Intrabodies targeting intracellular tau

Abstract

The present invention provides a method of binding intracellular tau in a cell, in which the method comprises contacting the cell with a vector comprising a polynucleotide encoding an intrabody that specifically binds tau. The present invention also provides a method of reducing tau aggregation in a cell, in which the method comprises contacting the cell with a vector comprising a polynucleotide encoding an intrabody that specifically binds tau. The intrabody may bind to an epitope comprising amino acid residues 151-158 of tau, amino acid residues pS199 / pS202 of tau, or amino acid residues 375-380 of tau, in which the numbering of the amino acid is with reference to the amino acid sequence set forth in SEQ ID NO: 1.

Description

TITLE INTRABODIES TARGETING INTRACELLULAR TAU CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.63 / 547,121, filed on 2 November 2023, which is incorporated herein by reference in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 1, 2024, is named JAB7214WOPCT1_SL.xml, and is 286,208 bytes in size. BACKGROUND OF THE INVENTION

[0001] Intracellular tau aggregation is a common feature of several neurodegenerative disorders, collectively referred to as tauopathies. For example, in Alzheimer’s disease (AD), the progressive accumulation of aggregated tau in the brain has been shown to correlate well with cognitive decline and neurodegeneration (Gordon et al., 2018; Lowe et al., 2019). Other tauopathies include Pick’s disease, marked by severe gross atrophy of the frontotemporal lobes and corresponding tau-positive intracellular inclusions; progressive supranuclear palsy, which features tau-positive glial inclusions in gray matter and “coiled-bodies” in oligodendrocytes in white matter; and corticobasal degeneration, which involves formation of diffuse tau-positive threads that resemble plaques along with white matter-coiled bodies and threads, tau-positive ballooned neurons, and neuronal tangles (Coughlin & Irwin, 2017). As a result, tau has become a frequent target for therapies that aim to treat or alleviate the effects of AD and other tauopathies.

[0002] One of the main therapeutic approaches targeting tau is immunotherapy. Immunotherapeutic strategies aim to halt disease progression by capturing extracellular forms of tau (Colin et al., 2019). As an example, monoclonal antibodies have been developed that target monomeric, aggregated forms, phospho-specific, or conformationally altered forms of tauprotein (Jadhav et al., 2019). However, because tau aggregation is an intracellular phenomenon, therapeutic modalities that can act inside the cell may be more effective.

[0003] In this regard, antisense oligonucleotides (ASOs) have been explored as a therapy for targeting intracellular tau. A small Phase I study showed that reducing intracellular tau levels using ASOs was safe and well-tolerated and reduced aggregated tau levels as measured by positron emission tomography (PET) in mild AD patients (Mummery et al., 2023). Yet, due to the size and highly-charged nature of ASOs, their use as a therapy may face challenges in terms of cellular uptake, stability, and susceptibility to degradation by nucleases, and, particularly with CNS targeted therapies, overcoming the blood-brain barrier (Jadhav et al., 2019).

[0004] Thus, there is a need in the art for strategies that target intracellular tau. The present invention addresses this need. SUMMARY OF INVENTION

[0005] Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.

[0006] The present invention is based, in part, on a series of important discoveries that are described in more detail in the Examples section of this patent specification. For example, three anti-tau intrabodies in the single-chain variable fragment (scFv) format were developed, derived from monoclonal antibodies (mAbs) PT51, PT77, and hTau21 (Vandermeeren et al., 2018). These scFvs bind distinct epitopes at the proline-rich domain (PRD) and C-terminus of the tau protein, including a phosphorylated epitope (pS199 / pS202). These scFv-intrabodies are shown to interfere with K18-mediated aggregation of human tau with the P301L mutation in primary mouse cortical neurons. Additionally, scFv PT77, which binds to an epitope around pS199 / pS202, was also able to reduce AD-seed-mediated aggregation of mouse tau in primaryneurons. Building on these discoveries, and other discoveries presented herein, the present invention provides a variety of new and improved methods targeting tau intracellularly.

[0007] Accordingly, in one aspect, the present invention relates to a method of binding intracellular tau in a cell, in which the method comprises contacting the cell with a vector comprising a polynucleotide encoding an intrabody that specifically binds tau. In another aspect, the present invention relates to a method of reducing tau aggregation in a cell, in which the method comprises contacting the cell with a vector comprising a polynucleotide encoding an intrabody that specifically binds tau.

[0008] In some embodiments, the vector comprises a viral vector. In certain embodiments, the viral vector comprises an adeno-associated-virus (AAV) vector. In particular embodiments, the AAV vector comprises an AAV serotype 6 vector.

[0009] In some embodiments, the method is in vitro.

[0010] In some embodiments, the tau is human tau.

[0011] In some embodiments, the cell is selected from a neuron, an astrocyte, and an oligodendrocyte. In certain embodiments, the cell is a neuron.

[0012] In some embodiments, the intrabody binds to an epitope comprising amino acid residues 151-158 of tau, amino acid residues pS199 / pS202 of tau, or amino acid residues 375-380 of tau, in which the numbering of the amino acid is with reference to the amino acid sequence set forth in SEQ ID NO: 1.

[0013] In some embodiments, the intrabody comprises a single chain variable fragment (scFv), in which the scFv comprises a heavy chain variable region and a light chain variable region interconnected by a linker. The heavy chain variable region may comprise heavy chain variable complementarity determining region (CDR)-1, CDR-2, and CDR-3, and the light chain variable region may comprise light chain variable CDR-1, CDR-2, and CDR-3.

[0014] In yet another aspect, the present invention relates to an anti-tau intrabody that binds specifically to tau. The intrabody comprises an scFv, in which the scFv comprises a heavy chain variable region and a light chain variable region interconnected by a linker. The heavy chainvariable region may comprise heavy chain variable CDR-1, CDR-2, and CDR-3, and the light chain variable region may comprise light chain variable CDR-1, CDR-2, and CDR-3.

[0015] In a further aspect, the present invention relates to a method of making an anti-tau intrabody for targeting tau intracellularly, in which the anti-tau intrabody comprises a single chain variable fragment. The method comprises (a) grafting CDRs of an anti-tau antibody to a scFv framework identified to be stable intracellularly, and (b) removing disulfide bonds in the scFv. The intrabody binds to an epitope comprising amino acid residues 151-158 of tau, amino acid residues pS199 / pS202 of tau, or amino acid residues 375-380 of tau, in which the numbering of the amino acid is with reference to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the CDRs comprise a heavy chain variable CDR-1, CDR-2, and CDR-3 and a light chain variable CDR-1, CDR-2, and CDR-3.

[0016] In some embodiments of the invention, the removing of the disulfide bonds in (b) comprises removing cysteine amino acids from the scFv. In certain embodiments, the removing of the disulfide bonds in (b) further comprises replacing the cysteine amino acids with a combination of alanine-valine.

[0017] According to embodiments of the invention, (a) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 4, 5, and 6, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (b) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 7, 8, and 6, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (c) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 9, 10, and 11, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 20, LVS, and 19, respectively; or(d) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 12, 13, and 6, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (e) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 14, 15, and 16, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively; or (f) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 4, 27, and 6, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (g) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 14, 28, and 16, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively; or (h) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 14, 31, and 16, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively; or (i) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 36, 37, and 38, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (j) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 39, 40, and 38, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or(k) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 41, 42, and 43, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 52, RMS, and 51, respectively; or (l) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 44, 45, and 38, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (m) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 46, 47, and 48, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 54, 55, and 56, respectively; or (n) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 36, 59, and 38, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (o) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 46, 60, and 48, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 54, 55, and 56, respectively; or (p) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 69, 70, and 71, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (q) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 72, 73, and 71, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or(r) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 74, 75, and 76, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 85, AAS, and 84, respectively; or (s) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 77, 78, and 71, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (t) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 79, 80, and 81, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively; or (u) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 69, 92, and 71, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (v) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 79, 93, and 81, respectively; and the light chain variable CDR-1, CDR- 2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively; or (w) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 79, 96, and 81, respectively; the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively.

[0018] In some embodiments of the invention, (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26; or(ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 29, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30; or (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 32, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30; or (iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 34; or (v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 34; or (vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 57, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 58; or (vii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62; or (viii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62; or (ix) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 64, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62; or (x) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 65, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 66; or(xi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 67, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 66; or (xii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 68, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 66; or (xiii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 90, and the light chain variable region variable region comprises the amino acid sequence of SEQ ID NO: 91; or (xiv) the heavy chain variable region variable region comprises the amino acid sequence of SEQ ID NO: 94, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 95; or (xv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 97, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 95; or (xvi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 98, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 99; or (xvii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 100, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 99.

[0019] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[0020] In another aspect, the present invention relates to a polynucleotide encoding the anti-tau intrabody.

[0021] In yet another aspect, the present invention relates to a vector comprising the polynucleotide. In some embodiments, the vector comprises a viral vector. In certainembodiments, the viral vector comprises an AAV vector. In particular embodiments, the AAV vector comprises an AAV serotype 6 vector.

[0022] In a further aspect, the present invention relates to a composition comprising a carrier and (a) an anti-tau antibody as described herein; or (b) a polynucleotide as described herein; or (c) a vector as described herein. BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0023] FIG.1 presents results demonstrating properties of the selected mAbs and characterization of derived scFvs, having a GS linker or a GSEK linker, expressed in the periplasm of E. coli and through the secretory pathway of HEK293 cells, as described in the Example. scFvs were expressed in the periplasm of E. coli and cleared cell lysates were used for characterization. Expression levels were determined by western blotting and arbitrary unit (A.U.) concentrations were determined based on western blotting quantification. Images shown in Panel A are representative of 2 independent experiments. All lysates were tested for binding against recombinant human tau on enzyme-linked immunosorbent assay (ELISA), starting from 1 A.U. of scFv. Plots of the results are shown in Panels B (lysates from expression of scFvs with a GS linker) and C (lysates from expression of scFvs with a GSEK linker). Detection in western blotting and ELISA was done with an anti-HA tag HRP-labelled antibody. Tau paired helical filaments (ePHF) coating was used for phospho-specific scFv PT77. Plots of the results are shown in Panels D (lysates from expression of scFvs with a GS linker) and E (lysates from expression of scFvs with a GSEK linker). Results are shown as mean of 2 independent experiments. scFvs were expressed as secreted protein from HEK293 cells and culture medium was used for characterization. Expression levels were determined by western blotting. Images of the western blots are shown in Panel F. All samples were tested against recombinant human tau on ELISA, starting from undiluted culture medium. Plots of the results are shown in Panels G (samples from expression of scFvs with a GS linker) and H (samples from expression of scFvs with a GSEK linker). ePHF coating was used for phospho-specific scFv PT77. Plots of the results are shown in Panels I (samples from expression of scFvs with a GS linker) and J (samples from expression of scFvs with a GSEK linker). Results are presented as mean ± SD of 3 independent experiments. Detection is done with an anti-FLAG-tag HRP-labelled antibody.

[0024] FIG.2 presents results demonstrating characterization of scFvs, having a GS linker or a GSEK linker, expressed and the cytoplasm of HEK293 cells, as described in the Example. scFvs were expressed in the cytoplasm of HEK293 cells and cleared cell lysates were used for characterization. Intrabody presence in the lysates was determined by western blotting, images of which are shown in Panel A. Cell lysates were tested on ELISA in serial dilution starting at 10 μg of total protein, against recombinant human tau. Plots of the results are shown in Panels B (lysates from expression of scFvs with a GS linker) and C (lysates from expression of scFvs with a GSEK linker). ePHF coating was used for phospho-specific scFv PT77. Plots of the results are shown in Panels D (samples from expression of scFvs with a GS linker) and E (samples from expression of scFvs with a GSEK linker). Results are shown as mean of 2 independent experiments. Detection is done with an anti-FLAG-tag HRP-labelled antibody.

[0025] FIG.3 presents results demonstrating intrabody solubility in the cytoplasm before and after CDR grafting, as described in the Example. Intrabodies were expressed in the cytoplasm of HEK293 cells and fixed 24 hrs after transfection. Intrabody detection is done via a C-terminal FLAG-tag. Panel A shows a schematic representation of the CDR grafting strategy. The CDRs from each chain of the original scFvs (represented in blue) are transferred to a new framework (represented in brown). Framework amino acids identified as important for binding are transferred as well (represented by blue stripes). Additionally, versions where cysteines (indicated by C) are replaced by the amino acid combination Val-Ala (indicated by V and A, respectively) were also designed. Designed with biorender.com Panel B shows images of immunocytochemistry evaluation of intrabody solubility in the cytoplasm. Images labeled B1- B6 show results with original intrabody sequences; images labeled B7-B9 shows results with intrabodies designed in the VL-VH orientation; images labeled B10-B22 shows results with CDR-grafted versions, with and without disulfide bonds (SS-). Images are representative of 3 independent experiments with 2 replicates each. Scale bar: 25 μm. SS-: scFv without the cysteines that participate in disulfide bonds.

[0026] FIG.4 presents results demonstrating evaluation of intrabody tau binding after CDR grafting into different frameworks, as described in the Example. Intrabodies of PT51 (Panel A), hTa21 (Panel B), and PT77 (Panel C) were expressed in HEK293A cells and cell lysates were tested in a serial dilution starting at 1:3 dilution for binding against recombinant human tau onELISA. ePHF coating was used for phospho-specific intrabody of PT77 (Panel D). Detection is done with an anti-FLAG-tag HRP-labelled antibody. SS-: scFv without the cysteines that participate in disulfide bonds. Results are represented as mean ± SD of 3 independent experiments.

[0027] FIG.5 presents results demonstrating evaluation of intrabody tau binding in the cytoplasm of HEK293A cells, as described in the Example. Panel A shows images of HEK293 cells co-transfected with a plasmid expressing the intrabody and a plasmid expressing either tau or Į-synuclein coupled to green fluorescent protein (GFP) and a nuclear translocation signal (NLS). When the intrabody is co-expressed with tau-NLS, it can be translocated to the nucleus only if it is capable of binding tau in the cytoplasmic environment. Co-expression with human Į-synuclein-NLS is used as negative control. Panel B shows plot of tau-NLS phosphorylated at S199 / S202 detected in cell lysates using a sandwich MSD assay with PT77 as capture antibody. Images are representative of 3 independent experiments with 2 biological replicates each. Scale bar: 50 μM. SS-: scFv without the cysteines that participate in disulfide bonds.

[0028] FIG.6 presents results demonstrating the effect of intrabodies on AD-seed mediated aggregation, as described in the Example. Primary mouse cortical neurons were transduced with AAV-intrabody on day in vitro (DIV) 7 followed by addition of AD-tau-seeds on DIV 10. Neurons were kept until DIV 17 after which they were lysed in RIPA buffer. Endogenous mouse tau aggregation was measured on cell lysates with sandwich MSD assays. Results for intrabodies PT51 (Panel A), PT77 S- 4D5 (Panel B), hTau21 (Panel C), and negative control scFv (Panel D) are shown as plots of the percentage of condition without intrabody expression after normalization to total mouse Į-synuclein levels, along with western blot image inserts. Expression levels were detected with western blotting using undiluted lysates. ȕ-actin was used as loading control. MSD results are shown as mean ± SD of 3 independent experiments. Statistical analysis done with the fitted mixed-effect model with Dunnett correction for multiple comparisons (**p-value ^ 0.01). Western blot images are representative of 3 independent experiments. SS-: scFv without the cysteines that participate in disulfide bonds. MOI: multiplicity of infection.

