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 a particular tau protein, particularly such antibodies specific for 4R or 3R tau protein isoforms. The antibodies can be used, for example, for detection, diagnosis and treatment.

Background Art

[0002] Microtubule-associated protein tau (tau) was discovered in 1975. It was initially characterized as an essential microtubule-binding protein for microtubule growth and stabilization within the axons of neurons. Binding to microtubules is achieved at the C-terminus of tubulin via the microtubule-binding region (MTBR) of tau (encoded via exons 9-12). Specifically, it has been shown by peptide-competition NMR that tau interacts with the interface between α-tubulin and β-tubulin by 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 premRNA results in six splice variant isoforms of tau in the central nervous system, ranging in length from 342 to 441 amino acids. The tau isoforms are classified into two categories based on the inclusion or exclusion of exon 10 (within the MTBR): four MTBRs containing exon 10 (4-repeat (4R) tau) and three MTBRs excluding exon 10 (3-repeat (3R) tau). Within each of these categories are three N-terminal variants of tau based on the splicing of exons 2 and 3. This results in 0N tau (containing neither exon 2 nor 3), 1N tau (containing only exon 2), and 2N tau (containing 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: N-terminal overhang (amino acids 1-165); proline-rich domain (amino acids 166-242); MTBR (amino acids 243-367); and C-terminal domain (amino acids 368-441). The presence or absence of the sequence encoded by exon 10 in the MTBR domain signifies 3R or 4R tau.

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

[0005] Although tau is a largely unstructured protein, FRET experiments suggest that its N-terminus and C-terminus are oriented in close proximity. Further studies using NMR confirmed that the N-terminus and C-terminus of tau fold into a “paperclip”-like structure on the central region of the protein. It has also been demonstrated that tau tends to form more complex, transient, localized secondary structures, specifically the β-chain in the MTBR and polyproline helices in the proline-rich domain.

[0006] Tau is primarily expressed in neurons, and is found to be expressed at low levels in oligodendrocytes and astrocytes. Originally identified in association with axonal microtubules, and to a lesser extent, in association with the plasma membrane, nucleus, and mitochondria. In healthy adult neurons, tau distribution to axons bridges tubulin, enabling interconnection with other cytoskeletal components such as neuronal filaments and actin. These interactions stabilize tubulin aggregates within microtubules, regulating their dynamically unstable nature and enabling the cytoskeletal reorganization necessary for axonal growth. Tau has also been shown to directly regulate microtubule binding of the motor transport proteins dynein and kinesin, which transport cargo retrogradely and anterogradely along the axon, respectively. This regulatory effect is thought to occur via direct competition with the aforementioned motor proteins for microtubule access. In tau overexpression systems, a greater effect on kinesin is observed, resulting in net retrograde transport and accumulation in the cell body rather than diffusion of cargo (such as mitochondria) downstream of the axon. However, this regulatory effect is not fully understood and may involve complementary mechanisms, as demonstrated in tau knockout mice where it does not affect axonal transport.

[0007] Tau proteins and their isoforms are involved in several diseases typically characterized by tau fibril deposition, and these conditions are collectively known as tauopathies. While the majority of tauopathies occur sporadically in the population, many MAPT mutation-associated tauopathies exhibit 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), characterized by neuronal tau inclusions with predominant cell death in the frontal and temporal lobes of the brain. Within these lobes, tau inclusions are considered the primary driving factor in the pathogenesis. A well-characterized example is progressive supranuclear palsy (PSP). In contrast, secondary tauopathy is a disorder in which tau lesions are observed in association with other brain lesions. A well-characterized example of secondary tauopathy is Alzheimer's disease (AD), in which the disease lesions are characterized by both tau fibrils in neurons and extracellular amyloid plaques, as defined by Alois Alzheimer in 1907. It is noteworthy that in AD, there is still a correlation between tau levels and cognitive decline, indicating that tau still plays a significant role in this multifactorial disease.

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

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

[0010] The present invention (a) Specifically binds to the 4R tau protein isoform in a physiological sample; or (b) Specifically binds 3R tau protein isoforms in physiological samples, The present invention provides an antibody or its antigen-binding fragment.

[0011] If an antibody or its antigen-binding fragment specifically binds to the 4R tau protein, then it is: (a) If the 4R tau protein has post-translational modifications at one or more of the amino acid positions 279, 280, 281, 285, and 289 of tau 4R, it binds to the amino acid region encoded by exon 10 of the 4R tau protein; (b) Binding to a peptide containing or consisting of amino acids 294-302 of the 4R tau protein; (c) Binding to a peptide corresponding to a single epitope from the amino acid sequence encoded by exon 10 of tau; or (d) Any of the antibodies or fragments from (a) to (c) can be cross-blocked, or cross-blocked by them.

[0012] Antibodies or their antigen-binding fragments can specifically bind to the 4R tau protein isoform in cell lysates from cells expressing the physiological 4R tau protein isoform, preferably in cell lysates from iPSC-derived nerve cells expressing the 4R tau protein isoform. Antibodies or antigen-binding fragments can detect the 4R tau protein isoform by immunofluorescence on or within cells expressing the 4R tau protein isoform.

[0013] Antibodies against 4R tau protein or their antigen-binding fragments are (a) comprising one or more light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 3, 5, and 7, and one or more 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) Including the light chain variable region and heavy chain variable region of Sequence IDs 1 and 9, (c) Preferably an intracellular expression antibody (intrabody) comprising the sequence of SEQ ID NO: 17, or a sequence having at least 95% sequence identity therewith, and still capable of specifically binding to the tau protein 4R isoform; or (d) Any of the antibodies or fragments from (a) to (c) can be cross-blocked or cross-blocked by them.

[0014] If an antibody or its antigen-binding fragment specifically binds to a 3R tau protein isoform, then it is: (a) A peptide comprising an amino acid sequence encoded by a region crosslinking exons 9 and 11 of tau, such as a cyclic peptide, which is bound to and / or (b) A peptide consisting of seven amino acids on either side of the boundary of the sequence encoded by exons 9 and 11 of tau, such as a cyclic peptide, can be bound to it.

[0015] Antibodies against 3R tau protein or their antigen-binding fragments are (a) comprising one or more light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 20, 22, and 24, and one or more 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) Including the light chain variable region and heavy chain variable region of Sequence IDs 18 and 26, (c) Preferably an intracellular expression antibody (intrabody) comprising the sequence of SEQ ID NO: 34, or a sequence having at least 95% sequence identity therewith, which can specifically bind to the tau protein 3R isoform; or (d) Any of the antibodies or fragments from (a) to (c) can be cross-blocked or cross-blocked by them.

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

[0017] The antibody or its antigen-binding fragment may be an intracellularly expressed antibody (intrabody) or a degrabody.

[0018] The present invention also provides one or more nucleic acids encoding the antibody or antigen-binding fragment of the present invention; one or more vectors comprising the nucleic acids; and host cells comprising the one or more nucleic acids or the one or more vectors.

[0019] The present invention also provides a pharmaceutical composition comprising an antibody of the present invention or an antigen-binding fragment thereof, one or more nucleic acids of the present invention, or one or more vectors of the present 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 of the present invention or an antigen-binding fragment thereof; and (b) detecting the 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 can be used for diagnosing tauopathies, preferably PSP, CBD, or Pick's disease.

[0021] An antibody or an antigen-binding fragment thereof can be used to treat tauopathies, such as tauopathies involving an imbalance between 4R tau protein isoforms and 3R tau protein isoforms. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] [Figure 1] Shows the tau protein sequences of exons 9-12. The TE9 / 11 peptide sequence is outlined within the solid-line frame. Potentially cross-reactive epitopes at the boundaries of exons 9-10, 10-11, and 11-12 are highlighted within the dashed-line frame. Amino acid differences between TE9 / 11 are highlighted with asterisks. [Figure 2] Shows the tau protein sequences of exons 9-12. The TE10 peptide sequence is outlined within the solid-line frame. Potentially cross-reactive epitopes within exons 9, 11, and 12 are highlighted within the dashed-line frame. Amino acid differences between TE10 are highlighted with asterisks. Documented post-translational modifications at N279, K280 / 281, and S285 / 289 are highlighted within the dotted-line frame immediately to the left of the region surrounded by the solid line. [Figure 3]A homogeneous fluorescence-based assay for screening primary B cell culture supernatant. Green dots represent cells selected for hitpicking, while red dots represent cells that were not selected. [Figure 4] The ELISA data for TE10 vs TE9 / 11 is shown as a magnification change relative to the background. Wells selected for 3R specificity are marked in red in the upper left of the graph, and wells for 4R specificity are marked in green in the lower right. [Figure 5] The ELISA data for 0N3R vs. 0N4R is shown as a magnification change relative to the background. Wells selected for 3R specificity are marked in red in the upper left, and wells selected for 4R specificity are marked in green in the lower right. [Figure 6] ELISA for binding of neat TAP IgG expression products, with one representative TAP expression per focus group. Values ​​are expressed as a magnification change relative to background binding. 3R and 4R specific antibodies selected for cloning are highlighted in red in the upper left and green in the lower right, respectively. Black dots represent wells containing antibodies not selected for cloning. [Figure 7] A. This figure shows the ELISA optical density (OD) at 630 nm during the cloning transient of each rabbit isoform-specific antibody. This represents the signal at 10 μg / ml for each clone. 4R-selective antibodies are shown in green, and 3R-selective antibodies are highlighted in red. Clone 2 is not shown because it could not be repeatedly expressed. Clone 14 (VR7081) and Clone 3 (VR7082) are circled in red and green, respectively, and were selected as preferred isoform-specific antibodies for further study. B. Heavy and light chain CDR3 sequencing of cloned rabbit antibodies. The CDR3s of antibodies selected as 3R and 4R specific by ELSA are highlighted in green and red boxes. [Figure 8]Complete titration of VR7081(A) and VR7082(B) against all tau isoforms directly coated on ELISA plates. VR7081 is selective for tau 3 isoforms, and VR7082 is selective for tau 4 isoforms. [Figure 9] Flow cytometry assays and titration of VR7081(A) and VR7082(B) against intracellularly expressed tau isoforms. [Figure 10] Western blots from single isoform overexpression cell lysates, probed with VR7081(A) or VR7082(B). [Figure 11] Western blots of 2N3R or 2N4R tau-containing lysates, blotted with non-tau-reactive rabbit (A) or mouse (A) IgG. [Figure 12] Western blots from single isoform overexpression 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-whole tau antibody (AF488). [Figure 14] Immunofluorescence staining of CHOK1 cells expressing 0N3R or 0N4R tau, co-stained with VR7082 (AF647) and polyclonal anti-whole tau antibody (AF488). [Figure 15] Immunofluorescence staining of iPSC-derived neurons with VR7081. Immunofluorescence staining of non-mutant (control) iPSC-derived neurons and monoallelic (single allele) 10+16 MAPT mutant neurons stained with VR7081(3R-tau)AF647. In the composite image, DAPI is shown in blue and tau staining is shown in red. Green arrows indicate examples of axon staining with VR7081. [Figure 16]Immunofluorescence staining of iPSC-derived neurons using VR7082. Immunofluorescence staining of non-mutant (control) iPSC-derived neurons and monoalelic 10+16 MAPT mutant neurons stained with VR7082(4R-tau)AF647. In the composite image, DAPI is shown in blue and tau staining is shown in red. Green arrows indicate examples of axon staining with VR7082. [Figure 17] Simple Western immunoblot probed with either VR7081(A) or VR7082(B). [Figure 18] Western blots of human brain lysates and recombinant Taurader probed with VR7081(A) and VR7082(B). [Figure 19] scFv mouse Fc conversion for VR7082 binding profile confirmation. Rabbit IgG (A) and scFv-Ms-Fc (B) format diagrams. 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 intracellular expression antibody (intrabody) test. A. Flow cytometry fluorescence of VR7082-scFv-GFP transfected cells and mock transfected cells. B. Western blots from four repeat transfections with VR7082-scFv-GFP. C. Western blots of lysate pulldowns from four repeat VR7082-scFv-GFP transfected cells pulled down with either TE10 or a control peptide. D. Western blot band densimetry from VR7082-scFv-GFP pulldowns. [Figure 21] Potential VR7082 degradation screening using degrading body constructs. A. Representative Western blots of total tau and GAPDH after cotransfection of 0N4R tau with a degrading body or a control intracellularly expressed antibody (intrabody) fusion. B. Western blot band densimetry N=3 Western blot. [Figure 22] Flow cytometry assay of degradation using VR7082-degradation bodies. A. Representative flow cytometry gating and histogram plots demonstrating the level of tau staining for GFP control, non-degradation intracellular expression antibody (intrabody) (A-1), degradation body (A-2), and degradation body (A-3) in the presence of MG132. B. ON4R and ON3R tau staining in the presence and absence of MG132, expressed as a percentage of the GFP non-degradation intracellular expression antibody (intrabody) control for all degradation body constructs. [Figure 23] VR7082-XIAP degradation study in iPSC-derived neurons. A. Gating strategies for GFP-based sorting. A-1 Identification of viable 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 blotting of GFP-negative sorted cells identified by polyclonal anti-tau antibody. C. Peggy Sue simple Western blotting of GFP-positive sorted cells identified by polyclonal anti-tau antibody. [Figure 24]VR7082-XIAP Degradable Mitochondrial Membrane Polarization Assay in iPSC-Derived Neurons. A. Representative gating strategies for mitochondrial polarization assays 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 Evaluation of mitochondrial membrane polarization. B. Representative mitochondrial membrane polarization histograms derived from the ratio of mitochondrial polarization to total mitochondrial mass. B-1 WT cell iPSC-derived neurons. B-2 10+16 monoallelic 10+16 MAPT mutant iPSC-derived neurons (10+16 mono). B-3 A12 biallelic (two-allelelic) MAPT mutant iPSC-derived neurons (10+16 biallelic). C. Mitochondrial polarization assays for each 3-iPSC-derived neuron line and the entire AAV-treated group. Statistical significance between groups is determined using ANOVA. D. Normalized 3R tau staining across all 3-iPSC-derived neuron lines and the entire AAV treatment group. Statistical significance between groups was determined using ANOVA. [Figure 25] A comparison of the structures of various tau isoforms. The exon sequences present in various isoforms of the tau protein are shown. [Figure 26]VR7082 antibody epitope analysis. The graph shows the relative binding of the VR7082 antibody to various tau variants, expressed as a percentage, compared to binding to ON4R tau. The results demonstrate that the binding of VR7082 to tau depends on K294;D295;N296; and I297, respectively. When any of these residues are independently mutated to alanine, the antibody bound to ON4R tau is completely cleaved. The results also show that binding is partially dependent on K298 and V300, as binding decreases by approximately 50% when their positions are mutated to alanine. The results indicate that H299, P301, and G302 are not involved in the binding of VR7082 to tau, as there is no difference between the observed binding levels between the alanine variant and ON4R tau when they are independently mutated to alanine. Finally, the results show that VR7082 can bind to both the P301S and P301L forms of the tau protein. [Modes for carrying out the invention]

