WO2026033422A1 - Methods to evaluate early-stage pre-tangle tau aggregates and treatment of alzheimer's disease - Google Patents

Methods to evaluate early-stage pre-tangle tau aggregates and treatment of alzheimer's disease

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Publication number
WO2026033422A1
WO2026033422A1 PCT/IB2025/057987 IB2025057987W WO2026033422A1 WO 2026033422 A1 WO2026033422 A1 WO 2026033422A1 IB 2025057987 W IB2025057987 W IB 2025057987W WO 2026033422 A1 WO2026033422 A1 WO 2026033422A1
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WIPO (PCT)
Prior art keywords
tau
binding reagent
binding
sample
nft
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French (fr)
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Thomas. K. KARIKARI
Milos Dragan IKONOMOVIC
Eric E. ABRAHAMSON
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University of Pittsburgh
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University of Pittsburgh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum

Definitions

  • NFT neurofibrillary tangle
  • Ser262 and/or Ser356 of a tau protein and detecting binding between the first binding reagent and the tau protein, wherein detecting binding between the first binding reagent and the tau protein indicates the presence of a pre-stage NFT in the patient sample.
  • Also provided herein is a method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample, including incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a composition comprising a first-binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein and detecting binding between the first binding reagent and the tau protein, wherein detecting binding between the first binding reagent and the tau protein indicates the presence of a pre-stage NFT in the patient sample.
  • NFT neurofibrillary tangle
  • Also provided herein is a method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample including incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a composition comprising a first-binding reagent and a second binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein and the second binding reagent is specific to Ser202/Thr205 of the tau protein and detecting binding between the first binding reagent and the tau protein and between the second binding reagent and the tau protein, wherein detecting binding between the first binding reagent and the tau protein and an absence of binding between the second binding reagent and the tau protein indicates the presence of a pre-NFT in the patient sample.
  • NFT neurofibrillary tangle
  • a method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample comprising: obtaining a sample from a patient suspected of having or at risk of developing a tauopathy; incubating the sample with a composition comprising a first binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein; and detecting binding between the first binding reagent and the tau protein, wherein detecting binding between the first binding reagent and the tau protein indicates the presence of a pre-stage NFT in the patient sample.
  • NFT neurofibrillary tangle
  • detecting binding between the first binding reagent and the tau protein comprises a step of eluting the first binding reagent and tau protein from the substrate, thereby generating a free protein-first binding reagent complex, and performing an immunoblotting assay or protein separation and immunodetection assay on the free protein-first binding reagent complex.
  • the human patient sample is a sample from the patient’s central nervous system, optionally a tissue sample, optionally from hippocampus, entorhinal cortex, or basal forebrain tissue.
  • a method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample comprising: incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a composition comprising a first- binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein; and detecting binding between the first binding reagent and the tau protein, wherein detecting binding between the first binding reagent and the tau protein indicates the presence of a pre-stage NFT in the patient sample.
  • NFT neurofibrillary tangle
  • a method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample comprising: incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a composition comprising a first- binding reagent and a second binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein and the second binding reagent is specific to Ser202/Thr205 of the tau protein; and detecting binding between the first binding reagent and the tau protein and between the second binding reagent and the tau protein, wherein:
  • detecting binding between the first binding reagent and the tau protein and an absence of binding between the second binding reagent and the tau protein indicates the presence of a pre-NFT in the patient sample.
  • FIG. 1 shows antibody optimization. Sections from the middle frontal gyrus from Case-1 , clinically diagnosed with Alzheimer’s disease (AD) and with autopsy- confirmed severe AD neuropathological change were stained with the pSer262 antibody (A-C) and the pSer356 antibody (D-F) using progressively higher dilutions of antibody (1 :250: A,D; 1 :500: B,E; 1 :1 ,000: C,F). The dilution yielding the optimal signal to noise ratio was 1 :250 for both antibodies.
  • IHC studies were then performed on fixed tissue sections obtained from the hippocampus from a series of cases (Cases 2-8) that spanned the range of severity of NFT pathology determined by Braak Stage.
  • the hippocampus was chosen as the region of interest due to its early involvement in the neuropathological progression of AD.
  • FIG. 2 shows IHC studies of hippocampus - Braak Stage study. Adjacent tissue sections were processed using IHC and antibodies AT8 (pSer202/pThr205; A1 - G1 ), pSer262 (A2-G2), and pSer356 (A3-G3). AT8 was used as a comparator to the pSer262 and pSer356 antibodies and was previously optimized for use in our laboratories.
  • tissue sections were obtained from the hippocampus from cases neuropathologically determined to be Braak Stage I (Case-2: A1 -A3), Braak Stage II (Case-3: B1 -B3), Braak Stage III (Case-4: C1 -C3), Braak Stage IV (Case-5: D1 -D3), Braak Stage V (Case-6: E1 -E3), and Braak Stage VI (Case-7: F1 - F3).
  • CA1 region one of the earliest sites of NFT development in the hippocampus CA fields
  • the CA1 region contained neurons immunoreactive to all three antibodies even at Braak Stage I, typically thought of as containing lesions confined to the allocortex.
  • Immunoreactive neurons were observed in progressively higher densities at progressively higher Braak Stages.
  • the pattern of pSer262 and pSer356 labeling differed markedly from patterns seen with AT8. Specifically, pSer262 and pSer356 labeling exhibited a distinct intracellular punctate appearance in contrast to the more homogeneous neuronal staining seen with antibody AT8, even at higher
  • the pSer262 and pSer356 antibodies did not label two types of neurofibrillary pathology, neuropil threads (p-tau containing distal dendrites) or neuritic (i.e., axons) structures in neuritic plaques. These observations strongly support the idea that the pSer262 and pSer356 antibodies identify neurofibrillary change at the earliest neuropathological stages of AD and are specific to NFT lesions, that is, neuronal cell bodies, destruction of which results in irreparable harm to the neural circuits to which they are connected.
  • FIG. 3 shows T riple-fluorescence labeling experiment combines pSer262 (A) with pSer202/pThr205 (AT8; B) and the pan amyloid binding dye X-34 (C, a marker of fibrillar aggregates) on hippocampal sections from a Braak Stage II case (Case-9) revealing that cells with granular pSer262 signal contain only small amounts of AT8 co-labeling and no X-34 labeling, indicating an early stage NFT, prior to fibrillization of tau and prior to robust pSer202/pThr205 tau phosphorylation (panel D merges panels A-C).
  • the punctate appearance of pSer262 immunolabeling was seen consistently and it replicates our observations obtained from chromogen-IHC experiments in FIG. 8A.
  • FIG. 4 shows Triple-fluorescence labeling experiments combining pSer262 with pSer202/pThr205 (AT8: B) and the pan amyloid binding dye X-34 (C) were performed on sections of hippocampus from a Braak Stage V case (Case-10). These experiments indicate that pSer262 signal, even in this late Braak Stage case marks newly developing NFT that have weak co-labeling with antibody AT8 and very little or no fibrillar tau (i.e., very little or no co-labeling with the dye, X-34). However, their abundance is significantly higher than seen in lower Braak Stages (see FIG. 3), complementing blood biomarker studies assaying this phospho-epitope.
  • FIG. 5 shows Triple-fluorescence labeling experiments combining pSer356 with pSer202/pThr205 (AT8: B) and the pan amyloid binding dye X-34 (C) were performed on sections of hippocampus from a Braak Stage V case (Case-10). These experiments indicate that even in this late Braak Stage case, pSer356 signal marks newly developing NFT that have weak co-labeling with antibody AT8 and very little or no fibrillar tau (i.e., very little or no co-labeling with the X-34 dye). Thus, like pSer262, the antibody targeting the pSer356 phospho-epitope labels very early forms of NFT.
  • FIGS. 6A-6D show (A) a schematic illustration of the principle of the tau- FRET assay. Simultaneous binding of the donor-antibody and acceptor-antibody
  • the boxplot center indicates the median, the box boundaries indicate the 25th and 75th percentiles, and the whiskers indicate extreme values outside the box boundaries (Q1 - 1.5 x IQR and Q3 + 1.5 x IQR), where Q1 , Q3 and IQR refer to the 25th percentile, 75th percentile and interquartile range, respectively.
  • the FRET assay recognizes soluble and solubilized tau assemblies from recombinant sources and from human brain tissues; it is selective for ST As in AD versus other tauopathies and the signal decrease is proportional to the relative amount of soluble and solubilized assemblies present after sample dilution.
  • FIGS. 7A-7J show (A-B) a schematic illustration of the epitopes of the anti- tau antibodies used in the IP experiments. These antibodies target defined nonphosphorylated tau sites;
  • C Flow diagram of the experimental procedure used to isolate TBS-soluble tau fractions from AD human brains. Aliquots of the soluble fraction underwent immunodepletion using a defined antibody, including those shown in a. Afterward, the depleted fraction was examined with the FRET assay described in Fig. 1 , while the precipitate portion was evaluated using immunoblotting. The diagram in the inset illustrates that fuzzy coat peptides are more accessible to antibodies and thus are first removed in the imunodepletion step using IP with those antibodies.
  • n 2 biological replicates.
  • E-F For three antibodies with epitopes in the N terminus (tau12), MTBR (77G7) and the extreme C terminus (tauAB), the corresponding immunoreactivity of the precipitate fractions to the selected antibodies using immunoblotting is shown. Representative images from three biological replicates.
  • G Schematic illustration of the epitopes of the set of ‘CT’ mAbs developed in this study against defined regions inside and outside the MTBR.
  • H Schematic illustration of the anti-tau antibodies used in this study that target the phosphorylation sites.
  • tau-FRET assay signals after immunodepletion of TBS-soluble brain fractions with anti-tau antibodies that target defined phosphorylation epitopes relative to a nondepleted control. Data are shown as the mean with individual data points overlaid, n 2 biological replicates. This figure shows that STAs in TBS-soluble AD brain tissue contain a core region that covers the peptide ⁇ tau258-368. IP followed by high- resolution MS analysis using several anti-tau antibodies identified that tau forms that contain this STA core region are long, near-full-length fragments that stretch from the N terminus or mid-region into the MTBR
  • FIGS. 8A-8C show chromogen immunohistochemistry (IHC) analyses of tau forms with phosphorylated epitopes inside (p-tau262, p-tau356) and outside (p- tau231 , p-tau202/205 (AT8)) the STA core region in postmortem human hippocampus, a-j, Photomicrographs of hippocampal tissue sections from an individual with Braak NFT stage II (a-e,a1-e1 ) and an individual with Braak NFT stage VI (f— j,f1— j1 ) immunohistochemically processed using antibodies directed against the p-tau262 epitope of the tau protein (a,a1 ,f,f1 ), the p-tau356 epitope (b,b1 ,g,g1 ), the p-tau231 epitope (clone AT 180; c,c1 , h , h 1 ) and the p-tau202
  • pan-amyloid binding dye X-34 was used to confirm the presence of fibrillar tau Immunolabeling and histofluorescence observed in the CA1 region, near the CA1/CA2 border, are illustrated at low (a-e,f-j) and higher (a1-e1 ,f1— j1 ) magnification. The locations of the higher-magnification images are indicated by dashed outlines in the low-magnification images.
  • the illustrated immunohistochemical staining was replicated in three sections per individual in each of three individuals with Braak II and five individuals with Braak VI.
  • the p-tau262 and p-tau356 immunostaining is localized mainly to granular structures inside hippocampal neurons,
  • FIGS. 9A-9B show dual immunofluorescence staining of the p-tau262 and p-tau356 sites in the STA core versus p-tau202/205 (AT8) in the fuzzy coat in human postmortem tissue at early and late Braak NFT stages, a-l, Tissue sections of the hippocampus from an individual at Braak NFT stage II (a-f) and an individual at Braak NFT stage VI (g-l) were processed using dual immunofluorescence to assess the codistribution of p-tau262 labeling with p-tau202/205 (AT8) labeling (Braak II: a-c; Braak VI: g-i) and p-tau356 labeling with p-tau202/205 (AT8) labeling (Braak II: d-f; Braak VI: j-l).
  • green fluorescence indicates p-tau262 (a,g) or p-tau356 (d,j); red fluorescence indicates p-tau202/205 (AT8) (b,e,h,k).
  • Merged images are shown in c,f,i,L The illustrated immunohistochemical staining was replicated in three sections per individual in each of three Braak II and five Braak VI individuals.
  • hippocampal neurons with confluent p-tau202/205 labeling also contained p-tau262 or p-tau356-labeled granular structures in a portion of the cell cytoplasm
  • braak NFT stage VI only a subset of hippocampal neurons with confluent p-tau202/205 labeling also contained p-tau262 or p-tau356 immunofluorescence.
  • FIGS. 10A-10J show (A) a schematic representation of recombinantly produced truncated tau species covering the putative STA core sequence (aa 258- 368), the insoluble fibril core peptide (aa 302-368) and the flanking N-terminal (aa 1- 124) and C-terminal (aa 368-441 ) peptide controls. (B) SPR profiles of the recombinant STA core peptide relative to the fibril core sequence, and the N-terminal and C-terminal peptides. The SPR sensorgrams illustrate the effectiveness of the CT19.1 antibody in recognizing the specific epitope within the tau sequence (aa 331 — 361 ).
  • Each ‘n’ in d-h represents a whole-cell patch clamp recording made from individual acute hippocampal brain slices incubated with one of the recombinant tau peptides (diluted in aCSF; Methods) or control aCSF.
  • E Representative example of membrane potential responses to naturalistic current injection86 for each of the three conditions described in (D).
  • FIGS. 11A-11G show (A) levels of the CSF tau STA and t-tau ratio in a well- characterized cohort with paired antemortem CSF samples and neuropathological diagnosis at postmortem (cohort 3).
  • the center line represents the median
  • the boundaries of the box are the 25th and 75th percentiles
  • the whiskers extend to the furthest data value 1.5 times the interquartile range (IQR).
  • IQR interquartile range
  • FDR Benjamini-Hochberg false discovery rate
  • the center of the box plot indicates the median, the box boundaries indicate the 25th and 75th percentiles, and the whiskers indicate the furthest value 1.5 times the IQR.
  • the plot in panel f is z-scored representation of the CSF STA/t-tau ratio normalized to the Braak 0 group as control.
  • FIG. 12 depicts pSer262 tau antibody-based IHC analyses of p-tau lesions in middle temporal gyrus based on Braak NFT Stages.
  • FIG. 13 depicts AT8 (pSer202/pThr205; A, a, b), pSer262 tau (B, c), and pSer356 (C, d) antibody-based IHC studies of CA1 hippocampus and adjacent fiber tracts in a case of AD (Braak Stage III) with a common non-AD related tau copathology termed aging-related tau astrogliopathy (ART AG).
  • FIG. 14 depicts AT8 (pSer202/pThr205; A, a, b), pSer262 tau (B, c), and pSer356 (C, d) antibody-based IHC studies of cortical white matter in a case of Alzheimer’s disease (Braak Stage III) with ART AG co-pathology.
  • FIG. 15 depicts pSer356 tau immunoreactivity (B and C, E and F) compared to immunoreactivity seen with antibody clone AT8 (pSer202/pThr205, A and D) in middle frontal cortex from a case neuropathologically diagnosed with the 4R tau tauopathy, corticobasal degeneration (CBD, A-C), and a case neuropathologically diagnosed with the primarily 3R tau tauopathy, Pick’s disease (PiD, D-F).
  • the term “comprising” is open-ended and may be synonymous with “including”, “containing”, or “characterized by”.
  • the term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • embodiments “comprising” one or more stated elements or steps also include, but are not limited to embodiments “consisting essentially of” and consisting of these stated elements or steps.
  • a "patient” or “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose).
  • a primate such as a human, a non-human primate, e.g., a monkey, and a chimpanzee
  • a non-primate such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a
  • patient typically refers to human patients, but does not require that the individual is under the care of a physician.
  • treating or “treatment” refer to a beneficial or desired result, such as improving one of more functions, or symptoms of a disease.
  • the "treatment” or “treating” of a patient means administration to a patient by any suitable dosage regimen, procedure and/or administration route of a composition, device, or structure with the object of achieving a desirable clinical/medical end-point, including but not limited to, any suitable treatment for AD or a tauopathy, and also includes monitoring the patient for development of AD or a tauopathy by any useful method, including by use of a method according to any aspect, embodiment, or example, provided herein.
  • An amount of any reagent or therapeutic agent, administered by any suitable route, effective to treat a patient is an amount capable of improving any symptom or physiological effect of AD or a tauopathy in a patient.
  • a therapeutic agent may be administered by any effective route.
  • a therapeutic agent may be administered as a single dose, at regular or irregular intervals, in amounts and intervals as dictated by any clinical parameter of a patient, or continuously.
  • a treatment method may include one or more steps of identification and/or quantification according to any aspect or embodiment provided herein.
  • “Therapeutically effective amount” or an “amount effective” as used herein is intended to include the amount of a therapeutic agent as described herein that, when administered to a subject having a disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease or one or more symptoms of disease).
  • the “therapeutically effective amount” may vary depending on the nature of the injury and its causes, how the therapeutic agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.
  • a “therapeutically-effective amount” also includes an amount of a therapeutic agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment.
  • An “amount effective" for treatment of a condition is an amount of an active agent or dosage form, such as a single dose or multiple doses, effective to achieve a determinable end-point.
  • the "amount effective” is preferably safe - at least to the extent the benefits of treatment outweighs the detriments, and/or the detriments are acceptable to one of ordinary skill and/or to an appropriate regulatory agency, such as the U.S. Food and Drug Administration.
  • a therapeutically effective amount of an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual.
  • prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).
  • a single bolus may be administered, several divided doses may be administered over time, or the composition may be administered continuously or in a pulsed fashion with doses or partial doses being administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120 minutes, every 2 through 12 hours daily, or every other day, etc., be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • Drug products, or pharmaceutical compositions comprising an active agent may be prepared by any method known in the pharmaceutical arts, for example, by bringing into association the active ingredient with the carrier(s) or excipient(s).
  • a “pharmaceutically acceptable excipient”, “carrier”, or “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof.
  • the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used in delivery systems, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are broadly-known to those skilled in the art. The preferred form may depend on the intended mode of administration and therapeutic application, which will in turn dictate the types of carriers/excipients. Suitable forms include, but are not limited to, liquid, semi-solid, and solid dosage forms.
  • compositions adapted for oral administration may be presented, for example and without limitation, in capsules, tablets, oral solutions, or the like, and include suitable carriers and coatings as are broadly-known in the pharmaceutical arts.
  • compositions adapted for parenteral administration may be presented, for example and without limitation, in syringes, vials, bottles, IV/infusion
  • Excipients include, for example and without limitation, water, saline, PBS, lactated Ringers, or any other injectable carriers. Suitable emulsifiers, lipids, surfactants, or the like may be utilized to maintain an active agent in solution.
  • compositions adapted for transdermal administration may be presented, for example and without limitation, as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time or electrodes for iontophoretic delivery.
  • compositions adapted for topical administration may be formulated, for example and without limitation, as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • sterile injectable solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with suitable carrier(s), followed by filter-sterilization.
  • An appropriate fluidity of a solution can be maintained, for example, by the use of a rheology modifier.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium, zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium, zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • the therapeutic agents described herein can be administered by any effective route.
  • delivery routes include, without limitation: topical, for example, epicutaneous, inhalational, enema, ocular, otic, and intranasal delivery; enteral, for example, orally, by gastric feeding tube, and rectally; and parenteral, such as, intravenous, intraarterial, intrathecally, intramuscular, intracardiac, subcutaneous, intraosseous, intradermal, intrathecal, intraperitoneal, transdermal, iontophoretic, transmucosal, epidural, and intravitreal, with intrathecal and oral approaches being preferred in many instances.
  • Suitable dosage forms may include single-dose, or multiple-dose vials or other containers, such as medical syringes, containing a
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).
  • a single bolus may be administered, several divided doses may be administered over time, or the therapeutic agent may be administered continuously or in a pulsed fashion with doses or partial doses being administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120 minutes, every 2 through 12 hours daily, or every other day, etc., be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the specification for the dosage unit forms may be dictated by and directly dependent on (a) the unique characteristics of the therapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic agent for the treatment of sensitivity in individuals.
  • the methods provided herein comprise imaging brain tissue for diagnosis of a condition related to tauopathies, e.g., Alzheimer's, e.g., pre-Alzheimer's, in a patient.
  • the brain tissue may be obtained by biopsy in a living patient, or by dissection in a deceased patient. While obtaining brain tissue from deceased patient may be used to determine the neuropathological diagnosis, or for research purposes such as drug discovery and optimization, obtaining tissue (biopsy) from a live patient to determine if Alzheimer's, or pre-Alzheimer's is present has significant therapeutic value in that early Alzheimer's (such as pre-Alzheimer's) may be treatable with an appropriate treatment regimen.
  • Tissue samples may be obtained from particular sections of the brain, such as from the hippocampus, entorhinal cortex, or basal forebrain, among other sections of the brain, or central nervous system (CNS), as appropriate. Once obtained, the tissue sample may be preserved by freezing after cryoprotection, paraffin-embedding after fixation, or otherwise. Tissue is then processed, e.g., as indicated, to identify in situ pSer262 or pSer356 epitopes and to evaluate their localization relative to X-34 staining of intracellular protein aggregates which display amyloid [3-sheet structure (tau fibrils) and optionally pSer202/pThr205 epitopes, if present. pSer262 and pSer356 binding signifies pretangles/immature NFTs prior to development of mature NFTs when neurons start to accumulate tau detected by
  • Pretangles are characteristic of an early AD state in the patient and, additionally, indicate that NFT pathogenesis is progressing.
  • Early AD with or without cognitive impairment may be treated with anti-amyloid drugs, such as, for example and without limitation, aducanumab (e.g., Aduhelm®, currently discontinued) donanemab (e.g., KisunlaTM), or lecanemab (e.g., Leqembi®), antiamyloid antibody intravenous infusion therapies.
  • anti-amyloid drugs such as, for example and without limitation, aducanumab (e.g., Aduhelm®, currently discontinued) donanemab (e.g., KisunlaTM), or lecanemab (e.g., Leqembi®), antiamyloid antibody intravenous infusion therapies.
  • Symptomatic AD treatment includes cholinesterase inhibitors, such as, for example and without limitation, galantamine, rivastigmine, and donepezil.
  • cholinesterase inhibitors such as, for example and without limitation, galantamine, rivastigmine, and donepezil.
  • an N-methyl-D-aspartate (NMDA) antagonist such as memantine, donepezil, combinations of memantine and donepezil, rivastigmine (e.g., patch), and/or brexpiprazole may be administered to a patient.
  • NMDA N-methyl-D-aspartate
  • Additional treatments many of which are in development, may be used to treat a patient with AD.
  • the Congo Red derivative dye X-34 stains pathological structures, including extracellular senile plaques (SP) and hyperphosphorylated tau in its insoluble state in the form of intracellular neurofibrillary tangles (NFT) consisting of twisted, paired helical filaments, recognized as representing mature NFT as opposed to pretangles which are composed of soluble tau assemblies recognized as an early step in the formation of NFT.
  • Dyes equivalent to X-34 in their ability to stain mature NFT include thioflavin derivative dyes disclosed, for example and without limitation, in U.S. Patent No. 7,270,800, incorporated herein by reference in its entirety for its disclosure of amyloid binding compounds.
  • Antibodies described herein for use in the described assays and methods are commercially available as polyclonal antibodies, with additional suitable polyclonal antibodies and monoclonal antibodies being commercially available, or readily prepared as polyclonal antibodies, by immunization of an animal, such as a chicken, rabbit, goat, horse, camelid, etc. by common methods, e.g., as described below.
  • Alzheimer's progression may be characterized neuropathologically by Braak NFT Stages, as is known to those of skill in the art.
  • Tau pathology follows a stereotypical pattern approximating the Braak stages defined post-mortem, where tau begins accumulating in mesial temporal regions (Braak l-ll), then spreads to limbic regions (Braak lll-IV), and lastly to the whole neocortical mantle (Braak V-VI). PET studies have confirmed this pattern in vivo. That said, Braak staging, and classic methods of such staging are useful for classifying AD cases based on AD tau NFT
  • an "isolated” or “purified” biological component refers to a component that has been substantially separated, produced apart from, or purified away from other components in a preparation or other biological components in a cell or in an organism in which the component occurs, that is, separated from other chromosomal and extrachromosomal DNA, RNA, proteins or other cellular, tissue, or organ constituents.
  • Nucleic acids, peptides and proteins that have been “isolated” or “purified” thus, include, for example and without limitation, nucleic acids and proteins purified by standard purification methods.
  • nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins.
  • isolated or purified does not require absolute purity; rather, it is intended as a relative term.
  • an isolated biological component is one in which the biological component is more enriched than the biological component is in its natural environment within a cell, or other production vessel.
  • a preparation may be purified such that the biological component represents at least 50%, such as
  • epitope refers to a physical structure or moiety on a molecule that interacts with an antibody or antibody fragment.
  • the primary amino acid sequence can define an epitope, but secondary and tertiary protein structure, as well as post-translational modifications (e.g., phosphorylation), can define an epitope, though secondary and tertiary structure typically follows from the primary amino acid sequence.
  • Antibodies or other binding reagents may be produced by any effective method, such as by hybridoma or they may be recombinantly or synthetically produced.
  • an antigen is introduced into an animal, and antibodies are purified from the animal's blood or other bodily fluid.
  • a suitable antigen may be a fragment of a tau protein that is phosphorylated at the identified location, e.g., pSer202/pThr205, pSer262, and/or pSer356 of tau.
  • An exemplary reference sequence for tau is NCBI Reference Sequence: NP_005901 .2 (microtubule- associated protein tau isoform 2 [Homo sapiens], see also, Goedert M, et al. Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease. Neuron. 1989 Oct;3(4):519-26), having the amino acid sequence of (SEQ ID NO: 1 ), or a sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, and/or 99% or greater, all values and subranges therebetween inclusive:
  • Numbering of tau and phosphorylation sites thereof is made in reference to SEQ ID NO: 1 , including pSer202/pThr205, pSer262, and pSer356.
  • Any fragment of tau, e.g., SEQ ID NO: 1 ) with the specified phosphorylated amino acid, e.g., pSer202/pThr205, pSer262, and pSer356, may be used to generate antibodies or antigen binding reagents for use in the methods provided herein.
  • Polyclonal antibodies may be monoclonal, but typically comprise antibodies directed to different epitopes, to the same epitope with differing avidity, and/or different antibody types. Polyclonal antibodies may be adsorbed, e.g., by affinity, to remove certain fractions, such as removing one or more specific classes of antibodies, or to purify a specific class, e.g., IgG, from a larger antibody pool. Antigens having crossreactivity to a target protein may be removed, e.g., by affinity purification, to yield a non-cross-reactive population of antibodies.
  • antibodies cross-reactive with a non-phosphorylated tau protein may be removed by adsorption to human nonphosphorylated tau protein, or a fragment of human tau in which an amino acid, such as amino acid Ser262, Ser356, Ser202, and/or Thr205 is/are not phosphorylated, where retained antibodies are removed.
  • Retaining antibodies that bind to human tau fragments comprising pSer262, pSer356, pSer202, and/or pThr205 may be effective to enrich a population of antibodies, such as polyclonal or monoclonal antibodies.