[0029] FIG.7 presents results demonstrating the effect of intrabodies on hTau-P301L aggregation upon K18-P301L seeds addition, as described in the Example. Primary mousecortical neurons were transduced with AAV-intrabody and AAV-hTauP301L on DIV 1, followed by addition of sonicated K18-P301L seeds on DIV 3. Neurons were kept until DIV 10 after which they were lysed in RIPA buffer. hTau-P301L aggregates were measured on cell lysates using sandwich MSD assays. Results for intrabodies PT51 (Panel A), PT77 S- 4D5 (Panel B), hTau21 (Panel C), and negative control scFv (Panel D) are shown as plots of the percentage of condition without intrabody expression after normalization to total mouse Į- synuclein levels. Expression levels were detected with western blotting using undiluted lysates. ȕ-actin was used as loading control. MSD results are shown as mean ± SD of 3 independent experiments. Statistical analysis done with the fitted mixed-effect model with Dunnett correction for multiple comparisons (**p-value ^ 0.01; ***p-value ^ 0.001). Western blot images are representative of 3 independent experiments.

[0030] FIG.8 presents results demonstrating an evaluation of the effect of anti-tau scFv- intrabodies in organotypic hippocampal slice cultures (OHSCs), as described in the Example. OHSCs were transduced with AAV6-intrabody at DIV 1 and 2, followed by addition of K18 seeds two weeks later. Slices were lysed at DIV 31. Panels A-D shows results of aggregated and phospho-aggregated tau levels measured using MSD assays. Two phospho-independent assays were used: one with a sandwich of hTau43 (Panel A), and another with a proprietary C- terminal tau antibody (Panel B). Phosphorylated aggregates were detected with AT8 / AT8 (S202 / T205 / S209) (Panel C) and PT3 / PT3 (T212 / T217) (Panel D). Results are shown as percentage of the signal / background of slices treated with K18 and no intrabody (K18 alone), calculated from interpolated values in arbitrary units (A.U.) with mean ± SD. Statistical analysis was done with the fitted mixed-effect model with Dunnett’s correction for multiple comparisons (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001). Note that for the AT8 / AT8 assay, no values could be interpolated for scFv-intrabody PT77. Thus, the values were set to the lowest limit of quantification. Panel E shows results of scFv-intrabody expression levels evaluated with anti-FLAG staining on western blotting. Panel F shows results of tau aggregates isolated from the brain of patients with AD, incubated with increasing amount of mAb PT77, prior to the measurements.DETAILED DESCRIPTION OF THE INVENTION

[0031] The practice of the present invention can employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.

[0032] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.

[0033] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0034] All references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art. Definitions

[0035] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0036] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0037] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone).Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0038] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and / or “consisting essentially of” are included.

[0039] Units, prefixes, and symbols are denoted in their Système International d’Unités (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Likewise, a disclosed range is a disclosure of each individual value (i.e., intermediate) encompassed by the range, including integers and fractions. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10 individually, and of 5.2, 7.5, 8.7, and so forth.

[0040] Unless otherwise indicated, the terms “at least” or “about” preceding a series of elements is to be understood to refer to every element in the series. The term “about” preceding a numerical value includes ± 10% of the recited value. For example, a concentration of about 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of about 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v).

[0041] Amino acids are referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.

[0042] The term “intrabody” refers to an antibody that has been designed to be expressed intracellularly and that recognizes and specifically binds to an antigen within a cell, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing, through at least one antigen recognition site within the variable region of the molecule. Most commonly, an intrabody is a single chain variable fragment, or “scFv,” comprising a heavy chain variable region and a light chain variable region interconnected by alinker. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0043] The heavy and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with regions that are more conserved, termed framework (FW) regions. The CDRs in each chain are held together in close proximity by the FW regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of intrabodies. Each heavy and light chain variable region is composed of three CDRs: CDR-1, CDR-2, and CDR-3.

[0044] There are at least two techniques for determining CDRs: (1) an approach based on cross- species sequence variability (Kabat et al., 1991); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., 1997). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0045] The amino acid position numbering as in Kabat, refers to the numbering system used for heavy chain variable domains or light chain variable domains (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FW or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a, according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc., according to Kabat) after heavy chain FW residue 82.

[0046] The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia & Lesk, 1987). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the KabatCDRs and Chothia structural loops, and are used by Oxford Molecular’ s AbM antibody modeling software.

[0047] IMGT (ImMunoGeneTics) also provides a numbering system for the variable regions, including the CDRs (see, e.g., Lefranc et al., 2003). The IMGT numbering system was based on an alignment of more than 5,000 sequences, structural data, and characterization of hypervariable loops and allows for easy comparison of the variable and CDR regions for all species. According to the IMGT numbering schema heavy chain variable region CDR-1 is at positions 26 to 35, heavy chain variable region CDR-2 is at positions 51 to 57, heavy chain variable region CDR-3 is at positions 93 to 102, light chain variable region CDR-1 is at positions 27 to 32, light chain variable region CDR-2 is at positions 50 to 52, and light chain variable region CDR-3 is at positions 89 to 97.

[0048] In addition, the variable regions can be delineated based on “Specificity Determining Residue Usage” (SDRU) (Almagro 2004), where SDR, refers to amino acid residues of an immunoglobulin that are directly involved in antigen contact. This SDRU concept was used to develop the “Contact” method of defining the CDRs, which renamed the SDRs as “contact residues” (MacCallum et al., 1996).

[0049] The term “germlining” means that amino acids at specific positions in an antibody are mutated back to those in the germ line.

[0050] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an intrabody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., intrabody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity intrabodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity intrabodies generally bind antigen faster and tend to remain bound longer.

[0051] The affinity or avidity of an intrabody for an antigen can be determined experimentally using any suitable method known in the art, e.g., flow cytometry, enzyme-linked immunosorbentassay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., KINEXA®or BIACORE™ or OCTET®analysis). Direct binding assays as well as competitive binding assay formats can be readily employed (see, e.g., Berzofsky et al., 1984; Kuby, 1992). The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH, temperature). Thus, measurements of affinity and other antigen-binding parameters (e.g., KDor Kd, Kon, Koff) are made with standardized solutions of antibody and antigen, and a standardized buffer, as known in the art.

[0052] The terms “reduce,” “inhibit,” “block,” and “suppress” are used interchangeably and refer to any statistically significant decrease in a given activity, including full blocking of the activity. For example, “reduction” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in activity. Accordingly, when the terms “reduction” or “inhibition” or “suppression” are applied to describe, e.g., an effect of an anti-tau intrabody, the terms may refer to the ability of an anti-tau intrabody to statistically significantly decrease: (a) binding of anti-tau intrabody to tau, or (b) activation of, or signaling by, tau, and the like. Inhibition may be determined relative to an untreated control—for example, a control not contacted with the anti- tau intrabody. In some embodiments, anti-tau intrabody can inhibit an activity of tau (such as those listed above) by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% or about 100%, as determined, for example, by flow cytometry, Western blotting, ELISA, proliferation assays, or other assays known to those of skill in the art.

[0053] The term “composition” refers to a preparation that is in such form as to permit the activity of the active ingredient to be effective and which contains no additional components that are unacceptably toxic to environment in which the composition would be delivered. Such composition can be sterile and can comprise a carrier, such as saline.

[0054] An “effective amount” of an intrabody as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.

[0055] The anti-tau intrabody of the invention can be naked or conjugated to other molecules such as toxins, labels, etc. The term “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to an intrabody, so as to generate a “labeled” intrabody. The label can be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, as in the case of, for instance, an enzymatic label, can catalyze chemical alteration of a substrate compound or composition that is detectable.

[0056] As used herein, the term “epitope” refers to a site on an antigen to which an immunoglobulin, antibody, or antigen-binding fragment thereof, specifically binds. Epitopes can be formed both from contiguous amino acids or from noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols, 1996).

[0057] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids and non-amino acids can interrupt it. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. In certain embodiments, the polypeptides can occur as single chains or associated chains.

[0058] A “polynucleotide,” as used herein can include one or more “nucleic acids,” “nucleic acid molecules,” or “nucleic acid sequences,” and refers to a polymer of nucleotides of any length, and includes DNA and RNA. The polynucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0059] The term “vector” means a construct, which is capable of delivering and, in some embodiments expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0060] An “isolated” polypeptide, intrabody, polynucleotide, or vector is in a form not found in nature. Isolated polypeptides, intrabodies, polynucleotides, or vectors include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, intrabody, polynucleotide, or vector that is isolated is substantially pure. When used herein, the term “substantially pure” refers to purity of greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%.

[0061] Other terms are defined elsewhere in this patent disclosure, or else are used in accordance with their usual meaning in the art. Anti-Tau Intrabodies

[0062] The present invention provides anti-tau intrabodies that specifically bind tau.

[0063] As used herein, the term “tau” or “tau protein”, also known as microtubule-associated protein tau, MAPT, neurofibrillary tangle protein, paired helical filament (PHF)-tau, MAPTL, or MTBT1, refers to an abundant central and peripheral nervous system protein having multiple isoforms. In the human central nervous system (CNS), six major tau isoforms ranging in size from 352 to 441 amino acids in length exist due to alternative splicing (Hanger et al., 2009). Examples of tau include, but are not limited to, tau isoforms in the CNS, such as the 441-amino acid longest tau isoform (2N4R), also named microtubule-associated protein tau isoform 2, that has four repeats and two inserts, such as the human tau isoform 2 having the amino acid sequence represented in SEQ ID NO: 1. Other examples of tau include the 352-amino acid long shortest (fetal) isoform (3R0N), also named microtubule-associated protein tau isoform 4, that has three repeats and no inserts, such as the human tau isoform 4 having the amino acid sequence represented in GenBank Accession No. NP_058525.1. Examples of tau also include the “big tau” isoform expressed in peripheral nerves that contains 300 additional residues (exon 4a)(Friedhoff et al., 2000). Examples of tau include a human big tau that is a 758 amino acid-long protein encoded by an mRNA transcript 6762 nucleotides long (NM_016835.4), or isoforms thereof. The amino acid sequence of the exemplified human big tau is represented in GenBank Accession No. NP_058519.3. As used herein, the term “tau” includes homologs of tau from species other than human, such as Macaca Fascicularis (cynomolgus monkey), rhesus monkeys or Pan troglodytes (chimpanzee). As used herein, the term “tau” includes proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions, and splice variants of full- length wild type tau. The term “tau” also encompasses post-translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, phosphorylation.

[0064] The anti-tau intrabody specifically binds to an epitope of tau. In some embodiments, the epitope comprises a domain of tau selected from the mid-term domain, the PRD, and the carboxy-terminus domain. In some embodiments, the epitope is a phosphorylated epitope.

[0065] In certain embodiments, the epitope comprises amino acid residues 151-158 of tau, amino acid residues pS199 / pS202 of tau, or amino acid residues 375-380 of tau, wherein the numbering of the amino acid is with reference to the amino acid sequence set forth in SEQ ID NO: 1.

[0066] In embodiments of the invention, the anti-tau intrabody of the present invention comprises an scFv. Thus, the anti-tau intrabody comprises a heavy chain variable region and a light chain variable region interconnected by a linker. In some embodiments, the linker comprises an amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 2) (referred to herein as a “GS linker”). In other embodiments, the linker comprises an amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 3) (referred to herein as a “GSEK linker”).

[0067] Exemplary anti-tau intrabodies of the present invention include an intrabody derived from monoclonal antibody PT51, referred to herein as “scFv PT51.” The amino acid sequences for the CDRs of scFv PT51 are presented in Table 1, which also provides SEQ ID NOs for each amino acid sequence.Table 1. Sequences for the heavy chain variable CDRs and light chain variable CDRs of scFv PT51. Kabat numbering scheme Variable Region CDR-1 CDR-2 CDR-3 TSWMN RIYPGDGDTNYNGKFKD SDWEGFAY Heavy Chain (SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 6) KSTKSLLNSDGFTYLD LVSNRFS FQTNYLPLT Light Chain (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) Chothia numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GYAFSTS YPGDGD SDWEGFAY Heavy Chain (SEQ ID NO: 7) (SEQ ID NO: 8) (SEQ ID NO: 6) KSTKSLLNSDGFTYLD LVSNRFS FQTNYLPLT Light Chain (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) IMGT numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GYAFSTSW IYPGDGDT TRSDWEGFAY Heavy Chain (SEQ ID NO: 9) (SEQ ID NO: 10) (SEQ ID NO: 11) KSLLNSDGFTY FQTNYL t Chain (SEQ ID NO: 20)LVPLT LighS(SEQ ID NO: 19) ABM numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GYAFSTSWMN RIYPGDGDTN SDWEGFAY Heavy Chain (SEQ ID NO: 12) (SEQ ID NO: 13) (SEQ ID NO: 6) KSTKSLLNSDGFTYLD LVSNRFS FQTNYLPLT Light Chain (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) Contact numbering scheme Variable Region CDR-1 CDR-2 CDR-3 STSWMN WIGRIYPGDGDTN TRSDWEGFA Heavy Chain (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) LNSDGFTYLDWY LLIYLVSNRF FQTNYLPL Light Chain (SEQ ID NO: 22) (SEQ ID NO: 23) (SEQ ID NO: 24)

[0068] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises: (a) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 4, 5, and 6, respectively; and a light chain variable CDR- 1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or(b) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 7, 8, and 6, respectively; and a light chain variable CDR- 1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; (c) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 9, 10, and 11, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 20, LVS, and 19, respectively; or (d) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 12, 13, and 6, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (e) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 14, 15, and 16, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively.

[0069] The heavy chain variable region and light chain variable region of scFv PT51 are presented in Table 2, which also provides SEQ ID NOs for each amino acid sequence. Table 2. Sequences for the heavy and light chain variable regions of scFv PT51. Variable Region Amino Acid Sequence QVQLQQSGPELVKPGASVKISCEASGYAFSTSWMNWVKQRPGK GLEWIGRIYPGDGDTNYNGKFKDKATLTADKSSSTVYMQLSSLT Heavy Chain SEDSAVYFCTRSDWEGFAYWGQGTLVTVSA (SEQ ID NO: 25) DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKP GQSPQLLIYLVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVY Light Chain YCFQTNYLPLTFGAGTKLELK (SEQ ID NO: 26)

[0070] Therefore, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0071] Other exemplary anti-tau intrabodies of the present invention include intrabodies derived from monoclonal antibody PT51, but grafted into a framework based on antibody germlines with high similarity to previously-described scFvs F8 and A48-4D5 (Donini et al., 2003; Wörn & Plückthun, 1998; Wörn & Plückthun, 1999), the resulting intrabodies of which is referred to herein as “scFv PT51 [F8]” or “scFv PT51 F8”, and “scFv PT51 [A48-4D5]” or “scFv PT51 A48-4D5”, respectively.

[0072] The amino acid sequences for the CDRs of scFv PT51 [F8] are presented in Table 3, which also provides SEQ ID NOs for each amino acid sequence. Table 3. Sequences for the heavy chain variable CDRs and light chain variable CDRs of scFv PT51 [F8]. Kabat numbering scheme Variable Region CDR-1 CDR-2 CDR-3 TSWMN RIYPGDGDTNYNDSVKG SDWEGFAY Heavy Chain (SEQ ID NO: 4) (SEQ ID NO: 27) (SEQ ID NO: 6) KSTKSLLNSDGFTYLD LVSNRFS FQTNYLPLT Light Chain (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) Chothia numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GYAFSTS YPGDGD SDWEGFAY Heavy Chain (SEQ ID NO: 7) (SEQ ID NO: 8) (SEQ ID NO: 6) KSTKSLLNSDGFTYLD LVSNRFS FQTNYLPLT Light Chain (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) IMGT numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GYAFSTSW IYPGDGDT TRSDWEGFAY Heavy Chain (SEQ ID NO: 9) (SEQ ID NO: 10) (SEQ ID NO: 11) KSLLNSDGFTYLVSFQTNYLPLT Light Chain (SEQ ID NO: 20)(SEQ ID NO: 19) ABM numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GYAFSTSWMN RIYPGDGDTN SDWEGFAY Heavy Chain (SEQ ID NO: 12) (SEQ ID NO: 13) (SEQ ID NO: 6) KSTKSLLNSDGFTYLD LVSNRFS FQTNYLPLT Light Chain (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) Contact numbering scheme Variable Region CDR-1 CDR-2 CDR-3 STSWMN WVSRIYPGDGDTN TRSDWEGFA Heavy Chain (SEQ ID NO: 14) (SEQ ID NO: 28) (SEQ ID NO: 16)LNSDGFTYLDWY LLIYLVSNRF FQTNYLPL Light Chain (SEQ ID NO: 22) (SEQ ID NO: 23) (SEQ ID NO: 24)

[0073] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 4, 27, and 6, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively. In other embodiments, the anti-tau intrabody comprises a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 14, 28, and 16, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively.

