Intrabodies targeting intracellular tau

Abstract

This invention provides a method for binding intracellular tau in cells, wherein the method includes contacting the cells with a vector containing a polynucleotide encoding an intracellular antibody that specifically binds tau. This invention also provides a method for reducing tau aggregation in cells, wherein the method includes contacting the cells with a vector containing a polynucleotide encoding an intracellular antibody that specifically binds tau. The intracellular antibody may bind to an epitope containing amino acid residues 151-158 of tau, amino acid residues pS199 / pS202 of tau, or amino acid residues 375-380 of tau, wherein the amino acid numbers refer to the amino acid sequence shown in SEQ ID NO:1.

Description

[0001] Cross-references to related applications This application claims the benefit of U.S. Provisional Application No. 63 / 547,121, filed November 2, 2023, the entire contents of which are incorporated herein by reference.

[0002] sequence list This application includes a sequence list that has been electronically submitted in XML format, and is incorporated herein by reference in its entirety. The XML copy was created on November 1, 2024, and is named JAB7214WOPCT1_SL.xml, with a size of 286,208 bytes. Background Technology

[0003] Intracellular tau accumulation is a common feature of several neurodegenerative diseases (collectively known as tau protein diseases). For example, in Alzheimer's disease (AD), the gradual accumulation of tau in the brain has been shown to be strongly correlated with cognitive decline and neurodegeneration (Gordon et al., 2018; Lowe et al., 2019). Other tau protein diseases include: Pick's disease, characterized by severe gross atrophy of the frontotemporal lobes and corresponding tau-positive intracellular inclusions; progressive supranuclear palsy, characterized by tau-positive glial inclusions in the gray matter and "coiled bodies" in oligodendrocytes in the white matter; and corticobasal degeneration, which involves the formation of diffuse tau-positive linear structures (patches) as well as white matter coiled bodies and linear structures, tau-positive ballooning neurons, and neuronal tangles (Coughlin & Irwin, 2017). Therefore, tau has become a common target for therapies aimed at treating or mitigating the effects of AD and other tau protein diseases.

[0004] One of the main treatment approaches targeting tau is immunotherapy. Immunotherapy strategies aim to halt disease progression by capturing extracellular forms of tau (Colin et al., 2019). For example, monoclonal antibodies targeting monomeric, aggregated, phosphorylation-specific, or conformationally altered forms of tau protein have been developed (Jadhav et al., 2019). However, because tau aggregation is an intracellular phenomenon, therapeutic modalities that can act intracellularly may be more effective.

[0005] In this regard, antisense oligonucleotides (ASOs) have been explored as therapies targeting intracellular tau. A small phase I study demonstrated that using ASOs to reduce intracellular tau levels was safe and well-tolerated, and reduced aggregated tau levels in patients with mild AD, as measured by positron emission tomography (PET) (Mummery et al., 2023). However, due to the size and high charge of ASOs, their use as a therapy may face challenges in terms of cellular uptake, stability, and sensitivity to nuclease degradation, particularly for CNS-targeted therapies, particularly in overcoming the blood-brain barrier (Jadhav et al., 2019).

[0006] Therefore, there is a need in the art for strategies to target intracellular tau. This invention addresses this need. Summary of the Invention

[0007] Some key aspects of the present invention are summarized below. Further aspects are described in the detailed description, embodiments, drawings, and claims of this disclosure. The descriptions in each part of this disclosure are intended to be read in conjunction with the other parts. Furthermore, the various embodiments described in each part of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the invention.

[0008] This invention is based in part on a series of important findings, which are described in more detail in the Embodiments section of this patent specification. For example, three single-chain variable fragment (scFv) forms of anti-tau intracellular antibodies have been developed, derived from monoclonal antibodies (mAbs) PT51, PT77, and hTau21 (Vandermeeren et al., 2018). These scFvs bind to the proline-enriched domain (PRD) and different epitopes at the C-terminus of the tau protein, including phosphorylated epitopes (pS199 / pS202). These scFv-intramural antibodies have been shown to interfere with K18-mediated accumulation of human tau with the P301L mutation in primary mouse cortical neurons. Additionally, scFv PT77, which binds to epitopes near pS199 / pS202, is also able to reduce AD ​​seed-mediated mouse tau accumulation in primary neurons. Based on these findings, and other findings presented herein, this invention provides new and improved methods for targeting tau intracellularly.

[0009] Therefore, in one aspect, the present invention relates to a method for binding intracellular tau in cells, wherein the method includes contacting the cells with a carrier comprising a polynucleotide encoding an intracellular antibody specifically binding tau. In another aspect, the present invention relates to a method for reducing tau aggregation in cells, wherein the method includes contacting the cells with a carrier comprising a polynucleotide encoding an intracellular antibody specifically binding tau.

[0010] In some embodiments, the vector includes a viral vector. In some embodiments, the viral vector includes an adeno-associated virus (AAV) vector. In a particular embodiment, the AAV vector includes an AAV serotype 6 vector.

[0011] In some implementations, the method is performed outside the body.

[0012] In some implementations, tau is human tau.

[0013] In some embodiments, the cells are selected from neurons, astrocytes, and oligodendrocytes. In some embodiments, the cells are neurons.

[0014] In some embodiments, the intracellular antibody binds to an epitope containing amino acid residues 151-158 of tau, amino acid residues pS199 / pS202 of tau, or amino acid residues 375-380 of tau, wherein the amino acid numbering refers to the amino acid sequence shown in SEQ ID NO: 1.

[0015] In some embodiments, the intracellular antibody 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. The heavy chain variable region may comprise heavy chain variable complementarity-determining regions (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.

[0016] In another aspect, the present invention relates to an intracellular anti-tau antibody that specifically binds to tau. The intracellular antibody comprises an scFv, wherein the scFv includes a heavy chain variable region and a light chain variable region interconnected by a linker. The heavy chain variable region may include heavy chain variable CDR-1, CDR-2, and CDR-3, and the light chain variable region may include light chain variable CDR-1, CDR-2, and CDR-3.

[0017] In another aspect, the present invention relates to a method for preparing an intracellular anti-tau antibody for intracellular targeting of tau, wherein the anti-tau intracellular antibody comprises a single-chain variable fragment. The method includes: (a) grafting a CDR of the anti-tau antibody into a scFv framework identified as intracellularly stable, and (b) removing disulfide bonds from the scFv. The intracellular antibody 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 amino acid numbering refers to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the CDR comprises heavy chain variable CDR-1, CDR-2, and CDR-3 and light chain variable CDR-1, CDR-2, and CDR-3.

[0018] In some embodiments of the invention, removing the disulfide bond in (b) includes removing the cysteine ​​amino acid from the scFv. In some embodiments, removing the disulfide bond in (b) further includes replacing the cysteine ​​amino acid with a combination of alanine and valine.

[0019] According to an embodiment of the present invention, (a) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; or (b) The heavy chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 6, respectively; and the light chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; or (c) The heavy chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; and the light chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 20, LVS, and 19, respectively; or (d) The heavy chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 6, respectively; and the light chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; or (e) The heavy chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; and the light chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24; or (f) The heavy chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 27, and SEQ ID NO: 6; and the light chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; or (g) The heavy chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 28, and SEQ ID NO: 16; and the light chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24; or (h) The heavy chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 31, and SEQ ID NO: 16; and the light chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24; or (i) The heavy chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; and the light chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively; or (j) The heavy chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 38; and the light chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or (k) The heavy chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively; and the light chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 52, RMS, and 51, respectively; or (l) The heavy chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 38; and the light chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or (m) The heavy chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48; and the light chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; or (n) The heavy chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 59, and SEQ ID NO: 38, respectively; and the light chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively; or (o) The heavy chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 46, SEQ ID NO: 60, and SEQ ID NO: 48, respectively; and the light chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively; or (p) The heavy chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively; and the light chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively; or (q) The heavy chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 71; and the light chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; or (r) The heavy chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76, respectively; and the light chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 85, AAS, and 84, respectively; or (s) The heavy chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 77, SEQ ID NO: 78, and SEQ ID NO: 71; and the light chain variants CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; or (t) The heavy chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively; and the light chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively; or (u) The heavy chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 92, and SEQ ID NO: 71, respectively; and the light chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively; or (v) The heavy chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 93, and SEQ ID NO: 81, respectively; and the light chain variants CDR-1, CDR-2, and CDR-3 contain the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively; or (w) The heavy chain variants CDR-1, CDR-2 and CDR-3 contain the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 96 and SEQ ID NO: 81, respectively; the light chain variants CDR-1, CDR-2 and CDR-3 contain the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89, respectively.

[0020] In some embodiments of the present invention (i) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 25, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 26; or (ii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 29, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 30; or (iii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 32, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 30; or (iv) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 33, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 34; or (v) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 35, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 34; or (vi) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 57, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 58; or (vii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 61, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 62; or (viii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 63, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 62; or (ix) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 64, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 62; or (x) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 65, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 66; or (xi) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 67, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 66; or (xii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 68, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 66; or (xiii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 90, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 91; or (xiv) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 94, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 95; or (xv) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 97, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 95; or (xvi) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 98, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 99; or (xvii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 100, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 99.

[0021] In some embodiments, the adapter comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[0022] In another respect, the present invention relates to a polynucleotide encoding an intracellular antibody against tau.

[0023] In another aspect, the present invention relates to a vector comprising the polynucleotide. In some embodiments, the vector comprises a viral vector. In some embodiments, the viral vector comprises an AAV vector. In a particular embodiment, the AAV vector comprises an AAV serotype 6 vector.

[0024] In another aspect, the present invention relates to a composition comprising a load and (a) an anti-tau antibody as described herein; or (b) a polynucleotide as described herein; or (c) a vector as described herein. Attached Figure Description

[0025] Figure 1The results demonstrate the characterization of the selected mAb and the expression of derived scFv (with GS or GSEK linkers) in the periplasm of *E. coli* and via the HEK293 cell secretion pathway, as described in the examples. scFv expression in the periplasm of *E. coli* was performed using clarified cell lysates. Expression levels were determined by Western blotting, and arbitrary unit (AU) concentrations were quantified based on Western blotting. The images shown in Figure A represent two independent experiments. All lysates were tested for binding to recombinant human tau, starting with 1 A.U. of scFv, by enzyme-linked immunosorbent assay (ELISA). The results are illustrated in Figure B (lysates from scFv expression with GS linkers) and Figure C (lysates from scFv expression with GSEK linkers). Detection was performed in Western blotting and ELISA using an anti-HA-tagged HRP-labeled antibody. Tau-paired helical filaments (ePHF) were used to phosphorylate the phosphorylated specific scFv PT77. The results are illustrated in Figures D (lysates from scFv expression with the GS linker) and E (lysates from scFv expression with the GSEK linker). Results are presented as the mean of two independent experiments. scFv expression is a secreted protein from HEK293 cells and was characterized using culture medium. Expression levels were determined by Western blotting. Images of the Western blotting are shown in Figure F. All samples were tested for recombinant human tau by ELISA, starting with undiluted culture medium. The results are illustrated in Figures G (samples from scFv expression with the GS linker) and H (samples from scFv expression with the GSEK linker). ePHF packets were used to phosphorylate the specific scFv PT77. The results are illustrated in Figures I (samples from scFv expression with the GS linker) and J (samples from scFv expression with the GSEK linker). Results are presented as the mean ± SD of three independent experiments. Detection was performed using an anti-FLAG-tagged HRP-labeled antibody.

[0026] Figure 2The results demonstrate the characterization of scFv expression (with GS or GSEK adapters) in the cytoplasm of HEK293 cells, as described in the examples. scFv was expressed in the cytoplasm of HEK293 cells and characterized using clear cell lysates. The presence of intracellular antibodies in the lysates was determined by Western blotting, and the image is shown in Figure A. Cell lysates were serially diluted starting at 10 μg of total protein and tested against recombinant human tau by ELISA. The results are illustrated in Figures B (from lysates of scFv expression with GS adapters) and C (from lysates of scFv expression with GSEK adapters). ePHF packets were used to phosphorylate the specific scFv PT77. The results are illustrated in Figures D (from samples of scFv expression with GS adapters) and E (from samples of scFv expression with GSEK adapters). The results are shown as the average of two independent experiments. Detection was performed using an anti-FLAG-tagged HRP-labeled antibody.

[0027] Figure 3 Results were presented demonstrating intracellular antibody solubility in the cytoplasm before and after CDR transplantation, as described in the examples. Intracellular antibodies were expressed in the cytoplasm of HEK293 cells and fixed 24 hours post-transfection. Intracellular antibody detection was performed via a C-terminal FLAG tag. Figure A shows a schematic diagram of the CDR transplantation strategy. CDRs from each strand of the original scFv (indicated in blue) were transferred to the new framework (indicated in brown). Framework amino acids identified as important for binding were also transferred (indicated by blue stripes). Additionally, a version was designed with cysteine ​​(indicated by C) replaced by the amino acid combination Val-Ala (indicated by V and A, respectively). Designed using biorender.com. Figure B shows an image of the immunocytochemical evaluation of intracellular antibody solubility in the cytoplasm. Images labeled B1-B6 show results using the original intracellular antibody sequence; images labeled B7-B9 show results using intracellular antibodies designed in the VL-VH orientation; images labeled B10-B22 show results using CDR-grafted versions (with and without disulfide bonds (SS-)). Images represent three independent experiments, each with two replicates. Scale bar: 25 μm. SS - scFv of cysteine ​​that does not participate in disulfide bonds.

