Anti-acetylated TAU antibodies and uses thereof

Abstract

The present disclosure provides anti-ac-tauK174 antibodies, or antigen-binding fragments thereof. In addition, methods of using the anti-ac-tauK174 antibodies, or antigen-binding fragments thereof, for the treatment of the progression of and / or prevention of Alzheimer's Disease and related tauopathies, including traumatic brain injury, are also provided. Methods of reducing clinical decline in a subject having early Alzheimer's Disease, reducing brain amyloid levels in a subject, and methods of preventing Alzheimer's Disease and related tauopathies are also provided, the methods comprising administering a composition comprising a therapeutically effective amount of at least one anti-ac-tauK174 antibody. In some embodiments, the subject is ApoE4-positive.

Description

Attorney Docket No. AETN0001-401-PCANTI-ACETYLATED TAU ANTIBODIES AND USES THEREOF

[0001] This application claims the benefit of priority of U.S. Patent Application No. 63 / 734,635, filed December 16, 2024, and U.S. Patent Application No. 63 / 837,541, filed July 2, 2025, the contents of which are incorporated by reference as if written herein in their entirety.

[0002] This application is being filed with a Sequence Listing in XML format, which is 89000 bytes, generated on December 16, 2025. The Sequence Listing XML file, submitted via the USPTO’s Patent Center electronic filing system on December 16, 2025, is incorporated by reference herein in its entirety.

[0003] The present disclosure relates to antibodies that selectively bind acetylated tau protein, the microtubule-associated protein tau (“MAPT”), and their use in treating tauopathies. Tauopathies are a group of neurodegenerative diseases characterized by brain deposition of neurofibrillary tangles (NFTs) of tau protein.

[0004] Tau is a soluble protein of the central nervous system formed by 352-441 amino acids and encoded by the MAPT gene on chromosome 17 which generates 6 isoforms (Andreadis et al., 1992). Tau is located in axons, dendrites, nuclei, cell membranes, and synapses of neurons (Imbimbo et al., 2023). The protein is also expressed to a lesser extent in astrocytes and oligodendrocytes, although its role in these cells has been little investigated. Id. Tau protein promotes tubulin assembly, microtubule stability, and cytoskeletal integrity (Asuni et al., 2007). The protein is also present in the interstitial fluid and can cross into the cerebrospinal fluid (CSF) and reach the systemic circulation. One function of tau is promoting the assembly and stabilization of microtubules in neuronal axons. Tau plays also a role in a range of other biological processes including myelination, neurogenesis, motor function, learning, and memory (Kent et al., 2020).

[0005] Acetylated tau is a toxic form of tau, which is elevated in tauopathy brains, and has been shown to play a role in pathogenesis and disease progression. The neuropathological hallmarks of tau-mediated neurodegeneration, including Alzheimer’s Disease (AD), consist of extracellular amyloid-beta (AP) plaques and intraneuronal aggregates of tau protein associated with neuritic plaques (NPs), neuropil threads (NTs), and neurofibrillary tangles (NFTs) (Goedert et al., 1988). Acetylated tau can adopt a misfolded beta-sheet conformation that aggregates into fibrils with a filament core that assemble into NFTs (Cleveland et al., 1977; Crowther et al., 1989; Fitzpatrick et al., 2017).Attorney Docket No. AETN0001-401-PC

[0006] Mutation of the tau (MAPT) gene results in Frontotemporal Dementia with Parkinsonism linked to chromosome 17 (FTDP-17) (Strang et al., 2019). Therefore, tau plays a central role in the neurodegenerative disease process and presents an attractive target for therapeutic intervention in AD and related tauopathies (Hutton et al., 1998). The objective of immunotherapy for tau pathology is that anti-tau antibodies can clear tau aggregates that may affect neuronal viability. In preclinical studies, inhibition of tau acetylation with a mouse monoclonal antibody protected against tau-mediated neurodegeneration and behavioral deficits.

[0007] Although anti-tau antibodies have been tested in different animal models of disease, human clinical trials have shown poor outcomes (Asuni et al., 2007; Gibbons et al., 2020; Imbimbo et al., 2023). These studies have targeted phosphorylated-tau or conformation-specific tau; however, while tau is to some extent normally acetylated, it becomes more extensively acetylated under pathological conditions (Cohen et al., 2011; Min et al., 2015; Min et al., 2010; Morris et al., 2015). Antibodies that target other disease- associated modifications of tau includes ac-tauK280 (Song et al., 2023), which targets tau when acetylated at lysine 280).

[0008] With respect to AD, current therapeutic agents include acetylcholinesterase inhibitors (AChEls), such as donepezil, and N-methyl-D-aspartate (NMD A) receptor antagonists, such as memantine. While such agents may improve the symptoms of AD, they have not been reported to alter the progression of the disease. With no cures available, tauopathies are considered too complex for a one-size-fits-all solution. Targeting acetylated lysine at position 174 in human tau (ac-tauK174), targeting acetylated lysine at position 274 in human tau (ac-tauK274), and / or targeting acetylated lysine at position 353 in human tau (ac-tauK353), all of which are elevated in the brains of tauopathy patients, may treat and / or protect against tau-mediated neurodegeneration and behavioral deficits. Thus, there is an unmet need for compositions and methods of treating the progression of and / or preventing AD and related tauopathies.

[0009] Antibodies disclosed herein target acetylated forms of tau such as ac-tauK174, ac- tauK274, and / or ac-tauK353 and have utility as therapeutics for tauopathy alone and in combination with other therapeutics for brain injury. The present antibodies bind human ac- tauK174, ac-tauK274, and / or ac-tauK353 with high affinity and specificity and / or reduce pathogenic tau spread and phosphorylated-tau (p-tau) accumulation. Treatment with anAttorney Docket No. AETN0001-401-PC antibody against acetylated tau mitigates neurob ehavi oral impairment and reduces tau pathology, including neuronal loss and brain atrophy.

[0010] Antibodies disclosed herein exhibit dose-dependent improvement in cognitive function, trend toward the rescue of hippocampal volume loss, and mitigate tau pathology in a model of tauopathy.BRIEF DESCRIPTION OF DRAWINGS

[0011] FIG. 1 A shows the sequence of the antigen peptide (human tau amino acids 163— 185) and workflow used to generate monoclonal antibodies against acetylated tau (ac-K174), prior to undertaking the characterization of new anti-acetylated tau (ac-tauK174) antibodies.

[0012] FIG. IB shows an immunoblot of HEK293T cells transfected with p300 and WT human tau (hTau), K174R, or K274R mutant. The ac-tauK174 signal is readily detected where WT hTau is co-transfected with p300, while K174R, but not K274R mutation blocks the AbM5 signal.

[0013] FIG. 1C shows an immunoblot of HEK293T cells transfected with p300 and WT human tau (hTau), K174R, or K274R mutant. The ac-tauK174 signal is readily detected where WT hTau is co-transfected with p300, while K174R, but not K274R mutation blocks the AbMl signal.

[0014] FIG. ID is an immunoblot of hippocampal lysate from age-ranged WT, PS 19, and Tau KO mice showing that AbM5 detected ac-tauK174 immunoreactivities. Human tau (hTau, arrows) migrates at a higher molecular mass than murine tau (~50 kDa). The asterisk indicates a non-specific band in AbM5.

[0015] FIG. IE is an immunoblot of hippocampal lysate from age-ranged WT, PS 19, and Tau KO mice showing that AbMl detected ac-tauK174 immunoreactivities. Human tau (hTau, arrows) migrates at a higher molecular mass than murine tau (~50 kDa). The asterisk indicates a non-specific band in AbMl.

[0016] FIG. IF shows the specific binding affinity of AbM5 for ac-tauK174.

[0017] FIG. 1G shows the specific binding affinity of AbMl for ac-tauK174.

[0018] FIG. 1H shows the dissociation constant between clones and ligand as measured by Surface Plasmon Resonance (SPR) binding using the ac-tauK175 peptide as the ligand, where Kon is the association rate; Kq / / is the dissociation rate; and KD is the dissociation constant.

[0019] FIG. 2A shows an experimental timeline for determining whether AbM5 of anti- ac-tauK174 antibody rescues neurob ehavi oral impairment in P301S (PS19) mice. As shownAttorney Docket No. AETN0001-401-PC in the schematic, at 6 months of age, male and female P301S mice were peripherally (IP) injected with AbM5 (25 mg / kg) or vehicle (PBS). Non-transgenic (WT) littermates were injected with PBS and are included as controls. All mice received a single injection weekly for a period of 15 weeks. Behavioral analysis was performed during the last 4 weeks of treatment. Mice were sacrificed at 10 mo following the last injection.

[0020] FIG. 2B shows the percentage of weight loss at the endpoint (week 15) among three groups of animals, normalized to the start point (week 1) where ***p < 0.001, *p < 0.05 by one-way ANOVA, Sidak’s multiple comparison test.

[0021] FIG. 2C shows that motor coordination impairment was measured using a hindlimb extension test. Each mouse was suspended by its tail for 10 s and its hind-limb posture was scored as 1, 0.5, or 0. The average score for each group is presented where *** / ?<0.001, **p < 0.01 by one-way ANOVA, Sidak’s multiple comparison test.

[0022] FIG. 2D shows a plot of spatial learning and memory as measured by Morris water maze (MWM).

[0023] FIG. 2E shows a learning curve during the training phase where ***p < 0.001, *p < 0.05 by two-way ANOVA, Tukey’s multiple comparison test.

[0024] FIG. 2F shows a graph of a probe trial 72 hr post-training performed on Day 8 where **p < 0.001, *p < 0.05 by paired t-test. n = 14 per group.

[0025] FIG. 3A shows that an anti-ac-tauK174 antibody ameliorates tau pathology and neurodegeneration in PS 19 mice, with representative Nissl-staining showing hippocampal morphology of WT mice treated with PBS (left), PS19 mice treated with PBS (middle), and PS 19 mice treated with AbM5 antibody (right) where the scale bar is 500 pm.

[0026] FIG. 3B is a bar graph showing the quantification of hippocampal volume by oneway ANOVA, Kruskal-Wallis test where **p<0.01.

[0027] FIG. 3C shows a Pearson correlation analysis of Morris water maze performance score (rank summary latency during learning) (y-axis) versus hippocampal volume (x-axis).

[0028] FIG. 3D is a representative immunohistochemistry staining of p-tau (AT8) in the hippocampi. Scale bar: 250 pm.

[0029] FIG. 3E shows the quantification of hippocampal AT8-positive area by one-way ANOVA, Kruskal -Wallis test where ***p<0.001.

[0030] FIG. 3F is a schematic diagram showing that the injection of tau fibrils to the hippocampus of PS 19 mice induces spreading of tau pathology from the ipsilateral (seeding) side to the contralateral (spreading) side of the brain.Attorney Docket No. AETN0001-401-PC

[0031] FIG. 3G is a representative immunostaining showing MCI-positive tau inclusions in the seeding side and the spreading side after treatment with IgG2a or anti-ac-tauK174 AbMl antibody. Scale bar: 100 pm. The images show that treatment with AbMl reduces the spread of tau pathology in neurons.

[0032] FIG. 3H shows the quantification of relative MCI-positive signal in IgG2a and AbMl -treated groups, normalizing the spreading side to the seeding side where n = 5 mice for both groups and ***p<0.001, STATA mixed model.

[0033] FIG. 4 A shows a schematic for investigating whether anti-ac-tauK174 immunotherapy could prevent trauma-induced neuropathological changes, transcriptomic alterations, and deficits in memory function. Specifically, 8-month PS 19 mice were exposed to sham or traumatic brain injury (closed head concussive) surgery. One day prior to trauma surgery animals received PBS or AbM5 administered intraperitoneally (IP). Treatment continued weekly throughout the duration of experimentation until termination 5 weeks postinjury.

[0034] FIG. 4B is a schematic diagram showing the injury site and areas of pathology analysis.

[0035] FIG. 4C is a graph showing the percentage of weight loss at Day 28 among three groups of PS 19 animals showing a trend of reduction in mice with TBI as compared to less weight loss in mice treated with antibody.

[0036] FIG. 4D is a representative immunohistochemistry staining of AT8-positive p- tau showing neuropathological changes among three sections adjacent to the injury site in animals, where PS 19 mice subjected to TBI showed increased AT8-positive phosphorylated- tau (p-tau) deposition in the cortex compared to a sham group and a third group treated with anti-ac-tauK174 AbM5 antibody, where AT8 is a phospho-tau antibody used to detect phosphorylated tau.

[0037] FIG. 4E is a bar graph showing the quantification of AT8 positive % area in the upper cortex among PS 19 mice with sham surgery or TBI, treated with PBS or AbM5 antibody.

[0038] FIG. 4F is a bar graph showing the quantification of AT8 positive % area in the lower cortex among PS 19 mice with sham surgery or TBI, treated with PBS or AbM5 antibody.

[0039] FIG. 4G is a representative immunohistochemistry staining of lb a- 1 -positive cells among three sections adjacent to the injury site in animals, showing that TBI mice gaveAttorney Docket No. AETN0001-401-PC increased Iba-1 signal in the cortex compared to a sham group and a third group treated with anti-ac-tauK174 AbM5 antibody, where the Iba-1 antibody is used to identify microglia.

[0040] FIG. 4H is a bar graph showing the quantification of Iba-1 positive % area in the upper cortex among PS 19 mice with sham surgery or TBI, treated with PBS or AbM5 antibody.

[0041] FIG. 41 is a bar graph showing the quantification of Iba-1 positive % area in the lower cortex among PS 19 mice with sham surgery or TBI, treated with PBS or AbM5 antibody.

[0042] FIG. 4J shoes the Pearson correlation analysis of AT8-positive p-tau levels (by western blot) and Iba-1 signal in the lower cortex, n = 12 per group.

[0043] Overall, FIG. 4 A through FIG. 4 J show that anti-ac-tauK174 treatment ameliorates behavioral impairments and neuropathology in P301S mice with TBI.

[0044] FIG. 5 A shows a bar graph of cell ratio per condition within each microglia (ML) cluster where **** / ? < 0.0001, *** / ? < 0.001, ** / ? < 0.01, and * / ? < 0.05 by one-way ANOVA with Tukey’s multiple comparisons correction within each subcluster. Microglial impairment in TBI is rescued by anti-ac-tauK174 immunotherapy (AbM5).

[0045] FIG. 5B shows a bar graph of cell ratio per condition within each oligodendrocyte (OL) cluster where ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 by one-way ANOVA with Tukey’s multiple comparisons correction within each subcluster.Oligodendrocyte myelination impairment in TBI is rescued by anti-ac-tauK174 immunotherapy (AbM5).

[0046] FIG. 6A is a schematic illustrating that trauma-induced memory deficits were measured by a novel object recognition (NOR) test. Three weeks post-injury mice were exposed to two identical objects, five minutes later one of the objects was replaced with a new object

[0047] FIG. 6B is a graph showing that memory deficits were calculated by decrease in time spent with the novel object graphed as a discrimination index where *p< 0.05 by oneway ANOVA, Sidak’s multiple comparison test. n=9 (Sham-PBS), 8 (TBI-PBS), 12 (TBI-Ab AbM5).

[0048] FIG. 6C is a plot showing a Pearson correlation analysis of NOR discrimination index (DI) and AT8-positive p-tau levels (by western blot) where n=12 per group.

[0049] FIG. 6D is a plot showing a Pearson correlation analysis of NOR discrimination index (DI) and Iba-1 signal (by western blot) where n=12 per group.Attorney Docket No. AETN0001-401-PC

[0050] FIG. 6E is a schematic illustrating that tau repeat domain (RD) P301S FRET Biosensor cells were treated with brain lysates and liposomes, incubated for 72 hours, and imaged to assess tau seeding activity by measuring % FRET+ cells.

[0051] FIG. 6F is a representative immunocytochemistry staining and quantification of the percentage of CFP / YFP FRET cells after incubation with PS 19 mouse lysate where n=4.

[0052] FIG. 6G is a graph showing a significant increase in CFP / YFP FRET signal with TBI and a downward trend with antibody treatment.

[0053] FIG. 6H is a representative immunoblot of acetylated tau (K174) (AbMl) in control (Normal) and TBI human plasma samples.

[0054] FIG. 61 shows the quantification of plasma ac-tau (KI 74) levels normalized by total protein levels. The mean level of ac-tau (K174) was significantly higher in the TBI cohort at 24 h in comparison to the controls (1.57 ± 0.48 versus 1.13 ± 0.27,***p< 0.001) where n=24 (normal), 44 (TBI). Overall, FIG. 6A through FIG. 61 shows that anti-ac- tauK174 treatment ameliorates behavioral impairments and reduces tau seeding in vitro.

[0055] FIG. 7 shows a plot of the affinity measurement of murine-human chimeric antibodies by direct binding ELISA. Biotinylated ac-tauK174 peptides were used to capture and an anti-human IgG antibody was used for detection. Concentration responsive curves of AbMl and AbMl and EC50 for each antibody were determined.

[0056] FIG. 8A shows direct binding using ELISA of the chimeric parental and first four (1-4) humanized variants against Tau peptide.

[0057] FIG. 8B shows direct binding using ELISA of the humanized variants five (5) through nine (9) against Tau peptide. Human IgG2 Fc antibody was used as negative control. Similar affinity was detected among all 9 clones.

[0058] FIG. 9A shows Western Blots that demonstrate that recombinant human wild-type tau / K174R tau was expressed in HEK293T cells together with acetyltransferase P300 to generate Ac-Tau / Ac-K174R hTau. A Western Blot shows the affinity of the 9 humanized cones to full-length ac-tauK174.

[0059] FIG. 9B shows that when lysine 174 is mutated to arginine (K174R), AT-01 (VH2+VL2) lost its binding capacity to acetylated hTau, indicating the epitope of AT-01 is acetylated lysine 174, which is the same as the AbMl clone. The presence of other acetylated tau species was confirmed by ac-K274 tau antibody AbM3. Total tau was detected by HT7 or Dako Tau antibody.

[0060] FIG. 10 shows that the leading humanized candidate AT-01 immuno-stained acetylated tau in PS 19 mice and human AD brains. Mouse monoclonal AbMl and humanizedAttorney Docket No. AETN0001-401-PC clone AT 01 recognized acetylated tau inclusions in PS 19 mouse and AD patient brain, but not in non-transgenic control mouse and healthy human. Absence of primary antibodies were used as negative control.

[0061] FIG. 11 is an immunoblot showing differential binding between AbMl and AbM5 with ac-K174-Tau from HEK lysates.

[0062] FIG. 12 provides an immunoblot (left) showing relative binding between AbM3 (anti-ac-K174-tau) and peptides ac-K174-tau and ac-K274-tau from PS19 lysates. When lysine 174 and 274 are mutated to arginine (lanes 3 and 4), respectively, reduced binding is visualized in lane 3 only, indicating that AbM3 is specific for anti-ac-K174-tau.Immunohistochemistry staining of AbM3 in PS 19 brain tissue is also shown (right).

[0063] FIG. 13 provides an immunoblot (left) showing relative binding between AbM2 (anti-ac-K174-tau) and peptides ac- ac-K174-tau and ac-K274-tau from PS19 lysates. When lysine 174 and 274 are mutated to arginine (lanes 3 and 4), respectively, reduced binding is visualized in lane 3 only, indicating that AbM2 is specific for anti-ac-K174-tau.Immunohistochemistry staining of AbM2 in PS 19 brain tissue is also shown (right).

[0064] FIG. 14 provides immunoblots (left and center) showing relative binding between AbM4 (anti-ac-K274-tau) and peptides ac-K174-tau and ac-K274-tau from PS19 lysates. When lysine 174 and 274 are mutated to arginine (lanes 3 and 4), respectively, reduced binding is seen in lane 4, indicating that AbM4 is specific for anti-ac-K274-tau.Immunohistochemistry staining of AbM4 in PS 19 brain tissue is also shown (right).

[0065] FIG. 15 shows staining of AbMl (anti-ac-K174-tau) in brain tissue from PS 19 mice (left) versus non-transgenic mice (right).

[0066] FIG. 16 is a Western Blot showing an increase of ac-K353-tau in lysates derived from two human brains afflicted with Alzheimer’s disease. Four different lysates were prepared for each of the two human brain regions, BM-9 and BM-22. For BM-9, 8834, 8900, 8910, 8912 are shown, and for BM-22, 8854, 8862, 8883 and 8887 are shown. Murine antibody AbM6 raised against ac-K353-tau specifically binds human acetylated tau (ac- K353-tau), which is thus elevated in brain tissue from Alzheimer’s patients.DETAILED DESCRIPTION

[0067] The following disclosure is intended to illustrate various aspects of the claims.

