid sequence changes compared to the sequences of SEQ ID NOs: 28, 30 and 32, and the antibody is still able to specifically bind 3R tau protein isoforms. In one embodiment, the antibody or antigen-binding fragment thereof comprises both such light and heavy chain CDRs, and in a preferred embodiment, the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 20, 22, and 24, and the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 28, 30, and 32. In one embodiment, the light chain of the antibody may comprise one or more of the FW1, FW2, FW3, and FW4 framework regions of SEQ ID NOs: 19, 21, 23, and 25. In one embodiment, it may comprise all four of the FW1, FW2, FW3, and FW4 of SEQ ID NOs: 19, 21, 23, and 25. In another embodiment, the heavy chain of the antibody may comprise one or more of the FW1, FW2, FW3, and FW4 framework regions of SEQ ID NOs: 27, 29, 31, and 33. In one embodiment, it may comprise all four of those framework regions. In another embodiment, it may comprise both such heavy and light chain variable regions. In one embodiment, an antibody or antigen-binding fragment thereof comprising the above sequences is humanized, e.g., such that all of the sequences of the antibody other than the CDR sequences are human sequences, and the antibody or antigen-binding fragment thereof is still able to specifically bind a 3R tau protein isoform.

[0045] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO: 18 or a light chain variable region having at least 90% sequence identity with SEQ ID NO: 18, where the antibody or antigen-binding fragment is still capable of specifically binding a 3R Tau protein isoform. In one embodiment, the light chain variable region has at least 95%, 98% or 99% sequence identity, or has 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid sequence changes compared to SEQ ID NO: 18, but is still capable of specifically binding a 3R Tau protein isoform. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region of SEQ ID NO: 26 or a heavy chain variable region having at least 90% sequence identity thereto, where the antibody or antigen-binding fragment is still capable of specifically binding a 3R Tau protein isoform. In one embodiment, the light chain variable region has at least 95%, 98% or 99% sequence identity or 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid sequence changes compared to SEQ ID NO: 18 or 26 and still can specifically bind 3R tau protein isoforms. In one embodiment, the sequence changes compared to the heavy and light chain sequences are only in the framework regions of the variable regions. In one embodiment, the antibody or antigen-binding fragment thereof comprises both the light chain variable region and the heavy chain variable region as described above. In one embodiment, the antibody of the antigen-binding fragment of the invention comprises any of the light and / or heavy chain variable region sequences as described above, but they are humanized such that all of the sequences other than the CDR sequences are human.

[0046] As discussed further below, the antibodies or antigen-binding fragments of the present invention may be provided in a variety of antibody and antibody fragment formats, and those specific for the 3R tau protein isoform may be provided in any of the antibody formats described herein, and they may also have any of the levels of sequence variation described herein.

[0047] Epitope In one embodiment, the antibody or antigen-binding fragment thereof of the present invention binds to the same epitope or at least substantially the same epitope as one of the antibodies or antigen-binding fragments disclosed herein. Specific regions or epitopes of tau can be identified by any suitable epitope mapping method known in the art in combination with any one of the antibodies provided by the present disclosure. An example of such a method includes screening peptides of various lengths derived from tau protein for binding to the tau-binding antibody or binding fragment thereof of the present disclosure with the smallest fragment capable of specifically binding to the antibody containing the sequence of the epitope recognized by the tau-binding antibody or binding fragment thereof. In a particularly preferred embodiment, residues forming part of the epitope of the antibody can be identified using mutational analysis, for example, by exchanging residues of tau protein to alanine and determining the effect on the binding of the antibody or antigen-binding fragment thereof to tau protein, particularly 4R tau protein.

[0048] In one embodiment, at least one of K294, D295, N296, I297, K298, and V300 may be present in the antibody epitope, particularly where those amino acid positions are defined relative to those of SEQ ID NO:35.

[0049] In a particularly preferred embodiment, an antibody or antigen-binding fragment thereof is provided that binds to an epitope comprising at least one of the amino acid residues K294, D295, N296 and I297. Preferably, an antibody or antigen-binding antibody is provided that binds to an epitope comprising at least two of the amino acid residues K294, D295, N296 and I297. Thus, more preferably, the antibody or antigen-binding fragment thereof binds to an epitope comprising at least three of the amino acid residues K294, D295, N296 and I297. In an even more preferred embodiment, the antibody or antigen-binding fragment thereof binds to an epitope comprising all four of the amino acid residues K294, D295, N296 and I297.

[0050] In a further preferred embodiment, the antibody or antigen-binding fragment thereof may bind to an epitope that further comprises amino acid residues K298 and V300 of tau protein. In one preferred aspect, the antibody or antigen-binding fragment thereof binds to an epitope that comprises all of residues K294, D295, N296, I297, K298 and V300 of tau protein.

[0051] In any of the above embodiments, the antibody or antigen-binding fragment thereof may preferably be a 4R-specific antibody. Thus, in one embodiment, the antibody or antigen-binding fragment thereof is a 4R-specific antibody that binds to an epitope comprising K294, D295, N296, and I297 of 4R tau protein. In another preferred embodiment, the antibody or antigen-binding fragment thereof is a 4R-specific antibody that binds to an epitope comprising K294, D296, N396, I297, K298, and V300 of 4R tau protein.

[0052] In one embodiment, the amino acids H299, P301 and G302 are not involved in binding of the provided antibody or antigen-binding fragment thereof to tau protein. In one embodiment, the antibody or antigen-binding fragment thereof is a 4R-specific antibody that binds to an epitope comprising K294, D296, N396, I297, K298 and V300 of 4R tau protein, while the H299, P301 and G302 residues are not involved in binding of the antibody or antigen-binding fragment thereof to tau protein.

[0053] In one embodiment, the entire epitope of the antibody or antigen-binding fragment thereof comprises amino acids in the region from residues K294 to V300 of the Tau4R protein. In one embodiment, the amino acids of the epitope of the antibody or antigen-binding fragment thereof are exclusively in that region. In another embodiment, at least the residues forming the epitope are in that region.

[0054] Any of the mentioned antibodies or antigen-binding fragments thereof can bind to such epitopes. In one embodiment, such antibodies can be chimeric, humanized or fully human monoclonal antibodies or can be used to obtain chimeric, humanized or fully human monoclonal antibodies. In a particularly preferred embodiment, the antibody or antigen-binding fragment thereof that binds to the epitope is a degrabody.

[0055] Antibody Format The antibody or antigen-binding fragment of the present invention may be provided in any suitable format. In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention may be an antibody or fragment thereof of the IgG class. In one embodiment, it may be an IgG1, IgG2, IgG3, or IgG4 isotype antibody, in particular IgG1. In another embodiment, the antibody or antigen-binding fragment thereof of the present invention may be an antibody of the IgA, IgE, IgD, or IgM class.

[0056] The present disclosure relates to antibodies and antibody fragments. Whenever an antibody is mentioned, an antibody fragment can be used unless otherwise specified. Thus, the antibody of the present invention can include a complete antibody having full-length heavy and light chains, or can be an antigen-binding fragment, such as a Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibody (e.g., VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibody, Bis-scFv, diabody, triabody, tetrabody, or epitope-binding fragment of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3),209-217). Methods for generating and producing antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include Fab and Fab' fragments, which are described in International Patent Applications, WO 2005 / 003169, WO 2005 / 003170 and WO 2005 / 003171. Multivalent antibodies may comprise multiple specificities, e.g. bispecificities, or may be monospecific (see, for example, WO 92 / 22853, WO 05 / 113605, WO 2009 / 040562 and WO 2010 / 035012).Examples of possible antibody formats are known in the art and are disclosed, for example, in the review articles "The coming of Age of Engineered Multivalent Antibodies", Nunez-Prado et al Drug Discovery Today Vol 20 Number 5 Mar 2015, pages 588-594, D. Holmes, Nature Rev Drug Disc Nov 2011:10;798, Chan and Carter, Nature Reviews Immunology vol.10, May 2010,301, which are incorporated herein by reference.

[0057] In one embodiment, antibody formats include those known in the art and described herein, for example, the antibody molecule format includes or is one selected from the group consisting of: diabody, BYbe, scdiabody, triabody, tribody, tetrabody, TrYbe, tandem scFv, FabFv, Fab'Fv, FabdsFv, Fab-scFv, Fab-dsscFv, Fab-(dsscFv)2, diFab, diFab', tandem scFv-Fc, scFv-Fc-scFv, scdiabody-Fc, scdiabody-CH3, Ig-scFv, scFv-Ig, V-Ig, Ig-V, Duobody, and DVDIg.

[0058] In one particularly preferred embodiment, the antibody of the invention is an scFv-Ms-Fc format antibody.

[0059] In one embodiment, the antibodies, or antigen-binding fragments thereof, of the invention are small enough to cross the blood-brain barrier (BBB).

[0060] In one embodiment, the antibody or antigen-binding fragment thereof is a bispecific or multispecific antibody, in which at least one of the antigen-binding sites is specific for the 4R or 3R tau protein isoform. Thus, in one preferred embodiment, it is such a bispecific antibody.

[0061] In a particularly preferred embodiment, the antibody or antigen-binding fragment thereof is one that can be expressed intracellularly, in particular an intrabody. An intrabody is an antibody or antibody fragment that is capable of expression, correct folding and antigen binding within a cell. Intrabodies are typically capable of correct folding in the reducing environment of the cytoplasm due to the lack of inter- and intrachain disulfide bonds. Intrabodies are an alternative format for therapeutic antibodies of the invention, as in some instances they may be easier to target to cells than administration of the antibody itself.

[0062] In one embodiment, the antibody of the present invention is a targeting intrabody. For example, in one embodiment, the ER-intrabody is targeted to the ER lumen using KDEL or SEKDEL sequences at its C-terminus (as described in Wheeler, Chen, and Sane 2003 Mol Ther, 8:355-66; Lewis and Pelham 1992 Cell, 68:353-64, both of which are incorporated by reference in their entirety). In one embodiment, the intrabody of the present invention is targeted to the cytoplasm, for example by removal of the leader sequence (such a particular class of intrabodies may be referred to as cellular intrabodies (intrabodies)). The intrabodies used in the examples of this application are expressed in the cytoplasm of cells. In another embodiment, an intrabody of the invention is targeted to the mitochondria or nucleus by addition of an appropriate targeting signal (e.g., as described in Biocca, Neuberger, and Cattaneo 1990 EMBO J, 9:101-8, incorporated by reference in its entirety).