[0074] The heavy and light chain variable regions of scFv PT51 [F8] are presented in Table 4, which also provides SEQ ID NOs for each amino acid sequence. Table 4. Sequences for the heavy and light chain variable regions of scFv PT51 [F8]. Variable Region Amino Acid Sequence QVQLVESGGGLVQPGGSLRLSCSASGYAFSTSWMNWVRQAPG KGLEWVSRIYPGDGDTNYNDSVKGRFTLSADKSKSTVYLQMN Heavy Chain SLRAEDTAVYFCTRSDWEGFAYWGQGTLVTVSS (SEQ ID NO: 29) DIQLTQSPSSLSASVGDRVTITCKSTKSLLNSDGFTYLDWYQQK PGQSPKLLIYLVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFAT Light Chain YYCFQTNYLPLTFGQGTKLEIK (SEQ ID NO: 30)

[0075] Hence, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30.

[0076] The amino acid sequences for the CDRs of scFv PT51 [A48-4D5] are presented in Table 5, which also provides SEQ ID NOs for each amino acid sequence. Table 5. Sequences for the heavy chain variable CDRs and light chain variable CDRs of scFv PT51 [A48-4D5].Kabat numbering scheme Variable Region CDR-1 CDR-2 CDR-3 TSWMN RIYPGDGDTNYNDSVKG SDWEGFAY Heavy Chain (SEQ ID NO: 4) (SEQ ID NO: 27) (SEQ ID NO: 6) KSTKSLLNSDGFTYLD LVSNRFS FQTNYLPLT Light Chain (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) Chothia numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GYAFSTS YPGDGD SDWEGFAY Heavy Chain (SEQ ID NO: 7) (SEQ ID NO: 8) (SEQ ID NO: 6) KSTKSLLNSDGFTYLD LVSNRFS FQTNYLPLT Light Chain (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) IMGT numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GYAFSTSW IYPGDGDT TRSDWEGFAY Heavy Chain (SEQ ID NO: 9) (SEQ ID NO: 10) (SEQ ID NO: 11) KSLLNSDGFTY FQTNYLPLT Light Chain(SEQ ID NO: 20)LVS(SEQ ID NO: 19) ABM numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GYAFSTSWMN RIYPGDGDTN SDWEGFAY Heavy Chain (SEQ ID NO: 12) (SEQ ID NO: 13) (SEQ ID NO: 6) KSTKSLLNSDGFTYLD LVSNRFS FQTNYLPLT Light Chain (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) Contact numbering scheme Variable Region CDR-1 CDR-2 CDR-3 STSWMN WVARIYPGDGDTN TRSDWEGFA Heavy Chain (SEQ ID NO: 14) (SEQ ID NO: 31) (SEQ ID NO: 16) LNSDGFTYLDWY LLIYLVSNRF FQTNYLPL Light Chain (SEQ ID NO: 22) (SEQ ID NO: 23) (SEQ ID NO: 24)

[0077] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 14, 31, and 16, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively.

[0078] The heavy and light chain variable regions of scFv PT51 [A48-4D5] are presented in Table 6, which also provides SEQ ID NOs for each amino acid sequence. Table 6. Sequences for the heavy and light chain variable regions of scFv PT51 [A48-4D5].Variable Region Amino Acid Sequence EVQLVESGGGLVQPGGSLRLSCAASGYAFSTSWMNWVRQAPG KGLEWVARIYPGDGDTNYNDSVKGRFTLSADKAKSSVYLQMN Heavy Chain SLRAEDTAVYFCTRSDWEGFAYWGQGTLVTVSS (SEQ ID NO: 32) DIQLTQSPSSLSASVGDRVTITCKSTKSLLNSDGFTYLDWYQQK PGQSPKLLIYLVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFAT Light Chain YYCFQTNYLPLTFGQGTKLEIK (SEQ ID NO: 30)

[0079] Therefore, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30.

[0080] Other exemplary anti-tau intrabodies of the present invention include intrabodies derived from monoclonal antibody PT51, grafted into a framework based on antibody germlines with high similarity to previously-described scFvs F8 and A48-4D5, and rendered cysteine-free by replacing cysteine with the amino acid combination alanine-valine, the resulting intrabodies of which are referred to herein as “scFv PT51-SS−[F8]”, and “scFv PT51-SS−[A48-4D5]”, respectively.

[0081] The amino acid sequences for the CDRs of scFv PT51-SS−[F8] are the same as the CDRs of scFv PT51 [F8] presented in Table 3. The heavy and light chain variable regions of scFv PT51-SS−[F8] are presented in Table 7, which also provides SEQ ID NOs for each amino acid sequence. Table 7. Sequences for the heavy and light chain variable regions of scFv PT51-SS−[F8]. Variable Region Amino Acid Sequence QVQLVESGGGLVQPGGSLRLSVSASGYAFSTSWMNWVRQAPG KGLEWVSRIYPGDGDTNYNDSVKGRFTLSADKSKSTVYLQMNS Heavy Chain LRAEDTAVYFATRSDWEGFAYWGQGTLVTVSS (SEQ ID NO: 33) DIQLTQSPSSLSASVGDRVTITVKSTKSLLNSDGFTYLDWYQQKP GQSPKLLIYLVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY Light Chain AFQTNYLPLTFGQGTKLEIK (SEQ ID NO: 34)

[0082] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 34.

[0083] The amino acid sequences for the CDRs of scFv PT51-SS−[A48-4D5] are the same as the CDRs of scFv PT51 [A48-4D5] presented in Table 5. The heavy and light chain variable regions of scFv PT51-SS−[A48-4D5] are presented in Table 8, which also provides SEQ ID NOs for each amino acid sequence. Table 8. Sequences for the heavy and light chain variable regions of scFv PT51-SS−[A48-4D5]. Variable Region Amino Acid Sequence EVQLVESGGGLVQPGGSLRLSVAASGYAFSTSWMNWVRQAPGK GLEWVARIYPGDGDTNYNDSVKGRFTLSADKAKSSVYLQMNSLR Heavy Chain AEDTAVYFATRSDWEGFAYWGQGTLVTVSS (SEQ ID NO: 35) DIQLTQSPSSLSASVGDRVTITVKSTKSLLNSDGFTYLDWYQQKPG QSPKLLIYLVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAF Light Chain QTNYLPLTFGQGTKLEIK (SEQ ID NO: 34)

[0084] Hence, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 34.

[0085] Yet other exemplary anti-tau intrabodies of the present invention include an intrabody derived from monoclonal antibody PT77, referred to herein as “scFv PT77.” The amino acid sequences for the CDRs of scFv PT77 are presented in Table 9, which also provides SEQ ID NOs for each amino acid sequence. Table 9. Sequences for the heavy chain variable CDRs and light chain variable CDRs of scFv PT77. Kabat numbering scheme Variable Region CDR-1 CDR-2 CDR-3 TYAMN RIRSKSDNYATYYADSVKD QDYYV Heavy Chain (SEQ ID NO: 36) (SEQ ID NO: 37) (SEQ ID NO: 38) RSSKSLLHSNGKTYLY RMSNLVS MQHLEYPLT Light Chain (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51) Chothia numbering schemeVariable Region CDR-1 CDR-2 CDR-3 GFSFNTY RSKSDNYA QDYYV Heavy Chain (SEQ ID NO: 39) (SEQ ID NO: 40) (SEQ ID NO: 38) RSSKSLLHSNGKTYLY RMSNLVS MQHLEYPLT Light Chain (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51) IMGT numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GFSFNTYA IRSKSDNYAT VRQDYYV Heavy Chain (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) KSLLHSNGKTY RMSMQHLEYPLT Light Chain (SEQ ID NO: 52)(SEQ ID NO: 51) ABM numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GFSFNTYAMN RIRSKSDNYATY QDYYV Heavy Chain (SEQ ID NO: 44) (SEQ ID NO: 45) (SEQ ID NO: 38) RSSKSLLHSNGKTYLY RMSNLVS MQHLEYPLT Light Chain (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51) Contact numbering scheme Variable Region CDR-1 CDR-2 CDR-3 NTYAMN WVARIRSKSDNYATY VRQDYY Heavy Chain (SEQ ID NO: 46) (SEQ ID NO: 47) (SEQ ID NO: 48) LHSNGKTYLYWF LLIYRMSNLV MQHLEYPL Light Chain (SEQ ID NO: 54) (SEQ ID NO: 55) (SEQ ID NO: 56)

[0086] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises: (a) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 36, 37, and 38, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (b) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 39, 40, and 38, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; (c) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 41, 42, and 43, respectively; and a light chain variableCDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 52, RMS, and 51, respectively; or (d) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 44, 45, and 38, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (e) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 46, 47, and 48, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 54, 55, and 56, respectively.

[0087] The heavy and light chains of scFv PT77 are presented in Table 10, which also provides SEQ ID NOs for each amino acid sequence. Table 10. Sequences for the heavy and light chain variable regions of scFv PT77. Variable Region Amino Acid Sequence EVKLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGK GLEWVARIRSKSDNYATYYADSVKDRFTISRDDSESMLYLQMNN Heavy Chain LKTEDTAMYYCVRQDYYVWGTGTSVTVSS (SEQ ID NO: 57) DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGKTYLYWFLQRPG QSPQLLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYY Light Chain CMQHLEYPLTFGAGTKLELK (SEQ ID NO: 58)

[0088] Therefore, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58.

[0089] Other exemplary anti-tau intrabodies of the present invention include intrabodies derived from monoclonal antibody PT77, but grafted into a framework based on antibody germlines with high similarity to previously-described scFvs F8, 4D5, and A48-4D5 (Donini et al., 2003; Wörn & Plückthun, 1998; Wörn & Plückthun, 1999), the resulting intrabodies of which is referred to herein as “scFv PT77 [F8]” or “scFv PT77 F8”, “scFv PT77 [4D5]” or “scFv PT774D5”, and “scFv PT77 [A48-4D5]” or “scFv PT77 A48-4D5”, respectively.

[0090] The amino acid sequences for the CDRs of scFv PT77 [F8] are presented in Table 11, which also provides SEQ ID NOs for each amino acid sequence. Table 11. Sequences for the heavy chain variable CDRs and light chain variable CDRs of scFv PT77 [F8]. Kabat numbering scheme Variable Region CDR-1 CDR-2 CDR-3 TYAMN RIRSKSDNYATYYADSVKG QDYYV Heavy Chain (SEQ ID NO: 36) (SEQ ID NO: 59) (SEQ ID NO: 38) RSSKSLLHSNGKTYLY RMSNLVS MQHLEYPLT Light Chain (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51) Chothia numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GFSFNTY RSKSDNYA QDYYV Heavy Chain (SEQ ID NO: 39) (SEQ ID NO: 40) (SEQ ID NO: 38) RSSKSLLHSNGKTYLY RMSNLVS MQHLEYPLT Light Chain (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51) IMGT numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GFSFNTYA IRSKSDNYAT VRQDYYV Heavy Chain (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) KSLLHSNGKTY RMSMQHLEYPLT Light Chain (SEQ ID NO: 52)(SEQ ID NO: 51) ABM numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GFSFNTYAMN RIRSKSDNYATY QDYYV Heavy Chain (SEQ ID NO: 44) (SEQ ID NO: 45) (SEQ ID NO: 38) RSSKSLLHSNGKTYLY RMSNLVS MQHLEYPLT Light Chain (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51) Contact numbering scheme Variable Region CDR-1 CDR-2 CDR-3 NTYAMN WVSRIRSKSDNYATY VRQDYY Heavy Chain (SEQ ID NO: 46) (SEQ ID NO: 60) (SEQ ID NO: 48) LHSNGKTYLYWF LLIYRMSNLV MQHLEYPL Light Chain (SEQ ID NO: 54) (SEQ ID NO: 55) (SEQ ID NO: 56)

[0091] Hence, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 36, 59, and 38, respectively; and a light chain variable CDR-1, CDR- 2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively.In other embodiments, the anti-tau intrabody comprises a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 46, 60, and 48, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 54, 55, and 56, respectively.

[0092] The heavy and light chain variable regions of scFv PT77 [F8] are presented in Table 12, which also provides SEQ ID NOs for each amino acid sequence. Table 12. Sequences for the heavy and light chain variable regions of scFv PT77 [F8]. Variable Region Amino Acid Sequence QVQLVESGGGLVQPGGSLRLSCSASGFSFNTYAMNWVRQAPG KGLEWVSRIRSKSDNYATYYADSVKGRFTISRDDSKSTLYLQM Heavy Chain NSLRAEDTAVYYCVRQDYYVWGQGTTVTVSS (SEQ ID NO: 61) DIQMTQSPSSLSASVGDRVTITCRSSKSLLHSNGKTYLYWFQQK PGQSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFAT Light Chain YYCMQHLEYPLTFGQGTKLEIK (SEQ ID NO: 62)

[0093] Therefore, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 61, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62.

[0094] The amino acid sequences for the CDRs of scFv PT77 [4D5] are the same as the CDRs of scFv PT77 [F8] presented in Table 11. The heavy and light chain variable regions of scFv PT77 [4D5] are presented in Table 13, which also provides SEQ ID NOs for each amino acid sequence. Table 13. Sequences for the heavy and light chain variable regions of scFv PT77 [4D5]. Variable Region Amino Acid Sequence EVQLVESGGGLVQPGGSLRLSCAASGFSFNTYAMNWVRQAPGK GLEWVSRIRSKSDNYATYYADSVKGRFTISRDDSKSTLYLQMNSL Heavy Chain RAEDTAVYYCVRQDYYVWGQGTTVTVSS (SEQ ID NO: 63) DIQMTQSPSSLSASVGDRVTITCRSSKSLLHSNGKTYLYWFQQKP GQSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY Light Chain CMQHLEYPLTFGQGTKLEIK (SEQ ID NO: 62)

[0095] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62.

[0096] The amino acid sequences for the CDRs of scFv PT77 [A48-4D5] are presented in Table 14, which also provides SEQ ID NOs for each amino acid sequence. Table 14. Sequences for the heavy chain variable CDRs and light chain variable CDRs of scFv PT77 [A48-4D5]. Kabat numbering scheme Variable Region CDR-1 CDR-2 CDR-3 TYAMN RIRSKSDNYATYYADSVKG QDYYV Heavy Chain (SEQ ID NO: 36) (SEQ ID NO: 59) (SEQ ID NO: 38) RSSKSLLHSNGKTYLY RMSNLVS MQHLEYPLT Light Chain (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51) Chothia numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GFSFNTY RSKSDNYA QDYYV Heavy Chain (SEQ ID NO: 39) (SEQ ID NO: 40) (SEQ ID NO: 38) RSSKSLLHSNGKTYLY RMSNLVS MQHLEYPLT Light Chain (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51) IMGT numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GFSFNTYA IRSKSDNYAT VRQDYYV Heavy Chain (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) KSLLHSNGKTYRMSMQHLEYPLT Light Chain (SEQ ID NO: 52)(SEQ ID NO: 51) ABM numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GFSFNTYAMN RIRSKSDNYATY QDYYV Heavy Chain (SEQ ID NO: 44) (SEQ ID NO: 45) (SEQ ID NO: 38) RSSKSLLHSNGKTYLY RMSNLVS MQHLEYPLT Light Chain (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51) Contact numbering scheme Variable Region CDR-1 CDR-2 CDR-3 NTYAMN WVARIRSKSDNYATY VRQDYY Heavy Chain (SEQ ID NO: 46) (SEQ ID NO: 47) (SEQ ID NO: 48) LHSNGKTYLYWF LLIYRMSNLV MQHLEYPL Light Chain (SEQ ID NO: 54) (SEQ ID NO: 55) (SEQ ID NO: 56)

[0097] The heavy and light chain variable regions of scFv PT77 [A48-4D5] are presented in Table 15, which also provides SEQ ID NOs for each amino acid sequence. Table 15. Sequences for the heavy and light chain variable regions of scFv PT77 [A48-4D5]. Variable Region Amino Acid Sequence EVQLVESGGGLVQPGGSLRLSCAASGFSFNTYAMNWVRQAPGK GLEWVARIRSKSDNYATYYADSVKGRFTISRDDAKSSLYLQMN Heavy Chain SLRAEDTAVYYCVRQDYYVWGQGTTVTVSS (SEQ ID NO: 64) DIQMTQSPSSLSASVGDRVTITCRSSKSLLHSNGKTYLYWFQQKP GQSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY Light Chain CMQHLEYPLTFGQGTKLEIK (SEQ ID NO: 62)

[0098] Therefore, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 64, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62.