  • Binding reagents may be synthetic, in that they do not comprise a naturally-occurring antibody sequence, including engineered versions and derivatives thereof, such as scFv versions thereof, or humanized versions thereof. Based on the present disclosure, any monoclonal or polyclonal antibody or binding reagent that is produced, may be tested in the assays described in the methods below for their ability to identify a biopsy or necropsy with pre-NFT tangles characteristic of early AD or pre-AD tauopathy.
  • NFTs neurofibrillary tangles
  • AD Alzheimer’s Disease
  • pre-AD pre-stage neurofibrillary tangles
  • Such methods allow for earlier diagnoses, and thus earlier implementation of treatments and/or other interventions, potentially slowing progression and/or increasing survival rates.
  • pre-NFT means a soluble, non-fibrillar, diffuse, aggregate of tau protein.
  • pre-NFT and soluble tau aggregate are used interchangeably.
  • matrix NFT means an insoluble, organized, fibrillar tau aggregate.
  • tau protein means a protein having, in a human, an amino acid sequence of SEQ ID NO: 1 , or a sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or
  • a method may include identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample.
  • the sample may be obtained directly from a patient and/or may be obtained from a sample repository.
  • the sample may be a tissue sample, a fluid sample, and/or any other suitable sample obtainable from a patient and likely to have a tau protein therein.
  • the sample is from a patient’s CNS (e.g., neural tissue (including one or more neurons, glial cells (e.g., astrocytes) from one or more of hippocampus, entorhinal cortex, or basal forebrain tissue, and/or the like, cerebral-spinal fluid (CSF), and/or any other neural sample).
  • CNS cerebral-spinal fluid
  • the sample may be a blood sample, a plasma sample, and/or the like.
  • the patient has been diagnosed with a tauopathy.
  • the patient is at risk of developing a tauopathy, based on genetics, one or more co-morbidities, age, family history, and/or the like.
  • a method may include, in non-limiting embodiments, incubating the sample with a composition that includes a first binding reagent.
  • binding reagent refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having an antigen-binding domain which is homologous or largely homologous to an immunoglobulin binding domain, and complexes thereof, which are typically covalently linked, as in immunoglobulin (see, e.g., Chailyan et al. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity. FEBS J. 2011 Aug;278(16):2858-66, U.S. Patent No. 1 1 ,578,428, and U.S. Patent Publication No.
  • An antigen-binding molecule may comprise a nucleic acid, as in the case of an aptamer.
  • the binding reagent operates as a ligand for its cognate antigen, which can be virtually any polypeptide or protein.
  • Natural antibodies typically comprise two heavy chains and two light chains and are bi-valent. The interaction between the variable regions of heavy and light chain forms a binding site (e.g., a paratope, defined by a set of CDRs) capable of specifically binding an antigen.
  • Antigen-binding molecules bind specifically to a target, e.g., an epitope and are therefore "target-specific".
  • target-specific or reference to the ability of one compound to bind another target compound specifically, it is meant that the compound binds to the target compound to the exclusion of others in a given reaction system, e.g., in vitro, or in vivo, to acceptable tolerances, permitting a sufficiently specific diagnostic or therapeutic effect according to the standards of a person of skill in the art, a medical community, and/or a regulatory authority, such as the U.S.
  • FDA Food and Drug Agency
  • Binding reagent also includes miniaturized antibodies or other engineered binding reagents, such as scFvs, that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope-binding (e.g., paratope) sequences and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition.
  • scFvs miniaturized antibodies or other engineered binding reagents, such as scFvs, that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope-binding (e.g., paratope) sequences and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition.
  • multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((ScFv)2 fragments), diabodies, triabodies, tetrabodies, which typically are covalently linked or otherwise stabilized (e.g., leucine zipper or helix stabilized) scFv fragments, bi-specific T-cell engager (BiTE, e.g., a DbTE), di-scFv (dimeric single-chain variable fragment), single-domain antibody (sdAb), or antibody binding domain fragments.
  • monospecific or bispecific antibodies such as disulfide stabilized Fv fragments, scFv tandems ((ScFv)2 fragments), diabodies, triabodies, tetrabodies, which typically are covalently linked or otherwise stabilized (e.g., leucine zipper or helix stabilized) scFv fragments, bi-specific T-cell engage
  • Antibody fragments also include miniaturized antibodies or other engineered binding reagents that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope-binding sequences (e.g., paratope) and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition.
  • one or more antigen-binding or epitope-binding sequences e.g., paratope
  • any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition.
  • scFv molecules may be manufactured using any suitable technology.
  • recombinant cells comprising genes for expressing scFv-containing polypeptides are engineered, e.g., according to decades-old methods using any of a variety of publicly- and commercially-available expression systems.
  • binding reagents any binding reagent reactive with the specified antigen (e.g., peptides comprising human tau pSer262 epitopes, human tau pSer356 epitopes, and human tau pSer202/pThr205 epitopes) may be utilized in the specified assays.
  • peptides comprising human tau pSer262 epitopes, human tau pSer356 epitopes, and human tau pSer202/pThr205 epitopes
  • DOCX Attorney Docket No. 06527-2501 181 may be evaluated in an affinity assay, such as an ELISA assay, bilayer interferometry (e.g., BLItz, see, e.g., Muller-Esparza H, Osorio-Valeriano M, Steube N, Thanbichler M, Randau L. Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes. Front Mol Biosci.
  • BLItz bilayer interferometry
  • the first binding reagent is specific to a region of the tau protein including positions 262 and/or 356 (e.g., Ser262 and/or Ser356 of the tau protein).
  • region it is meant a region encompassing 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more amino acids, all values and subranges therebetween inclusive.
  • the first binding reagent may be specific to a phosphorylated amino acid (e.g., phosphorylated Ser262 (pSer262) and/or phosphorylated Ser356 (pSer356)).
  • the method may include detecting binding between the first binding reagent and the tau protein, where such detecting indicates the presence of a pre-stage NFT in the patient sample.
  • Suitable methods of detecting binding may include, for example and without limitation, immunoprecipitation assays, including binding reagents that are themselves bound to a substrate, such as a bead, as are known in the art.
  • the binding reagent e.g., antibody or fragment thereof
  • the substrate may be a bead, for example an agarose bead or a magnetic bead.
  • Suitable substrates may be modified as is known in the art, for example with coatings that properly configure the binding reagent, that improve signaling during various blotting procedures, and/or that improve adhesion.
  • the method further includes eluting the washed, immunoprecipitated tau peptide (and substrate, e.g., beads), thereby generating free a protein/binding reagent complex (e.g., free of the beads).
  • the elution may be conducted with one or more solutions, for example, a glycine-containing buffer (e.g., a glycine elution buffer).
  • the first binding reagent is an antibody, a fragment thereof, and/or an ScFv that binds to a region of the tau protein including Ser262 and/or Ser356, and/or binds to Ser262 or Ser356, and/or to a phosphorylated equivalent thereof.
  • Suitable antibodies and/or fragments thereof are commercially available, for example from ThermoFisher Scientific.
  • the method may include an eluting step as described above.
  • the protein and binding reagent complex can be used in an assay.
  • an assay may include a mass spectrometry assay, a chromatography assay, a binding assay (e.g., a Western Blot), and/or any assay known to those of skill in the art useful for identification and/or quantification of proteins.
  • the immunoprecipitated protein may be stained, for example with a dye, for example with a dye that allows for localization of binding between the first binding reagent and the tau protein.
  • the dye is a dye is X-34 (e.g., a dye having the formula C24H18O6), available commercially from Cell Signaling Technology.
  • Other dyes such as a thioflavin derivative disclosed in U.S. Patent No. 7,270,800, incorporated herein by reference in its entirety, are within the scope of this disclosure.
  • the binding reagent may be labeled and the signal from that label may be compared to the staining from the dye.
  • the method may include incubating the sample with a second binding reagent (in a first, or a separate second, immunoprecipitation, for example).
  • the second binding reagent is specific to a region of the tau protein including positions 202 and/or 205 (e.g., Ser 202 and/or
  • the second binding reagent may be specific to a phosphorylated amino acid (e.g., phosphorylated Ser 202 (pSer202) and/or phosphorylated Thr205 (pThr205)).
  • the second binding reagent is specific to pSer202/pThr205.
  • the second binding reagent is a monoclonal antibody derived from clone AT8, commercially available from, for example, ThermoFisher Scientific.
  • the method may include incubating the sample with the first and second binding reagent, and in non-limiting embodiments performing immunoprecipitation.
  • an elution step may be performed.
  • the immunoprecipitated protein may be stained, for example with a dye, for example with a dye that allows for localization of binding between the first binding reagent, the second binding reagent, and the tau protein, as described above.
  • the method may include detecting binding between the second binding reagent and the tau protein, and detecting binding between the second binding reagent and the tau protein may indicate the presence a mature NFT in the patient sample.
  • the method may include detecting binding between the first binding reagent and the second binding reagent and the tau protein, and detecting binding between the first binding reagent and the tau protein and an absence of binding between the second binding reagent and the tau protein may indicate the presence of a pre-NFT in the patient sample. Accordingly, in non-limiting embodiments, methods disclosed herein may be useful for distinguishing pre-NFTs and mature NFTs.
  • the patient sample may be from a human patient (though, as noted above, other species are contemplated and fall within the scope of this disclosure).
  • the patient sample is from a living patient.
  • the patient sample is from a living patient and, when a pre-NFT is detected based on the methods described herein, the patient may be treated for early-stage tauopathy, for example early-stage AD, for example, with therapeutic agents such as cholinesterase inhibitors (e.g., donepezil/Aricept), glutamate receptor modulators (e.g., memantine), anti-A
  • cholinesterase inhibitors e.g., donepezil/Aricept
  • glutamate receptor modulators e.g., memantine
  • the method may include, in non-limiting embodiments, incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a composition that includes a first-binding reagent (which may be any binding reagent described herein).
  • a first-binding reagent which may be any binding reagent described herein.
  • the first binding reagent is specific to Ser262 (e.g., pSer262) and/or Ser356 (e.g., pSer356) of a tau protein.
  • the method may further include detecting binding between the first binding reagent and the tau protein (e.g., with an immunoprecipitation assay and/or any other assay described herein or known to those of skill in the art), wherein detecting binding between the first binding reagent and the tau protein indicates the presence of a pre-stage NFT in the patient sample.
  • the sample may be from a living patient, and the method may further include treating the patient with a specific treatment as described herein.
  • the method may include incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a first-binding reagent and a second binding reagent, each of which may be any binding reagent described herein.
  • the first binding reagent is specific to Ser262 (e.g., pSer262) and/or Ser356 (e.g., pSer356) of the tau protein and the second binding reagent is specific to Ser202/Thr205 (e.g., pSer202/pThr205) of the tau protein.
  • the method may include detecting binding between the first binding reagent and the tau protein and between the second binding reagent and the tau protein, where detecting binding between the first binding reagent and the tau protein and an absence of binding between the second binding reagent and the tau protein indicates the presence of a pre-NFT in the patient sample.
  • the sample may be from a living patient, and the method may further include treating the patient with a specific treatment as described herein.
  • detection of pre-NFTs as described herein may be combined with treatments, as a means for, for example, determining efficacy of a treatment and/or monitoring progression of a condition associated with pre-NFTs.
  • Single antibody chromogen-based immunohistochemistry used a published protocol (see, Ikonomovic MD, et al. Post-mortem correlates of in vivo PiB- PET amyloid imaging in a typical case of Alzheimer's disease. Brain. 2008 Jun ;131 (Pt 6):1630-45); this protocol uses a modification of the avidin-biotin/peroxidase technique of Hsu et al. (Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem.
  • Tissue sections (40 pm thick, free-floating) obtained from 4% paraformaldehyde fixed tissue blocks are washed in 0.1 M sodium phosphate buffer (PB, pH 7.4) and endogenous peroxidase activity is inhibited by immersing sections in 0.3% H2O2 made in 0.1 M Tris-buffered saline containing 0.25% Triton X- 100 (TBST) for 45 minutes.
  • PB sodium phosphate buffer
  • Tris-buffered saline containing 0.25% Triton X- 100
  • Sections are then rinsed in TBST and incubated in 3% goat serum made in TBST for 30 minutes followed by two 10-minute rinses in 1 % goat serum in TBST. Sections are incubated overnight at 4°C in rabbit polyclonal primary antibody diluted in TBST. Next, after washing in 1 % goat serum in TBST, sections are incubated for 1 hour in biotinylated anti-rabbit IgG made in goat diluted in 1 % normal serum in TBST, washed in TBST, and processed with the avidin-biotin Elite kit in TBST for 1 hour at room temperature.
  • IHC studies were then performed on fixed tissue sections obtained from the hippocampus from a series of cases (Cases 2-8) that spanned the range of severity of NFT pathology determined by Braak Stage.
  • the hippocampus was chosen as the region of interest due to its early involvement in the neuropathological progression of AD.
  • FIG. 2 Depicts IHC studies of hippocampus based on Braak Stage. Methods were essentially as described above. Adjacent tissue sections were processed using the same immunohistochemical procedure and antibodies AT8 (pSer202/pThr205; A1 -G1 ; Invitrogen, MN1020), pSer262 (A2-G2; Thermo, 44-750- G), and pSer356 (A3-G3; Thermo, 44-751 G). AT8 was used as a comparator to the pSer262 and pSer356 antibodies and was previously optimized for use in our laboratories.
  • AT8 was used as a comparator to the pSer262 and pSer356 antibodies and was previously optimized for use in our laboratories.
  • tissue sections were obtained from the hippocampus from cases neuropathologically determined to be Braak Stage I (Case-2: A1 -A3), Braak Stage II (Case-3: B1 -B3), Braak Stage III (Case-4: C1 -C3), Braak Stage IV (Case-5: D1 -D3), Braak Stage V (Case-6: E1 -E3), and Braak Stage VI (Case-7: F1 - F3).
  • CA1 region one of the earliest sites of NFT development in the hippocampus CA fields
  • pSer262 and pSer356 labeling differed markedly from patterns seen with AT8. Specifically, pSer262
  • Tissue sections were processed as described for chromogen-based immunohistochemical procedures as described above with the following differences: the step in which tissue sections are incubated in H2O2 was omitted, primary antibodies were prepared as cocktails (rabbit polyclonal IgG to pSer262 or pSer356 was diluted together with mouse monoclonal IgG clone AT8); a cocktail of fluorophore-conjugated secondary antibodies was used to achieve distinguishable colors (goat anti-rabbit conjugated to the Alexa488 fluorophore and goat anti-mouse conjugated to the Alexa594 fluorophore).
  • DOCX Attorney Docket No. 06527-2501 181 visualized using a violet filter (excitation peak 405 nm, beam splitter 440, emission peak 455; #1 1005, Chroma).
  • X-34 labels neurofibrillary pathology in neurons containing fibrillar aggregates of tau proteins (e.g., tau protein in its insoluble state, consisting of helical filaments) - these are recognized as representing mature NFT as opposed to pretangles which are composed of soluble, non-fibrillar tau assemblies recognized as an early step in the formation of NFT.
  • tau proteins e.g., tau protein in its insoluble state, consisting of helical filaments
  • the Braak NFT Stage classification scheme is by nature a neuropathological construct for characterization of the neuropathological severity of the disease and does not necessarily reflect an individual's clinical status, as cognitively normal elderly present with a range of Braak Stages from l-lll, and rarely Braak Stage IV.
  • traditional methods used in the original Braak studies included the modified Galiyas silver impregnation technique and immunohistochemical staining using the antibody clone AT8 (AT8 is an antibody directed against tau phospho-epitopes pSer202/pThr205); neither of these post-mortem techniques have pre-mortem biomarker equivalents.
  • the pathological substrates of the antibodies employed as blood-based biomarkers of brain pathology are characterized in post-mortem AD brain tissue; such innovative "blood-to-brain" strategy is an important theoretical basis of this invention.
  • Our histopathological studies described above support that the pSer262 and pSer356 epitopes are important phospho-sites involved in the earliest stage of tau accumulation in neurons in AD. The presence of these epitopes in granular, vesicle like structures, often appears to precede another early phosphorylation site (pSer202/pThr205) as well as tau fibrillization. Thus, these epitopes are highly likely to mark the earliest stage of tau pathology development, supporting their use as early biomarkers of the AD neuropathological process.
  • a dilution linearity test was performed to identify the most suitable fold dilution to use for the brain samples.
  • the TBS-soluble fraction from the AD and control brain samples was used in a test run, first brought to the same total protein concentration of 1.317 mg ml-1 , before a dilution series.
  • the samples were serially diluted 1 :100, 1 :50, 1 :10 and 1 :5 in TBS, incubated overnight with the labeled antibodies added according to the manufacturer’s protocol in 96-wells low-volume white microplates.
  • the AF percentage values were calculated using the ratio between the wavelength and the negative control provided with the kit.
  • the observed signals were multiplied by the fold dilution and compared with the expected signals to determine the linearity of dilution.
  • PCR fragments were cloned directly into the pET_SUMO vector (cohort 3: the Shiley-Marcos Alzheimer's Disease Research Center (ADRC), University of California, San Diego (UCSD)), an expression vector with a 6x His-SUMO tag N-terminally fused to the protein or peptide of interest. Constructs containing the 6x His-SUMO-tau fusion protein were sequenced and transformed into the Escherichia coli BL21 (DE3) strain for protein expression.
  • ADRC Shiley-Marcos Alzheimer's Disease Research Center
  • UCSD San Diego
  • E. coli BL21 (DE3) cells harboring the construct of interest were inoculated into 20 ml lysogeny broth (LB) medium supplemented with kanamycin at a concentration of 50 pg ml-1 and incubated overnight to obtain a starter culture. The overnight culture was used to inoculate 1 I of LB medium with kanamycin (50 pg ml-1 ) and incubated at 37 °C. When the optical density (OD)600 reached 0.5-0.7, protein expression was induced with 1.0 mM isopropyl B-d-1 -thiogalactopyranoside and grown overnight at 27 °C. The next morning, the culture was centrifuged at 7,000 rpm for 20 min at 4 °C and the dry weight was calculated. The pellet was stored at -20 °C until purification.
  • LB lysogeny broth
  • the pellet was gently thawed at room temperature and resuspended in 1 x native buffer containing 50 mM sodium phosphate, pH 8.0, and 0.5 M sodium chloride (Invitrogen) added at a ratio of 8 ml buffer to 1 g of dry weight of pellet. Lysozyme solution (Thermo Fisher Scientific) was added and the lysate was incubated on ice for 30 min, followed by sonication and then centrifugation at 12,000 rpm for 20 min at 4 °C, after which the supernatant was collected.
  • Lysozyme solution Thermo Fisher Scientific
  • the protein extract was added to Ni-NTA agarose columns (Novex) equilibrated with 10 mM imidazole in 1 x native buffer and incubated with gentle rotation at 4 °C for 1 h.
  • the Ni-NTA agarose column was washed with 1 x native buffer containing 20 mM imidazole, and the 6x His-SUMO-tau fusion protein of interest eluted with 250 mM imidazole in 1 x native buffer.
  • the eluted protein was dialyzed against 50 mM Tris-HCI, 150 mM NaCI, pH 7.8, for 1 h; the buffer was replenished with fresh supply and the process was repeated for another hour.
  • DTT Dithiothreitol
  • the Queen Square Brain Bank for Neurological Disorders has generic ethical approval from a London multicenter research ethics committee under a license from the Human Tissue Authority.
  • the Netherlands Brain Bank cohort was approved by the ethics committee of the Vrije Universiteit Medical Center, Amsterdam.
  • the research protocol for the UCSD cohort was reviewed and approved by the human subject review board at UCSD, while informed consent was obtained from all patients or their caregivers as consistent with California State law.
  • Biofluids including the CSF, were periodically collected from the participants who consented. In this study, we measured CSF samples from individuals with both neuropathological examination and antemortem CSF samples within 5 years of death. We included individuals with sporadic disease, excluding those with a family history of autosomal dominant AD, dominantly inherited mutations (such as PSEN1 , PSEN2 and APP mutations) or early-onset disease (under 50 years).
  • AD neuritic plaques, diffuse plaques and NFT were identified either with 1 % thioflavin S staining viewed with ultraviolet illumination and a 440 pm bandpass wavelength excitation filter, or with immunohistochemical staining using antibodies to A
  • 3 antibody 69D, rabbit polyclonal from E. Koo, 1 :1 ,200 dilution
  • PHF1 tau from P. Davies, 1 :600 dilution
  • Neuritic plaque density and NFT pathology were assessed according to CERAD and Braak staging, respectively.
  • pathological diagnosis of AD was made using the National Institute on Aging (NIA)- Alzheimer's Association (AA) consensus criteria.
  • NIA National Institute on Aging
  • AA Alzheimer's Association
  • Lewy body pathology was evaluated using hematoxylin and eosin staining in addition to immunostaining with antibodies against a-synuclein (p-synuclein 81 A from V. Lee, 1 :15,000 dilution).
  • Disease staging was performed in accordance with consensus LBD guidelines93.
  • TDP-43 pathology was identified using immunohistochemical staining (polyclonal, 1 :12,000 dilution, cat. no. 10782-2-AP, Proteintech).
  • TBS-soluble fraction The total protein concentration in the various TBS extracts was determined using the DC Protein Assay (Bio-Rad Laboratories).
  • the antibodies used in these experiments included Tau12, 95-108, HT7, BT2, Tau5, K9JA, 77G7, 4R, 368, 419, Tau46, TauAB, CT2, CT3, CT4, CT1 , CT5, and P-tau181 , which are commercially available from, for example, DAKO, BioLegend and ThermoFisher Scientific.
  • Various antibodies and sources thereof are listed below in Table 2:
  • the indicated anti-tau antibodies were conjugated to Dynabeads M-280 sheep anti-mouse or anti-rabbit IgG (Thermo Fisher Scientific), respectively, depending on the origin of the antibody, and according to the manufacturer’s recommended protocol. Briefly, 10 pg of total protein from the brain extract was incubated with the Dynabeads-antibody complex (that is, 4 pg antibody added to 50 pl beads in 1 x PBS) and incubated overnight at 4 °C with gentle rocking to enable even
  • the Dynabeads-antibody complex was recovered by using a magnetized rack, the supernatant was reincubated in new 50 pl antibody- Dynabeads conjugate and the immunoprecipation or depletion process repeated for 2 h at room temperature. Afterwards, the Dynabeads-antibody complex (the precipitate fraction) was recovered and the remaining sample (the depleted fraction) was used in the tau-homogeneous time-resolved fluorescence energy transfer assay, where 10 pl of each sample was analyzed and untreated TBS-soluble brain extract from the same patient was used as control for the depleted samples.
  • IP-MS experiments were performed at the MS facility in the Biofluid Biomarker Laboratory, Department of Psychiatry, University of Pittsburgh. Briefly, 30 pl of a pooled TBS-soluble fraction (7.1 mg ml-1 ) from the mid-temporal regions of postmortem brains was diluted to 1 ml with binding buffer (100 mM Tris-HCI, pH 7.4, 300 mM NaCI, 0.2% w/v n-dodecyl-B-d-maltoside, 0.2% w/v n-Nonyl-[3-d-thiomaltoside (cat. no.
  • binding buffer 100 mM Tris-HCI, pH 7.4, 300 mM NaCI, 0.2% w/v n-dodecyl-B-d-maltoside, 0.2% w/v n-Nonyl-[3-d-thiomaltoside (cat. no.
  • proteins bound to the beads were eluted twice with 100 pl glycine buffer (50 mM glycine, pH 2.8, 0.1 % n-dodecyl-B-d-maltoside). The combined eluates were then neutralized with 5.5 pl 2N NaOH. Two replicate IPs were performed for each tau antibody.
  • Proteins were digested using SP3-based trypsin digestion94, similarly as described previously. Briefly, 50 pl of nondepleted and depleted fractions and 160 pl of precipitated fractions were brought up to 200 pl with 100 mM Tris, pH 8.0, and 2% SDS. The samples were then reduced with 10 mM DTT at 56 °C for 10 min and
  • trypsin Sequencing Grade Modified Trypsin; cat. no. V51 1 1 , Promega Corporation
  • 100 pl of 50 mM ammonium bicarbonate with 1 mM CaCI2 was used to digest the proteins bound to the magnetic particles.
  • the samples were desalted with C18 spin cartridges (cat. no. SMM SS18V, The Nest Group), dried using a SpeedVac and then reconstituted in 0.1 % formic acid (20 pl for nondepleted and depleted fractions and 16 pl for the precipitate).
  • the reconstituted peptides were analyzed using reverse-phase LC- tandem MS (MS/MS) using a nanoflow LC (a Dionex UltiMate 3000 RSLCnano System) coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific).
  • the Xcalibur software (v.2.2 SP1.48, Thermo Fisher Scientific) was used to operate the LC-MS/MS system.
  • 1 pl peptides were directly injected onto a 5-cm Aurora series electrospray ionization column with a 150 pm ID filled with 1.6 pm reversed-phase C-18 packing material (120-A pore size) (lonOpticks).
  • Peptides were eluted using a linear gradient of 3-34% mobile phase B (0.1 % formic acid in acetonitrile) in 5.5 min, then to 90% B for an additional 1 min, all at a constant flow rate of 1 pl min-1.
  • Data acquisition parameters included a full MS scan from 350 to 1 ,600 m/z at a 30,000 resolution and an automatic gain control (AGC) target of 300%, followed by four data-dependent MS/MS scans at a 15,000 resolution and a standard AGC target, and a retention-time-scheduled PRM analysis of 18 tau peptides.
  • AGC automatic gain control
  • the PRM parameters included an Orbitrap resolution of 15,000, a standard AGC target, an automatic injection time, an isolation window of 2 m/z and a higher- energy C-trap dissociation-normalized collision energy of 30.
  • Supplementary Table 1 shows the targeted inclusion list with the retention-time-scheduled PRM scans. Each peptide sample was analyzed twice using LC-MS/MS.
  • Skyline (v.21 .2.0.568) was used to facilitate the extraction of peptide quantification data from the PRM scans.
  • the chromatogram peak selection for each PRM assay was based on the presence of at least ten coeluting fragment ions.
  • the final quantification of each peptide was based on the total area of
  • Sandwich ELISAs were used to validate the specificity of the p-tau262 and p-tau356 antibodies. For each measurement, 80 pl of the antibodies at 2 pg ml-1 in PBS, pH 7.2, was added to the well and incubated overnight at 4 °C. The well was then blocked with 200 pl PBS/0.1 % BSA (cat. no. 81 -053-3, Merck Millipore) for 1 h at room temperature. After blocking, the well was washed twice with 300 pl PBS with 0.05% Tween 20 (PBST).
  • PBST Tween 20
  • OD at 550 nm was used to determine color development.
  • the indicated synthetic peptides at a 0.1 pg ml-1 concentration were added to each well for competitive ELISA during the immunocomplex formation step.
  • the recombinant tau aggregate preparations (5 pl) were pipetted onto copper grids that had been glow-discharged and carbon-coated, allowed 1 min to adhere onto the grid surface and then rinsed with ultrapure distilled water. Next, the grids were treated with 0.75% uranyl formate (Electron Microscopy Sciences) for 30 s to enable negative staining.
  • TEM micrographs were taken on a Talos L120C 120 kV TEM microscope (Thermo Fisher Scientific) fitted with a BM-CETA camera- 4.096 x 4.096, 14-pm pixel complementary metal-oxide-semiconductor. Microscopic imaging was performed at the Centre for Cellular Imaging at the University of Gothenburg.