[0028] Figure 4Results were presented demonstrating the evaluation of intracellular antibody tau binding after CDR transplantation into different frameworks, as described in the examples. Intracellular antibodies against PT51 (Fig. A), hTa21 (Fig. B), and PT77 (Fig. C) were expressed in HEK293A cells, and cell lysates were serially diluted starting at a 1:3 dilution and tested for binding to recombinant human tau by ELISA. ePHF packets were used for phosphorylation-specific intracellular antibodies against PT77 (Fig. D). Detection was performed using anti-FLAG-tagged HRP-labeled antibodies. SS - scFv of cysteine ​​that is not involved in disulfide bonds. Results are expressed as the mean ± SD of three independent experiments.

[0029] Figure 5 Results are presented demonstrating the evaluation of intracellular antibody tau binding in the cytoplasm of HEK293A cells, as described in the examples. Figure A shows images of HEK293 cells co-transfected with plasmids expressing intracellular antibodies and plasmids expressing either tau or α-synuclein conjugated to green fluorescent protein (GFP) and nuclear translocation signal (NLS). When the intracellular antibody is co-expressed with tau-NLS, it translocates to the nucleus only if it is able to bind tau in the cytoplasmic environment. Co-expression with human α-synuclein-NLS was used as a negative control. Figure B shows a graph of phosphorylated tau-NLS detected at S199 / S202 in cell lysates as determined using a sandwich MSD assay with PT77 as the capture antibody. Images represent three independent experiments with two biological replicates per experiment. Scale bar: 50 μM. SS - scFv of cysteine ​​that does not participate in disulfide bonds.

[0030] Figure 6 Results were presented demonstrating the effect of intracellular antibodies on AD seed-mediated aggregation, as described in the examples. Primary mouse cortical neurons were transduced in vitro (DIV) with AAV-intramural antibodies on day 7, followed by AD-tau seeding at DIV 10. Neurons were maintained until DIV 17, after which they were lysed in RIPA buffer. Endogenous mouse tau aggregation was measured on the cell lysates using a sandwich MSD assay. Intracellular antibodies PT51 (Figure A), PT77 S -Results for 4D5 (Fig. B), hTau21 (Fig. C), and the negative control scFv (Fig. D) are shown as percentages of conditions with no intracellular antibody expression after normalization to total mouse α-synuclein levels, along with insets of Western blot images. Expression levels were detected by Western blot using undiluted lysates. β-actin was used as a loading control. MSD results are shown as mean ± SD of three independent experiments. Statistical analysis was performed using a fitted mixed-effects model, with Dunnett correction used for multiple comparisons (**p ≤ 0.01). Western blot images represent three independent experiments. SS - scFv of cysteine ​​that is not involved in disulfide bonds. MOI: Multiplicity of infection.

[0031] Figure 7 Results were presented demonstrating the effect of intracellular antibody on hTau-P301L aggregation after the addition of K18-P301L seed, as described in the examples. Primary mouse cortical neurons were transduced in DIV 1 with AAV-intracellular antibody and AAV-hTauP301L, followed by the addition of sonicated K18-P301L seed in DIV 3. Neurons were maintained until DIV 10, after which they were lysed in RIPA buffer. hTau-P301L aggregates were measured on the cell lysates using a sandwich MSD assay. Intracellular antibodies PT51 (Figure A), PT77 S - Results for 4D5 (Fig. B), hTau21 (Fig. C), and the negative control scFv (Fig. D) are shown as percentages of conditions with no intracellular antibody expression after normalization to total mouse α-synuclein levels. Expression levels were detected by Western blot using undiluted lysates. β-actin was used as a loading control. MSD results are shown as mean ± SD of three independent experiments. Statistical analysis was performed using a fitted mixed-effects model, with Dunnett correction used for multiple comparisons (**p ≤ 0.01; ***p ≤ 0.001). Western blot images represent three independent experiments.

[0032] Figure 8Results are presented demonstrating the evaluation of the role of anti-tau scFv intracellular antibody in organoid hippocampal slice cultures (OHSCs), as described in the examples. OHSCs were transduced with AAV6 intracellular antibody in DIV 1 and DIV 2, followed by K18 seeding two weeks later. Slices were lysed in DIV 31. Figures A through DD show the results of aggregated tau levels and phosphorylated aggregated tau levels as measured using MSD assays. Two phosphorylation-independent assays were used: one using an hTau43 sandwich (Figure A) and the other using a proprietary C-terminal tau antibody (Figure B). Phosphorylated aggregates were detected using AT8 / AT8 (S202 / T205 / S209) (Figure C) and PT3 / PT3 (T212 / T217) (Figure D). Results are shown as the percentage of signal / background for K18-treated slices without intracellular antibody (K18 only), calculated by interpolation in arbitrary units (AU), mean ± SD. Statistical analysis was performed using a fitted mixed-effects model, with Dunnett correction used for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001). Note that for AT8 / AT8 assays, no values ​​could be interpolated for scFv-intracellular antibody PT77. Therefore, values ​​were set to the lowest limit of quantitation. Figure E shows the results of scFv-intracellular antibody expression levels evaluated by anti-FLAG staining on Western blots. Figure F shows the results of tau aggregates isolated from the brains of patients with AD, incubated with increased amounts of mAb PT77 prior to measurement. Detailed Implementation

[0033] Unless otherwise stated, the present invention may be practiced using conventional techniques of cell biology, molecular biology, microbiology, recombinant DNA and immunology, which are within the scope of the art.

[0034] To facilitate a clearer understanding of the invention, certain terms are first defined. Additional definitions are set forth throughout this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to this invention.

[0035] Any headings provided herein are not intended to limit the various aspects or embodiments of the invention, which can be obtained by referring to the specification as a whole. Therefore, the terms defined thereafter are defined more fully by referring to the entire specification.

[0036] All references cited in this disclosure are incorporated herein by reference in their entirety. Furthermore, any manufacturer's specifications or catalogues of any products referenced or mentioned herein are incorporated by reference. Documents incorporated herein by reference, or any teachings therein, may be used in the practice of this invention. Documents incorporated herein by reference are not acknowledged as prior art.

[0037] definition The wording or terminology used in this disclosure is for descriptive purposes and not for limitation, and the terminology or terminology in this specification should be interpreted by those skilled in the art based on the teachings and instructions.

[0038] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural references. The term “a” (or “an”) and the terms “one or more” and “at least one” are used interchangeably.

[0039] Furthermore, "and / or" is considered to be a specific disclosure of each of two specified features or components with or without the other. Therefore, the term "and / or," as used in phrases such as "A and / or B," is intended to include A and B, A or B, A (alone) and B (alone). Similarly, the term "and / or," as used in phrases 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).

[0040] Any implementation described using the language "includes" includes other similar implementations described under the terms "consisting of" and / or "substantially consisting of".

[0041] Units, prefixes, and symbols are represented in their International System of Units (SI) accepted form. A numerical range includes the number that defines the range, and any single value provided herein can be used as an endpoint of a range that includes other single values ​​provided herein. For example, a set of values ​​such as 1, 2, 3, 8, 9, and 10 is also a disclosure of numerical ranges from 1 to 10, from 1 to 8, from 3 to 9, etc. Similarly, a disclosed range is a disclosure of each individual value (i.e., intermediate value) covered by that range, including integers and fractions. For example, the specified range of 5 to 10 is also a disclosure of 5, 6, 7, 8, 9, and 10 individually, as well as 5.2, 7.5, 8.7, etc., individually.

[0042] Unless otherwise stated, the terms "at least" or "about" preceding a series of elements should be understood to refer to each element in the series. The term "about" preceding a numerical value includes ±10% of the stated value. For example, a concentration of about 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of about 1% (w / v) to 10% (w / v) includes 0.9% (w / v) to 11% (w / v).

[0043] Amino acids are represented in this paper by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides are represented by their generally accepted single-letter codes.

[0044] The term "intracellular antibody" refers to an antibody designed to be expressed within cells and to recognize and specifically bind to intracellular antigens (such as proteins, polypeptides, peptides, carbohydrates, polynucleotides, lipids, or combinations thereof) through at least one antigen recognition site within the variable region of the molecule. Most commonly, intracellular antibodies are single-chain variable fragments, or "scFvs," which contain heavy chain and light chain variable regions interconnected by linkers. The variable regions of both the heavy and light chains contain binding domains that interact with the antigen.

[0045] The heavy chain variable region and light chain variable region can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework (FW) regions. The CDRs in each chain are tightly bound together by the FW regions and, together with the CDRs of the other chain, contribute to the formation of antigen-binding sites for intracellular antibodies. Each heavy chain variable region and light chain variable region consists of three types of CDRs: CDR-1, CDR-2, and CDR-3.

[0046] At least two techniques exist for determining CDRs: (1) methods based on cross-species sequence variability (Kabat et al., 1991); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., 1997). Furthermore, combinations of these two methods are sometimes used in the field to determine CDRs.

[0047] The amino acid position numbering in Kabat refers to a numbering system used for variable domains in the heavy or light chain (approximately residues 1-107 in the light chain and residues 1-113 in the heavy chain). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortened or inserted FW or CDRs of the variable domain. For example, a variable domain in the heavy chain may include a single amino acid insertion after residue 52 of H2 (according to residue 52a in Kabat) and inserted residues after FW residue 82 of the heavy chain (e.g., residues 82a, 82b, and 82c, etc., according to Kabat).

[0048] The Kabat residue number of a given antibody can be determined by comparing the homologous region of the antibody sequence with the "standard" Kabat numbering sequence. Chothia refers to the location of the structural loop (Chothia & Lesk, 1987). When using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the loop length (this is because the Kabat numbering scheme inserts 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 region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by the AbM antibody modeling software from Oxford Molecular.

[0049] IMGT (ImMunoGeneTics) also provides a numbering system for variable regions, including CDRs (see, for example, Lefranc et al., 2003). The IMGT numbering system is based on alignments, structural data, and characterization of hypervariable loops from over 5,000 sequences and allows for easy comparison of variable regions and CDRs across all species. According to the IMGT numbering scheme, heavy chain variable region CDR-1 is located at positions 26–35, heavy chain variable region CDR-2 at positions 51–57, heavy chain variable region CDR-3 at positions 93–102, light chain variable region CDR-1 at positions 27–32, light chain variable region CDR-2 at positions 50–52, and light chain variable region CDR-3 at positions 89–97.

[0050] Furthermore, variable regions can be partitioned based on the "Specific Determining Residue Use" (SDRU) (Almagro 2004), where SDRs refer to amino acid residues of immunoglobulins that are directly involved in antigen contact. This SDRU concept was used to develop a "contact" approach to define CDRs, which renamed SDRs as "contact residues" (MacCallum et al., 1996).

[0051] The term "germination" refers to the mutation of amino acids at specific locations in an antibody back into the germline amino acids.

[0052] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an intracellular antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., intracellular antibody and antigen). The affinity of molecule X for its partner Y can generally be determined by the dissociation constant (K). D( ) indicates affinity. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity intracellular antibodies typically bind antigens slowly and tend to dissociate easily, while high-affinity intracellular antibodies typically bind antigens more quickly and tend to remain bound for longer periods.

[0053] The affinity or cohesion of intracellular antibodies to antigens can be determined using any suitable method (e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA)) or kinetics (e.g., KINEXA) known in the art. ® Or BIACORE ™ Or OCTET ® (Analysis) is determined experimentally. Direct binding assays and competitive binding assays can be readily employed (see, for example, Berzofsky et al., 1984; Kuby, 1992). The affinity of a specific antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH, temperature). Therefore, affinity and other antigen-binding parameters (e.g., K0) will affect the measurement. D or K d K on K off The measurement results were obtained using standard solutions of antibodies and antigens, as well as standard buffer solutions (as known in the art).

[0054] The terms “reduction,” “inhibition,” “blocking,” and “suppression” are used interchangeably and refer to any statistically significant reduction in a given activity, including complete blockage of activity. For example, “reduction” can mean a reduction in activity of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Therefore, when the terms “reduction,” “inhibition,” or “suppression” are used to describe, for example, the effect of an anti-tau intracellular antibody, these terms can refer to the ability of the anti-tau intracellular antibody to statistically significantly reduce: (a) the binding of the anti-tau intracellular antibody to tau, or (b) the activation or signaling of tau, etc. Inhibition can be determined relative to an untreated control (e.g., a control not exposed to an anti-tau intracellular antibody). In some embodiments, anti-tau intracellular antibodies can inhibit the 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 assay, or other assays known to those skilled in the art.

[0055] The term "composition" refers to a formulation that is in a form that allows the active ingredient to be active and does not contain any additional components that would have unacceptable toxicity to the environment in which the composition is delivered. Such compositions may be sterile and may contain a carrier, such as saline.

[0056] The “effective amount” of intracellular antibodies as disclosed herein is an amount sufficient to carry out the stated purpose. Regarding the stated purpose, the “effective amount” can be determined empirically and in a conventional manner.

[0057] The anti-tau intracellular antibody of the present invention can be naked or conjugated with other molecules, such as toxins, markers, etc. The term "marker," as used herein, refers to a detectable compound or composition that conjugates directly or indirectly to an intracellular antibody to produce a "labeled" intracellular antibody. The marker can be self-detectable (e.g., a radioisotope marker or a fluorescent marker), or, in the case of an enzyme marker, catalyzes a detectable chemical change in the substrate compound or composition.

[0058] As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin, antibody, or its antigen-binding fragment specifically binds. Epitopes can be formed from either consecutive amino acids or from discontinuous amino acids juxtaposed by the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed from ternary folds are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a distinctive spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and 2D nuclear magnetic resonance (see, for example, Epitope Mapping Protocols, 1996).

[0059] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also covers amino acid polymers that have been naturally modified or modified by intervention; natural or interventional modifications include, for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art. In some embodiments, the polypeptide may exist as a single chain or an associated chain.