[0063] IgG-derived intrabodies can be made using the variable region domains of IgG linked via a Gly4Ser peptide linker to create scFv fragments (Bird et al. 1988 Science, 242:423-6, incorporated by reference). These can then be used directly as intrabodies since they no longer require disulfide bond formation for correct folding. In one embodiment, the intrabodies of the invention are single domain antibody intrabodies (intrabodies). For example, in one embodiment, the intrabodies of the invention are heavy chain only antibodies, such as camelid heavy chain only antibodies (HCabs) (Hamers-Casterman et al. 1993, Nature, 363:446-8, incorporated by reference in its entirety). The single variable region of HCabs (known as VHH) can be used as intrabodies.

[0064] In one embodiment, the antibodies or antigen-binding fragments of the present invention are non-disulfide stabilized scFvs and can be used as intrabody antibodies (intrabodies). In the scFv format, the heavy and light chain variable regions can be physically linked by a flexible linker. This ensures that the heavy and light chains pair correctly in the cell to form a binding moiety without the need for cysteine ​​bridges between the heavy and light chains.

[0065] In a preferred embodiment, the provided antibody or antigen-binding fragment thereof may comprise a degradation domain. Examples of possible degradation domains that may be used include the C-terminal sequence of ornithine decarboxylase (ODC), human FkBP-12 protein (FkBP), the C-terminus of Hsc70 interacting protein (CHIP), X-linked inhibitor of apoptosis protein (XIAP), von Hippel-Lindau protein (VHL), and the N-terminus of Simb protein (NSImb). The degradation domain may, in one embodiment, be dependent on the ubiquitin proteasome for degradation. In another embodiment, it is not so dependent. In one embodiment, the degradation domain is present as an N-terminal fusion to the rest of the antibody, e.g., a C-terminal fusion to an scFv. In another embodiment, it is an N-terminal fusion, e.g., an N-terminal fusion to an scFv.

[0066] In one embodiment, the antibody or antigen-binding fragment of the present invention is an intracellular degrading antibody, also known as a targeted degrading intrabody (intrabody) or degrabody. In the degrabody format, an intracellular antibody (intrabody) is linked to a degradation domain, so that the degrabody is in effect a specific type of intracellular antibody (intrabody). Thus, in a particularly preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof is a degrabody.

[0067] In one embodiment, the antibody or antigen-binding fragment thereof of the invention is a rabbit antibody or fragment thereof. In another embodiment, the antibody or antigen-binding fragment of the invention comprises rabbit variable regions, with the remaining sequences of the antibody being mouse sequences. In another embodiment, the CDRs of the antibody are rabbit CDRs, and the remainder of the antibody is mouse. In a further embodiment, the antibody is a fully human antibody. In a further embodiment, all sequences of the antibody other than the CDR sequences are human sequences.

[0068] variant In one embodiment, an antibody or antibody fragment provided by the present invention, rather than the specific sequences described herein, may have a certain level of sequence identity or number of amino acid sequence changes compared to the specific sequence, provided that the antibody or fragment is still capable of specifically binding to either the 4R or 3R tau protein isoform it is intended to be specific for. In another embodiment, a nucleic acid sequence may have a certain level of sequence identity to one of the specific sequences described herein, provided that it still encodes an antibody or fragment thereof, or a component thereof, that is still capable of specifically binding to either the 4R or 3R tau protein isoform it is intended to be specific for.

[0069] The degree of identity and similarity can be easily calculated (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; BLAST™ software available from NCBI (Altschul, S. F. al.,1990,J.Mol.Biol.215:403-410;Gish,W.&States,DJ1993,Nature Genet.3:266-272.Madden,TLet al.,1996,Meth.Enzymol.266:131-141;Altschul,SFet al.,1997,Nucleic Acids Res.25:3389-3402; Zhang, J. & Madden, TL1997, Genome Res.7:649-656,).

[0070] Thus, the disclosure extends to novel polypeptide sequences disclosed herein and sequences that are at least 80% similar or identical thereto, such as sequences that are 85% or more similar or identical, such as 90% or more, particularly 95%, 96%, 97%, 98% or 99% or more similar. As used herein, "identity" indicates that at any particular position in the aligned sequences, the amino acid residues are identical between the sequences. In one embodiment, the sequences can have one of these levels of sequence identity, so long as the encoded antibody or fragment is still able to specifically bind the 4R or 3R tau protein isoform.

[0071] A particular amino acid sequence may differ from one of the particular amino acid sequences described herein by up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid sequence changes, provided that it is still capable of specifically binding to a 4R or 3R tau protein isoform. In one embodiment, it may differ from a particular sequence by the number of sequence changes, and the sequence changes are conservative.

[0072] In addition to variant sequences defined by percentage identity or number of sequence changes, the present invention further provides antibodies or antigen-binding fragments defined by their ability to cross-block one of the specific antibodies or fragments described herein. The antibodies may also have one of the recited levels of sequence identity or number of sequence changes. Cross-blocking antibodies can be identified using any suitable method in the art, for example, by using competitive ELISA or BIAcore assays, where binding of the cross-blocking antibody to an antigen (a particular tau protein of interest, such as 4R or 3R, or one of the peptides from the peptides discussed herein) prevents binding of the antibody or antibody fragment of the present invention, or vice versa. In one embodiment, the antibody reduces binding of the specific antibody or antigen-binding fragment disclosed herein by at least 50%, 60%, 70%, 80%, 90% or more.

[0073] peptide The present invention also provides peptides that can be used to generate antibodies and antigen-binding fragments of such antibodies. In one embodiment, a peptide is provided that comprises a sequence bridging exons 9 and 10 of tau protein. For example, in one embodiment, a peptide is provided that comprises or consists of the sequence of SEQ ID NO: 36, also known as TE9 / 11 peptide. In another embodiment, the present invention provides a peptide sequence that corresponds to the amino acid sequence encoded by exon 10 of tau, indicating that the antibody is likely to specifically recognize the 4R tau protein isoform. In one embodiment, the peptide does not contain amino acids that represent post-translational modification sites in the naturally occurring 4R tau protein isoform. In one embodiment, a peptide is provided that comprises or consists of the amino acid sequence of SEQ ID NO: 37, also referred to as TE10 peptide.

[0074] In one embodiment, the peptide may contain additional amino acid residues not derived from tau protein, for example a terminal cysteine ​​that aids in conjugating the peptide to a carrier. In one embodiment, the peptide is conjugated to a carrier. For example, in one embodiment, the carrier protein is KLH, ovalbumin or BSA. In one embodiment, the peptide is conjugated to a carrier as a linear peptide, in particular the part of the peptide most likely to generate specific antibodies is further away from the carrier. In another embodiment, the peptide is a cyclic peptide, for example one that is conjugated to a carrier such that the amino acid residues most likely to generate antibodies specific to the 4R or 3R tau protein isoform are further away from the carrier.

[0075] In one embodiment, an antibody or an antigen-binding fragment thereof is provided that can be obtained by immunizing an animal with one of the above peptides, particularly one conjugated to a carrier. In one embodiment, the antibody or antibody fragment is obtained by such a method. In one embodiment, the immunized animal is a rabbit. Any suitable method can be used to identify the desired antibody from such an immunized animal, for example, screening by ELISA using a tau peptide or protein, or any of the other methods discussed herein.

[0076] In one embodiment, the antibody or antigen-binding fragment of the invention specific for the 3R tau protein isoform is not an antibody mentioned in de Silva et al. (2003) Neuropathology and Applied Neurobiology, 29:288-302. In one embodiment, the antibody or antigen-binding fragment is not the RD3 antibody disclosed in de Silva et al. In another embodiment, the antibody or antigen-binding fragment thereof is specific for the 4R tau protein isoform but is not an antibody mentioned in Croft et al. (2018) https: / / doi.org / 10.1371 / journal.pone.0195211. In one embodiment, the antibody or antigen-binding fragment thereof is specific for the 4R tau protein isoform but is not an antibody mentioned in either de Silva et al. (2013) or Croft et al. (2018).

[0077] Nucleic Acids, Vectors and Host Cells In a further aspect, there is provided a nucleotide sequence, e.g. a DNA sequence, encoding an antibody or antibody fragment of the invention as described herein. In one embodiment, there is provided a nucleotide sequence, e.g. a DNA sequence, encoding an antibody or fragment of the invention as described herein. In one embodiment, the nucleotide sequences are collectively present on a plurality of polynucleotides, which together can encode an antibody or antibody fragment of the invention.

[0078] The present invention also extends to a vector comprising a nucleotide sequence as defined above. The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One example of a vector is a "plasmid", which is a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be linked to the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell, where they are then replicated along with the host genome. In the present specification, the terms "plasmid" and "vector" can be used interchangeably, since the plasmid is the most commonly used form of vector. The general methods by which vectors can be constructed, transfected, and cultured are well known to those skilled in the art. In this regard, reference is made to the Maniatis Manual, Current Protocols in Molecular Biology, 1999, FMAusubel (ed), Wiley Interscience, New York, and Cold Spring Harbor Publishing.

[0079] The term vector as used herein also includes, for example, particles that contain the vector, such as LNP (lipid nanoparticle) particles, in particular LNP-mRNA particles. It also includes viral particles that are used to transfer the vectors of the invention.

[0080] The vector of the present invention may contain a selectable marker. The term "selectable marker" as used herein refers to a protein, the expression of which allows identification of cells transformed or transfected with a vector containing a marker gene. A wide range of selectable markers are known in the art. For example, typically, a selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector is introduced. A selectable marker may also be a visually identifiable marker, such as a fluorescent marker. Examples of fluorescent markers include rhodamine, FITC, TRITC, Alexa Fluors and various conjugates thereof. In one embodiment, a selectable marker may be flanked by sequences that allow removal of the marker, such as loxP or frt.

[0081] Host cells are also provided that contain one or more cloning or expression vectors that contain one or more DNA sequences encoding the antibodies or antigen-binding fragments thereof of the present invention. Any suitable host cell / vector system can be used for the expression of DNA sequences encoding the antibodies or fragments of the present invention. Bacterial, such as E. coli, and other microbial systems may be used, or eukaryotic, such as mammalian, host cell expression systems may be used. Suitable mammalian host cells include CHO, myeloma or hybridoma cells. Host cells that contain the nucleic acid molecules or vectors of the present invention are also provided.