  • TBS-soluble homogenates were first immunoprecipitated (following the procedures described above) with the tau12 antibody (which has been shown to enrich for tau forms that stretch into the MTBR; to
  • the SPR experiments were performed using a Biacore T100 biosensor (GE Healthcare). Immobilization of the CT19.1 antibody (epitope: aa 331-361 of tau44i) ligand on the surface of a CM5 chip was performed at a 5 pl min-1 flow rate to a level of 4,000 response units using standard amino coupling reagents (Cytiva). Thereafter, the analytes (that is, the truncated tau peptides) were injected at a flow rate of 20 pl min-1 , with the experiments being performed in PBS at 25 °C. The BIAevaluation and Prism 9 (Graph Pad Software) software programs were used for data processing and presentation, respectively.
  • the new library of anti-tau mAbs was generated by immunizing 8-week-old BALB/c mice with 100 pg of recombinant tau 241 -441 peptide in complete Freund’s adjuvant (Sigma-Aldrich). After 2-3 further dosages of the immunogen (100 pg per mouse) in incomplete Freund’s adjuvant (Sigma-Aldrich), mice were euthanized, the spleen was removed and B cells were fused with the SP2/0 myeloma cell line according to standard protocols. Approximately 10 days after fusion, direct ELISA experiments were performed to screen the cell medium for antibodies that react with full-length recombinant tau441 (2N4R) or tau24i ⁇ 4i.
  • a new polyclonal antibody specific against tau truncated at aa 368 was generated by immunizing rabbits with 200 pg of a peptide containing the tau36o-368 sequence (Casio ApS) in complete Freund’s adjuvant. After one more dose of the
  • Hippocampal tissue samples were obtained from autopsy cases at the University of Pittsburgh ADRC brain bank, including cases with NFT stage B1 or B2 and those with severe NFT stage B3 (ref. 96).
  • samples of the hippocampus were dissected at the level of lateral geniculate nucleus and placed in cold (4 °C), 4% paraformaldehyde (cat. no. 158127- 5006, Sigma-Aldrich) made in 0.01 M sodium phosphate buffer (pH 7.2) (sodium phosphate monobasic and dibasic, cat. nos.
  • Tissue sections were removed from the cryoprotectant solution and rinsed three times in 0.1 M Trizma-buffered saline containing 0.25% Triton X-100 (Trizma, pH 7.4, cat. no. T7693, Sigma-Aldrich; Triton X-100, cat. no. T9284, Sigma-Aldrich).
  • Chromogen-based IHC was performed as described previously using the VECTASTAIN Elite ABC-HRP Kit (cat. no. PK-6100, Vector Laboratories) with Ni- enhanced 3,3'-diaminobenzidine tetrahydrochloride (cat. no. D8001 , Sigma-Aldrich) as the chromogen.
  • the details of the primary antibodies including primary antibodies targeting an epitope at p-tau202/205 (clone AT8, 1 :3,000 dilution for chromogen-based IHC and 1 :500 dilution for the multi-immunofluorescence experiments, cat. no. MN1020, Thermo Fisher Scientific), an epitope at p-tau231 (clone AT180, 1 :1 ,000 dilution for chromogen-based IHC, cat. no.
  • Secondary antibodies are given in Supplementary Table 6. Secondary antibodies (all used at 1 :250 dilution) included a biotinylated goat anti-mouse IgG (cat. no. 1 15-065-146, Jackson ImmunoResearch), a biotinylated goat anti-rabbit IgG (cat. no. 1 1 1 -065-045, Jackson ImmunoResearch), an Alexa Fluor 594-conjugated goat anti-mouse IgG (cat. no. 115-585-146, Jackson ImmunoResearch) and an Alexa Fluor 488-conjugated goat anti-rabbit IgG (cat. no. 1 1 1 -585-144, Jackson ImmunoResearch). X-34 staining was performed as described previously.
  • Light microscopy analysis was performed using an Olympus BX53 microscope.
  • the immunofluorescence analysis was performed as described previously, using the Olympus BX53 microscope connected to a fluorescence illuminator (X-Cite 120Q).
  • the microscope was equipped with an Olympus DP72 digital camera connected to a Dell Precision T5500 Desktop Workstation running the Olympus cellSens Standard v.1 .12 imaging software, and with a U PLAN S-APO x4 objective (numerical aperture (NA) 0.16), a U PLAN S-APO x10 objective (NA 0.4) and a U PLAN S-APO x20 objective (NA 0.75).
  • the fluorescence of the Alexa594 fluorophore was visualized using a Texas red isothiocyanate-compatible filter (excitation peak 535 nm, beam splitter 565 nm, emission peak 610 nm; #41002, Chroma).
  • the brain was rapidly removed and acute parasagittal or horizontal brain slices (350-400 pM) were cut with a Microm HM 650V microslicer in cold (2-4 °C) high Mg2+, low Ca2+ aCSF, consisting of the following: 127 mM NaCI, 1 .9 mM KCI, 8 mM MgCI2, 0.5 mM CaCI2, 1 .2 mM KH2PO4, 26 mM NaHCO3 and 10 mM d-glucose (pH 7.4 when bubbled with
  • a slice was transferred to the recording chamber, submerged and perfused (2-3 ml min-1 ) with aCSF at 30 °C.
  • Slices were visualized using infrared IR differential interference contrast optics with an Olympus BX151 W microscope (Scientifica) and a charge-coupled device camera (Hitachi).
  • Whole-cell current clamp recordings were made from pyramidal cells in area CA1 of the hippocampus using patch pipettes (5- 10 mQ) manufactured from thick-walled glass (Harvard Apparatus). Pyramidal cells were identified by their position in the slice, morphology (from fluorescence imaging) and characteristics of the standard step current-voltage relationship.
  • Voltage recordings were made using an Axon Multiclamp 700B amplifier (Molecular Devices) and digitized at 20 kHz. Data acquisition and analysis were performed using pCIamp 10 (Molecular Devices). Recordings from neurons that had an RMP of between -55 and -75 mV at whole-cell breakthrough were accepted for analysis. Bridge balance was monitored throughout the experiments; any recordings where it changed by more than 20% were discarded.
  • a standard current-voltage relationship was constructed by injecting standard (step) currents from -200 pA, incrementing by either 50 or 100 pA (1 -s duration) until a regular firing pattern was induced.
  • step currents from -200 pA, incrementing by either 50 or 100 pA (1 -s duration) until a regular firing pattern was induced.
  • step current against voltage response around the resting potential was used to measure the infrared (from the gradient of the fitted line).
  • a dynamic-l-V curve defined by the average transmembrane current as a function of voltage during naturalistic activity, can be used to efficiently parameterize neurons.
  • This current waveform which mimics the stochastic actions of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and gamma- aminobutyric acid receptor channel activation, is injected into cells and the resulting voltage recorded (a fluctuating, naturalistic trace).
  • the voltage trace was used to measure the frequency of action potential firing and to construct a dynamic-l-V curve.
  • the FR was measured from voltage traces evoked by injecting a current waveform of the same gain for all recordings (to give an FR of ⁇ 2-3 Hz).
  • Action potentials were detected by a manually set threshold and the interval between action potentials was measured. All analyses were completed using either the MATLAB or Julia (v.1.7.3) software platforms.
  • a 400-pM parasagittal slice was transferred to the submerged recording chamber and perfused with aCSF at 4-6 ml min-1 (32 °C). The slice was placed on a grid allowing perfusion above and below the tissue; all tubing (Tygon) was gas-tight to prevent loss of oxygen.
  • fEPSPs field excitatory postsynaptic potentials
  • an aCSF-filled microelectrode was placed on the surface of the stratum radiatum in CA1 .
  • a bipolar concentric stimulating electrode (FHC) controlled by an isolated pulse stimulator model 2100 (AM Systems) was used to evoke fEPSPs at the Schaffer collateral-commissural pathway.
  • fEPSPs were evoked every 30 s (0.03 Hz). Stimulus input and output curves for the fEPSPs were generated using a stimulus strength of 2-80 mA for all slices (stimulus duration 200 ps). Signals were filtered at 3 kHz and digitized online (10 kHz) with a Micro CED (mark 2) interface controlled by the Spike
  • a rabbit polyclonal antibody targeting an end-specific truncation at aa 368 was coupled to paramagnetic beads (cat. no. 103207, Quanterix), while the detection antibody CT23.1 (epitope: aa 321-371 ) was conjugated to biotin (cat. no. A3959, Thermo Fisher Scientific) according to the manufacturer’s recommendations.
  • the resulting method was a three-step Simoa assay that combined the assay beads (that is, beads conjugated with the capture antibody) and the helper beads in a 70% to 30% ratio to give 20,000 beads per pl and 1 pg ml-1 of biotin- conjugated detection antibody with 100 pl of undiluted CSF. The average number of enzymes per bead signal for each sample was plotted against the concentration of the inputted biospecimen.
  • the 368-coated beads were used for IP of either the positive control or antigen (aa ITHVPGGGN (SEQ ID NO: 2) equivalent to aa 359-368 with truncation at aa 368) or the negative control (aa GSLDNITHVPGGGNKKIETHKLTFRE (SEQ ID NO: 3) 355-380 lacking truncation at aa 368) in PBS.
  • Beads and samples were transferred to a KingFisher magnetic particle processor (polypropylene tubes, Thermo Fisher Scientific) for automatic washing and elution of full-length and truncated peptides.
  • CN participants were defined as having an MMSE score greater than 24 and a CDR score of 0. This group included both young individuals (younger than 30 years) and older adults (older than 55 years). Participants with MCI had a CDR score of 0.5, with subjective and objective impairments in cognition, while their activities of daily living were preserved. Patients with AD dementia met the diagnostic criteria of the NIA and AA, and had a CDR score greater than or equal to 0.5.
  • [18F]AZD4694 and [18F]MK6240 PET were used to assess brain A
  • PET scans from each participant were combined with their magnetic resonance imaging data.
  • the cerebellar gray matter and the inferior cerebellar gray matter were used as reference regions for calculating the standard uptake value ratio (SUVR) for amyloid-[3 and tau-PET, respectively.
  • SUVR standard uptake value ratio
  • 3 positivity was determined as a global [18F]AZD4694 SUVR equal to or greater than 1 .55.
  • a global index of tau pathology was obtained by calculating the average SUVR in the temporal meta-region of interest. Tau positivity was then defined as an SUVR equal to or greater than 1 .24.
  • participants were categorized into PET-based Braak stages based on the topography of tau-PET abnormality, as described in previous studies.
  • Python v.3.1 1 .2 was used to perform nonimaging statistical analyses. For several data processing and statistical tasks, several additional packages were used. Pandas (v.1 .5.3) was used as a powerful data analysis tool, providing data structures like DataFrames and Series, which allowed for efficient data handling and transformation. NumPy (v.1 .24.2) was used for numerical computations, enabling the manipulation of multidimensional arrays and matrices. Scikit-learn (v.1.2.2) was used for regression, clustering and model evaluation. Statsmodels (v.0.13.5) was used for statistical modeling and hypothesis testing.
  • FRET fluorescence resonance energy transfer
  • the assay signal (background-subtracted percentage fluorescence signal increase over the negative control) is proportional to the number and sizes of STAs present.
  • FRET assay is specific to AD-type ST As
  • tau-FRET assay had a low coefficient of variation (that is, high precision) in both cohorts, there was remarkable heterogeneity in signals within groups despite all brain samples being at the advanced stages of pathology, suggesting inherent interindividual variability in STA levels. Together, these results show that the FRET assay is selective for AD-type ST As over those in other tauopathies.
  • AD tau filaments consist of a core region and an outer layer (fuzzy coat) binding adhesively to the Corel 6,31 ,32
  • STAs will have a similar organization.
  • immunodepletion with anti-tau antibodies will first remove the outer fuzzy coat layer before targeting the difficult-to-reach core region.
  • antibodies targeting epitopes outside the core region will be more efficient at removing the aggregation signal obtained in the FRET assay when that same antibody is used to immunoprecipitate tau from the sample before the FRET analysis, and vice versa.
  • the core region will be minimally affected by immunodepletion as it should be least accessible to antibodies.
  • the fuzzy coat contains N-terminal and C-terminal tau
  • Tau12 was efficient at depleting the tau-FRET signal (FIG. 7C). To estimate the length of tau12+ fragments, we screened antibodies covering neighboring sites. Tau12-precipitated tau stained positive for BT2 (aa 194- 198) but not 77G7 or tauAB, meaning that tau12-containing forms stretched into the mid-region but not into the far end recognized by 77G7 or the extreme C terminus where tauAB binds (FIG. 7D). In contrast, 77G7 was poor at depleting the tau-FRET signal (FIG. 7C), suggesting that it targets an epitope within the STA core.
  • 77G7- precipitated tau fractions were negative for HT7 (aa 159-163) and BT2, indicating that the tau fragment immunoprecipitated by the 77G7 antibody lacked the mid-region sequences recognized by HT7 and BT2 (FIG. 7D).
  • the tauAB-precipitated fraction did not stain strongly for the mid-region-targeting antibodies HT7 and BT2 or the MTBR-binding UGH (FIG. 7D).
  • IP paramagnetic bead-based immunoprecipitation
  • the mid-region-directed antibodies HT7, BT2 and tau5 were not as efficient as tau12 in precipitating N-terminal tau, but they were highly efficient in enriching midregion tau and MTBR sequences, including aa 231-240 and 282-290, but not any peptide C terminus to these.
  • 77G7 showed reduced efficiency in precipitating the N terminus and mid-region of tau but much higher efficiency for the MTBR and C terminus, particularly after aa 275 (the R2 MTBR region).
  • tau46 antibody precipitated fragments containing the MTBR-end species through the C terminus that is, aa 354-438
  • tau forms that contain the STA core included near-full-length sequences that can stretch from the N terminus to the MTBR and from the mid-region to the far C terminus.
  • P-tau262 and p-tau356 in the STA core detect early-stage NFTs
  • each antibody labeled isolated groups of CA1 pyramidal cells (FIG. 8, panels a-d, a1-d1 ), whereas at the high Braak NFT stages (for example, Braak stage VI; FIG. 8) they revealed high frequencies of immunolabeled cells throughout the CA1 region (FIG. 8, panels f-l, fl-il ).
  • the presence of fibrillar tau was confirmed using the pan-amyloid binding dye X-34 (FIG. 8, panels e, e1 , j, j1).
  • the p-tau262 and p-tau356 antibody immunostaining patterns differed from those produced by p-tau23i and p-tau202/205 antibodies in the overall appearance and extent of labeling neurons and neuritic pathology (dendritic tau pathology presenting as neuropil threads and axonal tau pathology presenting as dystrophic neurites in neuritic plaques).
  • the p-tau262- directed antibody labeled pre-NFTs with a predominantly granular, vesicle-like immunolabeling pattern in portions of the cell soma and, less frequently, a diffuse, confluent immunolabeling over the cell soma and proximal dendrites at both low and high Braak NFT stages (FIG. 8, panels a1 , f1), while p-tau262 antibody labeling of neuropil threads was rare or absent (FIG. 8, panels a, a1 , f, f1 ).
  • the p-tau356-directed antibody resulted in more instances of confluent staining in pyramidal cell bodies and portions of proximal dendritic processes, as well as granular and vesicle-like staining, in both low and high Braak NFT stages (FIG. 8, panels b, b1 , g, g1).
  • the p-tau356 antibody also labeled small numbers of neuropil threads and neuritic processes (FIG. 8, panels g, g1 ).
  • the p-tau231 -directed Compared with the p-tau262 and p-tau356 antibody immunostains, the p-tau231 -directed (FIG.
  • Dual immunofluorescence was used to assess the relationship of p-tau262 and p-tau356 with p-tau202/205 immunolabeling in CA1 pyramidal neurons from cases with low and high Braak NFTs.
  • low Braak NFTs many neurons with confluent p- tau202/205 immunolabeling throughout their soma and processes had p-tau262 or p- tausse immunolabeling restricted to only a portion of the cell cytoplasm, mostly in clusters of vesicle-like granular structures (FIG. 9, panels a-f).
  • p- tau262 and p-tau356 immunofluorescence were observed in only a subset of p-tau202/205- immunolabeled CA1 pyramidal neurons and was almost completely absent from p- tau202/205-immunolabeled neuropil threads (FIG. 9, panels g-l).
  • p-tau262 or p-tau356 immunofluorescence signals were localized to a portion of the cell soma in contrast to the confluent p-tau2O2/2os signal that extended throughout the cell body.
  • the proteins with the highest reactivity to both the p-tau262 and p-tau356 antibodies were those phosphorylated by CAMK2, BRSK2 and protein kinase A.
  • MS MS to compare the ratio of the phosphorylated and nonphosphorylated versions of tryptic peptides
  • the STA core peptide alters neuronal excitability
  • the STA core peptide alters synaptic transmission
  • the STA and t-tau ratio significantly differed according to Braak NFT stagingl (P ⁇ 0.0001 ) and was significantly lower in both individuals with Braak V and VI and Braak 111— IV staging versus Braak 0-II staging (P ⁇ 0.05; FIG. 11 B), with a sharp decrease between Braak III and IV (FIGS. 11C-11 D) suggesting that pathologically relevant changes occur in incipient AD, before isocortical association areas are affected in stages V and VI.
  • the STA and t-tau ratio was also examined across combined PET-based Braak NFT stages, including stages 0, I and II, III and IV, and V and VI.
  • the levels were significantly lower in the Braak V and VI versus I and II groups (P ⁇ 0.0001 ; FIG. 11 F).
  • regions associated with later Braak stages (V and VI) involving neocortical areas, including primary sensory regions, also displayed negative associations between [18F]MK6240 tau-PET and CSF tau STA and t-tau ratio (FIG. 11G).
  • MS showed, beside N-terminal forms, abundance of tau species containing not only defined peptides within the MTBR but also in the mid-region in brain TBS- soluble fractions, supporting previous results in human biospecimen and cell models.
  • the mid-region peptides but not the MTBR forms — remain abundant, supporting approved assays currently used in specialized clinics and recently developed mid-region-tau-targeting CSF assays.
  • tau forms bearing MTBR fragments were lacking in the CSF, we demonstrated in this study that their levels are indeed low among tau-PET- young adults and cognitively normal older adults.
  • p-tau262 and p-tau356 are early indicators of tau pathology in hippocampal pyramidal neurons; hyperphosphorylation promotes tau self-aggregation, with p-tau262 and p-tau356 being critical to initiate this pathological process. Furthermore, p-tau262 and p-tau356 have been reported in AD brain extracts from several studies; however, their relevance to NFT formation has remained unclear.
  • p-tau262 decreases tau binding to microtubules.
  • the p-tau356 site is shared by both the STA and fibril core regions and might thus be an indicator of a biochemical change from soluble to insoluble aggregation. Notably, Augustinak et al.
  • AD-type tau fibrils differs from those in PSP, PiD and CBD brains. Our results, supported by others, show that these differences extend to AD-type versus non-AD STAs. Moreover, both the soluble and insoluble tau forms are surrounded by N-terminal and C-terminal fuzzy coat structures. However, the faster aggregation and higher neurotoxicity of STA versus fibril core peptides suggest unique biochemical properties. The presence of the second MTBR repeat region containing the 275VQIINK280 aggregation-promoting hexapeptide motif and the p-tau262 site in the STA core but not the fibril core might, at least partly, explain these results.
  • the STA core peptide addresses a critical need in drug development, that is, the discovery of druggable therapeutic target(s) for early-stage tau aggregates in AD. As recently shown for A
  • the utility of the pSer262 tau-directed antibody in early detection of p-tau neuropathological change and staging of the progression of p-tau lesions in the brain during the course of Alzheimer’s disease (AD) was evaluated by IHC-based staining of fixed tissue sections of the middle temporal gyrus from the same cases used in the analysis of the hippocampus (see FIG. 2 for IHC-based staining of the hippocampus).
  • the pSer262 directed antibody and IHC methods used for the single-label chromogenbased IHC staining of middle temporal gyrus sections were the same as those used in the analysis of the hippocampus (see above).
  • FIG. 12 depicts pSer262 tau antibody-based IHC analyses of p-tau lesions in middle temporal gyrus based on Braak NFT Stages.
  • tissue sections were obtained from the middle temporal gyrus from cases neuropathologically determined to be Braak Stage I (A), II (not shown), III (not shown), IV (B), V (C), and VI (D).
  • the morphological appearance of tau pathology structures labeled with pSer262 IHC differed across Braak Stages. In Braak Stages I and II, pSer262 immunosignal was
  • FIG. 13 depicts AT8 (pSer202/pThr205; A, a, b), pSer262 tau (B, c), and pSer356 (C, d) antibody-based IHC studies of CA1 hippocampus and adjacent fiber tracts in a case of AD (Braak Stage III) with a common non-AD related tau copathology termed aging-related tau astrogliopathy (ART AG), which consists of p-tau accumulation in glial cells (primarily astroglia) at the pial surface, near the ependyma of the ventricles, in the white matter, and in the gray matter and appears to be associated with aging rather than specific known disease processes.
  • ART AG aging-related tau astrogliopathy
  • FIG. 14 depicts AT8 (pSer202/pThr205; A, a, b), pSer262 tau (B, c), and pSer356 (C, d) antibody-based IHC studies of cortical white matter in a case of Alzheimer’s disease (Braak Stage III) with ART AG co-pathology.
  • AT8 immunoreactive astroglia A, a
  • NFT A, b
  • the clusters of AT8 immunoreactive astroglia were not labeled with pSer262 tau (B) or pSer356 tau (C) antibodies, though rare small neurons were immunolabeled with
  • FIG. 15 depicts pSer356 tau immunoreactivity compared to immunoreactivity seen with antibody clone AT8 (pSer202/pThr205) in middle frontal cortex from a case neuropathologically diagnosed with the 4R tau tauopathy, corticobasal degeneration (CBD), and a case neuropathologically diagnosed with the primarily 3R tau tauopathy, Pick’s disease (PiD).
  • CBD corticobasal degeneration
  • SiD case neuropathologically diagnosed with the primarily 3R tau tauopathy
  • Braak Stages characterized by differential regional involvement with NFT can be viewed as three groups, Braak Stages l-ll where NFT are restricted to the allocortex (transentorhinal and entorhinal cortex) and rarely, CA1 hippocampus, Braak Stages lll-IV characterized by expansion of NFT into the hippocampus and limbic cortex, and Braak Stages V-VI characterized by presence of NFT in neocortical association areas.
  • Galiyas silver impregnation technique and/or IHC with antibody clone AT8 have been used traditionally in neuropathological evaluation of Alzheimer’s disease NFT pathology neither of which have fluid of PET biomarker equivalents, precluding pre-mortem classification of individuals into Braak Stages. This prevents identification of individuals at the early
  • middle temporal gyrus is a neocortical association area that integrates higher order sensory information with the brain default mode network for modulation of future, goal directed behavior.

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Abstract

Provided herein is a method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample, including obtaining a sample from a patient suspected of having or at risk of developing a tauopathy, incubating the sample with a composition comprising a first binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein, and detecting binding between the first binding reagent and the tau protein, wherein detecting binding between the first binding reagent and the tau protein indicates the presence of a pre-stage NFT in the patient sample.

Description

Attorney Docket No. 06527-2501 181
METHODS TO EVALUATE EARLY-STAGE PRE-TANGLE TAU AGGREGATES AND TREATMENT OF ALZHEIMER’S DISEASE
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent Application No. 63/679,361 , filed August 5, 2024, the disclosure of which is hereby incorporated by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant numbers AG014449, AG066468, and AG005133 awarded by the National Institutes of Health. The government has certain rights in the invention.
REFERENCE TO A SEQUENCING LISTING
[0003] The Sequence Listing associated with this application is filed in electronic format via Patent Center and is hereby incorporated by reference into the specification in its entirety. The name of the file containing the Sequence Listing is 2501 181. xml. The size of the file is 4,159 bytes, and the file was created on August 4, 2025.
BACKGROUND OF THE INVENTION
Field of the Invention
[0004] Provided herein are methods of identifying biomarkers of tauopathies, such as Alzheimer’s Disease.
Description of Related Art
[0005] Therapeutic targeting of Alzheimer’s disease (AD) patients with little or no quantifiable insoluble brain tau neurofibrillary tangle (NFT) pathology has demonstrated stronger clinical benefits than in those with advanced NFTs. To prevent NFT formation requires targeting early-stage soluble tau aggregates (STAs), the building blocks of NFTs. However, biochemical understanding and biomarkers of STAs are lacking.
SUMMARY OF THE INVENTION
[0006] Provided herein is a method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample, including obtaining a sample from a patient suspected of having or at risk of developing a tauopathy, incubating the sample with a composition comprising a first binding reagent, wherein the first binding reagent is specific to
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66A6790.DOCX Attorney Docket No. 06527-2501 181
Ser262 and/or Ser356 of a tau protein, and detecting binding between the first binding reagent and the tau protein, wherein detecting binding between the first binding reagent and the tau protein indicates the presence of a pre-stage NFT in the patient sample.
[0007] Also provided herein is a method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample, including incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a composition comprising a first-binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein and detecting binding between the first binding reagent and the tau protein, wherein detecting binding between the first binding reagent and the tau protein indicates the presence of a pre-stage NFT in the patient sample.
[0008] Also provided herein is a method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample including incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a composition comprising a first-binding reagent and a second binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein and the second binding reagent is specific to Ser202/Thr205 of the tau protein and detecting binding between the first binding reagent and the tau protein and between the second binding reagent and the tau protein, wherein detecting binding between the first binding reagent and the tau protein and an absence of binding between the second binding reagent and the tau protein indicates the presence of a pre-NFT in the patient sample.
[0009] Further non-limiting embodiments are set forth in the following numbered clauses:
[0010] 1. A method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample, comprising: obtaining a sample from a patient suspected of having or at risk of developing a tauopathy; incubating the sample with a composition comprising a first binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein; and detecting binding between the first binding reagent and the tau protein, wherein detecting binding between the first binding reagent and the tau protein indicates the presence of a pre-stage NFT in the patient sample.
[0011] 2. The method of clause 1 , wherein the first binding reagent is an antibody against Ser262 or Ser356.
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[0012] 3. The method of clause 1 or clause 2, wherein the first binding reagent is an antibody against phosphorylated Ser262 (pSer262) or phosphorylated Ser356 (pSer356).
[0013] 4. The method of any of clauses 1 -3, wherein the composition comprises the first binding reagent bound to a substrate.
[0014] 5. The method of any of clauses 1 -4, wherein the substrate is a bead.
[0015] 6. The method of any of clauses 1 -5, wherein the bead is a magnetic bead.
[0016] 7. The method of any of clauses 1 -6, wherein detecting binding between the first binding reagent and the tau protein comprises a step of eluting the first binding reagent and tau protein from the substrate, thereby generating a free protein-first binding reagent complex, and performing an immunoblotting assay or protein separation and immunodetection assay on the free protein-first binding reagent complex.
[0017] 8. The method of any of clauses 1 -7, further comprising staining the sample with a dye, optionally wherein the dye has the formula C24H1806.
[0018] 9. The method of any of clauses 1 -8, further comprising localizing binding of the first binding reagent to the tau protein based on localization of the binding reagent relative to the stain.
[0019] 10. The method of any of clauses 1 -9, further comprising incubating the sample with a second binding reagent, the second binding reagent specific to Ser202/Thr205.
[0020] 1 1 . The method of any of clauses 1 -10, wherein the second binding reagent is specific to phosphorylated Ser202 (pSer202) and phosphorylated Thr205 (pThr205), optionally wherein the second binding reagent is a monoclonal antibody, optionally wherein the second binding reagent is a monoclonal antibody derived from clone AT8.