[0060] As used herein, "polynucleotide" can include one or more "nucleic acids," "nucleic acid molecules," or "nucleic acid sequences," and refers to a polymer of nucleotides of any length, including DNA and RNA. 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 polymerases. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and their analogs. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0061] The term "vector" refers to a construct capable of being delivered to a host cell and, in some embodiments, expressing one or more genes or sequences of interest. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0062] "Isolated" peptides, intracellular antibodies, polynucleotides, or carriers are forms that do not exist in nature. Isolated peptides, intracellular antibodies, polynucleotides, or carriers include those that have been purified to the point that they are no longer in their naturally occurring form. In some embodiments, the isolated peptides, intracellular antibodies, polynucleotides, or carriers are substantially pure. When used herein, the term "substantially pure" means a purity greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%.

[0063] Other terms are defined elsewhere in this patent disclosure or used in accordance with their common meaning in the art.

[0064] Anti-Tau intracellular antibody This invention provides an intracellular anti-tau antibody that specifically binds to tau.

[0065] As used herein, the term “tau” or “tau protein,” also known as microtubule-associated protein tau, MAPT, neurofibrillary tangles, pairing helical filament (PHF)-tau, MAPTL, or MTBT1, refers to a rich central and peripheral nervous system protein with multiple isotypes. In the human central nervous system (CNS), due to alternative splicing, there are six major tau isotypes ranging in length from 352 to 441 amino acids (Hanger et al., 2009). Examples of tau include, but are not limited to, tau isotypes in the CNS, such as the longest tau isotype (2N4R) of 441 amino acids, also known as microtubule-associated protein tau isotype 2, which has four repeat sequences and two insert sequences, such as human tau isotype 2 with the amino acid sequence represented in SEQ ID NO: 1. Other examples of tau include the shortest (fetal) isotype (3R0N) of 352 amino acids, also known as microtubule-associated protein tau isotype 4, which has three repeating sequences and no inserts, such as human tau isotype 4 with the amino acid sequence represented in GenBank accession number NP_058525.1. Examples of tau also include the “large tau” isotype expressed in the peripheral nerves containing 300 additional residues (exon 4a) (Friedhoff et al., 2000). Examples of tau include human large tau, a 758-amino acid protein encoded by a 6762-nucleotide-long mRNA transcript (NM_016835.4) or its isotypes. The amino acid sequence of the exemplified human large tau is represented by GenBank accession number NP_058519.3. As used herein, the term "tau" includes homologs of tau from species other than humans, such as the cynomolgus monkey (Macaca Fascicularis), rhesus monkeys, or chimpanzees (Pantroglodytes). As used herein, the term "tau" includes proteins containing mutations of full-length wild-type tau, such as point mutations, fragments, insertions, deletions, and splice variants. The term "tau" also encompasses post-translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, phosphorylation.

[0066] Intracellular anti-tau antibodies specifically bind to tau epitopes. In some embodiments, the epitope comprises a tau domain selected from the intermediate domain, PRD, and C-terminal domain. In some embodiments, the epitope is a phosphorylated epitope.

[0067] In some 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 amino acid numbering refers to the amino acid sequence shown in SEQ ID NO: 1.

[0068] In embodiments of the present invention, the anti-tau intracellular antibody of the present invention comprises scFv. Therefore, the anti-tau intracellular antibody comprises a heavy chain variable region and a light chain variable region interconnected by a linker. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 2) (referred to herein as the “GS linker”). In other embodiments, the linker comprises the amino acid sequence GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 3) (referred to herein as the “GSEK linker”).

[0069] An exemplary anti-tau intracellular antibody of the present invention comprises an intracellular antibody derived from the monoclonal antibody PT51, referred to herein as "scFv PT51". The amino acid sequence of the CDR of scFv PT51 is presented in Table 1, which also provides the SEQ ID NO for each amino acid sequence.

[0070] Table 1. Sequences of heavy chain variable CDRs and light chain variable CDRs of scFv PT51 .

[0071] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises: (a) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; or (b) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 6, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; (c) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 20, LVS, and 19; or (d) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 6, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; or (e) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24.

[0072] The heavy chain variable region and light chain variable region of scFv PT51 are presented in Table 2, which also provides the SEQ ID NO for each amino acid sequence.

[0073] Table 2. Sequences of the heavy and light chain variable regions of scFv PT51 . Variable region amino acid sequence Heavy chain QVQLQQSGPELVKPGASVKISCEASGYAFSTSWMNWVKQRPGKGLEWIGRIYPGDGDTNYNGKFKDKATLTADKSSSTVYMQLSSLTSEDSAVYFCTRSDWEGFAYWGQGTLVTVSA (SEQ ID NO: 25) Light chain DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQTNYLPLTFGAGTKLELK (SEQ ID NO: 26)

[0074] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 26.

[0075] Other exemplary anti-tau intracellular antibodies of the present invention include intracellular antibodies derived from the monoclonal antibody PT51 but grafted into a framework based on antibody lineages that are highly similar to the previously described scFv F8 and A48-4D5 (Donini et al., 2003; Wörn & Plückthun, 1998; Wörn & Plückthun, 1999), the resulting intracellular antibodies referred herein as “scFv PT51 [F8]” or “scFv PT51 F8”, and “scFv PT51 [A48-4D5]” or “scFv PT51 A48-4D5”, respectively.

[0076] The amino acid sequences of the CDR of scFv PT51 [F8] are presented in Table 3, which also provides the SEQ ID NO for each amino acid sequence.

[0077] Table 3. Sequences of heavy chain variable CDRs and light chain variable CDRs of scFv PT51 [F8] .

[0078] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises: heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 27, and SEQ ID NO: 6, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively. In other embodiments, the anti-tau intracellular antibody comprises: heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 28, and SEQ ID NO: 16, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively.

[0079] The heavy and light chain variable regions of scFv PT51 [F8] are presented in Table 4, which also provides the SEQ ID NO for each amino acid sequence.

[0080] Table 4. Sequences of the heavy and light chain variable regions of scFv PT51 [F8] . Variable region amino acid sequence Heavy chain QVQLVESGGGLVQPGGSLRLSCSASGYAFSTSWMNWVRQAPGKGLEWVSRIYPGDGDTNYNDSVKGRFTLSADKSKSTVYLQMNSLRAEDTAVYFCTRSDWEGFAYWGQGTLVTVSS (SEQ ID NO: 29) Light chain DIQLTQSPSSSLSASVGDRVTITCKSTKSLLNSDGFTYLDWYQQKPGQSPKLLIYLVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQTNYLPLTFGQGTKLEIK (SEQ ID NO: 30)

[0081] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 30.

[0082] The amino acid sequences of the CDR of scFv PT51 [A48-4D5] are presented in Table 5, which also provides the SEQ ID NO for each amino acid sequence.

[0083] Table 5. Sequences of heavy chain variable CDRs and light chain variable CDRs of scFv PT51 [A48-4D5] .

[0084] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises: heavy chain variable CDR-1, CDR-2 and CDR-3 comprising the amino acid sequences of SEQ ID NO:14, SEQ ID NO:31 and SEQ ID NO:16 respectively; and light chain variable CDR-1, CDR-2 and CDR-3 comprising the amino acid sequences of SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24 respectively.

[0085] The heavy and light chain variable regions of scFv PT51 [A48-4D5] are presented in Table 6, which also provides the SEQ ID NO for each amino acid sequence.

[0086] Table 6. Sequences of the heavy and light chain variable regions of scFv PT51 [A48-4D5] . Variable region amino acid sequence Heavy chain EVQLVESGGGLVQPGGSLRLSCAASGYAFSTSWMNWVRQAPGKGLEWVARIYPGDGDTNYNDSVKGRFTLSADKAKSSVYLQMNSLRAEDTAVYFCTRSDWEGFAYWGQGTLVTVSS (SEQ ID NO: 32) Light chain DIQLTQSPSSSLSASVGDRVTITCKSTKSLLNSDGFTYLDWYQQKPGQSPKLLIYLVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQTNYLPLTFGQGTKLEIK (SEQ ID NO: 30)

[0087] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 32 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 30.

[0088] Other exemplary anti-tau intracellular antibodies of the present invention include intracellular antibodies derived from the monoclonal antibody PT51, transplanted into a framework based on antibody lineages highly similar to the previously described scFv F8 and A48-4D5, and made cysteine-free by replacing cysteine ​​with the amino acid combination alanine-valine. The resulting intracellular antibodies are referred to herein as “scFv PT51-SS”, respectively. - [F8]” and “scFv PT51-SS” - [A48-4D5]”.

[0089] scFv PT51-SS - The amino acid sequence of the CDR of [F8] is identical to that of scFv PT51 [F8] presented in Table 3. scFv PT51-SS - The heavy and light chain variable regions of [F8] are presented in Table 7, which also provides the SEQ ID NO for each amino acid sequence.

[0090] Table 7. scFv PT51-SS - Sequence of the heavy and light chain variable regions of [F8] . Variable region amino acid sequence Heavy chain QVQLVESGGGLVQPGGSLRLSVSASGYAFSTSWMNWVRQAPGKGLEWVSRIYPGDGDTNYNDSVKGRFTLSADKSKSTVYLQMNSLRAEDTAVYFATRSDWEGFAYWGQGTLVTVSS (SEQ ID NO: 33) Light chain DIQLTQSPSSSLSASVGDRVTITVKSTKSLLNSDGFTYLDWYQQKPGQSPKLLIYLVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAFQTNYLPLTFGQGTKLEIK (SEQ ID NO: 34)

[0091] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 34.

[0092] scFv PT51-SS - The amino acid sequence of the CDR of [A48-4D5] is identical to that of scFv PT51 [A48-4D5] presented in Table 5. scFv PT51-SS - The heavy and light chain variable regions of [A48-4D5] are presented in Table 8, which also provides the SEQ ID NO for each amino acid sequence.

[0093] Table 8. scFv PT51-SS - Sequence of the heavy and light chain variable regions of [A48-4D5] . Variable region amino acid sequence Heavy chain EVQLVESGGGLVQPGGSLRLSVAASGYAFSTSWMNWVRQAPGKGLEWVARIYPGDGDTNYNDSVKGRFTLSADKAKSSVYLQMNSLRAEDTAVYFATRSDWEGFAYWGQGTLVTVSS (SEQ ID NO: 35) Light chain DIQLTQSPSSSLSASVGDRVTITVKSTKSLLNSDGFTYLDWYQQKPGQSPKLLIYLVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAFQTNYLPLTFGQGTKLEIK (SEQ ID NO: 34)

[0094] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 35 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 34.

[0095] Other exemplary anti-tau intracellular antibodies of the present invention include intracellular antibodies derived from the monoclonal antibody PT77, referred to herein as "scFv PT77". The amino acid sequence of the CDR of scFv PT77 is presented in Table 9, which also provides the SEQ ID NO for each amino acid sequence.

[0096] Table 9. Sequences of heavy chain variable CDRs and light chain variable CDRs of scFv PT77 .

[0097] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises: (a) Heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively; or (b) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 38, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively; (c) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 52, RMS, and 51; or (d) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 38; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or (e) Heavy chain variants CDR-1, CDR-2, and CDR-3, each containing the amino acid sequences of SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48; and light chain variants CDR-1, CDR-2, and CDR-3, each containing the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56.

[0098] The heavy and light chains of scFv PT77 are presented in Table 10, which also provides the SEQ ID NO for each amino acid sequence.

[0099] Table 10. Sequences of the heavy and light chain variable regions of scFv PT77 . Variable region amino acid sequence Heavy chain EVKLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSDNYATYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVRQDYYVWGTGTSVTVSS (SEQ ID NO: 57) Light chain DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGKTYLYWFLQRPGQSPQLLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPLTFGAGTKLELK (SEQ ID NO: 58)

[0100] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 57 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 58.

[0101] Other exemplary anti-tau intracellular antibodies of the present invention include intracellular antibodies derived from the monoclonal antibody PT77 but grafted into a framework based on antibody lineages that are highly similar to the previously described scFv F8, 4D5, and A48-4D5 (Donini et al., 2003; Wörn & Plückthun, 1998; Wörn & Plückthun, 1999), the resulting intracellular antibodies referred herein as “scFv PT77 [F8]” or “scFv PT77 F8”, “scFv PT77 [4D5]” or “scFv PT77 4D5”, and “scFv PT77 [A48-4D5]” or “scFv PT77 A48-4D5”, respectively.

[0102] The amino acid sequence of the CDR of scFv PT77 [F8] is presented in Table 11, which also provides the SEQ ID NO for each amino acid sequence.

[0103] Table 11. Sequences of heavy chain variable CDRs and light chain variable CDRs of scFv PT77 [F8] .

[0104] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises: heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO:36, SEQ ID NO:59, and SEQ ID NO:38, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively. In other embodiments, the anti-tau intracellular antibody comprises: heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO:46, SEQ ID NO:60, and SEQ ID NO:48, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56, respectively.

[0105] The heavy and light chain variable regions of scFv PT77 [F8] are presented in Table 12, which also provides the SEQ ID NO for each amino acid sequence.

[0106] Table 12. Sequences of the heavy and light chain variable regions of scFv PT77 [F8] . Variable region amino acid sequence Heavy chain QVQLVESGGGLVQPGGSLRLSCSASGFSFNTYAMNWVRQAPGKGLEWVSRIRSKSDNYATYYADSVKGRFTISRDDSKSTLYLQMNSLRAEDTAVYYCVRQDYYVWGQGTTVTVSS (SEQ ID NO: 61) Light chain DIQMTQSPSSSLSASVGDRVTITCRSSKSLLHSNGKTYLYWFQQKPGQSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQHLEYPLTFGQGTKLEIK (SEQ ID NO: 62)

[0107] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 61 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 62.

[0108] The amino acid sequence of the CDR of scFv PT77 [4D5] is identical to that 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 the SEQ ID NO for each amino acid sequence.