[0068] Disclosed herein is an anti -acetyl ated-tau antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variableAttorney Docket No. AETN0001-401-PC region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3 polypeptides and the VL comprises LCDR1, LCDR2, and LCDR3 polypeptides which are chosen from: a) HCDR1 is GYTFTDYY (SEQ ID NO:38), HCDR2 is IFPGSDLI (SEQ ID NO:39), HCDR3 is ARGVYYDGGNFFDY (SEQ ID NO:40), LCDR1 is QDISSY (SEQ ID NO:41), LCDR2 is YTS (SEQ ID NO:42), and LCDR3 is QQYSKRPWT (SEQ ID NO:43); b) HCDR1 is GFNIKNTY (SEQ ID NO:44), HCDR2 is IDPSNGNT (SEQ ID NO:45), HCDR3 is ADD YAW (SEQ ID NO:46), LCDR1 is QSLLYTNGKTY (SEQ ID NO:47), LCDR2 is LSV (SEQ ID NO:48), and LCDR3 is LQSTHFPRT (SEQ ID NO:49); c) HCDR1 is GFTFGDYW (SEQ ID NO:50), HCDR2 is IKLKSDNYAT (SEQ ID NO:51), HCDR3 is TTIYYYDSRRFDY (SEQ ID NO:52), LCDR1 is QSVSYD (SEQ ID NO:53), LCDR2 is HAS (SEQ ID NO:54), and LCDR3 is HQEYSSPYT (SEQ ID NO: 55); or d) HCDR1 is GFTFTIYG (SEQ ID NO:62), HCDR2 is IDENGGA (SEQ ID NO:63), HCDR3 is ARDDGT (SEQ ID NO:64), LCDR1 is QSLFYSDGQTY (SEQ ID NO:65), LCDR2 is LSV (SEQ ID NO:66), and LCDR3 is VQGTHFPLT (SEQ ID NO: 67).

[0069] In some embodiments, the anti-acetylated-tau antibody, or an antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and the VL are polypeptides chosen from: a) VH of SEQ ID NO: 10 and VL of SEQ ID NO: 11; b) VH of SEQ ID NO: 12 and VL of SEQ ID NO: 13; c) VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15; d) VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17; e) VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; f) VH of SEQ ID NO:20 and VL of SEQ ID NO:21; g) VH of SEQ ID NO:22 and VL of SEQ ID NO:23; h) VH of SEQ ID NO:24 and VL of SEQ ID NO:25; i) VH of SEQ ID NO:26 and VL of SEQ ID NO:27; j) VH of SEQ ID NO:28 and VL of SEQ ID NO:29; k) VH of SEQ ID NO:30 and VL of SEQ ID NO:31; l) VH of SEQ ID NO:32 and VL of SEQ ID NO:33; m) VH of SEQ ID NO: 34 and VL of SEQ ID NO:35; orAttorney Docket No. AETN0001-401-PC n) VH of SEQ ID NO:36 and VL of SEQ ID NO:37.

[0070] In some embodiments, the anti-acetylated-tau antibody, or an antigen-binding fragment thereof, comprises a heavy chain (HC) and a light chain (LC), wherein the HC and LC are polypeptides chosen from: a) HC of SEQ ID NO: 74 and LC of SEQ ID NO: 75; and b) HC of SEQ ID NO:78 and LC of SEQ ID NO:79.

[0071] In some embodiments, the anti-acetylated-tau antibody, or an antigen-binding fragment thereof, comprises two light chains and two heavy chains wherein each light chain and each heavy chain are polypeptides chosen from: a) HC of SEQ ID NO: 74 and LC of SEQ ID NO: 75; and b) HC of SEQ ID NO:78 and LC of SEQ ID NO:79.

[0072] In some embodiments, the anti-acetylated-tau antibody, or an antigen-binding fragment thereof, is a monoclonal antibody. In some embodiments, the anti-acetylated-tau antibody, or an antigen-binding fragment thereof, has heavy and light chain constant regions of the IgGl, IgG2, or IgG4 subclasses. In some embodiments, the anti-acetylated-tau antibody, or an antigen-binding fragment thereof, is of the IgGl subclass. In some embodiments, the anti-acetylated-tau antibody, or an antigen-binding fragment thereof, has a heavy chain constant domain represented by the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the anti-acetylated-tau antibody, or an antigen-binding fragment thereof, has a mutated heavy chain constant domain as compared to the wildtype. In some embodiments, the anti-acetylated-tau antibody, or an antigen-binding fragment thereof, has a heavy chain represented by the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the antibody, or an antigen-binding fragment thereof, is a humanized or chimeric antibody. In some embodiments, the anti-acetylated-tau antibody, or an antigenbinding fragment thereof, has a light chain constant domain of the lambda (X) subtype.

[0073] The anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of any one of claims 1 to 11 which has a light chain constant domain of the kappa (K) subtype. In some embodiments, the anti-acetylated-tau antibody, or an antigen-binding fragment thereof, has a light chain constant domain represented by the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the antibody, or an antigen-binding fragment thereof, specifically binds acetylated lysine 174 on human tau (ac-tauK174). In some embodiments, the antibody, or an antigen-binding fragment thereof, is capable of binding with a KD of at least about IxlO'11M, at least about 1X10'12M, or at least about 2.5xlO'12M. In someAttorney Docket No. AETN0001-401-PC embodiments, the antibody, or an antigen-binding fragment thereof, specifically binds acetylated lysine 274 on human tau (ac-tauK274).

[0074] Also provided herein is a nucleic acid encoding an antibody, or an antigen-binding fragment thereof, as disclosed herein. Also provided is a nucleic acid encoding a heavy chain of an antibody, or an antigen-binding fragment thereof, where the heavy chain is represented by the amino acid sequence set forth in SEQ ID NO:78. In some embodiments, the nucleic acid is represented by the sequence set forth in SEQ ID NO:80. Also provided is a nucleic acid encoding a light chain of an antibody, where the light chain is represented by the amino acid sequence set forth in SEQ ID NO:79. In some embodiments, the nucleic acid is represented by the sequence set forth in SEQ ID NO:81.

[0075] Also provided herein is a vector comprising a nucleic acid as disclosed herein.

[0076] Also provided herein is a host cell comprising the vector as disclosed herein.

[0077] Also provided herein is a pharmaceutical composition comprising the antiacetyl ated-tau antibody, or an antigen-binding fragment thereof, as disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0078] Also provided herein is a method of treating a condition chosen from clinical or pre-clinical Alzheimer’s Disease, prodromal Alzheimer’s Disease, Down Syndrome, clinical or pre-clinical amyloid angiopathy (CAA), frontotemporal dementia (FTD) with TAR DNA- binding protein 43 (TDP-43), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick’s disease (PiD), argyrophilic grain disease (AGD), traumatic brain injury (TBI), and chronic traumatic encephalopathy (CTE), comprising administering to a human in need thereof, an antibody, or an antigen-binding fragment thereof, as disclosed herein, or a pharmaceutical composition comprising the foregoing as disclosed herein. In some embodiments the condition is Alzheimer’s Disease.

[0079] Also provided is a method of reducing clinical decline in a subject having early Alzheimer’s Disease comprising administering a composition comprising a therapeutically effective amount of at least one an anti -acetylated tau antibody, or an antigen-binding fragment thereof, as disclosed herein, or a pharmaceutical composition comprising the foregoing as disclosed herein. In some embodiments, the at least one anti -acetylated tau antibody is antagonistic. In some embodiments, the antibody specifically binds acetylated lysine 174 on human tau (ac-tauK174). In some embodiments, the at least one anti-acetylated tau antibody is chosen from AbMl, ABH1 or ABH5. In some embodiments, the at least one anti -acetylated tau antibody comprises three heavy chain complementarity determiningAttorney Docket No. AETN0001-401-PC regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:68 (HCDR1), SEQ ID NO:69 (HCDR2), and SEQ ID NO:70 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:71 (LCDR1), SEQ ID NO:72 (LCDR2), and SEQ ID NO:73 (LCDR3). In some embodiments, the subject having early Alzheimer’s Disease has been diagnosed as having mild cognitive impairment (MCI) due to Alzheimer’s Disease and / or has been diagnosed as having mild Alzheimer’s Disease dementia. In some embodiments, diagnosis comprises determining an increased level of aberrantly acetylated or hyperacetylated tau relative to a normal level from a non-diseased control.

[0080] In some embodiments, the amount of the anti-acetylated tau antibody administered in the methods disclosed herein comprises 5 mg / kg to 10 mg / kg of ABH1 or ABH5 relative to the weight of the subject. In some embodiments, the amount of the antiacetylated tau antibody comprises 10 mg / kg of ABH1 or ABH5 relative to the weight of the subject. In some embodiments, the at least one anti-acetylated tau antibody is administered once every 2 weeks or once every month. In some embodiments of the methods disclosed herein, clinical decline is reduced by at least 45% relative to placebo as determined by the Alzheimer’s Disease Composite Score (ADCOMS) after 12 months of administration of the composition.

[0081] In some embodiments of the methods disclosed herein, the subject is concomitantly administered at least one Alzheimer’s Disease medication other than ABH1 or ABH5. In some embodiments of the methods disclosed herein, the subject is not concomitantly administered at least one Alzheimer’s Disease medication other than ABH1 or ABH5.

[0082] In some embodiments of the methods disclosed herein, the administration of the anti -acetylated tau antibody, or an antigen-binding fragment thereof, results in a reduction of cerebrospinal fluid levels of acetylated K174 tau and / or acetylated K274 tau.

[0083] Also provided herein is a method of treating a subject having early Alzheimer’s Disease comprising administering a composition comprising a therapeutically effective amount of at least one anti-acetylated tau antibody, or an antigen-binding fragment thereof, wherein clinical decline of the subject is reduced by at least 35% relative to placebo as determined by ADCOMS after 6 months of administration of the composition, by at least 30% relative to placebo as determined by ADCOMS after 12 months of administration of the composition, and / or by at least 25% relative to placebo as determined by ADCOMS after 18 months of administration of the composition. In some embodiments, the at least one antiAttorney Docket No. AETN0001-401-PC acetylated tau antibody comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:68 (HCDR1), SEQ ID NO:69 (HCDR2), and SEQ ID NO:70 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:71 (LCDR1), SEQ ID NO:72 (LCDR2), and SEQ ID NO:73 (LCDR3). In some embodiments, the subject having early Alzheimer’s Disease has been diagnosed as having mild cognitive impairment due to Alzheimer’s Disease - intermediate likelihood and / or has been diagnosed as having mild Alzheimer’s Disease dementia.

[0084] Also provided herein is a method of treating a subject having early Alzheimer’s Disease comprising administering a composition comprising a therapeutically effective amount of at least one anti-acetylated tau antibody, or an antigen-binding fragment thereof, wherein the severity of at least one symptom associated with Alzheimer’s Disease is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% relative to the severity of the same symptom in subjects that received placebo. In some embodiments, the severity of at least one symptom associated with Alzheimer’s Disease is determined by ADCOMS, PET, MMSE, CDR-SB, and / or ADAS-Cog. In some embodiments, the at least one anti-acetylated tau antibody comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:68 (HCDR1), SEQ ID NO:69 (HCDR2), and SEQ ID NO:70 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:71 (LCDR1), SEQ ID NO:72 (LCDR2), and SEQ ID NO:73 (LCDR3). In some embodiments, the at least one symptom associated with Alzheimer’s Disease is clinical decline, and the clinical decline is reduced by at least 30% relative to placebo as determined by ADCOMS after 18 months of administration of the composition comprising the at least one anti-acetylated tau antibody, or an antigen-binding fragment thereof.

[0085] In some embodiments of the methods disclosed herein, the subject is ApoE4- positive. In some embodiments of the methods disclosed herein, the subject is ApoE4- negative.

[0086] Also provided herein is a stable liquid pharmaceutical formulation comprising at least one antibody that specifically binds to acetylated tau, wherein the antibody is an antibody comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:68, an HCDR2 comprising the amino acid sequence of SEQ ID NO:69, an HCDR3 comprising the amino acid sequence of SEQ ID NO:70, an LCDR1 comprising the amino acid sequence ofAttorney Docket No. AETN0001-401-PCSEQ ID N0:71, an LCDR2 comprising the amino acid sequence YTS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:73.

[0087] Additional embodiments follow and are disclosed throughout the specification.Enumerated Embodiments

[0088] Also disclosed herein are the following enumerated embodiments:

[0089] Embodiment 1. An anti-acetylated-tau antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3 polypeptides and the VL comprises LCDR1, LCDR2, and LCDR3 polypeptides chosen from the following sets, respectively: a) HCDR1 is GYTFTDYY (SEQ ID NO:38), HCDR2 is IFPGSDLI (SEQ ID NO:39), HCDR3 is ARGVYYDGGNFFDY (SEQ ID NO:40), LCDR1 is QDISSY (SEQ ID NO:41), LCDR2 is YTS (SEQ ID NO:42), and LCDR3 is QQYSKRPWT (SEQ ID NO:43); b) HCDR1 is GFNIKNTY (SEQ ID NO:44), HCDR2 is IDPSNGNT (SEQ ID NO:45), HCDR3 is ADD YAW (SEQ ID NO:46), LCDR1 is QSLLYTNGKTY (SEQ ID NO:47), LCDR2 is LSV (SEQ ID NO:48), and LCDR3 is LQSTHFPRT (SEQ ID NO:49); c) HCDR1 is GFTFGDYW (SEQ ID NO:50), HCDR2 is IKLKSDNYAT (SEQ ID NO:51), HCDR3 is TTIYYYDSRRFDY (SEQ ID NO:52), LCDR1 is QSVSYD (SEQ ID NO:53), LCDR2 is HAS (SEQ ID NO:54), and LCDR3 is HQEYSSPYT (SEQ ID NO:55); d) HCDR1 is GFTFGDYW (SEQ ID NO:56), HCDR2 is IKLKSDNYAT (SEQ ID NO:57), HCDR3 is TTIYYYDSRRFDY (SEQ ID NO:58), LCDR1 is QSVSYD (SEQ ID NO:59), LCDR2 is INPKYGDT (SEQ ID NO:60), and LCDR3 is AVIYYGFYTMDY (SEQ ID NO:61); e) HCDR1 is GFTFTIYG (SEQ ID NO:62), HCDR2 is IDENGGA (SEQ ID NO:63), HCDR3 is ARDDGT (SEQ ID NO:64), LCDR1 is QSLFYSDGQTY (SEQ ID NO:65), LCDR2 is LSV (SEQ ID NO:66), and LCDR3 is VQGTHFPLT (SEQ ID NO: 67); and f) HCDR1 is GYKFTEYY (SEQ ID NO:84), HCDR2 is INPKYGDT (SEQ ID NO:85), HCDR3 is AVIYYGFYTMDY (SEQ ID NO:86), LCDR1 is QSLLDISNQKNH (SEQAttorney Docket No. AETN0001-401-PCID NO:87), LCDR2 is FAS (SEQ ID NO:88), and LCDR3 is QQHYNIPRT (SEQ ID NO:89), wherein the numbering of amino acid residues is according to IMGT.

[0090] Embodiment 2. The anti -acetyl ated-tau antibody, or an antigen-binding fragment thereof, of Embodiment 1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and the VL are polypeptides chosen from: a) VH of SEQ ID NO: 10 and VL of SEQ ID NO: 11; b) VH of SEQ ID NO: 12 and VL of SEQ ID NO: 13; c) VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15; d) VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17; e) VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; f) VH of SEQ ID NO:20 and VL of SEQ ID NO:21; g) VH of SEQ ID NO:22 and VL of SEQ ID NO:23; h) VH of SEQ ID NO:24 and VL of SEQ ID NO:25; i) VH of SEQ ID NO:26 and VL of SEQ ID NO:27; j) VH of SEQ ID NO:28 and VL of SEQ ID NO:29; k) VH of SEQ ID NO:30 and VL of SEQ ID NO:31; l) VH of SEQ ID NO:32 and VL of SEQ ID NO:33; m) VH of SEQ ID NO:34 and VL of SEQ ID NO:35; n) VH of SEQ ID NO:36 and VL of SEQ ID NO:37; and o) VH of SEQ ID NO : 82 and VL of SEQ ID NO: 83.

[0091] Embodiment 3. The anti -acetyl ated-tau antibody, or an antigen-binding fragment thereof, of Embodiment 2, comprising a combination of a heavy chain (HC) polypeptide and a light chain (LC) polypeptide chosen from: a) HC of SEQ ID NO: 74 and LC of SEQ ID NO: 75; and b) HC of SEQ ID NO:78 and LC of SEQ ID NO:79.

[0092] Embodiment 4. The anti -acetyl ated-tau antibody, or an antigen-binding fragment thereof, of Embodiment 3, comprising two combinations of a light chain and a heavy chain wherein each combination of light chain polypeptide and heavy chain polypeptide is chosen from: a) HC of SEQ ID NO: 74 and LC of SEQ ID NO: 75; and b) HC of SEQ ID NO:78 and LC of SEQ ID NO:79.

[0093] Embodiment 5. The anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of any of Embodiments 1 to 4, which is a monoclonal antibody.Attorney Docket No. AETN0001-401-PC

[0094] Embodiment 6. The anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of any of Embodiments 1 to 5, which has heavy and light chain constant regions of the IgGl, IgG2, or IgG4 subclasses.

[0095] Embodiment 7. The anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of Embodiment 6, which is of the IgGl subclass.

[0096] Embodiment 8. The anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of Embodiment 7, which has a heavy chain constant domain represented by the amino acid sequence set forth in SEQ ID NO:4.

[0097] Embodiment 9. The anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of Embodiment 7, which has a mutated heavy chain constant domain as compared to the wildtype.

[0098] Embodiment 10. The anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of Embodiment 9, which has a heavy chain represented by the amino acid sequence set forth in SEQ ID NO: 5.

[0099] Embodiment 11. The anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of any of Embodiments 1 to 10, which is a humanized or chimeric antibody.

[0100] Embodiment 12. The anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of any of Embodiments 1 to 11, which has a light chain constant domain of the lambda ( / ) subtype.

[0101] Embodiment 13. The anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of any of Embodiments 1 to 11, which of any of Embodiments 1 to 11, which has a light chain constant domain of the kappa (K) subtype.

[0102] Embodiment 14. The anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of Embodiment 13, which has a light chain constant domain represented by the amino acid sequence set forth in SEQ ID NO: 8.

[0103] Embodiment 15. The anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of any of Embodiments 1 to 14, which specifically binds acetylated lysine 174 on human tau (ac-tauK174).

[0104] Embodiment 16. The anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of Embodiment 13, which is capable of binding with a KD of at least about IxlO'11M, at least about IxlO'12M, or at least about 2.5xl0'12M.Attomey Docket No. AETN0001-401-PC

[0105] Embodiment 17. The anti -acetyl ated-tau antibody, or an antigen-binding fragment thereof, of any of Embodiments 1 to 14, which specifically binds acetylated lysine 274 on human tau (ac-tauK274).

[0106] Embodiment 18. A nucleic acid encoding an antibody, or an antigen-binding fragment thereof, encoding the anti-acetylated-tau antibody, or an antigen-binding fragment thereof, of any of Embodiments 1-17, or as disclosed herein.

[0107] Embodiment 19. A nucleic acid encoding a heavy chain of an antibody, where the heavy chain is represented by the amino acid sequence set forth in SEQ ID NO:78.

[0108] Embodiment 20. The nucleic acid of Embodiment 19, wherein the nucleic acid is represented by the sequence set forth in SEQ ID NO:80.

[0109] Embodiment 21. A nucleic acid encoding a light chain of an antibody, wherein the light chain is represented by the amino acid sequence set forth in SEQ ID NO:79.

[0110] Embodiment 22. The nucleic acid of Embodiment 21, wherein the nucleic acid is represented by the sequence set forth in SEQ ID NO:81.

[0111] Embodiment 23. A vector comprising the nucleic acid of any of Embodiments 18-22, or encoding any of the nucleic acids as disclosed herein.

[0112] Embodiment 24. A host cell comprising the vector of Embodiment 23, or a vector comprising any of the nucleic acids as disclosed herein.

[0113] Embodiment 25. A pharmaceutical composition comprising any of the anti- acetylated-tau antibodies, or antigen-binding fragments thereof, of any of Embodiments 1- 17, or of the instant disclosure, plus a pharmaceutically acceptable carrier, diluent, or excipient; and optionally wherein the composition is a stable liquid formulation.

[0114] Embodiment 26. A method of treating a condition chosen from clinical or pre- clinical Alzheimer’s Disease, prodromal Alzheimer’s Disease, Down Syndrome, clinical or pre-clinical amyloid angiopathy (CAA), frontotemporal dementia (FTD) with TAR DNA- binding protein 43 (TDP-43), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick’s disease (PiD), argyrophilic grain disease (AGD), traumatic brain injury (TBI), and chronic traumatic encephalopathy (CTE) is provided, wherein the method comprises administering, to a human in need thereof, any of the antibodies, or antigen-binding fragments thereof, of any of Embodiments 1-17, or of the instant disclosure, or the pharmaceutical composition comprising either of them of Embodiment 25.

[0115] Embodiment 27. A method of treating Alzheimer’s Disease, wherein the method comprises administering, to a human in need thereof, any of the antibodies, or antigen-Attorney Docket No. AETN0001-401-PC binding fragments thereof, of any of Embodiments 1-17, or of the instant disclosure, or the pharmaceutical composition comprising either of them of Embodiment 25.

[0116] Embodiment 28. A method of reducing clinical decline in a subject having early Alzheimer’s Disease, wherein the method comprises administering, to a human in need thereof, any of the antibodies, or antigen-binding fragments thereof, of any of Embodiments 1-17, or of the instant disclosure, or the pharmaceutical composition comprising either of them of Embodiment 25.

[0117] Embodiment 29. The method of any of Embodiments 26-28, wherein the antibody is an anti-acetylated tau antibody.

[0118] Embodiment 30. The method of any of Embodiments 26-28, wherein the antiacetylated tau antibody, or antigen-binding fragments thereof, is antagonistic.

[0119] Embodiment 31. The method of any of Embodiments 26-28, wherein the antiacetylated tau antibody, or an antigen-binding fragment thereof, specifically binds acetylated lysine 174 on human tau (ac-tauK174).