[0021] 12. The method of any of clauses 1 -1 1 , further comprising detecting binding between the second binding reagent and the tau protein, wherein detecting binding between the second binding reagent and the tau protein indicates the presence a mature NFT in the patient sample.
[0022] 13. The method of any of clauses 1 -12, wherein detecting binding between the first binding reagent and the tau protein and an absence of binding between the second binding reagent and the tau protein indicates the presence of a pre-NFT in the patient sample.
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[0023] 14. The method of any of clauses 1 -13, wherein the tau protein is a human tau protein from a human patient sample.
[0024] 15. The method of any of clauses 1 -14, wherein the human patient sample is a sample from the patient’s central nervous system, optionally a tissue sample, optionally from hippocampus, entorhinal cortex, or basal forebrain tissue.
[0025] 16. The method of any of clauses 1 -15, wherein the human patient sample is a sample from the patient’s blood.
[0026] 17. The method of any of clauses 1 -16, wherein the sample is a from a living patient.
[0027] 18. The method of any of clauses 1 -17, further comprising treating the patient for early-stage AD if the sample comprises a pre-NFT.
[0028] 19. The method of any of clauses 1 -18, wherein the first binding reagent is a diluted binding reagent.
[0029] 20. The method of any of clauses 1 -19, wherein the first binding reagent is diluted 1 :250
[0030] 21. The method of any of clauses 1 -20, wherein the first binding reagent comprises an IgG antibody.
[0031] 22. A method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample, comprising: incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a composition comprising a first- binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein; and detecting binding between the first binding reagent and the tau protein, wherein detecting binding between the first binding reagent and the tau protein indicates the presence of a pre-stage NFT in the patient sample.
[0032] 23. A method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample, comprising: incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a composition comprising a first- binding reagent and a second binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein and the second binding reagent is specific to Ser202/Thr205 of the tau protein; and detecting binding between the first binding reagent and the tau protein and between the second binding reagent and the tau protein, wherein:
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[0033] detecting binding between the first binding reagent and the tau protein and an absence of binding between the second binding reagent and the tau protein indicates the presence of a pre-NFT in the patient sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 shows antibody optimization. Sections from the middle frontal gyrus from Case-1 , clinically diagnosed with Alzheimer’s disease (AD) and with autopsy- confirmed severe AD neuropathological change were stained with the pSer262 antibody (A-C) and the pSer356 antibody (D-F) using progressively higher dilutions of antibody (1 :250: A,D; 1 :500: B,E; 1 :1 ,000: C,F). The dilution yielding the optimal signal to noise ratio was 1 :250 for both antibodies. The immunoreactive structures were mainly pyramidal neurons with the appearance of neurofibrillary tangles (NFT), as expected for this case of severe AD neuropathology. Scale bars = 200 pm. IHC studies were then performed on fixed tissue sections obtained from the hippocampus from a series of cases (Cases 2-8) that spanned the range of severity of NFT pathology determined by Braak Stage. The hippocampus was chosen as the region of interest due to its early involvement in the neuropathological progression of AD.
[0035] FIG. 2 shows IHC studies of hippocampus - Braak Stage study. Adjacent tissue sections were processed using IHC and antibodies AT8 (pSer202/pThr205; A1 - G1 ), pSer262 (A2-G2), and pSer356 (A3-G3). AT8 was used as a comparator to the pSer262 and pSer356 antibodies and was previously optimized for use in our laboratories. In this experiment, tissue sections were obtained from the hippocampus from cases neuropathologically determined to be Braak Stage I (Case-2: A1 -A3), Braak Stage II (Case-3: B1 -B3), Braak Stage III (Case-4: C1 -C3), Braak Stage IV (Case-5: D1 -D3), Braak Stage V (Case-6: E1 -E3), and Braak Stage VI (Case-7: F1 - F3). In these IHC studies, we observed that the CA1 region (one of the earliest sites of NFT development in the hippocampus CA fields) contained neurons immunoreactive to all three antibodies even at Braak Stage I, typically thought of as containing lesions confined to the allocortex. Immunoreactive neurons were observed in progressively higher densities at progressively higher Braak Stages. Although all three antibodies labeled neurons across the Braak Stages, the pattern of pSer262 and pSer356 labeling differed markedly from patterns seen with AT8. Specifically, pSer262 and pSer356 labeling exhibited a distinct intracellular punctate appearance in contrast to the more homogeneous neuronal staining seen with antibody AT8, even at higher
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Braak Stages where hippocampal NFT pathology was abundant. Additionally, unlike AT8, the pSer262 and pSer356 antibodies did not label two types of neurofibrillary pathology, neuropil threads (p-tau containing distal dendrites) or neuritic (i.e., axons) structures in neuritic plaques. These observations strongly support the idea that the pSer262 and pSer356 antibodies identify neurofibrillary change at the earliest neuropathological stages of AD and are specific to NFT lesions, that is, neuronal cell bodies, destruction of which results in irreparable harm to the neural circuits to which they are connected.
[0036] FIG. 3 shows T riple-fluorescence labeling experiment combines pSer262 (A) with pSer202/pThr205 (AT8; B) and the pan amyloid binding dye X-34 (C, a marker of fibrillar aggregates) on hippocampal sections from a Braak Stage II case (Case-9) revealing that cells with granular pSer262 signal contain only small amounts of AT8 co-labeling and no X-34 labeling, indicating an early stage NFT, prior to fibrillization of tau and prior to robust pSer202/pThr205 tau phosphorylation (panel D merges panels A-C). The punctate appearance of pSer262 immunolabeling was seen consistently and it replicates our observations obtained from chromogen-IHC experiments in FIG. 8A.
[0037] FIG. 4 shows Triple-fluorescence labeling experiments combining pSer262 with pSer202/pThr205 (AT8: B) and the pan amyloid binding dye X-34 (C) were performed on sections of hippocampus from a Braak Stage V case (Case-10). These experiments indicate that pSer262 signal, even in this late Braak Stage case marks newly developing NFT that have weak co-labeling with antibody AT8 and very little or no fibrillar tau (i.e., very little or no co-labeling with the dye, X-34). However, their abundance is significantly higher than seen in lower Braak Stages (see FIG. 3), complementing blood biomarker studies assaying this phospho-epitope.
[0038] FIG. 5 shows Triple-fluorescence labeling experiments combining pSer356 with pSer202/pThr205 (AT8: B) and the pan amyloid binding dye X-34 (C) were performed on sections of hippocampus from a Braak Stage V case (Case-10). These experiments indicate that even in this late Braak Stage case, pSer356 signal marks newly developing NFT that have weak co-labeling with antibody AT8 and very little or no fibrillar tau (i.e., very little or no co-labeling with the X-34 dye). Thus, like pSer262, the antibody targeting the pSer356 phospho-epitope labels very early forms of NFT.
[0039] FIGS. 6A-6D show (A) a schematic illustration of the principle of the tau- FRET assay. Simultaneous binding of the donor-antibody and acceptor-antibody
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66A6790.DOCX Attorney Docket No. 06527-2501 181 complexes to a polyvalent analyte leads to excitation followed by energy transfer to a nearby acceptor-antibody that fluoresces at a 665-nm wavelength. (B) Linearity of decreases in tau-FRET signal in response to dilution of the tau-FRET assay in recombinant tau441 diluted 500, 1 ,000 or 2,000 times, n = 2 biological replicates, performed on different days. (C) Dilution linearity of the tau-FRET assay in brain tissues from four different Braak stage VI individuals with neuropathologically confirmed AD. Data are from three replicates. (D) Comparison of the tau-FRET signal in neuropathologically diagnosed AD, other neurodegenerative diseases (progressive supranuclear palsy, corticobasal degeneration and PiD) and controls (cohort 1 : n = 10 participants per group) with a Kruskal-Wallis test with Dunn’s multiple comparisons. Cohort 2 includes neuropathologically diagnosed individuals with AD and controls (n = 4 participants each); two-sided Mann-Whitney U-test = 0 (P = 0.0286). In each panel, data points are shown as the mean ± s.e.m. The boxplot center indicates the median, the box boundaries indicate the 25th and 75th percentiles, and the whiskers indicate extreme values outside the box boundaries (Q1 - 1.5 x IQR and Q3 + 1.5 x IQR), where Q1 , Q3 and IQR refer to the 25th percentile, 75th percentile and interquartile range, respectively. To summarize, the FRET assay recognizes soluble and solubilized tau assemblies from recombinant sources and from human brain tissues; it is selective for ST As in AD versus other tauopathies and the signal decrease is proportional to the relative amount of soluble and solubilized assemblies present after sample dilution.
[0040] FIGS. 7A-7J show (A-B) a schematic illustration of the epitopes of the anti- tau antibodies used in the IP experiments. These antibodies target defined nonphosphorylated tau sites; (C) Flow diagram of the experimental procedure used to isolate TBS-soluble tau fractions from AD human brains. Aliquots of the soluble fraction underwent immunodepletion using a defined antibody, including those shown in a. Afterward, the depleted fraction was examined with the FRET assay described in Fig. 1 , while the precipitate portion was evaluated using immunoblotting. The diagram in the inset illustrates that fuzzy coat peptides are more accessible to antibodies and thus are first removed in the imunodepletion step using IP with those antibodies. (D) tau-FRET signals obtained before (‘nondepleted’) and after (denoted by the name of the depleting antibody) immunodepleting tau content from the TBS-soluble brain tissue fraction from a patient with autopsy-verified Braak stage VI AD using the named antibodies covering the tau441 sequence. Data are shown as the mean with individual
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66A6790.DOCX Attorney Docket No. 06527-2501 181 data points overlaid, n = 2 biological replicates. (E-F) For three antibodies with epitopes in the N terminus (tau12), MTBR (77G7) and the extreme C terminus (tauAB), the corresponding immunoreactivity of the precipitate fractions to the selected antibodies using immunoblotting is shown. Representative images from three biological replicates. (G) Schematic illustration of the epitopes of the set of ‘CT’ mAbs developed in this study against defined regions inside and outside the MTBR. (H) Schematic illustration of the anti-tau antibodies used in this study that target the phosphorylation sites. (I) tau-FRET assay signals after immunodepletion of TBS- soluble brain fractions with the new CT mAbs relative to a nondepleted control. Data are shown as the mean with individual data points overlaid, n = 2 biological replicates. (J) tau-FRET assay signals after immunodepletion of TBS-soluble brain fractions with anti-tau antibodies that target defined phosphorylation epitopes relative to a nondepleted control. Data are shown as the mean with individual data points overlaid, n = 2 biological replicates. This figure shows that STAs in TBS-soluble AD brain tissue contain a core region that covers the peptide ~tau258-368. IP followed by high- resolution MS analysis using several anti-tau antibodies identified that tau forms that contain this STA core region are long, near-full-length fragments that stretch from the N terminus or mid-region into the MTBR
[0041] FIGS. 8A-8C show chromogen immunohistochemistry (IHC) analyses of tau forms with phosphorylated epitopes inside (p-tau262, p-tau356) and outside (p- tau231 , p-tau202/205 (AT8)) the STA core region in postmortem human hippocampus, a-j, Photomicrographs of hippocampal tissue sections from an individual with Braak NFT stage II (a-e,a1-e1 ) and an individual with Braak NFT stage VI (f— j,f1— j1 ) immunohistochemically processed using antibodies directed against the p-tau262 epitope of the tau protein (a,a1 ,f,f1 ), the p-tau356 epitope (b,b1 ,g,g1 ), the p-tau231 epitope (clone AT 180; c,c1 , h , h 1 ) and the p-tau202/205 epitope (clone AT8; d,d1 ,i,i1 ). The pan-amyloid binding dye X-34 was used to confirm the presence of fibrillar tau Immunolabeling and histofluorescence observed in the CA1 region, near the CA1/CA2 border, are illustrated at low (a-e,f-j) and higher (a1-e1 ,f1— j1 ) magnification. The locations of the higher-magnification images are indicated by dashed outlines in the low-magnification images. The illustrated immunohistochemical staining was replicated in three sections per individual in each of three individuals with Braak II and five individuals with Braak VI. In summary, the p-tau262 and p-tau356 immunostaining is localized mainly to granular structures inside hippocampal neurons,
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66A6790.DOCX Attorney Docket No. 06527-2501 181 whereas the p-tau231 and p-tau202/205 antibodies show a more diffuse pattern of immunolabeling in hippocampal neurons, and in neuropil threads, at both Braak II and VI NFT stages.
[0042] FIGS. 9A-9B show dual immunofluorescence staining of the p-tau262 and p-tau356 sites in the STA core versus p-tau202/205 (AT8) in the fuzzy coat in human postmortem tissue at early and late Braak NFT stages, a-l, Tissue sections of the hippocampus from an individual at Braak NFT stage II (a-f) and an individual at Braak NFT stage VI (g-l) were processed using dual immunofluorescence to assess the codistribution of p-tau262 labeling with p-tau202/205 (AT8) labeling (Braak II: a-c; Braak VI: g-i) and p-tau356 labeling with p-tau202/205 (AT8) labeling (Braak II: d-f; Braak VI: j-l). In each triplet, green fluorescence indicates p-tau262 (a,g) or p-tau356 (d,j); red fluorescence indicates p-tau202/205 (AT8) (b,e,h,k). Merged images are shown in c,f,i,L The illustrated immunohistochemical staining was replicated in three sections per individual in each of three Braak II and five Braak VI individuals. Together, in Braak NFT stage II, hippocampal neurons with confluent p-tau202/205 labeling also contained p-tau262 or p-tau356-labeled granular structures in a portion of the cell cytoplasm, whereas in Braak NFT stage VI, only a subset of hippocampal neurons with confluent p-tau202/205 labeling also contained p-tau262 or p-tau356 immunofluorescence.
[0043] FIGS. 10A-10J show (A) a schematic representation of recombinantly produced truncated tau species covering the putative STA core sequence (aa 258- 368), the insoluble fibril core peptide (aa 302-368) and the flanking N-terminal (aa 1- 124) and C-terminal (aa 368-441 ) peptide controls. (B) SPR profiles of the recombinant STA core peptide relative to the fibril core sequence, and the N-terminal and C-terminal peptides. The SPR sensorgrams illustrate the effectiveness of the CT19.1 antibody in recognizing the specific epitope within the tau sequence (aa 331 — 361 ). The N-terminal tau fragment (aa 1-224) and the C-terminal tau fragment (aa 368-441 ) fall outside the epitope region. Consequently, their respective sensorgrams closely resemble the baseline signal. In contrast, the sensorgrams for the soluble (aa 258-368) and insoluble (aa 302-368) core fragments exhibit higher signals. The SPR plots were generated from n = 3 replicates. (C) Representative negative stain transmission electron microscopy (TEM) images of the recombinant STA core, fibril core, and the N-terminal and C-terminal control peptides. Images are representative of n = 2 replicates. Scale bars, 500 nm. (D) Whole-cell patch clamp recordings were
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66A6790.DOCX Attorney Docket No. 06527-2501181 made from CA1 pyramidal neurons in acute mouse hippocampal brain slices. Representative examples of standard current-voltage responses for slices incubated in soluble aliquots of the recombinant tau peptides (N terminus, n = 12; STA core, n = 12; fibril core, n = 12; or C terminus, n = 12) or control aCSF (n = 12) for 1 h before electrophysiological recordings. Incubation with the STA core region peptide depolarized the RMP and increased the IR of the recorded neurons. Each ‘n’ in d-h represents a whole-cell patch clamp recording made from individual acute hippocampal brain slices incubated with one of the recombinant tau peptides (diluted in aCSF; Methods) or control aCSF. (E) Representative example of membrane potential responses to naturalistic current injection86 for each of the three conditions described in (D). (F) Incubation with the STA core peptide resulted in a significant depolarization of the RMP (mean RMP in slices incubated with the STA core of -58 ± 1.31 mV-1 (n = 12) compared with -65 ± 0.83 mV-1 in controls (n = 12)); Kruskal- Wallis test (Kruskal-Wallis statistic = 14.27, P = 0.0065) with Dunn’s multiple comparisons (P = 0.0400), which was not observed with the other tau truncations. Data are presented as the mean ± s.e.m., with individual data points overlaid. RMP depolarization with the STA core also significantly differed from that of the C-terminal peptide. (G) Incubation with the STA core peptide also significantly increased IR (mean IR in slices incubated with the STA core peptide was 220.7 ± 9.6 mfl (n = 12), compared with the mean IR in aCSF control slices of 159.3 ± .6 mfl (n = 12); Kruskal- Wallis test (Kruskal-Wallis statistic = 24.60, P < 0.0001 ) with Dunn’s multiple comparisons (P = 0.0008)), an effect which was also not observed with the other tau truncations. Data are presented as the mean ± s.e.m., with individual data points overlaid. (H) Incubation with the STA core peptide significantly increased the FR (a correlate of neuronal excitability). The mean FR in slices incubated with the STA core was 6.4 ± 0.85 Hz (n = 12), compared to the mean FR in aCSF control slices of 2.7 ± 0.32 Hz (n = 12); Kruskal- Wallis test (Kruskal-Wallis statistic = 25.66, P < 0.0001 ) with Dunn’s multiple comparisons (P = 0.0016). Incubation with the fibril core also significantly increased the FR compared to aCSF controls (mean FR in slices incubated with the fibril core was increased to 6.3 ± 0.62 Hz (n = 12); Kruskal-Wallis test with Dunn’s multiple comparisons (P = 0.0006). No change versus aCSF control was observed with N-terminal or C-terminal tau. Data are presented as the mean ± s.e.m., with individual data points overlaid. (I) Graph showing the mean paired-pulse ratio against interval for the STA core peptide versus the other tau truncations.
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Incubation with the STA core peptide or the fibril core peptide significantly enhanced paired-pulse facilitation compared with aCSF control slices. The mean paired-pulse ratio at a 100-ms interval was 1 .78 ± 0.12 in control aCSF (n = 9 slices) compared with the STA core (2.01 ± 0.09; P = 0.0190; n = 9 slices) and 2.03 ± 0.17 for the fibril core (n = 8 slices). There was a significant difference between these values; Kruskal-Wallis test (Kruskal-Wallis statistic = 21.05, P = 0.0003) with Dunn’s multiple comparisons (P = 0.0359). (J) Inset to (I): representative example traces of fEPSP waveforms for the 100-ms interval for each tau peptide. The first fEPSPs were normalized so that facilitation could be compared across conditions. *P < 0.05, **P < 0.01 , ***P < 0.001 . To summarize, the results demonstrate that the recombinant STA core region peptide has a strong in vitro aggregation propensity and robust impairment of neuronal excitability and functional modulation of neuronal and network functions in mouse hippocampal slices.
[0044] FIGS. 11A-11G show (A) levels of the CSF tau STA and t-tau ratio in a well- characterized cohort with paired antemortem CSF samples and neuropathological diagnosis at postmortem (cohort 3). The groups diagnosed with ADNC (n = 21 ) and those with ADNC plus concomitant neurodegenerative pathologies (ADNC + other, n = 19) each had significantly lower levels of the STA and t-tau ratio versus those with low tau pathology (probably age-related tau, n = 8) and non-ADNC (other pathology, n = 19). In the box plots, the center line represents the median, the boundaries of the box are the 25th and 75th percentiles, and the whiskers extend to the furthest data value 1.5 times the interquartile range (IQR). The displayed P values correspond to post hoc pairwise Mann-Whitney U-tests with Benjamini-Hochberg false discovery rate (FDR) multiple comparison adjustment, after a significant overall Kruskal-Wallis rank-sum test (chi-squared = 17.3, d.f. = 3, P = 6.1 x 10-4). (B) The CSF STA and t- tau ratio levels decreased with increasing Braak staging. The lowest levels were in those with the most advanced ADNC (Braak stages V and VI, n = 40), being significantly different from both the Braak III and IV (n = 13) and 0-II (n = 14) groups. As above, the center of the box plot indicates the median, the box boundaries indicate the 25th and 75th percentiles, and the whiskers indicate the furthest value 1 .5 times the IQR. The P values correspond to post hoc pairwise Mann-Whitney U-tests with FDR adjustment, after a significant overall Kruskal-Wallis rank-sum test (chi-squared = 18.7, d.f. = 2, P = 8.5 x 10-5). (C) Analyzing the STA and t-tau ratio levels in the cohort according to each of the six different Braak stages (n = 6 Braak stage 0; n = 4
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(I); n = 4 (II); n = 8 (III); n = 5 (IV); n = 9 (V); n = 31 (VI)) highlights a sharp decrease in the ratio between Braak stages III and IV, suggesting a pathology-dependent increase in the amount of soluble tau in human CSF once affected individuals reach this disease stage. As above, the center of the box plot indicates the median, the box boundaries indicate the 25th and 75th percentiles, and the whiskers indicate the furthest value 1 .5 times the IQR. The P values correspond to post hoc pairwise Mann- Whitney U-tests with FDR adjustment, after a significant overall Kruskal-Wallis ranksum test (chi-squared = 31 .6, d.f. = 6, P = 1 .9 x 10-5). (D) z-score model plots showing the CSF STA and t-tau ratio levels at each Braak stage relative to t-tau and STA separately, and p-tau181 and p-tau231 , in the same cohort as in (C). (E) CSF STA and t-tau ratio levels according to diagnostic group in the tau-PET cohort (cohort 4). The STA and t-tau ratio was lowest in tau-PET+ AD dementia/T+ (n = 17) and highest in tau-PET- young adults (young, n = 25), and tau-PET- older adults either with CN/T- (n = 92) or MCI/T- (n = 30). Tau-PET+ individuals with MCI/T (n = 16) and tau-PET- AD/T- (n = 6) showed intermediate values. As above, the center of the box plot indicates the median, the box boundaries indicate the 25th and 75th percentiles, and the whiskers indicate the furthest value 1.5 times the IQR. The P values correspond to post hoc pairwise Mann-Whitney U-tests with FDR adjustment, after a significant overall Kruskal-Wallis rank-sum test (chi-squared = 41 .8, d.f. = 5, P = 6.4 x 10-8). (F) The CSF STA and t-tau ratio decreased according to tau-PET-based Braak staging (n = 100 (Braak stage 0), n = 34 (stages I and II), n = 16 (stages III and IV) and n = 31 (stages V and VI)). As above, the center of the box plot indicates the median, the box boundaries indicate the 25th and 75th percentiles, and the whiskers indicate the furthest value 1 .5 times the IQR. The P values correspond to post hoc pairwise Mann- Whitney U-tests with FDR adjustment, after a significant overall Kruskal-Wallis ranksum test (chi-squared = 48.6, d.f. = 3, P = 1 .6 x 10-10). The plot in panel f is z-scored representation of the CSF STA/t-tau ratio normalized to the Braak 0 group as control. (G) Voxel-wise association analyses showed inverse correlation of the STA and t-tau ratio with regional tau-PET accumulation. The voxel-wise analyses were adjusted for A|3 PET uptake, age, sex and APOE s4 genotype. This figure demonstrates that there are only minute amounts of ST As in the CSF of young adults and cognitively normal older adults. However, the levels increase with disease severity such that the ratio of these tau forms to t-tau, accounting for interindividual variability in tau production and/or release, decreases with worsening NFT pathology as indexed by the Braak
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66A6790.DOCX Attorney Docket No. 06527-2501 181 staging both at autopsy using immunohistochemical analysis and in vivo using tau- PET. These changes are specific to individuals with high ADNC with or without mixed neurodegenerative pathologies.
[0045] FIG. 12 depicts pSer262 tau antibody-based IHC analyses of p-tau lesions in middle temporal gyrus based on Braak NFT Stages.
[0046] FIG. 13 depicts AT8 (pSer202/pThr205; A, a, b), pSer262 tau (B, c), and pSer356 (C, d) antibody-based IHC studies of CA1 hippocampus and adjacent fiber tracts in a case of AD (Braak Stage III) with a common non-AD related tau copathology termed aging-related tau astrogliopathy (ART AG).
[0047] FIG. 14 depicts AT8 (pSer202/pThr205; A, a, b), pSer262 tau (B, c), and pSer356 (C, d) antibody-based IHC studies of cortical white matter in a case of Alzheimer’s disease (Braak Stage III) with ART AG co-pathology.
[0048] FIG. 15 depicts pSer356 tau immunoreactivity (B and C, E and F) compared to immunoreactivity seen with antibody clone AT8 (pSer202/pThr205, A and D) in middle frontal cortex from a case neuropathologically diagnosed with the 4R tau tauopathy, corticobasal degeneration (CBD, A-C), and a case neuropathologically diagnosed with the primarily 3R tau tauopathy, Pick’s disease (PiD, D-F).
DESCRIPTION OF THE INVENTION
[0049] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word "about". In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values. As used herein "a" and "an" refer to one or more.
[0050] As used herein, the term "comprising" is open-ended and may be synonymous with "including", "containing", or "characterized by". The term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. As used herein, embodiments "comprising" one or more stated elements or steps also include, but are not limited to embodiments "consisting essentially of" and consisting of these stated elements or steps.
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[0051] As used herein, spatial or directional terms, such as "left", "right", "inner", "outer", "above", "below", "over", "under", and the like, relate to the invention as it is shown in the drawing figures are provided solely for ease of description and illustration, and do not imply directionality, unless specifically required for operation of the described aspect of the invention. It is to be understood that the invention can assume various alternative orientations and, accordingly, such terms are not to be considered as limiting.
[0052] As used herein, a "patient" or "subject" is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). The term "patient" typically refers to human patients, but does not require that the individual is under the care of a physician. As used herein, the terms "treating", or "treatment" refer to a beneficial or desired result, such as improving one of more functions, or symptoms of a disease. [0053] Unless stated otherwise, nucleotide sequences are recited herein in a 5' to 3' direction, and amino acid sequences are recited herein in an N-terminal to C- terminal direction according to convention.
[0054] As used herein, the "treatment" or "treating" of a patient means administration to a patient by any suitable dosage regimen, procedure and/or administration route of a composition, device, or structure with the object of achieving a desirable clinical/medical end-point, including but not limited to, any suitable treatment for AD or a tauopathy, and also includes monitoring the patient for development of AD or a tauopathy by any useful method, including by use of a method according to any aspect, embodiment, or example, provided herein. An amount of any reagent or therapeutic agent, administered by any suitable route, effective to treat a patient is an amount capable of improving any symptom or physiological effect of AD or a tauopathy in a patient. A therapeutic agent may be administered by any effective route. A therapeutic agent may be administered as a single dose, at regular or irregular intervals, in amounts and intervals as dictated by any clinical parameter of a patient, or continuously. A treatment method may include one or more steps of identification and/or quantification according to any aspect or embodiment provided herein.
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66A6790.DOCX Attorney Docket No. 06527-2501 181
[0055] "Therapeutically effective amount" or an "amount effective" as used herein, is intended to include the amount of a therapeutic agent as described herein that, when administered to a subject having a disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease or one or more symptoms of disease). The "therapeutically effective amount" may vary depending on the nature of the injury and its causes, how the therapeutic agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated. A "therapeutically-effective amount" also includes an amount of a therapeutic agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment.
[0056] An "amount effective" for treatment of a condition is an amount of an active agent or dosage form, such as a single dose or multiple doses, effective to achieve a determinable end-point. The "amount effective" is preferably safe - at least to the extent the benefits of treatment outweighs the detriments, and/or the detriments are acceptable to one of ordinary skill and/or to an appropriate regulatory agency, such as the U.S. Food and Drug Administration. A therapeutically effective amount of an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.