[0082] Tau Detection and Diagnosis The antibody and antigen-binding fragment thereof of the present invention can be used in a diagnostic / detection kit. In one embodiment, the antibody or antibody fragment of the present invention is immobilized on a solid surface. The solid surface can be, for example, a chip or an ELISA plate. The binding molecules, particularly the antibodies, of the present invention can be conjugated to, for example, a fluorescent marker that facilitates the detection of bound antibody-antigen complexes. They can be used for immunofluorescence microscopy. Alternatively, the antibody or antigen-binding fragment thereof can also be used for Western blotting or ELISA.

[0083] In a particularly preferred embodiment, the ability to bind to 3R or 4R tau protein isoforms is measured, where tau protein is present in the same form as it is present physiologically, e.g. not in a denatured form. In a preferred embodiment, it is present intracellularly. In one embodiment, the antibody or its antigen-binding fragment binds the protein when it is present on a cell, and can therefore be used to detect the protein when it is present on a cell. In one embodiment, the antibody or its antigen-binding fragment can bind the protein when it is present in the extracellular space, e.g. in the form of secreted fibrils.

[0084] Any suitable method can be used to determine the binding of a given antibody or antigen-binding fragment thereof to 4R tau compared to 3R tau. In one embodiment, ELISA is used: for example, binding to immobilized tau4R protein can be compared to that seen with immobilized 3R tau protein. Binding to the peptides discussed herein can also be measured and compared by ELISA.

[0085] In another embodiment, Western blotting can be used to detect the binding of a given antibody or its antigen fragment to 4R and 3R tau protein isoforms. Such Western blots can be performed on any suitable material, for example cell lysates of cells known to express 4R or 3R or both tau protein isoforms. In one embodiment, the cells used are iPSC cells, particularly such cells known to express specific tau proteins. In another embodiment, they are neurons differentiated from iPSCs. In one embodiment, Western blots are performed on tissue samples, for example brain tissue from a subject. In a preferred embodiment, Peggy Sue's Simple Western is used.

[0086] In another embodiment, the ability of an antibody or its antigen-binding fragment to bind a particular form of tau protein is measured by immunofluorescence. For example, the antibody or its antigen-binding fragment may be conjugated to a fluorescent dye itself, and the present invention also provides such a conjugated antibody or its antigen-binding fragment. Alternatively, the binding of an antibody or its antigen-binding fragment to tau protein may be identified and measured, for example, using a secondary antibody specific to the species of the primary antibody. In one embodiment, such immunofluorescence is performed on cells expressing 4R or 3R tau protein, for example CHO cells, particularly CHOK1 cells overexpressing the desired tau. In another embodiment, immunohistochemistry is performed on iPSC cells. In another embodiment, binding may be measured in tissue samples, for example by immunohistochemistry (IHC) by fluorescence or other means.

[0087] In a particularly preferred embodiment, iPSCs (induced pluripotent stem cells) are used to evaluate the antibodies or antigen-binding fragments of the invention. In another preferred embodiment, neurons are used, for example neurons obtained by differentiating iPSCs. In a preferred embodiment, cells are isolated from a subject with a particular disorder, for example any of the disorders mentioned herein, and used to evaluate the antibodies or antigen-binding fragments thereof of the invention. In one embodiment, fibroblasts are isolated from a subject with a tauopathy and used to generate iPSCs and then neurons. In one embodiment, the subject used to obtain cells is one that has a mutation in the tau gene (MAPT) gene, for example one of the mutations mentioned herein. For example, fibroblasts from such a subject can be used to generate iPSCs that will contain the same mutation in the tau gene as the subject. In a particularly preferred embodiment, such iPSCs are differentiated into neurons. In one embodiment, the neurons are cultured for at least 5, 6, 7, 8, or 9 months before being used. In one embodiment, they can be compared to a control cell line that does not have a tauopathy-associated mutation in the tau gene, for example cells isolated from a healthy subject that does not have such a mutation. In one embodiment, fibroblasts are isolated from healthy control subjects and used to generate iPSCs, which are then differentiated into neurons.

[0088] In another embodiment, rather than using cells from a subject with a particular disease mutation in the tau gene, a mutation associated with a tauopathy is engineered into a selected cell line, e.g., one that is an iPSC or is used to generate iPSCs, in one embodiment, the iPSCs are then differentiated into neurons that contain the mutation.

[0089] In one embodiment, the antibody or antigen-binding fragment of the invention is used to detect 4R tau protein isoforms or 3R tau protein isoforms. For example, the invention provides a method for detecting or measuring 4R tau protein isoforms, comprising: (a) contacting a test sample with an antibody or antigen-binding fragment thereof of the invention; and (b) detecting binding of the antibody. Any of the detection means discussed herein can be used, for example ELISA, immunofluorescence, flow cytometric analysis or immunohistochemistry can be used as detection means. In one embodiment, the test sample comprises, for example, a cell lysate or cells or tissue from the subject. In one embodiment, the test sample comprises neurons or lysates therefrom. In one embodiment, the test sample is brain tissue or lysate. In one embodiment, such a method may also include a positive control, for example one known to express 4R tau protein or one that expresses it at normal levels. Such a method may also include performing the method separately with an antibody that binds to all tau protein isoforms of the subject to obtain an indication of the amount of 4R tau protein compared to the total amount of tau protein present.

[0090] In another embodiment, an antibody or antigen-binding fragment thereof of the invention specific for a 4R tau protein isoform, but one specific for a 3R tau protein isoform, is used in the methods described above for a 4R tau protein isoform, excluding methods for the detection or measurement of a 3R tau protein isoform.

[0091] The method may include analyzing a test sample for both the level of 4R tau protein isoform and the level of 3R tau protein isoform. For example, the method may include testing two portions of the same test material, or staining simultaneously with both an antibody or fragment thereof specific for a 4R tau protein isoform and an antibody or fragment thereof specific for a 3R tau protein isoform. In one example, each is differentially labeled, for example with a different fluorescent dye, to allow direct comparison of the relative amounts of 4R tau protein and 3R tau protein.

[0092] Such methods for detecting the relative amounts of 4R and 3R tau protein isoforms can be used to detect an imbalance between the two, for example, by comparing samples from healthy subjects or samples from the same subjects before they develop a tauopathy. In one embodiment, the antibodies or fragments thereof of the present invention can be used to diagnose a tauopathy. In one preferred embodiment, the antibodies or fragments thereof of the present invention are used to diagnose conditions involving an imbalance between 4R and 3R tau protein isoforms. For example, frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD) are all believed to involve a predominance of 4R tau protein isoforms, and thus the present invention can be used as a method of diagnosing such conditions to detect such an imbalance. Conversely, Pick's disease is believed to be one in which there is more 3R tau protein than 4R tau protein. Thus, the present invention can also be used to diagnose the conditions based on identifying such an imbalance.

[0093] In a preferred embodiment, the antibody or antigen-binding fragment thereof is capable of specifically binding to the 4R tau protein isoform but not the 3R tau protein isoform in a Peggy Sue Simple Western. Others may exhibit such specificity when measured by flow cytometry, particularly for cells that overexpress tau. In another embodiment, such specificity is exhibited when used to analyze human brain samples, for example by Western blot. In a particularly preferred embodiment, the antibody or fragment is used to perform immunofluorescence on neurons, particularly those obtained from iPSCs that express the relevant tau protein at physiological levels.

[0094] In a preferred embodiment, the antibody or antigen-binding fragment thereof is capable of specifically binding to the 3R tau protein isoform but not the 4R tau protein isoform in a Peggy Sue simple Western blot. In others, it can show such specificity when measured by flow cytometry, particularly for cells that overexpress tau. In another embodiment, it shows such specificity when used to analyze human brain samples, for example by Western blot. In a particularly preferred embodiment, it shows such specificity when the antibody or fragment is used to perform immunofluorescence on neurons, particularly those obtained from iPSCs that express the relevant tau protein at physiological levels.

[0095] Pathological conditions In one embodiment, the antibody or antigen-binding fragment thereof can be used to treat or diagnose tauopathy. In one embodiment, the condition to be treated is a primary tauopathy. In one embodiment, the condition can include frontotemporal lobar degeneration (FTLD): for example, it can be characterized by neuronal tau inclusions with cell death predominantly in the frontal and temporal lobes of the brain. In another embodiment, the condition can be a secondary tauopathy, thus a condition in which tau pathology is observed in association with other brain pathologies.

[0096] The present invention may be used to treat or diagnose tauopathies in general, such as Alzheimer's disease (AD), and a range of other conditions including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease or frontotemporal dementia (FTD).

[0097] In one preferred embodiment of the invention, the condition to be treated or diagnosed is Alzheimer's Disease (AD), a secondary tauopathy.

[0098] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is used to shift the balance of 3R and 4R tau proteins in a subject with tauopathy. For example, an antibody specific for 3R tau protein can be used to shift the balance towards 4R tau protein. Alternatively, an antibody specific for 4R tau protein can be used to shift the balance towards 3R tau protein.

[0099] In a preferred embodiment, the condition treated or diagnosed by the present invention may be Pick's disease (PD). In a preferred embodiment, the antibody or antigen-binding fragment thereof specific for the 3R tau protein isoform may be used to treat Pick's disease. In a preferred embodiment, the antibody or antigen-binding fragment thereof may be used to shift the balance away from the 3R tau protein isoform. For example, such application of the present invention results in the reduction or elimination of Pick's globules. The antibody of the present invention specific for the 3R tau protein isoform may be used to eliminate, reduce or stabilize the loss of neurons and glia in the frontal, temporal and parietal lobes of the brain of a subject, particularly a subject with Pick's disease. In one embodiment, the 3R:4R ratio of tau protein isoforms in a subject may be shifted toward or to a level found in individuals without Pick's disease by treating the subject with the antibody of the present invention specific for the 3R tau protein isoform or antigen-binding fragment thereof.

[0100] In another embodiment, the antibody or antigen-binding fragment thereof of the present invention is used to treat conditions in which there is more 4R tau protein than in healthy subjects, particularly conditions in which the 4R:3R ratio of tau protein is shifted toward 4R tau protein. Thus, the antibody or antigen-binding fragment of the present invention specific for 4R tau protein can be used to treat 4R tauopathy. Examples of such conditions that can be treated include frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17); progressive supranuclear palsy (PSP); and corticobasal degeneration (CBD).