[0120] Embodiment 32. The method of any of Embodiments 26-28, wherein the antiacetylated tau antibody, or an antigen-binding fragment thereof, specifically binds acetylated lysine 274 on human tau (ac-tauK274).

[0121] Embodiment 33. The method of any of Embodiments 26-28, wherein the antiacetylated tau antibody is chosen from any of AbHl through AbH9.

[0122] Embodiment 34. The method of any of Embodiments 26-33, wherein the antiacetylated tau antibody comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:68 (HCDR1), SEQ ID NO:69 (HCDR2), and SEQ ID NO:70 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:71 (LCDR1), SEQ ID NO: 72 (LCDR2), and SEQ ID NO: 73 (LCDR3).

[0123] Embodiment 35. The method of Embodiment 34, wherein the subject having early Alzheimer’s Disease has been diagnosed as having mild cognitive impairment (MCI) due to Alzheimer’s Disease and / or has been diagnosed as having mild Alzheimer’s Disease dementia.

[0124] Embodiment 36. The method of any of Embodiments 26-35, wherein the diagnosis comprises determining an increased level of aberrantly acetylated or hyperacetylated tau relative to a normal level from a non-diseased control.Attorney Docket No. AETN0001-401-PC

[0125] Embodiment 37. The method of any of Embodiments 26-36, wherein the subject is concomitantly administered at least one Alzheimer’s Disease medication other than any of AbHl through AbH9.

[0126] Embodiment 38. The method of any of Embodiments 26-36, wherein the subject is not concomitantly administered at least one Alzheimer’s Disease medication other than any of AbHl through AbH9.

[0127] Embodiment 39. The method of any of Embodiments 30-38, wherein the administration of any of AbHl through AbH9 results in a reduction of cerebrospinal fluid levels of acetylated K174 tau and / or acetylated K274 tau.

[0128] Embodiment 40. A method of treating a subject having early Alzheimer’s Disease, wherein the method comprises administering a composition comprising a therapeutically effective amount of at least one of the antibodies, or antigen-binding fragments thereof, of any of Embodiments 1-17, or of the instant disclosure, or the pharmaceutical composition comprising either of them of Embodiment 25, and wherein clinical decline of the subject is reduced by at least 35% relative to placebo as determined by ADCOMS after 6 months of administration of the composition, by at least 30% relative to placebo as determined by ADCOMS after 12 months of administration of the composition, and / or by at least 25% relative to placebo as determined by ADCOMS after 18 months of administration of the composition.

[0129] Embodiment 41. The method of Embodiment 40, wherein the anti -acetylated tau antibody comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:68 (HCDR1), SEQ ID NO:69 (HCDR2), and SEQ ID NO:70 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:71 (LCDR1), SEQ ID NO:72 (LCDR2), and SEQ ID NO:73 (LCDR3).

[0130] Embodiment 42. The method of Embodiment 41, wherein the subject having early Alzheimer’s Disease has been diagnosed as having mild cognitive impairment due to Alzheimer’s Disease - intermediate likelihood and / or has been diagnosed as having mild Alzheimer’s Disease dementia.

[0131] Embodiment 43. A method of treating a subject having early Alzheimer’s Disease, comprising administering a composition comprising a therapeutically effective amount of at least one of the antibodies, or antigen-binding fragments thereof, of any of Embodiments 1-17, or of the instant disclosure, or the pharmaceutical composition comprising either of them of Embodiment 25, wherein the severity of at least one symptomAttorney Docket No. AETN0001-401-PC associated with Alzheimer’s Disease is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% relative to the severity of the same symptom in subjects that received placebo.

[0132] Embodiment 44. The method of Embodiment 43, wherein the severity of at least one symptom associated with Alzheimer’s Disease is determined by ADCOMS, PET, MMSE, CDR-SB, and / or ADAS-Cog.

[0133] Embodiment 45. The method of Embodiment 44, wherein the at least one antiacetylated tau antibody comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:68 (HCDR1), SEQ ID NO:69 (HCDR2), and SEQ ID NO:70 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:71 (LCDR1), SEQ ID NO:72 (LCDR2), and SEQ ID NO:73 (LCDR3).

[0134] Embodiment 46. The method of any of Embodiments 43-45, wherein the at least one symptom associated with Alzheimer’s Disease is clinical decline, wherein clinical decline may be shown to be reduced by at least about 30% relative to placebo as determined by ADCOMS after 18 months of administration of any of the anti-acetylated-tau antibodies of the instant disclosure.

[0135] Embodiment 47. The method of any of Embodiments 26-46, wherein the subject is ApoE4-positive.

[0136] Embodiment 48. The method of any of Embodiments 26-46, wherein the subject is ApoE4-negative.

[0137] Embodiment 49. A stable liquid pharmaceutical formulation comprising at least one antibody that specifically binds to acetylated tau, wherein the antibody is an antibody comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:68, an HCDR2 comprising the amino acid sequence of SEQ ID NO:69, an HCDR3 comprising the amino acid sequence of SEQ ID NO:70, an LCDR1 comprising the amino acid sequence of SEQ ID NO:71, an LCDR2 comprising the amino acid sequence YTS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:73, or is one of any of the antibodies, or antigen-binding fragments thereof, of any of Embodiments 1-17, or of the instant disclosure, or the pharmaceutical composition comprising either of them of Embodiment 25..

[0138] Embodiment 50. A method of manufacturing an antibody, or an antigen-binding fragment thereof, comprising:Attorney Docket No. AETN0001-401-PC a. expressing one or more polynucleotide molecule(s) encoding a VH and a VL chain of an antibody, or an antigen-binding fragment thereof, disclosed herein in a host cell; and b. purifying the antibody from the cell and / or a fluid medium in which the cell is disposed.

[0139] Embodiment 51. The method of Embodiment 50, wherein the host cell is chosen from a bacterial cell, a fungal cell, and a mammalian cell.

[0140] Embodiment 52. The method of Embodiment 51, wherein the host cell is chosen from an E. coli cell, a Saccharomyces cerevisiae cell, and a CHO cell.

[0141] Embodiment 53. An antibody, or an antigen-binding fragment thereof, that binds to an epitope of acetylated-tau, wherein the epitope is within the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3.

[0142] Embodiment 54. The antibody, or an antigen-binding fragment thereof, of Embodiment 53, wherein the antibody, or an antigen-binding fragment thereof, crosscompetes with a second antibody in a competitive binding assay for binding to tau, wherein the second antibody has an antigen binding domain that is structurally defined according to any of the sequences disclosed in Table 4, Table 5, Table 6, Table 7, Table 8, and / or Table 9.

[0143] Embodiment 55. A kit comprising an antibody, or an antigen-binding fragment thereof, of any Embodiments 1-17 or 53-54, and instructions for its use.

[0144] Embodiment 56. The kit of Embodiment 55, wherein the antibody, or an antigen-binding fragment thereof, is lyophilized.

[0145] Embodiment 57. The kit of Embodiment 56, further comprising a fluid for reconstitution of the lyophilized antibody, or an antigen-binding fragment thereof.

[0146] Embodiment 58. The kit of Embodiment 57, further comprising a device for subcutaneous or intravenous administration.

[0147] Embodiment 59. The antibody, or an antigen-binding fragment thereof, of any of Embodiments 1-17 or 53-54, wherein the antibody, or an antigen-binding fragment thereof, forms part of a multi-specific antibody.

[0148] Embodiment 60. The antibody, or an antigen-binding fragment thereof, of Embodiment 59 wherein the antibody, or an antigen-binding fragment thereof, forms part of a bispecific antibody.

[0149] Embodiment 61. The antigen-binding fragment of any of Embodiments 1-17, 53-54, or 59-60, wherein the antibody fragment is chosen from a single-variable domainAttorney Docket No. AETN0001-401-PC antibody, a single chain antibody, an scFv, a Fab, a F(ab')2, a minibody, a diabody, and a triabody.

[0150] Embodiment 62. The antigen-binding fragment of Embodiment 61, wherein the antibody fragment is an scFv.

[0151] Embodiment 63. The antibody, or an antigen-binding fragment thereof, of any of Embodiments 1-17, 53-54, or 59-62, wherein the antibody, or an antigen-binding fragment thereof, is aglycosylated.

[0152] Embodiment 64. The antibody, or an antigen-binding fragment thereof, of any of Embodiments 1-17, 53-54, or 59-63, wherein the antibody, or an antigen-binding fragment thereof, is operably linked to another compound.

[0153] Embodiment 65. The antibody, or an antigen-binding fragment thereof, of Embodiment 64, wherein the other compound is a therapeutic agent, label, or tag.

[0154] Embodiment 66. The antibody, or an antigen-binding fragment thereof, of Embodiment 65, wherein the therapeutic agent is a cytotoxic agent, a chemotherapeutic agent, a cytokine, an anti-angiogenic agent, a tyrosine kinase inhibitor, a toxin, a radioisotope, or other therapeutically active agent.

[0155] Embodiment 67. An antibody-drug conjugate (ADC) comprising the antibody, or an antigen-binding fragment thereof, of any of Embodiments 1-17, 53-54, or 59-66.AbbreviationsAttorney Docket No. AETN0001-401-PCDefinitions

[0156] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo or polynucleotide chemistry and hybridization disclosed herein are those well-known and commonly used in the art.

[0157] As used herein, the singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise.

[0158] The phrase “and / or,” as used herein, means “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases.

[0159] “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. The term “about,” as used herein, is intended to qualify the numerical values which it modifies,Attorney Docket No. AETN0001-401-PC denoting such a value as variable within a range. When no particular range, such as a margin of error or a standard deviation to a mean value given in a chart or table of data, is recited, the term “about” should be understood to mean the greater of the range which would encompass the recited value and the range which would be included by rounding up or down to that figure as well, taking into account significant figures, and the range which would encompass the recited value plus or minus a degree of error. Exemplary degrees of error are within about 20 percent (%), typically, within about 10%, and more typically, within about 5% of a given value or range of values.

[0160] The phrase “amyloid hypothesis” refers to the proposition that amyloid 0 (A0) peptides play a central role in the pathogenesis of AD. Specifically, it is hypothesized that neurodegeneration in AD may be caused by deposition of Ab plaques in brain tissue due to an imbalance between A0 production and Ab clearance, leading to formation of neurofibrillary tangles containing tau protein. A0 peptides generally exist in a dynamic continuum of conformational states such that species tend to progress from monomeric A0, to soluble A0 assemblies that include a range of low molecular weight oligomers to higher molecular weight protofibrils, and finally to insoluble fibrils (plaques). A number of immunotherapies have been developed with the intent to reduce the amount of insoluble A0 fibrils deposited in the brain. However, a simple correlation between the quantity and progressive accumulation of insoluble amyloid plaques and the clinical course of AD has not been determined. While therapeutic strategies continue to focus on removal of insoluble amyloid plaques, an additional approach to therapy may include reducing the toxic A0 aggregates, such as protofibrils, that may contribute to the neuronal degeneration characteristic of AD (Dodart & May, 2005; Englund et al., 2007; Gbtz et al., 2004).

[0161] “Early AD” or “early Alzheimer’s Disease,” as used herein, is a continuum of AD severity from mild cognitive impairment due to AD - intermediate likelihood to mild Alzheimer’s Disease dementia. Subjects with early AD include subjects with mild Alzheimer’s Disease dementia as defined herein and subject with MCI due to AD - intermediate likelihood as defined herein. In some embodiments, subjects with early AD have MMSE of 22 to 30 and CDR global range 0.5 to 1.0.

[0162] An intermediate likelihood of Alzheimer’s Disease can refer to a patient’s Amyloid Probability Score (APS) or the presence of only one type of biomarker for mild cognitive impairment (MCI) due to Alzheimer’s, an APS of 36 to 57 is consideredAttorney Docket No. AETN0001-401-PC intermediate and requires further evaluation. A high APS indicates a higher likelihood of having amyloid plaques on a PET scan.

[0163] Subjects with “mild Alzheimer’s Disease dementia,” as used herein, are subjects meet the NIA-AA core clinical criteria for probable Alzheimer’s Disease dementia (McKhann et al., 2011). Also included herein are subjects who have a CDR score of 0.5 to 1.0 and a Memory Box score of 0.5 or greater at screening and baseline.

[0164] Subjects with “MCI due to AD - intermediate likelihood,” as used herein are those identified as such in accordance with the NIA-AA core clinical criteria for mild cognitive impairment due to Alzheimer’s Disease - intermediate likelihood (see McKhann supra). For example, symptomatic but not demented AD subjects with evidence of brain amyloid pathology making them less heterogeneous and more similar to mild Alzheimer’s Disease dementia subjects in cognitive and functional decline as measured by the ADCOMS Composite Clinical Score defined herein. Also included are subjects who have a CDR score of 0.5 and a Memory Box score of 0.5 or greater at screening and baseline. Furthermore, subjects who report a history of subjective memory decline with gradual onset and slow progression over the last 1 year before screening, which is corroborated by an informant, are also included herein.

[0165] Biomarkers are not required for the diagnosis of MCI, although the diagnosis of MCI due to AD would require biomarker support (Ganguli et al., 2011). If only one type of biomarker is positive, the term “MCI due to AD - intermediate likelihood” is used. If biomarkers for both amyloid abnormalities and neurodegeneration are present, the term “MCI due to AD - high likelihood” is used.

[0166] As used herein, “ADCOMS” refers to Alzheimer’s Disease Composite Score, a composite clinical score based on an analysis of four ADAS-Cog items (delayed word recall, orientation, word recognition, and word finding difficulty), two MMSE items (orientation to time, and drawing), and all six CDR-SB items (personal care, community affairs, home and hobbies, memory, orientation, and judgment and problem solving), as discussed in the Examples and in (Wang et al., 2016). ADCOMS was developed to be particularly sensitive to disease progression during early stages of AD, i.e., prodromal and mild AD.

[0167] The term “antibody,” as used herein, refers to an antigen-binding molecule or molecular complex comprising a set of complementarity determining regions (CDRs) that specifically bind to or interact with a particular antigen (e.g., Ac-tauK174). The term “antibody,” as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, asAttorney Docket No. AETN0001-401-PC well as multimers thereof (e.g., IgM). In a typical antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as VH or HCVR) and a heavy chain constant region (HC). The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL or LCVR) and a light chain constant region (LC). The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FRs of the antibody (or antigen-binding portion thereof) may be identical to the human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0168] Unless specifically indicated otherwise, the term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, “antigen-binding domain,” and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR- grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.

[0169] As used herein the term “antibody” or “immunoglobulin” have the same meaning and will be used equally in the present disclosure. The term “antibody” as used herein refers to a protein, or polypeptide sequence derived from immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immuno-specifically binds an antigen. Antibodies can beAttorney Docket No. AETN0001-401-PC polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources.

[0170] The term “antibody fragment” refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hinderance, stabilizing / destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH) and camelid domains.

[0171] The term “antibody heavy chain,” refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.

[0172] The term “antibody light chain,” refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (k) and lambda (1) light chains refer to the two major antibody light chain isotypes.

[0173] The term “antigen” or “Ag” refers to a molecule that provokes an immune response. It is readily apparent that an antigen can be generated by synthesis or can be derived from a biological sample or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a brain sample, a cell or a fluid with other biological components, such as cerebral spinal fluid.

[0174] As used herein, “ApoE4-positive” subjects and “ApoE4 carriers” refer to subjects who harbor the e4 variant of the apolipoprotein gene. The e4 variant is one of several major alleles of the apolipoprotein gene. The gene is generally responsible for metabolism of fats. It has been found that carriers of the apolipoprotein e4 show significantly greater rates of amyloid retention when compared to non-carriers (Drzezga et al., 2009). In some embodiments, the subject is a heterozygous carrier of the apolipoprotein E e4 gene allele. In some embodiments, the subject is a homozygous carrier of the apolipoprotein E e4 gene allele.

[0175] As used herein, whether an early AD subject is “amyloid-positive” or “amyloidnegative” is determined based on whether or not the patient has a positive amyloid load as indicated by longitudinal PET assessment of an amyloid imaging agent uptake into the brain and / or a CSF assessment of the presence of amyloid pathology using assessments of markers such as Abi-42 (soluble CSF biomarker analysis). In some embodiments, a qualitative visual read of PET scans will be used to determine amyloid positive and amyloid negative byAttorney Docket No. AETN0001-401-PC categorizing subjects as having either “normal” or “abnormal” uptake on the basis of the PET image pattern. Readers will have been trained and certified to recognize brain PET images with abnormal or normal patterns of uptake, or the detection of amyloid is done through a semi-quantitative or quantitative approach.

[0176] The term “binding” as used herein refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank the strength of bimolecular interactions. The smaller the KD value, the greater the binding affinity of the ligand for its target. As used herein, the term “binding” in the context of the binding of an antibody to a predetermined target molecule (e.g., an antigen or epitope) typically is binding with an affinity corresponding to a KD of about 10'7M or less, such as about 10'8M or less, such as about 10'9M or less, about IO-10M or less, or about 10'11M or even less. The concepts of binding affinity, association constant, and dissociation constant are well known.

[0177] As used herein, the term “binding domain” or “antibody molecule” refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term “binding domain” or “antibody molecule” encompasses antibodies and antibody fragments.

[0178] The term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within an antibody of the disclosure can beAttorney Docket No. AETN0001-401-PC replaced with other amino acid residues from the same side chain family and the altered antibody can be tested using the functional assays disclosed herein.

[0179] The term “corresponding human germline sequence” refers to the nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. The corresponding human germline sequence can also refer to the human variable region amino acid sequence or subsequence with the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences. The corresponding human germline sequence can be framework regions only, complementarity determining regions only, framework and complementary determining regions, a variable segment (as defined above), or other combinations of sequences or subsequences that comprise a variable region. Sequence identity can be determined using the methods disclosed herein, for example, aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference variable region nucleic acid or amino acid sequence.

[0180] An “effective dose” or “therapeutically effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[0181] The term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0182] The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.

[0183] “Fc region” refers to the portion of a single immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C- terminus of the antibody. Accordingly, a complete Fc region comprises at least a hinge, a CH2 domain, and a CH3 domain. Two Fc regions that are dimerized are referred to as “Fc” or “Fc dimer.” An Fc region may be a naturally occurring Fc region, or a naturally occurring Fc region in which one or more amino acids have been substituted, added or deleted, provided that the Fc region has the desired biological properties. A desired biological activityAttorney Docket No. AETN0001-401-PC may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to that of the naturally occurring domain.

[0184] The term “humanized antibody” or “engineered antibody” refers to an antibody having variable region framework and constant regions from a human antibody but retaining the CDRs of a previous non-human antibody. In some embodiments, a humanized antibody contains minimal sequences derived from a non-human immunoglobulin. For the most part, humanized antibodies and antibody binding fragments thereof may be human immunoglobulins (recipient antibody or antibody fragment) in which residues from complementary determining regions (CDRs) of the recipient are replaced by residues from a CDR of non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.

[0185] In some embodiments, the antibody of the present disclosure is a humanized antibody. In some embodiments, the humanized antibody may also comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. The prefix “hum,” “hu,” “Hu,” or “h” is added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies. The humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions can be included to increase affinity, increase stability of the humanized antibody, remove a post-translational modification or for other reasons.

[0186] The term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. “Fully human” refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.

[0187] As used herein, “MMSE” refers to the Mini-Mental State Examination, a cognitive instrument commonly used for screening purposes, but also often measuredAttorney Docket No. AETN0001-401-PC longitudinally in AD clinical trials having a 30-point scale with higher scores indicating less impairment and lower scores indicating more impairment.

[0188] As used herein the term “modulate” relates to a capacity to alter an effect, result, or activity (e.g., signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (i.e., attenuating, but not abolishing) or it can completely abolish such activity (e.g., neutralizing). Modulation can include internalization of a receptor following binding of an antibody or a reduction in expression of a receptor on the target cell. Agonistic modulation can enhance or otherwise increase or enhance an activity (e.g., signal transduction). In a still further embodiment, such modulation can alter the nature of the interaction between a ligand and its cognate receptor so as to alter the nature of the elicited signal transduction. For example, the molecules can, by binding to the ligand or receptor, alter the ability of such molecules to bind to other ligands or receptors and thereby alter their overall activity. In some embodiments, such modulation will provide at least a 10% change in a measurable immune system activity, at least a 50% change in such activity, or at least a 2-fold, 5-fold, 10-fold, or at least a 100-fold change in such activity.

[0189] The term “parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or any other infusion techniques.

[0190] As used herein, the expression “pharmaceutical formulation” means a combination of at least one active ingredient (e.g., an antibody, small molecule, compound, etc. which is capable of exerting a biological effect in a human or non-human animal), and at least one inactive ingredient which, when combined with the active ingredient and / or one or more additional inactive ingredients, is suitable for therapeutic administration to a human or non-human animal. The term “formulation,” as used herein, means “pharmaceutical formulation” unless specifically indicated otherwise.

[0191] As used herein, a “pharmaceutically acceptable excipient” or “diagnostically acceptable excipient” includes but is not limited to, sterile distilled water, saline, phosphate buffered solutions, amino acid-based buffers, or bicarbonate buffered solutions. An excipient selected and the amount of excipient used will depend upon the mode of administration. Administration comprises an injection, infusion, or a combination thereof.