[0057] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the composition may be administered continuously or in a pulsed fashion with doses or partial doses being administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120 minutes, every 2 through 12 hours daily, or every other day, etc., be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some instances, it may be especially advantageous to formulate compositions, such as parenteral or inhaled compositions, in dosage unit form for ease of administration and uniformity of dosage. The specification for the
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66A6790.DOCX Attorney Docket No. 06527-2501 181 dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
[0058] Drug products, or pharmaceutical compositions comprising an active agent (e.g., drug), may be prepared by any method known in the pharmaceutical arts, for example, by bringing into association the active ingredient with the carrier(s) or excipient(s). As used herein, a "pharmaceutically acceptable excipient", "carrier", or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohol's such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the active agent. In certain aspects, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used in delivery systems, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are broadly-known to those skilled in the art. The preferred form may depend on the intended mode of administration and therapeutic application, which will in turn dictate the types of carriers/excipients. Suitable forms include, but are not limited to, liquid, semi-solid, and solid dosage forms.
[0059] Pharmaceutical formulations adapted for oral administration may be presented, for example and without limitation, in capsules, tablets, oral solutions, or the like, and include suitable carriers and coatings as are broadly-known in the pharmaceutical arts.
[0060] Pharmaceutical formulations adapted for parenteral administration may be presented, for example and without limitation, in syringes, vials, bottles, IV/infusion
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66A6790.DOCX Attorney Docket No. 06527-2501 181 bags, or the like, as are broadly-known to those of ordinary skill. Excipients include, for example and without limitation, water, saline, PBS, lactated Ringers, or any other injectable carriers. Suitable emulsifiers, lipids, surfactants, or the like may be utilized to maintain an active agent in solution.
[0061] Pharmaceutical formulations adapted for transdermal administration may be presented, for example and without limitation, as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time or electrodes for iontophoretic delivery.
[0062] Pharmaceutical formulations adapted for topical administration may be formulated, for example and without limitation, as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.
[0063] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. For example, sterile injectable solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with suitable carrier(s), followed by filter-sterilization. An appropriate fluidity of a solution can be maintained, for example, by the use of a rheology modifier. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
[0064] The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium, zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
[0065] The therapeutic agents described herein can be administered by any effective route. Examples of delivery routes include, without limitation: topical, for example, epicutaneous, inhalational, enema, ocular, otic, and intranasal delivery; enteral, for example, orally, by gastric feeding tube, and rectally; and parenteral, such as, intravenous, intraarterial, intrathecally, intramuscular, intracardiac, subcutaneous, intraosseous, intradermal, intrathecal, intraperitoneal, transdermal, iontophoretic, transmucosal, epidural, and intravitreal, with intrathecal and oral approaches being preferred in many instances. Suitable dosage forms may include single-dose, or multiple-dose vials or other containers, such as medical syringes, containing a
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66A6790.DOCX Attorney Docket No. 06527-2501 181 composition comprising the therapeutic agent useful for treatment of graft rejection as described herein.
[0066] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the therapeutic agent may be administered continuously or in a pulsed fashion with doses or partial doses being administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120 minutes, every 2 through 12 hours daily, or every other day, etc., be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some instances, it may be especially advantageous to formulate therapeutic agents in dosage unit form for ease of administration and uniformity of dosage. The specification for the dosage unit forms may be dictated by and directly dependent on (a) the unique characteristics of the therapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic agent for the treatment of sensitivity in individuals.
[0067] The methods provided herein comprise imaging brain tissue for diagnosis of a condition related to tauopathies, e.g., Alzheimer's, e.g., pre-Alzheimer's, in a patient. The brain tissue may be obtained by biopsy in a living patient, or by dissection in a deceased patient. While obtaining brain tissue from deceased patient may be used to determine the neuropathological diagnosis, or for research purposes such as drug discovery and optimization, obtaining tissue (biopsy) from a live patient to determine if Alzheimer's, or pre-Alzheimer's is present has significant therapeutic value in that early Alzheimer's (such as pre-Alzheimer's) may be treatable with an appropriate treatment regimen. Tissue samples may be obtained from particular sections of the brain, such as from the hippocampus, entorhinal cortex, or basal forebrain, among other sections of the brain, or central nervous system (CNS), as appropriate. Once obtained, the tissue sample may be preserved by freezing after cryoprotection, paraffin-embedding after fixation, or otherwise. Tissue is then processed, e.g., as indicated, to identify in situ pSer262 or pSer356 epitopes and to evaluate their localization relative to X-34 staining of intracellular protein aggregates which display amyloid [3-sheet structure (tau fibrils) and optionally pSer202/pThr205 epitopes, if present. pSer262 and pSer356 binding signifies pretangles/immature NFTs prior to development of mature NFTs when neurons start to accumulate tau detected by
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66A6790.DOCX Attorney Docket No. 06527-2501 181 antibodies against these phosphorylated epitopes but still lack X-34-stained tau fibrils and optionally pSer202/pThr205 binding. Pretangles are characteristic of an early AD state in the patient and, additionally, indicate that NFT pathogenesis is progressing. Early AD with or without cognitive impairment may be treated with anti-amyloid drugs, such as, for example and without limitation, aducanumab (e.g., Aduhelm®, currently discontinued) donanemab (e.g., Kisunla™), or lecanemab (e.g., Leqembi®), antiamyloid antibody intravenous infusion therapies. Symptomatic AD treatment includes cholinesterase inhibitors, such as, for example and without limitation, galantamine, rivastigmine, and donepezil. For moderate to severe AD, an N-methyl-D-aspartate (NMDA) antagonist, such as memantine, donepezil, combinations of memantine and donepezil, rivastigmine (e.g., patch), and/or brexpiprazole may be administered to a patient. Additional treatments, many of which are in development, may be used to treat a patient with AD.
[0068] The Congo Red derivative dye X-34 stains pathological structures, including extracellular senile plaques (SP) and hyperphosphorylated tau in its insoluble state in the form of intracellular neurofibrillary tangles (NFT) consisting of twisted, paired helical filaments, recognized as representing mature NFT as opposed to pretangles which are composed of soluble tau assemblies recognized as an early step in the formation of NFT. Dyes equivalent to X-34 in their ability to stain mature NFT, include thioflavin derivative dyes disclosed, for example and without limitation, in U.S. Patent No. 7,270,800, incorporated herein by reference in its entirety for its disclosure of amyloid binding compounds.
[0069] Antibodies described herein for use in the described assays and methods are commercially available as polyclonal antibodies, with additional suitable polyclonal antibodies and monoclonal antibodies being commercially available, or readily prepared as polyclonal antibodies, by immunization of an animal, such as a chicken, rabbit, goat, horse, camelid, etc. by common methods, e.g., as described below.
[0070] Alzheimer's progression may be characterized neuropathologically by Braak NFT Stages, as is known to those of skill in the art. Tau pathology follows a stereotypical pattern approximating the Braak stages defined post-mortem, where tau begins accumulating in mesial temporal regions (Braak l-ll), then spreads to limbic regions (Braak lll-IV), and lastly to the whole neocortical mantle (Braak V-VI). PET studies have confirmed this pattern in vivo. That said, Braak staging, and classic methods of such staging are useful for classifying AD cases based on AD tau NFT
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66A6790.DOCX Attorney Docket No. 06527-2501 181 lesion severity but are less useful for understanding the pathobiology of early tau pathology development in AD as well as patients suitable for treatments aiming to prevent the progression of tau pathology. The focus of the methods described herein are to identify tau pathology at its earliest stages, in people at risk for developing Alzheimer's disease and in Alzheimer's patients at earlier clinical stages, thus, facilitating earlier treatment, and to assist in the development of effective treatments.
[0071] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Unless otherwise indicated, polymer molecular weight is expressed as number-average molecular weight (Mn). Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0072] An "isolated" or "purified" biological component (such as a nucleic acid, peptide, protein, protein complex, or particle) refers to a component that has been substantially separated, produced apart from, or purified away from other components in a preparation or other biological components in a cell or in an organism in which the component occurs, that is, separated from other chromosomal and extrachromosomal DNA, RNA, proteins or other cellular, tissue, or organ constituents. Nucleic acids, peptides and proteins that have been "isolated" or "purified", thus, include, for example and without limitation, nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins. The term "isolated" or "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated biological component is one in which the biological component is more enriched than the biological component is in its natural environment within a cell, or other production vessel. A preparation may be purified such that the biological component represents at least 50%, such as
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66A6790.DOCX Attorney Docket No. 06527-2501 181 at least 70%, at least 90%, at least 95%, or greater, of the total biological component content of the preparation.
[0073] As used herein, the term "epitope" refers to a physical structure or moiety on a molecule that interacts with an antibody or antibody fragment. In terms of proteins or polypeptides, the primary amino acid sequence can define an epitope, but secondary and tertiary protein structure, as well as post-translational modifications (e.g., phosphorylation), can define an epitope, though secondary and tertiary structure typically follows from the primary amino acid sequence.
[0074] Antibodies or other binding reagents may be produced by any effective method, such as by hybridoma or they may be recombinantly or synthetically produced. In the case of polyclonal antibodies, an antigen is introduced into an animal, and antibodies are purified from the animal's blood or other bodily fluid. A suitable antigen may be a fragment of a tau protein that is phosphorylated at the identified location, e.g., pSer202/pThr205, pSer262, and/or pSer356 of tau. An exemplary reference sequence for tau is NCBI Reference Sequence: NP_005901 .2 (microtubule- associated protein tau isoform 2 [Homo sapiens], see also, Goedert M, et al. Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease. Neuron. 1989 Oct;3(4):519-26), having the amino acid sequence of (SEQ ID NO: 1 ), or a sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, and/or 99% or greater, all values and subranges therebetween inclusive:
1 maeprqefev medhagtygl gdrkdqggyt mhqdqegdtd aglkesplqt ptedgseepg 60 61 setsdakstp taedvtaplv degapgkqaa aqphteipeg ttaeeagigd tpsledeaag 120 121 hvtqarmvsk skdgtgsddk kakgadgktk iatprgaapp gqkgqanatr ipaktppapk 180 181 tppssgeppk sgdrsgyssp gspgtpgsrs rtpslptppt repkkvavvr tppkspssak 240 241 srlqtapvpm pdlknvkski gstenlkhqp gggkvqiink kldlsnvqsk cgskdnikhv 300 301 pgggsvqivy kpvdlskvts kcgslgnihh kpgggqvevk sekldfkdrv qskigsldni 360 361 thvpgggnkk iethkltfre nakaktdhga eivykspvvs gdtsprhlsn vsstgsidmv 420 421 dspqlatlad evsaslakqg I 441 .
[0075] Numbering of tau and phosphorylation sites thereof is made in reference to SEQ ID NO: 1 , including pSer202/pThr205, pSer262, and pSer356. Any fragment of tau, e.g., SEQ ID NO: 1 ) with the specified phosphorylated amino acid, e.g., pSer202/pThr205, pSer262, and pSer356, may be used to generate antibodies or antigen binding reagents for use in the methods provided herein. Methods of
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66A6790.DOCX Attorney Docket No. 06527-2501 181 producing polyclonal and monoclonal antibodies and antigen-binding reagents are broadly-known, and suitable antibodies for use in the described methods are commercially-available.
[0076] Polyclonal antibodies may be monoclonal, but typically comprise antibodies directed to different epitopes, to the same epitope with differing avidity, and/or different antibody types. Polyclonal antibodies may be adsorbed, e.g., by affinity, to remove certain fractions, such as removing one or more specific classes of antibodies, or to purify a specific class, e.g., IgG, from a larger antibody pool. Antigens having crossreactivity to a target protein may be removed, e.g., by affinity purification, to yield a non-cross-reactive population of antibodies. For example, antibodies cross-reactive with a non-phosphorylated tau protein may be removed by adsorption to human nonphosphorylated tau protein, or a fragment of human tau in which an amino acid, such as amino acid Ser262, Ser356, Ser202, and/or Thr205 is/are not phosphorylated, where retained antibodies are removed. Retaining antibodies that bind to human tau fragments comprising pSer262, pSer356, pSer202, and/or pThr205 may be effective to enrich a population of antibodies, such as polyclonal or monoclonal antibodies. Binding reagents may be synthetic, in that they do not comprise a naturally-occurring antibody sequence, including engineered versions and derivatives thereof, such as scFv versions thereof, or humanized versions thereof. Based on the present disclosure, any monoclonal or polyclonal antibody or binding reagent that is produced, may be tested in the assays described in the methods below for their ability to identify a biopsy or necropsy with pre-NFT tangles characteristic of early AD or pre-AD tauopathy.
[0077] Provided herein are methods that allow for highly sensitive and accurate detection of pre-stage neurofibrillary tangles (NFTs), which may allow for diagnosis of tauopathies, such as Alzheimer’s Disease (AD) or pre-AD, in a patient. Such methods allow for earlier diagnoses, and thus earlier implementation of treatments and/or other interventions, potentially slowing progression and/or increasing survival rates.
[0078] As used herein, the term “pre-NFT means a soluble, non-fibrillar, diffuse, aggregate of tau protein. In the present disclosure, pre-NFT and soluble tau aggregate (STA) are used interchangeably. As used herein, the term “mature NFT” means an insoluble, organized, fibrillar tau aggregate. As used herein a “tau protein” means a protein having, in a human, an amino acid sequence of SEQ ID NO: 1 , or a sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or
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66A6790.DOCX Attorney Docket No. 06527-2501 181 greater, 95% or greater, and/or 99% or greater sequence identity to SEQ ID NO: 1 , all values and subranges therebetween inclusive. Those of skill in the art will appreciate that tau proteins from other organisms, such as mouse, fall within the scope of this disclosure, and the sequences for such proteins are known and available.
[0079] In non-limiting embodiments a method may include identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample. The sample may be obtained directly from a patient and/or may be obtained from a sample repository. The sample may be a tissue sample, a fluid sample, and/or any other suitable sample obtainable from a patient and likely to have a tau protein therein. In non-limiting embodiments, the sample is from a patient’s CNS (e.g., neural tissue (including one or more neurons, glial cells (e.g., astrocytes) from one or more of hippocampus, entorhinal cortex, or basal forebrain tissue, and/or the like, cerebral-spinal fluid (CSF), and/or any other neural sample). In non-limiting embodiments, the sample may be a blood sample, a plasma sample, and/or the like. In non-limiting embodiments, the patient has been diagnosed with a tauopathy. In non-limiting embodiments, the patient is at risk of developing a tauopathy, based on genetics, one or more co-morbidities, age, family history, and/or the like.
[0080] A method may include, in non-limiting embodiments, incubating the sample with a composition that includes a first binding reagent.
[0081] The term "binding reagent", for ease of reference and unless otherwise specified, refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having an antigen-binding domain which is homologous or largely homologous to an immunoglobulin binding domain, and complexes thereof, which are typically covalently linked, as in immunoglobulin (see, e.g., Chailyan et al. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity. FEBS J. 2011 Aug;278(16):2858-66, U.S. Patent No. 1 1 ,578,428, and U.S. Patent Publication No. 2024/0158529, showing typical antibody structures, including humanized antibodies, each of which is incorporated herein by reference in its entirety). An antigen-binding molecule may comprise a nucleic acid, as in the case of an aptamer. As such, the binding reagent operates as a ligand for its cognate antigen, which can be virtually any polypeptide or protein. Natural antibodies typically comprise two heavy chains and two light chains and are bi-valent. The interaction between the variable regions of heavy and light chain forms a binding site (e.g., a paratope, defined by a set of CDRs) capable of specifically binding an antigen.
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66A6790.DOCX Attorney Docket No. 06527-2501 181
The term "VH" refers to a heavy chain variable region of an antibody. The term "VL" refers to a light chain variable region of an antibody. Antibodies may be derived from natural sources, or partly or wholly synthetically produced, and may be "humanized" to reduce immunogenicity, as is known in the related arts. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including, for example and without limitation, any of the human classes: IgG, IgM, IgA, IgD, and IgE.
[0082] Antigen-binding molecules bind specifically to a target, e.g., an epitope and are therefore "target-specific". By "target-specific" or reference to the ability of one compound to bind another target compound specifically, it is meant that the compound binds to the target compound to the exclusion of others in a given reaction system, e.g., in vitro, or in vivo, to acceptable tolerances, permitting a sufficiently specific diagnostic or therapeutic effect according to the standards of a person of skill in the art, a medical community, and/or a regulatory authority, such as the U.S. Food and Drug Agency (FDA), in aspects, in the context of use of anti-human tau pSer262 or pSer356 antibodies or binding reagents with X-34 dye for detection of pre-tangles in CNS tissue samples according to various aspects, embodiments, or examples of the methods provided herein.
[0083] The binding reagent may optionally be a single chain antibody fragment. Alternatively, the binding reagent may comprise multiple chains which are linked together, for instance, by disulfide linkages. The binding reagent may also optionally be a multi-molecular complex. A functional binding reagent may consist of at least about 50 amino acids or at least about 200 amino acids. Binding reagent also includes miniaturized antibodies or other engineered binding reagents, such as scFvs, that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope-binding (e.g., paratope) sequences and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition.
[0084] A binding reagent or complexes thereof may be, for example and without limitation, a monoclonal antibody, a polyclonal antibody, including fragments, derivatives, or analogs thereof, or complexes thereof, including without limitation: Fab, Fab?, Fv fragments, single chain Fv (scFv) fragments, dsFv, Fab1 fragments, F(ab’)2 fragments, single domain antibodies, camelized (camelid) antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of
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66A6790.DOCX Attorney Docket No. 06527-2501 181 the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((ScFv)2 fragments), diabodies, triabodies, tetrabodies, which typically are covalently linked or otherwise stabilized (e.g., leucine zipper or helix stabilized) scFv fragments, bi-specific T-cell engager (BiTE, e.g., a DbTE), di-scFv (dimeric single-chain variable fragment), single-domain antibody (sdAb), or antibody binding domain fragments. Antibody fragments also include miniaturized antibodies or other engineered binding reagents that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope-binding sequences (e.g., paratope) and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition.
[0085] scFv molecules may be manufactured using any suitable technology. Typically, recombinant cells comprising genes for expressing scFv-containing polypeptides are engineered, e.g., according to decades-old methods using any of a variety of publicly- and commercially-available expression systems. Huston J. S., M. Mudgett-Hunter, M. S. Tai et al., "Protein engineering of single-chain Fv analogs and fusion proteins, "Methods in Enzymology, vol. 203, pp. 46-88, 1991 ; Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. scFv antibody: principles and clinical application. Clin Dev Immunol. 2012;2012:980250; Gaciarz A, Ruddock LW. Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv. PLoS One. 2017 Dec 18;12(12):e0189964; Sandomenico A, Sivaccumar JP, Ruvo M. Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments. Int J Mol Sci. 2020 Aug 31 ;21 (17):6324; Petrus MLC, Kiefer LA, Puri P, Heemskerk E, Seaman MS, Barouch DH, Arias S, van Wezel GP, Havenga M. A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli. Appl Microbiol Biotechnol. 2019 Nov;103(21 -22):8875- 8888; and Toleikis L, Frenzel A. Cloning single-chain antibody fragments (ScFv) from hybridoma cells. Methods Mol Biol. 2012;907:59-71 ; see, also, kbdna co /cjaninq-sciv.
[0086] While specific types of binding reagents are described specifically herein, any binding reagent reactive with the specified antigen (e.g., peptides comprising human tau pSer262 epitopes, human tau pSer356 epitopes, and human tau pSer202/pThr205 epitopes) may be utilized in the specified assays. Effective binding
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66A6790.DOCX Attorney Docket No. 06527-2501 181 may be evaluated in an affinity assay, such as an ELISA assay, bilayer interferometry (e.g., BLItz, see, e.g., Muller-Esparza H, Osorio-Valeriano M, Steube N, Thanbichler M, Randau L. Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes. Front Mol Biosci. 2020 May 27;7:98), singlemolecule array (e.g., SIMOA), surface plasmon resonance (SPR), oblique-incidence reflectivity difference (OI-RD) binding affinity, or cell binding assay, among other antibody specificity, affinity, and/or avidity assay methods. Binding reagents with high binding affinities may bind to their corresponding antigen with a KD of 10 pM or less, 500 nM or less, 100nM or less, 75nM or less, 50nM or less, or 25 nM or less.
[0087] Continuing with the method, in non-limiting embodiments the first binding reagent is specific to a region of the tau protein including positions 262 and/or 356 (e.g., Ser262 and/or Ser356 of the tau protein). By region it is meant a region encompassing 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more amino acids, all values and subranges therebetween inclusive. In non-limiting embodiments, the first binding reagent may be specific to a phosphorylated amino acid (e.g., phosphorylated Ser262 (pSer262) and/or phosphorylated Ser356 (pSer356)). In non-limiting embodiments the first binding reagent may be an IgG. In non-limiting embodiments, the first binding reagent may be diluted prior to incubation with the sample, for example, diluted 1 :250. Dilutions of the first binding reagent for optimal signal to noise ratio may be optimized as is known in the art.
[0088] In non-limiting embodiments, the method may include detecting binding between the first binding reagent and the tau protein, where such detecting indicates the presence of a pre-stage NFT in the patient sample. Suitable methods of detecting binding may include, for example and without limitation, immunoprecipitation assays, including binding reagents that are themselves bound to a substrate, such as a bead, as are known in the art. In non-limiting embodiments the binding reagent (e.g., antibody or fragment thereof) may be conjugated to a substrate that may, for example, increase the ability to isolate the bound protein. In non-limiting embodiments, the substrate may be a bead, for example an agarose bead or a magnetic bead. Suitable substrates may be modified as is known in the art, for example with coatings that properly configure the binding reagent, that improve signaling during various blotting procedures, and/or that improve adhesion.
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[0089] In non-limiting embodiments, the method further includes washing the immunoprecipitated tau protein. Washing may be performed with any suitable solvent and/or buffer. In non-limiting embodiments, the washing is conducted with saline, for example a buffered saline such as phosphate-buffered saline (PBS).
[0090] In non-limiting embodiments, the method further includes eluting the washed, immunoprecipitated tau peptide (and substrate, e.g., beads), thereby generating free a protein/binding reagent complex (e.g., free of the beads). In nonlimiting embodiments, the elution may be conducted with one or more solutions, for example, a glycine-containing buffer (e.g., a glycine elution buffer).
[0091] In non-limiting embodiments the first binding reagent is an antibody, a fragment thereof, and/or an ScFv that binds to a region of the tau protein including Ser262 and/or Ser356, and/or binds to Ser262 or Ser356, and/or to a phosphorylated equivalent thereof. Suitable antibodies and/or fragments thereof are commercially available, for example from ThermoFisher Scientific.
[0092] In non-limiting embodiments, the method may include an eluting step as described above. In non-limiting embodiments, following elution with a suitable solution, the protein and binding reagent complex can be used in an assay. For example, and without limitation, such an assay may include a mass spectrometry assay, a chromatography assay, a binding assay (e.g., a Western Blot), and/or any assay known to those of skill in the art useful for identification and/or quantification of proteins.
[0093] In non-limiting embodiments, the immunoprecipitated protein may be stained, for example with a dye, for example with a dye that allows for localization of binding between the first binding reagent and the tau protein. In non-limiting embodiments, the dye is a dye is X-34 (e.g., a dye having the formula C24H18O6), available commercially from Cell Signaling Technology. Other dyes, such as a thioflavin derivative disclosed in U.S. Patent No. 7,270,800, incorporated herein by reference in its entirety, are within the scope of this disclosure. In such embodiments, the binding reagent may be labeled and the signal from that label may be compared to the staining from the dye.
[0094] In non-limiting embodiments, the method may include incubating the sample with a second binding reagent (in a first, or a separate second, immunoprecipitation, for example). In non-limiting embodiments, the second binding reagent is specific to a region of the tau protein including positions 202 and/or 205 (e.g., Ser 202 and/or
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Thr205 of the tau protein). By region it is meant a region encompassing 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more amino acids, all values and subranges therebetween inclusive. In non-limiting embodiments, the second binding reagent may be specific to a phosphorylated amino acid (e.g., phosphorylated Ser 202 (pSer202) and/or phosphorylated Thr205 (pThr205)). In nonlimiting embodiments the second binding reagent is specific to pSer202/pThr205. In non-limiting embodiments, the second binding reagent is a monoclonal antibody derived from clone AT8, commercially available from, for example, ThermoFisher Scientific.
[0095] In non-limiting embodiments the method may include incubating the sample with the first and second binding reagent, and in non-limiting embodiments performing immunoprecipitation. In non-limiting embodiments, an elution step may be performed. In non-limiting embodiments, the immunoprecipitated protein may be stained, for example with a dye, for example with a dye that allows for localization of binding between the first binding reagent, the second binding reagent, and the tau protein, as described above.
[0096] In non-limiting embodiments, the method may include detecting binding between the second binding reagent and the tau protein, and detecting binding between the second binding reagent and the tau protein may indicate the presence a mature NFT in the patient sample. In non-limiting embodiments, the method may include detecting binding between the first binding reagent and the second binding reagent and the tau protein, and detecting binding between the first binding reagent and the tau protein and an absence of binding between the second binding reagent and the tau protein may indicate the presence of a pre-NFT in the patient sample. Accordingly, in non-limiting embodiments, methods disclosed herein may be useful for distinguishing pre-NFTs and mature NFTs.
[0097] In non-limiting embodiments, the patient sample may be from a human patient (though, as noted above, other species are contemplated and fall within the scope of this disclosure). In non-limiting embodiments, the patient sample is from a living patient. In non-limiting embodiments, the patient sample is from a living patient and, when a pre-NFT is detected based on the methods described herein, the patient may be treated for early-stage tauopathy, for example early-stage AD, for example, with therapeutic agents such as cholinesterase inhibitors (e.g., donepezil/Aricept), glutamate receptor modulators (e.g., memantine), anti-A|3 monoclonal antibodies
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(e.g., lecanemab or donanemab), and/or cognitive/behavioral interventions, such as cognitive rehabilitation, exercise, and/or the like.
[0098] Also provided herein is a method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample. The method may include, in non-limiting embodiments, incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a composition that includes a first-binding reagent (which may be any binding reagent described herein). In non-limiting embodiments the first binding reagent is specific to Ser262 (e.g., pSer262) and/or Ser356 (e.g., pSer356) of a tau protein. In non-limiting embodiments, the method may further include detecting binding between the first binding reagent and the tau protein (e.g., with an immunoprecipitation assay and/or any other assay described herein or known to those of skill in the art), wherein detecting binding between the first binding reagent and the tau protein indicates the presence of a pre-stage NFT in the patient sample. In non-limiting embodiments, as described herein, the sample may be from a living patient, and the method may further include treating the patient with a specific treatment as described herein.
[0099] Also provided herein is a method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample. The method may include incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a first-binding reagent and a second binding reagent, each of which may be any binding reagent described herein. In non-limiting embodiments, the first binding reagent is specific to Ser262 (e.g., pSer262) and/or Ser356 (e.g., pSer356) of the tau protein and the second binding reagent is specific to Ser202/Thr205 (e.g., pSer202/pThr205) of the tau protein. In non-limiting embodiments, the method may include detecting binding between the first binding reagent and the tau protein and between the second binding reagent and the tau protein, where detecting binding between the first binding reagent and the tau protein and an absence of binding between the second binding reagent and the tau protein indicates the presence of a pre-NFT in the patient sample. In non-limiting embodiments, as described herein, the sample may be from a living patient, and the method may further include treating the patient with a specific treatment as described herein. In non-limiting embodiments, detection of pre-NFTs as described herein may be combined with treatments, as a means for, for example, determining efficacy of a treatment and/or monitoring progression of a condition associated with pre-NFTs.