[0101] In one embodiment, the subject to be treated may have a MAPT mutation, i.e. a mutation in the tau gene. In one embodiment, the subject may have a splice mutation in or near intron 10, which results in excessive inclusion of exon 10 (MTBR R3) and an increase in 4R tau. In a preferred embodiment, the tauopathy involves a 10+16 mutation. In other words, in one embodiment, the mutation is an IVS 10+16 CT mutation. In one embodiment, the subject has a MAPT mutation that results in an increase in mRNA of 4R tau protein. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention specific for 4R tau protein can be used to treat such conditions. In one embodiment, the subject to be treated has FTDP-17, particularly early-onset FTDP-17.

[0102] The present invention can be applied to treat, prevent or diagnose tauopathies resulting from other mutations that may affect the inclusion of exon 10 and thus lead to an increase in 4R tau mRNA. These can be present within stem-loop structures, such as S305N and S305I, which destabilize the stem-loop in a similar manner to intronic mutations, leading to an increase in the inclusion of exon 10 (Hasegawa et al. 1998, FEBS letters, 443(2), 93-96; Kovacs et al., 2008, protein-based neuropathology and molecular classification of human neurodegenerative diseases: 251-272; Stanford et al., 2000, Brain, 123(5), 857-859).

[0103] Alternatively, mutations within the regulatory elements of exon 10 have been demonstrated to increase exon 10 inclusion. The mutations N279K and L284L strengthen the enhancer region within exon 10, leading to increased inclusion and excessive 4R tau expression (D'Souza and Schellenberg, 2002, Journal of Biological Chemistry, 277(29), 26587-99; D'Souza et al., 1999, Proceedings of the National Academy of Sciences, 96(10), 5598-603; Hasegawa et al., 1999, supra). Other mutations in exon 10 that have been demonstrated to increase 4R tau expression include N296N and N296H, which are hypothesized to either disrupt a silencer region (D'Souza and Schellenberg, 2002, supra) or create a new enhancer (Andrew Grover et al., 2002, Neuroscience letters, 323(1), 33-36). Somewhat counterintuitively, mutations in exons 12 and 13 (E342V and N410H, respectively) have been reported to increase expression of 4R tau in neurons (Lippa et al. 2000, Annals of neurology, 48(6), 850-58). Again, the present invention may be used to treat tauopathies associated with such mutations.

[0104] Mitochondrial impairment may also be a feature of tauopathies. In one embodiment, treatment with the antibody or antigen-binding fragment thereof of the present invention may help restore mitochondrial function in tauopathies. For example, in such tauopathies, the mitochondrial membrane potential may be affected, and treatment with the antibody or antigen-binding fragment thereof may help restore the mitochondrial membrane potential to or toward normal levels. In one embodiment, the subject to be treated is a subject exhibiting mitochondrial dysfunction. In one embodiment, the subject may be a subject exhibiting an altered mitochondrial membrane potential. In one embodiment, the subject exhibits a reduced membrane potential. In a preferred embodiment, the subject exhibits an elevated membrane potential, and in particular exhibits such an elevated membrane potential and has a 10+16 MAPT mutation. In one embodiment, such a subject may exhibit an excess of 4R tau protein isoforms. In another embodiment, the subject may have a P301L mutation and in particular exhibit a hyperpolarized mitochondrial membrane. In one embodiment, using the present invention may mean that the mitochondrial membrane potential may return to normal, or at least become close to normal.

[0105] In a particularly preferred embodiment, an antibody or antigen-binding fragment thereof capable of crossing the blood-brain barrier can be used. In a further preferred embodiment, rather than administering an antibody or antigen-binding fragment itself, one or more nucleic acids or one or more vectors of the invention can be administered, in one preferred embodiment, encoding an intracellular antibody (intrabody) or a degrabody, can be administered to the subject. In another embodiment, a host cell of the invention is used, capable of expressing an antibody or antigen-binding fragment of the invention.

[0106] Pharmaceutical Compositions In one aspect, a pharmaceutical composition is provided that comprises: (a) an antibody or antigen-binding fragment thereof, one or more nucleic acid molecules, or one or more vectors of the invention; and (b) a pharma- ceutically acceptable carrier or diluent. In a preferred embodiment, the pharmaceutical composition comprises an antibody or an antigen-binding fragment. The composition may be in solid or liquid form, inter alia, in the form of a powder, tablet, solution, or aerosol.

[0107] Also provided is an antibody or antigen-binding fragment thereof, one or more nucleic acid molecules, one or more vectors, or pharmaceutical composition of the invention for use in a method of treatment or diagnosis of the human or animal body. Further provided is the use of an antibody or antigen-binding fragment thereof, one or more nucleic acid molecules, one or more vectors, or pharmaceutical composition of the invention for the manufacture of a medicament for the treatment of a pathological condition or disorder. In one embodiment in which a therapeutic agent of the invention is administered to a subject who is also administered a second therapeutic agent, the two may be administered, for example, simultaneously, sequentially, or separately. In one embodiment, the two are provided in the same pharmaceutical composition. In another embodiment, the two are provided in separate pharmaceutical compositions.

[0108] The composition of the present invention is usually supplied as a sterile pharmaceutical composition.The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable adjuvant.In another embodiment, such an adjuvant is not present in the composition of the present invention.The present invention also provides a process for preparing a pharmaceutical or diagnostic composition, comprising adding and mixing the binding molecule of the present invention, particularly the antibody, together with one or more of a pharmaceutically acceptable excipient, diluent or carrier.

[0109] The term "pharmaceutical acceptable excipient" as used herein refers to a pharmaceutical acceptable formulation carrier, solution or additive for enhancing the desired characteristics of the composition of the present invention. Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer and bicarbonate buffer), amino acids, urea, alcohol, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol and glycerol. The solution or suspension can be encapsulated in liposomes or biodegradable microspheres. The formulation is generally provided in a substantially sterile form using a sterile manufacturing process. This may include manufacturing and sterilizing by filtration of the buffer solvent solution used in the formulation, aseptically suspending the antibody in the sterile buffer solvent solution, and dispensing the formulation into a sterile container by methods well known to those skilled in the art.

[0110] A pharma- ceutically acceptable carrier should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic.Suitable carriers can be large macromolecules that are slowly metabolized, such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.

[0111] Pharmaceutically acceptable salts can be used, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate and sulfate, or organic acid salts such as acetate, propionate, malonate and benzoate. Pharmaceutically acceptable carriers in therapeutic compositions can further contain liquids such as water, saline, glycerol and ethanol. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions for ingestion by the patient.

[0112] A thorough discussion of pharma- ceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, New Jersey, NJ 1991).

[0113] The term "therapeutically effective amount" as used herein refers to the amount of a therapeutic agent required to treat, ameliorate or prevent a target disease or condition, or to exhibit a detectable therapeutic or prophylactic effect. For any binding molecule, particularly an antibody, the therapeutically effective amount can be initially estimated in either cell culture assays or animal models, usually rodents, rabbits, dogs, pigs or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful dosages and routes of administration in humans. The precise therapeutically effective amount for a human subject will depend on the severity of the disease state, the subject's general health, the subject's age, weight and sex, diet, time and frequency of administration, drug combinations, reaction sensitivities, and tolerance / response to treatment. This amount can be determined by routine experimentation and is within the judgment of the clinician. In general, the therapeutically effective amount is between 0.01 mg / kg and 50 mg / kg per day, for example, between 0.1 mg / kg and 20 mg / kg per day. Alternatively, the dosage may be 1-500 mg / day, for example 10-100, 200, 300 or 400 mg / day. The pharmaceutical composition may conveniently be provided in a unit dosage form containing a predetermined amount of the active agent of the present invention. In one embodiment, the amount in a given dosage is at least sufficient to provide a particular function.

[0114] The present invention can be administered to a subject using any of a number of routes of administration, including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intracerebroventricular, transdermal, transdermal (see, e.g., WO 98 / 20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal or rectal routes. A hypospray can also be used to administer the pharmaceutical composition of the present invention. Direct delivery of the composition is generally achieved by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The composition can also be administered into a specific tissue of interest. In one embodiment, administration is to the brain. The dosage regimen can be a single dose schedule or a multiple dose schedule. If the product is for injection or infusion, it can take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and can contain formulatory agents such as suspending agents, preservatives, stabilizing agents and / or dispersing agents. Alternatively, the pharmaceutical composition of the present invention may be in a dry form for reconstitution with a suitable sterile liquid before use. When the composition is administered by a route using the digestive tract, the composition may contain an agent that protects the antibody from degradation but releases the antibody or its antigen-binding fragment once absorbed from the digestive tract. The nebulizable formulation according to the present invention may be provided as a single-dose unit (e.g., a sealed plastic container or vial) packaged, for example, in a foil envelope. Each vial contains a unit dose in a volume, for example, 2 ml, of solvent / solution buffer.

[0115] In one embodiment, an antibody or antigen-binding fragment, nucleic acid, vector, host cell, or pharmaceutical composition of the invention is administered to the brain or administered such that it has access to the brain.

[0116] The present invention also provides a process for preparing a pharmaceutical or diagnostic composition, comprising adding and mixing an antibody or antigen-binding fragment thereof, particularly an antibody, of the present invention together with one or more pharma- ceutically acceptable excipients, diluents or carriers. The antibody or antigen-binding molecule of the present invention may be the only active ingredient in the pharmaceutical or diagnostic composition, or may be associated with other active ingredients, including antibody or non-antibody components, such as steroids or other drug molecules. The pharmaceutical composition suitably comprises a therapeutically effective amount of the antibody or antigen-binding fragment thereof. As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic agent required to treat, ameliorate or prevent a target disease or condition, or to exhibit a detectable therapeutic or prophylactic effect.

[0117] The pharmaceutical composition can be conveniently provided in a unit dose form containing a predetermined amount of the active agent of the present invention per dose.The pharmaceutical composition of the present invention can be provided in a container that provides a means for administration to a subject.The pharmaceutical composition of the present invention can be provided in a pre-filled syringe.The present invention therefore provides such a filled syringe.Also provides an automatic injector filled with the pharmaceutical composition of the present invention.

[0118] It is also envisaged that the binding molecules of the invention, particularly antibodies, may be administered by using gene therapy. To achieve this, when the binding molecule is an antibody, DNA sequences encoding the heavy and light chains of the antibody molecule under the control of appropriate DNA elements are introduced into the patient, whereby the antibody chains are expressed from the DNA sequences and assembled in situ. In another particularly preferred embodiment, sequences encoding the intrabodies of the invention may be administered to the subject.