[0023] Tau protein The antibodies and antigen-binding fragments provided by the present invention bind to tau protein. In one particularly preferred embodiment, the tau protein referred to herein is human tau protein. The convention of amino acid numbering based on the largest human isoform of tau protein, i.e., 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 to either the 4R tau protein isoform or the 3R tau protein isoform.

[0024] Antibodies specific to certain tau proteins The present invention provides antibodies and antigen-binding fragments specific to either the 4R tau protein isoform or the 3R tau protein isoform. As will be further discussed below, typically, antibodies and antigen-binding fragments specific to 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), and that region is specific to the 4R tau protein isoform. Conversely, typically, antibodies or antigen-binding fragments of the present invention specific to the 3R tau protein isoform recognize a portion of the protein that includes a cross-linking 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 its antigen-binding fragment binds to the 4R or 3R tau protein isoform in a physiological state, for example, when the protein is not in a denatured form. For example, the antibody or its antigen-binding fragment of the present invention can specifically bind to the 4R or 3R tau protein isoform in intact cells, as determined, for example, by immunofluorescence. In one embodiment, the protein can be detected in flow cytometry analysis of cells. In another embodiment, it can be used to detect the protein in tissue samples, for example, by immunofluorescence. Alternatively, it may be possible to detect the protein by immunohistochemistry. In a preferred embodiment, it may be possible to detect the tau protein via Western blotting, particularly by Peggy Sue's simple Western blotting.

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

[0027] As shown in Figure 25, 4R tau protein isoforms represent a group of isoforms that all have an amino acid sequence encoded by exon 10 of the tau gene, while 3R tau protein isoforms represent a group of isoforms that all lack the amino acid sequence encoded by exon 10. These characteristics indicate how tau proteins are grouped into 4R and 3R tau protein isoforms, but within each of these names, there are different isoforms depending on whether or not the amino acid sequences encoded by exons 2 and 3 are present. For example, 4R tau protein isoforms include 2N4R, which has sequences encoded by exons 2 and 3; 1N4R, which has a sequence encoded by exon 2 but not by exon 3; and 0N4R, which has neither a sequence encoded by exon 2 nor an exon 3. Since all three, 2N4R, 1N4R, and 0N4R, contain a sequence encoded by exon 10, they are called 4R tau proteins. Therefore, when referring herein to an antibody or its antigen-binding fragment that specifically binds to 4R tau protein isoforms, it typically means that the antibody or fragment binds to all three isoforms 2N4R, 1N4R, and 0N4R, but not to any of the 3R tau protein isoforms 2N3R, 1N3R, and 0N3R. In embodiments where the antibody or antigen-binding fragment specifically binds to 3R tau protein isoforms, the reverse is also true; the antibody will bind to 2N3R, 1N3R, and ON3R, but not to 2N4R, 1N4R, and 0N4R.

[0028] In one embodiment, the affinity of the antibody or its antigen-binding fragment to either the specific 4R or 3R tau protein isoform is about 100 nM or higher, for example, about 50 nM, 20 nM, 10 nM, 1 nM, 500 pM, 250 pM, 200 pM, or 100 pM or higher. In one embodiment, the binding affinity is 50 pM or higher. In one embodiment, the affinity of the antibody specific to either the 4R or 3R tau protein may 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, Kd is about 0.1 pM to about 1 μM.

[0029] The tau protein contains 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, the antibody or antigen-binding fragment of the present invention binds only to the repeat containing its epitope and not to the other repeats. For example, in one embodiment, the antibody or antigen-binding fragment of the present invention binds to the repeat containing the junction between exons 9 and 11, but not to the other repeats, and is therefore specific to 3R tau. In another embodiment, the antibody or antibody fragment of the present invention binds to the sequence in the repeat present in the sequence encoded by exon 10 of the tau gene, but not to the sequences of the other three repeats.

[0030] Tau 4R-specific antibody In one particularly preferred embodiment, the antibody or its antigen-binding fragment is specific to the tau 4R protein isoform. In one embodiment, the antibody or its antigen-binding fragment specifically binds to the tau 4R protein isoform but not to the 3R tau protein isoform. For example, it may bind to all isoforms of the tau 4R protein of a given species or subject, but not to any isoform of the tau 3R protein of that species or subject. Thus, in one embodiment, it may bind to all 2N4R, 1N4R, and 0N4R isoforms of the tau 4R protein, but not to any of the 2N3R, 1N3R, and 0N3R isoforms of the tau 3R protein. In one embodiment, the antibody or its antigen-binding fragment specifically binds to tau 4R protein isoforms derived from humans, rats, and mice, but not to 3R tau protein isoforms derived from those species.

[0031] A general difference between the 4R tau protein isoform and the 3R tau protein isoform is that the latter lacks the amino acid sequence encoded by exon 10 of the tau gene. Therefore, in one embodiment, an antibody or antigen-binding fragment specific to the 4R tau protein isoform binds to a region specific to the 4R tau protein isoform, and thus to the region encoded by exon 10. In a preferred embodiment, an antibody or antigen-binding fragment specific to the 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, the antibody or antigen-binding fragment of the present invention binds to a region within the amino acid sequence encoded by exon 10 of the tau gene, and does not bind to the 3R tau protein isoform in particular.

[0032] In one embodiment, the antibody or antigen-binding fragment of the present invention, which is specific to the 4R tau protein isoform, can bind a peptide containing 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 long. In one embodiment, it is less than 14, 13, 12, 11, or 10 amino acids long. In one embodiment, the antibody or antigen-binding fragment binds a peptide sequence that is 9 amino acids long. In a preferred embodiment, a peptide consisting of amino acids 294 to 302 of the 4R tau protein isoform can be bound. In one embodiment, the antibody or antigen-binding fragment can bind to a peptide if the peptide is in a linear form. In one embodiment, the antibody or antigen-binding fragment of the present invention can bind to a peptide corresponding to the amino acid sequence encoded by exon 10 of the tau gene, of a length in which only one epitope exists. In one embodiment, an antibody or its antigen-binding fragment can bind to a carrier, such as KLH, ovalbumin, or BSA, particularly if the peptide is in a linear form.

[0033] In one preferred embodiment, an antibody or its antigen-binding fragment can specifically bind to a tau 4R protein isoform regardless of whether the protein has any post-translational modifications at one or more of the amino acid positions 279, 280, 281, 285, and 289. In one embodiment, it can specifically bind to a tau 4R protein isoform regardless of whether the protein is modified at any or all of the 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 modifications at positions 281 and 289 are acetylation, and the most frequent post-translational modifications at positions 285 and 289 are phosphorylation. In one embodiment, the antibody or its antigen-binding fragment of the present invention can bind to a tau 4R protein isoform regardless of whether there is glycosylation, acetylation, or phosphorylation at those positions. In one embodiment, the antibody or its antigen-binding fragment of the present invention can bind the tau 4R protein isoform 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 the tau 4R 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 to the 4R tau isoform binds to repeat 2 in 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 rather than to the sequence encoded by exons 9, 11, and 12 of the 4R tau protein isoform.

[0035] In one embodiment, an antibody or antigen-binding fragment specific to the 4R tau protein is: (a) If the tau 4R protein has post-translational modifications at one or more of the amino acid positions 279, 280, 281, 285, and 289 of tau 4R, it binds to the amino acid region encoded by exon 10 of tau; (b) bound to a peptide containing or consisting of amino acids 294-302 of the tau 4R protein; and / or (c) Binding to a peptide corresponding to a single epitope from the amino acid sequence encoded by exon 10 of tau; or (d) Either crossblocks any of the antibodies or fragments from (a) to (c), or is crossblocked by them.

[0036] In one embodiment, the antibody or antigen-binding fragment of the present invention is (a) comprising one or more light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 3, 5, and 7, and one or more 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) Including the light chain variable region and heavy chain variable region of Sequence IDs 1 and 9, (c) Preferably an intracellular expression antibody (intrabody) comprising the sequence of SEQ ID NO: 17, or a sequence having at least 95% sequence identity therewith, and still capable of specifically binding to the tau protein 4R isoform, or (d) Either the antibody or fragment of (a) to (c) is cross-blocked, or is cross-blocked by them.