[0109] Table 13. Sequences of the heavy and light chain variable regions of scFv PT77 [4D5] . Variable region amino acid sequence Heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFSFNTYAMNWVRQAPGKGLEWVSRIRSKSDNYATYYADSVKGRFTISRDDSKSTLYLQMNSLRAEDTAVYYCVRQDYYVWGQGTTVTVSS (SEQ ID NO: 63) Light chain DIQMTQSPSSSLSASVGDRVTITCRSSKSLLHSNGKTYLYWFQQKPGQSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQHLEYPLTFGQGTKLEIK (SEQ ID NO: 62)

[0110] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 63 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 62.

[0111] The amino acid sequence of the CDR of scFv PT77 [A48-4D5] is presented in Table 14, which also provides the SEQ ID NO for each amino acid sequence.

[0112] Table 14. Sequences of heavy chain variable CDRs and light chain variable CDRs of scFv PT77 [A48-4D5] .

[0113] The heavy and light chain variable regions of scFv PT77 [A48-4D5] are presented in Table 15, which also provides the SEQ ID NO for each amino acid sequence.

[0114] Table 15. Sequences of the heavy and light chain variable regions of scFv PT77 [A48-4D5] . Variable region amino acid sequence Heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSDNYATYYADSVKGRFTISRDDAKSSLYLQMNSLRAEDTAVYYCVRQDYYVWGQGTTVTVSS (SEQ ID NO: 64) Light chain DIQMTQSPSSSLSASVGDRVTITCRSSKSLLHSNGKTYLYWFQQKPGQSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQHLEYPLTFGQGTKLEIK (SEQ ID NO: 62)

[0115] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 64 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 62.

[0116] Other exemplary anti-tau intracellular antibodies of the present invention include intracellular antibodies derived from the monoclonal antibody PT77, grafted into a framework based on antibody lineages highly similar to the previously described scFv F8, 4D5, and A48-4D5, and made cysteine-free by replacing cysteine ​​with the amino acid combination alanine-valine. The resulting intracellular antibodies are referred to herein as “scFv PT77-SS”, respectively. -[F8]”, “scFv PT51-SS” - [4D5]” and “scFv PT51-SS” - [A48-4D5]”.

[0117] scFv PT77-SS - The amino acid sequence of the CDR of [F8] is identical to that of scFv PT77 [F8] presented in Table 11. scFv PT77-SS - The heavy and light chain variable regions of [F8] are presented in Table 16, which also provides the SEQ ID NO for each amino acid sequence.

[0118] Table 16. scFv PT77-SS - Sequence of the heavy and light chain variable regions of [F8] . Variable region amino acid sequence Heavy chain QVQLVESGGGLVQPGGSLRLSVSASGFSFNTYAMNWVRQAPGKGLEWVSRIRSKSDNYATYYADSVKGRFTISRDDSKSTLYLQMNSLRAEDTAVYYAVRQDYYVWGQGTTVTVSS (SEQ ID NO: 65) Light chain DIQMTQSPSSSLSASVGDRVTITVRSSKSLLHSNGKTYLYWFQQKPGQSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAMQHLEYPLTFGQGTKLEIK (SEQ ID NO: 66)

[0119] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 65 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 66.

[0120] scFv PT77-SS - The amino acid sequence of the CDR of [4D5] is identical to that of the CDR of scFv PT77 [F8] presented in Table 11. scFv PT51-SS - The heavy and light chain variable regions of [4D5] are presented in Table 17, which also provides the SEQ ID NO for each amino acid sequence.

[0121] Table 17. scFv PT51-SS - Sequence of the heavy and light chain variable regions of [4D5] . Variable region amino acid sequence Heavy chain EVQLVESGGGLVQPGGSLRLSVAASGFSFNTYAMNWVRQAPGKGLEWVSRIRSKSDNYATYYADSVKGRFTISRDDSKSTLYLQMNSLRAEDTAVYYAVRQDYYVWGQGTTVTVSS (SEQ ID NO: 67) Light chain DIQMTQSPSSSLSASVGDRVTITVRSSKSLLHSNGKTYLYWFQQKPGQSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAMQHLEYPLTFGQGTKLEIK (SEQ ID NO: 66)

[0122] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 67 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 66.

[0123] scFv PT77-SS - The amino acid sequence of the CDR of [A48-4D5] is identical to that of scFv PT77 [A48-4D5] presented in Table 14. scFv PT77-SS - The heavy and light chain variable regions of [A48-4D5] are presented in Table 18, which also provides the SEQ ID NO for each amino acid sequence.

[0124] Table 18. scFv PT77-SS - Sequence of the heavy and light chain variable regions of [A48-4D5] . Variable region amino acid sequence Heavy chain EVQLVESGGGLVQPGGSLRLSVAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSDNYATYYADSVKGRFTISRDDAKSSLYLQMNSLRAEDTAVYYAVRQDYYVWGQGTTVTVSS (SEQ ID NO: 68) Light chain DIQMTQSPSSSLSASVGDRVTITVRSSKSLLHSNGKTYLYWFQQKPGQSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAMQHLEYPLTFGQGTKLEIK (SEQ ID NO: 66)

[0125] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 68 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 66.

[0126] Other exemplary anti-tau intracellular antibodies of the present invention include intracellular antibodies derived from the monoclonal antibody hTau21, referred to herein as "scFv hTau21". The amino acid sequence of the CDR of scFv hTau21 is presented in Table 19, which also provides the SEQ ID NO for each amino acid sequence.

[0127] Table 19. Sequences of heavy chain variable CDRs and light chain variable CDRs of scFv hTau21 .

[0128] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises: (a) Heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively; or (b) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 71, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively; (c) Heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 85, AAS, and 84, respectively; or (d) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 77, SEQ ID NO: 78, and SEQ ID NO: 71; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; or (e) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89.

[0129] The heavy and light chain variable regions of scFv hTau21 are presented in Table 20, which also provides the SEQ ID NO for each amino acid sequence.

[0130] Table 20. Sequences of the heavy and light chain variable regions of scFv hTau21 . Variable region amino acid sequence Heavy chain EVQLQQSGAELVKPGASGKLSCTASGCNIKDTYIHWVKQRPEQGLEWIGRIDPANGNSKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCAHHDGYWGQGTLVTVSA (SEQ ID NO: 90) Light chain DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKAGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEAPYTFGGGTRLEIK (SEQ ID NO: 91)

[0131] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 90 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 91.

[0132] Other exemplary anti-tau intracellular antibodies of the present invention include intracellular antibodies derived from the monoclonal antibody hTau21 but grafted into a framework based on antibody lineages that are highly similar to the previously described scFv F8 and A48-4D5 (Donini et al., 2003; Wörn & Plückthun, 1998; Wörn & Plückthun, 1999), the resulting intracellular antibodies referred to herein as “scFv hTau21 [F8]” and “scFv hTau21 [A48-4D5]”, respectively.

[0133] The amino acid sequence of the CDR of scFv hTau21 [F8] is presented in Table 21, which also provides the SEQ ID NO for each amino acid sequence.

[0134] Table 21. Sequences of heavy chain variable CDRs and light chain variable CDRs of scFv hTau21 [F8] .

[0135] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises: heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO:69, SEQ ID NO:92, and SEQ ID NO:71, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84, respectively. In other embodiments, the anti-tau intracellular antibody comprises: heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO:79, SEQ ID NO:93, and SEQ ID NO:81, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89, respectively.

[0136] The heavy and light chains of scFv hTau21 [F8] are presented in Table 22, which also provides the SEQ ID NO for each amino acid sequence.

[0137] Table 22. Sequences of the heavy and light chain variable regions of scFv hTau21 [F8] . Variable region amino acid sequence Heavy chain QVQLVESGGGLVQPGGSLRLSCSASGCNIKDTYIHWVRQAPGKGLEWVSRIDPANGNSKYDDSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAHHDGYWGQGTLVTVSS (SEQ ID NO: 94) Light chain DIQLTQSPSSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEAPYTFGGGTKVEIK (SEQ ID NO: 95)

[0138] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 94 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 95.

[0139] The amino acid sequence of the CDR of scFv hTau21 [A48-4D5] is presented in Table 23, which also provides the SEQ ID NO for each amino acid sequence.

[0140] Table 23. Sequences of heavy chain variable CDRs and light chain variable CDRs of scFv hTau21 [A48-4D5]. .

[0141] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises: heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO:79, SEQ ID NO:96, and SEQ ID NO:81, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89, respectively.

[0142] The heavy and light chains of scFv hTau21 [A48-4D5] are presented in Table 24, which also provides the SEQ ID NO for each amino acid sequence.

[0143] Table 24. Sequences of the heavy and light chain variable regions of scFv hTau21 [A48-4D5] . Variable region amino acid sequence Heavy chain EVQLVESGGGLVQPGGSLRLSCAASGCNIKDTYIHWVRQAPGKGLEWVARIDPANGNSKYDDSVKGRFTISADTAKNSAYLQMNSLRAEDTAVYYCAHHDGYWGQGTLVTVSS (SEQ ID NO: 97) Light chain DIQLTQSPSSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEAPYTFGGGTKVEIK (SEQ ID NO: 95)

[0144] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 97 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 95.

[0145] Other exemplary anti-tau intracellular antibodies of the present invention include intracellular antibodies derived from the monoclonal antibody hTau21, transplanted into a framework based on antibody lineages highly similar to the previously described scFv F8 and A48-4D5, and made cysteine-free by replacing cysteine ​​with the amino acid combination alanine-valine. The resulting intracellular antibodies are referred to herein as “scFv hTau21-SS”, respectively. - [F8]” and “scFv hTau21-SS” - [A48-4D5]”.

[0146] scFv hTau21-SS - The amino acid sequence of the CDR of [F8] is identical to that of scFv hTau21 [F8] presented in Table 21. scFv PT51-SS - The heavy and light chain variable regions of [F8] are presented in Table 25, which also provides the SEQ ID NO for each amino acid sequence.

[0147] Table 25. scFv hTau21-SS - Sequence of the heavy and light chain variable regions of [F8] . Variable region amino acid sequence Heavy chain QVQLVESGGGLVQPGGSLRLSVSASGCNIKDTYIHWVRQAPGKGLEWVSRIDPANGNSKYDDSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYAAHHDGYWGQGTLVTVSS (SEQ ID NO: 98) Light chain DIQLTQSPSSSLSASVGDRVTITVKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAQQSNEAPYTFGGGTKVEIK (SEQ ID NO: 99)

[0148] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 98 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 99.

[0149] scFv hTau21-SS - The amino acid sequence of the CDR of [A48-4D5] is identical to that of the CDR of scFv hTau21[A48-4D5] presented in Table 23. scFv hTau21-SS - The heavy and light chain variable regions of [A48-4D5] are presented in Table 26, which also provides the SEQ ID NO for each amino acid sequence.

[0150] Table 26. scFv hTau21-SS - Sequence of the heavy and light chain variable regions of [A48-4D5] . Variable region amino acid sequence Heavy chain EVQLVESGGGLVQPGGSLRLSVAASGCNIKDTYIHWVRQAPGKGLEWVARIDPANGNSKYDDSVKGRFTISADTAKNSAYLQMNSLRAEDTAVYYAAHHDGYWGQGTLVTVSS (SEQ ID NO: 100) Light chain DIQLTQSPSSSLSASVGDRVTITVKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAQQSNEAPYTFGGGTKVEIK (SEQ ID NO: 99)

[0151] Therefore, according to some embodiments of the present invention, the anti-tau intracellular antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 100 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 99.

[0152] In some embodiments, this disclosure provides a composition comprising the anti-tau intracellular antibody of the present invention, the composition optionally further comprising one or more carriers, and optionally one or more diluents, excipients or other additives.

[0153] Polynucleotides encoding anti-Tau scFv, their preparation and expression This disclosure provides certain polynucleotides containing nucleic acid sequences encoding intracellular antibodies against tau. The polynucleotides of this invention can be in RNA or DNA form. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single-stranded, can be a coding strand or a non-coding (antisense) strand.

[0154] In some embodiments, the polynucleotide may be isolated. In some embodiments, the polynucleotide may be substantially pure. In some embodiments, the polynucleotide may be cDNA or derived from cDNA. In some embodiments, the polynucleotide may be recombinantly generated. In some embodiments, the polynucleotide may comprise a coding sequence for a mature polypeptide fused in the same reading frame to a polynucleotide that facilitates, for example, expression and optional secretion of the polypeptide from a host cell (e.g., a promoter or other regulatory sequence, a leader sequence that acts as a secretory sequence controlling the transport of the polypeptide from the cell). The polypeptide having the leader sequence is a preprotein and may have a leader sequence that can be cleaved by the host cell to form the mature form of the polypeptide.

[0155] This disclosure provides an isolated polynucleotide comprising a nucleic acid encoding an anti-tau intracellular antibody comprising an amino acid sequence from the heavy chain and / or light chain having 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity to the amino acid sequence shown herein, and / or relative to the amino acid sequence shown herein (such as 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], scFvPT77 [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]、scFvhTau21-SS - The sequence [A48-4D5] contains 1, 2, 3, 4, 5 or more amino acid substitutions, such as conserved substitutions.

[0156] Polynucleotide variants are also provided. Polynucleotide variants may contain alterations to coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions but do not change the properties or activity of the encoded polypeptide. In some embodiments, polynucleotide variants are produced through silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for various reasons, such as to optimize codon expression for a specific host (changing codons in human mRNA to codons preferred by bacterial hosts such as *E. coli*).