[0192] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.Attorney Docket No. AETN0001-401-PCPolypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof. “Protein” may also refer to a three-dimensional structure of the polypeptide. “Denatured protein” refers to a partially denatured polypeptide, having some residual three-dimensional structure or, alternatively, to an essentially random three-dimensional structure, as is the case in a totally denatured protein. Polypeptide variants can be produced by glycosylation, phosphorylation, sulfation, disulfide bond formation, deamidation, isomerization, cleaving points in signal or leader sequence processing, covalent and non-covalently bound cofactors, oxidized variants, and the like. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).

[0193] As used herein, the terms “percent sequence identity” and “% sequence identity” refer to the percentage of sequence similarity found by a comparison or alignment of two or more amino acid or nucleic acid sequences. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. An algorithm for calculating percent identity is the Smith-Waterman homology search algorithm.

[0194] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi -stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidineAttorney Docket No. AETN0001-401-PC bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiments being described, single-stranded (such as sense or anti sense) and double-stranded polynucleotides.

[0195] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0196] As used herein, the term “prevent” refers to obtaining beneficial or desired results including, but not limited to, prophylactic benefit. For prophylactic benefit, the composition may be administered to a subject at risk of developing Alzheimer’s Disease, to a subject having one or more preclinical symptoms but not clinical symptoms of Alzheimer’s Disease, or to a subject reporting one or more of the physiological symptoms of Alzheimer’s Disease, even though a clinical diagnosis of having Alzheimer’s has not been made. As used herein “prevention” may further include therapeutic benefit, by which is meant eradication or amelioration of the underlying condition being treated or of one or more of the physiological symptoms associated therewith.

[0197] Pre-AD biomarker levels that may suggest the development of Alzheimer’s Disease include, but are not limited to, brain amyloid level, cerebrospinal fluid level of Abi- 42, cerebrospinal fluid level of total tau, cerebrospinal fluid level of neurogranin, and cerebrospinal fluid level of neurofilament light chain.

[0198] The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.

[0199] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includesAttorney Docket No. AETN0001-401-PC subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.

[0200] The term “recombinant antibody,” as used herein, is intended to include all antibodies that are prepared, expressed, created, or isolated by recombinant means, using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art. The term includes, but is not limited to, antibodies expressed using a recombinant expression vector transfected into a host cell (e.g., Chinese hamster ovary (CHO) cell) or cellular expression system, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies isolated from a non-human animal (e.g., a mouse, such as a mouse that is transgenic for human immunoglobulin genes (Taylor et al., 1992). In some embodiments, the recombinant antibody is a recombinant human antibody. In some embodiments, recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0201] As used herein, “sample” refers to a sample from a human, animal, placebo, or research sample, such as a cell, tissue, organ, fluid, gas, aerosol, slurry, colloid, or coagulated material. The “sample” may be tested in vivo, (i.e. without removal from the human or animal), or it may be tested in vitro. The sample may be tested after processing, such as by histological methods. “Sample” also refers to a cell comprising a fluid or tissue sample, or a cell separated from a fluid or tissue sample. “Sample” may also refer to a cell, tissue, organ, or fluid that is freshly taken from a human or animal, or to a cell, tissue, organ, or fluid that is processed or stored. One example is cerebral spinal fluid.

[0202] Antibody “specificity” is defined according to the paratope-epitope interaction. When specificity is low, an antibody may bind several different epitopes. Specificity indicates that an antibody binds almost exclusively to one particular antigen or epitopeAttorney Docket No. AETN0001-401-PC thereon, with minimal binding to anything else, even closely related antigens. Thus, specific binding relates to an antibody’s ability to target a particular epitope on an antigen, and implies a very precise interaction, whereas selective binding means favoring one antigen over others, even if there may be minor cross-reactivity with similar antigens present in a mixture. Essentially, “specific” is a stricter definition than “selective” in the context of antibody binding.

[0203] The term “specifically binds,” or the like, means that an antibody or an antigenbinding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 x 106M or less, e.g., 107M, 10xM, 109M, 1010M, 101 1M, or 1012M (a smaller KD denotes a tighter binding). Methods for determining whether an antibody specifically binds to an antigen are known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., BIACORE™), bio-layer interferometry assay (e.g., Octet® HTX biosensor), solution-affinity ELISA, and the like. In some embodiments, specific binding is measured in a surface plasmon resonance assay, e.g., at 25° C. or 37° C. An antibody or antigen-binding fragment that specifically binds an antigen from one species may or may not have cross-reactivity to other antigens, such as an orthologous antigen from another species.

[0204] Specific binding can also mean, e.g., that the binding compound, nucleic acid ligand, antibody, or binding composition derived from the antigen-binding site of an antibody, of the contemplated method binds to its target with an affinity that is often at least 25% greater, more often at least 50% greater, most often at least 100% (2-fold) greater, normally at least ten times greater, more normally at least 20-times greater, and most normally at least 100-times greater than the affinity with any other binding compound.

[0205] As used herein, the term “subject” refers to a human or non-human organism. Thus, the methods and compositions disclosed herein are applicable to both human and veterinary disease. In certain embodiments, subjects are “patients,” such as living humans that are receiving medical care for a disease or condition. This includes persons with no defined illness who are being investigated for signs of pathology.

[0206] The term “substantially,” as used in the context of binding or exhibited effect, is intended to denote that the observed effect is physiologically or therapeutically relevant. Thus, for example, a molecule is able to substantially block an activity of a ligand or receptor if the extent of blockage is physiologically or therapeutically relevant (for example if such extent is greater than 60% complete, greater than 70% complete, greater than 75% complete,Attorney Docket No. AETN0001-401-PC greater than 80% complete, greater than 85% complete, greater than 90% complete, greater than 95% complete, or greater than 97% complete). Similarly, a molecule is said to have substantially the same immunospecificity and / or characteristic as another molecule, if such immunospecificities and characteristics are greater than 60% identical, greater than 70% identical, greater than 75% identical, greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical, or greater than 97% identical).

[0207] As used herein, the term “therapeutically effective amount” is defined as an amount of a reagent or pharmaceutical composition that is sufficient to induce a desired immune response specific for encoded heterologous antigens to show a patient benefit (e.g. to cause a decrease, prevention, or amelioration of the symptoms of the condition being treated). When the agent or pharmaceutical composition comprises a diagnostic agent, a “diagnostically effective amount” is defined as an amount that is sufficient to produce a signal, image, or other diagnostic parameter. Effective amounts of the pharmaceutical formulation will vary according to factors such as the degree of susceptibility of the individual, the age, gender, and weight of the individual, and idiosyncratic responses of the individual (U.S. Pat. No. 5,888,530).

[0208] The term “treating” (or “treat” or “treatment”) refers to processes involving a slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related symptoms, conditions, or disorders associated with an anti-ac-tauK174 antibody or an anti-ac-tauK274 of the disclosure.

[0209] As used herein, the term “variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0210] In one embodiment “functional variants” of a reference antibody show sequence variation at one or more CDRs when compared to corresponding reference CDR sequences. Thus, a functional antibody variant may comprise a functional variant of a CDR. Where theAttorney Docket No. AETN0001-401-PC term “functional variant” is used in the context of a CDR sequence, this means that the CDR has at most 2, preferably at most 1 amino acid difference when compared to a corresponding reference CDR sequence, and when combined with the remaining 5 CDRs (or variants thereof) enables the variant antibody to bind to the same target antigen as the reference antibody.

[0211] In one embodiment a variant antibody comprises: a light chain CDR1 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR2 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR3 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR1 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR2 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR3 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; wherein the variant antibody binds to the same target antigen as the reference antibody.

[0212] Preferably, a variant antibody comprises: a light chain CDR1 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a light chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a light chain CDR3 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a heavy chain CDR1 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a heavy chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a heavy chain CDR3 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; wherein the variant antibody binds to the same target antigen as the reference antibody.

[0213] For example, a variant antibody may comprise: a light chain CDR1 having at most 2 amino acid differences when compared to SEQ ID NO:71; a light chain CDR2 having at most 2 amino acid differences when compared to SEQ ID NO: 72; a light chain CDR3 having at most 2 amino acid differences when compared to SEQ ID NO: 73; wherein the variant antibody binds to ac-tauK174.

[0214] For example, a variant antibody may comprise: a light chain CDR1 having at most 1 amino acid differences when compared to SEQ ID NO:71; a light chain CDR2 having at most 1 amino acid differences when compared to SEQ ID NO: 72; a light chain CDR3 havingAttorney Docket No. AETN0001-401-PC at most 1 amino acid differences when compared to SEQ ID NO: 73; wherein the variant antibody binds to ac-tauK174.

[0215] For example, a variant antibody may comprise: a heavy chain CDR1 having at most 2 amino acid difference when compared to SEQ ID NO:68; a heavy chain CDR2 having at most 2 amino acid difference when compared to SEQ ID NO: 69; a heavy chain CDR3 having at most 2 amino acid difference when compared to SEQ ID NO:70; wherein the variant antibody binds to ac-tauK174.

[0216] For example, a variant antibody may comprise: a heavy chain CDR1 having at most 1 amino acid difference when compared to SEQ ID NO:68; a heavy chain CDR2 having at most 1 amino acid difference when compared to SEQ ID NO: 69; a heavy chain CDR3 having at most 1 amino acid difference when compared to SEQ ID NO:70; wherein the variant antibody binds to ac-tauK174.Bioequivalents

[0217] The present disclosure includes formulations comprising antibodies that are bioequivalent to any of the exemplary antibodies set forth herein. Two antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple doses. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.

[0218] In one embodiment, two antibodies are bioequivalent if a patient can be switched one or more times between the first antibody (e.g., reference product) and the second antibody (e.g., biological product) without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.

[0219] Bioequivalence may be demonstrated by in vivo and in vitro methods. Nonlimiting examples of bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data;Attorney Docket No. AETN0001-401-PC(c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and(d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.

[0220] A full-length antibody is an immunoglobulin molecule comprising 2 heavy (H) chains and 2 light (L) chains interconnected by disulfide bonds. The amino terminal portion of each chain includes a variable region of about 100-110 amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein. The carboxy -terminal portion of each chain defines a constant region primarily responsible for effector function.

[0221] The CDRs are interspersed with regions that are conserved, termed framework regions (FR). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light chain are referred to as “LCDR1, LCDR2, and LCDR3” and the 3 CDRs of the heavy chain are referred to as “HCDR1, HCDR2, and HCDR3.” The CDRs contain most of the residues which form specific interactions with the antigen.

[0222] As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (X) and kappa (K).

[0223] The constant domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).

[0224] Light chains are classified as kappa or lambda and are characterized by a particular constant region as known in the art. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the isotype of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively. IgG antibodies can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, or IgG4. Each heavy chain type is characterized by a particular constant region with a sequence well known in the art. In some embodiments, the anti-ac-tau antibodies of the present disclosure comprises a heavy chain constant region that is an Ig gamma- 1 chain C regionAttorney Docket No. AETN0001-401-PC(e.g., NCBI ACCESSION: P01857), and a light chain constant region that is an Ig kappa chain C region (e.g., NCBI ACCESSION: P01834).

[0225] As used herein, the term “monoclonal antibody” (Mab) refers to an antibody that is derived or isolated from a single copy or clone including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Mabs of the present disclosure preferably exist in a homogeneous or substantially homogeneous population. Complete Mabs contain 2 heavy chains and 2 light chains. The phrase “antigen-binding fragments” includes, for example, Fab fragments, Fab’ fragments, F(ab’)2 fragments, and single chain Fv fragments. Monoclonal antibodies of the present disclosure and antigenbinding fragments thereof can be produced, for example, by recombinant technologies, phage display technologies, synthetic technologies, e.g., CDR-grafting, or combinations of such technologies, or other technologies known in the art. For example, mice can be immunized with human Anti-N3pGlu AB or fragments thereof, the resulting antibodies can be recovered and purified, and determination of whether they possess binding and functional properties similar to or the same as the antibody compounds disclosed herein can be assessed by the methods disclosed essentially as described in Examples below. Antigen-binding fragments can also be prepared by conventional methods. Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapters 5-8 and 15, ISBN 0-87969-314-2.

[0226] The phrase “human engineered antibodies” refers to monoclonal antibodies that have framework regions that are substantially human or fully human surrounding CDRs derived from a non-human antibody. “Antigen-binding fragments” of such human engineered antibodies include, for example, Fab fragments, Fab’ fragments, F(ab’)2 fragments, and single chain Fv fragments. “Framework region” or “framework sequence” refers to any one of framework regions 1 to 4. Human engineered antibodies and antigen-binding fragments thereof encompassed by the present disclosure include molecules wherein any one or more of framework regions 1 to 4 is substantially or fully human, i.e., wherein any of the possible combinations of individual substantially or fully human framework regions 1 to 4., is present. For example, this includes molecules in which framework region 1 and framework region 2, framework region 1 and framework region 3, framework region 1, 2, and 3, etc., are substantially or fully human. Substantially-human frameworks are those that have at least about 80% sequence identity to a known human germline framework sequence. Preferably,Attorney Docket No. AETN0001-401-PC the substantially human frameworks have at least about 85%, about 90%, about 95%, or about 99% sequence identity to a known human germline framework sequence.

[0227] Fully human frameworks are those that are identical to a known human germline framework sequence. Human framework germline sequences can be obtained from ImMunoGeneTics (IMGT) via their website imgt.cines.fr, or from The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, ISBN 012441351. For example, germline light chain frameworks can be chosen from the group consisting of: Al l, A17, A18, A19, A20, A27, A30, LI, Li l, L12, L2, LS, L15, L6, L8, 012, 02, and 08, and germline heavy chain framework regions can be chosen from the group consisting of: VH2-5, VH2-26, VH2-70, VH3-20, VH3-72, VHI-46, VH3- 9, VH3-66, VH3- 74, VH4-31, VHI-18, VHI-69, VI-13-7, VH3-11, VH3-15, VH3-21, VH3-23, VH3-20, VH3- 48, VH4-39, VH4-59, and VH5-5I.

[0228] Human engineered antibodies in addition to those disclosed herein exhibiting similar functional properties according to the present disclosure can be generated using several different methods. The specific antibody compounds disclosed herein can be used as templates or parent antibody compounds to prepare additional antibody compounds. In one approach, the parent antibody compound CDRs are grafted into a human framework that has a high sequence identity with the parent antibody compound framework. The sequence identity of the new framework will generally be at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the sequence of the corresponding framework in the parent antibody compound. This grafting may result in a reduction in binding affinity compared to that of the parent antibody. If this is the case, the framework can be back-mutated to the parent framework at certain positions based on specific criteria (Co et al., 1991). Additional references describing methods useful in humanizing mouse antibodies include U.S. Patent Nos. 4,816,397; 5,225,539, and 5,693,761; computer programs AB MOD and ENCAD (Levitt, 1983); and the method of Winter and coworkers (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988).

[0229] The identification of residues to consider for back-mutation can be carried out as follows: When an amino acid falls under the following category, the framework amino acid of the human germ-line sequence that is being used (the “acceptor framework”) is replaced by a framework amino acid from a framework of the parent antibody compound (the “donor framework”): the amino acid in the human framework region of the acceptor framework is unusual for human frameworks at that position, whereas the corresponding amino acid in the donor immunoglobulin is typical for human frameworks at that position; the position of theAttorney Docket No. AETN0001-401-PC amino acid is immediately adjacent to one of the CDRs; or any side chain atom of a framework amino acid is within about 5-6 angstroms (center-to-center) of any atom of a CDR amino acid in a three dimensional immunoglobulin model.

[0230] When each of the amino acids in the human framework region of the acceptor framework and a corresponding amino acid in the donor framework is generally unusual for human frameworks at that position, such amino acid can be replaced by an amino acid typical for human frameworks at that position. This back-mutation criterion enables one to recover the activity of the parent antibody compound.

[0231] Another approach to generating human engineered antibodies exhibiting similar functional properties to the antibody compounds disclosed herein involves randomly mutating amino acids within the grafted CDRs without changing the framework and screening the resultant molecules for binding affinity and other functional properties that are as good as or better than those of the parent antibody compounds. Single mutations can also be introduced at each amino acid position within each CDR, followed by assessing the effects of such mutations on binding affinity and other functional properties. Single mutations producing improved properties can be combined to assess their effects in combination with one another. Further, a combination of both of the foregoing approaches is possible. After CDR grafting, one can back-mutate specific framework regions in addition to introducing amino acid changes in the CDRs (Wu et al., 1999).

[0232] Applying the teachings of the present disclosure, a person skilled in the art can use common techniques, e.g., site-directed mutagenesis, to substitute amino acids within the presently disclosed CDR and framework sequences and thereby generate further variable region amino acid sequences derived from the present sequences. All alternative naturally occurring amino acids can be introduced at a specific substitution site. The methods disclosed herein can then be used to screen these additional variable region amino acid sequences to identify sequences having the indicated in vivo functions. In this way, further sequences suitable for preparing human engineered antibodies and antigen-binding portions thereof in accordance with the present disclosure can be identified. Preferably, amino acid substitution within the frameworks is restricted to one, two, or three positions within any one or more of the 4 light chain and / or heavy chain framework regions disclosed herein. Preferably, amino acid substitution within the CDRs is restricted to one, two, or three positions within any one or more of the 3 light chain and / or heavy chain CDRs. Combinations of the various changes within these framework regions and CDRs described above are also possible.Attomey Docket No. AETN0001-401-PC

[0233] The antibodies of the present disclosure can be used as medicaments in human medicine, administered by a variety of routes. Most preferably, such compositions are for parenteral administration. Such pharmaceutical compositions can be prepared by methods well known in the art and comprise an antibody as disclosed herein or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.Pharmaceutical Compositions and Methods of Treatment

[0234] The presently disclosed subject matter provides compositions comprising a presently disclosed anti-ac-tau antibody or an antigen-binding fragment thereof. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0235] Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which may enhance the shelf life or effectiveness of binding proteins. The compositions of an injection can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to a subject.

[0236] Any suitable method or route can be used to administer an anti-ac-tau antibody or binding fragment thereof. Routes of administration include, but are not limited to, intravenous, subcutaneous, intramuscular, intrathecal, pleural, intrapleural, topical, and direct administration. In certain embodiments, the administration is intravenous. In certain embodiments, the intravenous administration is by injection. In certain embodiments, the intravenous administration is by infusion.

[0237] The presently disclosed subject matter provides various methods of using the anti- ac-tau antibodies or antigen-binding fragments thereof, and the composition disclosed herein. For example, the presently disclosed subject matter provides methods for treating or ameliorating a disease or disorder in a subject. In certain embodiments, the method comprises administering one or more of the anti-ac-tau antibodies or antigen-binding fragments thereof, or the composition disclosed herein to the subject. In certain embodiments, the disease or disorder is associated with atetylated tau. In certain embodiments, the disease or disorder is associated with overexpression or overabundance of ac-tau.

[0238] In certain embodiments, the disease or disorder is a tauopathy. In certain embodiments, the tauopathy is a primary tauopathy. In certain embodiments, the primary tauopathy is chosen from frontotemporal dementia (FTD), for example FTD with TAR DNA-Attorney Docket No. AETN0001-401-PC binding protein 43 (TDP-43), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick’s disease (PiD) and argyrophilic grain disease (AGD). In certain embodiments, the tauopathy is a secondary tauopathy. In certain embodiments, the secondary tauopathy is chosen from Alzheimer’s Disease (AD), cerebral amyloid angiopathy (CAA), traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), and Down Syndrome.

[0239] It was hypothesized that anti-ac-tauK174 and / or anti-ac-tauK274 antibodies could be used to slow AD progression in subjects at early stages of the disease when amyloid had been deposited in the brain but where the downstream neurodegenerative cascade thought to be triggered by the amyloid deposition was still relatively early in its course (i.e., limited brain tissue loss has been produced and associated clinical deficits are at a minimum). In certain embodiments, the tauopathy to be treated is early Alzheimer’s Disease.

[0240] As disclosed herein, using an anti-ac-tauK174 antibody as an exemplary antiacetyl ated-tau antibody, a novel method of reducing brain amyloid levels in amyloid positive early AD subjects is disclosed. Also disclosed herein is a novel method of converting amyloid positive early AD subjects to amyloid negative comprising administering a composition comprising a therapeutically effective amount of at least one anti -acetylated tau antibody. A method of reducing clinical decline in amyloid positive early AD subjects comprising administering a composition comprising a therapeutically effective amount of at least one anti-acetylated-tau antibody is also disclosed herein. Beneficial results may be achieved when the subjects are ApoE4 positive.

[0241] Also disclosed is a method that prevents and / or delays onset of tauopathy or AD associated with TBI (TBI AD) in subjects comprising administering a composition comprising a therapeutically effective amount of at least one anti-ac-tau antibody. In some embodiments, the subject is ApoE4 positive. In some embodiments, the at least one anti- acetylated-tau antibody is an anti-ac-tauK174 antibody.

[0242] The results of the following assays demonstrate that the monoclonal antibodies and antigen-binding fragments thereof of the present disclosure are useful for treating a condition associated with aberrant tau or hyperacetylated tau activity such as Alzheimer’s Disease, Down’s syndrome, and CAA. Ac-tauK174 plays a critical role in the pathophysiology of tauopathy and TBI. Treatment with anti-ac-tauK174 antibodies yielded promising results in reducing tau pathology, improving cognitive function, and mitigating inflammatory responses. The following presents evidence for anti-acK174 immunotherapy as a promising therapeutic for the treatment of tauopathy-driven TBI in humans.Attorney Docket No. AETN0001-401-PCSequences of Proteins of Immunological Interest

[0243] In some embodiments, the at least one anti-ac-tau antibody is an ac-tauK174 antibody and comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:5 (HCDR1), SEQ ID NO:6 (HCDR2), and SEQ ID NO:7 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:8 (LCDR1), SEQ ID NO:9 (LCDR2), and SEQ ID NO: 10 (LCDR3).