[0099] Yet other exemplary anti-tau intrabodies of the present invention include intrabodies derived from monoclonal antibody PT77, grafted into a framework based on antibody germlines with high similarity to previously-described scFvs F8, 4D5, and A48-4D5, and rendered cysteine-free by replacing cysteine with the amino acid combination alanine-valine, the resulting intrabodies of which are referred to herein as “scFv PT77-SS−[F8]”, “scFv PT51-SS−[4D5]”, and “scFv PT51-SS−[A48-4D5]”, respectively.

[0100] The amino acid sequences for the CDRs of scFv PT77-SS−[F8] are the same as the CDRs of scFv PT77 [F8] presented in Table 11. The heavy and light chain variable regions of scFv PT77-SS−[F8] are presented in Table 16, which also provides SEQ ID NOs for each amino acid sequence. Table 16. Sequences for the heavy and light chain variable regions of scFv PT77-SS−[F8].Variable Region Amino Acid Sequence QVQLVESGGGLVQPGGSLRLSVSASGFSFNTYAMNWVRQAPGK GLEWVSRIRSKSDNYATYYADSVKGRFTISRDDSKSTLYLQMNSL Heavy Chain RAEDTAVYYAVRQDYYVWGQGTTVTVSS (SEQ ID NO: 65) DIQMTQSPSSLSASVGDRVTITVRSSKSLLHSNGKTYLYWFQQKP GQSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY Light Chain AMQHLEYPLTFGQGTKLEIK (SEQ ID NO: 66)

[0101] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 65, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 66.

[0102] The amino acid sequences for the CDRs of scFv PT77-SS−[4D5] are the same as the CDRs of scFv PT77 [F8] presented in Table 11. The heavy and light chain variable regions of scFv PT51-SS−[4D5] are presented in Table 17, which also provides SEQ ID NOs for each amino acid sequence. Table 17. Sequences for the heavy and light chain variable regions of scFv PT51-SS−[4D5]. Variable Region Amino Acid Sequence EVQLVESGGGLVQPGGSLRLSVAASGFSFNTYAMNWVRQAPGK GLEWVSRIRSKSDNYATYYADSVKGRFTISRDDSKSTLYLQMNSL Heavy Chain RAEDTAVYYAVRQDYYVWGQGTTVTVSS (SEQ ID NO: 67) DIQMTQSPSSLSASVGDRVTITVRSSKSLLHSNGKTYLYWFQQKPG QSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYA Light Chain MQHLEYPLTFGQGTKLEIK (SEQ ID NO: 66)

[0103] Hence, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 66.

[0104] The amino acid sequences for the CDRs of scFv PT77-SS−[A48-4D5] are the same as the CDRs of scFv PT77 [A48-4D5] presented in Table 14. The heavy and light chain variable regions of scFv PT77-SS−[A48-4D5] are presented in Table 18, which also provides SEQ ID NOs for each amino acid sequence.Table 18. Sequences for the heavy and light chain variable regions of scFv PT77-SS−[A48- 4D5]. Variable Region Amino Acid Sequence EVQLVESGGGLVQPGGSLRLSVAASGFSFNTYAMNWVRQAPGK GLEWVARIRSKSDNYATYYADSVKGRFTISRDDAKSSLYLQMNS Heavy Chain LRAEDTAVYYAVRQDYYVWGQGTTVTVSS (SEQ ID NO: 68) DIQMTQSPSSLSASVGDRVTITVRSSKSLLHSNGKTYLYWFQQKP GQSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY Light Chain AMQHLEYPLTFGQGTKLEIK (SEQ ID NO: 66)

[0105] Therefore, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 68, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 66.

[0106] Yet other exemplary anti-tau intrabodies of the present invention include an intrabody derived from monoclonal antibody hTau21, referred to herein as “scFv hTau21.” The amino acid sequences for the CDRs of scFv hTau21 are presented in Table 19, which also provides SEQ ID NOs for each amino acid sequence. Table 19. Sequences for the heavy chain variable CDRs and light chain variable CDRs of scFv hTau21. Kabat numbering scheme Variable Region CDR-1 CDR-2 CDR-3 DTYIH RIDPANGNSKYDPKFQG HDGY Heavy Chain (SEQ ID NO: 69) (SEQ ID NO: 70) (SEQ ID NO: 71) KASQSVDYDGDSYMN AASNLES QQSNEAPYT Light Chain (SEQ ID NO: 82) (SEQ ID NO: 83) (SEQ ID NO: 84) Chothia numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GCNIKDT DPANGN HDGY Heavy Chain (SEQ ID NO: 72) (SEQ ID NO: 73) (SEQ ID NO: 71) KASQSVDYDGDSYMN AASNLES QQSNEAPYT Light Chain (SEQ ID NO: 82) (SEQ ID NO: 83) (SEQ ID NO: 84) IMGT numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GCNIKDTY IDPANGNS AHHDGY Heavy Chain (SEQ ID NO: 74) (SEQ ID NO: 75) (SEQ ID NO: 76)QSVDYDGDSY )AAQQSNEAPYT Light Chain (SEQ ID NO: 85S(SEQ ID NO: 84) ABM numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GCNIKDTYIH RIDPANGNSK HDGY Heavy Chain (SEQ ID NO: 77) (SEQ ID NO: 78) (SEQ ID NO: 71) KASQSVDYDGDSYMN AASNLES QQSNEAPYT Light Chain (SEQ ID NO: 82) (SEQ ID NO: 83) (SEQ ID NO: 84) Contact numbering scheme Variable Region CDR-1 CDR-2 CDR-3 KDTYIH WIGRIDPANGNSK AHHDG Heavy Chain (SEQ ID NO: 79) (SEQ ID NO: 80) (SEQ ID NO: 81) DYDGDSYMNWY LLIYAASNLE QQSNEAPY Light Chain (SEQ ID NO: 87) (SEQ ID NO: 88) (SEQ ID NO: 89)

[0107] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises: (a) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 69, 70, and 71, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (b) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 72, 73, and 71, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; (c) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 74, 75, and 76, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 85, AAS, and 84, respectively; or (d) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 77, 78, and 71, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or(e) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 79, 80, and 81, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively.

[0108] The heavy and light chain variable regions of scFv hTau21are presented in Table 20, which also provides SEQ ID NOs for each amino acid sequence. Table 20. Sequences for the heavy and light chain variable regions of scFv hTau21. Variable Region Amino Acid Sequence EVQLQQSGAELVKPGASGKLSCTASGCNIKDTYIHWVKQRPEQ GLEWIGRIDPANGNSKYDPKFQGKATITADTSSNTAYLQLSSLT Heavy Chain SEDTAVYYCAHHDGYWGQGTLVTVSA (SEQ ID NO: 90) DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQK AGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAAT Light Chain YYCQQSNEAPYTFGGGTRLEIK (SEQ ID NO: 91)

[0109] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 90, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 91.

[0110] Other exemplary anti-tau intrabodies of the present invention include intrabodies derived from monoclonal antibody hTau21, but grafted into a framework based on antibody germlines with high similarity to previously-described scFvs F8 and A48-4D5 (Donini et al., 2003; Wörn & Plückthun, 1998; Wörn & Plückthun, 1999), the resulting intrabodies of which is referred to herein as “scFv hTau21 [F8]” and “scFv hTau21 [A48-4D5]”, respectively.

[0111] The amino acid sequences for the CDRs of scFv hTau21 [F8] are presented in Table 21, which also provides SEQ ID NOs for each amino acid sequence. Table 21. Sequences for the heavy chain variable CDRs and light chain variable CDRs of scFv hTau21 [F8]. Kabat numbering scheme Variable Region CDR-1 CDR-2 CDR-3 DTYIH RIDPANGNSKYDDSVKG HDGY Heavy Chain (SEQ ID NO: 69) (SEQ ID NO: 92) (SEQ ID NO: 71)KASQSVDYDGDSYMN AASNLES QQSNEAPYT Light Chain (SEQ ID NO: 82) (SEQ ID NO: 83) (SEQ ID NO: 84) Chothia numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GCNIKDT DPANGN HDGY Heavy Chain (SEQ ID NO: 72) (SEQ ID NO: 73) (SEQ ID NO: 71) KASQSVDYDGDSYMN AASNLES QQSNEAPYT Light Chain (SEQ ID NO: 82) (SEQ ID NO: 83) (SEQ ID NO: 84) IMGT numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GCNIKDTY IDPANGNS AHHDGY Heavy Chain (SEQ ID NO: 74) (SEQ ID NO: 75) (SEQ ID NO: 76) QSVDYDGDSY QQSN t Chain (SEQ ID NO: 85)AEAPYT LighAS(SEQ ID NO: 84) ABM numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GCNIKDTYIH RIDPANGNSK HDGY Heavy Chain (SEQ ID NO: 77) (SEQ ID NO: 78) (SEQ ID NO: 71) KASQSVDYDGDSYMN AASNLES QQSNEAPYT Light Chain (SEQ ID NO: 82) (SEQ ID NO: 83) (SEQ ID NO: 84) Contact numbering scheme Variable Region CDR-1 CDR-2 CDR-3 KDTYIH WVSRIDPANGNSK AHHDG Heavy Chain (SEQ ID NO: 79) (SEQ ID NO: 93) (SEQ ID NO: 81) DYDGDSYMNWY LLIYAASNLE QQSNEAPY Light Chain (SEQ ID NO: 87) (SEQ ID NO: 88) (SEQ ID NO: 89)

[0112] Hence, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 69, 92, and 71, respectively; and a light chain variable CDR-1, CDR- 2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively. In other embodiments, the anti-tau intrabody comprises a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 79, 93, and 81, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively.

[0113] The heavy and light chains of scFv hTau21 [F8] are presented in Table 22, which also provides SEQ ID NOs for each amino acid sequence.Table 22. Sequences for the heavy and light chain variable regions of scFv hTau21 [F8]. Variable Region Amino Acid Sequence QVQLVESGGGLVQPGGSLRLSCSASGCNIKDTYIHWVRQAPGK GLEWVSRIDPANGNSKYDDSVKGRFTISADTSKNTAYLQMNSL Heavy Chain RAEDTAVYYCAHHDGYWGQGTLVTVSS (SEQ ID NO: 94) DIQLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQK PGQPPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT Light Chain YYCQQSNEAPYTFGGGTKVEIK (SEQ ID NO: 95)

[0114] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 95.

[0115] The amino acid sequences for the CDRs of scFv hTau21 [A48-4D5] are presented in Table 23, which also provides SEQ ID NOs for each amino acid sequence. Table 23. Sequences for the heavy chain variable CDRs and light chain variable CDRs of scFv hTau21 [A48-4D5]. Kabat numbering scheme Variable Region CDR-1 CDR-2 CDR-3 DTYIH RIDPANGNSKYDDSVKG HDGY Heavy Chain (SEQ ID NO: 69) (SEQ ID NO: 92) (SEQ ID NO: 71) KASQSVDYDGDSYMN AASNLES QQSNEAPYT Light Chain (SEQ ID NO: 82) (SEQ ID NO: 83) (SEQ ID NO: 84) Chothia numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GCNIKDT DPANGN HDGY Heavy Chain (SEQ ID NO: 72) (SEQ ID NO: 73) (SEQ ID NO: 71) KASQSVDYDGDSYMN AASNLES QQSNEAPYT Light Chain (SEQ ID NO: 82) (SEQ ID NO: 83) (SEQ ID NO: 84) IMGT numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GCNIKDTY IDPANGNS AHHDGY Heavy Chain (SEQ ID NO: 74) (SEQ ID NO: 75) (SEQ ID NO: 76) QSVDYDGDSYAASQQSNEAPYT Light Chain (SEQ ID NO: 85)(SEQ ID NO: 84)ABM numbering scheme Variable Region CDR-1 CDR-2 CDR-3 GCNIKDTYIH RIDPANGNSK HDGY Heavy Chain (SEQ ID NO: 77) (SEQ ID NO: 78) (SEQ ID NO: 71) KASQSVDYDGDSYMN AASNLES QQSNEAPYT Light Chain (SEQ ID NO: 82) (SEQ ID NO: 83) (SEQ ID NO: 84) Contact numbering scheme Variable Region CDR-1 CDR-2 CDR-3 KDTYIH WVARIDPANGNSK AHHDG Heavy Chain (SEQ ID NO: 79) (SEQ ID NO: 96) (SEQ ID NO: 81) DYDGDSYMNWY LLIYAASNLE QQSNEAPY Light Chain (SEQ ID NO: 87) (SEQ ID NO: 88) (SEQ ID NO: 89)

[0116] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 79, 96, and 81, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively.

[0117] The heavy and light chains of scFv hTau21 [A48-4D5] are presented in Table 24, which also provides SEQ ID NOs for each amino acid sequence. Table 24. Sequences for the heavy and light chain variable regions of scFv hTau21 [A48-4D5]. Variable Region Amino Acid Sequence EVQLVESGGGLVQPGGSLRLSCAASGCNIKDTYIHWVRQAPGK GLEWVARIDPANGNSKYDDSVKGRFTISADTAKNSAYLQMNSL Heavy Chain RAEDTAVYYCAHHDGYWGQGTLVTVSS (SEQ ID NO: 97) DIQLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQK PGQPPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT Light Chain YYCQQSNEAPYTFGGGTKVEIK (SEQ ID NO: 95)

[0118] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 97, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 95.

[0119] Other exemplary anti-tau intrabodies of the present invention include intrabodies derived from monoclonal antibody hTau21, grafted into a framework based on antibody germlines withhigh similarity to previously-described scFvs F8 and A48-4D5, and rendered cysteine-free by replacing cysteine with the amino acid combination alanine-valine, the resulting intrabodies of which are referred to herein as “scFv hTau21-SS−[F8]” and “scFv hTau21-SS−[A48-4D5]”, respectively.

[0120] The amino acid sequences for the CDRs of scFv hTau21-SS−[F8] are the same as the CDRs of scFv hTau21 [F8] presented in Table 21. The heavy and light chain variable regions of scFv PT51-SS−[F8] are presented in Table 25, which also provides SEQ ID NOs for each amino acid sequence. Table 25. Sequences for the heavy and light chain variable regions of scFv hTau21-SS−[F8]. Variable Region Amino Acid Sequence QVQLVESGGGLVQPGGSLRLSVSASGCNIKDTYIHWVRQAPGKG LEWVSRIDPANGNSKYDDSVKGRFTISADTSKNTAYLQMNSLRA Heavy Chain EDTAVYYAAHHDGYWGQGTLVTVSS (SEQ ID NO: 98) DIQLTQSPSSLSASVGDRVTITVKASQSVDYDGDSYMNWYQQKP GQPPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY Light Chain AQQSNEAPYTFGGGTKVEIK (SEQ ID NO: 99)

[0121] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 98, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 99.