[0037] In one embodiment, the antibody or antigen-binding fragment of the present invention comprises light chain CDR1, CDR2, and CDR3, or CDRs, corresponding to the sequences of SEQ ID NOs. 3, 5, and 7, in which case each CDR has two or fewer amino acid sequence changes compared to the sequences of SEQ ID NOs. 3, 5, and 7, and the antibody is still capable of specifically binding to the 4R tau protein isoform. In one embodiment, the sequence changes are conservative amino acid sequence changes. In another embodiment, the antibody or antigen-binding fragment of the present invention comprises heavy chain CDR1, CDR2, and CDR3 sequences, or CDRs, corresponding to the sequences of SEQ ID NOs. 11, 13, and 15, in which case each CDR has two or fewer amino acid sequence changes compared to the sequences of SEQ ID NOs. 11, 13, and 15, and the antibody is still capable of specifically binding to the 4R tau protein isoform. In one embodiment, the antibody or its antigen-binding fragment comprises both such light chain and heavy chain CDRs, and in a preferred embodiment, comprises 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 regions 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 chain framework regions and light chain framework regions. In one embodiment, an antibody or its antigen-binding fragment containing the above sequence is humanized such that, for example, all of the antibody sequence except the CDR sequence is a human sequence, and the antibody or its antigen-binding fragment still specifically binds to the 4R tau protein isoform. In another embodiment, all of the antibody sequence may be human, apart from the variable region.

[0038] In one embodiment, the antibody or its antigen-binding fragment includes the light chain variable region of SEQ ID NO: 1 or a light chain variable region having at least 90% sequence identity thereto, where the antibody or antigen-binding fragment can still specifically bind to the 4R tau protein isoform. In one embodiment, the light chain variable region has at least 95%, 98%, or 99% sequence identity with respect to SEQ ID NO: 1, or has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid sequence change, but can still specifically bind to the 4R tau protein isoform. In one embodiment, the antibody or its antigen-binding fragment includes the heavy chain variable region of SEQ ID NO: 9 or a heavy chain variable region having at least 90% sequence identity thereto, where the antibody or antigen-binding fragment can still specifically bind to the 4R tau protein isoform. In one embodiment, the light chain variable region has at least 95%, 98%, or 99% sequence identity with respect to SEQ ID NO: 9, or has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid sequence change, and still specifically binds to the 4R tau isoform. In one embodiment, the sequence change compared to the heavy chain and light chain sequences is only present in the framework region of the variable region. In one embodiment, the antibody or its antigen-binding fragment includes both the light chain variable region and the heavy chain variable region. In one embodiment, the antibody or antigen-binding fragment of the present invention includes either of the light chain and / or heavy chain variable region sequences, but they are humanized such that all sequences other than the CDR sequence are human.

[0039] As will be further discussed below, the antibodies or antigen-binding fragments of the present invention may be provided in various antibody and antibody fragment formats, and those specific to the 4R tau protein isoform may be provided in any of the antibody formats described herein. They may also have any of the levels of sequence mutation described herein and any of the properties described herein.

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

[0041] A general difference between 3R tau protein and those of 4R tau is that the former lacks the amino acid sequence encoded by exon 10 of the tau gene. Therefore, in one embodiment, an antibody or antigen-binding fragment specific to the 3R tau protein isoform binds to a region specific to the 3R tau protein isoform, particularly a region containing the 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 present invention binds to an epitope containing the junction between exons 9 and 11.

[0042] In one embodiment, the antibody or antigen-binding fragment of the present invention, which is specific to the 3R tau protein isoform, can bind a peptide containing 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 consisting of 14 amino acids, including 7 amino acids from each side of the junction between exons 9 and 11. In one embodiment, the antibody or antigen-binding fragment of the present invention, which is specific to the 3R tau protein isoform, can bind a protein containing amino acids 268-274 and 306-312 of SEQ ID NO: 35, but not amino acids 275-305 of SEQ ID NO: 35. In one embodiment, the antibody or antigen-binding fragment of the present invention, which is specific to the 3R tau protein isoform, can bind a peptide containing the amino acid sequence of SEQ ID NO: 36. In one embodiment, the antibody or antigen-binding fragment can bind to a peptide if the peptide is in a cyclic form, particularly if it is conjugated to a carrier such as KLH, ovalbumin, or BSA.

[0043] In one embodiment, an antibody or antigen-binding fragment specific to the 3R tau protein isoform is (a) comprising one or more light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 20, 22, and 24, and one or more 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) Including the light chain variable region and heavy chain variable region of Sequence IDs 18 and 26, (c) Preferably an intracellular expression antibody (intrabody) comprising the sequence of SEQ ID NO: 34, or a sequence having at least 95% sequence identity therewith, and still capable of specifically binding to the tau protein 3R isoform. (d) Either the antibody or fragment of (a) to (c) is cross-blocked, or is cross-blocked by them.

[0044] In one embodiment, the antibody or antigen-binding fragment of the present invention comprises light chain CDR1, CDR2, and CDR3, or CDR, corresponding to the sequences of SEQ ID NOs. 20, 22, and 24, in which case each CDR has two or fewer amino acid sequence changes compared to the sequences of SEQ ID NOs. 20, 22, and 24, and the antibody is still capable of specifically binding to the 3R tau protein isoform. In one embodiment, the sequence changes are conservative amino acid sequence changes. In another embodiment, the antibody or antigen-binding fragment of the present invention comprises heavy chain CDR1, CDR2, and CDR3, or CDR, corresponding to the sequences of SEQ ID NOs. 28, 30, and 32, in which case each CDR has two or fewer amino acid sequence changes compared to the sequences of SEQ ID NOs. 28, 30, and 32, and the antibody is still capable of specifically binding to the 3R tau protein isoform. In one embodiment, the antibody or its antigen-binding fragment comprises both such light chain and heavy chain CDRs, and in a preferred embodiment, comprises 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 regions 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 chain variable regions and light chain variable regions. In one embodiment, an antibody or its antigen-binding fragment containing the above sequence is humanized such that, for example, all of the antibody sequences other than the CDR sequence are human sequences, and the antibody or its antigen-binding fragment can still specifically bind to the 3R tau protein isoform.

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

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

[0047] Epitope In one embodiment, the antibody or antigen-binding fragment 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. The specific region or epitope of tau can be identified in combination with any suitable epitope mapping method known in the art by any one of the antibodies provided by this disclosure. An example of such a method is to screen peptides of various lengths derived from tau protein for binding to the tau-conjugating antibody or its binding fragment of this disclosure using the smallest fragment that can specifically bind to the antibody containing the sequence of the epitope recognized by the tau-conjugating antibody or its binding fragment. In one particularly preferred embodiment, residues that form part of the antibody epitope can be identified by using mutagenesis, for example, by replacing residues of the tau protein with alanine and determining the effect on the binding of the antibody or its antigen-binding fragment to the tau protein, particularly the 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, where in particular their amino acid positions are defined in relation to those in SEQ ID NO: 35.

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

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

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

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

[0053] In one embodiment, the overall epitope of the antibody or its antigen-binding fragment contains amino acids within the region of residues K294-V300 of the tau 4R protein. In another embodiment, the amino acids of the epitope of the antibody or its antigen-binding fragment are located only in that region. In yet another embodiment, the residues forming the epitope are located in at least that region.

[0054] Either the antibody or its antigen-binding fragment mentioned can bind to such an epitope. In one embodiment, such an antibody may be a chimeric, humanized, or fully human monoclonal antibody, or can be used to obtain a chimeric, humanized, or fully human monoclonal antibody. In one particularly preferred embodiment, the antibody or its antigen-binding fragment that binds to the epitope is a degra body.

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

[0056] This disclosure relates to antibodies and antibody fragments. Whenever an antibody is referred to, an antibody fragment may be used unless otherwise specified. Accordingly, the antibodies of the present invention may include complete antibodies having full-length heavy and light chains, or antigen-binding fragments, e.g., Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabody, triabody, tetrabody, or any of the above epitope-binding fragments (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for preparing and manufacturing 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 the Fab and Fab' fragments described in international patent applications, international publications WO2005 / 003169, 2005 / 003170, and 2005 / 003171. Polyvalent antibodies may include polyspecificity, such as bispecificity, or monospecificity (see, for example, international publications 92 / 22853, 05 / 113605, 2009 / 040562, and 2010 / 035012).Examples of possible antibody formats are publicly known in the art and are disclosed, for example, in the review article “The coming of Age of Engineered Multivalent Antibodies,” Nunez-Prado et al., Drug Discovery Today Vol 20 Number 5 Mar 2015, pp. 588–594, D. Holmes, Nature Rev Drug Disc Nov 2011:10;798, and Chan and Carter, Nature Reviews Immunology vol.10, May 2010, 301, which are incorporated herein by reference.

[0057] In one embodiment, the antibody format includes those known in the Art and those described herein, for example, the antibody molecular format includes or includes 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, or one selected from the group consisting of these.

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

[0059] In one embodiment, the antibody or antigen-binding fragment of the present invention is small enough to cross the blood-brain barrier (BBB).

[0060] In one embodiment, the antibody or its antigen-binding fragment is a bispecific or polyspecific antibody in which at least one antigen-binding site is specific to a 4R tau protein isoform or a 3R tau protein isoform. Thus, in one preferred embodiment, it is such a bispecific antibody.

[0061] In a particularly preferred embodiment, the antibody or its antigen-binding fragment can be expressed intracellularly, and is especially an intracellularly expressed antibody (intrabody). An intracellularly expressed antibody (intrabody) is an antibody or antibody fragment that is capable of intracellular expression, correct folding, and antigen binding. Intracellularly expressed antibodies (intrabodies) are typically capable of correct folding in the reducing environment of the cytoplasm, due to the absence of interchain and intrachain disulfide bonds. In some cases, intracellularly expressed antibodies (intrabodies) may be easier to target cells than the antibody itself, and therefore represent an alternative format for the therapeutic antibody of the present invention.

[0062] In one embodiment, the antibody of the present invention is a targeted intracellular expression antibody (intrabody). For example, in one embodiment, the ER-intracellular expression antibody (intrabody) is targeted to the ER lumen using a KDEL or SEKDEL sequence 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 in their entirety by reference). In one embodiment, the intracellular expression antibody (intrabody) of the present invention targets the cytoplasm, for example, by the removal of a leader sequence (such a particular class of intracellular expression antibodies (intrabodies) may be called cellular intrabodies (intrabodies)). The intracellular expression antibodies (intrabodies) used in the examples of this application are expressed in the cytoplasm of a cell. In another embodiment, the intracellular expression antibody (intrabody) of the present invention targets mitochondria or the nucleus by the addition of an appropriate targeting signal (for example, as described in Biocca, Neuberger, and Cattaneo 1990 EMBO J, 9:101-8, the whole of which is incorporated by reference).

[0063] IgG-derived intracellular expression antibodies (intrabodies) can be prepared using the variable region domain 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 intracellular expression antibodies (intrabodies) as they no longer require disulfide bond formation for correct folding. In one embodiment, the intracellular expression antibody (intrabody) of the present invention is a single-domain antibody intrabody. For example, in one embodiment, the intracellular expression antibody (intrabody) of the present invention is a heavy chain-only antibody, e.g., a camelid heavy chain-only antibody (HCabs) (Hamers-Casterman et al. 1993, Nature, 363:446-8, the whole thereof incorporated by reference). A single variable region of HCab (known as VHH) can be used as an intracellular expression antibody (intrabody).