[0244] The residues in antibody variable domains may be conventionally numbered according to a system devised by Kabat and colleagues (Kabat, 1991). In certain embodiments, the CDRs are identified using the IMGT numbering system (the international ImMunoGeneTics information system®) as described on the website “www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefmition.htmr’, also accessible at “www.imgt.org” (accessed on November 3, 2024). The IMGT numbering system is used in the present specification.

[0245] The Kabat and IMGT residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the Kabat and IMGT numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure.

[0246] The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (HCDR1), residues 50-65 (HCDR2) and residues 95-102 (HCDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (LCDR1 ), residues 50-56 (LCDR2) and residues 89-97 (LCDR3) according to the Kabat numbering system.

[0247] The CDRs of the heavy chain variable domain are located at residues 27-38 (HCDR1), residues 56-65 (HCDR2) and residues 105-117 (HCDR3) according to the IMGT numbering system. The CDRs of the light chain variable domain are located at residues 27-38 (LCDR1 ), residues 56-65 (LCDR2) and residues 105-117 (LCDR3) according to the IMGT numbering system.

[0001] The CDRs of the heavy chain variable domain are located at residues 27-38 (HCDR1), residues 56-65 (HCDR2) and residues 105-117 (HCDR3) according to the IMGT numberingAttorney Docket No. AETN0001-401-PC system. The CDRs of the light chain variable domain are located at residues 27-38 (LCDR1), residues 56-65 (LCDR2) and residues 105-117 (LCDR3) according to the IMGT numbering system.

[0002] The following general rules disclosed in www.bioinf.org.uk: Prof. Andrew C.R. Martin’s Group and reproduced in Table 11 below may be used to define the CDRs in an antibody sequence that includes those amino acids that specifically interact with the amino acids comprising the epitope in the antigen to which the antibody binds. There are rare examples where these generally constant features do not occur; however, the Cys residues are the most conserved feature.Table 11. Table of CDR Definitions1 Some of these definitions (particularly for Chothia loops) may vary'.2 Any of the numbering schemes can be used for these CDR definitions, except the contact definition uses the Chothia or Martin (Enhanced Chothia) definition.3 The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. (This is because the Kabat numbering scheme places insertions at H35A and H35B).* If neither H35A nor H35B is present, the loop ends at H32• If only H35A is present, the loop ends at H33Attorney Docket No. AETN0001-401-PC• If both H35A and H35B are present, the ioop ends at H34

[0248] It is understood that the molecules of the present disclosure may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions. Table 1 shows exemplary conservative amino acid substitutions. In some embodiments, the antibody constant domain can comprise an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgGl antibody and differ by one or more substitution(s).Table 1 - Conservative amino acid substitutionsAttorney Docket No. AETN0001-401-PC

[0249] In some embodiments, the at least one anti-ac-tau antibody comprises a human constant region. In some embodiments, the human constant region of the at least one anti-ac- tau antibody comprises a heavy chain constant region chosen from IgGl, lgG2, lgG3, lgG4, IgM, IgA, IgE, and any allelic variation thereof known in the art. Any one or more of such sequences may be used in the present disclosure. In some embodiments, the heavy chain constant region is chosen from IgGl and allelic variations thereof.

[0250] Exemplary polypeptide chains of heavy and light chain constant regions of the IgGl, IgG2, and IgG4 subclasses are shown below in SEQ ID NO:4 through SEQ ID NO:9 in Table 2. An example of an IgGl mutated sequence has the amino acid sequence set forth in SEQ ID NO:5.

[0251] Table 2 - Exemplary Constant DomainsAttorney Docket No. AETN0001-401-PC

[0252] In some embodiments, the anti-ac-tau antibody of the present disclosure comprises two heavy chains, each heavy chain comprising a variable domain and a constant domain, the heavy chain variable domain comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, and two light chains, each light chain comprising a variable domain and a constant domain, the light chain variable domain comprising complementarity determining regions LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-ac-tau antibody of the present disclosure comprises a heavy chain constant region that is an Ig gamma-1 chain C region (e.g., NCBI ACCESSION: P01857), and a light chain constant region that is an Ig kappa chain C region (e.g., NCBI ACCESSION: P01834).

[0253] The amino acid sequence of human IgGl constant region is known in the art and set forth in SEQ ID NO:4.

[0254] In some embodiments, the human constant region of the at least one anti-ac-tau antibody comprises a light chain constant region chosen from K-l-chain constant regions and any allelic variation thereof as discussed in the Kabat report. Any one or more of such sequences may be used in the present disclosure. In some embodiments, the light chain constant region is chosen from k and allelic variations thereof. The amino acid sequence of human K chain constant region is known in the art and set out in SEQ ID NO:8.Attorney Docket No. AETN0001-401-PC

[0255] In some embodiments, the at least one anti-ac-tau antibody comprises human heavy and light chain variable region frameworks. In some embodiments, the at least one anti-ac-tau antibody is an anti-ac-tauK174 antibody and comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:28, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:29.

[0256] In some embodiments, the at least one anti-ac-tau antibody an anti-ac-tauK174 antibody and comprises a human IgGl heavy chain constant region, and a human Ig kappa light chain constant region. In some embodiments, the at least one anti-ac-tauK174 antibody comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO:3, and a light chain constant region comprising an amino acid sequence of SEQ ID NO:4.

[0257] In some embodiments, an anti-ac-tauK174 antibody comprises (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:20 and (b) a light chain variable domain comprising the amino acid sequence of SEQ ID NO:21. The full length sequence of an anti-ac-tauK174 antibody heavy chain is set forth in SEQ ID NO:78 and the full length sequence of an anti-ac-tauK174 antibody light chain is set forth in SEQ ID NO:79.

[0258] Other non-limiting examples of suitable antibodies for use as the at least one anti- ac-tauK174 antibody in the present disclosure include AbMl, AbM2, AbM3, AbM4, AbM5, AbHl, AbH2, AbH3, AbH4, AbH5, AbH6, AbH7, AbH8 or AbH9.Therapeutically effective amount of anti-ac-tau antibodies

[0259] The methods of the present disclosure comprise administering to a subject a composition comprising a therapeutically effective amount of at least one anti-ac-tau antibody. As used herein, the term a “therapeutically effective amount” refers to an amount of a compound or pharmaceutical composition sufficient to product a desired therapeutic effect.

[0260] One of ordinary skill in the art will understand that the therapeutically effective amount of the at least one anti-ac-tau antibody administered to a subject may depend upon a number of factors including pharmacodynamic characteristics, route of administration, frequency of treatment, and health, age, and weight of the subject to be treated and, with the information disclosed herein, will be able to determine the appropriate amount for each subject.Anti-Ac-tau antibodies and antigen-binding fragments thereof

[0261] In some embodiments, an anti-ac-tau antibody or an antigen-binding fragment thereof comprises one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and / or light chain variable domains as compared to the corresponding germline sequences from which the individual antibodies were derived.Attorney Docket No. AETN0001-401-PCSuch mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germ line sequences available from, for example, public antibody sequence databases. The antibodies of the present disclosure may comprise antigen-binding fragments which are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues within the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germ line sequence from which the antibody was originally derived). Furthermore, the antibodies or antigen-binding fragments may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germ line sequence while certain other residues that differ from the original germ line sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies or antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties, reduced immunogenicity, etc. Antibodies and antigenbinding fragments obtained in this general manner are encompassed within the present disclosure.

[0262] The present disclosure also includes antibodies or antigen-binding fragments that comprise variants of any of the VH, VL, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includesAttorney Docket No. AETN0001-401-PC antibodies or antigen-binding fragments comprising VH, VL, and / or CDR amino acid sequences with, e.g., 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 conservative amino acid substitution(s) relative to any of the VH, VL, and / or CDR amino acid sequences disclosed herein. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamateaspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix (Gonnet et al., 1992). A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0263] The present disclosure also includes antibodies or antigen-binding fragments comprising a VH, VL, and / or CDR amino acid sequence that is substantially identical to any of the VH, VL, and / or CDR amino acid sequences disclosed herein. In some embodiments, an antigen-binding molecule comprises VH, VL, and / or CDR amino acid sequence having at least 85% sequence identity, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, to a sequence disclosed in Table 1. In some embodiments, an antigen-binding molecule comprises VH, VL, and / or CDR amino acid sequence having at least 85% sequence identity, e.g, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, to a sequence disclosed in Table 5 or Table 6, wherein the differences in the amino acid residue(s) relative to the sequence disclosed in Table 5 or Table 6 are conservative substitutions or moderately conservative substitutions.

[0264] For formulations comprising two or more anti-ac-tau antibodies, in some embodiments each antibody is present in an amount from about 1 mg / mL to about 250Attorney Docket No. AETN0001-401-PC mg / mL, e.g., about 5 mg / mL to about 200 mg / mL, about 10 mg / mL to about 150 mg / mL, about 20 mg / mL to about 100 mg / mL, about 50 mg / mL to about 150 mg / mL, or about 50 mg / mL to about 100 mg / mL. In some embodiments, each antibody is present in an amount of about 1 mg / mL; about 2 mg / mL; about 5 mg / mL; about 10 mg / mL; about 15 mg / mL; about 20 mg / mL; about 25 mg / mL; about 30 mg / mL; about 35 mg / mL; about 40 mg / mL; about 45 mg / mL; about 50 mg / mL; about 55 mg / mL; about 60 mg / mL; about 65 mg / mL; about 70 mg / mL; about 75 mg / mL; about 80 mg / mL; about 85 mg / mL; about 90 mg / mL; about 95 mg / mL; about 100 mg / mL; about 105 mg / mL; about 110 mg / mL; about 115 mg / mL; about 120 mg / mL; about 125 mg / mL; about 130 mg / mL; about 135 mg / mL; about 140 mg / mL; about 145 mg / mL; about 150 mg / mL; about 155 mg / mL; about 160 mg / mL; about 165 mg / mL; about 170 mg / mL; about 175 mg / mL; about 180 mg / mL; about 185 mg / mL; about 190 mg / mL; about 195 mg / mL; or about 200 mg / mL. In certain embodiments, the pharmaceutical formulations contain about 25±3 mg / mL of each antibody, about 50±5 mg / mL of each antibody, about 75±7.5 mg / mL of each antibody, or about 100±10 mg / mL of each antibody. In some embodiments, each antibody is present in the formulation in the same amount. In some embodiments, the antibodies are present in the formulation in different amounts. In some embodiments, the antibodies (e.g., REGN1908 and REGN1909) are present in a molecular ratio of 1 : 1 or a molecular ratio of about 1 : 1. In some embodiments, the antibodies e.g., AbMl or AbM2) are present in a molecular ratio of about 1.5: 1 or about 1 : 1.5. In some embodiments, the antibodies (e.g., AbMl and AbM2) are present in a molecular ratio of about 2: 1 or about 1 :2.EXAMPLESExample 1: Production of AntibodiesGeneration of new anti -acetylated tau tac-tauK I 74) monoclonal antibodies:

[0265] Mouse monoclonal antibodies against ac-K174 tau were developed by testing candidates against the acetylated peptide and counter-testing against a non-acetylated control peptide. An example of one isoform of human microtubule-associated protein tau is set forth in SEQ ID NO: 1 (e.g., NCBI ACCESSION: NM_005910.6). Synthetic human tau peptide antigens were used for immunization (amino acids 163-185) with acetylated lysine 174 (SEQ ID NO:2 and SEQ ID NO:3), or acetylated lysine 274.

[0266] Animals were immunized with at least the following peptides:

[0267] AbM5 (Biotin-KGQANATRIPA(Ac)KTPPAPKTPPSS-amide) SEQ ID NO:2

[0268] AbMl (Ac-CKGQANATRIPA(KAc)TPPAPKTPPSS-amide) SEQ ID NO:3Attorney Docket No. AETN0001-401-PC

[0269] The ac-K174 peptide was injected into either wild-type mice or tau knockout mice to enhance immune reaction. 10 pg / peptide was immunized using first complete and then incomplete Freund’s adjuvant on day 0, 14, and 28. On day 63, mice were immunized with 2 pg of each peptide. For AbMl, female BALB / c, Fl, and CD1 mice were immunized with a cocktail of synthetic peptides coupled to Keyhole Limpet Hemocyanin (KLH) to increase their immunogenicity and trigger a stronger immune response. Spleens of 6 animals were removed and fused by electroporation 3 days later. After splenectomy and hybridoma fusion, positive clones were validated for targeting peptide binding by ELISA and Western.Supernatants of dozens of subclones were screened for specificity against ac-K174 tau, where AbMl and AbM5 were identified as ac-K174-tau-specific antibodies. Specifically, after 1- day of rest in tissue culture media, cells were plated into semi-solid media containing the peptides conjugated to phycoerythrin. Cells were cultured until colonies were observed. Colonies positive for PE were picked with a clone PIX2 colony-picking robot, deposited into 96-well plates, and screened by flow cytometry for binding to bead-conjugated peptides.

[0270] ELISA-based testing of specificity and immunoreactivity allowed for selection of two top antibodies: AbM5 was generated in wild-type mice, and AbMl was generated in tau knockout (KO) mice (FIG. 1 A). Both AbMl and AbM5 detected strong ac-tau signal in HEK293T cells co-transfected with wild-type (WT) human tau (hTau) and p300, the acetyltransferase that acetylates tau (Min et al., 2015; Min et al., 2010). Thus, the two anti-ac- tauK175 antibodies used in this study were independently generated. AbM5 was generated from wild-type mice before AbMl and was used in comprehensive animal studies. AbMl was applied to biomarker and seeding assays.

[0271] When a K174R point mutation was introduced, this blocked AbMl and AbM5 signals, while introducing the K274R mutation did not (FIG. IB, FIG. 1C). The impact of given residues on antibody binding helped to identify the characteristics, and the sequence required for antibody recognition.

[0272] The antibodies from AbMl and AbM5 were then tested in PS 19 mice, which are hTau transgenic mice that carry the P301S mutation that has been shown to cause frontotemporal lobar degeneration (FTLD) in humans (Lopez-Gonzalez et al., 2015; Yoshiyama et al., 2007).

[0273] Both AbMl and AbM5 detected ac-tauK174 immunoreactivities in the hippocampal lysate of PS 19 mice at different ages, at the same molecular weight as hTau (FIG.Attomey Docket No. AETN0001-401-PCID, FIG. E). The signal was absent in WT mice and tau KO mice (FIG. ID, FIG. IE). Antibodies AbMl, AbM2, AbM3 and AbM4 in Table 5 were made by this process.

[0274] RNA was extracted from monoclonal hybridomas for discovery of the sequences at the variable region of the heavy and light chains. RT-PCR was conducted via gene-specific primers from Meyer et al. (2019). Amplicons were cloned into TOPO-TA sequencing vectors. Resulting colonies were Sanger sequenced.Example 2: Binding Affinity to soluble ac-tauK174 peptides

[0275] Surface plasmon resonance (SPR) was performed by Genscript® in a conventional format, and measurements were conducted with the BIACORE® 2000 instrument to measure binding of anti-ac-tauK174 antibodies with ac-tauK174 synthetic peptides.

[0276] All measurements were performed at 25°C. Peptides were dissolved in PBS at 5mg / ml prior to dilution for binding experiments. Samples were dissolved in HBS-EP buffer (150 mM sodium chloride, 3 mM EDTA, 0.005% (w / v) surfactant P-20, and 10 mM HEPES, pH 7.4). Binding was evaluated using multiple analytical cycles of antibody capture, peptide injection / association, prolonged buffer flow for dissociation, and surface regeneration. For the antibody capture step, a Series S Sensor Chip CM5 was immobilized with either AbM5 or AbMl peptides. Antibodies were injected onto the CM5 Chip and immobilized with antimouse antibody as Capture. Lys-Ac antigen was diluted and injected over the surface of flow cells 1 and 2 (association phase), followed by injection of running buffer (dissociation phase). The flow rate was 50 pL / min, and the chip surface was regenerated using 20 pL of 10 mM glycine hydrochloride, pH 1.5. The binding affinity (KD) was then obtained from association and dissociation rates for each cycle using a 1 : 1 binding model in the BIA evaluation analysis software.

[0277] The SPR assay revealed that the ac-tauK174 antibodies recognize ac-tauK174 specifically, with the dissociation constant (KD) for the ac-tauK174 synthetic peptide being 1.15 x 109for AbM5 (FIG. IF, FIG. H) and 7.62 x 1010for AbMl (FIG. 1G, FIG. H). Together, these data demonstrated the specificity and affinity of the monoclonal ac-tauK174 antibodies disclosed herein.Example 3: Anti-ac-tauK174 antibody rescues neurobehavioral impairment and ameliorates neuropathology in PS19 mice

[0278] The potential therapeutic effect of anti-ac-tauK174 antibody was investigated by testing AbM5 in PS 19 mice. At 25 mg / kg, AbM5 was injected intraperitoneally (IP) in 6-Attorney Docket No. AETN0001-401-PC month-old (mo) PS 19 mice, and WT littermates, once a week for 16 weeks; PBS was injected as control. Mice at 9 months of age were subjected to behavioral and cognitive tests to assess motor and memory functions, while treatment was continued (FIG. 2A). At the end of the treatment, PS19-PBS mice exhibited significant percent weight loss (16.38%), compared to WT littermates injected with PBS. By contrast, antibody-treated PS19 mice showed significantly less percent weight loss (7.46%) (FIG. 2B), suggesting an overall positive effect on animal health.

[0279] Next, motor strength and coordination was assessed through the hindlimb extension reflex test. WT mice responded to tail suspension by immediately extending both hindlimbs, which is considered normal behavior (FIG. 2C, left). As disease progressed, PS 19 mice exhibited abnormally clasped hindlimbs when suspended by the tail (FIG. 2C, right). Intermediary phenotypes ranging from normal to abnormal (0.75, 0.5, 0.25) were scored. The abnormal clasping behavior was prevented when PS 19 mice were treated with the anti-ac- K174 antibody from AbM5 (FIG. 2D).

[0280] Spatial learning and memory was then assessed by Morris water maze. Mice were trained to find a platform hidden under opaque water. The time required to find the platform across multiple days was considered a direct measure of learning. During the training period, PBS-treated PS 19 mice found the escape platform more slowly than PBS-treated WT mice (FIG. 2E). PS19 mice treated with the anti-ac-K174 antibody from AbM5, however, located the escape location more quickly, indicating improved learning (FIG. 2E).

[0281] At 72 h post-training (Day 8), spatial memory was assessed by removing the escape platform and measuring the time mice spent searching in its previous location, termed the target quadrant. WT-PBS mice spent more time in the target quadrant, whereas PS19-PBS mice showed no preference for this region (FIG. 2F), indicating impaired memory. Treatment with the anti-ac-K174 antibody from AbM5 prevented memory impairment in PS19 mice (FIG. 2F).

[0282] The neuropathology of the animals was examined at 10 months of age. PS19-PBS mice showed significant hippocampal volume loss compared to WT-PBS mice (FIG. 3A, FIG. 3B). Anti-ac-K174 antibody (AbM5) treatment yielded a trend towards reduced neuronal atrophy (FIG. 3 A, FIG. 3B, / ? = 0.09) and decreased accumulation of pathological p- tau (S202 / T205, AT8) deposition in the hippocampus (FIG. 3D, FIG. 3E, / ? = 0.08). A correlation analysis of the relationship between pathology and behavioral phenotypes was then conducted via rank-summary score method, with raw scores replaced by quantile scores for each trial and then averaged for each subject. This showed that the rank summary scoreAttorney Docket No. AETN0001-401-PC based on latency in the learning phase of Morris water maze of individual animals negatively correlates with hippocampal volume (FIG. 3C). Taken together, these data indicate that anti- ac-tauK174 immunotherapy reduced tau-induced motor and cognitive impairment.Example 4: Anti-ac-tauK174 antibody reduces fibril-induced tan spreading in PS19 mice

[0283] Whether anti-ac-tauK174 antibody could affect pathological tau spreading was investigated by testing a second ac-K174 antibody (AbMl) in a fibril-induced tau spreading model in vivo. In this model, inoculation of exogenous tau seeds induced profound tau spread in 3.5-month-old PS19 mice within one month. K18-P301L tau fibrils were injected in the dentate gyrus of the hippocampus of PS19 mice (AP=-2.5, ML = 2, DV = 1.8) to induce tau spreading (FIG. 3F). AbMl and control mouse IgG2a were IP injected weekly at 25 mg / kg for 4 weeks. Injection of tau fibrils induced tau aggregates (MCI -positive) that spread to the contralateral hippocampus within 4 weeks (FIG. 3G). AbMl treatment significantly reduced the amount of aggregated tau in the contralateral side, providing evidence that anti-ac- tauK174 immunotherapy reduces tau spreading in vivo (FIG. 3G, FIG. 3H).Example 5: Anti-ac-tauK174 immunotherapy prevents TBI-induced neuropathology in PS19 mice

[0284] Traumatic Brain Injury (TBI) is associated with exacerbated tau pathology (see, e.g., (Zanier et al., 2018), and ac-tau is established as the first blood biomarker of post-TBI neurodegeneration in both mice and humans that directly reflects the abundance of a therapeutic target in the brain (Shin et al., 2021). Whether anti-ac-tauK174 immunotherapy could prevent trauma-induced neurop athologi cal changes, transcriptomic alterations, and deficits in memory function was investigated (FIG. 4A).