[0157] In some implementations, the nucleotide sequence encoding an anti-tau intracellular antibody can be constructed via chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and by selecting those codons preferred in the host cell that will produce the recombinant polypeptide of interest. Standard methods can be applied to synthesize isolated polynucleotide sequences encoding isolated polypeptides of interest. For example, the complete amino acid sequence can be used to construct genes for reverse translation. Furthermore, nucleotide oligomers containing nucleotide sequences encoding specific isolated polypeptides can be synthesized. For example, several small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then linked together. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0158] Once assembled (through synthesis, site-directed mutagenesis, or another method), the multinucleotide sequence encoding a specific polypeptide of interest can be inserted into the expression vector and operatively linked to expression control sequences suitable for protein expression in the desired host. Correct assembly can be confirmed, for example, by nucleotide sequencing, restriction mapping, and / or expression of the bioactive polypeptide in a suitable host. To achieve high expression levels of the transfected gene in the host, the gene can be operatively linked or associated with transcriptional and translational expression control sequences that function in the selected expression host.

[0159] This invention includes vectors comprising the aforementioned polynucleotides. Suitable vectors are described elsewhere herein and are known to those skilled in the art. In some embodiments, a polynucleotide comprising a nucleic acid encoding a portion of a heavy chain variable region or thereof and a polynucleotide comprising a nucleic acid encoding a portion of a light chain variable region or thereof may reside in a single vector or may reside in separate vectors. In some embodiments, a polynucleotide comprising nucleic acids encoding portions of heavy chain and light chain CDR-1, CDR-2, and CDR-3 may reside in a single vector or may reside in separate vectors. In some embodiments, a polynucleotide comprising nucleic acids encoding portions of heavy chain and light chain variable regions or thereof may reside in a single vector or may reside in separate vectors. Therefore, this disclosure provides one or more vectors comprising the aforementioned polynucleotides.

[0160] In some embodiments, the vector is a reproducible DNA construct having a synthetic or cDNA-derived DNA fragment encoding a polypeptide chain encoding an anti-tau intracellular antibody, operatively linked to a suitable transcriptional or translational regulatory element derived from a mammalian, microbial, viral, or insect gene. A transcription unit typically comprises an assembly of: (1) one or more genetic elements that play a regulatory role in gene expression, such as a transcription promoter or enhancer; (2) a structural or coding sequence transcribed into mRNA and translated into a protein; and (3) suitable transcription and translation initiation and termination sequences, as detailed below. Such regulatory elements may include operon sequences to control transcription. Additional incorporation may be made of host replication capacity typically conferred by the origin of replication, as well as selection genes that aid in the identification of transformants. DNA regions are operatively linked when they are functionally related to each other. For example, if the DNA of a signal peptide (secretion leader sequence) is expressed as a precursor involved in polypeptide secretion, then that DNA is operatively linked to the DNA of the polypeptide; if a promoter controls transcription of a coding sequence, then that promoter is operatively linked to that sequence; or if a ribosome binding site is localized to allow translation, then that ribosome binding site is operatively linked to the coding sequence. Structural elements intended for use in yeast expression systems include leader sequences that enable the host cell to secrete translated proteins extracellularly. Alternatively, when the recombinant protein is expressed without a leader or transport sequence, the protein may contain an N-terminal methionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to provide the final product.

[0161] The choice of expression control sequence and expression vector will depend on the cell seeking expression. Various expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from *E. coli*, including pADL-22c, pCR1, pBR322, pMB9, and their derivatives, broader host-scope plasmids such as M13, and filamentous single-stranded DNA phages. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an AAV vector. In a particular embodiment, the AAV vector is an AAV serotype 6 vector.

[0162] In some embodiments, this disclosure provides a composition comprising the polynucleotide or carrier as described above, and optionally further comprising one or more carriers, diluents, excipients or other additives.

[0163] The method of the present invention This invention relates to the use of intracellular anti-tau antibodies for intracellular targeting of tau. As genetically encoded proteins, intracellular antibodies can be advantageously expressed in a cell- or tissue-specific manner and can be developed to target specific conformations and post-translational modifications (Marschall et al., 2015).

[0164] To deliver anti-tau intracellular antibodies into cells, in some embodiments, the anti-tau intracellular antibodies are preferably encoded by polynucleotides of the present invention, for example, polynucleotides encoding intracellular antibodies that bind to epitopes 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 amino acid numbers refer to the amino acid sequence shown in SEQ ID NO: 1; or polynucleotides encoding intracellular antibodies 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 polynucleotides encoding intracellular antibodies comprising heavy chain variable regions and light chain variable regions as described herein; or polynucleotides encoding 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]、scFvPT77-SS - [4D5]、scFv PT77-SS - [A48-4D5], scFv hTau21, scFv hTau21 [F8], scFvhTau21 [A48-4D5], scFv hTau21-SS - [F8]、scFv hTau21-SS - [A48-4D5] intracellular antibody polynucleotide. Furthermore, the polynucleotide is preferably delivered to cells via the vector of the present invention.

[0165] Therefore, in one aspect, the present invention provides a method for binding intracellular tau in cells, the method comprising contacting the cells with a carrier of the present invention, the carrier comprising a polynucleotide encoding an intracellular antibody against tau. In another aspect, the present invention provides a method for reducing tau aggregation in cells, the method comprising contacting the cells with a carrier of the present invention, the carrier comprising a polynucleotide encoding an intracellular antibody against tau.

[0166] Examples of cells may include, but are not limited to, neurons, astrocytes, and oligodendrocytes. In a preferred embodiment, the neuron is a neuron.

[0167] This invention also relates to methods for improving the properties of intracellular anti-tau antibodies. As shown in the examples, transplanting a CDR from the scFv of interest into a framework described as stable in the cytoplasmic environment can improve the stability of intracellular antibodies against scFvs. Furthermore, designing disulfide-free scFvs can also improve intracellular properties, as the inability of disulfide bonds to form under reducing conditions is one of the main factors contributing to misfolding and aggregation of intracellular antibodies against scFvs.

[0168] Therefore, in one aspect, the present invention provides a method for increasing the binding of an intracellular anti-tau antibody to intracellular tau and / or increasing the intracellular solubility of the anti-tau intracellular antibody, wherein the anti-tau intracellular antibody comprises an scFv, the method comprising: (a) grafting a CDR of the anti-tau antibody into a scFv framework identified as intracellularly stable, and (b) removing disulfide bonds in the scFv. In another aspect, the present invention provides a method for preparing an intracellular antibody for intracellular targeting of tau, wherein the anti-tau intracellular antibody comprises an scFv, the method comprising: (a) grafting a CDR of the anti-tau antibody into a scFv framework identified as intracellularly stable, and (b) removing disulfide bonds in the scFv.

[0169] Increased binding of anti-tau intracellular antibodies to intracellular tau can be determined using methods known in the art, such as by evaluating binding affinity.

[0170] In some implementations, the ported CDR can be a CDR as described herein, such as scFv PT51, scFvPT51 [F8], scFv PT51 [A48-4D5], scFv PT51-SS - [F8]、scFv PT51-SS - [A48-4D5], scFvPT77, 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], scFvhTau21 [A48-4D5], scFv hTau21-SS - [F8]、scFv hTau21-SS -CDR of [A48-4D5].

[0171] In some implementations, the scFv framework can be identified as stable based on methods known in the art, such as evaluating the framework's melting temperature, aggregate formation, overall yield, etc. Examples of scFv frameworks include, but are not limited to, those used in intracellular antibodies described by Donini et al. (2003) or those used in intracellular antibodies described herein, namely scFvPT51, scFv PT51 [F8], scFv PT51 [A48-4D5], and 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 the scFv framework include, but are not limited to, those used in intracellular antibodies described by Donini et al. (2003), Wörn & Plückthun (1998), and Wörn & Plückthun (1999).

[0172] In some embodiments, removing the disulfide bond includes removing the cysteine ​​amino acid from the scFv. In some embodiments, removing the disulfide bond includes replacing the cysteine ​​amino acid in the scFv with a combination of alanine and valine.

[0173] In some implementations, tau is human tau.

[0174] In some embodiments, the method of the present invention is performed outside the body.

[0175] Kits containing polynucleotides, vectors, or combinations thereof This disclosure also provides a kit comprising a polynucleotide encoding the anti-tau intracellular antibody of the present invention, a vector comprising the polynucleotide encoding the anti-tau intracellular antibody of the present invention, and / or a combination thereof, which can be used to perform the methods described herein. In some embodiments, the kit comprises at least one purified polynucleotide, vector, or composition of the present invention in one or more containers. In some embodiments, the kit contains one or more components necessary and / or sufficient to perform the methods of the present invention. Those skilled in the art will readily recognize that the disclosed polynucleotide, vector, or composition can be readily incorporated into any established kit form well known in the art.

[0176] The embodiments of this disclosure can be further described and understood with reference to the following non-limiting "Examples," which describe the preparation of certain exemplary anti-tau intracellular antibodies, some exemplary characterization of such intracellular antibodies, and some exemplary methods of using such intracellular antibodies. It will be apparent to those skilled in the art that numerous modifications can be made to the specific descriptions provided in the examples without undue experimentation and without departing from the scope of this disclosure.

[0177] Example This embodiment describes the development of an intracellular anti-tau antibody for targeting tau within cells.

[0178] method scFv selection and design Monoclonal antibodies (mAbs) were selected from the previously characterized anti-tau antibody group based on epitope location, mAb and / or Fab fragment affinity, and in vitro and in vivo potency in interfering with tau aggregation.

[0179] scFvs were designed in the VH-VL orientation using DNA sequences of the variable domains of each previously identified mAb. Two variants of each scFv were prepared using two different adapters to ligate the variable domains: the GGGGSGGGGSGGGGS (SEQ ID NO: 2) adapter and the GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 3) adapter described by Bird et al. (Bird et al., 1988). These adapters are referred to herein as the GS adapter and the GSEK adapter, respectively.

[0180] scFv expression in the periplasm of Escherichia coli cDNA of each scFv with each adapter was cloned into the periplasmic expression vector pADL-22c, which included an N-terminal His6HA tag. Overexpression of all constructs was performed in MC1061F' E. coli cells (Biosearch Technologies, Novato, CA). Precultures were prepared from stock glycerol in 2YT medium (Sigma, Y1003) supplemented with 100 μg / mL carbenicillin (Thermo Fisher, 10177012) and incubated overnight at 37°C with stirring at 400 rpm. 50 μL of the preculture was then inoculated into 5 mL of 2YT medium supplemented with carbenicillin and incubated at 37°C for 3 h. Then, 1 mM IPTG (Merck, D48784) was added, and the culture was incubated overnight again, followed by harvesting the next day by centrifugation at 2200 g for 15 min. The pellet was rapidly frozen on dry ice and thawed in warm water. The precipitate was then resuspended in BugBuster HT protein extraction reagent (Merck, D49036) supplemented with 0.2 mg / ml chicken lysozyme (Sigma, L3790) and incubated with vigorous shaking for 30 minutes. Cell debris and insoluble matter were removed by centrifugation at 500g for 2 minutes, and the supernatant was collected.

[0181] scFv expression in the QBI-HEK293 cell line cDNA from all scFvs with each adapter was cloned into the internally designed mammalian expression vector pUNDER (PCT Publication No. WO 2010002785 A1) and placed 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.

[0182] Human embryonic kidney-derived QBI-HEK293A cells (QBiogene) were seeded in 12-well plates (Falcon, 353043) for secretory expression or in 6-well plates (Falcon, 353046) for cytoplasmic expression. The culture medium was 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 culture medium was replaced with antibiotic-free DMEM, and plasmid DNA was transfected using FuGENE6 (Promega, E2691) according to the manufacturer's instructions. Forty-eight hours after transfection, culture medium was collected from cells that secreted scFv, while cells expressing scFv in the cytoplasm were lysed in RIPA (Sigma, R0278) containing phosphatase and protease inhibitors (Roche, 4906837001; 11836170001).

[0183] Protein blot Dilute the sample with NuPAGE ™ LDS sample buffer and reducing agent (Thermo Scientific, NP0008, NP0009) were added and heated with shaking at 75°C for 10 minutes, then loaded onto a BisTris gel (4% to 12%) (Invitrogen WG1403BOX). After separation, the gel was imprinted onto a nitrocellulose membrane (BioRad, 1704159) using a Trans-Blot Turbo system (BioRad, Hercules, CA). The membrane was then dissolved in Tris-buffered saline + 0.1% Tween. ® 20 (TBS-T) blocked with 5% skim milk powder, and with anti-HAHRP (abcam, ab1190) (for E. coli lysates) used at a concentration of 0.5 μg / ml in TBS-T containing 5% skim milk powder, or anti-FLAG used at a 1:500 dilution. ® M2-HRP (Sigma, A8592) (for QBI-HEK293 medium and lysates) was incubated at room temperature (RT) for 1 hour. SuperSignal was used. ™ West Dura (Thermo Scientific) ™ The bands were developed using a 34076 and imaged on an Amersham Imager 600 (GE Healthcare, Chicago, IL).

[0184] Prior to Western blotting, total protein in QBI-HEK293 cell lysates was determined using a dioctoctanic acid assay (BCA) (Sigma, BCA1-1KT), and 6 μg of total protein was loaded onto a gel. Bacterial cell lysates and culture medium from QBI-HEK293 cells were loaded directly without dilution.

[0185] Enzyme-linked immunosorbent assay (ELISA) Nunc MaxiSorp ™ Flat-bottomed 96-well plates (Thermo Fisher Scientific, 430341) were coated with 50 μl of coating buffer (10 mM NaCl, 10 mM Tris-HCl, pH 8.6) containing 1 μg / ml full-length recombinant human tau (hTau), 1 μg / ml full-length recombinant human α-synuclein, or AD brain-derived tau paired helical fibers (ePHF) at a 1:500 dilution, and incubated overnight at 4°C. The next day, the plates were washed five times with 200 μl of wash buffer (0.05% Tween-20 in PBS solution) and then incubated at room temperature for 2 hours with 150 μl of blocking buffer (0.1% casein in PBS solution). After another wash, 50 μl of sample was added at serial dilution. After incubation at room temperature for 2 hours, the plates were washed, and the detection antibodies anti-HA HRP (abcam, ab1190) or anti-FLAG, diluted 1:2500 in blocking buffer, were added. ® M2-HRP (Sigma, A8592) was incubated at room temperature for 2 hours. After incubation, the plate was washed, and 50 μl of TMB (Thermo Scientific, 34029) was added to the wells. The enzymatic reaction was terminated with 50 μl of 2NH2SO4. Immediately after incubation, the plate was dried in EnVision. ® The data was read from a 2102 multi-label plate reader (PerkinElmer, Waltham, Massachusetts) and analyzed using GraphPad Prism 9 software.