[0119] The present invention also extends to kits comprising an antibody or antigen-binding fragment thereof of the invention, optionally together with instructions for administration. In yet another embodiment, the kit further comprises one or more reagents for carrying out one or more of the assays or methods discussed herein.

[0120] In one embodiment, the molecules of the invention, including the antibodies or antigen-binding fragments thereof, of the invention are provided for use as laboratory reagents.

[0121] "Purified form" as used above is intended to refer to a purity of at least 90%, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w / w or more.

[0122] In the context of this specification, "comprising" shall be interpreted as "including." Aspects of the invention comprising certain elements are also intended to extend to alternative embodiments that "consist of" or "consist essentially of" the relevant elements.

[0123] Positively recited embodiments herein may be used as the basis for a disclaimer.

[0124] Where the singular is referred to herein, the plural is also included unless specifically stated or apparent. In particular, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0125] All references mentioned herein are specifically incorporated by reference.

[0126] The subheadings herein are used to aid in the organization of the specification and are not intended to be used to frame the meaning of the technical terms herein.

[0127] The sequences of the present invention are provided below. EXAMPLES

[0128] Example 1 Peptide immunogen design 3R tau The amino acid sequence of tau MTBR, exons 9-12, is shown in Figure 1. This splicing of exon 10 creates a unique exon boundary between exon 9 and exon 11, found only in 3R tau. This epitope was used as a means to generate an antibody that confers specificity for 3R tau over 4R tau. To drive an immune response against this epitope, a peptide was designed to encompass amino acids 268-311 of tau, as shown by the solid box in Figure 1 and the TE9 / 11 sequence in Table 1 (the peptide is designated TE9 / 11). [Table 1]

[0129] TE9 / 11 shares homology with other regions of tau that reside at exon boundaries between all exons of the tau MTBR. These potential cross-reactive epitopes are highlighted with dashed boxes (Figure 1), with asterisks highlighting sequence differences. For a clearer comparison, Table 1 shows the exact peptide alignment of the potential cross-reactive epitopes.

[0130] The first of these is the exon boundary between exons 10 and 11 (tau residues 299-312 in Figure 1), which differs from TE9 / 11 by only two amino acids (peptide residues 2 and 7 (Table 1)). The second region of similarity is at the boundary between exons 9 and 10 (tau residues 268-281 (Figure 1)), which differs by three amino acids (peptide residues 11, 12, and 14 (Table 1)). Finally, there is sequence homology at the interface between exons 11 and 12 (tau residues 330-343 (Figure 1)). However, there are eight differences (peptide residues 2, 7, and 9-14 (Table 1)) that represent 50% of the epitope space, thus reducing the possibility of driving nonspecific antibody binding.

[0131] Example 2 Peptide immunogen design 4R tau As mentioned above, a unique feature of 4R tau over 3R tau is the presence of exon 10 in 4R tau. This represents an epitope for antibodies that confers specificity to 4R tau over 3R tau. It is worth noting that it may be possible to achieve 4R specificity by targeting the exon boundary between exon 9 / 10 and exon 10 / 11. In both cases, there will be only one amino acid to target (I278 or S301, respectively). It is also important to consider that there are several sites of known post-translational modifications within exon 10, N279, K280 / 281 and S285 / 289 (Ercan et al. 2017, Mol Neurodegener, 12(1), 87; Kontaxi et al. 2017, Front Mol Biosci, 4, 56; Mair et al. 2016, Anal Chem, 88(7), 3704-14) (shown in the dashed box within exon 10 in Figure 2). To ensure that the antibody is specific for 4R tau and will bind regardless of the post-translational modification state of tau, peptide TE10 was designed to encompass tau amino acids 294-302 (Figure 22) because this is within and in the region of exon 10 and avoids the post-translational modification sites. Figure 2 shows the location of TE10 within the four exons of tau MTBR in a solid box. The TE10 line in Table 2 shows the exact peptide sequence. [Table 2]

[0132] Given the repetitive nature of MTBR, TE10 shares sequence homology with three other exons of MTBR (highlighted by dashed boxes in Figure 2 and aligned in Table 2). The first of these is within exon 12 (tau residues 357-365 (Figure 2)) and differs from the TE10 peptide by two amino acids (peptide residues 1 and 5 (Table 2)). The second region of similarity is present within exon 9 (tau residues 263-271 (Figure 2)) and differs by four amino acids (peptide residues 1, 2, 5 and 7 (Table 2)). Finally, there is sequence homology within exon 11 (tau residues 325-333 (Figure 2)) and differs by four amino acids (peptide residues 1, 2, 4 and 7 (Table 2)).

[0133] Example 3 Peptide conjugation for immunization To elicit an immune response, peptides were conjugated to the carrier proteins keyhole limpet hemocyanin (KLH), ovalbumin (OVA) and bovine serum albumin (BSA). All peptides were conjugated to biotin to allow screening by streptavidin capture onto beads or plates (performed by CRO Peptide Synthetics).

[0134] The immunodominant position of the peptide immunogen is placed furthest from the carrier protein as this is the position most exposed to the immune system throughout immunization. Therefore, TE9 / 11 was conjugated as a cyclic peptide to ensure that the predicted key residues 2, 7, 11 and 12 (Table 2) were present in the most immunodominant position. TE10 (designed to confer a 4R tau-specific immune response) was designed as a linear peptide attached to the carrier protein via its C-terminus. This was to ensure that the lysine residues at positions 1 and 5 of the peptide, the only two amino acids unique to TE10 (Table 2), were in immunodominant positions. For both peptides, a single cysteine ​​was added to the peptide for linkage to a maleimide linker and carrier protein or biotin-PEG-maleimide via the thiol group. Cyclization of the peptide was achieved by N- to C-terminal amide bond formation to generate a cyclic peptide loop.

[0135] Example 4 Identification of 3R- and 4R-specific antibodies from rabbits The conjugated peptides were used to immunize rabbits. B cells from rabbits 6170 and 6171 were activated to antibody-secreting B cells in 400×96-well plate cultures. Culture supernatants were primary screened for binding. This homogeneous fluorescent assay was against a mixture of biotinylated TE9 / 11 and TE10 peptides captured on streptavidin beads. Figure 3 shows the primary screening data from the 50×384-well assay screening plate. Highlighted in green are wells where bead-associated fluorescence was observed, indicating antibodies with specificity to either TE10 or TE9 / 11. In effect, the colonies highlighted in green roughly exceed 1000 bead-associated fluorescence in the graph. These hits were selected based on a binding value threshold of 1000 bead-associated fluorescence units. This threshold gives high confidence in the binding profile and allows for immobilization of samples to the 12×80-well master plate.

[0136] Example 5 Rabbit B cell culture secondary screening Following identification of supernatants that showed reactivity against either TE10 or TE9 / 11 (Figure 3) and cryopreservation of activated B cells, a secondary supernatant screening was performed to identify wells with antibodies exhibiting isoform specificity. This was accomplished by ELISA using each peptide antigen (Figure 4) as well as ELISA against recombinant 0N3R and 0N4R tau (Figure 5).

[0137] Importantly, all four remaining tau isoforms are identical within the MTBR, and there are no other potential cross-reactive epitopes within the remainder of the protein.

[0138] The data obtained are shown in Figure 4 for peptide ELISA results and in Figure 5 for protein ELISA results. All ELISA data are plotted as fold change over background for each data point. Supernatants from wells that showed binding to a single peptide and at least 4-fold higher binding to the desired tau 0N isoform were selected for B cell isolation and variable region gene recovery. In each case, wells selected as 3R-specific are highlighted in red and 4R-specific wells in green. It is interesting to note that some wells appear to show cross-reactivity to both peptides. These likely recognize a common epitope on the peptide. The lack of sequence homology between the two tau peptides suggests that the cross-reactivity is likely due to the linker conjugating the peptide to a biotin molecule or a carrier protein or polyreactive antibodies within the developing immune repertoire of the rabbit.

[0139] Example 6 B cell isolation, RT-PCR and transcriptional activity PCR recombinant transient screening Fluorescent B cell isolation was performed on all of the selected wells highlighted in red or green in Figures 4 and 5. Reverse transcription and three rounds of PCR were performed to recover the variable region genes and generate linear expression cassettes ready for transfection. Table 3 shows the recovery levels from B cell isolation, the PCRs from each well and the number of successful PCRs, along with the specificity of that well for either 3R or 4R tau. [Table 3]

[0140] In general, the results showed good B cell isolation efficiency with fluorescent foci occurring in 83% of wells. Variable region recovery was observed in 97% of vH PCRs and 99% of vK PCRs, representing an overall V region pair recovery rate of 96%.

[0141] Linear expression cassettes generated by tertiary TAP PCR were directly transfected into Expi293F cells. After expression, antibody-containing supernatants were assayed for IgG expression by ELISA. IgG expression was observed in all cases where V-region pairs were recovered by PCR. A total of 77 samples out of 90 wells contained IgG. All wells were then assayed for binding to either 0N3R or 0N4R tau by ELISA. ELISA data with one representative TAP expression per focus group are shown in Figure 6. Wells selected for cloning and further testing are highlighted in Figure 6 in green and red for 4R and 3R specific antibodies, respectively.

[0142] It is interesting to note that all picked focus wells except one retained their binding specificity for 3R or 4R tau after TAP expression. For the single antibody highlighted in black that no longer appears to bind either 3R or 4R tau, this was a false positive in the B cell supernatant screening ELISA with the protein (Figure 5) and likely bound to a non-tau representative epitope on the TE10 peptide. This explains how it was possible to generate fluorescent foci when performed with the peptide, which then shows no binding to tau protein since TAP is transient.

[0143] Example 7 Transient cloning, screening and sequencing of rabbit IgG Variable region fragments generated by PCR were cloned as full-length rabbit IgG. All cloned antibodies were sequenced and expressed as 30ml transients in Expi293F cells and the resulting culture supernatants were assayed to determine the concentration of IgG they contained. All supernatants were assayed by ELISA against 0N3R and 0N4R tau at 10ug / ml IgG to determine whether they had retained their specificity profile after cloning. Figure 7A shows the ELISA data for all cloned antibodies and Figure 7B shows the CDR3 sequences of these antibodies.