[0064] In one embodiment, the antibody or antigen-binding fragment of the present invention is a non-disulfide-stabilized scFv and can therefore be used as an intracellularly expressed antibody (intrabody). In the scFv format, the heavy chain and light chain variable regions may be physically linked by a flexible linker. This ensures that the heavy chain and light chain pair up precisely within the cell and form a binding site without the need to form a cysteine ​​crosslink between the heavy chain and light chain.

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

[0066] In one embodiment, the antibody or antigen-binding fragment of the present invention is an intracellularly degraded antibody, also known as a targeted degradation intracellular expression antibody (intrabody) or degrading body. In the degrading body format, the intracellular expression antibody (intrabody) is linked to a degradation domain, so the degrading body is effectively a specific type of intracellular expression antibody (intrabody). Therefore, in one particularly preferred embodiment of the present invention, the antibody or its antigen-binding fragment is a degrading body.

[0067] In one embodiment, the antibody or antigen-binding fragment of the present invention is a rabbit antibody or a fragment thereof. In another embodiment, the antibody or antigen-binding fragment of the present invention includes a rabbit variable region, and the rest of the antibody sequence is a mouse sequence. In yet another embodiment, the CDR of the antibody is a rabbit CDR, and the rest of the antibody is mouse. In a further embodiment, the antibody is a fully human antibody. In a further embodiment, all of the antibody sequences other than the CDR sequence are human sequences.

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

[0069] The degree of identity and similarity can be easily calculated (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, 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., Stockton Press, New York, 1991, BLAST™ software available from NCBI (Altschul, SFet 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] Accordingly, this disclosure extends to novel polypeptide sequences disclosed herein and sequences that are at least 80% similar or identical thereto, for example, sequences with similarity or identity of 85% or more, for example, 90% or more, and particularly 95%, 96%, 97%, 98%, or 99% or more. As used herein, “identity” means that at any particular position in the aligned sequences, amino acid residues are identical between sequences. In one embodiment, a sequence may have one of those levels of sequence identity, insofar as the encoded antibody or fragment can still specifically bind to a 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 change, provided that it can still specifically bind to a 4R or 3R tau protein isoform. In one embodiment, it may differ from a particular sequence by the number of sequence changes, the sequence changes being conserved.

[0072] In addition to variant sequences defined by identity percentage or sequence change count, 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. Antibodies may also have one of the enumerated levels of sequence identity or sequence change count. Cross-blocking antibodies can be identified using any suitable method in the art, for example, by using a competitive ELISA or BIAcore assay, where the binding of the cross-blocking antibody to the antigen (a specific tau protein of interest, e.g., 4R or 3R, or one of the peptides from the peptides discussed herein) prevents the binding of the antibody or antibody fragment of the present invention, or vice versa. In one embodiment, the antibody reduces the 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 antibodies and peptides that can be used to generate antigen-binding fragments of such antibodies. In one embodiment, a peptide is provided comprising a sequence that crosslinks exons 9 and 10 of the tau protein. For example, in one embodiment, a peptide comprising or comprising the sequence of SEQ ID NO: 36, also known as the TE9 / 11 peptide, is provided. In another embodiment, the present invention provides a peptide sequence corresponding to an amino acid sequence encoded by exon 10 of tau, such that the antibody is likely to specifically recognize the 4R tau protein isoform. In one embodiment, the peptide does not contain amino acids representing post-translational modification sites in the native 4R tau protein isoform. In one embodiment, a peptide comprising or comprising the amino acid sequence of SEQ ID NO: 37, also known as the TE10 peptide, is provided.

[0074] In one embodiment, the peptide may include additional amino acid residues not derived from the tau protein, such as terminal cysteine ​​residues that help conjugate the peptide to the carrier. In one embodiment, the peptide is conjugated to the carrier. For example, in one embodiment, the carrier protein is KLH, ovalbumin, or BSA. In one embodiment, the peptide is conjugated to the carrier as a linear peptide, with the portion of the peptide most likely to produce a specific antibody further away from the carrier. In another embodiment, the peptide is conjugated to the carrier such that the amino acid residues most likely to produce an antibody specific to a cyclic peptide, such as a 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, which 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. The desired antibody can be identified from such an immunized animal using any suitable method, for example, screening by ELISA using tau peptides or proteins, or any of the other methods discussed herein.

[0076] In one embodiment, the antibody or antigen-binding fragment of the present invention, which is specific to the 3R tau protein isoform, is not the 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 its antigen-binding fragment is specific to the 4R tau protein isoform, but is not the antibody mentioned in Croft et al (2018) https: / / doi.org / 10.1371 / journal.pone.0195211. In one embodiment, the antibody or its antigen-binding fragment is specific to the 4R tau protein isoform, but is not the antibody mentioned in either de Silva et al (2013) or Croft et al (2018).

[0077] Nucleic acids, vectors, and host cells In further embodiments, nucleotide sequences, for example, DNA sequences encoding the antibody or antibody fragment of the present invention as described herein, are provided. In one embodiment, nucleotide sequences, for example, DNA sequences encoding the antibody or antibody fragment of the present invention as described herein, are provided. In one embodiment, the nucleotide sequences are collectively present on a plurality of polynucleotides, but together the nucleotide sequences can encode the antibody or antibody fragment of the present invention.

[0078] The present invention also extends to vectors containing the nucleotide sequences defined above. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. An example of a vector is a “plasmid,” which is a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector, in which an additional DNA segment can be ligated to a viral genome. Certain vectors can autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomatic mammalian vectors). Other vectors (e.g., non-episomatic mammalian vectors) can be incorporated into the genome of a host cell, after which they replicate together with the host genome. In this specification, the terms “plasmid” and “vector” may be used interchangeably, as plasmids are the most commonly used form of vector. General methods for constructing vectors, transfection methods, and culture methods are well known to those skilled in the art. For further information, see the Maniatis Manual, “Current Protocols in Molecular Biology,” 1999, FMAusubel (ed), Wiley Interscience, New York, and produced by Cold Spring Harbor Publishing.

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

[0080] The vectors of the present invention may include selectable markers. As used herein, the term “selectable marker” refers to a protein whose expression can identify 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 onto host cells into which the vector has been introduced. Selectable markers can also be visually identifiable markers, such as fluorescent markers. Examples of fluorescent markers include rhodamine, FITC, TRITC, Alexa Fluors, and their various conjugates. In one embodiment, the selectable marker may be adjacent to a sequence that allows for marker removal, such as loxP or frt.

[0081] Host cells comprising one or more cloning vectors or expression vectors comprising one or more DNA sequences encoding the antibody or antigen-binding fragment of the present invention are also provided. Any suitable host cell / vector system can be used for the expression of the DNA sequences encoding the antibody or fragment of the present invention. Bacteria, e.g., Escherichia coli, and other microbial systems may be used, or eukaryotes, e.g., mammals, and host cell expression systems may be used. Suitable mammalian host cells include CHO, myeloma, or hybridoma cells. Host cells comprising nucleic acid molecules or vectors of the present invention are also provided.

[0082] Tau detection and diagnosis The antibodies and antigen-binding fragments of the present invention can be used in diagnostic / detection kits. In one embodiment, the antibody or antibody fragment of the present invention is immobilized on a solid surface. The solid surface may be, for example, a chip or an ELISA plate. The binding molecules of the present invention, particularly the antibodies, can be conjugated, for example, to fluorescent markers that facilitate the detection of the bound antibody-antigen complex. They can be used in immunofluorescence microscopy. Alternatively, the antibodies or antigen-binding fragments can also be used in Western blotting or ELISA.

[0083] In one particularly preferred embodiment, the ability to bind to a 3R or 4R tau protein isoform is measured, where the tau protein is present in the same form as it is physiologically present, rather than, for example, a denatured form. In one preferred embodiment, it is present within a cell. In one embodiment, an antibody or its antigen-binding fragment may be used to detect a protein when it is present on a cell, since it binds to the protein when it is present on a cell. In one embodiment, an antibody or its antigen-binding fragment may bind to a protein when it is present in the extracellular space, for example, in the form of secreted fibrils.

[0084] The binding of a given antibody or its antigen-binding fragment to 4R tau compared to 3R tau can be determined using any suitable method. In one embodiment, ELISA is used: for example, binding to immobilized tau 4R protein can be compared to that observed on immobilized 3R tau protein. Binding to 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 antigenic fragment to 4R and 3R tau protein isoforms. Such a Western blot may 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, in particular such cells known to express specific tau proteins. In another embodiment, they are neurons differentiated from iPSCs. In one embodiment, the Western blot is performed on a tissue sample, for example, brain tissue from a subject. In a preferred embodiment, Peggy Sue's simple Western blotting is used.

[0086] In another embodiment, the ability of an antibody or its antigen-binding fragment to bind to 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 can 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, e.g., CHO cells, particularly CHOK1 cells overexpressing a desired tau. In another embodiment, immunohistochemistry is performed on iPSc cells. In another embodiment, binding can be measured in tissue samples, for example, by immunohistochemistry (IHC) by fluorescence or other means.

[0087] In one particularly preferred embodiment, iPSCs (induced pluripotent stem cells) are used to evaluate the antibody or antigen-binding fragment of the present invention. In another preferred embodiment, neurons, for example, neurons obtained by differentiating iPSCs, are used. In one preferred embodiment, cells are isolated from a subject having a specific disorder, e.g., one of the disorders mentioned herein, and used to evaluate the antibody or its antigen-binding fragment of the present invention. In one embodiment, fibroblasts are isolated from a subject having tauopathy and used to produce iPSCs, and then neurons. In one embodiment, the subject used to obtain the cells has a mutation in the tau gene (MAPT) gene, e.g., one of the mutations mentioned herein. For example, fibroblasts from such a subject can be used to produce iPSCs that will contain the same tau gene mutation 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 use. In one embodiment, they can be compared to a control cell line that does not have a tauopathy-related mutation in the tau gene, e.g., cells isolated from a healthy subject without such a mutation. In one embodiment, fibroblasts are isolated from a healthy control subject and used to create iPSCs, which are then differentiated into neurons.

[0088] In another embodiment, instead of using cells from a subject with a specific disease mutation in the tau gene, the tauopathy-related mutation is manipulated into a selected cell line, such as an iPSC, or one used to generate iPSCs. In one embodiment, the iPSCs are then differentiated into neurons containing the mutation.

[0089] In one embodiment, the antibody or antigen-binding fragment of the present invention is used to detect a 4R tau protein isoform or a 3R tau protein isoform. For example, the present invention provides a method for detecting or measuring a 4R tau protein isoform, comprising (a) contacting a test sample with the antibody or its antigen-binding fragment; and (b) detecting the binding of the antibody. Any of the detection means discussed herein may be used, for example, ELISA, immunofluorescence, flow cytometry, or immunohistochemistry. In one embodiment, the test sample comprises, for example, cell lysates or cells or tissue derived from a subject. In one embodiment, the test sample comprises neurons or lysates from them. In one embodiment, the test sample is brain tissue or lysates. 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 a normal level. Such a method may also include performing the method separately with antibodies that bind to all tau protein isoforms of a subject to obtain an index 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 of the present invention, rather than one specific to the 4R tau protein isoform, that is specific to the 3R tau protein isoform is used in the methods described above for the 4R tau protein isoform, excluding the method for detecting or measuring the 3R tau protein isoform.