[0122] The amino acid sequences for the CDRs of scFv hTau21-SS−[A48-4D5] are the same as the CDRs of scFv hTau21 [A48-4D5] presented in Table 23. The heavy and light chain variable regions of scFv hTau21-SS−[A48-4D5] are presented in Table 26, which also provides SEQ ID NOs for each amino acid sequence.Table 26. Sequences for the heavy and light chain variable regions of scFv hTau21-SS−[A48- 4D5]. Variable Region Amino Acid Sequence EVQLVESGGGLVQPGGSLRLSVAASGCNIKDTYIHWVRQAPGKG LEWVARIDPANGNSKYDDSVKGRFTISADTAKNSAYLQMNSLRA Heavy Chain EDTAVYYAAHHDGYWGQGTLVTVSS (SEQ ID NO: 100) DIQLTQSPSSLSASVGDRVTITVKASQSVDYDGDSYMNWYQQKP GQPPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY Light Chain AQQSNEAPYTFGGGTKVEIK (SEQ ID NO: 99)

[0123] Thus, according to some embodiments of the invention, the anti-tau intrabody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 100, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 99.

[0124] In some embodiments, the disclosure provides a composition comprising an anti-tau intrabody of the invention, optionally further comprising one or more carriers, and optionally one or more diluents, excipients, or other additives. Polynucleotides Encoding Anti-Tau scFv, Preparation and Expression Thereof

[0125] This disclosure provides certain polynucleotides comprising nucleic acid sequences that encode anti-tau intrabodies. The polynucleotides of the invention can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and, if single-stranded, can be the coding strand or non- coding (anti-sense) strand.

[0126] In certain embodiments, the polynucleotide can be isolated. In certain embodiments, the polynucleotide can be substantially pure. In certain embodiments, the polynucleotide can be cDNA or are derived from cDNA. In certain embodiments, the polynucleotide can be recombinantly produced. In certain embodiments, the polynucleotide can comprise the coding sequence for a mature polypeptide, fused in the same reading frame to a polynucleotide which aids, for example, in expression and optionally, secretion, of a polypeptide from a host cell (e.g., a promoter or other regulatory sequence, a leader sequence that functions as a secretory sequence for controlling transport of a polypeptide from the cell). The polypeptide having a leadersequence is a pre-protein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.

[0127] The disclosure provides an isolated polynucleotide comprising a nucleic acid encoding an anti-tau intrabody comprising an amino acid sequence from a heavy chain and / or light chain having 85%, 90%, 95%, 96%, 97%, 98% or 99% similarity to an amino acid sequence set forth herein, and / or comprising 1, 2, 3, 4, 5 or more amino acid substitutions, e.g., conservative substitutions, relative to an amino acid sequence set forth herein, such as a sequence from scFv PT51, scFv PT51 [F8], scFv PT51 [A48-4D5], scFv PT51-SS−[F8], scFv PT51-SS−[A48-4D5], scFv PT77, scFv PT77 [F8], scFv PT77 [4D5], scFv PT77 [A48-4D5], scFv PT77-SS−[F8], scFv PT77-SS−[4D5], scFv PT77-SS−[A48-4D5], scFv hTau21, scFv hTau21 [F8], scFv hTau21 [A48-4D5], scFv hTau21-SS−[F8], scFv hTau21-SS−[A48-4D5].

[0128] Polynucleotide variants are also provided. Polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).

[0129] In some embodiments a nucleotide sequence encoding an anti-tau intrabody can be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a nucleotide oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5’ or 3’ overhangs for complementary assembly.

[0130] Once assembled (by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed, e.g., by nucleotide sequencing, restriction mapping, and / or expression of a biologically active polypeptide in a suitable host. In order to obtain high expression levels of a transfected gene in a host, the gene can be operatively linked to or associated with transcriptional and translational expression control sequences that are functional in the chosen expression host.

[0131] The invention includes vectors comprising the polynucleotides described above. Suitable vectors are described elsewhere herein, and are known to those of ordinary skill in the art. In some embodiments, a polynucleotide comprising a nucleic acid encoding a heavy chain variable region or portion thereof, and a polynucleotide comprising a nucleic acid encoding a light chain variable region or portion thereof, can reside in a single vector, or can be on separate vectors. In some embodiments, polynucleotides comprising nucleic acids encoding heavy and light chain CDR-1, CDR-2, and CDR-3, or portions thereof, can reside in a single vector, or can be on separate vectors. In some embodiments, polynucleotides comprising nucleic acids encoding a heavy chain variable region and a light chain variable region, or portions thereof, can reside in a single vector, or can be on separate vectors. Accordingly, the disclosure provides one or more vectors comprising the polynucleotides described above.

[0132] In certain embodiments, the vectors are replicable DNA constructs that have synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of an anti-tau intrabody, operatively linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences, as described in detail below. Such regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are operatively linked whenthey are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where a recombinant protein is expressed without a leader or transport sequence, the protein can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

[0133] The choice of expression control sequence and expression vector will depend upon the cell in which expression is sought. A wide variety of expression host / vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pADL-22c, pCR 1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13, and filamentous single-stranded DNA phages. In some embodiments, the vector is a viral vector. In certain embodiments, the viral vector is an AAV vector. In particular embodiments, the AAV vector is an AAV serotype 6 vector.

[0134] In certain embodiments, the disclosure provides a composition comprising a polynucleotide or vector as described above, optionally further comprising one or more carriers, diluents, excipients, or other additives. Methods of the Invention

[0135] The invention relates to the use of anti-tau intrabodies to target tau intracellularly. As genetically encodable proteins, intrabodies advantageously can be expressed in a cell or tissue- specific manner and can be developed to target specific conformations and post-translational modifications (Marschall et al., 2015).

[0136] To deliver an anti-tau intrabody into the cell, in some embodiments, the anti-tau intrabody is preferably encoded by a polynucleotide of the present invention, for example, a polynucleotide that encodes an intrabody that binds to an epitope comprising amino acid residues 151-158 of tau, amino acid residues pS199 / pS202 of tau, or amino acid residues 375-380 of tau, wherein the numbering of the amino acid is with reference to the amino acid sequence set forth in SEQ ID NO: 1; or an intrabody comprising heavy chain variable CDR-1, CDR-2, and CDR-3 and light chain variable CDR-1, CDR-2, and CDR-3 as described herein; or an intrabody comprising a heavy chain variable region and a light chain variable region as described herein; or an intrabody selected from scFv PT51, scFv PT51 [F8], scFv PT51 [A48-4D5], scFv PT51-SS−[F8], scFv PT51-SS−[A48-4D5], scFv PT77, scFv PT77 [F8], scFv PT77 [4D5], scFv PT77 [A48-4D5], scFv PT77-SS−[F8], scFv PT77-SS−[4D5], scFv PT77-SS−[A48-4D5], scFv hTau21, scFv hTau21 [F8], scFv hTau21 [A48-4D5], scFv hTau21-SS−[F8], scFv hTau21-SS−[A48-4D5]. Further, the polynucleotide is preferably delivered to the cell via a vector of the present invention.

[0137] Thus, in one aspect, the present invention provides a method of binding intracellular tau in a cell, the method comprising contacting the cell with a vector of the invention that comprises a polynucleotide encoding an anti-tau intrabody. In another aspect, the present invention provides a method of reducing tau aggregation in a cell, the method comprising contacting the cell with a vector of the invention that comprises a polynucleotide encoding an anti-tau intrabody.

[0138] Examples of a cell may include, but are not limited to, a neuron, an astrocyte, and an oligodendrocyte. In preferred embodiments, the neuronal cell is a neuron.

[0139] The present invention also relates to methods of improving properties of anti-tau intrabodies. As shown in the Example, grafting the CDRs from scFvs of interest into frameworks that have been described as stable in the cytoplasmic environment can improve scFv intrabody stability. In addition, designing scFvs that are disulfide-free may also improve intracellular properties, as the inability of disulfide bonds to form in reducing conditions is one of the main factors for scFv intrabody misfolding and aggregation.

[0140] Thus, in one aspect, the present invention provides a method of increasing binding of an anti-tau intrabody to intracellular tau and / or increasing intracellular solubility of an anti-tau intrabody, in which the anti-tau intrabody comprises a scFv, the method comprising: (a) grafting CDRs of an anti-tau antibody to a scFv framework identified to be stable intracellularly, and (b) removing disulfide bonds in the scFv. In yet another aspect, the present invention provides a method of preparing an intrabody for targeting tau intracellularly, in which the anti-tau intrabody comprises a scFv, the method comprising: (a) grafting CDRs of an anti-tau antibody to a scFv framework identified to be stable intracellularly, and (b) removing disulfide bonds in the scFv.

[0141] An increase in binding of an anti-tau intrabody to intracellular tau can be determined using methods known in the art, for example, by evaluating binding affinity.

[0142] In some embodiments, the grafted CDRs may be CDRs as described herein, e.g., the CDRs of scFv PT51, scFv PT51 [F8], scFv PT51 [A48-4D5], scFv PT51-SS−[F8], scFv PT51- SS−[A48-4D5], scFv PT77, scFv PT77 [F8], scFv PT77 [4D5], scFv PT77 [A48-4D5], scFv PT77-SS−[F8], scFv PT77-SS−[4D5], scFv PT77-SS−[A48-4D5], scFv hTau21, scFv hTau21 [F8], scFv hTau21 [A48-4D5], scFv hTau21-SS−[F8], scFv hTau21-SS−[A48-4D5].

[0143] In some embodiments, an scFv framework may be identified as stable based on methods known in the art, for example, by evaluating melting temperature of the framework, formation of aggregates, total yield, etc. Examples of scFv frameworks include, but are not limited to, those used in the intrabodies described in Donini et al. (2003) or those used in the intrabodies described herein, i.e., scFv PT51, scFv PT51 [F8], scFv PT51 [A48-4D5], scFv PT51-SS−[F8], scFv PT51-SS−[A48-4D5], scFv PT77, scFv PT77 [F8], scFv PT77 [4D5], scFv PT77 [A48- 4D5], scFv PT77-SS−[F8], scFv PT77-SS−[4D5], scFv PT77-SS−[A48-4D5], scFv hTau21, scFv hTau21 [F8], scFv hTau21 [A48-4D5], scFv hTau21-SS−[F8], scFv hTau21-SS−[A48- 4D5]. Other examples of scFv frameworks include, but are not limited to, those used in the intrabodies described in Donini et al. (2003), Wörn & Plückthun (1998), and Wörn & Plückthun (1999).

[0144] In some embodiments, removing disulfide bonds comprises removing cysteine amino acids from the scFv. In certain embodiments, removing disulfide bonds comprises replacing cysteine amino acids in the scFv with a combination of alanine-valine.

[0145] In some embodiments, the tau is human tau.

[0146] In some embodiments, the methods of the invention are in vitro. Kits Comprising Polynucleotides, Vectors, or Compositions Thereof

[0147] This disclosure further provides kits that comprise polynucleotides encoding an anti-tau intrabody of the invention, a vector comprising the polynucleotide encoding an anti-tau intrabody of the invention, and / or composition thereof, which can be used to perform the methods described herein. In certain embodiments, a kit comprises at least one purified polynucleotide, vector, or composition of the invention, in one or more containers. In some embodiments, the kit contains one or more of the components necessary and / or sufficient to perform a method of the invention. One skilled in the art will readily recognize that the disclosed polynucleotide, vector, or composition can be readily incorporated into any of the established kit formats that are well known in the art.

[0148] Embodiments of the present disclosure can be further described and understood by reference to the following non-limiting “Examples,” which describe in the preparation of certain exemplary anti-tau intrabodies, some exemplary characterization of such intrabodies, and some exemplary methods for using such intrabodies. It will be apparent to those skilled in the art that many modifications to the specific description provided in the Examples can be practiced without undue experimentation and without departing from the scope of the present disclosure. EXAMPLES

[0149] The present Example describes the development of anti-tau intrabodies to target tau intracellularly. Methods scFv selection and design

[0150] Monoclonal antibodies (mAb) were selected from a previously characterized panel of anti-tau antibodies, based on epitope location, mAb and / or Fab fragment affinity, and in vitro and in vivo potency to interfere with tau aggregation.

[0151] Previously identified DNA sequences of the variable domains of each mAb were used to design scFvs in the VH-VL orientation. Two variants of each scFv were made using two different linkers to connect the variable domains, the GGGGSGGGGSGGGGS (SEQ ID NO: 2) linker and the GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 3) linker described by Bird and colleagues (Bird et al., 1988). These linkers are referred to herein as GS and GSEK linker, respectively. scFv expression in the periplasm of E. coli

[0152] cDNA of each scFv with each linker was cloned into the periplasmic expression vector pADL-22c, which includes an N-terminal His6HA-tag. Overexpression of all constructs was carried out in MC1061F’ E. coli cells (Biosearch Technologies, Novato, CA). Pre-cultures were prepared from glycerol stocks in 2YT-medium (Sigma, Y1003) supplemented with 100 μg / mL carbenicillin (ThermoFisher, 10177012) incubated at 37 °C overnight with agitation of 400 rpm. 50 μL of the pre-culture was subsequently used to inoculate 5 mL 2YT-medium supplemented with carbenicillin followed by incubation for 3 hrs at 37 ஈC. Then, 1 mM IPTG (Merck, D48784) was added, and cultures were further incubated overnight and subsequently harvested the next day by centrifugation at 2200 g for 15 min. Pellets were quickly frozen on dry ice and thawed on tepid water. Pellets were then resuspended in BugBuster HT Protein Extraction Reagent (Merck, D49036) supplemented with 0.2 mg / ml Chicken Lysozyme (Sigma, L3790) and left for 30 min with vigorous shaking. Cell debris and insoluble material were removed by 500 g spin of 2 min and supernatant was collected. scFv expression in QBI-HEK293 cell line

[0153] cDNA of all scFvs with each linker was cloned into a mammalian expression vector that was designed internally, pUNDER (PCT Publication No. WO 2010002785 A1), under the control of the cytomegalovirus promoter. A FLAG-tag (DYKDDDDK) (SEQ ID NO: 101) was added to the C-terminus of each scFv for detection purposes in downstream assays. For cytoplasmic expression the secretion signal sequence was removed.

[0154] Human embryonic kidney-derived QBI-HEK293A cells (QBiogene) were seeded in 12 well plates (Falcon, 353043) for secreted expression, or 6-well plates (Falcon, 353046) for cytoplasmic expression, in DMEM (Sigma, D5796) supplemented with 10% Fetal Bovine Serum (Biowest, S1810-500), 2 mM L-glutamine (Sigma, G7513), 1 mM sodium pyruvate (Sigma, S8636), and Penicillin-Streptomycin, 100 U / ml and 100 μg / ml respectively, (Sigma, P4333). The next day the medium was changed to DMEM without antibiotics prior to plasmid DNA transfection with FuGENE6 (Promega, E2691), following manufacturer’s instructions. 48 hrs after transfection, culture medium was collected from the cells secreting scFvs, whereas cells expressing scFvs in the cytoplasm were lysed in RIPA (Sigma, R0278) with phosphatase and protease inhibitors (Roche, 4906837001; 11836170001). Western Blotting

[0155] Samples were diluted in NuPAGE™ LDS Sample Buffer and Reducing Agent (Thermo Scientific, NP0008, NP0009) and heated for 10 min at 75 ஈC with shaking, followed by loading on BisTris gel (4-12%) (Invitrogen WG1403BOX). After separation, the gel was blotted on a nitrocellulose membrane (BioRad, 1704159) using Trans-Blot Turbo system (BioRad, Hercules, CA). The membrane was blocked with 5% non-fat dry milk dissolved in Tris-Buffered Saline + 0.1% Tween®20 (TBS-T) and incubated with Anti-HA HRP (abcam, ab1190) at 0.5 μg / ml (for the E. coli lysates) or Anti-FLAG®M2-HRP (Sigma, A8592) 1:500 (for the QBI-HEK293 culture medium and lysates) in TBS-T with 5% non-fat dry milk for 1 hr at room temperature (RT). SuperSignal™ West Dura (Thermo Scientific™, 34076) was used to reveal the bands and imaging was done on Amersham Imager 600 (GE Healthcare, Chicago, IL).