[0144] As can be seen from Figure 7, the group of antibodies containing similar or identical antibody sequences clustered very closely in their ELISA values. There was a clear separation between 3R-specific and 4R-specific antibodies, with 4R-specificities generally showing higher cross-reactivity. Two antibody sequences, clone 3 (VR7082) (4R-specific) and clone 14 (VR7081) (3R-specific), appeared to have a suitable level of isoform selectivity (no measurable unwanted cross-reactivity) to justify larger-scale expression / purification and more detailed characterization. The sequences of the two antibodies and the sequences of the scFv intrabodies subsequently generated are shown in the table below, along with other related sequences. [Table 4-1] [Table 4-2] [Table 4-3]

[0145] Example 8 Characterization of 3R- and 4R-specific antibodies Following the identification of the 3R and 4R tau specific antibodies, VR7081 and VR7082, their suitability for any further studies was assessed by characterization in a range of assays against recombinant and naturally expressed tau.

[0146] (a) Recombinant tau binding and characterization assays As previously described, VR7081 and VR7082 demonstrated excellent specificity for 0N3R and 0N4R tau at 10 μg / ml by ELISA ( FIG. 7 ). It was important to ensure that this specificity was retained following purification, and therefore VR7081 and VR7082 were tested against all six recombinantly expressed isoforms of tau by ELISA ( FIGS. 8A and B ).

[0147] After confirming the 3R and 4R selectivity profile of VR7081 and VR7082 by ELISA, FIG. 8, it was important to confirm the specificity of these antibodies for tau in a cellular environment. Data from this flow cytometry assay is shown in FIG. 9. FIG. 9 demonstrates that both VR7081 (A) and VR7082 (B) show absolute specificity for tau in a cellular environment by flow cytometry. To confirm that this specificity was retained in the Western blot, lysates were generated from cells overexpressing all isoforms of tau and verified using a polyclonal anti-total tau antibody. Western blots containing VR7081 and VR7082 can be seen in FIG. 10A and FIG. 10B, respectively. Peptide TE9 / 11 and peptide TE10 represent linear epitopes spanning the tau exon 9 / 11 boundary unique to 3R tau and within exon 10 unique to 4R tau. Thus, these epitopes were unlikely to be affected by the sample reduction performed for the Western blot. FIG. 10 shows that both VR7081 (FIG. 10A) and VR7082 (FIG. 10B) retain their specificity profile for 3R and 4R tau, respectively, by Western blot.

[0148] Both antibodies also appeared to show reactivity with non-tau bands of approximately 49 kDa (VR7081) or 60 kDa (VR7082). It is unclear what is driving this extra band and it could be the result of rabbit IgG, a secondary HRP-conjugated antibody, or it could be driven by off-target variable region binding.

[0149] To address this issue, western blots were performed using 2N3R and 2N4R tau-containing lysates as well as recombinant tau ladder and mock lysates, and probed with non-tau reactive rabbit or mouse IgG, and the results are shown in Figure 11. The western blot in Figure 11A shows a similar non-specific banding pattern to VR7081 and VR7082, with non-specific bands at approximately 60 kDa and 42 kDa for non-tau reactive rabbit IgG. However, these bands are not observed with non-tau reactive mouse IgG in Figure 11B. The results clearly show that any non-tau bands observed in Figure 10 are due to rabbit IgG constant regions or HRP indications, rather than tau-specific variable regions.

[0150] Further experiments were then performed to confirm that the antibody has a more general utility. To confirm that this was possible, the rabbit IgG was converted to a mouse IgG with rabbit variable regions. This was done because the non-tau reactive mouse IgG showed no banding in the tau overexpressing lysates (Figure 11B). The resulting chimeric antibodies were then assayed by Western blot against cell lysates overexpressing each of the tau isoforms. The results of these blots can be seen in Figure 12. From Figure 12, it is clear that conversion to a mouse chimeric antibody with both VR7081 and VR7082 removed the non-tau bands from the blot. This data, along with the banding seen with the non-tau antibodies in Figure 11A, demonstrates that this non-tau reactive band was the result of the rabbit IgG constant region or secondary HRP-conjugated antibody used in the Western blotting. It is important to note that this non-tau reactivity is not observed in the same cells in a flow cytometry assay, so this is a Western blot artifact, likely due to presentation of linear epitopes of other cellular proteins.

[0151] Conversion to mouse IgG was demonstrated by Western blot to very successfully aid in the specificity of these antibodies, but as mentioned above, this non-tau reactive band is not an issue for experiments so performed unless otherwise stated all data was generated using rabbit IgG versions of these antibodies.

[0152] Another important area where it was important to evaluate these antibodies was in immunofluorescence. To evaluate these antibodies, 0N3R and 0N4R tau were overexpressed in adherent CHOK1 cells, followed by fixation, permeabilization, and co-staining with either VR7081 / VR7082 and a polyclonal anti-total tau antibody. The resulting immunofluorescence data can be seen in Figure 13 for VR7081 and Figure 14 for VR7082. In each case of VR7081 and VR7082 staining with that antibody was only seen on cells expressing the tau protein for which the antibody is specific. The data shown in Figures 13 and 14 clearly demonstrate by immunofluorescence that both VR7081 and VR7082 retain excellent specificity for 3R and 4R tau, respectively. Polyclonal anti-total tau antibody staining can visualize all cells expressing tau within these populations. It is therefore important to note that in both cases staining with either VR7081 or VR7082 overlaps with all polyclonal anti-total tau antibody staining for the appropriate tau isoform.

[0153] Thus, the data obtained clearly demonstrate that VR7081 and VR7082 are highly specific for 3R and 4R tau, respectively: in all assays performed, both antibodies showed no off-target binding to undesired tau isoforms.

[0154] (b) Native tau binding and characterization assays We then demonstrated binding to tau expressed in native systems of iPSC-derived neurons or from human brain samples.

[0155] The binding of VR7081 and VR7082 was first assessed by immunofluorescence in iPSC-derived neurons. Both control neurons without tau mutations (expressing only 0N3R tau) or monoallelic 10+16 MAPT mutant neurons (expressing both 0N3R and 0N4R tau) were used. The resulting images can be seen in Figure 15 for VR7081 and Figure 16 for VR7082.

[0156] As expected, VR7081 binds to 3R tau in both non-mutated control cells and 10+16 MAPT mutant neurons (Figure 15). VR7082 demonstrates binding to 4R tau in 10+16 mutant neurons (Figure 16), and as expected, does not bind to non-mutated control neurons, as these do not express 4R tau.

[0157] Specificity was also confirmed by immunoblotting using lysates from ExpHEK-293F cells overexpressing 0N3R or 0N4R tau, as well as tau-negative cell lysates and lysates from control, 10+16 monoallelic and biallelic MAPT mutant neurons via Peggy Sue simple western together with a recombinant tau ladder containing all tau isoforms (Figure 17). From the blots in Figures 17A and 17B, it can be seen that both VR7081 and VR7082 retain their specificity for both 3R and 4R, respectively, in the overexpression lysates. VR7081 can be seen to bind to the 0N3R tau size band in all three of the iPSC lysates, as expected. As previously noted at this age, non-mutated neurons do not express 4R tau. Thus, as expected, VR7082 demonstrates binding to the 0N4R tau size band only in 10+16 mutant iPSC-derived neurons.

[0158] It is interesting to note that the non-tau bands observed in Figure 10 for both VR7081 and VR7082 are no longer visible with this method. This blot used a different secondary antibody than the standard Western blot, which appears to have resolved the non-tau banding. This further indicates that the non-tau bands observed with both VR7081 / VR7082 and the non-tau rabbit antibody (Figures 10 and 11) were the result of a secondary HRP-conjugated antibody.

[0159] Tau can undergo significant post-translational modifications (Ercan et al., 2017, supra; Kontaxi et al., 2017, supra; Mair et al., 2016, supra). It was therefore important to confirm that both VR7081 and VR7082 were able to bind 3R and 4R tau, respectively, regardless of the post-translational modification state of tau. Aged brain lysates contain a range of tau species with many post-translational modifications and tau truncations. It therefore represented a good source of material to test in Western blots and confirm that both VR7081 and VR7082 were able to bind many forms of tau (Figure 18).

[0160] Multiple bands are observed in either the VR7081 or VR7082 blots (Figure 18), however, importantly, there are three distinct bands for the VR7081 and VR7082 tau ladders. The multiple banding with brain lysates is likely due to multiple phosphorylation states of tau, high molecular weight aggregates of tau and / or truncated tau variants. This data is consistent with other data using these lysates. This data provides high confidence that both VR7081 and VR7083 can bind to all post-translational modification states of 3R and 4R tau.

[0161] The data presented in the "Native Tau Binding and Characterization Assays" section demonstrates that both VR7081 and VR7082 can bind naturally expressed tau in a range of assays. Figure 17 also demonstrates that the non-tau banding observed in standard Western blots is due to a secondary HRP-conjugated antibody. Finally, Figure 18 demonstrates that both VR7081 and VR7082 can bind tau in a range of post-translational modification states.

[0162] Example 9 Generation of 4R-cleaved tau degrabody Derivatives such as IgG or Fab fragments cannot be used as intracellular antibodies because their disulfide bonds do not form correctly in the reducing environment of the cytoplasm. This ensures that the heavy and light chains do not associate and therefore do not form binding moieties. However, IgG can be reformatted into a non-disulfide stabilized scFv that allows for use as an intracellular antibody (intrabody). In the scFv format, the heavy and light chain variable regions are physically linked by a flexible linker. This ensures that the heavy and light chains pair correctly inside the cell to form the binding moiety. To ensure that such reformatted VR7082 retains its binding properties, the IgG was first reformatted into scFv-msFc. This is a screening construct in which the scFv is fused to a mouse Fc to allow detection with an anti-mouse secondary antibody; schematics of IgG and scFv-Fc can be seen in Figures 19A and 19B, respectively. VR7082 scFv-Ms-Fc was tested. The results of the VR7082 scFv-Ms-Fc conversion can be seen in Figure 19. The conversion was tested by ELISA (Figure 19D), flow cytometry (Figure 19F) and Western blot (Figure 19H). For comparison purposes, the data seen in Figures 19C and 19E are ELISA and flow cytometry data, respectively. Figure 19G is Western blot data utilizing VR7082 IgG for comparison with the scFv format of VR7082.