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Example 1
[00100] Single antibody chromogen-based immunohistochemistry (IHC) used a published protocol (see, Ikonomovic MD, et al. Post-mortem correlates of in vivo PiB- PET amyloid imaging in a typical case of Alzheimer's disease. Brain. 2008 Jun ;131 (Pt 6):1630-45); this protocol uses a modification of the avidin-biotin/peroxidase technique of Hsu et al. (Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981 Apr;29(4):577-80) and is highly sensitive to low abundance antigens (Ikonomovic MD, et al. AMPA-selective glutamate receptor subtype immunoreactivity in the aged human hippocampal formation. J Comp Neurol. 1995 Aug 21 ;359(2):239-52). Tissue sections (40 pm thick, free-floating) obtained from 4% paraformaldehyde fixed tissue blocks are washed in 0.1 M sodium phosphate buffer (PB, pH 7.4) and endogenous peroxidase activity is inhibited by immersing sections in 0.3% H2O2 made in 0.1 M Tris-buffered saline containing 0.25% Triton X- 100 (TBST) for 45 minutes. Sections are then rinsed in TBST and incubated in 3% goat serum made in TBST for 30 minutes followed by two 10-minute rinses in 1 % goat serum in TBST. Sections are incubated overnight at 4°C in rabbit polyclonal primary antibody diluted in TBST. Next, after washing in 1 % goat serum in TBST, sections are incubated for 1 hour in biotinylated anti-rabbit IgG made in goat diluted in 1 % normal serum in TBST, washed in TBST, and processed with the avidin-biotin Elite kit in TBST for 1 hour at room temperature. After washing repeatedly in imidazole acetate buffer (IAB), sections are exposed to a solution containing 0.05% 3’,3-diaminobenzidine tetra-hydrochloride (DAB)/0.0015% H2O2 for four minutes, washed in IAB, mounted on silane-coated slides, dehydrated in alcohols, cleared in xylene, and coverslipped with Permount. Immunohistochemical procedures for detection of human phospho-tau epitopes pSer262 and pSer356 used commercially available rabbit polyclonal antihuman pSer262 and rabbit polyclonal anti-human pSer356 IgG antibodies. Further details of the primary antibodies used in the study are provided in Table 1. Three different lots of each antibody were purchased and tested over a 6-month period. Optimal antibody dilution was determined by performing a three-point dilution curve for each of the two antibodies (FIG. 1 ).
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Table 1 - Antibody source and details.
[00101] IHC studies were then performed on fixed tissue sections obtained from the hippocampus from a series of cases (Cases 2-8) that spanned the range of severity of NFT pathology determined by Braak Stage. The hippocampus was chosen as the region of interest due to its early involvement in the neuropathological progression of AD.
[00102] FIG. 2 Depicts IHC studies of hippocampus based on Braak Stage. Methods were essentially as described above. Adjacent tissue sections were processed using the same immunohistochemical procedure and antibodies AT8 (pSer202/pThr205; A1 -G1 ; Invitrogen, MN1020), pSer262 (A2-G2; Thermo, 44-750- G), and pSer356 (A3-G3; Thermo, 44-751 G). AT8 was used as a comparator to the pSer262 and pSer356 antibodies and was previously optimized for use in our laboratories. In this experiment, tissue sections were obtained from the hippocampus from cases neuropathologically determined to be Braak Stage I (Case-2: A1 -A3), Braak Stage II (Case-3: B1 -B3), Braak Stage III (Case-4: C1 -C3), Braak Stage IV (Case-5: D1 -D3), Braak Stage V (Case-6: E1 -E3), and Braak Stage VI (Case-7: F1 - F3).
[00103] In these IHC studies, we observed that the CA1 region (one of the earliest sites of NFT development in the hippocampus CA fields) contained neurons immunoreactive to all three antibodies even at Braak Stage I, typically thought of as containing lesions confined to the allocortex. Immunoreactive neurons were observed in progressively higher densities at progressively higher Braak Stages. Although all three antibodies labeled neurons across the Braak Stages, the pattern of pSer262 and pSer356 labeling differed markedly from patterns seen with AT8. Specifically, pSer262
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66A6790.DOCX Attorney Docket No. 06527-2501 181 and pSer356 labeling exhibited a distinct intracellular punctate appearance in contrast to the more homogeneous neuronal staining seen with antibody AT8, even at higher Braak Stages where hippocampal NFT pathology was abundant. Additionally, unlike AT8, the pSer262 and pSer356 antibodies did not label two types of neurofibrillary pathology, neuropil threads (p-tau containing distal dendrites) or neuritic (e.g., axons) structures in neuritic plaques. These observations strongly support the idea that the pSer262 and pSer356 antibodies identify neurofibrillary change at the earliest neuropathological stages of AD and are specific to NFT lesions, that is, neuronal cell bodies, destruction of which results in irreparable harm to the neural circuits to which they are connected.
[00104] Multi-immunofluorescence (IF) experiments using established protocols (Mi Z, et al. Tenascin-C Is Associated with Cored Amyloid-[3 Plaques in Alzheimer Disease and Pathology Burdened Cognitively Normal Elderly. J Neuropathol Exp Neurol. 2016 Sep;75(9):868-76. Erratum in: J Neuropathol Exp Neurol. 2016 Dec;75(12):1 190 and Mizukami K, et al. Immunohistochemical analysis of ubiquilin-1 in the human hippocampus: association with neurofibrillary tangle pathology. Neuropathology. 2014 Feb;34(1 ):11 -8) were then performed on tissue sections of hippocampus from a neuropathologically early (Braak Stage II) case and from a neuropathologically end-stage (Braak Stage VI) case using a cocktail of pSer262 and AT8 primary antibodies (sourced as described above). Tissue sections were processed as described for chromogen-based immunohistochemical procedures as described above with the following differences: the step in which tissue sections are incubated in H2O2 was omitted, primary antibodies were prepared as cocktails (rabbit polyclonal IgG to pSer262 or pSer356 was diluted together with mouse monoclonal IgG clone AT8); a cocktail of fluorophore-conjugated secondary antibodies was used to achieve distinguishable colors (goat anti-rabbit conjugated to the Alexa488 fluorophore and goat anti-mouse conjugated to the Alexa594 fluorophore). After immunofluorescence procedures were completed, sections were mounted onto silane- coated slides and stained with the pan-amyloid binding compound X-34 to achieve three markers, each a different color, on the same tissue sections. The Alexa488 fluorophore was visualized using a FITC-compatible filter (excitation peak 480 nm, beam splitter 505 nm, emission peak 535 nm; #41001 , Chroma). Alexa594 fluorophore was visualized using a TRITC-compatible filter (excitation peak 535 nm, beam splitter 565 nm, emission peak 610 nm; #41002, Chroma). Fluorescence of X-34 was
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66A6790.DOCX Attorney Docket No. 06527-2501 181 visualized using a violet filter (excitation peak 405 nm, beam splitter 440, emission peak 455; #1 1005, Chroma). X-34 labels neurofibrillary pathology in neurons containing fibrillar aggregates of tau proteins (e.g., tau protein in its insoluble state, consisting of helical filaments) - these are recognized as representing mature NFT as opposed to pretangles which are composed of soluble, non-fibrillar tau assemblies recognized as an early step in the formation of NFT.
[00105] The punctate appearance of pSer262 immunolabeling in FIG. 3 was seen consistently and it replicates our observations obtained from chromogen-based immunohistochemical experiments (illustrated in FIG. 1 ). Referring to FIG. 4, these experiments indicate that pSer262 signal, even in this late Braak Stage case marks newly developing NFT that have variable co-labeling with antibody AT8 but lack fibrillar tau (that is, very little or no co-labeling with the dye, X-34). However, their abundance is significantly higher than seen in lower Braak Stages (see FIG. 3), complementing blood biomarker studies assaying this phospho-epitope.
[00106] Similar results were observed when the same multi-immunofluorescence experiments were performed with the pSer356 antibody in a Braak Stage II case (not shown) and a Braak Stage V case (FIG. 5). These experiments indicate that even in this late Braak Stage case, pSer356 signal marks newly developing NFT that have weak co-labeling with antibody AT8 and very little or no fibrillar tau (i.e., very little or no co-labeling with the X-34 dye). Thus, like pSer262, the antibody targeting the pSer356 phospho-epitope labels very early forms of NFT.
[00107] This histopathological analysis corroborates a previous study which reported p-tau262 immunolabeled vesicle-like clusters in hippocampal neurons morphologically assessed as pre-tangles, however, another antibody against both the p-tau262 and p-tau356 sites (12E8, not available commercially) failed to detect the vesicular patterns of pre-tangles, and identified only classic intra-neuronal NFT (Augustinak et al. Specific tau phosphorylation sites correlate with severity of neuronal cytopathology in Alzheimer's disease. Acta Neuropathol. 2002 Jan;103(1 ):26-35).
[00108] Interpretation and significance to Alzheimer's disease. Historically, cases of Alzheimer's disease (AD) have been stratified into Braak NFT Stages that reflect the neuropathological severity of the disease. The Braak staging scheme was developed as a diagnostic neuropathology method of providing order and classification to neurofibrillary pathology, specifically NFT a hallmark lesion of AD, and "based chiefly on the topographical expansion of the lesions". These studies describe six
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66A6790.DOCX Attorney Docket No. 06527-2501 181 stages characterized by differential regional involvement and can be viewed as three groups, Braak Stages l-ll where NFT are restricted to the allocortex (transentorhinal and entorhinal cortex) and rarely, CA1 hippocampus, Braak Stages lll-IV characterized by expansion of NFT into the hippocampus and limbic cortex, and Braak Stages V-VI characterized by presence of NFT in neocortical association areas. The Braak NFT Stage classification scheme is by nature a neuropathological construct for characterization of the neuropathological severity of the disease and does not necessarily reflect an individual's clinical status, as cognitively normal elderly present with a range of Braak Stages from l-lll, and rarely Braak Stage IV. Furthermore, traditional methods used in the original Braak studies included the modified Galiyas silver impregnation technique and immunohistochemical staining using the antibody clone AT8 (AT8 is an antibody directed against tau phospho-epitopes pSer202/pThr205); neither of these post-mortem techniques have pre-mortem biomarker equivalents. Although attempts have been made using PET imaging ligands reactive to highly fibrillized phospho-tau (marked post-mortem by the modified Galiyas procedure), these imaging methodologies are not reliable markers of early, pre-NFT changes in the brain. There are also no known blood biomarkers of tau phosphorylated at pSer202/pThr205, the targets of the AT8 antibody used post-mortem. However, to further advance the discovery of tau-based biomarkers, the current strategies for pre- mortem evaluations of AD predisposition, onset, and severity need to be strengthened by assessing the ability of blood-based biomarkers of tau phospho-epitopes to identify people at the earliest stages of AD and also to identify AD neuropathological change exclusive of other tauopathies (primary tauopathies). In this regard, it is important that the pathological substrates of the antibodies employed as blood-based biomarkers of brain pathology are characterized in post-mortem AD brain tissue; such innovative "blood-to-brain" strategy is an important theoretical basis of this invention. Our histopathological studies described above support that the pSer262 and pSer356 epitopes are important phospho-sites involved in the earliest stage of tau accumulation in neurons in AD. The presence of these epitopes in granular, vesicle like structures, often appears to precede another early phosphorylation site (pSer202/pThr205) as well as tau fibrillization. Thus, these epitopes are highly likely to mark the earliest stage of tau pathology development, supporting their use as early biomarkers of the AD neuropathological process.
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Example 2
Methods
[00109] Tau-FRET aggregation assay
[00110] We used the homogeneous time-resolved fluorescence energy transfer tau aggregation assay (originally from Cisbio, now Revvity) which contains a tau-specific antibody conjugated to either Tb cryptate or d2, generating a FRET signal when the labeled antibodies are in proximity. The resulting signal, which is proportional to the number and complexity of aggregates in the sample, was read at the 665 nm and 615 nm wavelengths on a VICTORX4 plate reader (PerkinElmer). A negative control consisting of the two labeled antibodies in diluent (without sample) was used to calculate the AF percentage, a value that reflects the signal to background of the assay.
[00111] Initially, a dilution linearity test was performed to identify the most suitable fold dilution to use for the brain samples. The TBS-soluble fraction from the AD and control brain samples was used in a test run, first brought to the same total protein concentration of 1.317 mg ml-1 , before a dilution series. The samples were serially diluted 1 :100, 1 :50, 1 :10 and 1 :5 in TBS, incubated overnight with the labeled antibodies added according to the manufacturer’s protocol in 96-wells low-volume white microplates. The AF percentage values were calculated using the ratio between the wavelength and the negative control provided with the kit. The observed signals were multiplied by the fold dilution and compared with the expected signals to determine the linearity of dilution.
[00112] Expression and purification of recombinant a-synuclein and tau constructs [00113] The DNA sequence for full-length a-synuclein and Tau441 (UniProt ID: P10636-8) and the tau peptides representing the STA (~tau258-36s) and fibril (tau302- 368) core peptides, as well as the N-terminal and C-terminal ends (taui-224 and tauses- 441, respectively) were amplified using PCR with primers representing the 5' and 3' sequence of each fragment, respectively, with the complementary DNA for full-length tau44i (cat. no. RC213312, Origene) as the template. The PCR fragments were cloned directly into the pET_SUMO vector (cohort 3: the Shiley-Marcos Alzheimer's Disease Research Center (ADRC), University of California, San Diego (UCSD)), an expression vector with a 6x His-SUMO tag N-terminally fused to the protein or peptide of interest. Constructs containing the 6x His-SUMO-tau fusion protein were sequenced and transformed into the Escherichia coli BL21 (DE3) strain for protein expression.
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[00114] To express the SUMO fusion proteins and peptides, E. coli BL21 (DE3) cells harboring the construct of interest were inoculated into 20 ml lysogeny broth (LB) medium supplemented with kanamycin at a concentration of 50 pg ml-1 and incubated overnight to obtain a starter culture. The overnight culture was used to inoculate 1 I of LB medium with kanamycin (50 pg ml-1 ) and incubated at 37 °C. When the optical density (OD)600 reached 0.5-0.7, protein expression was induced with 1.0 mM isopropyl B-d-1 -thiogalactopyranoside and grown overnight at 27 °C. The next morning, the culture was centrifuged at 7,000 rpm for 20 min at 4 °C and the dry weight was calculated. The pellet was stored at -20 °C until purification.
[00115] The pellet was gently thawed at room temperature and resuspended in 1 x native buffer containing 50 mM sodium phosphate, pH 8.0, and 0.5 M sodium chloride (Invitrogen) added at a ratio of 8 ml buffer to 1 g of dry weight of pellet. Lysozyme solution (Thermo Fisher Scientific) was added and the lysate was incubated on ice for 30 min, followed by sonication and then centrifugation at 12,000 rpm for 20 min at 4 °C, after which the supernatant was collected. The protein extract was added to Ni-NTA agarose columns (Novex) equilibrated with 10 mM imidazole in 1 x native buffer and incubated with gentle rotation at 4 °C for 1 h. The Ni-NTA agarose column was washed with 1 x native buffer containing 20 mM imidazole, and the 6x His-SUMO-tau fusion protein of interest eluted with 250 mM imidazole in 1 x native buffer. The eluted protein was dialyzed against 50 mM Tris-HCI, 150 mM NaCI, pH 7.8, for 1 h; the buffer was replenished with fresh supply and the process was repeated for another hour. Dithiothreitol (DTT) (1 mM) and a SUMO protease containing a 6x His tag was added and cleavage continued overnight at 4 °C. To remove the SUMO tag and the protease, an Ni-NTA column equilibrated with 20 mM imidazole in 1 x native buffer was added and incubation proceeded for 1 h at 4 °C. The flowthrough was collected and the remaining bound protein was eluted separately after adding 20 mM imidazole in 1 x native buffer. Eluate fractions were dialyzed against 1 x PBS; aliquots of these samples were examined using gel electrophoresis on 4-12% NuPAGE SDS gel (Invitrogen) and stained with Imperial Protein Stain (Thermo Fisher Scientific). Fractions that showed high immunoreactivity for the constructs of interest were pooled and stored at -80 °C until use, in line with previously described protocols. Where necessary, the protein or peptide constructs were further polished using size exclusion chromatography according to published methods.
[00116] Preparation of recombinant tau aggregates
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[00117] The monomeric forms of the recombinant tau peptides prepared according to the procedures described above and frozen at -80 °C in 1 x PBS were used to generate the aggregates. Each tau protein variant was diluted to a final concentration of 46 pM in 1 x PBS supplemented with 2 mM EDTA and incubated for 72 h on a shaking incubator (Thermomixer comfort, Eppendorf) at 350 rpm at 37 °C.
[00118] Human postmortem tissue and CSF studies (cohorts 1-3)
[00119] Human brain tissue and CSF specimens were obtained under permission and used in accordance with the Declaration of Helsinki 2013 and the relevant ethical boards at the respective institutions. The samples were from the following sources: cohort 1 , the Queen Square Brain Bank for Neurological Disorders, Department of Clinical and Movement Neurosciences, Institute of Neurology, University College London, London, UK; cohort 2, the Netherlands Brain Bank, Amsterdam, the Netherlands; and cohort 3, the Shiley-Marcos ADRC, UCSD. Ethical approval for these studies was provided by the institutional review boards (IRBs) at the participating institutions, with written consent sought for and provided by the participants or their close family members if deemed to be incapable of making such decisions at that time in accordance with IRB requirements. The Queen Square Brain Bank for Neurological Disorders has generic ethical approval from a London multicenter research ethics committee under a license from the Human Tissue Authority. The Netherlands Brain Bank cohort was approved by the ethics committee of the Vrije Universiteit Medical Center, Amsterdam. The research protocol for the UCSD cohort was reviewed and approved by the human subject review board at UCSD, while informed consent was obtained from all patients or their caregivers as consistent with California State law.
[00120] For cohort 1 , we used frontal gray matter tissue samples from n = 50 patients, including n = 10 each from AD, PSP, CBD, PiD and controls to enable pathological comparison across tauopathies. Neuropathological diagnosis followed established guidelines.
[00121] For cohort 2, the samples were taken from the superior parietal gyrus. Participants with AD were at Braak stages V and VI while controls were at Braak 0, fulfilling the criteria of Braak staging. Complete demographic information has been published previously29. Briefly, age at death (~64 years), sex distribution (25% males) and the postmortem interval (~6-7 h) were similar between the two groups.
[00122] Detailed methodological description of cohort 3 has been provided previously. Briefly, the individuals included received clinical assessment for cognitive
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66A6790.DOCX Attorney Docket No. 06527-2501 181 changes annually until death and signed to allow for neuropathological examination after their death. The evaluation results were carefully assessed at a consensus conference of experts to give a research diagnosis and determine the overall evaluation of cognition (normal, MCI; diagnosed after standard criteria, or dementia). [00123] Biofluids, including the CSF, were periodically collected from the participants who consented. In this study, we measured CSF samples from individuals with both neuropathological examination and antemortem CSF samples within 5 years of death. We included individuals with sporadic disease, excluding those with a family history of autosomal dominant AD, dominantly inherited mutations (such as PSEN1 , PSEN2 and APP mutations) or early-onset disease (under 50 years).
[00124] The autopsy procedures followed established protocols. For pathological diagnosis of AD, neuritic plaques, diffuse plaques and NFT were identified either with 1 % thioflavin S staining viewed with ultraviolet illumination and a 440 pm bandpass wavelength excitation filter, or with immunohistochemical staining using antibodies to A|3 (antibody 69D, rabbit polyclonal from E. Koo, 1 :1 ,200 dilution) and PHF1 tau (from P. Davies, 1 :600 dilution). Neuritic plaque density and NFT pathology were assessed according to CERAD and Braak staging, respectively. For more recent cases, pathological diagnosis of AD was made using the National Institute on Aging (NIA)- Alzheimer's Association (AA) consensus criteria. The National Alzheimer’s Coordinating Center Neuropathology Working Group recommendations were followed to stage the severity of cerebral amyloid angiopathy, grading from 0 (absent) to 3 (severe).
[00125] Lewy body pathology was evaluated using hematoxylin and eosin staining in addition to immunostaining with antibodies against a-synuclein (p-synuclein 81 A from V. Lee, 1 :15,000 dilution). Disease staging was performed in accordance with consensus LBD guidelines93. TDP-43 pathology was identified using immunohistochemical staining (polyclonal, 1 :12,000 dilution, cat. no. 10782-2-AP, Proteintech).
[00126] Homogenization and characterization of brain tissue isolates
[00127] The procedure used for cohorts 1 and 2 has been described previously29. Briefly, frozen brain tissue from each autopsy-verified case was dissected from the indicated region and 100 mg were dissolved in 0.5 ml TBS buffer (20 mM Tris-HCI, 137 mM NaCI, pH 7.6) containing complete protease inhibitor cocktail (Roche Diagnostic). Tissue homogenization was performed on ice with TissueLyser II
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(QIAGEN) under the following conditions: 200-Hz frequency for 2 min. The homogenate was transferred to a new 0.5 ml TBS buffer and centrifuged at 27,000g for 20 min at 4 °C. The supernatant (referred to as the TBS-soluble fraction) was aliquoted and stored frozen at -80 °C. The total protein concentration in the various TBS extracts was determined using the DC Protein Assay (Bio-Rad Laboratories).
[00128] The brain tissue homogenization protocol used for the MS and immunoblotting experiments followed a protocol described in Islam et al.30. Both this method and the one used for cohorts 1 and 2 (which involved centrifugation of brain extracts at 135,000g and 27,000g, respectively) led to the separation of tau oligomers and tangle-free filaments (sedimentable at 235,000g) from monomers.
[00129] IP and depletion of brain tau
[00130] The antibodies used in these experiments included Tau12, 95-108, HT7, BT2, Tau5, K9JA, 77G7, 4R, 368, 419, Tau46, TauAB, CT2, CT3, CT4, CT1 , CT5, and P-tau181 , which are commercially available from, for example, DAKO, BioLegend and ThermoFisher Scientific. Various antibodies and sources thereof are listed below in Table 2:
Table 2
[00131] For precipitation and depletion of tau from TBS-soluble fractions of AD brain isolates, the indicated anti-tau antibodies were conjugated to Dynabeads M-280 sheep anti-mouse or anti-rabbit IgG (Thermo Fisher Scientific), respectively, depending on the origin of the antibody, and according to the manufacturer’s recommended protocol. Briefly, 10 pg of total protein from the brain extract was incubated with the Dynabeads-antibody complex (that is, 4 pg antibody added to 50 pl beads in 1 x PBS) and incubated overnight at 4 °C with gentle rocking to enable even
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66A6790.DOCX Attorney Docket No. 06527-2501 181 mixing. The next morning, the Dynabeads-antibody complex was recovered by using a magnetized rack, the supernatant was reincubated in new 50 pl antibody- Dynabeads conjugate and the immunoprecipation or depletion process repeated for 2 h at room temperature. Afterwards, the Dynabeads-antibody complex (the precipitate fraction) was recovered and the remaining sample (the depleted fraction) was used in the tau-homogeneous time-resolved fluorescence energy transfer assay, where 10 pl of each sample was analyzed and untreated TBS-soluble brain extract from the same patient was used as control for the depleted samples.
[00132] Tau forms precipitated on the Dynabeads captured in the Dynabeads- antibody complex were eluted from the Dynabeads with 50 pl of 0.1 M citrate buffer, pH 2.75, into tubes containing 15 pl of 1 M Tris buffer, pH 9.0, for neutralization. The Dynabead-free tau samples were analyzed using immunoblotting, a capillary-based protein separation and immunodetection assay, to detect the different tau fragments present according to the manufacturer’s recommendations.
[00133] IP-MS
[00134] The IP-MS experiments were performed at the MS facility in the Biofluid Biomarker Laboratory, Department of Psychiatry, University of Pittsburgh. Briefly, 30 pl of a pooled TBS-soluble fraction (7.1 mg ml-1 ) from the mid-temporal regions of postmortem brains was diluted to 1 ml with binding buffer (100 mM Tris-HCI, pH 7.4, 300 mM NaCI, 0.2% w/v n-dodecyl-B-d-maltoside, 0.2% w/v n-Nonyl-[3-d-thiomaltoside (cat. no. N373, Dojindo Laboratories)), supplemented with 10% v/v Neurology Panel 4-PLEX E CSF sample diluent (cat. no. 103727, Quanterix) to minimize nonspecific binding. Tau protein forms were immunoprecipitated using 50 pl of Dynabeads (M-270 Epoxy, cat. no. 14301 ; cohort 3: Shiley-Marcos ADRC, UCSD) conjugated with 1 .25 pg of the specified antibodies (tau12, HT7, BT2, tau5, 77G7 and tau46), incubated overnight at 4 °C with rotation. The supernatant was then removed and the beads were washed twice with 0.5 ml PBS. After the removal of all residual liquid, proteins bound to the beads were eluted twice with 100 pl glycine buffer (50 mM glycine, pH 2.8, 0.1 % n-dodecyl-B-d-maltoside). The combined eluates were then neutralized with 5.5 pl 2N NaOH. Two replicate IPs were performed for each tau antibody.
[00135] Proteins were digested using SP3-based trypsin digestion94, similarly as described previously. Briefly, 50 pl of nondepleted and depleted fractions and 160 pl of precipitated fractions were brought up to 200 pl with 100 mM Tris, pH 8.0, and 2% SDS. The samples were then reduced with 10 mM DTT at 56 °C for 10 min and
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66A6790.DOCX Attorney Docket No. 06527-2501 181 alkylated with 20 mM iodoacetamide at room temperature in the dark for 1 h. Subsequently, 1 ml of 100% ethanol and 30 pl of 20 mg ml-1 Sera-Mag SpeedBeads Carboxylate-Modified Magnetic Particles (equal mix of hydrophobic and hydrophilic beads; cat. no. 65152105050250 and cat. no. 45152105050250, GE Healthcare) were added to each sample. The mixtures were incubated at room temperature with shaking at 1 ,400 rpm for 20 min, followed by three washes with 80% ethanol. After removing all liquid, 1 pg of trypsin (Sequencing Grade Modified Trypsin; cat. no. V51 1 1 , Promega Corporation) in 100 pl of 50 mM ammonium bicarbonate with 1 mM CaCI2 was used to digest the proteins bound to the magnetic particles. After digestion, the samples were desalted with C18 spin cartridges (cat. no. SMM SS18V, The Nest Group), dried using a SpeedVac and then reconstituted in 0.1 % formic acid (20 pl for nondepleted and depleted fractions and 16 pl for the precipitate).
[00136] The reconstituted peptides were analyzed using reverse-phase LC- tandem MS (MS/MS) using a nanoflow LC (a Dionex UltiMate 3000 RSLCnano System) coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific). The Xcalibur software (v.2.2 SP1.48, Thermo Fisher Scientific) was used to operate the LC-MS/MS system. For each analysis, 1 pl peptides were directly injected onto a 5-cm Aurora series electrospray ionization column with a 150 pm ID filled with 1.6 pm reversed-phase C-18 packing material (120-A pore size) (lonOpticks). Peptides were eluted using a linear gradient of 3-34% mobile phase B (0.1 % formic acid in acetonitrile) in 5.5 min, then to 90% B for an additional 1 min, all at a constant flow rate of 1 pl min-1. Data acquisition parameters included a full MS scan from 350 to 1 ,600 m/z at a 30,000 resolution and an automatic gain control (AGC) target of 300%, followed by four data-dependent MS/MS scans at a 15,000 resolution and a standard AGC target, and a retention-time-scheduled PRM analysis of 18 tau peptides. The PRM parameters included an Orbitrap resolution of 15,000, a standard AGC target, an automatic injection time, an isolation window of 2 m/z and a higher- energy C-trap dissociation-normalized collision energy of 30. Supplementary Table 1 shows the targeted inclusion list with the retention-time-scheduled PRM scans. Each peptide sample was analyzed twice using LC-MS/MS.