[0285] PS 19 male and female mice at 8 months of age were subjected to TBI or sham injury and treated with AbM5 or PBS via IP injection started one day prior to surgery and continued weekly for 5 weeks (FIG. 4A, B). AbM5 treatment in PS 19 mice produced a trend of reduced weight loss compared to both sham and TBI-PBS treated groups (FIG. 4C). We examined neuropathological changes in these animals, where PS 19 mice subjected to TBI showed increased AT8-positive phosphorylated-tau (p-tau) deposition in the cortex compared to sham group (FIG. 4D-F). We next assessed microgliosis in these animals by immunohistochemistry for Iba-1. As expected, TBI mice showed increased Iba-1 signal in the cortex (FIG. 4G, H). Anti-acK174 antibody treatment, however, significantly reduced Iba-1 signal (FIG. 4G-I), and this signal positively correlated with AT8 signal in the lower cortices (FIG. 4J).Attorney Docket No. AETN0001-401-PCExample 6: Single nuclei RNA-seq reveals microglial activation and oligodendrocyte myelination impairment in TBI are rescued by anti-ac-tauK174 immunotherapy

[0286] To characterize the therapeutic effects of anti-ac-tauK174 (AbM5) immunotherapy in PS 19 mice subjected to traumatic brain injury (TBI) at the transcriptomic level, single nuclei RNA-seq (snRNA-seq) using cortical tissues from 6 to 7 mo PBS-injected (PS 19- Sham -PBS), TBI + PBS-injected (PS19-TBI-PBS), and TBI + Clone 1 -injected (PS19- TBI-Ab) was performed with PS 19 transgenic male mice and WT littermates from FIG. 4. Following an established snRNA-seq protocol, 120,090 nuclei from all four conditions (n = 4 per condition) were sequenced. After removal of potential multiples using DoubletFinder™ and filtering for low-quality nuclei determined by thresholding gene counts, UMI counts, and percentage mitochondrial genes per nuclei, 99,168 nuclei were selected for downstream analysis. Using reference gene markers for annotations, major cell types in the brain that were similarly represented within each group and individual mouse were identified.

[0287] 2,718 microglial cells (841 from WT, 630 from PS19-Sham-PBS, 562 from PS19-TBI-PBS, 685 from PS19-TBI-Ab) were re-clustered to investigate how anti-ac-tauK174 altered microglial activation states in PS 19 mice with TBI. The microglial population from the four conditions exhibited five clearly defined subclusters (FIG. 5A). Analyses of the distribution of these five subclusters revealed that bulk sequencing of TBI-specific microglia dominated the composition of subcluster 1 (MG1) and that immunotherapy significantly reduced MG1 cell ratio to levels similar to WT and PS19-Sham-PBS mice (FIG. 5B). Ingenuity Pathway Analysis (IP A) revealed that many upstream regulators of MG1 were associated with pro-inflammatory immune responses, including cytokines (IL2, IL12, TNF, IL10) and transcription regulators (SPIB, SPI1, REL, STA T!) (FIG. 5C).

[0288] Microglia reactivity is a pathological hallmark of TBI, with chronic microglial activation having been reported in human patients after moderate-to-severe TBI. A mixed microglial phenotype following TBI in which reactive microglia simultaneously express pro- inflammatory and anti-inflammatory markers is known in the art. The expression levels of disease-associated microglial (DAM) genes between conditions by pseudo-bulk analysis were compared. Expression levels of Tyrobp, B2m, and Clqa were elevated in PS19 mice in both TBI and sham-injury conditions and were lowered by antibody treatment (FIG. 5D). Thus, treatment with anti-ac-tauK174 antibody blunted pathological microglia reactivity.

[0289] In is known in the art that TBI contributes to neuropsychiatric impairment and chronic neuronal cell death through progressive axonal degeneration. As TBI causes cerebral demyelination and impairment of remyelination mechanisms through secondaryAttorney Docket No. AETN0001-401-PC oligodendrocyte dysfunction, 13,761 oligodendrocytes (3,395 from WT, 4,555 from PS19- Sham-PBS, 2,894 from PS19-TBI-PBS, 2,917 from PS19-TBI-Ab) were re-clustered to investigate whether anti-ac-tauK174 could alter oligodendrocyte-mediated myelination in PS 19 mice with TBI. The oligodendrocyte population from the four conditions exhibited six clearly defined subclusters (FIG. 5E, Sup. Table 2), and cluster 5 (OL5) was unique to PS 19- TBI-Ab brains (FIG. 5F). OL5 markers were compared to a recently characterized disease- associated oligodendrocyte (DAO) gene signature from a meta-analysis of AD and multiple sclerosis single-cell RNA-seq (scRNA-seq) datasets. A strong positive correlation (R = 0.4, / ? = 2.7e-6) between the cluster-agnostic gene markers associated with DAOs and OL5 markers (FIG. 5G) was found, suggesting OL5 is comprised of oligodendrocytes with a disease- associated activation state. Thus, anti-ac-tauK174 treatment significantly reversed the TBI- induced states enriched with DAOs. Expression levels of established myelination-related genes between conditions by pseudo-bulk analysis were compared (FIG. 5H). Antibody treatment in TBI restored the low expression level of myelin basic protein (Mbp) to normal WT levels, indicating protection from TBI-induced axonal demyelination. Levels of both SoxlO and Tcf7!2, which cooperate during oligodendrocyte maturation to promote myelination, were also decreased by TBI and rescued by anti-ac-tauK174 immunotherapy.

[0290] Proinflammatory cytokines, including interferon (IFN) gamma, have been reported as key mediators of neuroinflammation in both tauopathy and TBI. It is known in the art that type I IFNs also contribute to aberrant inflammatory responses in tauopathy and after TBI, and that Arid5b, Bpgm, Auts2, and Uspl8 are human hallmark IFN gamma genes. Small IFN-responsive oligodendrocyte subpopulations have been found to be characterized by the expression of several IFN response-related genes, including Ifi27l2a and B2m. PS19-TBI oligodendrocytes exhibited higher levels of IRO genes (Bpgm, Ifi27l2a, B2m) and IFN- gamma genes (Arid5b, Auts2), which were all reduced with anti-ac-tauK174 treatment (FIG. 5H). Uspl8 is a negative regulator of IFN, and its deletion causes myelin pathology. Expression levels of Uspl8 are increased with anti-ac-tauK174 treatment after a TBI-induced depletion (FIG. 5H).

[0291] Together, expression levels of these markers indicate that anti-ac-tauK174 rescues TBI-induced transcriptomic shifts in oligodendrocyte function and activation. This transcriptomic profiling of the PS19-TBI model supports the therapeutic potential of anti-ac- tauK174 as a treatment for restoring oligodendrocyte remyelination capabilities and ameliorating disease-associated microglial responses induced by TBI.Example 7: Anti-ac-tauK174 treatment ameliorates behavioral impairments andAttorney Docket No. AETN0001-401-PC reduces tau seeding in vitro

[0292] Whether anti-ac-tauK174 (AbM5) immunotherapy could prevent trauma-induced deficits in memory function in TBI-subjected WT and PS 19 mice was investigated. To test for cortical-dependent short-term memory, the novel object recognition (NOR) task was utilized 3 weeks post-injury. In this task, animals were exposed to two identical objects, and then 5 min later one of the objects was replaced with a novel object (FIG. 6 A). The animal’s preference for the novel object (discrimination index, as measured by times spent with each object) provided a measure of short-term recognition memory because mice are inherently more interested in a new object. Sham animals spent more time exploring the novel object, which indicated intact short-term memory for the objects to which they had previously been exposed (FIG. 6B). TBI mice, however, displayed significantly impaired novel object discrimination, denoting deficits in short-term memory (FIG. 6B). Remarkably, anti-ac- tauK174 treatment of TBI mice completely preserved normal memory in this task (FIG. 6B). Upon examining neuropathological changes, mice with TBI displayed significantly elevated levels of ac-tau and t-tau, but not p-tau, by western blot compared to those that received the sham injury (data not shown).

[0293] AbM5 treatment did not significantly affect levels of ac-tau, p-tau, or total tau (data not shown). While no marked difference in p-tau was detected, there was a negative correlation between p-tau levels and memory performance with respect to individual animals (FIG. 6C). Iba-1 signal in the lower cortex (LOI 2) also negatively correlated with memory performance (FIG. 6D), as expected since NOR involves the entorhinal cortex.

[0294] To investigate whether anti-ac-tauK174 antibody could affect pathological tau spreading associated with TBI, a Tau RD P301S FRET biosensor assay was utilized. In vivo antibody treatment efficacy in reducing tau seeding activity in vitro was assessed (FIG. 6E). Specifically, Tau RD HEK293T cells were seeded with PS19 lysate. A significant increase in CFP / YFP FRET signal with TBI and a downward trend with antibody treatment was observed (p = 0.0634) (FIG. 6F, FIG. 6G). These findings highlight the functional benefits of anti-ac-tauK174 immunotherapy, which both prevents TBI-induced memory deficits in PS19 mice and reduces tau seeding and spreading in human cells.

[0295] To further explore the relevance of lowering ac-tauK174 levels by immunotherapy in humans, it was examined whether specific acetylation of tau at K174 is increased in TBI subjects, as detected by AbMl. Notably, plasma ac-tauK174 was increased by 50% within 24 h of TBI, compared to controls (p < 0.001), and remained consistently elevated across all time points (FIG. 6H, FIG. 61). Taken together, the data suggest that ac-tauK174 represents aAttomey Docket No. AETN0001-401-PC potential early therapeutic target after TBI that may mitigate the initial inflammatory responses associated with pathological tau acetylation.Example 8: Selection of murine ac-tauK174 antibody for humanization

[0296] Two murine anti-ac-tauK174 antibodies, AbMl and AbM2, were developed as potential candidates for humanization. Both antibodies have similar KD in the nanomolar range (AbMl: 1.59 x 10'9M vs AbM2: 1.82 x 10-9M). To further evaluate their potentials for humanization, murine-human chimeras of AbMl and AbM2 were generated. The variable regions of murine AbMl and AbM2 were cloned and sequenced by RACE (Rapid Amplification of cDNA Ends). Positive clones were selected and the DNA and amino acid sequences for VH and VL were analyzed. The construct expressing VH and VL regions were cloned into a proprietary high expression mammalian vector (human IgG2 expression vectors). The expression constructs were transfected in HEK293 cells, and the transient product of chimeric antibodies were purified by Protein A purification.

[0297] Both murine-human chimeric antibodies have very tight binding to ac-K174 tau peptide with a KD (1 / S) <1.0X10-07, which is below the detection limit of Octet. To compare the affinity of these two chimeric antibodies, a direct binding ELISA was used. The ELISA assay covers a broader range of antibody concentration and is ideal when the KD is too small to be determined by Octet system. AbMl had an EC50 value of ~3 ng / ml, indicating a higher affinity than AbM2 (~17 ng / ml) (FIG. 7). Together with additional evidence, including that AbMl specifically detected ac-tau tangles in AD patients by immunohistochemistry (IHC) and extracellular ac-tau in PS 19 mice and iPSC-derived human neurons by ELISA, and efficiently reduced tau spreading in vitro and in vivo, AbMl was selected as lead candidate for humanization.Example 9: Humanization of AbMl against ac-tauK174

[0298] Because of the high affinity and specificity of the mouse AbMl and its efficacy in blocking tau seeding and spread, AbMl was humanized with the goal of developing the monoclonal antibody as a therapeutic for treating AD and other tauopathies. Nine (9) humanized clones AbHl- AbH9 were constructed based on parental mouse AbMl and produced in a HEK293 system (Table 5) for selection based on the rank of affinity and potency. To generate the clones, AbMl was sequenced and used for homology modeling. The 3D structure of AbMl was used to select two heavy chain (HC) and two light chain (LC) acceptor frameworks based on the overall sequence identity across the framework, matching interface position, similarly classed CDR canonical positions, and presence of N- glycosylation sites that would have to be removed. Three humanized heavy chains (HC1-Attorney Docket No. AETN0001-401-PCHC3) and light chains (LC1-LC3) were designed by fusing select parts of the AbMl sequence with the human framework sequences. Using the 3D structure, these humanized sequences were methodically analyzed by eye and computer modeling to maximize the amount of human sequence in the final humanized antibodies while retaining the original antibody specificity. The humanness of humanized chains was calculated by analyzing the primary sequences of the variable regions with a T20 “humanness” score system (LakePharma) (Gao et al., 2013). Humanized antibodies with a T20 score over 79 (heavy chain) and 85 (light chain) were accepted, to reduce the immunogenicity risk of the humanized antibodies. The humanized heavy and light chains were then combined to create 9 variant fully humanized antibodies. Full-length antibody genes were constructed by first synthesizing the variable region sequences. The sequences were optimized for expression in mammalian cells. These variable region sequences were then cloned into expression vectors that already contain human Fc domains. Plasmids for the indicated heavy and light chains were transfected into suspension HEK293 cells using chemically defined serum free media for antibody production. Full length antibodies were then purified from conditioned media using Mab Select™ SuRe Protein A resin (GE Healthcare). Assessment of the antibody expression levels is shown in Table 3.Table 3 - Binding kinetics, humanness T20 scores and expression levels of 9 humanized antibodies versus their parental murine clone.Attorney Docket No. AETN0001-401-PCExample 10: Affinity measurement of nine humanized clones

[0299] ELISA was performed to compare the affinities of 9 humanized clones to ac- tauK174 peptides. Tau peptide was coated onto a 96-well plate at 2 ug / mL overnight at 4°C. The plate was then blocked with 2% BSA in PBS for 1 hour at room temperature. An 8-point, 1 :4 dilution series of the antibodies, starting at 20 ug / mL, were added and incubated for 1 hour at room temperature. A goat-anti-human HRP secondary antibody was added and incubated for 1 hour at room temperature. All 9 humanized variants were observed to have comparable binding as the chimeric parental AbMl (FIG. 8).

[0300] Additional affinity measurements of the 9 clones were then performed using Octet. Binding experiments were performed on Octet HTX at 25°C. Biotin-peptide (0.1 pg / mL) was loaded onto Streptavidin (SA) biosensors. Loaded sensors were dipped into a serial dilution of 9 human IgG2 clones (start 100 nM, 1 :2 dilution, 7 points). Kinetic constants were calculated using a monovalent (1 : 1) binding model (Table 2). VH2+VL1 or VL2, VH3+VL1 or VL2 have the lower ends of KD(M) and were selected for further evaluation.Example 11: AbH5 clone specifically detected full length ac-tauK174 with high immunoreactivity

[0301] To evaluate binding capacity of 9 humanized clones to full length ac-tauK174, recombinant human tau was expressed in HEK293 cells together with p300, an acetyltransferase to acetylate tau at K174. Consistent with the Octet results, clones with low KD(M) showed strong immunoreactivity with ac-tauK174 (FIG. 9 A). Among them, AbH5 had the strongest immunoreactivity, similar to AbMl. When the lysine was mutated to an arginine (K174R), labeling by AbH5 was lost (FIG. 9B) indicating that AbH5 selectively detected tau acetylated at KI 74 like AbMl. In contrast, the antibody AbM3, which detects tau acetylated at lysine K274 effectively detected ac-tauK174R and the general tau antibody HT7 detected both acetylated and non-acetylated tau. Thus, the humanized anti-ac-tauK174 monoclonal antibody disclosed herein detects ac-tauK174 with high affinity and selectively similar to the original mouse monoclonal antibody, AbMl.Attorney Docket No. AETN0001-401-PCExample 12: AbH5 detected endogenous ac-TauK174 in brains from AD patients and PS19 mice

[0302] It was established that AbH5 detected ac-tauK174 endogenously expressed in the brains of human AD patients and in PS 19 mice. Human on human IHC kit (HRP / DAB) (Abeam) was used to avoid high background caused by the presence of human IgG in human AD brains. This is an indirect detection method that is designed for staining human primary antibodies on human tissues without background staining. AbH5 detected ac-tau inclusions in human AD brains and PS 19 brains in a similar manner as AbMl labeling and no AbH5 was detected in either healthy human brains or brains from a non-transgenic (NTG) control mouse (FIG. 10). These findings indicate that humanized AbH5 has similar binding affinity and selectivity for ac-tauK174 as mouse AbMl, which was derived from and also selectively binds to endogenous human ac-tau in brain tissue.Detailed MethodologyMice

[0303] Custom animal collections include TAU KO mice (#007251, Jackson), in addition to female BALB / c, CD1, and C57B6xBALB Fl strains. Mice were assigned into gender- and age-matched treatment groups in a randomized manner. The sample size for each experiment was determined based on previous experience with each of the animal models used. Male and female PS 19 mice at 2-3 months of age were purchased from the Jackson Laboratory and housed in a pathogen-free barrier facility at the University of California, San Francisco (UCSF) with a 12-h light / 12-h dark cycle and ad libitum access to food and water. For non- TBI immunotherapy experiments, all behavior experiments were performed during daylight hours. For TBI experiments, the behavior experiments were performed during dark hours.Surgical procedures

[0304] All animals were randomly assigned to each TBI or sham surgery group. Male and female animals were equally divided between groups. Animals were anesthetized and maintained at 2-2.5% isoflurane for the duration of surgery.

[0305] Sham surgery Sham with sutures were performed as previously described (Chou et al., 2017). Briefly, animals were secured to a stereotaxic frame with nontraumatic ear bars. A midline incision was made to expose the skull and the scalp was sutured.

[0306] Traumatic Brain Injury: Closed Head Injury. TBI surgery was performed as previously described (Chou et al., 2017; Krukowski et al., 2020).Attorney Docket No. AETN0001-401-PC

[0307] Briefly, animals were secured to a stereotaxic frame with nontraumatic ear bars, and the head of the animal was supported with foam before injury. Contusion was induced using a 5-mm convex tip attached to an electromagnetic impactor (Leica) at the following coordinates: anteroposterior, - 1.50 mm, and mediolateral, 0 mm with respect to bregma. The contusion was produced with an impact depth of 1 mm from the surface of the skull with a velocity of 5.0 m / s sustained for 300 ms. Any animals that had a fractured skull after injury were excluded from the study. Following impact the scalp was sutured. After all surgeries, the animal recovered in an incubation chamber set to 37 °C. Animals were returned to their home cage after showing normal walking and grooming behavior.Behavioral analysis Morris water maze

[0308] Experimenters were blind to the genotypes or treatments of the mice for all behavioral analyses. The water maze consisted of a pool (122 cm in diameter) containing opaque water (20 ± 1 °C) and a platform (14 cm in diameter) submerged 1.5 cm under the water. Hidden platform training (days 1-5) consisted of 10 sessions (two per day, 2 h apart), of three trials each. The mouse was placed into the pool at alternating drop locations for each trial. A trial ended when the mouse located the platform. The maximum trial time was 60 s. Mice that failed to find the platform within 60 s were led to it and placed on it for 15 s. For the probe trial, 72 h after the final reversal training trial, mice were returned to the pool with a new drop location in the absence of a hidden platform. Performance was measured with an Etho Vision™ video-tracking system (Noldus Information Technology). Visible platform training, where the platform was cued with a mounted black-and-white striped mast, was conducted for four trials after completion of probe trials. Swimming speed during the probe trials was recorded. The pre-established exclusion criterion was that mice that floated or did not swim would be excluded from analysis.Novel object recognition

[0309] All surgical sutures were healed prior to behavioral analysis. For one week prior to behavioral analysis, animals were handled for habituation to investigators and room settings. Behaviors were performed in dark rooms during the animals’ wake cycle. All behaviors were recorded using an overhead camera. All animal behaviors were performed, and videos were manually scored by investigators blinded to groups. Cortical-dependent short-term memory function was measured at three weeks post-surgery using a mouse novel object recognition (NOR) assay. The test environment consisted of an open field arena under dim lighting. Mice were allowed to explore the arena for two 10-minute periods for twoAttorney Docket No. AETN0001-401-PC consecutive days (habituation phase). On day three (training phase), two identical objects (red Lego™ blocks) were secured to the floor in opposite comers of the arena using magnets, and mice were allowed to explore the arena and objects for 5 min. Five minutes later (testing phase), one of the objects was replaced with a novel object (orange Lego™ flower) of similar dimensions and texture. Mice were allowed to explore for 5 min. The objects and arena were cleaned with 70% ethanol between trials and animals. Trials were recorded and exploratory behavior was defined as the time the animal spent directing its nose toward an object. Video analysis was performed by investigators blinded to injury and treatment. Data was expressed as discrimination index = (time with novel object - time with familiar object) / (total object exploration time). Mice that had less than 5 s of exploration time during either training or testing were excluded from analysis.Hind limb extension reflex test

[0310] Hindlimb extension reflexes were evaluated according to the procedure and scoring system described previously. Briefly, mice were suspended by the tail, and the degree of motor deficit was scored on a 0 to 1 scale: a normal extension reflex in both hindlimbs was scored as 1; imbalanced extension in the hindlimbs as 0.75; extension reflex in only one hindlimb as 0.5; the absence of any hindlimb extension as 0.25; and total paralysis as 0.Wire hang