[0163] To allow intracellular cytoplasmic expression of antibody fragments or any other protein, expression vectors are designed that lack any specific leader sequence. Since tau is mainly present in the cytoplasm of cells, for intrabody studies it was important that the intrabody is also expressed in the cytoplasm. Therefore, expression constructs were designed without leader or localization signals. Despite the successful conversion of VR7082 to scFv format (Figure 19), it was important to ensure cytoplasmic expression of the scFv fragment and the possibility of specific binding to the immunizing peptide. To allow testing of VR7082 as an scFv intrabody, a fusion construct with GFP was designed by fusing GFP to VR7082-scFv with a flexible linker. GFP was chosen because it can be used as a surrogate for future fusions and allows detection of expression in live cells by fluorescence and detection of intrabodies by Western blot via anti-GFP antibodies.

[0164] The ensuing expression constructs were transfected into HEK-293F cells (n=4). These were then assessed for GFP fluorescence by flow cytometry (Figure 20A) and the cells were lysed. The lysates were then assayed by Western blot (Figure 20B) to confirm that the intrabody GFP fusions were intact and of the correct molecular weight. Finally, these lysates were used in pull-down experiments in which intracellular antibodies (intrabodies) were pulled down using the TE10, 4R tau-specific peptide prior to subsequent Western blot, and this was compared to pull-down with an irrelevant mixture of peptide controls (Figures 20C and 20D).

[0165] The data show that the intrabody-GFP fusion was expressed in HEK-293F cells and that GFP folded correctly as indicated by GFP fluorescence (Figure 20A). To verify that VR7082 was correctly fused to GFP, a Western blot of transfected HEK-293F cells with GFP antibody was performed. Intact, the scFv-GFP fusion intrabody should be observed at a molecular weight of approximately 53 kDa (scFv 26 kDa + linker 1 kDa + GFP 26 kDa). Western blotting of cell lysates (Figure 20B) shows that the construct expressed an intact fusion protein with a single band indicated at 53 kDa. Finally, the intrabody was assessed for binding to its immunizing peptide. Western blot (Figure 20C) shows that when VR7082-scFv-GFP lysates are pulled down with TE10-coated beads (lanes 1-4), a band the size of VR7082-scFv-GFP is observed. However, when VR7082-scFv-GFP lysates are pulled down with irrelevant peptide-coated beads (lanes 7-10) or mock-transfected lysates are pulled down with TE10-coated beads (lanes 5-6), no band is observed (Figure 20C). Band densitometry from this blot (Figure 20D) showed that the scFv-GFP intrabody construct is still able to specifically bind the TE10 peptide.

[0166] This stage of the study demonstrated the successful conversion of VR7082, a 4R tau specific rabbit IgG, into scFv-Ms-Fc and scFv-GFP intrabodies, both of which still demonstrated specific binding to 4R tau or peptides of 4R tau. The next stage of the study was to focus on converting this intrabody into a targeted degradation intrabody, i.e., a degrabody.

[0167] Example 10 Generation of degrabodies from VR7082-scFv intrabodies To convert the VR7082-scFv intrabody into a degrabody capable of degrading 4R tau, the GFP domain was replaced with one of six degradation domains selected from the literature. These domains were fused as N- or C-terminal fusions to VR7082-scFv, depending on the orientation suggested by the literature (Table 5). [Table 5]

[0168] All degrabody fusion constructs as well as GFP N- and C-terminal fusions (as non-degraded intrabody controls) were co-transfected into HEK-293F cells containing either 0N3R or 0N4R tau. 48 hours after transfection, cells were lysed and analyzed by Western blot utilizing a polyclonal total tau antibody (Dako). Band densitometry was then performed and values ​​expressed as a percentage of the GFP fusion control. A representative Western blot (Figure 21A) and n=3 densitometry data from the tau Western blot (Figure 21B) can be seen.

[0169] To allow for accurate comparison of degradation, each construct is expressed as % of tau compared to the non-degraded VR7082-GFP control (fused to the N- or C-terminus, respectively) (Figure 21B). This was important as it shows the effect of degrabodies, not just binding, but non-degrading intrabodies.

[0170] The data shown in Figure 21 demonstrates that co-transfection of VR7082 degrabody with 0N4R tau induces degradation of 0N4R compared to the non-degraded VR7082-GFP intrabody control. It is also seen that this is not the case when the degrabody construct is co-transfected with 0N3R tau.

[0171] Following this study, it was of interest to determine whether the presence of MG132 (a proteasome inhibitor) would halt the degradation of 4R tau. This should be true for all degrabodies, except for ODC, which has previously been demonstrated to be less dependent on the ubiquitin proteasome (Erales and Coffino, 2014, Biochim Biophys Acta, 1843(1), 216-21). Degrabody constructs were co-transfected with 0N4R or 0N3R tau in HEK-293F cells, and after 6 hours of incubation, MG132 was added to half of the cultures. All cultures were then incubated for an additional 18 hours before being analyzed by flow cytometry. In these experiments, flow cytometry rather than western blotting was used because it allows for higher throughput screening of all degrabody constructs against 0N3R and 0N4R tau, with or without MG132 treatment. While technically possible with Western blotting, the number of blots required meant that this technique was much less practical when testing many conditions and constructs. It was also important to ensure that any degradation was not an artifact of Western blotting and that it could be confirmed by multiple techniques. Representative flow cytometry plots and comparative assay data can be seen in Figure 22A, and the geometric mean of all single cell populations co-transfected with VR7082 degrabody, normalized to VR7082-GFP levels, can be seen in Figure 22B.

[0172] The gating strategy for this assay was to first allow identification of HEK-293F cells (Figure 22A-1), followed by identification of single cells by forward scatter area compared to height (Figure 22A-2). Single cells were then assessed for intracellular tau staining with a polyclonal total tau antibody (Dako) in either 0N4R- (Figure 22A-3) or 0N3R- (Figure 22A-4) transfected cells.

[0173] In Figure 22B, all tau levels are expressed as % of VR7082-GFP (binding but non-degrading intrabody) control as described above for Figure 21B. The data in Figure 22 clearly demonstrate that in another assay system, all degrabody constructs are able to degrade 0N4R tau while preserving the levels of 0N3R tau in HEK-293F cells. For all constructs, there is a statistically significant reduction in 4R tau levels compared to 3R tau levels (by T-test) (Figure 22B). All fusions except ODC are proteasome-dependent, as MG132 treatment blocked the degradation of 4R tau (Figure 22B), and there was a statistically significant difference (by T-test) for all 0N4R cotransfections with or without the addition of MG132 (Figure 22B). Again, this was not the case for 0N3R tau transfected cells, which showed no statistical difference between MG132-treated and untreated groups (Figure 22B), indicating that treatment with MG132 results only in proteasomal degradation and not in general tau proteostasis in the cells.

[0174] The data shown in Figures 21 and 22 clearly demonstrate that the VR7082-scFv intrabody was successfully converted into an scFv degrabody capable of specific degradation of 0N4R tau.

[0175] Example 11 Degradation of 4R tau using the VR7082 degrabody in iPSC-derived neurons Transfection of neurons with lipid-based transfection methodologies results in a negligible percentage of cells receiving the transfected DNA (Karra and Dahm 2010). For this reason, viral transfection using adeno-associated virus (AAV) was selected as the delivery method for degrabodies and controls for this project. Three AAV constructs were obtained from GeneCopeia, two of which contained VR7082-scFv fused to either the XIAP degradation domain or a Halo tag (non-degradation control), as well as an AAV vector negative control that contained a construct lacking it. VR7082-XIAP constructs were also obtained with and without IRES expression of the GFP transfection marker.

[0176] VR7082-XIAP+IRES GFP AAV was first used to transfect 10+16 biallelic MAPT mutant neurons 90 days after induction. Cells were analyzed 7 days after transfection to account for expression of degrabody and GFP transfection markers. Two cell populations were then identified and sorted using flow cytometry based on GFP positive or negative signals (Figure 23A-3). These two populations were lysed and tau protein levels were assessed using Peggy Sue's Simple Western (Figure 23B and Figure 23C). The complete gating strategy can be seen in Figure 23A.

[0177] Live cells were first identified by the absence of ToPro3 staining (Figure 23A-1). Intact cells were identified by FSC-A v SSC-A (Figure 23A-1-2). It is interesting to note that there are many events that are ToPro3 negative but very small and in the debris region of the FSC-A v SSC-A plot (bottom left of the plot). They could be cell debris that formed small lipid micelles. Such debris would theoretically have an intact lipid bilayer membrane and thus exclude ToPro3 staining (as live cells would). Finally, cells were sorted in two ways based on GFP expression (Figure 23A-3).

[0178] After sorting, the two cell populations were lysed and then run on Peggy Sue's Simple Western. The resulting traces can be seen from sorted GFP-negative cells (Figure 23B) and sorted GFP-positive cells (Figure 23C). The trace of GFP-negative cells shows two peaks, a large one representing 0N3R and a small one corresponding to 0N4R tau (Figure 23B). In contrast, GFP-positive cells had a peak representing 0N3R tau, but no peak representing 0N4R tau was detected (Figure 23B). This suggests that 0N4R was degraded in XIAP-transduced cells.

[0179] Taken together, the data shown in FIG. 23 demonstrates that it is possible to deliver degrabodies to iPSC-derived neurons and degrade 4R tau protein from these cells.

[0180] Example 12 Phenotypic effects of 4R tau ablation on iPSC-derived neurons Previous studies have shown that GFP can be toxic to neuronal cells (Detrait et al. 2002, Mol Ther, 5(6), 723-30). Because the effect of GFP on the mitochondrial polarization assay is unknown, in this study, we used AAV vectors without GFP. For the same reason, we used the VR7082-Halo tag intrabody as a non-degraded control, rather than the previously used VR7082-GFP intrabody.

[0181] Neurons were differentiated from WT, 10+16 monoallelic and 10+16 biallelic MAPT mutant iPSC line cell lines. On days 80-90, they were treated with AAV containing VR7082-XIAP, VR7082-Halo or negative AAV constructs and incubated for another 7 days. After incubation, cells were run in a mitochondrial membrane polarization assay. Here, mitochondrial membrane polarization was assessed by staining with MitoTracker Deep Red CMX ROS and normalized to total mitochondrial mass by staining with MitoTracker deep red. The data obtained are shown in Figure 24.