[00137] Skyline (v.21 .2.0.568) was used to facilitate the extraction of peptide quantification data from the PRM scans. To ensure accuracy, the chromatogram peak selection for each PRM assay was based on the presence of at least ten coeluting fragment ions. The final quantification of each peptide was based on the total area of
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66A6790.DOCX Attorney Docket No. 06527-2501 181 the top three high-quality fragment ions. Replicate injections were averaged before further data analysis. The normalized intensity area ratio of peptides from the precipitate to the nondepleted fractions was used to compare the relative enrichment efficiency of tau peptides from different regions. This was achieved by dividing each ratio by the sample’s mean ratio.
[00138] MS characterization of recombinant tau441 phosphorylation
[00139] Recombinant tau441 proteins, phosphorylated by various kinases, underwent in-solution trypsin digestion as detailed below: 1 pg of each protein was brought up to a final volume of 90 pl using 50 mM ammonium bicarbonate. This was followed by a reduction with 10 mM DTT at 56 °C for 10 min and alkylation with 20 mM iodoacetamide at room temperature in the dark for 1 h. Then, 0.25 pg of trypsin was added to each sample and the mixture was incubated overnight at 37 °C. After digestion, peptides were desalted using C18 spin cartridges, dried using SpeedVac, and reconstituted in 36 pl of 0.1 % formic acid. MS analysis proceeded in a manner similar to the IP-MS experiment, with the exception that different tau peptides were targeted for the PRM analysis. The Skyline software was used to extract quantitative data, similar to the IP-MS experiments.
[00140] Biochemical characterization of the p-tau262 and p-tau356 antibodies
[00141] Sandwich ELISAs were used to validate the specificity of the p-tau262 and p-tau356 antibodies. For each measurement, 80 pl of the antibodies at 2 pg ml-1 in PBS, pH 7.2, was added to the well and incubated overnight at 4 °C. The well was then blocked with 200 pl PBS/0.1 % BSA (cat. no. 81 -053-3, Merck Millipore) for 1 h at room temperature. After blocking, the well was washed twice with 300 pl PBS with 0.05% Tween 20 (PBST). Subsequently, 50 pl of recombinant tau441 at concentrations ranging from 0 to 400 ng ml-1 were added, followed by the addition of 50 pl of PBST with 2% milk and 50 pl biotinylated tau12 (specific to tau441 aa 6-18) at 1 pg ml-1 in PBST. Immunocomplex formation proceeded for 1 h with gentle shaking at 300 rpm at room temperature. After incubation, the wells were washed with 300 pl PBST five times. Pierce High Sensitivity Streptavidin-HRP (cat. no. 21 130, Thermo Fisher Scientific) was added and incubated for 1 h at room temperature, followed by five washes with 300 pl PBST. The 3,3',5,5'-tetramethylbenzidine substrate (cat. no. 34022, Thermo Fisher Scientific) was added and allowed to incubate for 30 min before stopping the reaction with 100 pl of Stop solution (cat. no. N600, Thermo Fisher Scientific). The OD at 450 nm with subtraction of the background
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OD at 550 nm was used to determine color development. The indicated synthetic peptides at a 0.1 pg ml-1 concentration were added to each well for competitive ELISA during the immunocomplex formation step.
[00142] Immunoblotting
[00143] Samples (5 pg for untreated and depleted, or precipitate from 5 pg tissue lysates after the IP procedures described above, using either PBS or the IP-MS buffer with 10% Neurology Plex 4E CSF sample diluent as the IP buffer) complemented with 1 x Laemmli sample buffer (cat. no. 161 -0747, Bio-Rad Laboratories) were loaded on 4-12% NuPAGE Bis-Tris gradient gels (cat. no. NP0322B0X, Thermo Fisher Scientific) and separated for 3 h at room temperature in 1 x NuPAGE MOPS Running Buffer (cat. no. NP0001 , Thermo Fisher Scientific) at 100 V. Separated proteins were then transferred to nitrocellulose membranes (Invitrogen iBIot 2 Transfer Stacks, cat. no. IB23002, Thermo Fisher Scientific) using the iBlot2 Western Blot Transfer System (cat. no. IB21001 , Thermo Fisher Scientific) at 20 V for 5 min at room temperature. Membranes were incubated for 1 h in Intercept (PBS) Blocking Buffer (cat. no. 927- 70001 , LI-COR Biosciences), then overnight in the presence of the anti-tau antibody tau12 (1 :1 ,000 dilution, cat. no. 806501 , BioLegend) in Intercept (PBS) Blocking Buffer with 0.2% Tween 20. Membranes were then incubated with Cy3 goat anti-mouse antibody (cat. no. 115-165-166, Jackson ImmunoResearch), diluted 700x in Intercept (PBS) Blocking Buffer with 0.2% Tween 20. Immunoblots were then dried and scanned using the ChemiDoc MP system (Bio-Rad Laboratories).
[00144] TEM
[00145] The recombinant tau aggregate preparations (5 pl) were pipetted onto copper grids that had been glow-discharged and carbon-coated, allowed 1 min to adhere onto the grid surface and then rinsed with ultrapure distilled water. Next, the grids were treated with 0.75% uranyl formate (Electron Microscopy Sciences) for 30 s to enable negative staining. TEM micrographs were taken on a Talos L120C 120 kV TEM microscope (Thermo Fisher Scientific) fitted with a BM-CETA camera- 4.096 x 4.096, 14-pm pixel complementary metal-oxide-semiconductor. Microscopic imaging was performed at the Centre for Cellular Imaging at the University of Gothenburg.
[00146] For the AD brain samples, TBS-soluble homogenates were first immunoprecipitated (following the procedures described above) with the tau12 antibody (which has been shown to enrich for tau forms that stretch into the MTBR; to
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66A6790.DOCX Attorney Docket No. 06527-2501 181 enrich for STAs. The resulting precipitated fractions were negatively stained by treating with 1 % uranyl acetate, allowed to dry and analyzed on a JEM-1400Flash TEM Microscope (JEOL) at x25,000 direct magnification, at the Center for Biological Imaging, University of Pittsburgh.
[00147] SPR
[00148] The SPR experiments were performed using a Biacore T100 biosensor (GE Healthcare). Immobilization of the CT19.1 antibody (epitope: aa 331-361 of tau44i) ligand on the surface of a CM5 chip was performed at a 5 pl min-1 flow rate to a level of 4,000 response units using standard amino coupling reagents (Cytiva). Thereafter, the analytes (that is, the truncated tau peptides) were injected at a flow rate of 20 pl min-1 , with the experiments being performed in PBS at 25 °C. The BIAevaluation and Prism 9 (Graph Pad Software) software programs were used for data processing and presentation, respectively.
[00149] Generation and characterization of CT antibodies
[00150] The new library of anti-tau mAbs was generated by immunizing 8-week-old BALB/c mice with 100 pg of recombinant tau 241 -441 peptide in complete Freund’s adjuvant (Sigma-Aldrich). After 2-3 further dosages of the immunogen (100 pg per mouse) in incomplete Freund’s adjuvant (Sigma-Aldrich), mice were euthanized, the spleen was removed and B cells were fused with the SP2/0 myeloma cell line according to standard protocols. Approximately 10 days after fusion, direct ELISA experiments were performed to screen the cell medium for antibodies that react with full-length recombinant tau441 (2N4R) or tau24i^4i. Positive clones were grown further, subcloned and subsequently frozen in liquid nitrogen. Antibody specificity was verified and the isotype determined using the Pierce Rapid Isotyping Kit-Mouse. Thereafter, mAbs were purified using a Hitrap Protein G column (Cytiva) according to the manufacturer’s instructions. Epitope mapping for each mAb was performed using direct ELISA against five custom-designed overlapping peptides spanning the tau24i- 441 sequence (Casio ApS), specifically tau24i-29i, tau28i-33i, tau32i-37i, tau36i^m, and taU401-441.
[00151] Generation and characterization of polyclonal antibody specific for truncation at aa 368
[00152] A new polyclonal antibody specific against tau truncated at aa 368 was generated by immunizing rabbits with 200 pg of a peptide containing the tau36o-368 sequence (Casio ApS) in complete Freund’s adjuvant. After one more dose of the
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66A6790.DOCX Attorney Docket No. 06527-2501 181 immunogen (200 ng per mouse) in incomplete Freund’s adjuvant, and two more doses of the immunogen (100 pg per mouse) in incomplete Freund’s adjuvant, the rabbits were euthanized and standard antibody generation procedures followed.
[00153] IHC and immunofluorescence studies
[00154] Cases and brain tissue samples
[00155] Studies were approved by the University of Pittsburgh Committee for Oversight of Research and Clinical Training Involving Decedents. Hippocampal tissue samples were obtained from autopsy cases at the University of Pittsburgh ADRC brain bank, including cases with NFT stage B1 or B2 and those with severe NFT stage B3 (ref. 96). At autopsy, samples of the hippocampus were dissected at the level of lateral geniculate nucleus and placed in cold (4 °C), 4% paraformaldehyde (cat. no. 158127- 5006, Sigma-Aldrich) made in 0.01 M sodium phosphate buffer (pH 7.2) (sodium phosphate monobasic and dibasic, cat. nos. S374 and S1319, respectively, Thermo Fisher Scientific) for 48 h. After fixation, samples were sequentially immersed for 48 h in each of 15% and 30% sucrose (cat. no. S512, Thermo Fisher Scientific) solutions made in sodium phosphate buffer. Samples were sliced on a freezing, sliding microtome (model 860, American Optical Corporation) into 40-pm-thick sections that were stored at -20 °C in cryoprotectant solution.
[00156] IHC and immunofluorescence
[00157] Tissue sections were removed from the cryoprotectant solution and rinsed three times in 0.1 M Trizma-buffered saline containing 0.25% Triton X-100 (Trizma, pH 7.4, cat. no. T7693, Sigma-Aldrich; Triton X-100, cat. no. T9284, Sigma-Aldrich). Chromogen-based IHC was performed as described previously using the VECTASTAIN Elite ABC-HRP Kit (cat. no. PK-6100, Vector Laboratories) with Ni- enhanced 3,3'-diaminobenzidine tetrahydrochloride (cat. no. D8001 , Sigma-Aldrich) as the chromogen. The details of the primary antibodies, including primary antibodies targeting an epitope at p-tau202/205 (clone AT8, 1 :3,000 dilution for chromogen-based IHC and 1 :500 dilution for the multi-immunofluorescence experiments, cat. no. MN1020, Thermo Fisher Scientific), an epitope at p-tau231 (clone AT180, 1 :1 ,000 dilution for chromogen-based IHC, cat. no. MN1040, Thermo Fisher Scientific), an epitope at p-tau262 (1 :250 dilution for chromogen-based IHC and 1 :200 dilution for the multi-immunofluorescence experiments, cat. no. 44-750G, Thermo Fisher Scientific) and an epitope at p-tau356 (1 :250 dilution for chromogen-based IHC and 1 :250 dilution for the multi-immunofluorescence experiments, cat no. 44-751 G, Thermo
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Fisher Scientific) are given in Supplementary Table 6. Secondary antibodies (all used at 1 :250 dilution) included a biotinylated goat anti-mouse IgG (cat. no. 1 15-065-146, Jackson ImmunoResearch), a biotinylated goat anti-rabbit IgG (cat. no. 1 1 1 -065-045, Jackson ImmunoResearch), an Alexa Fluor 594-conjugated goat anti-mouse IgG (cat. no. 115-585-146, Jackson ImmunoResearch) and an Alexa Fluor 488-conjugated goat anti-rabbit IgG (cat. no. 1 1 1 -585-144, Jackson ImmunoResearch). X-34 staining was performed as described previously. Light microscopy analysis was performed using an Olympus BX53 microscope. The immunofluorescence analysis was performed as described previously, using the Olympus BX53 microscope connected to a fluorescence illuminator (X-Cite 120Q). The microscope was equipped with an Olympus DP72 digital camera connected to a Dell Precision T5500 Desktop Workstation running the Olympus cellSens Standard v.1 .12 imaging software, and with a U PLAN S-APO x4 objective (numerical aperture (NA) 0.16), a U PLAN S-APO x10 objective (NA 0.4) and a U PLAN S-APO x20 objective (NA 0.75). The fluorescence of the Alexa Fluor 488 fluorophore was visualized using a fluorescein isothiocyanate- compatible filter (excitation peak = 480 nm, beam splitter = 505 nm, emission peak = 535 nm; cat. no. 41001 , Chroma). The fluorescence of the Alexa594 fluorophore was visualized using a Texas red isothiocyanate-compatible filter (excitation peak 535 nm, beam splitter 565 nm, emission peak 610 nm; #41002, Chroma). The fluorescence of X-34 was visualized using a violet filter set (excitation peak = 405 nm, dichroic mirror DM440, emission peak = 455 nm; cat. no. 11005, Chroma).
[00158] Electrophysiology experiments
[00159] Preparation of mouse brain slices
[00160] All animal care and experimental procedures were reviewed and approved by the institutional animal welfare and ethical review body at the University of Warwick. Animals were kept in standard housing with littermates, provided with food and water ad libitum and maintained on a 12:12 (light-dark) cycle. Male and female 3-4-week- old C57BL/6 mice were euthanized using cervical dislocation and decapitated in accordance with the UK Animals (Scientific Procedures) Act 1986. The brain was rapidly removed and acute parasagittal or horizontal brain slices (350-400 pM) were cut with a Microm HM 650V microslicer in cold (2-4 °C) high Mg2+, low Ca2+ aCSF, consisting of the following: 127 mM NaCI, 1 .9 mM KCI, 8 mM MgCI2, 0.5 mM CaCI2, 1 .2 mM KH2PO4, 26 mM NaHCO3 and 10 mM d-glucose (pH 7.4 when bubbled with
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95% 02 and 5% C02, 300 mOsm). Slices were stored at 34 °C in standard aCSF (1 mM Mg2+ and 2 mM Ca2+) for 1-8 h.
[00161] Incubation of acute brain slices with recombinant tau truncations
[00162] After at least 1 h of recovery, slices were either incubated in aCSF (control) or in 444 nM recombinant tau (1-224; N terminus, 258-368; STA core, 302-368; fibril core or 368-441 ; C terminus) in aCSF for 1 h, in bespoke incubation chambers at room temperature. The incubation chambers consisted of small, raised grids (to allow perfusion of slices from above and below) placed in the wells of a 24-well plate (Falcon), which were bubbled with 95% 02, 5% CO2 using microloaders (Eppendorf). Slices were placed into the incubation chambers one at a time (minimum volume 1 .5 ml to cover the raised grid). Individual slices were then placed on the recording rig and perfused with regular aCSF throughout the recording period, so the recombinant tau was only present for the 1 -h incubation, as shown previously.
[00163] Whole-cell patch clamp recording from single hippocampal CA1 pyramidal neurons
[00164] A slice was transferred to the recording chamber, submerged and perfused (2-3 ml min-1 ) with aCSF at 30 °C. Slices were visualized using infrared IR differential interference contrast optics with an Olympus BX151 W microscope (Scientifica) and a charge-coupled device camera (Hitachi). Whole-cell current clamp recordings were made from pyramidal cells in area CA1 of the hippocampus using patch pipettes (5- 10 mQ) manufactured from thick-walled glass (Harvard Apparatus). Pyramidal cells were identified by their position in the slice, morphology (from fluorescence imaging) and characteristics of the standard step current-voltage relationship. Voltage recordings were made using an Axon Multiclamp 700B amplifier (Molecular Devices) and digitized at 20 kHz. Data acquisition and analysis were performed using pCIamp 10 (Molecular Devices). Recordings from neurons that had an RMP of between -55 and -75 mV at whole-cell breakthrough were accepted for analysis. Bridge balance was monitored throughout the experiments; any recordings where it changed by more than 20% were discarded.
[00165] Stimulation protocols
[00166] To extract the electrophysiological properties of recorded neurons, both step and naturalistic, fluctuating currents were injected.
[00167] Standard IV protocol
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[00168] A standard current-voltage relationship was constructed by injecting standard (step) currents from -200 pA, incrementing by either 50 or 100 pA (1 -s duration) until a regular firing pattern was induced. A plot of step current against voltage response around the resting potential was used to measure the infrared (from the gradient of the fitted line).
[00169] Dynamic IV protocol
[00170] A dynamic-l-V curve, defined by the average transmembrane current as a function of voltage during naturalistic activity, can be used to efficiently parameterize neurons. The method has been described previously. Briefly, a current waveform, designed to provoke naturalistic fluctuating voltages, was constructed using the summed numerical output of two Ornstein-Uhlenbeck processes with time constants tfast = 3 ms and tslow = 10 ms. This current waveform, which mimics the stochastic actions of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and gamma- aminobutyric acid receptor channel activation, is injected into cells and the resulting voltage recorded (a fluctuating, naturalistic trace). The voltage trace was used to measure the frequency of action potential firing and to construct a dynamic-l-V curve. The FR was measured from voltage traces evoked by injecting a current waveform of the same gain for all recordings (to give an FR of ~2-3 Hz). Action potentials were detected by a manually set threshold and the interval between action potentials was measured. All analyses were completed using either the MATLAB or Julia (v.1.7.3) software platforms.
[00171] Extracellular recording of synaptic transmission
[00172] A 400-pM parasagittal slice was transferred to the submerged recording chamber and perfused with aCSF at 4-6 ml min-1 (32 °C). The slice was placed on a grid allowing perfusion above and below the tissue; all tubing (Tygon) was gas-tight to prevent loss of oxygen. To record field excitatory postsynaptic potentials (fEPSPs), an aCSF-filled microelectrode was placed on the surface of the stratum radiatum in CA1 . A bipolar concentric stimulating electrode (FHC) controlled by an isolated pulse stimulator model 2100 (AM Systems) was used to evoke fEPSPs at the Schaffer collateral-commissural pathway. fEPSPs were evoked every 30 s (0.03 Hz). Stimulus input and output curves for the fEPSPs were generated using a stimulus strength of 2-80 mA for all slices (stimulus duration 200 ps). Signals were filtered at 3 kHz and digitized online (10 kHz) with a Micro CED (mark 2) interface controlled by the Spike
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66A6790.DOCX Attorney Docket No. 06527-2501 181 software (v.6.1 ) (Cambridge Electronic Design). The fEPSP slope was measured from a 1 -ms linear region following the fiber volley.
[00173] Development and analytical validation of the CSF STA assay
[00174] To capture ST As, a rabbit polyclonal antibody targeting an end-specific truncation at aa 368 was coupled to paramagnetic beads (cat. no. 103207, Quanterix), while the detection antibody CT23.1 (epitope: aa 321-371 ) was conjugated to biotin (cat. no. A3959, Thermo Fisher Scientific) according to the manufacturer’s recommendations. The resulting method was a three-step Simoa assay that combined the assay beads (that is, beads conjugated with the capture antibody) and the helper beads in a 70% to 30% ratio to give 20,000 beads per pl and 1 pg ml-1 of biotin- conjugated detection antibody with 100 pl of undiluted CSF. The average number of enzymes per bead signal for each sample was plotted against the concentration of the inputted biospecimen.
[00175] The specificity of the capture antibody to the truncation at tau368 was confirmed with MALDI MS. In this experiment, 8 pg of the polyclonal 368 antibody was added to 50 pl M-280 Dynabeads (sheep anti-rabbit IgG, Invitrogen) per sample according to the manufacturer’s product description. The 368-coated beads were used for IP of either the positive control or antigen (aa ITHVPGGGN (SEQ ID NO: 2) equivalent to aa 359-368 with truncation at aa 368) or the negative control (aa GSLDNITHVPGGGNKKIETHKLTFRE (SEQ ID NO: 3) 355-380 lacking truncation at aa 368) in PBS. Beads and samples were transferred to a KingFisher magnetic particle processor (polypropylene tubes, Thermo Fisher Scientific) for automatic washing and elution of full-length and truncated peptides. Eluted samples were collected and dried in a vacuum centrifuge and redissolved in 5 pl 0.1 % formic acid in 20% acetonitrile and subsequently analyzed using a Bruker Daltonics UltrafleXtreme MALDI/ionization time-of-flight/time-of-flight mass spectrometer (Bruker Daltonics).
[00176] Clinical validation was performed using CSF samples from the CSF-to- autopsy and tau-PET studies (cohorts 3 and 4).
[00177] Tau-PET cohort
[00178] Study participants
[00179] Participants (n = 185) included in the study were selected from the Translational Biomarkers of Aging and Dementia (TRIAD) cohort, McGill University, Canada43,105. These participants had undergone A|3 and tau-PET imaging, the core
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CSF biomarker (A[342/40, p-tau181 and t-tau) analyses and had a CSF sample available for tau aggregate quantification.
[00180] In the TRIAD cohort, CN participants were defined as having an MMSE score greater than 24 and a CDR score of 0. This group included both young individuals (younger than 30 years) and older adults (older than 55 years). Participants with MCI had a CDR score of 0.5, with subjective and objective impairments in cognition, while their activities of daily living were preserved. Patients with AD dementia met the diagnostic criteria of the NIA and AA, and had a CDR score greater than or equal to 0.5.
[00181] All participants provided written informed consent; the research protocol was approved by the Montreal Neurological Institute PET working committee and the Douglas Mental Health University Institute Research Ethics Board.
[00182] Brain imaging
[00183] [18F]AZD4694 and [18F]MK6240 PET were used to assess brain A|3 and tau pathologies, respectively. Imaging was acquired at two time points, that is, 40- 70 min and 90-1 10 min after injection. PET scans were conducted using a Siemens High Resolution Research Tomograph (Siemens Medical Solutions).
[00184] To process the imaging data, PET scans from each participant were combined with their magnetic resonance imaging data. The cerebellar gray matter and the inferior cerebellar gray matter were used as reference regions for calculating the standard uptake value ratio (SUVR) for amyloid-[3 and tau-PET, respectively.
[00185] A|3 positivity was determined as a global [18F]AZD4694 SUVR equal to or greater than 1 .55. For tau-PET, a global index of tau pathology was obtained by calculating the average SUVR in the temporal meta-region of interest. Tau positivity was then defined as an SUVR equal to or greater than 1 .24. Moreover, participants were categorized into PET-based Braak stages based on the topography of tau-PET abnormality, as described in previous studies.
[00186] Statistical methods, data analysis and software
[00187] All statistical tests were two-sided.
[00188] Tau-FRET studies
[00189] Data were presented as the mean ± s.e.m. Two groups were compared with the Mann-Whitney U-test, whereas a Kruskal-Wallis analysis of variance with Dunn’s multiple comparison test was used to examine three or more groups in Prism 9.
[00190] Electrophysiology studies
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[00191] Prism 9 was used. Because of the small sample sizes (n < 15), statistical analysis was performed using nonparametric methods, that is, Kruskal-Wallis analysis of variance, Mann-Whitney U-test and Wilcoxon signed-rank test as required. All data are presented as the mean ± s.e.m. with individual experiments represented by single data points. For all experiments, significance was set at P < 0.05. Data points for each experimental condition were derived from a minimum of four individual animals.
[00192] CSF-to-autopsy study (cohort 3)
[00193] Statistical analyses were performed using R v.4.3.1 . The nonparametric Kruskal-Wallis rank-sum test was used for comparisons between group categories in demographic tables and figures alike, with significant results followed by post hoc pairwise Mann-Whitney U-tests with Benjamini-Hochberg FDR adjustment for multiple comparisons. Categorical variables were compared using a chi-squared test, with significant results followed by pairwise chi-squared tests with FDR adjustment for multiple comparisons.
[00194] Tau-PET cohort studies (cohort 4)
[00195] Python v.3.1 1 .2 was used to perform nonimaging statistical analyses. For several data processing and statistical tasks, several additional packages were used. Pandas (v.1 .5.3) was used as a powerful data analysis tool, providing data structures like DataFrames and Series, which allowed for efficient data handling and transformation. NumPy (v.1 .24.2) was used for numerical computations, enabling the manipulation of multidimensional arrays and matrices. Scikit-learn (v.1.2.2) was used for regression, clustering and model evaluation. Statsmodels (v.0.13.5) was used for statistical modeling and hypothesis testing.
[00196] To assess the overall differences among the multiple diagnostic categories, a Kruskal-Wallis test was performed. If the Kruskal-Wallis test yielded a significant result, indicating that there were overall differences among the groups, post hoc pairwise comparisons were conducted using a Mann-Whitney U-test. To control the increased risk of type I error because of multiple pairwise comparisons, a Bonferroni correction was applied to adjust the significance threshold (alpha) for each comparison and maintain an overall alpha level for the entire set of tests.
[00197] Correlation analyses were performed to examine the relationships between a set of selected variables. Specifically, the Pearson correlation coefficient was calculated to measure the linear associations between continuous variables. To control the risk of type I errors arising from multiple comparisons, FDR correction was
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66A6790.DOCX Attorney Docket No. 06527-2501 181 applied to adjust the P values. Only correlations with an FDR-corrected P < 0.05 were considered statistically significant and included in the final correlation matrix. Significant correlations were visualized using a heatmap, with color intensity representing the strength and direction of the associations between variables.
[00198] Neuroimaging analyses were carried out using the VoxelStats toolbox (https://github.com/sulantha2006/VoxelStats), a MATLAB-based analytical framework that allows for the execution of multimodal voxel-wise neuroimaging analyses'! 09. Using this toolbox, a voxel-wise linear model was constructed to assess the relationship between the CSF STA and t-tau ratio and [18F]MK6240 PET SUVR while correcting for age, sex, the presence of APOE s4 and [18F]AZD4694 PET SUVR, resulting in a t-map to display the strength of this relationship in a voxel-wise manner across different brain regions.
Results
[00199] Validation of the biochemical assay for STAs
[00200] We used a fluorescence resonance energy transfer (FRET) assay to quantify STAs in human autopsied frontal gray matter brain tissues from well- characterized individuals (FIG. 6A). The same anti-tau mAb, targeting a defined sequence in the proline-rich region and initial sequences of the MTBR, was coupled separately to the donor and acceptor molecules. As both antibody conjugates bind to an identical epitope, only one antibody can recognize any given tau monomer. Hence, the donor and receptor conjugates do not reach sufficient proximity to generate a signal. On the other hand, the polyvalency of STAs allows multiple binding events to occur. This leads to excitation of the antibody-donor complex followed by energy transfer to a nearby antibody acceptor that fluoresces at a wavelength of 665 nm. The assay signal (background-subtracted percentage fluorescence signal increase over the negative control) is proportional to the number and sizes of STAs present.
[00201] To test assay specificity, we compared equal concentrations of recombinant tau44i and a-synuclein as they both aggregate via [3-pleated sheet polymerization. Tau44i produced higher FRET signals than a-synuclein. Next, we performed dose titrations to evaluate FRET signal equivalence to the STAs present. First, recombinant tau44i showed decreasing signals proportional to the dilution fold (FIG. 6B). Then, we examined STAs from AD brain frontal gray matter tissue isolated according to established protocols. We used NFT-free TBS-soluble extract of AD brain homogenates. The tau-FRET signal decreased linearly across dilutions (fivefold,
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66A6790.DOCX Attorney Docket No. 06527-2501 181 tenfold or 50-fold) for four independent cases of identical starting total protein concentration, but with a wide range of starting FRET signals (FIG. 6C).