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

[0092] Such methods for detecting the relative amounts of 4R tau protein isoforms and 3R tau protein isoforms can be used to detect imbalances between the two, for example, by comparing a sample from a healthy subject with a sample from the same subject before the development of tauopathy. In one embodiment, tauopathy can be diagnosed using the antibody or fragment thereof of the present invention. In one preferred embodiment, a condition accompanied by an imbalance of 4R and 3R tau protein isoforms can be diagnosed using the antibody or fragment thereof of the present invention. For example, frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD) are all thought to be accompanied by a dominance of the 4R tau protein isoform, so the present invention can be used as a method for diagnosing such conditions to detect such imbalances. Conversely, Pick's disease is thought to be characterized by a greater presence of 3R tau protein than 4R tau protein. Therefore, the present invention can also be used to diagnose the condition based on the identification of such an imbalance.

[0093] In one preferred embodiment, the antibody or its antigen-binding fragment can specifically bind to the 4R tau protein isoform rather than the 3R tau protein isoform in Peggy Sue's simple Western blot. In another embodiment, such specificity can be observed, particularly for cells overexpressing tau, as measured by flow cytometry. In yet another embodiment, such specificity is observed when used, for example, to analyze human brain samples by Western blotting. In a particularly preferred embodiment, such specificity is observed when the antibody or fragment is used for immunofluorescence against neurons, particularly those obtained from iPSCs expressing the relevant tau protein at a physiological level.

[0094] In one preferred embodiment, the antibody or its antigen-binding fragment can specifically bind to the 3R tau protein isoform rather than the 4R tau protein isoform in Peggy Sue's simple Western blot. In another embodiment, such specificity can be observed, particularly for cells overexpressing tau, as measured by flow cytometry. In yet another embodiment, such specificity is observed when used, for example, to analyze human brain samples by Western blotting. In a particularly preferred embodiment, such specificity is observed when the antibody or fragment is used for immunofluorescence against neurons, particularly those obtained from iPSCs expressing the relevant tau protein at a physiological level.

[0095] Pathological condition In one embodiment, an antibody or its antigen-binding fragment can be used to treat or diagnose tauopathy. In one embodiment, the condition being treated is primary tauopathy. In one embodiment, the condition may include frontotemporal lobar degeneration (FTLD): for example, it may be characterized by tau inclusions in neurons with predominant cell death in the frontal and temporal lobes of the brain. In another embodiment, the condition may be secondary tauopathy, and thus a condition in which tau lesions are observed in connection with other brain lesions.

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

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

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

[0099] In one preferred embodiment, the condition treated or diagnosed by the present invention may be Pick's disease (PD). In one preferred embodiment, Pick's disease can be treated using an antibody or antigen fragment of the present invention that is specific to the 3R tau protein isoform. In one preferred embodiment, the antibody or antigen-binding fragment can be used to alter the balance away from the 3R tau protein isoform. For example, applying the present invention in this manner may result in a reduction or removal of Pick globules. By using an antibody of the present invention that is specific to the 3R tau protein isoform, neuronal and glial loss in the frontal, temporal, and parietal lobes of the brain of a subject, particularly a subject with Pick's disease, can be eliminated, reduced, or stabilized. In one embodiment, by treating a subject with an antibody or antigen-binding fragment of the present invention that is specific to the 3R tau protein isoform, the 3R:4R ratio of tau protein isoforms in the subject can be shifted toward or to the level seen in individuals without Pick's disease.

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

[0101] In one embodiment, the subject being 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, resulting in excessive inclusion of exon 10 (MTBR R3) and an increase in 4R tau. In a preferred embodiment, the tauopathy is accompanied by 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 resulting in an increase in 4R tau protein mRNA. In one embodiment, such a condition can be treated using an antibody of the present invention or its antigen-binding fragment that is specific to the 4R tau protein. In one embodiment, the subject being treated has FTDP-17, particularly early-onset FTDP-17.

[0102] The present invention can be applied to treat, prevent, or diagnose tauopathy resulting from other mutations that affect exon 10 inclusion and thus lead to an increase in 4R tau mRNA. These mutations can reside within stem-loop structures such as S305N and S305I and destabilize the stem-loop in a similar manner to intronic mutations, causing an increase in exon 10 inclusion (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 element of exon 10 have been shown to increase exon 10 inclusion. Mutations N279K and L284L enhance the enhancer region within exon 10, resulting in 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, cited above). Other mutations within exon 10 that have been shown to increase 4R tau expression include N296N and N296H, which are hypothesized to either disrupt the silencer region (D'Souza and Schellenberg, 2002, cited above) or create a new enhancer (Andrew Grover et al., 2002, Neuroscience Letters, 323(1), 33-36). Somewhat counterintuitively, mutations within exons 12 and 13 (E342V and N410H, respectively) have been reported to increase 4R tau expression in neurons (Lippa et al. 2000, Annals of Neurology, 48(6), 850-58). In this case as well, the present invention may be used to treat tauopathy associated with such mutations.

[0104] Mitochondrial dysfunction can also be a characteristic of tauopathy. In one embodiment, treatment with the antibody or its antigen-binding fragment of the present invention may help restore mitochondrial function in tauopathy. For example, in such tauopathy, the mitochondrial membrane potential may be affected, and treatment with the antibody or its antigen-binding fragment may help restore the mitochondrial membrane potential to or toward normal levels. In one embodiment, the subject being treated is a subject exhibiting mitochondrial dysfunction. In one embodiment, the subject may be a subject exhibiting altered mitochondrial membrane potential. In one embodiment, the subject exhibits a decreased membrane potential. In a preferred embodiment, the subject exhibits an elevated membrane potential, particularly an elevated membrane potential, and has the 10+16 MAPT mutation. In one embodiment, such a subject may exhibit an excess of the 4R tau protein isoform. In another embodiment, the subject may have the P301L mutation, particularly exhibiting a hyperpolarized mitochondrial membrane. In one embodiment, using the present invention may mean that the mitochondrial membrane potential returns to normal, or at least becomes close to normal.

[0105] In one particularly preferred embodiment, an antibody or its antigen-binding fragment that can cross the blood-brain barrier can be used. In a further preferred embodiment, instead of administering the antibody or antigen-binding fragment itself, one or more nucleic acids or one or more vectors of the present invention may be administered, and in one preferred embodiment, the subject may be administered something encoding an intracellularly expressed antibody (intrabody) or degrabody. In another embodiment, a host cell of the present invention capable of expressing the antibody or antigen-binding fragment of the present invention is used.

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

[0107] Also provided are antibodies or antigen-binding fragments thereof, one or more nucleic acid molecules, one or more vectors, or pharmaceutical compositions of the present invention for use in methods of treating or diagnosing the body of a human or animal. Further provided are uses of antibodies or antigen-binding fragments thereof, one or more nucleic acid molecules, one or more vectors, or pharmaceutical compositions of the present invention for the manufacture of pharmaceuticals for the treatment of pathological conditions or disorders. In one embodiment in which the therapeutic agent of the present 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 given in the same pharmaceutical composition. In another embodiment, the two are given in separate pharmaceutical compositions.

[0108] The compositions of the present invention are typically supplied as sterile pharmaceutical compositions. The pharmaceutical compositions of the present invention may further contain 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 conjugating molecule of the present invention, in particular an antibody, together with one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0109] As used herein, the term “pharmaceutically acceptable excipient” refers to a pharmaceutically 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, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions can be encapsulated in liposomes or biodegradable microspheres. Formulations are generally provided in a substantially sterile form using a sterile manufacturing process. This may include manufacturing and sterilization by filtration of the buffer solvent solution used in the formulation, sterile suspension of the antibody in a sterile buffer solvent solution, and dispensing of the formulation into sterile containers by methods well known to those skilled in the art.

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

[0111] Pharmaceutically acceptable salts, such as mineral salts like hydrochloride, hydrobromide, phosphate, and sulfate, or salts of organic acids like acetate, propionate, malonate, and benzoate, can be used. The pharmaceutically acceptable carrier in the therapeutic composition may further contain liquids such as water, saline, glycerol, and ethanol. With such a carrier, the pharmaceutical composition can be formulated as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, and suspensions for patient ingestion.

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

[0113] As used herein, the term “therapeutic dose” refers to the amount of therapeutic agent required to treat, improve or prevent a target disease or condition, or to demonstrate a detectable therapeutic or preventive effect. For any binding molecule, particularly antibodies, the therapeutic dose can be initially estimated using cell culture assays or animal models, typically 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 effective doses and routes of administration in humans. The exact therapeutic dose for human subjects depends on the severity of the disease condition, the subject’s overall health status, the subject’s age, weight and sex, diet, timing and frequency of administration, drug combinations, sensitivity to response, and tolerance / response to treatment. This amount can be determined by conventional experiments and is within the scope of clinician judgment. Generally, the therapeutic dose is 0.01 mg / kg to 50 mg / kg per day, for example, 0.1 mg / kg to 20 mg / kg. Alternatively, the dose may be 1 to 500 mg / day, for example, 10 to 100, 200, 300, or 400 mg / day. The pharmaceutical composition may be conveniently provided in unit dose form containing a predetermined amount of the active agent of the present invention. In one embodiment, the amount in a given dose is at least sufficient to produce a specific 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, intraarterial, intramedullary, intrathecal, intraventricular, percutaneous, percutaneous (see, for example, International Publication No. 98 / 20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, or rectal routes. The pharmaceutical composition of the present invention can also be administered using a hypospray. Direct delivery of the composition is generally achieved by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered into the interstitial space of a tissue. The composition can also be administered into a specific tissue of interest. In one embodiment, administration is made to the brain. The drug therapy may be a single-dose schedule or a multi-dose schedule. If the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and may contain formulary agents such as suspending agents, preservatives, stabilizers and / or dispersants. Alternatively, the pharmaceutical compositions of the present invention may be in a dry form for reconstitution with a suitable sterile liquid before use. When the composition is administered via a route using the gastrointestinal 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 gastrointestinal tract. The sprayable formulations of the present invention may be provided, for example, as single-dose units packaged in a foil envelope (e.g., a sealed plastic container or vial). Each vial contains a unit dose in a certain volume, for example, 2 ml of solvent / solution buffer.

[0115] In one embodiment, the antibody or antigen-binding fragment, nucleic acid, vector, host cell, or pharmaceutical composition of the present invention is administered to the brain or administered in a manner that allows access to the brain.

[0116] The present invention also provides a process for preparing a pharmaceutical or diagnostic composition, comprising adding and mixing the antibody or antigen-binding fragment thereof, particularly the antibody, with one or more pharmaceutically acceptable excipients, diluents, or carriers. The antibody or antigen-binding molecule of the present invention may be the sole active ingredient in the pharmaceutical or diagnostic composition, or it may be accompanied by other active ingredients, such as antibody or non-antibody components, including steroids or other drug molecules. The pharmaceutical composition appropriately contains a therapeutically effective amount of the antibody or antigen-binding fragment thereof. As used herein, the term “therapeutically effective amount” means the amount of therapeutic agent required to treat, improve or prevent a target disease or condition, or to exhibit a detectable therapeutic or preventive effect.

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

[0118] It is also conceivable that the conjugating molecules of the present invention, particularly antibodies, may be administered by the use of gene therapy. To achieve this, if the conjugating molecule is an antibody, DNA sequences encoding the heavy and light chains of the antibody molecule are introduced into the patient under the control of appropriate DNA components, thereby causing the antibody chain to be expressed from the DNA sequence and assembled in situ. In another particular preferred embodiment, sequences encoding the intrabodies of the present invention may be administered to a subject.

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

[0120] In one embodiment, a molecule of the present invention, comprising the antibody of the present invention or its antigen-binding fragment, is provided for use as an experimental reagent.

[0121] As used above, "purified form" is intended to refer to a purity of at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, or 99% w / w or higher.