[0156] Total protein in QBI-HEK293 cell lysates was determined with the bicinchoninic acid assay (BCA) (Sigma, BCA1-1KT) prior to western blotting and 6 μg of total protein was loaded in the gels. Bacterial cell lysates and culture medium from QBI-HEK293 cells were loaded undiluted. Enzyme-linked Immunosorbent Assay (ELISA)

[0157] Nunc MaxiSorp™ flat-bottom 96-well plates (Thermo Fisher Scientific, 430341) were coated with 50 μl containing 1 μg / ml of full-length recombinant human tau (hTau), 1 μg / ml of full-length recombinant human Į-synuclein or AD-brain derived tau paired helical filaments(ePHF) 1:500 in coating buffer (10 mM NaCl, 10 mM Tris-HCl, pH=8.6), and left overnight at 4 °C. The next day, the plates were washed 5x with 200 μl wash buffer (0.05% Tween-20 in PBS) followed by 2 hr incubation at RT with 150 μl blocking buffer (0.1% casein in PBS). After another wash, 50 μl of sample were added in a serial dilution. After 2 hr incubation at RT, the plates were washed and the detection antibody Anti-HA HRP (abcam, ab1190) or Anti-FLAG®M2-HRP (Sigma, A8592), diluted 1:2500 in blocking buffer, was incubated for 2 hr at RT. Following incubation, the plates were washed and 50 ^l TMB (ThermoScientific, 34029) was added to the wells. The enzymatic reaction was stopped with 50 ^l of 2N H2SO4. Plates were read immediately on EnVision®2102 Multilabel plate reader (PerkinElmer, Waltham, Massachusetts) and data was analyzed with GraphPad Prism 9 software.

[0158] To correct for concentration differences in bacterial cell lysates, band intensity on western blotting was quantified with ImageQuantTL software and converted to arbitrary units so that each scFv was tested at approximately the same concentration. For QBI-HEK293 cell lysates, samples were analyzed in a serial dilution starting at 100 μg / ml total protein. Culture medium samples were not corrected for protein content. Nuclear translocation (NLS) assay

[0159] Human embryonic kidney-derived QBI-HEK293A cells (QBiogene) were cultured on 96 well plates (Greiner Bio ONE, 655090) as described above. Plasmid DNA co-transfection was done 24 hrs after plating, with Lipofectamine 2000 (Invitrogen, 11668), according to the manufacturer’s instructions. 48 hrs after transfection, the cells were fixed in 4% paraformaldehyde and permeabilized with TBS containing 0.3% Triton X-100. scFvs were detected with primary antibody Anti-FLAG®M2 (Sigma, F3165) and secondary antibody goat anti-mouse IgG Alexa Fluor 555 (ThermoFisher, A-21424). Imaging was done on an Opera Phenix instrument (PerkinElmer, Waltham, Massachusetts) equipped with a 40× water immersion objective. Captured images were visually analyzed. scFv sequence engineering

[0160] To improve cytoplasmic solubility and stability, scFv sequences were modified using complementarity-determining region (CDR) grafting into two (scFvs PT51 and hTau21) or three (scFv PT77) different scFv frameworks. The selected frameworks were based on antibodygermlines with high similarity to previously described scFvs F8, 4D5, and A48-4D5, and this nomenclature was kept to identify the grafts. In order to prevent the formation of disulfide bonds, cysteine-free versions were also made for the CDR-grafted versions, by replacing the cysteines with the amino acid combination Ala-Val (Proba et al., 1998). Purification of AD seeds and paired-helical filaments (ePHF) from human brain

[0161] Human brain tissues from histologically confirmed sporadic AD patients with abundant tau pathology (Braak staging V / VI) were provided by the Center for Neurodegenerative Disease Research brain bank at the University of Pennsylvania and by the Newcastle Brain tissue resource with informed consent from next of kin.

[0162] Purification of AD seeds and ePHF from brain sections were performed as described in Soares et al. (2021) and Vandermeeren et al. (2018), respectively. Both protocols were executed in accordance with relevant ethical guidelines.

[0163] ePHF corresponds to the sarkosyl-insoluble fraction of homogenates from non-dissected human brain blocks. AD seeds are purified from gray matter only and correspond to a purer version of sarkosyl-insoluble tau that undergoes sonication. Generation of K18-P301L seeds

[0164] Recombinant K18-P301L (truncated human tau protein corresponding to the longest isoform between residues Q244 and E372) was produced in E. coli. Fibrils were generated by incubating 40 ^M K18-P301L protein with 40 ^M low-molecular-weight heparin and 2 mM DTT in 100 mM sodium acetate at 37 °C. After 10 days the solution was centrifuged at 100,000 g for 1 hr at 4 °C. The pellet was resuspended in PBS.Primary mouse cortical neurons and aggregation assays

[0165] Mouse primary cortical neurons were isolated from E19 C57Bl / 6J (Janvier) embryos in accordance with relevant ethical guidelines. Neurons were plated in 96 well plates (greiner Bio ONE, 655946) previously coated with poly-L-lysine (Sigma Aldrich, P1274) at 40,000 cells per well. Neurons were kept at 37 °C and 5% CO2in B-27 (Invitrogen, 17504044) and GlutaMax (Invitrogen, 35050-038) supplemented Neurobasal medium (Gibco, 10888022).

[0166] cDNA sequences of scFvs PT51 (GS), PT77 (SS- 4D5 GSEK), hTau21 (GSEK), and a scFv against a non-relevant neuronal protein were cloned into an adeno-associated-virus (AAV) vector and packaged into the AAV6 serotype (Sirion Biotech and Vector Builder).

[0167] The neuronal aggregation assay using human AD-tau-seeds was performed as described by Soares et al. (2021). Briefly, neurons were transduced with adeno-associated virus (AAV) - intrabody on day in vitro (DIV) 7 followed by addition of AD-tau-seeds on DIV 10. Neurons were kept until DIV 17 after which they were lysed in RIPA buffer.

[0168] The neuronal aggregation using K18-P301L seeds was based on Guo and Lee (2011) with some modifications. Briefly, primary mouse neurons were transduced with AAV-intrabody and AAV-hTauP301L on DIV 1, followed by addition of sonicated K18-P301L seeds on DIV 3. Neurons were kept until DIV 10 after which they were lysed in RIPA buffer with phosphatase and protease inhibitors.

[0169] scFv expression levels were evaluated using western blotting as described above. Samples were loaded in the gel undiluted. scFvs were detected using Anti-FLAG®M2-HRP (Sigma, A8592) 1:500. For ȕ-actin staining, the membranes were stripped with Restore™ PLUS Western Blot Stripping Buffer (Thermo Scientific™, 46430) for 15 min, followed by blocking and incubation with Monoclonal Anti-ȕ-Actin−Peroxidase (Sigma, A3854) 1:20000. OHSCs with K18-induced aggregation

[0170] OHSC were prepared from P301S mice at postnatal day 8. Briefly, pups were sacrificed by decapitation. Brains from pups were bisected into hemi-brains and the hippocampus was isolated and sliced into 410 μm-thick transverse sections. These sections were then transferred to ice-cold MEM (Gibco, 31095-029) supplemented with HEPES (Sigma-Aldrich, H0867), Tris(Sigma-Aldrich, 93350), and Penicillin-Streptomycin (Sigma-Aldrich, P4333), and then separated. After 1 hr incubation at 4 °C, the slices were transferred into previously prepared culture plates, with 3 slices being added per well, and immersed in warm culture medium (MEM (Gibco, 31095-029) supplemented with HEPES (Sigma-Aldrich, H0867), Tris (Sigma-Aldrich, 93350), Penicillin-Streptomycin (Sigma-Aldrich, P4333), HBSS (ThermoFisher Scientific, 20420), sodium pyruvate (Sigma-Aldrich, S8636), NaHCO3(Sigma-Aldrich, S5761), and horse serum (Gibco, 26050088)). AAV-intrabody was added at a concentration of 2x1010VG / ml to each well and the plates were incubated overnight at 37 °C and 5% CO2. As a negative control, an AAV expressing a scFv binding to Į-synuclein was used. The next day medium was renewed and AAV-intrabody at the same concentration was added. Plates were put back at 37 °C until DIV 4, after which temperature was changed to 35 °C for the remaining of the experiment. To induce the aggregation of tau, 333 μM of K18 seeds were added at DIV 14, on top of each slice. At DIV 31, slices were lysed in RIPA buffer with phosphatase and protease inhibitors. Throughout the experiment, culture medium was changed twice a week. Meso Scale Discovery (MSD)

[0171] 96-well Multi-Array®plates (Meso Scale Discovery, L15XA-3) were incubated overnight at 4 °C with coating antibodies diluted in PBS. After overnight incubation plates were washed 5 times with wash buffer (0.05% Tween in PBS) and then incubated with blocking buffer (0.1% casein in PBS) for 2 hrs at RT with agitation at 400 rpm. Next, the plates were washed again, and cell lysates were added to the plates, in serial dilutions in blocking buffer. Plates were then sealed and incubated overnight at 4 °C. The next day, the plates were washed and incubated with the respective detection antibody diluted in blocking buffer for 2 hrs at RT with agitation at 400 rpm. After incubation, the plates are washed and 150 ^l of MSD Read Buffer T with surfactant (Meso Scale Discovery, R92TC) 2x diluted in distilled water was added to each well. Plates were immediately read using the MSD SECTOR Imager 6000 (Meso Scale Discovery, Gaithersburg, MD).

[0172] All MSD assays used in this work were developed at Janssen R&D using internally developed antibodies. In the total mouse Į-synuclein assay, two commercial reagents weresequentially used for detection, biotinylated antibody D37A6 (Cell Signaling, 74184) and sulfo- labelled streptavidin (Meso Scale Discovery, R32AD-1).

[0173] When mentioned, statistical analyses were conducted using the R software version 4.2.1. Results Monoclonal antibodies were successfully converted to scFvs

[0174] Monoclonal antibodies PT51, PT77 and hTau21 (Table 27), all three developed from the same immunization campaign (Vandermeeren et al., 2018), were converted to scFvs. All three scFvs were expressed in E. coli periplasm or secreted from HEK293 cells. Analysis of E. coli lysates and culture medium from HEK293 cells in western blot, did not show major differences in scFv expression levels between the different linkers used (FIG. 1, Panels A and F), with a few exceptions. In the E. coli expression system, when the variable domains were connected with the GS linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 2), scFv hTau21 is detected mostly as a single band, with only a very faint band of potential dimers. When the GSEK linker (GGSEGKSSGSGSESKSTGGS) (SEQ ID NO: 3) is used, scFv hTau21 is detected as multiple bands, with the strongest bands being around 28 and 49 kDa, suggesting that it may be present as dimers. With the GS linker, some potential dimer formation is observed for scFvs PT51 and PT77, albeit with a low signal intensity.

[0175] Independent of the expression system, scFvs hTau21 and PT51 retained binding to recombinant tau (FIG. 1, Panels B, C, G, and H) without exhibiting non-specific binding to a negative control protein (data not shown). However, after periplasmic expression in E. coli, scFv PT77, which was derived from a phospho-tau-specific mAb, reacted with both recombinant tau and ePHF (FIG. 1, Panels D and E) with a very pronounced signal observed with the GSEK linker. Reaction with recombinant tau, i.e., indicative for an apparent loss of phospho- specificity, was unexpected. However, upon expression and secretion from HEK293 cells, scFv PT77 bound only to ePHF with no observed cross-reactivity to recombinant tau (FIG.1, Panels I and J), indicating that phospho-specificity was retained.Table 27. Summary of Fab (or mAb) affinity against human tau and tau paired helical filaments (PHF) from AD brains; efficacy in immunoprecipitating (IP) tau with aggregation capabilities from brain homogenates of P301S mice and AD patients. Fab affinity Fab affinity P301S AD brain Antibody Epitope (nM) (nM) homogenates % homogenates % 2N4R tau PHF seeding capacity seeding capacity after IP after IP PT51153TPRGAA15823.3 29.43 0.324 15.014 PT77 pS199 / pS202 / 0.054 1.079 9.822 hTau21375KLTRFE38082.8 (mAb) 1.640 (mAb) 5.298 82.826 scFvs PT51 and hTau21 retained tau binding as an intrabody

[0176] After expression as an intrabody in the cytoplasm of HEK293 cells, expression levels were again assessed via western blotting. Intrabodies PT51 and hTau21 were detected in both linker formats, while intrabody PT77 could not be detected (FIG.2, Panel A).

[0177] Evaluation of the cell lysates on ELISA for binding revealed that intrabodies hTau21 with the GSEK linker, and PT51 with both linkers, retained tau binding on ELISA, while no binding was detected for intrabody PT77 (FIG.2, Panels B-E). Low expression levels for intrabody PT77 could account for the absence of signal on ELISA. On the other hand, intrabody hTau21 with the GS linker was well expressed but did not react on ELISA, illustrating that other factors such as stability and folding may also be involved.

[0178] To evaluate if loss of binding might be the result of misfolding and / or aggregation due to the suboptimal environment of the cytoplasm, cytoplasmic solubility was evaluated via confocal microscopy. Intrabodies PT51 and PT77 showed a punctate staining potentially indicative of aggregation (FIG. 3, Panel B). A diffuse and even distribution in the cell was observed for intrabody hTau21, independently of the linker used, potentially indicative of soluble expression (FIG. 3, Panel B). Linker influence on solubility was seen for intrabody PT77, which appears to be soluble when the GSEK linker is used to connect the variable domains and shows some puncta when the GS linker is used (FIG. 3, Panel B).CDR grafting can rescue intrabody binding and cytoplasmic solubility

[0179] To overcome low solubility and stability of the intrabodies in the cytoplasm, the three intrabodies were CDR-grafted into different frameworks. These frameworks were selected based on previous reports describing their higher stability and evidence of intracellular activity (Donini et al., 2003; Wörn & Plückthun, 1998; Wörn & Plückthun, 1999). The intrabodies were redesigned in the VL-VH orientation and only the GSEK linker was used, based on its better performance on ELISA binding. Additionally, versions without the cysteines participating in disulfide bonds, were also evaluated. Intrabody PT51 became soluble in the VL-VH orientation, with solubility being maintained for all four CDR-grafts (FIG.3, Panel B). As for intrabodies PT77 and hTau21, solubility was maintained after switching the orientation of the variable domains and the CDR-grafting (FIG. 3, Panel B). Replacement of the cysteines participating in the disulfide bonds did not seem to affect solubility as assessed by confocal microscopy.

[0180] Binding was evaluated on ELISA and using a nuclear translocation assay based on Zhou et al. (2004). CDR-grafting did not improve ELISA binding for intrabody PT51, having a negative impact instead (FIG. 4, Panel A). However, in the NLS assay three of the PT51 grafted versions (F8, A48-4D5, and A48-4D5 SS−) showed a clear nuclear localization in the presence of tau-NLS (FIG.5, Panel A). With intrabody PT77 the opposite was observed. Virtually all six CDR-grafted variants rescued binding to ePHF on ELISA (FIG. 4, Panel C), while only the 4D5 SS−graft showed some nuclear localization (FIG.5, Panel A). Lack of binding in the NLS assay was not due to absence of phosphorylated tau-NLS, as its presence was confirmed with an MSD assay (FIG. 5, Panel B). Regarding intrabody hTau21, CDR-grafting did not improve binding on ELISA (FIG.4, Panel B), nor on the NLS assay (not shown). For this intrabody, only the original construct was positive for binding on both assays (FIG.4, Panel B; FIG. 5, Panel A). Intrabody PT77 SS−4D5 interferes with mouse tau aggregation induced by AD-tau seeds

[0181] To evaluate if the intrabodies can interfere with tau aggregation, primary mouse cortical neurons were transduced with AAV6 intrabody prior to inducing endogenous tau aggregation by addition of AD-brain derived tau seeds.