[0311] Muscle strength was measured by the inverted wire hang test. Each mouse was first placed with all four paws firmly grasping on a wire grid. The grid was then carefully inverted so that the mouse hung over a cage filled with bedding. The latency to fall was recorded. The maximum latency is 180 s. Mice were tested for a total of three trials, with 1 h between each trial.Rotarod

[0312] Motor coordination was measured using a rotarod (Med Associates Inc., Vermont, USA). The apparatus was equipped with infrared beams that automatically detect when the mouse has fallen off the rotating rod. On the first day (training), up to five mice of the same sex were simultaneously placed on the rotarod apparatus with the rod already rotating at the constant speed of 16 rpm (rotations per minute). The trial ended when the mouse fell off the rod or when 5 min elapsed. The mice were tested on three individual trials with an inter-trial interval between 15 and 20 min. On the second and third day of testing, five mice of the same sex were simultaneously placed on the rotarod apparatus with the rod rotating at an accelerating speed, from 4 to 40 rpm. The rotation speed increased by 4 rpm every 30 s. The trial ended when the mouse fell off the rod or when 5 min elapsed. The mice were given fourAttorney Docket No. AETN0001-401-PC trials each day for three days. The intertrial interval was between 15 and 20 min, and there was a 2 h interval between the AM and PM sessions.Open Field

[0313] Spontaneous activity in the open field was measured with an automated Flex Field Open Field Photobeam Activity System (San Diego Instruments). Mice were tested for 15 min in a clear plastic chamber (41 cm by 41 cm by 30 cm) equipped with two 16 by 16 photobeam arrays for the detection of horizontal and vertical movements. Total movements, movements made in the center and the periphery of the arena were recorded automatically for subsequent analysis.Stereotaxic injection

[0314] Mice were anesthetized with 2% isoflurane by inhalation for the duration of surgery and secured on a stereotaxic frame (Kopf Instruments). 3-4-month-old PS 19 mice were injected 2 pL of 3.5 mg / ml synthetic tau fibrils (K18- P301L) stereotaxically at a rate of 0.5 pl / min into the dentate gyrus of left hippocampus. The following coordinates were used for dentate gyrus (anterior-posterior - 2.1, medial-lateral - 1.7, dorsal-ventral - 2.1).Termination

[0315] All mice were lethally overdosed using a mixture of ketamine (10 mg / ml) and xylazine (1 mg / ml). Once animals were completely anesthetized, the chest cavity was opened, and blood was obtained by cardiac puncture. Following cardiac puncture animals were perfused with IX phosphate buffer solution, pH 7.4 (Gibco, Big Cabin OK, 70011-044) until the livers were clear (~ 1-2 min).Hippocampal volume quantification and immunostaining

[0316] Investigators were blinded to the genotypes or treatment of the mice. For quantification of hippocampal volume, mice hemibrains were cut at 30 pm coronally, and all hippocampi-including sections were collected. Brain sections were mounted on microscope slides (Fisher Scientific) in an anterior-to-posterior order, starting from the section where the hippocampal structure first becomes visible (first section) to the section where hippocampal structure just disappears (last section). Mice with missing sections were excluded from the analyses, a pre-established criterion. Mounted brain sections were dried at room temperature for 24 h and stained with cresyl violet (Nissl staining). After being defatted for 15 min in 100% xylene and 10 min in 100% ethanol then rehydrated, sections were stained in 0.1% cresyl violet solution and mounted in DePeX mounting medium (VWR). Images were acquired with a Keyence BZ-9000 microscope. Hippocampal volume was estimated using ImageJ (NIH) Volumest™ Plugin Image Analysis (Ferreiro et al., 2024).Attorney Docket No. AETN0001-401-PC

[0317] 10-12 hippocampal-containing sections were typically used for each analysis. For immunostaining, floating sections were permeabilized and incubated in blocking solution (10% NGS in 0.3% Triton X-100 TBST) at room temperature for 1 h. After incubation with AT8 (MN1020, Thermo Scientific), Iba-1 (Wako), or MCI (gift from Dr. Peter Davis), immunoreactive structures were detected with either Alexa Fluor 488- or Alexa Fluor 555- conjugated secondary antibodies (Invitrogen). After overnight incubation, the sections were incubated with secondary antibodies including Cy3-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch) and fluorescein-labeled goat anti-mouse IgG (Vector Laboratories). All images were acquired by DM5000B microscope (Leica) or CSU-W1 spinning disk confocal microscope (Nikon) with 60* oil immersion objective lens and analyzed by either MicroManager software (UCSF) or ImageJ (NUT).Homogenization of cells and tissues for immunoblot analyses

[0318] HEK293T cells or mouse brain tissues were homogenized in RIPA buffer containing protease inhibitor cocktail (Sigma), ImM phenylmethyl sulfonyl fluoride, phosphatase inhibitor cocktail (Sigma), 5mM nicotinamide (Sigma) and 1 pM trichostatin-A (Sigma). Mouse brain tissues were sonicated after homogenization. Lysates were centrifuged at 14,000 RPM at 4 °C for 15 min. Supernatants were collected and protein concentrations were determined by the BCA assay (Thermo Fisher). The same amount of protein was resolved on a 4-12% SDS-PAGE gel (Invitrogen), transferred to nitrocellulose membrane (Bio-Rad), and probed with appropriate antibodies. Bands in immunoblots were visualized by enhanced chemiluminescence (Pierce) and quantified by densitometry and ImageJ software (NIH). Representative blots from the same gel / membrane are shown and compared in the same figure. Samples from non-adjacent lanes were separated by a line.Human plasma study

[0319] Acetylated tau was measured in human plasma as previously described (Shin et al., 2021). Plasma was depleted of albumin and immunoglobulin according to manufacturer’s instructions (Bio-Rad Laboratories, Inc., 732-6701). In summary, aurum serum protein columns were washed two times with Aurum serum protein binding buffer to remove storage buffer. Sixty microliters of human plasma were diluted in 180 pL of Aurum serum binding protein buffer. 200 pL of the diluted plasma sample was loaded onto the resin, incubated for 15 min, and centrifuged through the column to collect the depleted plasma. The resin was washed with 200 pL of binding buffer, and eluate was added to the final depleted plasma sample. Protein concentration was measured using bicinchoninic acid assay (Thermo Scientific, A53225). For western blotting, depleted plasma was mixed with Laemmli SampleAttorney Docket No. AETN0001-401-PCBuffer (Bio-Rad Laboratories, Inc., #1,610,737) with beta-mercaptoethanol (Bio-Rad Laboratories, Inc., #1,610,710) and boiled for 5 min. Proteins were loaded in 4-20% Criterion TGX Stain-Free gels (Bio-Rad Laboratories, Inc., #5,678,095). Total plasma proteins were visualized using ultraviolet light in the ChemiDoc™ MP Imaging system (Bio-Rad Laboratories, Inc.). The Trans-Blot turbo system (Bio-Rad Laboratories, Inc.) was used to transfer proteins to 0.2 pm polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc., #1,704,157). Membranes were blocked in 5% milk in tris-buffered saline-tween 20 (TBST) for 1 h at room temperature. Membranes were then incubated with primary antibodies at 4 °C overnight. Membranes were washed in TBST (3 x 5 min) and incubated in horseradish peroxidase-conjugated secondary antibody. The BioSpectrum 810 Imaging System (UVP, Upland, CA) was used to detect blots developed in SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific, #34,096). Densitometry quantification of western blot signal was conducted by ImageJ2 version 2.9.0 software (National Institutes of Health, Bethesda, MD).Tau biosensor assay

[0320] Tau RD P301 S FRET Biosensor cells were purchased from ATCC (#CRL- 3275™). 125,000 cells were plated on PDL-coated coverslips in a 24-well plate. The following day, the media was changed, and cells were seeded with brain lysate. Brain lysate was prepared by homogenizing tissue in 10 times the volume of PBS with 0.02% NaN3, protease and phosphatase inhibitors, and de-acetylase inhibitors (5 mM nicotinamide and 1 pm trichostatin A). After homogenization, lysates were centrifuged at 21,000 g for 15 min at 4 degrees Celsius. The supernatant was collected, and protein concentration was measured using the bicinchoninic acid assay (Thermo Scientific, A53225). Lysates were diluted in PBS to a concentration of 1 mg / mL. Cells were seeded with brain lysate using Lipofectamine 3000 according to manufacturer instructions. Briefly, 7 pL of brain lysate (7 ug of protein) was mixed with OPTI-MEM, Lipofectamine 3000, and P3000 and incubated at room temperature for 20 min. The solution was added to each well in droplets. After 72 h of incubation, cells were fixed in 4% PF A, stained with DAP I, and mounted on slides with Vectashield. Images were acquired using Zeiss Axiolmager.M2 with a monochromatic digital camera (Zeiss AxioCam MRm Rev. 3). Images were analyzed using QuPath (Bankhead et al., 2017).Isolation of nuclei from frozen mouse brain tissue

[0321] The protocol for isolating nuclei from frozen brain tissue was adapted from a previous study with modifications. All procedures were done on ice or at 4 °C. In brief,Attorney Docket No. AETN0001-401-PC mouse brain tissue was placed in 1500 pl of nuclei PURE lysis buffer (Sigma, NUC201-1KT) and homogenized with a Dounce tissue grinder (Sigma, D8938-1SET) with 15 strokes with pestle A and 15 strokes with pestle B. The homogenized tissue was filtered through a 35 pm cell strainer and was centrifuged at 600 x g for 5 min at 4 °C and washed three times with 1 ml of PBS containing 1% BSA, 20 mM DTT, and 0.2 U pl - 1 recombinant RNase inhibitor. Then the nuclei were centrifuged at 600 * g for 5 min at 4 °C and resuspended in 500 pl of PBS containing 0.04% BSA and l x DAP I, followed by FACS sorting to remove cell debris. The FACS-sorted suspension of DAPI-stained nuclei was counted and diluted to a concentration of 1000 nuclei per microliter in PBS containing 0.04% BSA.Droplet-based single-nuclei RNA-seq

[0322] For droplet-based snRNA-seq, libraries were prepared with Chromium Single Cell 3’ Reagent Kits v3.1 (10x Genomics, PN- 1,000, 268) according to the manufacturer’s protocol. cDNA and library fragment analysis were performed using the Agilent Fragment Analyzer systems. The snRNA-seq libraries were sequenced on the NovaSeq™ 6000 sequencer (Illumina) with PE 2 x 50 paired-end kits by using the following read length: 28 cycles Read 1, 10 cycles i7 index, 10 cycles i5 index and 90 cycles Read 2.Analysis of droplet-based single-nuclei RNA-seq

[0323] Gene counts were obtained by aligning reads to the mouse genome (mm 10) with Cell Ranger software (v.6.1.2) (10x Genomics). Cell Ranger 6.1.2 default parameters were used to call cell barcodes. We further removed genes expressed in no more than 3 cells, cells with a unique gene count over 4000 or less than 300, and cells with a high fraction of mitochondrial reads (> 1%). Potential doublet cells were predicted and removed using DoubletFinder™ for each sample. Normalization and clustering were done with the Seurat package v4.0.0. In brief, counts for all nuclei were scaled by the total library size multiplied by a scale factor (10,000) and transformed to log space. A set of 2000 highly variable genes was identified with the FindVariableFeatures function based on a variance stabilizing transformation (vst). Principal component analysis (PCA) was done on all genes, and t-SNE was run with the top 15 PCs. Cell clusters were identified with the Seurat functions FindNeighbors (using the top 15 PCs) and FindClusters (resolution = 0.1). Uniform Manifold Approximation and Projection (UMAP) was performed with the top 15 PCs. For each cluster, a cell-type label was assigned using statistical enrichment for sets of marker genes and manual evaluation of gene expression for small sets of known marker genes. The subset function from Seurat was used to subset oligodendrocytes and microglia, separately.Attorney Docket No. AETN0001-401-PCDifferential gene expression analysis was done using the FindMarkers function and MAST (Finak et al., 2015).Statistical analysis

[0324] Data were analyzed with GraphPad™ Prism v.5 (GraphPad™) or STATA12 (StataCorp LP). Differences between means were assessed with paired or unpaired Student’s t-test, one-way or two-way analysis of variance (ANOVA), followed by post hoc testing of all pairwise comparisons among genotypes (with Tukey -Kramer correction or Dunnett’s test for one-way ANOVA and Bonferroni correction for two-way ANOVA), or by mixed effects model, as indicated. Pearson’s correlation coefficients were used to quantify the linear relationship between two variables. The Shapiro-Wilk test of normality was applied to all data sets, and in cases where the data did not demonstrate a normal distribution, nonparametric tests were used to analyze statistical differences. The Mann-Whitney test was used for unpaired t-tests, the Wilcoxon matched pairs test was used for paired comparisons, and the Kruskal-Wallis test was used for ANOVAs. Outliers were pre-established as data outside of mean ± 2 S.D. All samples or animals were included for statistical analysis unless otherwise noted in pre-established criteria.Production of selected humanized antibodies

[0325] Antibodies can be made and purified essentially as follows: An appropriate host cell, such as HEK 293 EBNA or CHO, is either transiently or stably transfected with an expression system for secreting antibodies using an optimal predetermined HC:LC vector ratio or a single vector system encoding both HC, such as SEQ ID NO:56, and SEQ ID NO:43, and LC, such as SEQ ID NO:55.

[0326] Clarified media, into which the antibody has been secreted, may be purified using any of many commonly- used techniques. For example, the medium may be conveniently applied to a Protein A or G Sepharose FF column that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4). The column is washed to remove nonspecific binding components. The bound antibody is eluted, for example, by pH gradient (such as 0.1 M sodium phosphate buffer pH 6.8 to 0. IM sodium citrate buffer pH 2.5). Antibody fractions are detected, such as by SDS-PAGE, and then are pooled. Further purification is optional, depending on the intended use. The antibody may be concentrated and / or sterile filtered using common techniques. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The purity of the antibody after these chromatography steps is greater than 99%. The product may be immediately frozen at -Attorney Docket No. AETN0001-401-PC70°C or may be lyophilized. The amino acid sequences for various antibodies of the present disclosure are provided below.Table 4 - Exemplary Antibody SEQ ID Nos.Table 5 - Antibody VH / VL DomainsAttorney Docket No. AETN0001-401-PCAttorney Docket No. AETN0001-401-PCAttorney Docket No. AETN0001-401-PCTable 6 - Antibody CDRsAttorney Docket No. AETN0001-401-PCAttorney Docket No. AETN0001-401-PCTable 7 - Anti-ac-tauK174 CDRsTable 8 - Anti-ac-tauK274 CDRs (AbM3 and AbM4)Table 9 - Anti-ac-tauK353 CDRs (AbM6)Attorney Docket No. AETN0001-401-PCTable 10 - Exemplary Antibody HC / LC DomainsAttorney Docket No. AETN0001-401-PCAttorney Docket No. AETN0001-401-PCAttorney Docket No. AETN0001-401-PCAttomey Docket No. AETN0001-401-PCCITATIONSAndreadis, A., Brown, W. M., & Kosik, K. S. (1992). Structure and novel exons of the human, tau. gene. Biochemistry, 37(43), 10626-10633.Asuni, A. A., Boutajangout, A., Quartermain, D., & Sigurdsson, E. M. (2007).Immunotherapy targeting pathological tau conformers in a tangle mouse model reduces brain pathology with associated functional improvements. Journal of Neuroscience, 27(34), 9115-9129.Bankhead, P., Loughrey, M. B., Fernandez, J. A., Dombrowski, Y., McArt, D. G., Dunne, P. D., McQuaid, S., Gray, R. T., Murray, L. J., & Coleman, H. G. (2017). QuPath: Open source software for digital pathology image analysis. Scientific reports, 7(1), 1-7.Chou, A., Krukowski, K., Jopson, T., Zhu, P. J., Costa-Mattioli, M., Walter, P., & Rosi, S. (2017). Inhibition of the integrated stress response reverses cognitive deficits after traumatic brain injury. Proceedings of the National Academy of Sciences, 114(31), E6420-E6426.Cleveland, D. W., Hwo, S.-Y., & Kirschner, M. W. (1977). Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly. Journal of molecular biology, 116(2), 227-247.Co, M. S., Deschamps, M., Whitley, R. J., & Queen, C. (1991). Humanized antibodies for antiviral therapy. Proceedings of the National Academy of Sciences, 88(7), 2869-Attorney Docket No. AETN0001-401-PC2873.Cohen, T. J., Guo, J. L., Hurtado, D. E., Kwong, L. K., Mills, I. P., Trojanowski, J. Q., & Lee, V. M. (2011). The acetylation of tau inhibits its function and promotes pathological tau aggregation. Nature communications, 2(1), 252.Crowther, T., Goedert, M., & Wischik, C. M. (1989). The repeat region of microtubule- associated protein tau forms part of the core of the paired helical filament of Alzheimer's disease. Annals of medicine, 27(2), 127-132.Dodart, J. C., & May, P. (2005). Overview on rodent models of Alzheimer's disease. Current Protocols in Neuroscience, 33(1), 9.22. 21-29.22. 16.Drzezga, A., Grimmer, T., Henriksen, G., Muhlau, M., Pemeczky, R., Miederer, I., Praus, C., Sorg, C., Wohlschlager, A., & Riemenschneider, M. (2009). Effect of APOE genotype on amyloid plaque load and gray matter volume in Alzheimer disease. Neurology, 72(f7), 1487-1494.Englund, H., Sehlin, D., Johansson, A. S., Nilsson, L. N., Gellerfors, P., Paulie, S., Lannfelt, L., & Pettersson, F. E. (2007). Sensitive ELISA detection of amyloid-P protofibrils in biological samples. Journal of neurochemistry, 103(f), 334-345.Ferreiro, E., Rodriguez-Iglesias, N., Cardoso, J., & Valero, J. (2024). VolumestJ: A New Method and Tool for Volumetric Estimation of Brain Structures After Serial Sectioning. In Advances in Stereology for Neuroscience (pp. 129-176). Springer.Finak, G., McDavid, A., Yajima, M., Deng, J., Gersuk, V., Shalek, A. K., Slichter, C. K., Miller, H. W ., McElrath, M. J., & Prlic, M. (2015). MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Genome biology, 16, 1-13.Fitzpatrick, A. W., Falcon, B., He, S., Murzin, A. G., Murshudov, G., Garringer, H. J., Crowther, R. A., Ghetti, B., Goedert, M., & Scheres, S. H. (2017). Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature, 547(1662), 185-190.Ganguli, M., Snitz, B. E., Saxton, J. A., Chang, C.-C. H., Lee, C.-W., Vander Bilt, J., Hughes, T. F., Loewenstein, D. A., Unverzagt, F. W ., & Petersen, R. C. (2011). Outcomes of mild cognitive impairment by definition: a population study. Archives of neurology, 68(6), 761-767.Gao, S. H., Huang, K., Tu, H., & Adler, A. S. (2013). Monoclonal antibody humanness score and its applications. BMC biotechnology, 13, 1-12.Gibbons, G. S., Kim, S.-J., Wu, Q., Riddle, D. M., Leight, S. N., Changolkar, L., Xu, H., Meymand, E. S., O’Reilly, M., & Zhang, B. (2020). Conformation-selective tau monoclonal antibodies inhibit tau pathology in primary neurons and a mouse model of Alzheimer’ s disease. Molecular Neurodegeneration, 15, 1-19.Goedert, M., Wischik, C., Crowther, R., Walker, J., & Klug, A. (1988). Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: identification as the microtubule-associated protein tau. Proceedings of the National Academy of Sciences, 5( 1 1), 4051-4055.Gonnet, G. H., Cohen, M. A., & Benner, S. A. (1992). Exhaustive matching of the entire protein sequence database. Science, 256(5062), 1443-1445.Gbtz, J., Streffer, J., David, D., Schild, A., Hoemdli, F., Pennanen, L., Kurosinski, P., & Chen, F. (2004). Transgenic animal models of Alzheimer's disease and related disorders: histopathology, behavior and therapy. Molecular psychiatry, 9(7), 664-683.Hutton, M., Lendon, C. L., Rizzu, P., Baker, M., Froelich, S., Houlden, H., Pickering-Brown, S., Chakraverty, S., Isaacs, A., & Grover, A. (1998). Association of missense and 5'- splice-site mutations in tau with the inherited dementia FTDP-17. Nature, 393(6686), 702-705.Imbimbo, B. P., Balducci, C., Ippati, S., & Watling, M. (2023). Initial failures of anti-tauAttorney Docket No. AETN0001-401-PC antibodies in Alzheimer’s disease are reminiscent of the amyloid-P story. Neural regeneration research, 18(1), 117-118.Jones, P. T., Dear, P. H., Foote, J., Neuberger, M. S., & Winter, G. (1986). Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature, 327(6069), 522-525.Kabat, E. A. (1991). Sequences of proteins of immunological interest. US Department of Health and Human Services, Public Health Service, National ....Kent, S. A., Spires-Jones, T. L., & Durrant, C. S. (2020). The physiological roles of tau and AP: implications for Alzheimer’s disease pathology and therapeutics. Acta neuropathologica, 140(A), 417-447.Krukowski, K., Nolan, A., Frias, E. S., Grue, K., Becker, M., Ureta, G., Delgado, L., Bemales, S., Sohal, V. S., & Walter, P. (2020). Integrated stress response inhibitor reverses sex-dependent behavioral and cell-specific deficits after mild repetitive head trauma. Journal of Neurotrauma, 37(11), 1370-1380.Levitt, M. (1983). Molecular dynamics of native protein. I. Computer simulation of trajectories. Journal of molecular biology, 168(3), 595-617.Lopez-Gonzalez, I., Aso, E., Carmona, M., Armand-Ugon, M., Blanco, R., Naudi, A., Cabre, R., Portero-Otin, M., Pamplona, R., & Ferrer, I. (2015). Neuroinflammatory gene regulation, mitochondrial function, oxidative stress, and brain lipid modifications with disease progression in tau P301S transgenic mice as a model of frontotemporal lobar degeneration-tau. Journal of neuropathology and experimental neurology, 74(10), 975-999.McKhann, G. M., Knopman, D. S., Chertkow, H., Hyman, B. T., Jack Jr, C. R., Kawas, C.H., Klunk, W. E., Koroshetz, W. J., Manly, J. J., & Mayeux, R. (2011). The diagnosis of dementia due to Alzheimer’s disease: Recommendations from the National Institute on Aging- Alzheimer’ s Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimer's & dementia, 7(3), 263-269.Min, S.-W., Chen, X., Tracy, T. E., Li, Y., Zhou, Y., Wang, C., Shirakawa, K., Minami, S. S., Defensor, E., & Mok, S. A. (2015). Critical role of acetylation in tau-mediated neurodegeneration and cognitive deficits. Nature medicine, 21(10), 1154-1162.Min, S.-W., Cho, S.-H., Zhou, Y., Schroeder, S., Haroutunian, V., Seeley, W. W ., Huang, E. J., Shen, Y., Masliah, E., & Mukheijee, C. (2010). Acetylation of tau inhibits its degradation and contributes to tauopathy. Neuron, 67(6), 953-966.Morris, M., Knudsen, G. M., Maeda, S., Trinidad, J. C., loanoviciu, A., Burlingame, A. L., & Mucke, L. (2015). Tau post-translational modifications in wild-type and human amyloid precursor protein transgenic mice. Nature neuroscience, 18(8), 1183-1189.Riechmann, L., Clark, M., Waldmann, H., & Winter, G. (1988). Reshaping human antibodies for therapy. Nature, 332(6162), 323-327.Shin, M.-K., Vazquez-Rosa, E., Koh, Y., Dhar, M., Chaubey, K., Cintron-Perez, C. J., Barker, S., Miller, E., Franke, K., & Noterman, M. F. (2021). Reducing acetylated tau is neuroprotective in brain injury. Cell, 184( 6), 2715-2732. e2723.Song, H.-L., Kim, N.-Y., Park, J., Kim, M. I., Jeon, Y.-N., Lee, S.-J., Cho, K., Shim, Y.-L., Lee, K.-H., & Mun, Y.-S. (2023). Monoclonal antibody Y01 prevents tauopathy progression induced by lysine 280-acetylated tau in cell and mouse models. The Journal of Clinical Investigation, 133(8).Strang, K. H., Golde, T. E., & Giasson, B. I. (2019). MAPT mutations, tauopathy, and mechanisms of neurodegeneration. Laboratory investigation, 99(7), 912-928.Taylor, L. D., Carmack, C. E., Schramm, S. R., Mashayekh, R., Higgins, K. M., Kuo, C.-C., Woodhouse, C., Kay, R. M., & Lonberg, N. (1992). A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins.Attorney Docket No. AETN0001-401-PCNucleic acids research, 20(23), 6287-6295.Verhoeyen, M., Milstein, C., & Winter, G. (1988). Reshaping human antibodies: grafting an antilysozyme activity. Science, 239(^iM), 1534-1536.Wang, J., Logovinsky, V., Hendrix, S. B., Stanworth, S. H., Perdomo, C., Xu, L., Dhadda, S., Do, I., Rabe, M., & Luthman, J. (2016). ADCOMS: a composite clinical outcome for prodromal Alzheimer's disease trials. Journal of Neurology, Neurosurgery & Psychiatry, 87(9), 993-999.Wu, H., Nie, Y., Huse, W. D., & Watkins, J. D. (1999). Humanization of a murine monoclonal antibody by simultaneous optimization of framework and CDR residues. Journal of molecular biology, 294( \ ), 151-162.Yoshiyama, Y., Higuchi, M., Zhang, B., Huang, S.-M., Iwata, N., Saido, T. C., Maeda, J., Suhara, T., Trojanowski, J. Q., & Lee, V. M.-Y. (2007). Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model. Neuron, 53(3), 337- 351.Zanier, E. R., Bertani, I., Sammali, E., Pischiutta, F., Chiaravalloti, M. A., Vegliante, G., Masone, A., Corbelli, A., Smith, D. H., & Menon, D. K. (2018). Induction of a transmissible tau pathology by traumatic brain injury. Brain, 141(9), 2685-2699.