[0182] The gating strategy for this assay is to identify intact neuronal cells that can be assayed for mitochondrial membrane polarization and total mitochondrial mass (Figure 24A). Representative examples of each treatment group for each iPSC-derived neuronal cell line (Figure 24B) show that in WT control neurons (Figure 24B-1), there was no effect of either the control AAV, VR7082-Halo or VR7082-XIAP treatment groups. However, when these treatments were applied to 10+16 mononeurons (Figure 24B-2) or 10+16 biallelic neurons (Figure 24B-3), treatment with VR7082-XIAP degrabody reduced the levels of mitochondrial membrane polarization. This was observed in n=5 neuronal inductions for each genotype and treatment group (Figure 24C). As expected, in 10+16 MAPT mutant neurons, no statistically significant difference in mitochondrial polarization was observed between control AAV particle treatment and VR7082-Halo intrabody for 10+16 monoallelic or 10+16 biallelic lines (p=0.145+p=0.922, ANOVA). This is because VR7082-Halo intrabody binds to 4R tau in these cells but does not induce 4R tau degradation. However, when cells were transfected with VR7082-XIAP degrabody, a statistically significant decrease in mitochondrial polarization was observed in both 10+16 monoallelic and 10+16 biallelic mutant cell lines from the control AAV (p=<0.0001+p=0.0052, ANOVA) and from the VR7082-Halo treatment group (p=<0.0001+p=0.0018, ANOVA) (Figure 24C). It is interesting to note that while only a slight reduction in the mutant phenotype is observed in 10+16 biallelic neurons, in 10+16 monoallelic neurons the level of mitochondrial polarization is reduced to a level that is not significantly different from WT neurons (p=0.567, ANOVA) (Figure 24C).

[0183] Because the VR7082-RbIgG 4R-specific antibody has the same epitope as the VR7082-scFv degrabody, 4R tau levels could not be assessed in these experiments. However, it has been previously demonstrated that VR7082-XIAP specifically induced 4R tau degradation, but not 3R degradation (Figures 21, 22, and 23). No change in 3R tau levels was observed between any of the treatment groups, suggesting that there was no off-target degradation effect in these experiments (Figure 24D).

[0184] Here, we successfully demonstrated that the VR7082 degrabody can be applied to 10+16 MAPT mutant iPSC-derived neurons to restore non-mutated levels of mitochondrial membrane polarization.

[0185] Example 13 Epitope characterization of the VR7082 antibody Intracellular tau expression constructs were designed with a single alanine point mutation across the peptide epitope TE10, as well as the P301S and L mutations common in disease models of tauopathies. Constructs were synthesized by TWIST bioscience into vectors suitable for intracellular expression of tau.

[0186] Once the constructs were obtained, single constructs were transiently transfected into Expi293 cells (Thermo-Fisher) according to the manufacturer's instructions. The cells were incubated at 38°C with shaking at 220 RPM in a humidified 5% CO2 environment. After 48 hours of incubation, the cells were incubated at 350 G for 1 h. (av)Cells were harvested by centrifugation at 4°C for 10 min. Cells were fixed and permeabilized using the Invitrogens Fix and Perm kit according to the manufacturer's instructions. Cells were then stained with the non-VR7082 cross-blocking antibody HT7 labeled with alexa-488 for 30 min on ice, washed with PBS+1% BSA, and then seeded at 20,000 cells / well. Each well was then stained with 10 μg / ml VR7082 for 30 min on ice, cells were washed once with PBS+1% BSA, and secondary anti-rabbit Fc-Alexa647 (Jackson Laboratories) was added at a 1:1000 dilution. Cells were incubated on ice for 30 min, washed with PBS+1% BSA, and resuspended in 50 μl / well. Cells were run on IQue3, tau-containing cells were gated from HT7 by alexa-488 fluorescence, and tau binding was assessed by alexa-647 staining. Data were normalized and expressed as a percentage of the binding signal observed for unmutated tau.

[0187] The results obtained are shown in Figure 26. The data in Figure 26 demonstrate the knockdown of tau binding signal in the presence of single specific alanine mutations across the peptide binding site (second to tenth bars from left to right), binding to P301S tau (eleventh bar from left to right), binding to P301L tau (twelfth bar from left to right) and binding to mock transfected cells (last bar from left to right). All values ​​are expressed as a percentage of binding to non-mutated 0N4R tau (first left bar). The results show that mutations of both K294; D295, N296 and I297 to alanine almost completely abolish binding of the VR7082 antibody to tau. Mutations of both K298 and V300 to alanine almost halve binding of the VR7082 antibody to tau. Mutations at H299, P301, and G302 did not affect the binding of VR7082 to tau. The results also demonstrate that VR7082 can bind to both P301S and P301L, indicating that it can be used in animal or cell models of tauopathies that contain these mutations.

Claims

1. (a) specifically binds to a 4R tau protein isoform in a physiological sample; or (b) specifically binds to 3R tau protein isoforms in a physiological sample; An antibody or an antigen-binding fragment thereof.

2. The antibody or antigen-binding fragment thereof of claim 1 , which specifically binds to a 4R tau protein isoform.

3. (a) binds to an amino acid region encoded by exon 10 of tau, where the 4R tau protein contains a post-translational modification at one or more of amino acid positions 279, 280, 281, 285, and 289 of tau4R; (b) binds to a peptide comprising or consisting of amino acids 294-302 of 4R tau protein; (c) binds to a peptide corresponding to a single epitope from the amino acid sequence encoded by exon 10 of tau; or (d) cross-blocks or is cross-blocked by any of the antibodies or fragments of (a)-(c); The antibody or antigen-binding fragment thereof described in claim 2.

4. (a) the antibody or antigen-binding fragment thereof specifically binds to a 4R Tau protein isoform in a cell lysate from a cell expressing a physiological 4R Tau protein isoform, preferably in a cell lysate from an iPSC-derived neuronal cell expressing a 4R Tau protein isoform; and / or (b) the antibody or antigen-binding fragment thereof is capable of detecting a 4R tau protein isoform by immunofluorescence on or in cells expressing the 4R tau protein isoform; The antibody or antigen-binding fragment thereof described in claim 2.

5. The antibody or antigen-binding fragment thereof, (a) comprising one or more of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 3, 5 and 7, and one or more of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 11, 13 and 15, preferably comprising the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 3, 5 and 7, and the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 11, 13 and 15; (b) comprising the light chain variable region and the heavy chain variable region of SEQ ID NOs: 1 and 9; (c) an intrabody, preferably an intrabody comprising the sequence of SEQ ID NO: 17, or a sequence having at least 95% sequence identity thereto, and still capable of specifically binding to the Tau 4R isoform; or (d) cross-blocks or is cross-blocked by any of the antibodies or fragments of (a)-(c); The antibody or antigen-binding fragment thereof described in claim 2.

6. The antibody or antigen-binding fragment thereof of claim 2, which binds to an epitope of 4R tau protein comprising amino acids K294, D295, N296 and I297.

7. The antibody or antigen-binding fragment thereof of claim 2, wherein the epitope further comprises K298 and V300 of 4R tau protein.

8. The antibody or antigen-binding fragment thereof of claim 1, which specifically binds to a 3R tau protein isoform.

9. The antibody or antigen-binding fragment thereof, (a) binds to a peptide comprising an amino acid sequence encoded by a region spanning exons 9 and 11 of tau; and / or (b) binds to a peptide of seven amino acids on either side of the boundary between the sequence encoded by exons 9 and 11 of tau; The antibody or antigen-binding fragment thereof described in claim 8.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the peptide is a cyclic peptide.

11. The antibody or antigen-binding fragment thereof, (a) comprising one or more of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 20, 22 and 24, and one or more of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 28, 30 and 32, preferably comprising the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 20, 22 and 24, and the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 28, 30 and 32; (b) comprising the light chain variable region and the heavy chain variable region of SEQ ID NOs: 18 and 26; (c) an intrabody, preferably an intrabody comprising the sequence of SEQ ID NO: 34, or a sequence having at least 95% sequence identity thereto, capable of specifically binding to the Tau 3R isoform; or (d) cross-blocks or is cross-blocked by any of the antibodies or fragments of (a)-(c); An antibody or an antigen-binding fragment thereof according to any one of claims 8 to 10.

12. An antibody or antigen-binding fragment thereof that specifically binds 4R Tau protein, wherein the antibody or antigen-binding fragment thereof binds to an epitope of 4R Tau protein that includes amino acids K294, D295, N296, and I297, and optionally the epitope further includes K298 and V300 of 4R Tau protein.

13. The antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody is an intrabody or a degrabody.

14. 10. One or more nucleic acids encoding the antibody or antigen-binding fragment of claim 1.

15. 15. One or more vectors comprising one or more nucleic acids of claim 14.

16. A host cell comprising one or more nucleic acids according to claim 14.

17. (a) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, one or more nucleic acids according to claim 14, or one or more vectors according to claim 15; and (b) Pharmaceutical Carriers or Excipients 13. A pharmaceutical composition comprising:

18. (a) contacting a test sample with the antibody or antigen-binding fragment thereof of any one of claims 2 to 7 and 12; and (b) detecting binding of the antibody or antigen-binding fragment thereof. A method for detecting a 4R tau protein isoform, comprising:

19. (a) contacting a test sample with the antibody or antigen-binding fragment thereof of any one of claims 8 to 11; and (b) detecting binding of the antibody or antigen-binding fragment thereof. A method for detecting a 3R tau protein isoform, comprising: (i) contacting a test sample with the antibody or antigen-binding fragment thereof of any one of claims 2 to 7 and 12; and Detecting binding of the antibody or antigen-binding fragment thereof A method for detecting a 4R tau protein isoform, comprising: (ii) contacting the test sample with the antibody or antigen-binding fragment thereof of any one of claims 8 to 11; and Detecting binding of the antibody or antigen-binding fragment thereof A method for detecting 3R tau protein isoforms comprising: A method for measuring levels of 4R tau protein isoforms and 3R tau protein isoforms, comprising carrying out:

21. 20. The method of claim 19, further comprising determining whether the relative amounts of 4R and 3R tau protein isoforms differ from that expected for the subject, optionally by comparing with the relative amounts of 4R and 3R in a sample from a healthy subject.

22. 21. The method of claim 20, used to diagnose a tauopathy, preferably PSP, CBD, or Pick's disease.

23. (a) Detection is via immunofluorescence; (b) detection is via Peggy Sue simple western blot; (c) detection is via ELISA; or (c) the test sample comprises a cell lysate, a cell, or a tissue; 21. The method of claim 20.

24. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 13, one or more nucleic acids according to claim 14, or one or more vectors according to claim 15, for treating a tauopathy.

25. 25. The pharmaceutical composition of claim 24, wherein the tauopathy comprises an imbalance between 4R and 3R tau protein isoforms.

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