[0186] To correct for concentration variations in bacterial cell lysates, band intensities on Western blots were quantified using ImageQuantTL software and converted to arbitrary units, ensuring that each scFv was tested at approximately the same concentration. For QBI-HEK293 cell lysates, samples were analyzed using serial dilutions starting at 100 μg / ml total protein. Culture medium samples were not corrected for protein content.

[0187] Nuclear translocation (NLS) determination As described above, human embryonic kidney-derived QBI-HEK293A cells (QBiogene) were cultured in 96-well plates (Greiner Bio ONE, 655090). Following the manufacturer's instructions, plasmid DNA was co-transfected with Lipofectamine 2000 (Invitrogen, 11668) 24 hours after plating. Forty-eight hours post-transfection, cells were fixed in 4% paraformaldehyde and permeabilized with TBS containing 0.3% Triton X-100. Cells were then treated with primary antibody anti-FLAG. ® scFv was detected using M2 (Sigma, F3165) and the secondary antibody goat anti-mouse IgG Alexa Fluor 555 (ThermoFisher, A-21424). Imaging was performed on an OperaPhenix instrument (PerkinElmer, Waltham, Massachusetts) equipped with a 40x water immersion objective. The acquired images were visually analyzed.

[0188] scFv sequence engineering To improve cytoplasmic solubility and stability, the scFv sequences were modified by transplanting the complementarity-determining region (CDR) into two (scFvPT51 and hTau21) or three (scFvPT77) different scFv frames. The selected frames were based on antibody lineages highly similar to previously described scFv F8, 4D5, and A48-4D5, and this nomenclature was retained for graft identification. To prevent disulfide bond formation, cysteine-free versions of the CDR-transplanted versions were also prepared by replacing cysteine ​​with the amino acid combination Ala-Val (Proba et al., 1998).

[0189] Purification of AD seeds and paired helical fibers (ePHF) from the human brain Human brain tissue from sporadic AD patients (Braak stage V / VI) with histologically confirmed rich tau pathology was provided by the Brain Bank of the Center for Neurodegenerative Diseases at the University of Pennsylvania and the Newcastle Brain Tissue Resource Center with informed consent from close relatives.

[0190] The purification of AD seeds and ePHF from brain slices was performed as described by Soares et al. (2021) and Vandermeeren et al. (2018), respectively. Both procedures were carried out in accordance with relevant ethical guidelines.

[0191] ePHF corresponds to the sarcodyl-insoluble fraction of a homogenate from an undissected human brain mass. AD seeds are purified only from ash and correspond to a purer version of sarcodyl-insoluble tau after sonication.

[0192] Generation of K18-P301L seeds Recombinant K18-P301L (corresponding to a truncated human tau protein between residues Q244 and E372 in the longest isotype) was generated in *E. coli*. Fibrouss were generated by incubating 40 μM K18-P301L protein with 40 μM 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 hour at 4 °C. The precipitate was resuspended in PBS.

[0193] Primary mouse cortical neurons and aggregation assay In accordance with relevant ethical guidelines, primary mouse cortical neurons were isolated from E19 C57Bl / 6J (Janvier) embryos. Neurons were seeded at 40,000 cells / well in 96-well plates (greiner Bio ONE, 655946) pre-coated with poly-L-lysine (Sigma Aldrich, P1274). Neurons were maintained at 37°C and 5% CO2 in Neurobasal medium (Gibco, 10888022) supplemented with B-27 (Invitrogen, 17504044) and GlutaMax (Invitrogen, 35050-038).

[0194] scFv PT51 (GS) and PT77 (SS) - The cDNA sequences of 4D5 GSEK, hTau21 (GSEK), and scFv targeting unrelated neuronal proteins were cloned into an adeno-associated virus (AAV) vector and packaged into the AAV6 serotype (SirionBiotech and Vector Builder).

[0195] Neuronal aggregation assays using human AD-tau seeds were performed as described by Soares et al. (2021). Briefly, neurons were transduced in vitro (DIV) with an adeno-associated virus (AAV)-intracellular antibody on day 7, followed by AD-tau seeding at DIV 10. Neurons were maintained until DIV 17, after which they were lysed in RIPA buffer.

[0196] Neuronal aggregation using K18-P301L seeds is based on Guo and Lee (2011) with slight modifications. In short, primary mouse neurons were transduced with AAV-intracellular antibody and AAV-hTauP301L in DIV 1, followed by the addition of sonicated K18-P301L seeds in DIV 3. Neurons were maintained until DIV 10, after which they were lysed in RIPA buffer containing phosphatase and protease inhibitors.

[0197] As described above, Western blotting was used to evaluate scFv expression levels. Samples were loaded directly into the gel undiluted. Anti-FLAG antibodies were used at a 1:500 dilution. ® M2-HRP (Sigma, A8592) was used to detect scFv. For β-actin staining, the membrane was treated with Restore. ™ PLUS Protein Blot Stripping Buffer (Thermo Scientific) ™ (46430) was stripped for 15 minutes, then blocked and incubated with a monoclonal anti-β-actin-peroxidase (Sigma, A3854) at a dilution of 1:20000.

[0198] OHSCs with K18-induced aggregation OHSCs were prepared from P301S mice on day 8 postnatal. Briefly, the pups were euthanized by decapitation. The brains from the pups were split in half, the hippocampus was isolated and cut 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 dissected. After incubating at 4°C for 1 hour, the slides were transferred to pre-prepared culture plates, with 3 slides added to each well, and immersed in warm 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 intracellular antibody was added at 2 × 10⁻⁶. 10 A concentration of VG / ml was added to each well, and the plate was incubated overnight at 37°C and 5% CO2. As a negative control, AAV expressing scFv that binds to α-synuclein was used. The next day, the medium was replaced, and the same concentration of AAV-intracellular antibody was added. The plate was returned to 37°C until DIV 4, after which the temperature was changed to 35°C for the remaining experiments. To induce tau aggregation, 333 μM K18 seeds were added to the top of each slice at DIV 14. At DIV 31, the slices were lysed in RIPA buffer with phosphatase and protease inhibitors. The medium was changed twice weekly throughout the experiments.

[0199] Meso Scale Discovery (MSD) 96-hole Multi-Array ® The plate (Meso Scale Discovery, L15XA-3) was incubated overnight at 4°C with the coated antibody diluted in PBS. After overnight incubation, the plate was washed five times with washing buffer (0.05% Tween in PBS solution) and then incubated at room temperature with blocking buffer (0.1% casein in PBS solution) at 400 rpm for 2 hours. Next, the plate was washed again, and cell lysates were added to the plate at serial dilutions in blocking buffer. The plate was then sealed and incubated overnight at 4°C. The next day, the plate was washed and incubated for 2 hours at room temperature with the corresponding detection antibody diluted in blocking buffer at 400 rpm. After incubation, the plate was washed, and 150 μl of MSD read buffer T (Meso Scale Discovery, R92TC) with surfactant diluted 2-fold in distilled water was added to each well. The plate was immediately read using an MSD SECTOR Imaging System 6000 (Meso Scale Discovery, Gaithersburg, MD).

[0200] All MSD assays used in this work were established at Janssen R&D using in-house developed antibodies. In the total mouse α-synuclein assay, two commercial reagents were used sequentially: biotinylated antibody D37A6 (CellSignaling, 74184) and sulfonated streptavidin (Meso Scale Discovery, R32AD-1).

[0201] When discussing statistical analysis, R software version 4.2.1 is used.

[0202] result The monoclonal antibody was successfully converted into scFv. Monoclonal antibodies PT51, PT77, and hTau21 (Table 27), all derived from the same immunization activity (Vandermeeren et al., 2018), were converted to scFv. All three scFvs were expressed in the periplasm of *E. coli* or secreted from HEK293 cells. Analysis of *E. coli* lysates and culture medium from HEK293 cells in Western blots did not reveal significant differences in scFv expression levels among the different adapters used. Figure 1(Figures A and F), with a few exceptions. In the *E. coli* expression system, when the variable domain was linked with the GS adapter (GGGGSGGGGSGGGGS) (SEQ ID NO: 2), scFv hTau21 was mainly detected as a single band, with only one very weak potential dimer band. When the GSEK adapter (GGSEGKSSGSGSESKSTGGS) (SEQ ID NO: 3) was used, scFv hTau21 was detected as multiple bands, with the strongest bands at approximately 28 kDa and 49 kDa, indicating that it may exist as a dimer. For the GS adapter, some potential dimer formation was observed for scFv PT51 and PT77, although the signal intensity was low.

[0203] Independent of the expression system, scFv hTau21 and PT51 retain binding with recombinant tau ( Figure 1 Figures B, C, G, and H show that the protein did not exhibit nonspecific binding to the negative control protein (data not shown). However, after periplasmic expression in E. coli, scFv PT77, derived from phosphorylated tau-specific mAb, responded to both recombinant tau and ePHF. Figure 1 (Figures D and E) A very clear signal was observed using the GSEK linker. The reaction with recombinant tau, indicating a significant loss of phosphorylation specificity, was unexpected. However, after expression and secretion from HEK293 cells, scFv PT77 bound only to ePHF, and no cross-reactivity with recombinant tau was observed. Figure 1 (Figures I and J) show that phosphorylation specificity is preserved.

[0204] Table 27. Summary of Fab (or mAb) affinity for human tau and tau-paired helical filaments (PHF) from AD brain; from Immunoprecipitation (IP) in brain homogenates from P301S mice and AD patients showed tau-like efficacy in aggregating. . Antibody Fab affinity (nM) 2N4Rtau Fab affinity (nM) PHF P301S inoculation rate after homogenization and IP (%) Inoculation rate after AD brain homogenate IP % PT51 <![CDATA[ 153 TPRGAA 15 8]]> 23.3 29.43 0.324 15.014 PT77 pS199 / pS202 / 0.054 1.079 9.822 hTau21 <![CDATA[ 375 KLTRFE 38 0]]> 82.8 (mAb) 1.640 (mAb) 5.298 82.826

[0205] scFv PT51 and hTau21 act as intracellular antibodies to retain tau binding. After being expressed as intracellular antibodies in the cytoplasm of HEK293 cells, the expression level was assessed again by Western blotting. Intracellular antibodies PT51 and hTau21 were detected in both adaptor forms, but intracellular antibody PT77 was not detected. Figure 2 (Figure A).

[0206] Evaluation of cell lysate binding by ELISA showed that intracellular antibodies hTau21 with a GSEK linker and PT51 with both linkers retained tau binding on ELISA, while intracellular antibody PT77 showed no binding. Figure 2(Figures B to E). The low expression level of the intracellular antibody PT77 may explain the lack of signal on ELISA. On the other hand, the intracellular antibody hTau21 with the GS linker expressed well but did not react on ELISA, suggesting that other factors, such as stability and folding, may also be involved.

[0207] To evaluate whether the loss of binding might be a result of misfolding and / or aggregation due to an unfavorable cytoplasmic environment, cytoplasmic solubility was assessed via confocal microscopy. Intracellular antibodies PT51 and PT77 showed punctate staining, possibly indicating aggregation. Figure 3 (Figure B). For the intracellular antibody hTau21, regardless of the linker used, diffuse and uniform distribution was observed in cells, possibly indicating soluble expression ( Figure 3 (Figure B). For the intracellular antibody PT77, the effect of the linker on solubility was observed; it appeared to be soluble when the variable domain was linked using the GSEK linker, while some dot-like structures were observed when the GS linker was used. Figure 3 (Figure B).

[0208] CDR transplantation can salvage intracellular antibody binding and cytoplasmic solubility. To overcome the low solubility and stability of intracellular antibodies in the cytoplasm, three intracellular antibody CDRs were grafted into different frames. These frames were selected based on previous reports describing evidence of their higher stability and intracellular activity (Donini et al., 2003; Wörn & Plückthun, 1998; Wörn & Plückthun, 1999). The intracellular antibodies were redesigned in the VL-VH orientation and, based on their better performance in ELISA binding, only GSEK linkers were used. Additionally, versions without disulfide bonds involving cysteine ​​residues were evaluated. Intracellular antibody PT51 became soluble in the VL-VH orientation, and the solubility of all four CDR grafts was maintained. Figure 3 (Figure B). As for the intracellular antibodies PT77 and hTau21, their solubility was maintained after switching the orientation of the variable domain and CDR transplantation. Figure 3 (Figure B). Replacing the cysteine ​​involved in the disulfide bond does not appear to affect solubility, as assessed by confocal microscopy.

[0209] Binding was evaluated by ELISA using a nuclear translocation assay based on Zhou et al. (2004). CDR transplantation did not improve ELISA binding of intracellular antibody PT51; in fact, it had a negative effect. Figure 4 (Figure A). However, in NLS assays, the three PT51 transplant versions (F8, A48-4D5, and A48-4D5 SS) showed similar results. - Clear nuclear localization was observed in the presence of tau-NLS. Figure 5 (Figure A). The opposite was observed with the intracellular antibody PT77. Almost all six CDR transplant variants rescued binding to ePHF on ELISA ( Figure 4 (Figure C), but only 4D5 SS - The graft showed some nuclear localization ( Figure 5 (Figure A). The lack of binding in the NLS assay is not due to the absence of phosphorylated tau-NLS, as its presence was confirmed by MSD assay (…). Figure 5 (Figure B). Regarding the intracellular antibody hTau21, CDR was transplanted into ELISA ( Figure 4 Binding was not improved on either the original construct (Figure B) or the NLS assay (not shown). For this intracellular antibody, only the original construct was positive for binding in both assays (Figure B). Figure 4 Figure B; Figure 5 (Figure A).