Claims

Attorney Docket No. AETN0001-401-PCCLAIMS[00327] What is claimed is:

1. An anti-acetylated-tau antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3 polypeptides and the VL comprises LCDR1, LCDR2, and LCDR3 polypeptides chosen from the following sets, respectively: a) HCDR1 is GYTFTDYY (SEQ ID NO:38), HCDR2 is IFPGSDLI (SEQ ID NO:39), HCDR3 is ARGVYYDGGNFFDY (SEQ ID NO:40), LCDR1 is QDISSY (SEQ ID NO:41), LCDR2 is YTS (SEQ ID NO:42), and LCDR3 is QQYSKRPWT (SEQ ID NO:43); b) HCDR1 is GFNIKNTY (SEQ ID NO:44), HCDR2 is IDPSNGNT (SEQ ID NO:45), HCDR3 is ADD YAW (SEQ ID NO:46), LCDR1 is QSLLYTNGKTY (SEQ ID NO:47), LCDR2 is LSV (SEQ ID NO:48), and LCDR3 is LQSTHFPRT (SEQ ID NO:49); c) HCDR1 is GFTFGDYW (SEQ ID NO:50), HCDR2 is IKLKSDNYAT (SEQ ID NO:51), HCDR3 is TTIYYYDSRRFDY (SEQ ID NO:52), LCDR1 is QSVSYD (SEQ ID NO:53), LCDR2 is HAS (SEQ ID NO:54), and LCDR3 is HQEYSSPYT (SEQ ID NO: 55); d) HCDR1 is GFTFGDYW (SEQ ID NO:56), HCDR2 is IKLKSDNYAT (SEQ ID NO:57), HCDR3 is TTIYYYDSRRFDY (SEQ ID NO:58), LCDR1 is QSVSYD (SEQ ID NO:59), LCDR2 is INPKYGDT (SEQ ID NO:60), and LCDR3 is AVIYYGFYTMDY (SEQ ID NO:61); e) HCDR1 is GFTFTIYG (SEQ ID NO:62), HCDR2 is IDENGGA (SEQ ID NO:63), HCDR3 is ARDDGT (SEQ ID NO:64), LCDR1 is QSLFYSDGQTY (SEQ ID NO:65), LCDR2 is LSV (SEQ ID NO:66), and LCDR3 is VQGTHFPLT (SEQ ID NO: 67); or f) HCDR1 is GYKFTEYY (SEQ ID NO:84), HCDR2 is INPKYGDT (SEQ ID NO:85), HCDR3 is AVIYYGFYTMDY (SEQ ID NO:86), LCDR1 is QSLLDISNQKNH (SEQ ID NO:87), LCDR2 is FAS (SEQ ID NO:88), and LCDR3 is QQHYNTPRT (SEQ ID NO: 89), wherein the numbering of amino acid residues is according to IMGT.Attorney Docket No. AETN0001-401-PC2. The antibody, or an antigen-binding fragment thereof, of claim 1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and the VL are polypeptides chosen from: a) VH of SEQ ID NO: 10 and VL of SEQ ID NO: 11; b) VH of SEQ ID NO: 12 and VL of SEQ ID NO: 13; c) VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15; d) VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17; e) VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; f) VH of SEQ ID NO:20 and VL of SEQ ID NO:21; g) VH of SEQ ID NO:22 and VL of SEQ ID NO:23; h) VH of SEQ ID NO:24 and VL of SEQ ID NO:25; i) VH of SEQ ID NO:26 and VL of SEQ ID NO:27; j) VH of SEQ ID NO:28 and VL of SEQ ID NO:29; k) VH of SEQ ID NO:30 and VL of SEQ ID NO:31; l) VH of SEQ ID NO:32 and VL of SEQ ID NO:33; m) VH of SEQ ID NO:34 and VL of SEQ ID NO:35; n) VH of SEQ ID NO:36 and VL of SEQ ID NO:37; and o) VH of SEQ ID NO: 82 and VL of SEQ ID NO: 83.

3. The antibody, or an antigen-binding fragment thereof, of claim 2 comprising a combination of a heavy chain (HC) and a light chain (LC), wherein the HC and LC are polypeptides chosen from: a) HC of SEQ ID NO: 74 and LC of SEQ ID NO: 75; and b) HC of SEQ ID NO:78 and LC of SEQ ID NO:79.

4. The antibody, or an antigen-binding fragment thereof, of claim 3 comprising two combinations of a light chain and a heavy chain, wherein each combination of a light chain and a heavy chain are polypeptide is chosen from: a) HC of SEQ ID NO: 74 and LC of SEQ ID NO: 75; and b) HC of SEQ ID NO:78 and LC of SEQ ID NO:79.

5. The antibody, or an antigen-binding fragment thereof, of any one of claims 1 to 4, wherein the antibody, or an antigen-binding fragment thereof, is a monoclonal antibody.Attorney Docket No. AETN0001-401-PC6. The antibody, or an antigen-binding fragment thereof, of any one of claims 1 to 5, having heavy and light chain constant regions of the IgGl, IgG2, or IgG4 subclasses.

7. The antibody, or an antigen-binding fragment thereof, of claim 6 which is of the IgGl subclass.

8. The antibody, or an antigen-binding fragment thereof, of claim 7 which has a heavy chain constant domain represented by the amino acid sequence set forth in SEQ ID NO:4.

9. The antibody, or an antigen-binding fragment thereof, of claim 7 which has a mutated heavy chain constant domain as compared to the wildtype.

10. The antibody, or an antigen-binding fragment thereof, of claim 9 which has a heavy chain represented by the amino acid sequence set forth in SEQ ID NO: 5.

11. The antibody, or an antigen-binding fragment thereof, of any one of claims 1 to 10, which is a humanized or chimeric antibody.

12. The antibody, or an antigen-binding fragment thereof, of any one of claims 1 to 11 which has a light chain constant domain of the lambda (X) subtype.

13. The antibody, or an antigen-binding fragment thereof, of any one of claims 1 to 11 which has a light chain constant domain of the kappa (K) subtype.

14. The antibody, or an antigen-binding fragment thereof, of claim 13 having a light chain constant domain represented by the amino acid sequence set forth in SEQ ID NO: 8.

15. The antibody, or an antigen-binding fragment thereof, of any one of claims 1 to 14, which specifically binds acetylated lysine 174 on human tau (ac-tauK174).

16. The antibody, or an antigen-binding fragment thereof, of claim 15 capable of binding with a KD of at least about IxlO'11M, at least about 1X10'12M, or at least about 2.5xl0'12M.

17. The antibody, or an antigen-binding fragment thereof, of any one of claims 1 to 14, which specifically binds acetylated lysine 274 on human tau (ac-tauK274).

18. A nucleic acid encoding an antibody, or an antigen-binding fragment thereof, of any one of claims 1-17.

19. A nucleic acid encoding a heavy chain of an antibody, where the heavy chain is represented by the amino acid sequence set forth in SEQ ID NO:78.

20. The nucleic acid of claim 19, where the nucleic acid is represented by the sequence set forth in SEQ ID NO:80.

21. A nucleic acid encoding a light chain of an antibody, where the light chain is represented by the amino acid sequence set forth in SEQ ID NO:79.Attorney Docket No. AETN0001-401-PC22. The nucleic acid of claim 21, where the nucleic acid is represented by the sequence set forth in SEQ ID N0:81.

23. A vector comprising the nucleic acid of any one of claims 19-22.

24. A host cell comprising the vector of claim 23.

25. A pharmaceutical composition comprising the antibody, or an antigen-binding fragment thereof, of any one of claims 1-17 and a pharmaceutically acceptable carrier, diluent, or excipient.

26. A method of treating a condition chosen from clinical or pre-clinical Alzheimer’s Disease, prodromal Alzheimer’s Disease, Down Syndrome, clinical or pre-clinical amyloid angiopathy (CAA), frontotemporal dementia (FTD) with TAR DNA-binding protein 43 (TDP-43), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick’s disease (PiD), argyrophilic grain disease (AGD), traumatic brain injury (TBI), and chronic traumatic encephalopathy (CTE), comprising administering to a human in need thereof, any one of the antibodies, or an antigen-binding fragment thereof, of any one of claims 1-17.

27. A method of treating Alzheimer’s Disease, comprising administering to a human in need thereof the antibody, or an antigen-binding fragment thereof, of any one of claims 1 to 17.

28. A method of treating a condition chosen from clinical or pre-clinical Alzheimer’s Disease, prodromal Alzheimer’s Disease, Down Syndrome, clinical or pre-clinical amyloid angiopathy (CAA), frontotemporal dementia (FTD) with TAR DNA-binding protein 43 (TDP-43), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick’s disease (PiD), argyrophilic grain disease (AGD), traumatic brain injury (TBI), and chronic traumatic encephalopathy (CTE), comprising administering to a human in need thereof the pharmaceutical composition of claim 25.

29. A method of treating Alzheimer’s Disease, comprising administering to a human in need thereof the pharmaceutical composition of claim 25.

30. A method of reducing clinical decline in a subject having early Alzheimer’s Disease comprising administering a composition comprising a therapeutically effective amount of at least one anti -acetylated tau antibody, or an antigen-binding fragment thereof, of any one of claims 1-17.

31. The method of claim 30, wherein the at least one anti-acetylated tau antibody is antagonistic.Attorney Docket No. AETN0001-401-PC32. The method of claim 31, wherein the antibody, or an antigen-binding fragment thereof, specifically binds acetylated lysine 174 on human tau (ac-tauK174).

33. The method of claim 32, wherein the at least one anti-acetylated tau antibody is chosen from AbMl, ABH1 or ABH5.

34. The method of claim 33, wherein the at least one anti-acetylated tau antibody, or an antigen-binding fragment thereof, comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:68 (HCDR1), SEQ ID NO:69 (HCDR2), and SEQ ID NO:70 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:71 (LCDR1), SEQ ID NO:72 (LCDR2), and SEQ ID NO:73 (LCDR3).

35. The method of claim 34, wherein the subject having early Alzheimer’s Disease has been diagnosed as having mild cognitive impairment (MCI) due to Alzheimer’s Disease and / or has been diagnosed as having mild Alzheimer’s Disease dementia.

36. The method of any one of claims 26 to 35, wherein diagnosis comprises determining an increased level of aberrantly acetylated or hyperacetylated tau relative to a normal level from a non-diseased control.

37. The method of any one of claims 26 to 36, wherein the subject is concomitantly administered at least one Alzheimer’s Disease medication other than ABH1 or ABH5.

38. The method of any one of claims 26 to 36, wherein the subject is not concomitantly administered at least one Alzheimer’s Disease medication other than ABH1 or ABH5.

39. The method of any one of claims 26 to 38, wherein said administration results in a reduction of cerebrospinal fluid levels of acetylated K174 tau and / or acetylated K274 tau.

40. A method of treating a subject having early Alzheimer’s Disease comprising administering a composition comprising a therapeutically effective amount of at least one anti -acetylated tau antibody, or an antigen-binding fragment thereof, wherein clinical decline of the subject is reduced by at least 35% relative to placebo as determined by ADCOMS after 6 months of administration of the composition, by at least 30% relative to placebo as determined by ADCOMS after 12 months of administration of the composition, and / or by at least 25% relative to placebo as determined by ADCOMS after 18 months of administration of the composition.

41. The method of claim 40, wherein the at least one anti-acetylated tau antibody comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:68 (HCDR1), SEQ ID NO:69Attorney Docket No. AETN0001-401-PC(HCDR2), and SEQ ID NO:70 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:71 (LCDR1), SEQ ID NO:72 (LCDR2), and SEQ ID NO:73 (LCDR3).

42. The method of claim 41, wherein the subject having early Alzheimer’s Disease has been diagnosed as having mild cognitive impairment due to Alzheimer’s Disease - intermediate likelihood and / or has been diagnosed as having mild Alzheimer’s Disease dementia.

43. A method of treating a subject having early Alzheimer’s Disease comprising administering a composition comprising a therapeutically effective amount of at least one anti -acetylated tau antibody, wherein the severity of at least one symptom associated with Alzheimer’s Disease is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% relative to the severity of the same symptom in subjects that received placebo.

44. The method of claim 43, wherein the severity of at least one symptom associated with Alzheimer’s Disease is determined by ADCOMS, PET, MMSE, CDR-SB, and / or ADAS- Cog.

45. The method of claim 44, wherein the at least one anti-acetylated tau antibody comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:68 (HCDR1), SEQ ID NO:69 (HCDR2), and SEQ ID NO:70 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:71 (LCDR1), SEQ ID NO:72 (LCDR2), and SEQ ID NO:73 (LCDR3).

46. The method of any one of claims 43 to 45, wherein the at least one symptom associated with Alzheimer’s Disease is clinical decline, and wherein the clinical decline is reduced by at least 30% relative to placebo as determined by ADCOMS after 18 months of administration of the composition comprising the at least one ac-tauK174 antibody.

47. The method of any one of claims 26 to 46, wherein the subject is ApoE4-positive.

48. The method of any one of claims 26 to 46, wherein the subject is ApoE4-negative.

49. A stable liquid pharmaceutical formulation comprising at least one antibody that specifically binds to acetylated tau, wherein the antibody is an antibody comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:68, an HCDR2 comprising the amino acid sequence of SEQ ID NO:69, an HCDR3 comprising the amino acid sequence of SEQ ID NO:70, an LCDR1 comprising the amino acid sequence of SEQ IDAttorney Docket No. AETN0001-401-PCN0:71, an LCDR2 comprising the amino acid sequence YTS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 73.

50. A method of manufacturing an antibody, or an antigen-binding fragment thereof, comprising: a) expressing one or more polynucleotide molecule(s) encoding a VH and a VL chain of an antibody, or an antigen-binding fragment thereof, of any one of claims 1-17 in a host cell; and b) purifying the antibody from the cell and / or a fluid medium in which the cell is disposed.

51. The method of claim 50, wherein the host cell is chosen from a bacterial cell, a fungal cell, and a mammalian cell.

52. The method of claim 51, wherein the host cell is chosen from an A. coli cell, a Saccharomyces cerevisiae cell, and a CHO cell.

53. An antibody, or an antigen-binding fragment thereof, that binds to an epitope of acetylated-tau, wherein the epitope is within the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:3.

54. The antibody, or an antigen-binding fragment thereof, of claim 53, wherein the antibody, or an antigen-binding fragment thereof, cross-competes with a second antibody in a competitive binding assay for binding to tau, wherein the second antibody has an antigen binding domain that is structurally defined according to any of the sequences disclosed in Table 4, Table 5, Table 6, Table 7, Table 8, and / or Table 9.

55. A kit comprising an antibody, or an antigen-binding fragment thereof, of any one of the preceding claims, and instructions for its use.

56. The kit of claim 55, wherein the antibody, or an antigen-binding fragment thereof, is lyophilized.

57. The kit of claim 56, further comprising a fluid for reconstitution of the lyophilized antibody, or an antigen-binding fragment thereof.

58. The kit of claim 57, further comprising a device for subcutaneous or intravenous administration.

59. The antibody, or an antigen-binding fragment thereof, of any one of claims 1 to 17 or claims 53 to 54, wherein the antibody, or an antigen-binding fragment thereof, forms part of a multi-specific antibody.Attorney Docket No. AETN0001-401-PC60. The antibody, or an antigen-binding fragment thereof, of claim 59 wherein the antibody, or an antigen-binding fragment thereof, forms part of a bispecific antibody.

61. The antigen-binding fragment of any one of claims 1 to 17, 53 to 54, or 59 to 60, wherein the antibody fragment is chosen from a single-variable domain antibody, a single chain antibody, an scFv, a Fab, a F(ab')2, a minibody, a diabody, and a triabody.

62. The antigen-binding fragment of claim 61, wherein the antibody fragment is an scFv.

63. The antibody, or an antigen-binding fragment thereof, of any one of claims 1 to 17, 53 to 54, or 59 to 62, wherein the antibody, or an antigen-binding fragment thereof, is aglycosylated.

64. The antibody, or an antigen-binding fragment thereof, of any one of claims 1 to 17, 53 to 54, or 59 to 63, wherein the antibody, or an antigen-binding fragment thereof, is operably linked to another compound.

65. The antibody, or an antigen-binding fragment thereof, of claim 64, wherein the other compound is a therapeutic agent, label, or tag.

66. The antibody, or an antigen-binding fragment thereof, of claim 65, wherein the therapeutic agent is a cytotoxic agent, a chemotherapeutic agent, a cytokine, an anti- angiogenic agent, a tyrosine kinase inhibitor, a toxin, a radioisotope, or other therapeutically active agent.

67. An antibody-drug conjugate (ADC) comprising the antibody, or an antigen-binding fragment thereof, of any one of claims 1 to 17, 53 to 54, or 59 to 66.

Images