[0122] In the context of this specification, “comprising” shall be interpreted as “including.” Aspects of the invention that include a particular element are also intended to extend to alternative embodiments that “consist of” or “essentially consist of” the relevant element.

[0123] In this specification, embodiments listed positively may be used as the basis for disclaimers.

[0124] Where the singular form is mentioned herein, the plural form is also included unless otherwise specified or made clear. In particular, the singular forms "a," "an," and "the," etc., refer to multiple objects unless the context explicitly indicates otherwise.

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

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

[0127] The sequence of the present invention is 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 forms a unique exon boundary between exon 9 and exon 11, found only in 3R tau. This epitope was used as a means to produce antibodies that confer specificity to 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 named TE9 / 11). [Table 1]

[0129] TE9 / 11 shares homology with other regions of tau located at exon boundaries between all exons of the tau MTBR. These potential cross-reactive epitopes are highlighted with dashed boxes (Figure 1), and asterisks highlight sequence differences. For a clearer comparison, Table 1 shows the exact peptide alignments 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 similarity region is located 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 boundary between exon 11 and exon 12 (tau residues 330-343 (Figure 1)). However, there are eight differences (peptide residues 2, 7, and 9-14 (Table 1)) which account for 50% of the epitope space, thus reducing the likelihood of driving nonspecific antibody binding.

[0131] Example 2 Peptide Immunogen Design 4R Tau As mentioned earlier, the unique characteristic of 4R tau over 3R tau is the presence of exon 10 in 4R tau. This represents an epitope for the antibody that gives specificity to 4R tau over 3R tau. It is noteworthy that 4R specificity may be achieved by targeting the exon boundary between exons 9 / 10 and exons 10 / 11. In either case, there would likely be only one target amino acid (I278 or S301, respectively). It is also important to consider that there are several known sites of post-translational modification within exons 10, N279, K280 / 281, and S285 / 289 (Ercanet 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 to 4R tau and binds regardless of the post-translational modification status of tau, peptide TE10 was designed to contain tau amino acids 294-302 (Figure 22). This is because these are located within and in the region of exon 10, thus avoiding the post-translational modification sites. Figure 2 shows the locations of TE10 within the four exons of the tau MTBR in solid boxes. The line labeled TE10 in Table 2 shows the exact peptide sequence. [Table 2]

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

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

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

[0135] Example 4 Identification of 3R and 4R-specific antibodies derived from rabbits Rabbits were immunized with conjugated peptides. B cells from rabbits 6170 and 6171 were activated into antibody-secreting B cells in 400 × 96 well plate cultures. The culture supernatant was screened for binding. This homogeneous fluorescence assay was against a mixture of biotinylated TE9 / 11 and TE10 peptides captured on streptavidin beads. Figure 3 shows the primary screening data from a 50 × 384 well assay screening plate. Wells highlighted in green are those where bead-associated fluorescence was observed, indicating antibodies with specificity to either TE10 or TE9 / 11. In effect, colonies highlighted in green roughly exceed 1000 bead-associated fluorescence units in the graph. These hits were selected based on a binding threshold of 1000 bead-associated fluorescence units. This threshold provides high reliability for the binding profile and allows for sample fixation to a 12 × 80 well master plate.

[0136] Example 5 Rabbit B cell culture secondary screening Following the identification of supernatants showing reactivity to either TE10 or TE9 / 11 (Figure 3) and cryopreservation of activated B cells, secondary supernatant screening was performed to identify wells containing antibodies exhibiting isoform specificity. This was achieved by ELISA using each peptide antigen (Figure 4), as well as ELISA for recombinant ON3R and ON4R tau (Figure 5).

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

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

[0139] Example 6 B cell isolation, RT-PCR, and transcriptional activity PCR recombinant transient screening B cells were isolated by fluorescence imaging for all selected wells highlighted in red or green in Figures 4 and 5. Reverse transcription and three rounds of PCR were performed to recover variable region genes and prepare linear expression cassettes for transfection. Table 3 shows the recovery levels from B cell isolation, the number of 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 fluorescence focusing occurring from 83% of the wells. Variable region recovery was observed from 97% of vH PCR and 99% of vK PCR. This represented an overall recovery rate of 96% of V region pairs.

[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 a V-region pair was recovered by PCR. A total of 77 samples from 90 wells contained IgG. Subsequently, all wells were assayed by ELISA for binding to either ON3R or ON4R tau. ELISA data containing one representative TAP expression per focus group are shown in Figure 6. Wells selected for cloning and further testing are highlighted in green and red in Figure 6 for 4R and 3R-specific antibodies, respectively.

[0142] It is interesting to note that all but one of the picked focus wells retained their binding specificity to 3R or 4R tau after TAP expression. For the single antibody highlighted in black, which no longer appears to bind to either 3R or 4R tau, this was likely a false positive in the B cell supernatant screening ELISA on the protein (Figure 5) and probably bound to a non-tau representative epitope on the TE10 peptide. This explains how a fluorescent focus could be generated when performed using the peptide, and subsequently does not show binding to the tau protein because TAP is transient.

[0143] Example 7 Clone transient 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 transients in 30 ml of Expi293F cells. The resulting culture supernatants were assayed to determine the IgG concentrations they contained. All supernatants were assayed by ELISA with 10 ug / ml of IgG against 0N3R and 0N4R tau to determine whether they retained their specificity profiles 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, groups 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 specificity generally exhibiting higher cross-reactivity. The two antibody sequences, clone 3 (VR7082) (4R-specific) and clone 14 (VR7081) (3R-specific), were considered to possess a level of isoform selectivity (no measurable undesirable cross-reactivity) suitable for larger-scale expression / purification and more detailed characterization. The sequences of the two antibodies and the subsequent generated scFv intrabodies are shown in the table below, along with other relevant sequences. [Table 4-1] [Table 4-2] [Table 4-3]

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

[0146] (a) Recombinant tau binding and characterization assay As previously mentioned, VR7081 and VR7082 showed excellent specificity for 0N3R and 0N4R tau at 10 μg / ml by ELISA (Figure 7). It was important to ensure that this specificity was maintained after purification, and therefore, VR7081 and VR7082 were tested against all six recombinant tau expression isoforms by ELISA (Figures 8A and B).

[0147] After confirming the selectivity profiles of VR7081 and VR7082 to 3R and 4R tau using ELISA (Figure 8), it was important to confirm the specificity of these antibodies against tau in the cellular environment. The data from this flow cytometry assay are shown in Figure 9. Figure 9 demonstrates that both VR7081(A) and VR7082(B) exhibit absolute specificity for tau in the cellular environment by flow cytometry. To confirm that this specificity was retained in Western blotting, lysates were generated from cells overexpressing all isoforms of tau and validated using polyclonal anti-whole tau antibodies. Western blots containing VR7081 and VR7082 can be seen in Figures 10A and 10B, respectively. Peptides TE9 / 11 and TE10 represent linear epitopes within the tau exon 9 / 11 boundary specific to 3R tau and within exon 10 specific to 4R tau. Therefore, these epitopes were unlikely to be affected by the sample reduction performed for Western blotting. Figure 10 shows that both VR7081 (Figure 10A) and VR7082 (Figure 10B) retain their specificity profiles for 3R and 4R tau, respectively, by Western blotting.

[0148] Both antibodies also appeared to resonate with non-tau bands of approximately 49 kDa (VR7081) or 60 kDa (VR7082). It is unclear what drives these extra bands; they may be a result of rabbit IgG, secondary HRP conjugated antibodies, or driven by out-of-target variable region binding.

[0149] To address this issue, Western blotting was performed using 2N3R and 2N4R tau-containing lysates, recombinant tau ladder, and mock lysates, and probing was performed with non-tau-reactive rabbit or mouse IgG. The results are shown in Figure 11. The Western blot in Figure 11A shows nonspecific bands of approximately 60 kDa and 42 kDa in non-tau-reactive rabbit IgG, exhibiting a nonspecific banding pattern similar to VR7081 and VR7082. However, these bands are not observed in non-tau-reactive mouse IgG in Figure 11B. The results clearly indicate that any non-tau bands observed in Figure 10 are attributable to the constant region of rabbit IgG or to HRP, rather than to the tau-specific variable region.

[0150] Subsequent experiments confirmed that the antibody possessed more general utility. To confirm this, rabbit IgG was converted to mouse IgG containing the rabbit variable region. This was done because the non-tau-reactive mouse IgG did not show banding in tau-overexpressing lysates (Figure 11B). The resulting chimeric antibody was then assayed by Western blotting against cell lysates overexpressing each tau isoform. The results of these blots can be seen in Figure 12. From Figure 12, it is clear that conversion to a mouse chimeric antibody containing both VR7081 and VR7082 removed the non-tau band from the blot. This data, along with the banding observed in the non-tau antibody in Figure 11A, demonstrates that this non-tau-reactive band was a result of the rabbit IgG constant region or secondary HRP-conjugated antibody used in Western blotting. It is important to note that this non-tau reactivity is not observed in the same cells in flow cytometry assays, suggesting it is an artificial result of Western blotting, likely due to the presentation of linear epitopes of other cellular proteins.

[0151] Western blotting has shown that conversion to mouse IgG is very successful in improving the specificity of these antibodies; however, as mentioned above, this non-tau-reactive band is not problematic for experiments performed in such a way, unless it is specifically stated that all data were generated using the rabbit IgG version of these antibodies.

[0152] Another important area where evaluating these antibodies was crucial was immunofluorescence. To evaluate these antibodies, 0N3R and 0N4R tau were overexpressed in adherent CHOK1 cells, then fixed, permeabilized, and co-stained with either VR7081 / VR7082 and polyclonal anti-whole tau antibody. The resulting immunofluorescence data can be seen in Figure 13 for VR7081 and in Figure 14 for VR7082. In each case of VR7081 and VR7082 staining with these antibodies, it was observed only in cells expressing the tau protein to 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-whole tau antibody staining allows visualization of all cells expressing tau within these populations. Therefore, it is important to note that in both cases, staining with either VR7081 or VR7082 overlaps with staining with all polyclonal anti-whole tau antibodies for the appropriate tau isoforms.

[0153] Therefore, the obtained data clearly demonstrate that VR7081 and VR7082 are highly specific to 3R and 4R tau, respectively. In all assays performed, neither antibody showed off-target binding to undesired tau isoforms.

[0154] (b) Binding and characterization assay of natural tau Next, we demonstrated the binding of tau expressed in native systems of iPSC-derived neurons or from human brain samples.

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

[0156] As expected, VR7081 binds to 3R tau in both non-mutant 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-mutant control neurons because these do not express 4R tau.

[0157] Specificity was also confirmed by immunoblotting using lysates from control, 10+16 monoallelic, and biallelic MAPT mutant neurons via Peggy Sue's simple Western blotting with lysates from ExpHEK-293F cells overexpressing 0N3R or 0N4R tau, as well as tau-negative cell lysates and 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 to both 3R and 4R, respectively, in the overexpression lysates. As expected, VR7081 can be seen to bind to the 0N3R tau-sized band in all three iPSC lysates. As previously mentioned at this age, non-mutant neurons do not express 4R tau. Therefore, as expected, VR7082 demonstrates binding to the 0N4R tau-sized band only in neurons derived from 10+16 mutant iPSCs.

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

[0159] Tau can undergo significant posttranslational modifications (Ercan et al., 2017, cited above; Kontaxi et al., 2017, cited above; Mair et al., 2016, cited above). Therefore, it was important to confirm that both VR7081 and VR7082 were able to bind 3R and 4R tau, respectively, regardless of the posttranslational modification status of tau. Lysates from aged brains contain a range of tau species with many posttranslational modifications and tau cleavages. It therefore represented a good material source for testing by Western blotting to confirm that both VR7081 and VR7082 can bind to many forms of tau (Figure 18).