[0210] Intracellular antibody PT77 SS - 4D5 interference with AD-tau seed-induced mouse tau aggregation To evaluate whether intracellular antibodies could interfere with tau aggregation, primary mouse cortical neurons were transduced with AAV6 intracellular antibody before inducing endogenous tau aggregation by adding AD brain-derived tau seeds.

[0211] Intracellular antibody PT77 SS expressed in an MOI-dependent manner - After 4D5, a significant reduction in tau aggregation was observed. Figure 6 (Figure B). Importantly, total mouse tau and α-synuclein levels remained stable (data not shown) and were independent of the presence of AAV or AD-tau seeds, indicating that the observed reduction in tau aggregation was not a result of neuronal death. The other two intracellular antibodies, PT51 GS and hTau21 GSEK, had no effect on aggregation levels in this model. Figure 6 (See Figures A and C, respectively). Intracellular antibody PT77 SS was detected by Western blotting at least at the highest MOI. - 4D5, hTau21, GSEK, and negative control intracellular antibodies. Intracellular antibody PT51 was undetectable.

[0212] Intracellular antibodies PT51 GS, PT77 SS - 4D5 and hTau21 GSEK interference induced by K18-P301L seeds Tau gathering of mutants Three intracellular antibodies were also tested in 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 aggregation induced by K18-P301L tau seeding.

[0213] All three intracellular antibodies interfered with mutant human tau aggregation in an MOI-dependent manner. Figure 7(Figures A to C). At the highest MOI, some reduction in mutant human tau aggregation was also observed with the negative control intracellular antibody, although it was not statistically significant. This result is considered a non-specific effect of intracellular antibody overexpression, as no dose-dependent reduction was observed with this intracellular antibody. Full-length human tau and total mouse α-synuclein levels remained stable (data not shown), independent of the presence of AAV or K18 seeds, again indicating that the observed reduction in aggregation was not due to neuronal death. PT77 SS - Intracellular antibodies 4D5, hTau21GSEK, and the negative control were all detected by Western blot at at least the highest MOI, while intracellular antibody PT51 was not detected.

[0214] scFv intracellular antibody interferes with tau aggregation in K18-seeded organoid hippocampal slice cultures (OHSCs). OHSCs were prepared from transgenic mice overexpressing P301S tau, and K18 seeds were used to induce the aggregation of mutant human tau, similar to the primary mouse cortical neuron model. Intracellular antibody PT77 and the negative control were well expressed and detected by Western blotting, although some differences were observed for a few sections. Figure 8 (Figures A to D). As for the intracellular antibodies PT51 and hTau21, the expression levels were low, and only a few sections showed detectable bands on Western blotting. Figure 8 (Figure E).

[0215] Aggregated tau was evaluated using sandwich immunoassays that recognize the N- or C-terminus of human tau, while phosphorylated aggregated tau was detected using 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 using phosphorylation-independent assays, no intracellular antibody resulted in a significant reduction. Figure 8 (Figures A and B). However, when observing phosphorylated tau aggregates, all three intracellular antibodies were able to reduce AT8 phosphorylated aggregates, while for PT3 phosphorylated aggregates, only the scFv-intracellular antibody PT77 showed a significant reduction (Figures A and B). Figure 8 (Figures C and D). It should be noted that in the case of the intracellular antibody PT77, its epitope on tau (S199 / S202) partially overlaps with the epitope of the AT8 antibody used in one of the assays for measuring phosphorylated aggregated tau. mAb PT77 interferes with the binding of AT8 to tau aggregates ( Figure 8(Figure F), therefore, some interference from intracellular antibodies in the AT8 assay cannot be completely ruled out. Regarding the PT3 assay, PT77 interference appears to be negligible (Figure F). Figure 8 (Figure F). The presence of K18 seeds or AAV had no effect on total mouse α-synuclein levels (data not shown), indicating that the observed decrease in aggregated tau levels was not due to neuronal loss caused by toxicity.

[0216] discuss In this study, mAbs PT51, PT77, and hTau21 were successfully converted into scFv intracellular antibodies. scFv PT51 and hTau21 retained their binding to tau, using at least one linker, while scFv PT77 only retained its binding after the CDR was transplanted into a more stable framework.

[0217] Differences in binding were observed between scFv expressed in the periplasm of *E. coli* and scFv secreted from QBI-HEK293 cells. scFv PT77, derived from an mAb that specifically recognizes phosphorylated epitopes on tau, lost its phosphorylation specificity when expressed in *E. coli*, but retained it when secreted from HEK293 cells. Differences in affinity, specificity, and folding between scFvs produced by prokaryotes and eukaryotes (including *E. coli* and mammalian cells) have been previously reported (Lemeulle et al., 1998; Yusakul et al., 2018; Vendel et al., 2012; Lee et al., 2017). These differences may be a result of differences in eukaryotic and prokaryotic molecular chaperones and saturation of periplasmic input mechanisms, leading to poor folding, different secondary structures, and consequently altered epitope-complementary site interactions (Schlegel et al., 2013; Hermann & Riemer, 2014; Baumgarten et al., 2018). This then explains the binding differences observed in this work and underscores the importance of confirming the key characteristics of scFv derived from well-characterized monoclonal antibodies in appropriate cellular systems.

[0218] scFv PT51 and PT77 exhibited some degree of aggregation when expressed as intracellular antibodies in the cytoplasm, at least when using one of the linkers. This suggests that these two intracellular antibodies did not fold into their correct conformation, possibly due to a lack or mismatch of intradomain disulfide bonds in the reducing environment of the cytoplasm (Wörn & Plückthun, 1998; Proba et al., 1998). However, soluble cytoplasmic expression alone was insufficient to produce intracellular antibody activity, as hTau21 retained binding only when using the GSEK linker, although it is soluble in both versions. Other factors such as intrinsic sequence stability and binding affinity also need to be considered to obtain optimal intracellular activity. For example, in a recent publication, scFvs were also designed from mAbs and compared as secreted proteins with intracellular antibodies; the authors introduced specific mutations in the intracellular antibody sequences to promote intracellular stability, and all three intracellular antibodies retained binding on ELISA (Goodwin et al., 2012). Although the authors do not mention the behavior of the intracellular antibody before the sequence change, based on this work, it is reasonable to assume that the binding is either very weak or non-existent.

[0219] Although several strategies have been described to improve the selection of cytoplasmic stable constructs and engineer scFvs for cytoplasmic function, the development of intracellular antibodies remains challenging. One such approach involves designing disulfide-free scFvs, as the inability of these bonds to form under reducing conditions is one of the main factors contributing to misfolding and aggregation of intracellular antibodies from scFvs. To this end, the amino acid combination Val-Ala has been successfully used to obtain disulfide-free scFvs for cytoplasmic expression (Wörn & Plückthun, 1998; Proba et al., 1998). Additionally, transplanting CDRs from scFvs of interest into frameworks described as stable in the cytoplasmic environment has also been suggested as a potential strategy to improve the stability of scFv-intracellular antibodies (Ewert et al., 2004). However, to our knowledge, CDR transplantation has not been used to develop therapeutic scFv-intramural antibodies; its application has largely focused on cytoplasmic expression of scFv for further purification or evaluation of scFv-intramural antibody activity in *E. coli* and yeast models (Jung & Plückthun, 1997; Wörn et al., 2000; Donini et al., 2003). Combining these two strategies enabled the rescue of the solubility and binding of the intracellular antibody PT77. On the other hand, intracellular antibodies PT51 and hTau21 did not benefit from this strategy, suggesting that CDR transplantation may not be a direct, universal solution for intracellular antibody development. Furthermore, although donor framework residues identified as important for binding function are preserved during CDR transplantation, for intracellular antibodies PT51 and hTau21, other residues from their original framework appear to be important for antigen contact and / or proper loop folding.

[0220] Binding was also evaluated using nuclear translocation assays (Zhou et al., 2004). This assay is frequently used to confirm intracellular binding for 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; instead, other methods such as co-immunoprecipitation and yeast or phage display were used. In all cases, NLS assays predicted binding in other assays. In these cases, the results were not always correlated with ELISA binding. No further investigation has been conducted into why this is the case, but it is speculated that the observed differences may be related to binding kinetics, scFv folding, and differences in epitope display between assays.

[0221] To evaluate the effect of the developed intracellular antibody on tau aggregation, two primary mouse cortical neuron models were first used: one model, which induced the aggregation of endogenous mouse tau by AD brain-derived tau seeds (Soares et al., 2021); and another model, which overexpressed human tau with the P301L mutation and induced its aggregation by K18-P301L seeds (41). Additionally, the intracellular antibody was evaluated in OHSC, where the aggregation of endogenously expressed human tau with the P301S mutation was achieved by adding K18-P301L seeds (42). Of the three intracellular antibodies described in this paper, only PT177 SS… - 4D5 can interfere with AD seed-mediated mouse tau accumulation in primary neurons. Regarding K18-inoculated mutant human tau accumulation, all three intracellular antibodies scFv PT51GS, hTau21 GSEK, and PT77 SS... - All 4D5 antibodies reduced aggregation. Importantly, in the K18 seeding model, no scFv-intracellular antibody bound to the K18 seed, implying that the observed effect was against de novo aggregation. In all three models, the intracellular antibody PT77 SS... - 4D5 outperforms both PT51 GS and hTau21 GSEK. This is even more evident in OHSC, where PT77 SS... - 4D5 resulted in a greater reduction in phosphorylated tau aggregates than the other two intracellular antibodies. The parental mAb PT77 (150 kDa) competitively binds to AT8 in the immunoassay; however, this was not tested with scFv-PT77 (approximately 25 kDa). Even so, antibody fragments tend to have lower affinity than their mAb counterparts, so any interference from the presence of scFv is not expected to be at the same level as that of the mAb. Nevertheless, it cannot be completely ruled out that if the scFv-intracellular antibody binds to tau aggregates, it might prevent mAb AT8 binding and lead to an overestimation of the effect of AT8 phosphorylated tau aggregates. However, by PT3 phosphorylation assays, scFv-intracellular antibody PT77 did not interfere with PT3 binding and still confirmed that scFv-intracellular antibody PT77 strongly reduces phosphorylated tau aggregates.

[0222] The differences observed in the ability of scFv-intracellular antibodies PT51 GS and hTau21 GSEK to interfere with tau aggregation may be related to structural differences in the aggregates formed in each model. The ability of these parental mAbs of scFv to immunize-depleted tau aggregates from AD brain homogenates and P301S mouse brain homogenates has been previously evaluated. Interestingly, both mAbs were more efficient at removing inoculating aggregates from P301S brain homogenates than from AD brain homogenates (Vandermeeren et al., 2018). This is consistent with results observed with intracellular antibodies, which are more potent against mutant human tau aggregates.

[0223] The intracellular antibody studied in this paper was not coupled to the degradation determinant, and no change in total tau levels was observed in either model. Therefore, it is hypothesized that the mechanism by which the scFv-intracellular antibody interferes with aggregation is through steric hindrance preventing newly formed aggregates from recruiting monomeric tau. This could be achieved by binding oligomers and small aggregates or the monomeric tau itself. Previous reports on other “naked” anti-tau intracellular antibodies 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 decreased only when the scFv-intracellular antibody fused with a mutant form of ubiquitin, targeting it to the proteasome or lysosomal degradation pathway (Gallardo et al., 2019).

[0224] This is the first report demonstrating the effectiveness of anti-tau intracellular antibodies in an in vitro neuronal model of tau aggregation and in OHSC. Previous publications have used HEK293 cell lines overexpressing mutant tau or HEK293 biosensor models, 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 in this paper require further practical work and offer lower throughput. However, they are closer to physiological / disease conditions because tau aggregation occurs in a neuronal environment, and in the case of AD seed models, disease-related forms of tau are used as templates. Additionally, the importance of using different tau aggregation models in parallel is shown, as aggregate structures can differ between models, leading to varying efficacy results.

[0225] In tau immunotherapy, the current consensus is that targeting the central region of tau is more likely to effectively prevent extracellular tau spread and subsequent intracellular accumulation (Vandermeeren et al., 2018; Courade et al., 2018; Albert et al., 2019). However, in the case of directly targeting intracellular tau, all tau domains appear to be effective epitopes. Previous publications have reported successful results with intracellular antibodies targeting the N-terminus and microtubule-binding repeat sequences of tau. The work presented here demonstrates that targeting the PRD and C-terminus of tau with intracellular antibodies reduces the level of mutant human tau accumulation in vitro. Furthermore, wild-type mouse tau accumulation is reduced when pS199 / pS202 is targeted. A side-by-side comparison of intracellular antibodies targeting different domains is needed to further elucidate which domain is more effective in preventing and / or clearing intracellular tau aggregates.

[0226] In summary, this paper demonstrates that monoclonal antibodies can be successfully converted into scFv-intracellular antibodies, even with some sequence engineering required. CDR transplantation has been shown to be a viable method for rescuing unstable scFv-intracellular antibodies, and the need for well-thought-out scFv design and selection of appropriate cell models is highlighted.