[0182] A significant reduction in tau aggregation was seen upon expression of intrabody PT77 SS−4D5 in a MOI-dependent manner (FIG. 6, Panel B). Importantly, total mouse tau and Į- synuclein levels remained stable (data not shown), independent of the presence of AAV or AD- tau seeds, suggesting that the observed decrease in tau aggregation is not a result of neuronal death. The other two intrabodies, PT51 GS and hTau21 GSEK, did not have an effect on aggregation levels in this model (FIG.6, Panels A and C, respectively). Intrabodies PT77 SS−4D5, hTau21 GSEK, and the negative control intrabody were all detected on western blotting at least at the highest MOI. Intrabody PT51 could not be detected Intrabodies PT51 GS, PT77 SS−4D5, and hTau21 GSEK interfere with mutant human tau aggregation induced by K18-P301L seeds

[0183] The three intrabodies were also tested on a K18-induced aggregation model. In this model, human tau with the P301L mutation was overexpressed in primary mouse cortical neurons via AAV transduction, followed by induction of aggregation with K18-P301L tau seeds.

[0184] All three intrabodies interfered with mutant human tau aggregation in a MOI-dependent manner (FIG.7, Panels A-C). At the highest MOI, some decrease in mutant human tau aggregation is also observed with the negative control intrabody, albeit without statistical significance. This result is believed to be a non-specific effect of intrabody overexpression, as no dose-dependent decrease was seen with this intrabody. Full-length human tau and total mouse Į-synuclein levels remained stable (data not shown), independent of the presence of AAV or K18 seeds, once again indicating that the observed reduction in aggregation was not due to neuronal death. PT77 SS−4D5, hTau21 GSEK, and the negative control intrabody were all detected on western blotting at least at the highest MOI, while intrabody PT51 could not be detected. scFv-intrabodies interfere with K18-seeded tau aggregation in organotypic hippocampal slice cultures (OHSCs)

[0185] OHSCs were prepared from transgenic mice overexpressing P301S tau, and similar to the primary mouse cortical neuronal model, K18 seeds were used to induce the aggregation of mutant human tau. Intrabody PT77 and the negative control were both well expressed and detected on western blotting, even if some differences are seen for a few slices (FIG. 8, Panel A-D). As for intrabodies PT51 and hTau21, expression levels were lower and only a few slices have detectable bands on western blotting (FIG.8, Panel E).

[0186] Aggregated tau was evaluated with sandwich immunoassays recognizing the N- or the C- terminus of human tau, while phosphorylated, aggregated tau was detected with AT8 and PT3 sandwich assays. AT8 recognizes tau phosphorylated at S202 / T205 / S208 (Vandermeeren et al., 2018; Malia et al., 2016), while PT3 recognizes tau phosphorylated at T212 / T217 (Van Kolen et al., 2020). Regarding aggregated tau measured with phosphorylation-independent assays, none of the intrabodies resulted in a significant reduction (FIG. 8, Panels A and B). However, when looking at phosphorylated tau aggregates, all three intrabodies were able to reduce AT8- phosphorylated aggregates, whereas for PT3-phosphorylated aggregates, only scFv-intrabody PT77 showed a significant reduction (FIG.8, Panels C and D). It should be noted that in the case of intrabody PT77, its epitope on tau (S199 / S202) partially overlaps with the epitope of the AT8 antibody used in one of the assays to measure phosphorylated, aggregated tau. mAb PT77 interferes with AT8-binding to tau aggregates (FIG. 8, Panel F), thus some interference of the intrabody in the AT8 assay cannot be fully excluded. Regarding the PT3 assay, PT77 interference seems to be neglectable (FIG.8, Panel F). Total mouse Į-synuclein levels were not affected by the presence of K18-seeds or AAV (data not shown), suggesting that the reductions observed in aggregated tau levels are not due to loss of neurons subsequent to toxicity. Discussion

[0187] In this study, mAbs PT51, PT77 and hTau21were successfully converted into scFv- intrabodies. scFvs PT51 and hTau21 retained binding to tau, with at least one of the linkers used, while scFv PT77 retained binding only after CDR-grafting into more stable frameworks.

[0188] Differences in binding between scFvs expressed in the E. coli periplasm versus secretion from QBI-HEK293 were observed. scFv PT77, which is derived from a mAb that specifically recognizes a phosphorylated epitope on tau, lost its phospho-specificity when expressed in E. coli, while it was retained when secreted from HEK293 cells. Differences in affinity, specificity, and folding between scFvs produced by prokaryotic versus eukaryotic organisms have also been previously reported, including E. coli versus mammalian cells (Lemeulle et al., 1998; Yusakul et al., 2018; Vendel et al., 2012; Lee et al., 2017). These could be the result of differences ineukaryotic and prokaryotic molecular chaperones, as well as saturation of the periplasm import machinery, leading to suboptimal folding, resulting in different secondary structures and consequent changes in epitope-paratope interaction (Schlegel et al., 2013; Hermann & Riemer, 2014; Baumgarten et al., 2018). This could then explain the differences in binding observed in this work and stresses the importance of confirming critical properties of a scFv derived from a well-characterized monoclonal antibody in a proper cell system.

[0189] scFvs PT51 and PT77 showed some degree of aggregation when expressed in the cytoplasm as intrabodies, at least with one of the linkers. This suggested that these two intrabodies were not folding into their correct conformation, likely due to the lack of or mispairing of intradomain disulfide bonds in the reducing environment of the cytoplasm (Wörn & Plückthun, 1998; Proba et al., 1998). Nevertheless, soluble cytoplasmic expression alone is also not enough for intrabody activity, since hTau21 only retained binding with the GSEK linker, even though it is soluble in both versions. Other factors such as intrinsic sequence stability and binding affinity also need to be considered for optimal intracellular activity. For instance, in a recent publication where scFvs were also designed from mAbs and compared as a secreted protein versus intrabody, the authors introduce specific mutations in the intrabody sequence to promote intracellular stability, and all three intrabodies retained binding on ELISA (Goodwin et al., 2012). Even though the authors do not refer to how the intrabodies performed before the sequence changes, based on this work it is fair to assume that binding was either very weak or absent.

[0190] Intrabody development remains challenging, even though several strategies have been described to improve selection of cytoplasmic stable constructs and engineer scFvs for cytoplasmic function. One of such approaches consists in designing disulfide-free scFvs, as the inability of these bonds to form in reducing conditions is one of the main factors for scFv intrabody misfolding and aggregation. To this end, the amino acid combination Val-Ala has been successfully employed to obtain disulfide-free scFvs for cytoplasmic expression (Wörn & Plückthun, 1998; Proba et al., 1998). Additionally, grafting the CDRs from scFvs of interest into frameworks that have been described as stable in the cytoplasmic environment has also been suggested as a potential strategy to improve scFv-intrabody stability (Ewert et al., 2004). However, to our knowledge CDR-grafting has not been used for the development of therapeuticscFv-intrabodies, with most of its application having been focused on cytoplasmic expression of scFvs for further purification or evaluation of scFv-intrabody activity in E. coli and yeast models (Jung & Plückthun, 1997; Wörn et al., 2000; Donini et al., 2003). Combining these two strategies, solubility and binding for intrabody PT77 was able to be rescued. On the other hand, intrabodies PT51 and hTau21 did not benefit from this strategy, demonstrating that CDR- grafting may not be a straightforward universal solution for intrabody development. Moreover, even though donor framework residues identified as important for binding function were kept during CDR-grafting, for intrabodies PT51 and hTau21, it appears that other residues from their original frameworks may be important for antigen contact and / or proper loop folding.

[0191] Binding was also evaluated with a nuclear translocation assay (Zhou et al., 2004). This assay has been frequently used to confirm intracellular binding of several scFvs (Zhou et al., 2004; Lecerf et al., 2001; Lynch et al., 2008; Martinelli et al., 2014; Dingus et al., 2022). In most reports, ELISA binding was not directly compared to NLS assay binding, with other methods such as co-immunoprecipitation and yeast or phage-display used instead. In all cases, the NLS assay was predictive of binding in other assays. In this case, the results did not always correlate with ELISA binding. It was not investigated further why this was the case, but it is speculated that the differences observed could be related to binding kinetics, scFv folding and differences in epitope presentation between assays.

[0192] To evaluate the effect of the developed intrabodies on tau aggregation, two primary mouse cortical neuron models were first used: one, in which aggregation of endogenous mouse tau was induced by AD-brain derived tau seeds (Soares et al., 2021); and one, in which human tau with the P301L mutation was overexpressed and its aggregation was induced by K18-P301L seeds (41). Additionally, the intrabodies were evaluated in OHSCs, in which endogenously expressed human tau with the P301S mutation was aggregated by the addition of K18-P301L seeds (42). Of the three intrabodies described here, only PT177 SS- 4D5 was able to interfere with AD-seed-mediated mouse tau aggregation in primary neurons. As for K18-seeded mutant human tau aggregation, all three intrabodies scFv PT51 GS, hTau21 GSEK, and PT77 SS−4D5 were able to reduce aggregation. Importantly, in the K18-seeded models, none of the scFv- intrabodies can bind to the K18 seeds, meaning that the observed effect is on de novo aggregation. Intrabody PT77 SS- 4D5 outperformed PT51 GS and hTau21 GSEK in all threemodels. This was more evident in OHSC, where PT77 SS- 4D5 led to a larger reduction in phosphorylated tau aggregates than the other two intrabodies. The parental mAb PT77 (150 kDa) competed with AT8 for binding in immunoassays, however, this was not tested with scFv- PT77 (~25 kDa). Even so, antibody fragments tend to have lower affinity than their mAb counterparts, so it would be expected that any interference resultant of the presence of a scFv would not be at the same level as with a mAb. Still, it cannot be completely excluded that, if the scFv-intrabody is bound to the tau aggregates, that it may prevent mAb AT8 from binding and result in an overestimation of the effect on AT8-phosphorylated tau aggregates. Nevertheless, with the PT3 phosphorylation assay, scFv-intrabody PT77 did not interfere with PT3 binding, and still confirmed the strong reduction of phosphorylated tau aggregates by scFv-intrabody PT77.

[0193] It is hypothesized that the differences seen in the ability of scFv-intrabodies PT51 GS and hTau21 GSEK to interfere with tau aggregation are likely related to structural differences in the aggregates that are formed in each model. The parent mAbs of these scFvs were previously evaluated for their capacity to immunodeplete tau aggregates from AD brain homogenates and P301S mice brain homogenates. Interestingly, both mAbs were more efficient at removing seeding-competent aggregates from P301S brain homogenates than from AD-brain homogenates (Vandermeeren et al., 2018). This is in line with what was observed with the intrabodies, where efficacy was higher against mutated human tau aggregation.

[0194] The intrabodies studied herein were not coupled to a degron and no changes were observed in total tau levels in both models. Thus, it is hypothesized that the mechanism by which the scFv-intrabodies were interfering with aggregation was by preventing newly formed aggregates from recruiting monomeric tau via steric hindrance. This could be by either by binding to oligomers and small aggregates or monomeric tau itself. Previous reports of other “naked” anti-tau intrabodies, have also observed a reduction in insoluble or phosphorylated tau, without affecting soluble tau levels (Goodwin et al., 2021; Danis et al., 2022). Additionally, another report showed that total tau levels were only reduced when the scFv-intrabody was fused to a mutated form of ubiquitin that would either target it to the proteasomal or the lysosomal degradation pathways (Gallardo et al., 2019).

[0195] To the best of knowledge, this is the first report showing the effectiveness of anti-tau intrabodies in in vitro neuronal models of tau aggregation, as well as in OHSCs. In previous publications, HEK293 cells lines overexpressing mutant forms of tau or HEK293 biosensor models were used, with only one report using primary mouse neurons in addition to HEK293 cells (Gallardo et al., 2019; Goodwin et al., 2021; Danis et al., 2022). The neuronal models and OHSC described here require more hands-on work and provide a smaller throughput. However, they are closer to physiological / disease conditions, as tau aggregation occurs in a neuronal environment, and in the case of the AD-seed model, a disease-related form of tau is used as template. Additionally, it is shown that the importance of using different models of tau aggregation in parallel, as aggregate structure will be different between models, resulting in different efficacy outcomes.

[0196] In tau immunotherapy, the current consensus is that targeting the mid-region of tau is more likely to be effective in preventing extracellular tau spreading and consequent intracellular aggregation (Vandermeeren et al., 2018; Courade et al., 2018; Albert et al., 2019). However, in the case of directly targeting intracellular tau, it seems for now that all tau domains are effective epitopes. Previous publications have reported successful results with intrabodies targeting the N- terminal and microtubule-binding repeats of tau. The work presented here demonstrated that targeting the PRD and the C-terminus of tau with intrabodies can reduce aggregation levels of mutated human tau in vitro. Moreover, wild type mouse tau aggregation was reduced when targeting pS199 / pS202. Side-by side comparisons between intrabodies targeting different domains are needed to further clarify whether one is more effective to prevent and / or clear intracellular tau aggregates.