[0160] Multiple bands are observed in both VR7081 and VR7082 blots (Figure 18); however, importantly, there are three distinct and different bands for the tau ladder of VR7081 and VR7082. The multiple banding by the brain lysates is likely due to multiple phosphorylation states of tau, high molecular weight aggregates of tau, and / or cleaved tau variants. This data is consistent with other data using these lysates. This data gives 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 "Natural Tau Binding and Characterization Assays" section demonstrate that both VR7081 and VR7082 can bind to naturally expressed tau in a series of assays. Figure 17 also demonstrates that non-tau banding observed in standard Western blotting is attributable to the secondary HRP-conjugated antibody. Finally, Figure 18 demonstrates that both VR7081 and VR7082 can bind to tau in a range of post-translational modification states.

[0162] Example 9 Generation of a 4R decomposed Taudegra body IgG or derivatives such as 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 the binding site does not form. However, IgG can be reformatted to a non-disulfide-stabilized scFv, which enables its use as an intracellular expression 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 accurately pair up in the cell and form the binding site. To ensure that VR7082 thus reformatted retains its binding properties, IgG was first reformatted to scFv-msFc. This is a screening construct in which scFv is fused to mouse Fc, enabling detection by an anti-mouse secondary antibody. Schematic diagrams 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 blotting (Figure 19H). For comparison purposes, the data shown in Figures 19C and 19E are ELISA and flow cytometry data, respectively. Figure 19G shows Western blotting data using VR7082 IgG for comparison with the scFv format of VR7082.

[0163] To enable intracellular cytoplasmic expression of antibody fragments or any other protein, expression vectors lacking any specific leader sequence are designed. Since tau is primarily present in the cytoplasm of cells, it was important that intracellularly expressed antibodies (intrabodies) were also expressed in the cytoplasm for intracellular antibody (intrabody) studies. Therefore, the expression constructs were designed without a leader or localization signal. Despite the successful conversion of VR7082 to scFv format (Figure 19), it was important to ensure the possibility of cytoplasmic expression of scFv fragments and specific binding to immunoassay peptides. To enable 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 substitute for future fusions and allows for fluorescence-based detection of expression in living cells, as well as detection of intracellularly expressed antibodies (intrabodies) by Western blotting via an anti-GFP antibody.

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

[0165] This data shows that the intrabody-GFP fusion was expressed in HEK-293F cells and that GFP was correctly folded, as indicated by GFP fluorescence (Figure 20A). To demonstrate that VR7082 was correctly fused to GFP, Western blotting of HEK-293F cells transfected with GFP antibody was performed. In an intact state, the scFv-GFP fusion intrabody should be observed with 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 this construct expressed an intact fusion protein with a single band indicated at 53 kDa. Finally, the intracellularly expressed antibody (intrabody) was evaluated for binding to its immunizing peptide. Western blotting (Figure 20C) shows that when VR7082-scFv-GFP lysates were pulled down with TE10-coated beads (lanes 1-4), bands the size of VR7082-scFv-GFP were observed. However, when VR7082-scFv-GFP lysates were pulled down with unrelated peptide-coated beads (lanes 7-10), or when mock transfect lysates were pulled down with TE10-coated beads (lanes 5-6), no bands were observed (Figure 20C). Band densitometry from this blot (Figure 20D) showed that the scFv-GFP intrabody construct was still able to specifically bind to 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 demonstrate specific binding to 4R tau or 4R tau peptides. The next stage of the study focused on the conversion of this intracellularly expressed antibody (intrabody) into a targeted degradation intrabody, i.e., a degraded intrabody.

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

[0168] All degradable body fusion constructs, as well as GFP N and C-terminal fusions (as undegraded intrabody controls), were co-transfected into HEK-293F cells containing either 0N3R tau or 0N4R tau. 48 hours after transfection, cells were lysed and analyzed by Western blotting using polyclonal whole tau antibody (Dako). Band densimetry was then performed, and values ​​were expressed as percentages of the GFP fusion control. Representative Western blots (Figure 21A) and n=3 densitometry data from tau Western blots (Figure 21B) are shown.

[0169] To enable accurate comparison of degradation, each construct is expressed as a percentage of tau compared to an undegraded VR7082-GFP control (fused to the N or C terminus, respectively) (Figure 21B). This was important because it shows the effect of degradation bodies, not just binding, in addition to the undegraded intra-body.

[0170] The data shown in Figure 21 demonstrates that cotransfection of VR7082 degraded bodies with 0N4R tau induces the degradation of 0N4R compared to an undegraded VR7082-GFP intra-body control. This is not the case when cotransfection of the degraded body construct with 0N3R tau.

[0171] Following this study, it was interesting to determine whether the presence of MG132 (a proteasome inhibitor) halts the degradation of 4R tau. This should apply to all degra body constructs except for ODC, which has been previously demonstrated to be less dependent on the ubiquitin proteasome (Erales and Coffino, 2014, Biochim Biophys Acta, 1843(1), 216-21). Degra body constructs were cotransfected 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 a further 18 hours and analyzed by flow cytometry. Flow cytometry was used instead of Western blotting in these experiments because it allows for higher throughput screening of all degra body constructs for 0N3R and 0N4R tau, with or without MG132 treatment. While technically possible with Western blotting, the sheer number of blots required to test numerous states and constructs made this technique far less practical. It was also crucial to ensure that any degradation was not an artificial result of Western blotting and that this 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 degraded bodies, normalized to VR7082-GFP levels, can be seen in Figure 22B.

[0172] The gating strategy for this assay was to first enable the identification of HEK-293F cells (Figure 22A-1), and then enable the identification of single cells by forward scattering area compared to height (Figure 22A-2). Subsequently, single cells were evaluated for intracellular tau staining with polyclonal whole tau antibody (Dako) in either 0N4R- (Figure 22A-3) or 0N3R- (Figure 22A-4) transfected cells.

[0173] In Figure 22B, as described above for Figure 21B, all tau levels are expressed as a percentage of the VR7082-GFP (binding but non-degradable intrabody) control. The data in Figure 22 clearly demonstrate that, in a different assay system, all degradable body constructs can degrade 0N4R tau while maintaining 0N3R tau levels in HEK-293F cells. For all constructs, 4R tau levels were statistically significantly reduced compared to 3R tau levels (by t-test) (Figure 22B). Since MG132 treatment blocked 4R tau degradation (Figure 22B), all fusions except ODC were proteasome-dependent and statistically significantly different for all 0N4R cotransfections with and without MG132 addition (by t-test) (Figure 22B). Again, this does not apply to ON3R tau-transfected cells, which do not show a statistically significant difference between the MG132-treated and untreated groups (Figure 22B), indicating that treatment with MG132 results only in proteasomal degradation and not in general intracellular tau proteostasis.

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

[0175] Example 11 4R tau decomposition using the VR7082 degrading body in iPSC-derived neurons In neuronal transfection using lipid-based transfection methodologies, the percentage of cells that accept the transfected DNA is very small (Karra and Dahm 2010). Therefore, viral transfection using adeno-associated virus (AAV) was selected as the delivery method for degradation bodies and controls for this project. Three AAV constructs were obtained from GeneCopeia, two of which included VR7082-scFv fused to either the XIAP degradation domain or a Halo tag (non-degradation control), as well as an AAV vector-negative control lacking this domain. The VR7082-XIAP construct was also obtained with or without IRES expression of the GFP transfection marker.

[0176] 10+16 biallelic MAPT mutant neurons were transfected 90 days post-induction using VR7082-XIAP+IRES GFP AAV. Cells were analyzed 7 days after transfection to account for the expression of degradation bodies 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 west-blood sterol (Figures 23B and 23C). The complete gating strategy can be seen in Figure 23A.

[0177] Live cells were initially 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 the presence of many events that are ToPro3 negative but very small and located in the debris region of the FSC-A v SSC-A plot (lower left of the plot). These may be cellular debris forming small lipid micelles. Such debris would theoretically have an intact lipid bilayer membrane and therefore would exclude ToPro3 staining (as live cells do). 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 blotting. Traces obtained can be seen from the sorted GFP-negative cells (Figure 23B) and the sorted GFP-positive cells (Figure 23C). The traces of the GFP-negative cells show two peaks: a large peak representing 0N3R and a small peak corresponding to 0N4R tau (Figure 23B). In contrast, the 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 cells transduced with XIAP.

[0179] In summary, the data shown in Figure 23 demonstrates that it is possible to deliver Degrabodies to iPSC-derived neurons and degrade the 4R tau protein from these cells.

[0180] Example 12 Phenotypic effects of 4R tau removal on iPSC-derived neurons Previous studies have shown that GFP may be toxic to neurons (Detrait et al. 2002, Mol Ther, 5(6), 723-30). Because the effect of GFP on mitochondrial polarization assays is unknown, this study used an AAV vector without GFP. For the same reason, VR7082-Halo-tagged intrabodies were used as a non-degradation control rather than the previously used VR7082-GFP intrabodies.

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

[0182] The gating strategy for this assay was to identify intact neurons that could 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 no effect in WT control neurons (Figure 24B-1) from any of 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 degrading bodies reduced the level 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 intrabodies in 10+16 monoallelic or 10+16 biallelic cell lines (p=0.145+p=0.922, ANOVA). This is because VR7082-Halo intrabodies bind to 4R tau in these cells but do not induce 4R tau degradation. However, when cells were transfected with VR7082-XIAP degrabodies, a statistically significant reduction in mitochondrial polarization was observed in both 10+16 monoallelic and 10+16 biallelic mutant cell lines from 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, the level of mitochondrial polarization in 10+16 monoallelic neurons decreases to a level that is not significantly different from that of WT neurons (p=0.567, ANOVA) (Figure 24C).

[0183] Since the VR7082-RbIgG 4R-specific antibody has the same epitope as the VR7082-scFv degrading body, it was not possible to evaluate 4R tau levels in these experiments. However, it has been previously demonstrated that VR7082-XIAP specifically induces 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 is no off-target degradation effect in these experiments (Figure 24D).

[0184] Here, the inventors successfully demonstrated that applying the VR7082 degra body to neurons derived from 10+16 MAPT mutant iPSCs can restore non-mutant levels of mitochondrial membrane polarization.

[0185] Example 13 Epitope characterization of VR7082 antibody We designed intracellular tau expression constructs to have a single alanine point mutation across the entire peptide epitope TE10, as well as the P301S and L mutations common in disease models of tauopathy. These constructs were synthesized into vectors suitable for intracellular tau expression using TWIST bioscience.