[0227] sequence list SEQ ID NO: 1 MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGD TPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPT REPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHH KPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL 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 GYAFSTSW SEQ 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 KSLLNSDGFTY LVS SEQ ID NO: 22 LNSDGFTYLDWY SEQ ID NO: 23 LLIYLVSNRF SEQ ID NO: 24 FQTNYLPL SEQ ID NO: 25 QVQLQQSGPELVKPGASVKISCEASGYAFSTSWMNWVKQRPGKGLEWIGRIYPGDGDTNYNGKFKDKATLTADKSSSTVYMQLSSLTSEDSAVYFCTRSDWEGFAYWGQGTLVTVSA SEQ ID NO: 26 DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQTNYLPLTFGAGTKLELK SEQ ID NO: 27 RIYPGDGDTNYNDSVKG SEQ ID NO: 28 WVSRIYPGDGDTN SEQ ID NO: 29 QVQLVESGGGLVQPGGSLRLSCSASGYAFSTSWMNWVRQAPGKGLEWVSRIYPGDGDTNYNDSVKGRFTLSADKSKSTVYLQMNSLRAEDTAVYFCTRSDWEGFAYWGQGTLVTVSS SEQ ID NO: 30 DIQLTQSPSSLSASVGDRVTITCKSTKSLLNSDGFTYLDWYQQKPGQSPKLLIYLVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQTNYLPLTFGQGTKLEIK SEQ ID NO: 31 WVARIYPGDGDTN SEQ ID NO: 32 EVQLVESGGGLVQPGGSLRLSCAASGYAFSTSWMNWVRQAPGKGLEWVARIYPGDGDTNYNDSVKGRFTLSADKAKSSVYLQMNSLRAEDTAVYFCTRSDWEGFAYWGQGTLVTVSS SEQ ID NO: 33 QVQLVESGGGLVQPGGSLRLSVSASGYAFSTSWMNWVRQAPGKGLEWVSRIYPGDGDTNYNDSVKGRFTLSADKSKSTVYLQMNSLRAEDTAVYFATRSDWEGFAYWGQGTLVTVSS SEQ ID NO: 34 DIQLTQSPSSLSASVGDRVTITVKSTKSLLNSDGFTYLDWYQQKPGQSPKLLIYLVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAFQTNYLPLTFGQGTKLEIK SEQ ID NO: 35 EVQLVESGGGLVQPGGSLRLSVAASGYAFSTSWMNWVRQAPGKGLEWVARIYPGDGDTNYNDSVKGRFTLSADKAKSSVYLQMNSLRAEDTAVYFATRSDWEGFAYWGQGTLVTVSS SEQ ID NO: 36 TYAMN SEQ ID NO: 37 RIRSKSDNYATYYADSVKD SEQ ID NO: 38 QDYYV SEQ ID NO: 39 GFSFNTY SEQ ID NO: 40 RSKSDNYA SEQ 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 MQHLEYPLT SEQ ID NO: 52 KSLLHSNGKTY RMS SEQ ID NO: 54 LHSNGKTYLYWF SEQ ID NO: 55 LLIYRMSNLV SEQ ID NO: 56 MQHLEYPL SEQ ID NO: 57 EVKLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSDNYATYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVRQDYYVWGTGTSVTVSS SEQ ID NO: 58 DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGKTYLYWFLQRPGQSPQLLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPLTFGAGTKLELK SEQ ID NO: 59 RIRSKSDNYATYYADSVKG SEQ ID NO: 60 WVSRIRSKSDNYATY SEQ ID NO: 61 QVQLVESGGGLVQPGGSLRLSCSASGFSFNTYAMNWVRQAPGKGLEWVSRIRSKSDNYATYYADSVKGRFTISRDDSKSTLYLQMNSLRAEDTAVYYCVRQDYYVWGQGTTVTVSS SEQ ID NO: 62 DIQMTQSPSSLSASVGDRVTITCRSSKSLLHSNGKTYLYWFQQKPGQSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQHLEYPLTFGQGTKLEIK SEQ ID NO: 63 EVQLVESGGGLVQPGGSLRLSCAASGFSFNTYAMNWVRQAPGKGLEWVSRIRSKSDNYATYYADSVKGRFTISRDDSKSTLYLQMNSLRAEDTAVYYCVRQDYYVWGQGTTVTVSS SEQ ID NO: 64 EVQLVESGGGLVQPGGSLRLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSDNYATYYADSVKGRFTISRDDAKSSLYLQMNSLRAEDTAVYYCVRQDYYVWGQGTTVTVSS SEQ ID NO: 65 QVQLVESGGGLVQPGGSLRLSVSASGFSFNTYAMNWVRQAPGKGLEWVSRIRSKSDNYATYYADSVKGRFTISRDDSKSTLYLQMNSLRAEDTAVYYAVRQDYYVWGQGTTVTVSS SEQ ID NO: 66 DIQMTQSPSSLSASVGDRVTITVRSSKSLLHSNGKTYLYWFQQKPGQSPKLLIYRMSNLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAMQHLEYPLTFGQGTKLEIK SEQ ID NO: 67 EVQLVESGGGLVQPGGSLRLSVAASGFSFNTYAMNWVRQAPGKGLEWVSRIRSKSDNYATYYADSVKGRFTISRDDSKSTLYLQMNSLRAEDTAVYYAVRQDYYVWGQGTTVTVSS SEQ ID NO: 68 EVQLVESGGGLVQPGGSLRLSVAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSDNYATYYADSVKGRFTISRDDAKSSLYLQMNSLRAEDTAVYYAVRQDYYVWGQGTTVTVSS SEQ ID NO: 69 DTYIH SEQ ID NO: 70 RIDPANGNSKYDPKFQG SEQ 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 AHHDG SEQ ID NO: 82 KASQSVDYDGDSYMN SEQ ID NO: 83 AASNLES SEQ ID NO: 84 QQSNEAPYT SEQ ID NO: 85 QSVDYDGDSY AAS SEQ ID NO: 87 DYDGDSYMNWY SEQ ID NO: 88 LLIYAASNLE SEQ ID NO: 89 QQSNEAPY SEQ ID NO: 90 EVQLQQSGAELVKPGASGKLSCTASGCNIKDTYIHWVKQRPEQGLEWIGRIDPANGNSKYDPKFQGKATIDTSSNTAYLQLSSLTSEDTAVYCAHHDGYWGQGTLVTVSA SEQ ID NO: 91 DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKAGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEDAATYYCQQSNEAPYTFGGGTRLEIK SEQ ID NO: 92 RIDING PANGSKYDDSVKG SEQ ID NO: 93 WVSRIDPANGNSK SEQ ID NO: 94 QVQLVESGGGLVQPGGSLRLSCSASGCNIKDTYIHWVRQAPGKGLEWVSRIDPANGNSKYDDSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAHHDGYWGQGTLVTVSS SEQ ID NO: 95 DIQLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEAPYTFGGGTKVEIK SEQ ID NO: 96 WVARIDPANGNSK SEQ ID NO: 97 EVQLVESGGGLVQPGGSLRLSCAASGCNIKDTYIHWVRQAPGKGLEWVARIDPANGNSKYDDSVKGRFTISADTAKNSAYLQMNSLRAEDTAVYYCAHHDGYWGQGTLVTVSS SEQ ID NO: 98 QVQLVESGGGLVQPGGSLRLSVSASGCNIKDTYIHWVRQAPGKGLEWVSRIDPANGNSKYDDSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYAAHHDGYWGQGTLVTVSS SEQ ID NO: 99 DIQLTQSPSSLSASVGDRVTITVKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAQQSNEAPYTFGGGTKVEIK SEQ ID NO: 100 EVQLVESGGGLVQPGGSLRLSVAASGCNIKDTYIHWVRQAPGKGLEWVARIDPANGNSKYDDSVKGRFTISADTAKNSAYLQMNSLRAEDTAVYYAAHHDGYWGQGTLVTVSS SEQ ID NO: 101 DYKDDDDK References Al-Lazikani B et al. 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Claims

1. A method for binding intracellular tau in cells, the method comprising contacting the cells with a vector containing a polynucleotide encoding an intracellular antibody that specifically binds tau. The intracellular antibody binds to epitopes containing amino acid residues 151-158 of tau, amino acid residues pS199 / pS202 of tau, or amino acid residues 375-380 of tau. The amino acids are numbered according to the amino acid sequence shown in SEQ ID NO:

1.

2. A method for reducing tau accumulation in cells, the method comprising contacting the cells with a vector containing a polynucleotide encoding an intracellular antibody that specifically binds to tau. The intracellular antibody binds to epitopes containing amino acid residues 151-158 of tau, amino acid residues pS199 / pS202 of tau, or amino acid residues 375-380 of tau. The amino acids are numbered according to the amino acid sequence shown in SEQ ID NO:

1.

3. The method according to 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 the AAV serotype 6 vector.

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

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

8. The method according to any one of claims 1 to 7, wherein the intracellular antibody 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 comprise the following: (a) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; or (b) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 6, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; or (c) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 20, LVS, and 19; or (d) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 6, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; or (e) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or (f) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 27, and SEQ ID NO: 6, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; or (g) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 28, and SEQ ID NO: 16, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or (h) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 31, and SEQ ID NO: 16, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or (i) heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or (j) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 38; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or (k) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 52, RMS, and 51, respectively; or (l) heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 38, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively; or (m) heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; or (n) heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 59, and SEQ ID NO: 38; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or (o) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 46, SEQ ID NO: 60, and SEQ ID NO: 48; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; or (p) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; or (q) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 71; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; or (r) heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences of SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences of SEQ ID NO: 85, AAS, and 84; or (s) heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 77, SEQ ID NO: 78, and SEQ ID NO: 71, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively; or (t) heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89; or (u) heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 92, and SEQ ID NO: 71; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; or (v) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 93, and SEQ ID NO: 81, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively; or (w) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 96, and SEQ ID NO: 81; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO:

89.

10. The method according to claim 8 or 9, wherein: (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 (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 contains the amino acid sequence of SEQ ID NO: 100, and the light chain variable region contains the amino acid sequence of SEQ ID NO:

99.

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

3.

12. The method according to any one of claims 1 to 11, wherein the cells are selected from neurons, astrocytes, and oligodendrocytes.

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

14. A method for preparing an intracellular anti-tau antibody for intracellular targeting of tau, wherein the anti-tau intracellular antibody comprises a single-chain variable fragment, the method comprising: (a) Transplantation of the complementarity-determining region (CDR) of anti-tau antibody into a scFv framework identified as intracellularly stable, and (b) Remove the disulfide bonds in the scFv. The anti-tau antibody binds to epitopes containing amino acid residues 151-158 of tau, amino acid residues pS199 / pS202 of tau, or amino acid residues 375-380 of tau. The amino acids are numbered according to the amino acid sequence shown in SEQ ID NO:

1.

15. The method of claim 14, wherein the CDR comprises heavy chain variable CDR-1, CDR-2 and CDR-3 and light chain variable CDR-1, CDR-2 and CDR-3, wherein: (a) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively comprise the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively comprise the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; or (b) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively comprise the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 6; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively comprise the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; or (c) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 20, LVS, and 19; or (d) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 6; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; or (e) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively comprise the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively comprise the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24; or (f) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 27, and SEQ ID NO: 6; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; or (g) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively comprise the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 28, and SEQ ID NO: 16; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively comprise the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24; or (h) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively comprise the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 31, and SEQ ID NO: 16; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively comprise the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24; or (i) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or (j) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 38; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or (k) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 52, RMS, and 51; or (l) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 38; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or (m) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; or (n) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 59, and SEQ ID NO: 38; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or (o) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 46, SEQ ID NO: 60, and SEQ ID NO: 48; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; or (p) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; or (q) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 71; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; or (r) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 85, AAS, and 84; or (s) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 77, SEQ ID NO: 78, and SEQ ID NO: 71; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; or (t) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89; or (u) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 92, and SEQ ID NO: 71; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; or (v) The heavy chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 93, and SEQ ID NO: 81; and the light chain variable CDR-1, CDR-2, and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89; or (w) The heavy chain variable CDR-1, CDR-2 and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 96 and SEQ ID NO: 81; the light chain variable CDR-1, CDR-2 and CDR-3 respectively contain the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO:

89.

16. The method according to any one of claims 14 to 15, wherein removing the disulfide bond in (b) comprises removing the cysteine ​​amino acid from the scFv.

17. The method of claim 16, wherein removing the disulfide bond in (b) further comprises replacing the cysteine ​​amino acid with a combination of alanine and valine.

18. An intracellular antibody against tau that specifically binds to tau, wherein the intracellular antibody 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; The heavy chain variable region and the light chain variable region include the following: (a) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; or (b) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 6, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; or (c) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 20, LVS, and 19; or (d) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 6, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; or (e) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or (f) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 27, and SEQ ID NO: 6, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; or (g) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 28, and SEQ ID NO: 16, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or (h) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 31, and SEQ ID NO: 16, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or (i) heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or (j) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 38; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or (k) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 52, RMS, and 51, respectively; or (l) heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 38, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively; or (m) heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; or (n) heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 59, and SEQ ID NO: 38; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or (o) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 46, SEQ ID NO: 60, and SEQ ID NO: 48; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; or (p) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; or (q) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 71; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; or (r) heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences of SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences of SEQ ID NO: 85, AAS, and 84; or (s) heavy chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 77, SEQ ID NO: 78, and SEQ ID NO: 71, respectively; and light chain variable CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively; or (t) heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89; or (u) heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 92, and SEQ ID NO: 71; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, comprising the amino acid sequences of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84; or (v) Heavy chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 93, and SEQ ID NO: 81, respectively; and light chain variants CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively; or (w) Heavy chain variants CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences of SEQ ID NO: 79, SEQ ID NO: 96, and SEQ ID NO: 81; and light chain variants CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO:

89.

19. The anti-tau intracellular antibody according to claim 18, wherein: (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 (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 contains the amino acid sequence of SEQ ID NO: 100, and the light chain variable region contains the amino acid sequence of SEQ ID NO:

99.

20. The anti-tau intracellular antibody according to 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 an anti-tau intracellular antibody according to any one of claims 18 to 20.

22. A vector comprising the polynucleotide according to claim 21.

23. The vector according to 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) an anti-tau intracellular antibody according to any one of claims 18 to 20; or (b) the polynucleotide according to claim 21; or (c) The carrier according to any one of claims 22 to 24; and carrier.

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