[0197] To conclude, it has been shown here that monoclonal antibodies can be successfully converted into scFv-intrabodies, even if some sequence engineering is required. It was demonstrated that CDR-grafting was a viable approach to rescue unstable scFv-intrabodies and highlight the need for thoughtful scFv design as well as selection of the appropriate cellular models used.SEQUENCE LISTING SEQ ID NO: 1 MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEP GSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEA AGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPP APKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSP SSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKD NIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKI GSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSS TGSIDMVDSPQLATLADEVSASLAKQGL SEQ ID NO: 2 GGGGSGGGGSGGGGS SEQ ID NO: 3 GGSEGKSSGSGSESKSTGGS SEQ ID NO: 4 TSWMN SEQ ID NO: 5 RIYPGDGDTNYNGKFKD SEQ ID NO: 6 SDWEGFAY SEQ ID NO: 7 GYAFSTS SEQ ID NO: 8 YPGDGD SEQ ID NO: 9 GYAFSTSWSEQ ID NO: 10 IYPGDGDT SEQ ID NO: 11 TRSDWEGFAY SEQ ID NO: 12 GYAFSTSWMN SEQ ID NO: 13 RIYPGDGDTN SEQ ID NO: 14 STSWMN SEQ ID NO: 15 WIGRIYPGDGDTN SEQ ID NO: 16 TRSDWEGFA SEQ ID NO: 17 KSTKSLLNSDGFTYLD SEQ ID NO: 18 LVSNRFS SEQ ID NO: 19 FQTNYLPLT SEQ ID NO: 20 KSLLNSDGFTYSEQ ID NO: 22 LNSDGFTYLDWY SEQ ID NO: 23 LLIYLVSNRF SEQ ID NO: 24 FQTNYLPL SEQ ID NO: 25 QVQLQQSGPELVKPGASVKISCEASGYAFSTSWMNWVKQRPGKGLEWIGRIYPGDGDTN YNGKFKDKATLTADKSSSTVYMQLSSLTSEDSAVYFCTRSDWEGFAYWGQGTLVTVSA SEQ ID NO: 26 DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQTNYLPLTFGAGTKLELK SEQ ID NO: 27 RIYPGDGDTNYNDSVKGSEQ ID NO: 29 QVQLVESGGGLVQPGGSLRLSCSASGYAFSTSWMNWVRQAPGKGLEWVSRIYPGDGDTN YNDSVKGRFTLSADKSKSTVYLQMNSLRAEDTAVYFCTRSDWEGFAYWGQGTLVTVSS SEQ ID NO: 30 DIQLTQSPSSLSASVGDRVTITCKSTKSLLNSDGFTYLDWYQQKPGQSPKLLIYLVSNR FSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQTNYLPLTFGQGTKLEIKSEQ ID NO: 31 WVARIYPGDGDTN SEQ ID NO: 32 EVQLVESGGGLVQPGGSLRLSCAASGYAFSTSWMNWVRQAPGKGLEWVARIYPGDGDTN YNDSVKGRFTLSADKAKSSVYLQMNSLRAEDTAVYFCTRSDWEGFAYWGQGTLVTVSS SEQ ID NO: 33 QVQLVESGGGLVQPGGSLRLSVSASGYAFSTSWMNWVRQAPGKGLEWVSRIYPGDGDTN YNDSVKGRFTLSADKSKSTVYLQMNSLRAEDTAVYFATRSDWEGFAYWGQGTLVTVSS SEQ ID NO: 34 DIQLTQSPSSLSASVGDRVTITVKSTKSLLNSDGFTYLDWYQQKPGQSPKLLIYLVSNR FSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAFQTNYLPLTFGQGTKLEIK SEQ ID NO: 35 EVQLVESGGGLVQPGGSLRLSVAASGYAFSTSWMNWVRQAPGKGLEWVARIYPGDGDTN YNDSVKGRFTLSADKAKSSVYLQMNSLRAEDTAVYFATRSDWEGFAYWGQGTLVTVSS SEQ ID NO: 36 TYAMN SEQ ID NO: 37 RIRSKSDNYATYYADSVKD SEQ ID NO: 38 QDYYV SEQ ID NO: 39 GFSFNTY SEQ ID NO: 40 RSKSDNYASEQ ID NO: 41 GFSFNTYA SEQ ID NO: 42 IRSKSDNYAT SEQ ID NO: 43 VRQDYYV SEQ ID NO: 44 GFSFNTYAMN SEQ ID NO: 45 RIRSKSDNYATY SEQ ID NO: 46 NTYAMN SEQ ID NO: 47 WVARIRSKSDNYATY SEQ ID NO: 48 VRQDYY SEQ ID NO: 49 RSSKSLLHSNGKTYLY SEQ ID NO: 50 RMSNLVS SEQ ID NO: 51 MQHLEYPLTSEQ ID NO: 52 KSLLHSNGKTY RMS SEQ ID NO: 54 LHSNGKTYLYWF SEQ ID NO: 55 LLIYRMSNLV SEQ ID NO: 56 MQHLEYPL SEQ ID NO: 57 EVKLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSDNYA TYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVRQDYYVWGTGTSVTVSS SEQ ID NO: 58 DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGKTYLYWFLQRPGQSPQLLIYRMSNL VSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPLTFGAGTKLELK SEQ ID NO: 59 RIRSKSDNYATYYADSVKG SEQ ID NO: 60 WVSRIRSKSDNYATY SEQ ID NO: 61 QVQLVESGGGLVQPGGSLRLSCSASGFSFNTYAMNWVRQAPGKGLEWVSRIRSKSDNYA TYYADSVKGRFTISRDDSKSTLYLQMNSLRAEDTAVYYCVRQDYYVWGQGTTVTVSSSEQ ID NO: 62 DIQMTQSPSSLSASVGDRVTITCRSSKSLLHSNGKTYLYWFQQKPGQSPKLLIYRMSNL VSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQHLEYPLTFGQGTKLEIK SEQ ID NO: 63 EVQLVESGGGLVQPGGSLRLSCAASGFSFNTYAMNWVRQAPGKGLEWVSRIRSKSDNYA TYYADSVKGRFTISRDDSKSTLYLQMNSLRAEDTAVYYCVRQDYYVWGQGTTVTVSS SEQ ID NO: 64 EVQLVESGGGLVQPGGSLRLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSDNYA TYYADSVKGRFTISRDDAKSSLYLQMNSLRAEDTAVYYCVRQDYYVWGQGTTVTVSS SEQ ID NO: 65 QVQLVESGGGLVQPGGSLRLSVSASGFSFNTYAMNWVRQAPGKGLEWVSRIRSKSDNYA TYYADSVKGRFTISRDDSKSTLYLQMNSLRAEDTAVYYAVRQDYYVWGQGTTVTVSS SEQ ID NO: 66 DIQMTQSPSSLSASVGDRVTITVRSSKSLLHSNGKTYLYWFQQKPGQSPKLLIYRMSNL VSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAMQHLEYPLTFGQGTKLEIK SEQ ID NO: 67 EVQLVESGGGLVQPGGSLRLSVAASGFSFNTYAMNWVRQAPGKGLEWVSRIRSKSDNYA TYYADSVKGRFTISRDDSKSTLYLQMNSLRAEDTAVYYAVRQDYYVWGQGTTVTVSS SEQ ID NO: 68 EVQLVESGGGLVQPGGSLRLSVAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSDNYA TYYADSVKGRFTISRDDAKSSLYLQMNSLRAEDTAVYYAVRQDYYVWGQGTTVTVSS SEQ ID NO: 69 DTYIH SEQ ID NO: 70 RIDPANGNSKYDPKFQGSEQ ID NO: 71 HDGY SEQ ID NO: 72 GCNIKDT SEQ ID NO: 73 DPANGN SEQ ID NO: 74 GCNIKDTY SEQ ID NO: 75 IDPANGNS SEQ ID NO: 76 AHHDGY SEQ ID NO: 77 GCNIKDTYIH SEQ ID NO: 78 RIDPANGNSK SEQ ID NO: 79 KDTYIH SEQ ID NO: 80 WIGRIDPANGNSK SEQ ID NO: 81 AHHDGSEQ ID NO: 82 KASQSVDYDGDSYMN SEQ ID NO: 83 AASNLES SEQ ID NO: 84 QQSNEAPYT SEQ ID NO: 85 QSVDYDGDSY SEQ ID NO: 87 DYDGDSYMNWY SEQ ID NO: 88 LLIYAASNLE SEQ ID NO: 89 QQSNEAPY SEQ ID NO: 90 EVQLQQSGAELVKPGASGKLSCTASGCNIKDTYIHWVKQRPEQGLEWIGRIDPANGNSK YDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCAHHDGYWGQGTLVTVSA SEQ ID NO: 91 DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKAGQPPKLLIYAASNLE SGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEAPYTFGGGTRLEIKSEQ ID NO: 93WVSRIDPANGNSK SEQ ID NO: 94 QVQLVESGGGLVQPGGSLRLSCSASGCNIKDTYIHWVRQAPGKGLEWVSRIDPANGNSK YDDSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAHHDGYWGQGTLVTVSS SEQ ID NO: 95 DIQLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLE SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEAPYTFGGGTKVEIK SEQ ID NO: 96 WVARIDPANGNSK SEQ ID NO: 97 EVQLVESGGGLVQPGGSLRLSCAASGCNIKDTYIHWVRQAPGKGLEWVARIDPANGNSK YDDSVKGRFTISADTAKNSAYLQMNSLRAEDTAVYYCAHHDGYWGQGTLVTVSS SEQ ID NO: 98 QVQLVESGGGLVQPGGSLRLSVSASGCNIKDTYIHWVRQAPGKGLEWVSRIDPANGNSK YDDSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYAAHHDGYWGQGTLVTVSS SEQ ID NO: 99 DIQLTQSPSSLSASVGDRVTITVKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLE SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAQQSNEAPYTFGGGTKVEIK SEQ ID NO: 100 EVQLVESGGGLVQPGGSLRLSVAASGCNIKDTYIHWVRQAPGKGLEWVARIDPANGNSK YDDSVKGRFTISADTAKNSAYLQMNSLRAEDTAVYYAAHHDGYWGQGTLVTVSS SEQ ID NO: 101 DYKDDDDKREFERENCES Al-Lazikani B, et al. 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Claims

WHAT IS CLAIMED IS 1. A method of binding intracellular tau in a cell, the method comprising contacting the cell with a vector comprising a polynucleotide encoding an intrabody that specifically binds tau, wherein the intrabody binds to an epitope comprising amino acid residues 151-158 of tau, amino acid residues pS199 / pS202 of tau, or amino acid residues 375-380 of tau, and wherein the numbering of the amino acid is with reference to the amino acid sequence set forth in SEQ ID NO:

1.

2. A method of reducing tau aggregation in a cell, the method comprising contacting the cell with a vector comprising a polynucleotide encoding an intrabody that specifically binds tau, wherein the intrabody binds to an epitope comprising amino acid residues 151-158 of tau, amino acid residues pS199 / pS202 of tau, or amino acid residues 375-380 of tau, and wherein the numbering of the amino acid is with reference to the amino acid sequence set forth in SEQ ID NO:

1.

3. The method of claim 1 or 2, wherein the vector comprises a viral vector.

4. The method of claim 3, wherein the viral vector comprises an adeno-associated- virus (AAV) vector.

5. The method of claim 4, wherein the AAV vector comprises an AAV serotype 6 vector.

6. The method of any one of claims 1-5, wherein the method is in vitro.

7. The method of any one of claims 1-6, wherein the tau is human tau.

8. The method of any one of claims 1-7, wherein the intrabody comprises a single chain variable fragment (scFv), wherein the scFv comprises a heavy chain variable region and a light chain variable region interconnected by a linker.

9. The method of claim 8, wherein the heavy chain variable region and the light chain variable region comprises the following: (a) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 4, 5, and 6, respectively; and a light chain variable CDR- 1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (b) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 7, 8, and 6, respectively; and a light chain variable CDR- 1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (c) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 9, 10, and 11, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 20, LVS, and 19, respectively; or (d) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 12, 13, and 6, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (e) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 14, 15, and 16, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively; or (f) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 4, 27, and 6, respectively; and a light chain variable CDR- 1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or(g) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 14, 28, and 16, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively; or (h) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 14, 31, and 16, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively; or (i) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 36, 37, and 38, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (j) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 39, 40, and 38, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (k) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 41, 42, and 43, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 52, RMS, and 51, respectively; or (l) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 44, 45, and 38, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (m) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 46, 47, and 48, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 54, 55, and 56, respectively; or (n) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 36, 59, and 38, respectively; and a light chain variableCDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (o) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 46, 60, and 48, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 54, 55, and 56, respectively; or (p) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 69, 70, and 71, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (q) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 72, 73, and 71, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (r) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 74, 75, and 76, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 85, AAS, and 84, respectively; or (s) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 77, 78, and 71, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (t) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 79, 80, and 81, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively; or (u) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 69, 92, and 71, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or(v) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 79, 93, and 81, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively; or (w) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 79, 96, and 81, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively.

10. The method of claim 8 or 9, wherein: (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26; or (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30; or (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30; or (iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 34; or (v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 34; or (vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 58; or (vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62; or (vii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62; or (viii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62; or (ix) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 66; or(x) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 67, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 66; or (xi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 68, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 66; or (xii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 90, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 91; or (xiii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 94, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 95; or (xiv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 97, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 95; or (xv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 98, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 99; or (xvi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 100, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

99.

11. The method of any one of claims 8-10, wherein the linker comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

12. The method of any one of claims 1-11, wherein the cell is selected from a neuron, an astrocyte, and an oligodendrocyte.

13. The method of claim 12, wherein the cell is a neuron.

14. A method of making an anti-tau intrabody for targeting tau intracellularly, wherein the anti-tau intrabody comprises a single chain variable fragment, the method comprising: (a) grafting complementarity determining regions (CDRs) of an anti-tau antibody to a scFv framework identified to be stable intracellularly, and (b) removing disulfide bonds in the scFv, wherein the anti-tau antibody binds to an epitope comprising amino acid residues 151-wherein the numbering of the amino acid is with reference to the amino acid sequence set forth in SEQ ID NO:

1.

15. The method of claim 14, wherein the CDRs comprise a heavy chain variable CDR-1, CDR-2, and CDR-3 and a light chain variable CDR-1, CDR-2, and CDR-3, wherein: (a) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 4, 5, and 6, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (b) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 7, 8, and 6, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (c) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 9, 10, and 11, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 20, LVS, and 19, respectively; or (d) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 12, 13, and 6, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (e) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 14, 15, and 16, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively; or (f) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 4, 27, and 6, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (g) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 14, 28, and 16, respectively; and the light chain variableCDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively; or (h) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 14, 31, and 16, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively; or (i) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 36, 37, and 38, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (j) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 39, 40, and 38, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (k) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 41, 42, and 43, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 52, RMS, and 51, respectively; or (l) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 44, 45, and 38, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (m) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 46, 47, and 48, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 54, 55, and 56, respectively; or (n) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 36, 59, and 38, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or(o) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 46, 60, and 48, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 54, 55, and 56, respectively; or (p) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 69, 70, and 71, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (q) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 72, 73, and 71, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (r) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 74, 75, and 76, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 85, AAS, and 84, respectively; or (s) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 77, 78, and 71, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (t) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 79, 80, and 81, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively; or (u) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 69, 92, and 71, respectively; and the light chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (v) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 79, 93, and 81, respectively; and the light chain variableCDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively; or (w) the heavy chain variable CDR-1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 79, 96, and 81, respectively; the light chain variable CDR- 1, CDR-2, and CDR-3 comprises the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively.

16. The method of any one of claims 14-15, wherein the removing of the disulfide bonds in (b) comprises removing cysteine amino acids from the scFv.

17. The method of claim 16, wherein the removing of the disulfide bonds in (b) further comprises replacing the cysteine amino acids with a combination of alanine-valine.

18. An anti-tau intrabody that binds specifically to tau, wherein the intrabody comprises a single chain variable fragment (scFv), the scFv comprising a heavy chain variable region and a light chain variable region interconnected by a linker; wherein the heavy chain variable region and the light chain variable region comprises the following: (a) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 4, 5, and 6, respectively; and a light chain variable CDR- 1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (b) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 7, 8, and 6, respectively; and a light chain variable CDR- 1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (c) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 9, 10, and 11, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 20, LVS, and 19, respectively; or(d) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 12, 13, and 6, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (e) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 14, 15, and 16, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively; or (f) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 4, 27, and 6, respectively; and a light chain variable CDR- 1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 17, 18, and 19, respectively; or (g) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 14, 28, and 16, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively; or (h) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 14, 31, and 16, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 22, 23, and 24, respectively; or (i) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 36, 37, and 38, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (j) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 39, 40, and 38, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (k) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 41, 42, and 43, respectively; and a light chain variableCDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 52, RMS, and 51, respectively; or (l) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 44, 45, and 38, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (m) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 46, 47, and 48, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 54, 55, and 56, respectively; or (n) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 36, 59, and 38, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 49, 50, and 51, respectively; or (o) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 46, 60, and 48, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 54, 55, and 56, respectively; or (p) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 69, 70, and 71, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (q) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 72, 73, and 71, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (r) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 74, 75, and 76, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 85, AAS, and 84, respectively; or(s) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 77, 78, and 71, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (t) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 79, 80, and 81, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively; or (u) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 69, 92, and 71, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 82, 83, and 84, respectively; or (v) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 79, 93, and 81, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively; or (w) a heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 79, 96, and 81, respectively; and a light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively.

19. The anti-tau intrabody of claim 18, wherein: (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26; or (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30; or (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30; or (iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 34; or(v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 34; or (vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 57, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 58; or (vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62; or (vii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62; or (viii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 64, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62; or (ix) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 65, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 66; or (x) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 67, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 66; or (xi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 68, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 66; or (xii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 90, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 91; or (xiii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 94, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 95; or (xiv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 97, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 95; or (xv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 98, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 99; or (xvi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 100, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

99.

20. The anti-tau intrabody of claim 19 or 20, wherein the linker comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

21. A polynucleotide encoding the anti-tau intrabody of any one of claims 18-20.A vector comprising the polynucleotide of claim 21.

23. The vector of claim 22, wherein the vector comprises a viral vector.

24. The vector of claim 23, wherein the viral vector comprises an adeno-associated- virus (AAV) vector.

25. The vector of claim 24, wherein the AAV vector comprises an AAV serotype 6 vector.

26. A composition comprising: (a) the anti-tau intrabody of any one of claims 18-20; or (b) the polynucleotide of claim 21; or (c) the vector of any one of claims 22-24; and a carrier.

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