[0186] Once the construct was obtained, a single construct was 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 transferred to 350G. (av)Cells were recovered by centrifugation for 10 minutes. Following the manufacturer's instructions, cells were fixed and permeabilized using the Invitrogens Fix and Perm kit. Cells were then stained with non-VR7082 cross-blocking antibody HT7 labeled with alexa-488 for 30 minutes on ice, washed with PBS + 1% BSA, and seeded at 20,000 cells / well. Each well was then stained with 10 μg / ml VR7082 for 30 minutes on ice, and cells were washed once with PBS + 1% BSA. Secondary anti-rabbit Fc-Alexa647 (Jackson Laboratories) indicates that the antibody was added at a 1:1000 dilution. After incubating cells on ice for 30 minutes, they were washed with PBS + 1% BSA and resuspended in 50 μl / well. Cells were electrophoresed on IQue3, tau-containing cells were gated from HT7 by alexa-488 fluorescence, and tau binding was assessed by alexa-647 staining. The data was 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 knockdown of the tau binding signal in the presence of a single specific alanine mutation across the entire peptide binding site (bars 2 to 10 from left to right), binding to P301S tau (bar 11 from left to right), binding to P301L tau (bar 12 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-mutant 0N4R tau (leftmost bar). The results show that any of the K294;D295, N296, and I297 mutations to alanine almost completely eliminate the binding of the VR7082 antibody to tau. Any of the K298 and V300 mutations to alanine almost halve the binding of the VR7082 antibody to tau. Mutations in 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 its potential use in animal or cell models of tauopathy containing these mutations. The present invention includes the following preferred embodiments. (1) (a) Specifically binds to the 4R tau protein isoform in a physiological sample; or (b) Specifically binds to the 3R tau protein isoform in the physiological sample, An antibody or its antigen-binding fragment. (2) The antibody described in (1) or its antigen-binding fragment, which specifically binds to the 4R tau protein isoform. (3) (a) If the 4R tau protein has post-translational modifications at one or more of the amino acid positions 279, 280, 281, 285, and 289 of tau 4R, it binds to the amino acid region encoded by exon 10 of tau; (b) Binding to a peptide containing or consisting of amino acids 294-302 of the 4R tau protein; (c) Binding to a peptide corresponding to a single epitope from the amino acid sequence encoded by exon 10 of tau; or (d) Crossblock any of the antibodies or fragments from (a) to (c), or be crossblocked by them. (2) The antibody or antigen-binding fragment thereof as described in (2). (4) (a) an antibody or its antigen-binding fragment specifically binds to the 4R tau protein isoform in cell lysates from cells expressing the physiological 4R tau protein isoform, preferably in cell lysates from iPSC-derived nerve cells expressing the 4R tau protein isoform; and / or (b) The antibody or its antigen-binding fragment can detect the 4R tau protein isoform by immunofluorescence on or within cells expressing the 4R tau protein isoform. (2) or (3) the antibody or its antigen-binding fragment. (5) An antibody or its antigen-binding fragment (a) comprising one or more light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 3, 5, and 7, and one or more 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) Including the light chain variable region and heavy chain variable region of Sequence IDs 1 and 9; (c) an intrabody, preferably the sequence of Sequence ID No. 17, or an intrabody having at least 95% sequence identity thereto and still capable of specifically binding to the tau protein 4R isoform; or (d) Crossblock any of the antibodies or fragments from (a) to (c), or be crossblocked by them. (2) to (4) The antibody or antigen-binding fragment thereof as described in any one of the above items. (6) An antibody or antigen-binding fragment thereof, as described in any one of (2) to (5), that binds to an epitope of the 4R tau protein containing amino acids K294, D295, N296, and I297. (7) An antibody or antigen-binding fragment thereof according to any one of (2) to (5), wherein the epitope further comprises K298 and V300 of the 4R tau protein. (8) The antibody described in (1) or its antigen-binding fragment, which specifically binds to the 3R tau protein isoform. (9) An antibody or its antigen-binding fragment (a) To bind to a peptide comprising an amino acid sequence encoded by a region crosslinking exons 9 and 11 of tau, and / or (b) A peptide consisting of seven amino acids on either side of the boundary of the sequence encoded by exons 9 and 11 of tau, (8) The antibody or antigen-binding fragment thereof as described in (8). (10) The antibody or antigen-binding fragment thereof as described in (9), wherein the peptide is a cyclic peptide. (11) An antibody or its antigen-binding fragment (a) comprising one or more light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 20, 22, and 24, and one or more 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) Including the light chain variable region and heavy chain variable region of Sequence IDs 18 and 26; (c) an intrabody, preferably an intrabody comprising the sequence of Sequence ID No. 34, or a sequence having at least 95% sequence identity thereto and capable of specifically binding to the tau protein 3R isoform; or (d) Crossblock any of the antibodies or fragments from (a) to (c), or be crossblocked by them. An antibody or antigen-binding fragment thereof as described in any one of items (8) to (10). (12) An antibody or antigen-binding fragment thereof that specifically binds to the 4R tau protein, wherein the antibody or antigen-binding fragment binds to an epitope of the 4R tau protein containing amino acids K294, D295, N296, and I297, and optionally the epitope further contains K298 and V300 of the 4R tau protein. (13) An antibody or antigen-binding fragment thereof as described in any one of items (1) to (12), wherein the antibody is an intrabody or a degrabody. (14) One or more nucleic acids encoding an antibody or antigen-binding fragment as described in any one of items (1) to (13). (15) One or more vectors comprising one or more nucleic acids as described in (14). (16) A host cell containing one or more nucleic acids described in (14), or one or more vectors described in (15). (17) (a)An antibody or antigen-binding fragment thereof as described in any one of items (1) to (13), one or more nucleic acids as described in (14), or one or more vectors as described in (15); and (b) Pharmaceutical carrier or excipient A pharmaceutical composition containing the above. (18) (a) Contacting the test sample with the antibody or antigen-binding fragment thereof described in any one of paragraphs (2) to (7) or (12); and (b) Detection of the binding of an antibody or its antigen-binding fragment. A method for detecting 4R tau protein isoforms, including those mentioned above. (19) (a) Contacting the test sample with the antibody or antigen-binding fragment thereof described in any one of items (8) to (11); and (b) Detection of the binding of an antibody or its antigen-binding fragment. A method for detecting 3R tau protein isoforms, including those mentioned above. (20) A method for determining the levels of 4R tau protein isoform and 3R tau protein isoform, comprising performing the methods described in both (17) and (18). (21) The method according to (19), further comprising determining whether the relative amounts of 4R and 3R tau protein isoforms differ from those expected for a subject by, optionally, comparing them to the relative amounts of 4R and 3R in a sample from a healthy subject. (22) The method according to (19) or (20), used for diagnosing tauopathy, preferably PSP, CBD, or Pick's disease. (23) (a) Detection is mediated by immunofluorescence; (b) Detection is via Peggy Sue's simple Western blot; (c) Detection is via ELISA; or (c) If the test sample contains cell lysates, cells, or tissue, The method described in any one of items (18) to (22). (24) An antibody or antigen-binding fragment thereof as described in any one of (1) to (13), one or more nucleic acids as described in (14), one or more vectors as described in (15), or a pharmaceutical composition as described in (16), for use in a method of treating tauopathy. (25) An antibody, an antigen-binding fragment thereof, one or more nucleic acids, one or more vectors, or a pharmaceutical composition for use in the method of (24), wherein the tauopathy involves an imbalance between 4R tau protein isoforms and 3R tau protein isoforms. (26) A method for treating tauopathy, comprising administering to a subject having tauopathy an antibody or antigen-binding fragment thereof as described in any one of (1) to (13), one or more nucleic acids as described in (14), one or more vectors as described in (15), or a pharmaceutical composition as described in (15). (27) The method according to (26), wherein the tauopathy involves an imbalance between the 4R tau protein isoform and the 3R tau protein isoform. (28) Use of an antibody or antigen-binding fragment thereof as described in any one of (1) to (13), one or more nucleic acids as described in (14), one or more vectors as described in (15), or a pharmaceutical composition as described in (16) in the manufacture of a pharmaceutical product for use in the treatment of tauopathy. (29) The use of (28) described above, wherein tauopathy involves an imbalance between 4R tau protein isoforms and 3R tau protein isoforms.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to a 4R tau protein isoform in a physiological sample, The antibody or its antigen-binding fragment (a) 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) Including the light chain variable region and heavy chain variable region of Sequence IDs 1 and 9; (c) an intrabody of the antibody of (a) or (b) or its antigen-binding fragment; or (d) Crossblock any of the antibodies (a) to (c) or their antigen-binding fragments, or be crossblocked by them. An antibody or its antigen-binding fragment.

2. (a) If the 4R tau protein has post-translational modifications at one or more of the amino acid positions 279, 280, 281, 285, and 289 of tau 4R, it binds to the amino acid region encoded by exon 10 of tau; (b) Binding to a peptide containing or consisting of amino acids 294–302 of the 4R tau protein; (c) Binding to a peptide corresponding to a single epitope from the amino acid sequence encoded by exon 10 of tau; or (d) Crossblock any of the antibodies (a) to (c) or their antigen-binding fragments, or be crossblocked by them. The antibody or antigen-binding fragment thereof according to claim 1.

3. (a) an antibody or its antigen-binding fragment specifically binds to the 4R tau protein isoform in cell lysates from cells expressing the physiological 4R tau protein isoform; and / or (b) The antibody or its antigen-binding fragment can detect the 4R tau protein isoform by immunofluorescence on or within cells expressing the 4R tau protein isoform. The antibody or antigen-binding fragment thereof according to claim 1.

4. The antibody or antigen-binding fragment according to claim 3, which specifically binds to the 4R tau protein isoform in a cell lysate from iPSC-derived nerve cells expressing the 4R tau protein isoform.

5. The antibody or antigen-binding fragment according to claim 1, which binds to an epitope of a 4R tau protein containing amino acids K294, D295, N296, and I297.

6. The antibody or antigen-binding fragment according to claim 5, wherein the epitope further comprises K298 and V300 of the 4R tau protein.

7. The antibody or antigen-binding fragment according to claim 1, which binds to a TE10 peptide having the amino acid sequence of SEQ ID NO:

37.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises the sequence of SEQ ID NO: 17 or a sequence having at least 95% sequence identity therewith, and is still a degradable body capable of specifically binding to the tau protein 4R isoform.

9. One or more nucleic acids encoding the antibody or antigen-binding fragment thereof as described in claim 1.

10. One or more vectors comprising one or more nucleic acids as described in claim 8.

11. A host cell comprising one or more nucleic acids as described in claim 9.

12. (a) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, one or more nucleic acids according to claim 9, or one or more vectors according to claim 10; and (b) Pharmaceutical carrier or excipient A pharmaceutical composition containing the following:

13. (a) Contacting the test sample with the antibody or antigen-binding fragment thereof described in any one of claims 1 to 8; and (b) Detection of the binding of an antibody or its antigen-binding fragment. A method for detecting 4R tau protein isoforms, including [specific protein name].

14. (i) Contacting the test sample with the antibody or antigen-binding fragment thereof described in any one of claims 1 to 7, and Detecting the binding of an antibody or its antigen-binding fragment. A method for detecting 4R tau protein isoforms, including, (ii) Contacting the test sample with an antibody or its antigen-binding fragment that can specifically bind to the 3R tau protein isoform in the physiological sample, and Detecting the binding of an antibody or its antigen-binding fragment. A method for detecting 3R tau protein isoforms, including A method for measuring the levels of 4R tau protein isoforms and 3R tau protein isoforms, including performing the following.

15. The method of claim 14, further comprising providing information for determining whether the relative amounts of 4R and 3R tau protein isoforms differ from those expected for a subject by comparing them with the relative amounts of 4R and 3R in a sample from a healthy subject.

16. The method according to claim 15, wherein the measured levels provide information for diagnosing tauopathy, including PSP, CBD, or Pick's disease.

17. (a) Detection is mediated by immunofluorescence; (b) Detection is via Peggy Sue's simple Western blot; (c) Detection is via ELISA; or (c) The test sample contains cell lysates, cells, or tissues. The method according to claim 14.

18. A pharmaceutical composition for treating tauopathy, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, one or more nucleic acids according to claim 9, or one or more vectors according to claim 10.

19. The pharmaceutical composition according to claim 18, wherein the tauopathy comprises an imbalance between the 4R tau protein isoform and the 3R tau protein isoform.

20. The pharmaceutical composition according to claim 19, wherein the tauopathy is PSP, CBD, or Pick's disease.

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