[00202] FRET assay is specific to AD-type ST As
[00203] We hypothesized that like filament heterogeneity, ST As from different tauopathies will have different tau-FRET assay binding. We examined TBS-soluble fractions of frontal gray matter tissue from autopsied cases (cohort 1 : n = 50; consisting of AD (n = 10) and non-AD tauopathies, including corticobasal degeneration (CBD) (n = 10), progressive supranuclear palsy (PSP) (n = 10) and Pick’s disease (PiD) (n = 10), as well as unaffected controls (n = 10). The tau-FRET signal (samples diluted to the same total protein concentration) was strongest in the AD group, being -300- fold higher than controls (P < 0.0001 ) and 15-43 times higher than the primary tauopathies (P = 0.0003 versus PSP and P = 0.0048 versus PiD; FIG. 6D). In a validation cohort of autopsy-confirmed AD and control cases without dementia who contributed parietal cortex tissue samples (cohort 2: (ref. 29) n = 8), the tau-FRET signal was again ~300-fold higher in AD versus control tissues (P = 0.0286; FIG. 6D). While the tau-FRET assay had a low coefficient of variation (that is, high precision) in both cohorts, there was remarkable heterogeneity in signals within groups despite all brain samples being at the advanced stages of pathology, suggesting inherent interindividual variability in STA levels. Together, these results show that the FRET assay is selective for AD-type ST As over those in other tauopathies.
[00204] The core region of AD brain STAs
[00205] We applied the tau-FRET technique to biochemically characterize STAs in AD brains. As AD tau filaments consist of a core region and an outer layer (fuzzy coat) binding adhesively to the Corel 6,31 ,32, we hypothesized that STAs will have a similar organization. We reasoned that immunodepletion with anti-tau antibodies will first remove the outer fuzzy coat layer before targeting the difficult-to-reach core region. Thus, antibodies targeting epitopes outside the core region will be more efficient at removing the aggregation signal obtained in the FRET assay when that same antibody is used to immunoprecipitate tau from the sample before the FRET analysis, and vice versa. In effect, the core region will be minimally affected by immunodepletion as it should be least accessible to antibodies.
[00206] We evaluated approximately two dozen antibodies targeting tau epitopes spanning the 441 -amino acid (aa) full-length tau sequence (FIGS. 7A-7B). Depleting TBS-soluble AD brain tissue homogenates with the N-terminus-targeting antibodies
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66A6790.DOCX Attorney Docket No. 06527-2501 181 tau12 and tau95-108 (epitopes: aa 6-18 and aa 95-108, respectively) as well as the mid-region antibody tau5 (aa 210-230), each led to complete removal of the tau-FRET signal (FIGS. 7B-7C), which taken together suggests the presence of long, near-fulllength tau species. Similar results were obtained with the C-terminal antibodies tau46 (aa 404-441 ), tauAB (aa 425-441 ) and tau419 (aa 419-433) (FIG. 7C). However, depletion with the MTBR-targeting polyclonal antibody K9JA led to a 70.4% decrease in signal (FIG. 7C). When using mAbs against defined MTBR epitopes, four-repeat- tau (~aa 275-291 ) removed a similar amount of the FRET signal (67.3%) while 77G7 (aa 315-355) removed less (42.7%) (FIG. 7C). However, tau368 (neospecific for truncation at aa 368 (ref. 33)) targeting the end of the MTBR removed much more FRET signal (88.9%). These findings could be caused by a decrease in the accessibility of the antibodies to the MTBR through which tau aggregates. The results further suggest that the STA core is located mostly in the R2-R4 region, as application of antibodies targeting this region produced the smallest depletion effect, which corresponds to the weakest attenuation of the tau-FRET signal.
[00207] To probe this further, we generated a set of antibodies covering the entire MTBR and the C terminus (FIGS. 7E-7F). The least depletion was obtained with CT4 (aa 321-371 ; 66.8% of the signal removed) followed by CT3 (aa 281-331 ; 76.6% removed) and CT1 (aa 361-411 ; 78.3% removed) (FIG. 7G). In agreement with FIG. 7C, CT2 (aa 241 -294; targeting mostly the R1 region) and CT5 (aa 401—441 ; directed at the extreme C terminus) were the most efficient at signal depletion (98-100% of the signal removed; FIG. 7G These results further indicate that the STA core may be located principally in the R2-R4 region.
[00208] Next, we examined phosphorylation (p-tau) sites in the STA core (FIG. 7F). Immunodepleting with p-tau262 and p-tau356 (serine residues in the KXGS motifs in the R1 and R4 repeats, respectively) showed limited antibody accessibility, suggesting that these epitopes are integral to the core (FIG. 7H). Corroborating the results in FIGS. 7C, 7G, immunodepleting with p-tau antibodies in the N terminus to mid-region (p-tau i, p-tau202, p-tau2i2, p-tau2i7, p-tau23i, and p-tau23s) or C terminus (p-tau396 and p-tau416) removed -100% of the tau-FRET signal (Fig. 7H). Together, given that the STA core is prominently phosphorylated at serine-262 and contains the R2 region (four-repeat-tau), we conclude that it starts from approximately aa 258 and ends at aa 368 where there is a major NFT-promoting pathological truncation.
[00209] The fuzzy coat contains N-terminal and C-terminal tau
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[00210] To further verify the STA core and the molecular forms of tau that make up the fuzzy coat, we used immunoblotting capillary electrophoresis to probe the antigenantibody-protein G-precipitated fractions from the immunodepletion experiments in FIGS. 7B-7C. Antibodies that efficiently depleted STAs (thus leading to high decreases in tau-FRET signals) had these signals consolidated in the precipitate, resulting in high immunoblotting immunoreactivity, and vice versa (FIGS. 7B-7D). Moreover, we hypothesized that antibodies that efficiently deplete most of the signal will cover the nonaggregating fuzzy coat region because the STA core should be least accessible to antibodies (FIG. 7B). Tau12 was efficient at depleting the tau-FRET signal (FIG. 7C). To estimate the length of tau12+ fragments, we screened antibodies covering neighboring sites. Tau12-precipitated tau stained positive for BT2 (aa 194- 198) but not 77G7 or tauAB, meaning that tau12-containing forms stretched into the mid-region but not into the far end recognized by 77G7 or the extreme C terminus where tauAB binds (FIG. 7D). In contrast, 77G7 was poor at depleting the tau-FRET signal (FIG. 7C), suggesting that it targets an epitope within the STA core. 77G7- precipitated tau fractions were negative for HT7 (aa 159-163) and BT2, indicating that the tau fragment immunoprecipitated by the 77G7 antibody lacked the mid-region sequences recognized by HT7 and BT2 (FIG. 7D). When considering the C terminus, the tauAB-precipitated fraction did not stain strongly for the mid-region-targeting antibodies HT7 and BT2 or the MTBR-binding UGH (FIG. 7D).
[00211] Molecular identity of tau forms that contain the STA core
[00212] We performed paramagnetic bead-based immunoprecipitation (IP) of a pooled TBS-soluble AD brain homogenate using six antibodies together stretching the tau441 sequence, comparing tau forms in the nondepleted (before IP), depleted (supernatant after IP) and precipitate (immunoprecipitated; tau forms pulled by the antibody of interest and thus enriched on the beads) fractions by MS using a parallel reaction monitoring (PRM) assay on a high-resolution quadrupole Orbitrap hybrid instrument (Orbitrap Exploris 480) online coupled with nanoflow LC. We monitored the levels of 18 tau peptides spanning aa 6-438 of tau 2N4R and quantified peptide intensity using the sum intensity area of the top three quality fragments of each PRM assay. Generally, the intensity area for the precipitate fraction was much stronger than the same for the nondepleted and depleted fractions, demonstrating efficient IP. No tau peptide was detected in the precipitate when a mouse IgG isotype control was used for the IP.
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[00213] We compared the enrichment efficiency of tau peptides across several regions of the protein by assessing the normalized precipitate to nondepleted ratios, with the mean ratio set to 1 to standardize IP with different antibodies. We observed that IP with the N-terminus-targeting antibody tau12 strongly enriched the N-terminal and mid-region tau forms in the precipitate, except for isoform-specific sequences in the 1 N and 2N regions (aa 45-103) for which results were driven by isoform abundance. However, there was a sharp decrease in peptides enriched in the precipitate after aa 209 but not for the second proline-rich region and the MTBR sequences aa 231-240 and aa 282-290, respectively, which remained abundant in the precipitate fraction. These results agree with the independent findings in FIG. 7 that did not include MS data. Together, the results indicate that tau12-containing tau forms can stretch not only from the N terminus to the mid-region but also into the N- terminal parts of the MTBR. This explains why tau12-precipitated tau forms stained positive for the mid-region-targeting antibody BT2 (epitope: aa 194-198) but not 77G7 (epitope: aa 316-355), which recognizes sequences toward the C terminus of the MTBR (FIG. 7D). Moreover, it clarifies the efficiency of tau12 (and by extension the antibody 95-108) to deplete the tau-FRET signal (Fig. 7C) as they bind tau forms covering the region which the antibody used in the FRET assay recognizes.
[00214] The mid-region-directed antibodies HT7, BT2 and tau5 were not as efficient as tau12 in precipitating N-terminal tau, but they were highly efficient in enriching midregion tau and MTBR sequences, including aa 231-240 and 282-290, but not any peptide C terminus to these. Compared to tau12, 77G7 showed reduced efficiency in precipitating the N terminus and mid-region of tau but much higher efficiency for the MTBR and C terminus, particularly after aa 275 (the R2 MTBR region). Finally, the tau46 antibody precipitated fragments containing the MTBR-end species through the C terminus (that is, aa 354-438), and some of the proline-rich region and N-terminal MTBR sequences, including aa 231-240 and 282-290, and the mid-region peptides 156-163. Together, tau forms that contain the STA core included near-full-length sequences that can stretch from the N terminus to the MTBR and from the mid-region to the far C terminus.
[00215] Furthermore, immunoblotting showed that tau12 prominently stained the nondepleted brain tissue sample. For the tau12 immunoprecipitated samples, the signal was much stronger in the precipitate versus depleted fractions when probed with tau12. This explains the efficiency of tau12 at immunoprecipitating STAs in the
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FRET (FIG. 7) and MS experiments. Together, the immunoblotting findings, further corroborating the results from the FRET and MS experiments, highlight the diversity of tau forms that contain the STA core region.
[00216] P-tau262 and p-tau356 in the STA core detect early-stage NFTs
[00217] We performed immunohistochemical analyses of autopsy-verified cases with either low (Braak NFT stages 0— II) or high (Braak NFT stages V and VI) tau pathology, focusing on the CA1 region of the hippocampus, which is highly vulnerable to AD tau pathology. We immunostained for p-tau262 and p-tau356, within the STA core, as well as p-tau23i (AT180) and AT8 (p-tau2O2/2os), outside of the core region.
[00218] At the low Braak NFT stages (for example, Braak stage II; FIG. 8), each antibody labeled isolated groups of CA1 pyramidal cells (FIG. 8, panels a-d, a1-d1 ), whereas at the high Braak NFT stages (for example, Braak stage VI; FIG. 8) they revealed high frequencies of immunolabeled cells throughout the CA1 region (FIG. 8, panels f-l, fl-il ). The presence of fibrillar tau was confirmed using the pan-amyloid binding dye X-34 (FIG. 8, panels e, e1 , j, j1). The p-tau262 and p-tau356 antibody immunostaining patterns differed from those produced by p-tau23i and p-tau202/205 antibodies in the overall appearance and extent of labeling neurons and neuritic pathology (dendritic tau pathology presenting as neuropil threads and axonal tau pathology presenting as dystrophic neurites in neuritic plaques). The p-tau262- directed antibody labeled pre-NFTs with a predominantly granular, vesicle-like immunolabeling pattern in portions of the cell soma and, less frequently, a diffuse, confluent immunolabeling over the cell soma and proximal dendrites at both low and high Braak NFT stages (FIG. 8, panels a1 , f1), while p-tau262 antibody labeling of neuropil threads was rare or absent (FIG. 8, panels a, a1 , f, f1 ). The p-tau356-directed antibody resulted in more instances of confluent staining in pyramidal cell bodies and portions of proximal dendritic processes, as well as granular and vesicle-like staining, in both low and high Braak NFT stages (FIG. 8, panels b, b1 , g, g1). In cases at the high Braak NFT stages, the p-tau356 antibody also labeled small numbers of neuropil threads and neuritic processes (FIG. 8, panels g, g1 ). Compared with the p-tau262 and p-tau356 antibody immunostains, the p-tau231 -directed (FIG. 8, panels c, c1 , h, hi) and p-tau202/205-directed (FIG. 8, panels d, d1, i, 11) antibodies yielded the most robust staining, primarily confluent, of pre-NFTs and mature NFTs in the low and high Braak NFT cases; in the latter, they also revealed a dense network of neuropil threads (FIG.
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8, panels h, hi , I, 11) and dystrophic neurites (for example, p-tau23i-immunolabeled neuritic plaque is present in the middle bottom of FIG. 8, panel h).
[00219] Dual immunofluorescence was used to assess the relationship of p-tau262 and p-tau356 with p-tau202/205 immunolabeling in CA1 pyramidal neurons from cases with low and high Braak NFTs. In low Braak NFTs, many neurons with confluent p- tau202/205 immunolabeling throughout their soma and processes had p-tau262 or p- tausse immunolabeling restricted to only a portion of the cell cytoplasm, mostly in clusters of vesicle-like granular structures (FIG. 9, panels a-f). In high Braak NFTs, p- tau262 and p-tau356 immunofluorescence was observed in only a subset of p-tau202/205- immunolabeled CA1 pyramidal neurons and was almost completely absent from p- tau202/205-immunolabeled neuropil threads (FIG. 9, panels g-l). In instances of intracellular codistribution, p-tau262 or p-tau356 immunofluorescence signals were localized to a portion of the cell soma in contrast to the confluent p-tau2O2/2os signal that extended throughout the cell body.
[00220] To determine the relationship of the p-tau262 and p-tau356 immunosignal with p-tau202/205 immunolabeling in pre-NFTs that lack tau fibrils and in NFTs that contain fibrillar tau, we performed triple fluorescence labeling experiments combining p-tau262 or p-tau356 antibodies with the p-tau202/205 antibody and the pan-amyloid binding dye X-34, which labels tau fibrils in NFT and A|3 fibrils in plaques and cerebral amyloid angiopathy. These experiments demonstrated that X-34-labeled NFTs frequently contained the p-tau2O2/2os signal but did not have appreciable p-tau262 or p- tausse immunosignal.
[00221] Specificity of the p-tau262 and p-tau356 antibodies
[00222] Previous studies reported tau phosphorylation by Ca2+ and calmodulindependent protein kinase II (CAMK2) at serine-262 and at serine-262 and serine-356 jointly, as well as by BR serine/threonine kinase 2 (BRSK2) at serine-262. Protein kinase A was reportedly involved in tau phosphorylation at serine-262. We performed sandwich enzyme-linked immunosorbent assays (ELISAs) with six different recombinant tau441 proteins in vitro phosphorylated by these kinases, which were commercially sourced from the same vendor. In sandwich ELISA, the proteins with the highest reactivity to both the p-tau262 and p-tau356 antibodies were those phosphorylated by CAMK2, BRSK2 and protein kinase A. To assess if the varying levels of reactivity aligned with phosphorylation levels, we used MS to compare the ratio of the phosphorylated and nonphosphorylated versions of tryptic peptides
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66A6790.DOCX Attorney Docket No. 06527-2501 181 covering the serine-262 and serine-356 epitopes across different recombinant tau441 proteins. The MS data largely mirrored the ELISA findings, with CAMK2- phosphorylated and BRSK2-phosphorylated tau44i having high p-tau262/tau262 and p- tausse/tausse ratios.
[00223] To further confirm antibody specificity, we performed competitive sandwich ELISAs using the highly reactive CAMK2-phosphorylated tau44i protein, which reacted dose-dependently when titrated against the p-tau262 or p-tau356 antibody. The signals were substantially decreased when synthetic peptides phosphorylated specifically at serine-262 or serine-356 were added in the reaction mixture to compete with the antibodies for target engagement. These results show that the p-tau262 and p-tau356 antibodies are specific to tau forms phosphorylated at serine-262 or serine-356.
[00224] In vitro aggregation of the STA core peptide
[00225] To investigate the functional effects of the STA core peptide in vitro, we expressed and purified its recombinant form (aa 258-368) and compared its characteristics with those of the fibril core previously identified using cryo-EM17 (~aa 302-368) and N-terminal and C-terminal controls (aa 1-224 and 368-441 , respectively; FIG. 10A).
[00226] In the surface plasmon resonance (SPR) experiments, an anti-tau antibody (CT19.1 ; aa 331-361 ) was immobilized onto a CM5 chip surface and peptide binding responses were recorded in cycles of binding and regeneration. The STA and fibril core peptides gave the highest SPR signals while the N-terminal and C-terminal sequences had no appreciable SPR increases from baseline (FIG. 10B). As the SPR signal is equivalent to the mass and number of molecules of the analyte (that is, the aggregates formed in this case) bound to the surface41 , these results suggest that the STA core peptide aggregates faster than the fibril core; however, the fuzzy coat structures do not form aggregates. In agreement, electron microscopy analysis showed that the STA and fibril cores formed aggregates, with structures from the latter appearing larger (FIG. 10C). As expected, the N-terminal and C-terminal control sequences did not show aggregate formation (FIG. 10C).
[00227] The STA core peptide alters neuronal excitability
[00228] We previously used an in vitro electrophysiology approach to quantify the functional effects of well-characterized recombinant tau species on neuronal and network function. This approach enables control over the structure and concentration of tau forms and permits studies of different tau forms, including truncated peptides as
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66A6790.DOCX Attorney Docket No. 06527-2501 181 well as monomers and oligomeric assemblies. In this study, acute mouse hippocampal slices were incubated in identical concentrations of recombinant tau peptides covering the STA core region identified in this study (the ~aa 258-368 STA core), the ~aa 302- 368 AD fibril core identified previously by cryo-EM, N terminus (aa 1-224) and C terminus (368-441 ) control peptides contained in the fuzzy coat peptides or in control buffer (artificial CSF (aCSF)) (FIG. 10A) for 1 h before electrophysiological recording. Whole-cell patch clamp recordings were made from mouse hippocampal CA1 pyramidal cells. Neuronal function was measured using stepwise and dynamic current injection protocols (FIGS. 10D-10E). Incubation with the STA core peptide led to significant depolarization (7 mV) of the resting membrane potential (RMP) versus aCSF control slices (P = 0.0400; FIG. 10F) and significant increases in input resistance (IR) versus aCSF control slices (reflecting a decrease in whole-cell conductance, P = 0.0008; FIG. 10G). There was no significant effect on RMP or IR in slices incubated with the other tau fragments, including the fibril core region. Given that the STA core depolarizes the RMP and increases the IR, we predicted that it should also increase the action potential firing rate (FR). Incubation with the STA core peptide significantly increased the FR versus aCSF control slices (P = 0.0016; FIG. 10H), so did the fibril core peptide versus aCSF controls (P = 0.0006; FIG. 10H). Thus, the STA core peptide impairs neuronal excitability in mouse hippocampal slices.
[00229] The STA core peptide alters synaptic transmission
[00230] Next, we investigated whether the STA core induced changes in synaptic transmission. We found no significant differences to the stimulus input and output responses across the different tau peptides. We then examined whether there were changes to the degree of paired-pulse facilitation. In slices incubated with either the STA or the fibril core peptide, the degree of facilitation was significantly increased compared with aCSF control (P = 0.0190 and P = 0.0359 at the 100-ms interval, respectivelyFIGS. 10I-10J). Together, these observations show that the STA core can modulate neuronal and network function.
[00231] An assay for ST As in human CSF
[00232] We next used our findings to develop an assay to measure ST As in human CSF, using single-molecule array (Simoa) technology as we have done for other tau- based biomarkers. The immunoassay paired two new mAbs: tau368 (neospecific for pathological truncation at aa 368) as capture and CT23.1 (aa 321-371 ) as detector. The epitopes of both antibodies fall within the STA core. Matrix-assisted laser
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66A6790.DOCX Attorney Docket No. 06527-2501 181 desorption/ionization (MALDI) MS analysis demonstrated that the tau368 antibody is end-specific for the truncation at aa 368.
[00233] ST As in the CSF separate AD from other tauopathies
[00234] We evaluated the clinical performance of the CSF assay in cohort 3 (n = 67) where each participant provided antemortem CSF samples on average 4.4 (s.d. = 2.1 ) years before death. To account for interindividual heterogeneity in STA levels (as in FIG. 6E), we took a ratio of STA to t-tau similar to the US Food and Drug Administration-approved A[342 and A[340 ratio assay, where A[340 normalizes for individual differences in aggregation-prone A[342 in the CSF49. The STA and t-tau ratio differed according to clinical diagnosis (P = 0.04), with post hoc comparisons demonstrating a significant difference between those with AD dementia and participants without dementia (P = 0.03). Based on standardized and expert neuropathological scoring50, individuals differed according to pathological diagnosis at autopsy (P = 0.0006), with post hoc comparisons showing that those with high AD neuropathological change (ADNC) at autopsy had similarly low CSF STA and t-tau ratio levels as those with high ADNC plus other neurodegenerative pathologies (P = 0.40); however, both groups had lower ratios than the participants with low ADNC pathology or other (non-ADNC) pathologies alone (P < 0.05 each; FIG. 11 A). These results indicate that the presence of a co-pathology did not affect the results. Expanding the ‘other’ groups into the constituent diseases did not change the outcome. The STA and t-tau ratio significantly differed according to Braak NFT stagingl (P < 0.0001 ) and was significantly lower in both individuals with Braak V and VI and Braak 111— IV staging versus Braak 0-II staging (P < 0.05; FIG. 11 B), with a sharp decrease between Braak III and IV (FIGS. 11C-11 D) suggesting that pathologically relevant changes occur in incipient AD, before isocortical association areas are affected in stages V and VI.
[00235] In multivariable regression models, the STA and t-tau ratio was significantly associated with Braak NFT staging dichotomized as Braak stages 0— III (‘low’) and Braak stages IV-VI (‘high’; t = -7.5, P < 0.0001 ) but not with age (t = 1 .3, P = 0.18), sex (t = — 1.2, P = 0.24) or the time interval between CSF collection and death (t = 1.9, P = 0.06). Adding the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) score for neuritic plaques to the models did not change the correlation results (Braak IV-VI, t = — 5.4, P < 0.0001 ; neuritic plaques, t = 0.649, P = 0.51903). Similarly, adding the Thai phase of A|3 deposition in place of CERAD did not change
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66A6790.DOCX Attorney Docket No. 06527-2501 181 the correlation results (Braak IV-VI, t = -5.9, P < 0.0001 ; Thai phase of A|3 deposition, t = 0.66, P = 0.52), suggesting that the ratio reflects central nervous system-derived CSF STA in AD independent of A|3 status.
[00236] ST As in CSF associate with in vivo tangle pathology
[00237] We further assessed the CSF tau STA assay in an antemortem cohort with in vivo tau-PET quantification using the high-affinity [18F]MK6240 tracer51 ,52 (cohort 4: n = 185 participants). There were six groups including tau-PET- young individuals (-20-25 years old; young/T-), cognitively normal (CN) older adults (CN/T-), and individuals with mild cognitive impairment (MCI) (MCI/T-) and AD dementia (AD/T-). The tau-PET+ groups included individuals with MCI (MCI/T+) and AD dementia (AD/T+). The STA and t-tau ratio was statistically different between groups (P < 0.0001 ; FIG. 11 E). Multiple comparisons demonstrated significantly lower (P < 0.05) ratio levels in AD/T+ versus each of the young/T-, CN/T- and MCI/T- groups. Additionally, there were significantly lower STA and t-tau ratio levels in the MCI/T+ group versus both the CN/T- and MCI/T- groups.
[00238] The STA and t-tau ratio was also examined across combined PET-based Braak NFT stages, including stages 0, I and II, III and IV, and V and VI. A Kruskal- Wallis test (P = 1 .60 x 10-10) and subsequent pairwise comparisons indicated significant between-group differences. Notably, the STA and t-tau ratio was significantly higher in Braak 0 individuals compared with Braak III and IV (P = 0.009) and V and VI (P < 0.0001 ) individuals. Furthermore, the levels were significantly lower in the Braak V and VI versus I and II groups (P < 0.0001 ; FIG. 11 F).
[00239] Significant and robust negative correlations were observed between the STA and t-tau ratio and the tau-PET radioligand [18F]MK6240 uptake across Braak NFT stages while accounting for A|3 PET uptake, age, sex and APOE s4 genotype. These correlations were primarily observed in the entorhinal cortex, amygdala, inferior and middle temporal gyri, fusiform gyrus and parahippocampal gyrus — regions including Braak stages I and IV (FIG. 11 G). Furthermore, regions associated with later Braak stages (V and VI) involving neocortical areas, including primary sensory regions, also displayed negative associations between [18F]MK6240 tau-PET and CSF tau STA and t-tau ratio (FIG. 11G). These findings suggest an inverse relationship between the STA and t-tau ratio and insoluble (fibrillar) brain tau pathology.
[00240] STAs in the CSF associate with cognition
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[00241] In cohort 3, the STA and t-tau ratio was positively associated with three different clinical measures of cognitive performance, that is, the Mini Mental State Examination (MMSE) (r = 0.41 , P = 0.0007), the Dementia Rating Scale (r = 0.36, P = 0.003) and the Clinical Dementia Rating (CDR)-Sum of Boxes (r = -0.32, P = 0.01 ). Similarly, in cohort 4, the STA and t-tau ratio was associated with the MMSE scores (r = 0.43, P < 0.001 ).
Discussion
[00242] Targeting early-stage STAs is a potentially effective approach to develop anti-tau diagnostics and therapies. We have shown that tau oligomers and related prefibrillar assemblies in the soluble fractions of autopsy-verified AD brains are biochemically different from those in other (primary) tauopathy brains. Next, we identified a minimal core STA peptide (~aa 258-368), revealing p-tau262 and p- tau356 aggregation-relevant phosphorylation sites. These findings have potential biomarker and therapeutic values. STA levels were higher at more advanced Braak NFT stages and correlated with a PET-detectable insoluble NFT burden specific to AD. Functional in vitro electrophysiology experiments demonstrated that a recombinantly produced STA core peptide alters synaptic transmission and neuronal excitability more potently than the insoluble fibril aggregate core, supporting the hypotheses that soluble tau might be more cytotoxic than fibrils. Finally, we used these insights to develop a CSF biomarker of tau pre-NFT pathology and verified its clinical performance in an autopsy-verified cohort with paired antemortem CSF samples and in an antemortem cohort with [18F]MK6240 tau-PET imaging.
[00243] Current plasma and CSF p-tau biomarkers have modest association with brain tau pathology assessed using tau-PET15,43,53, suggesting different pools of tau species in brain parenchyma versus the periphery. Therefore, methods for the direct quantification of aggregation-prone tau forms in body fluids are needed. The identified STA core provides an accessible CSF biomarker that can detect small nonf ibrillar tau species. Importantly, our findings indicate that p-tau sites detectable in currently used blood-based biomarker assays (for example, p-tau i , p-tau2os, p-tau2i2, p-tau2i7 and p-tau23i) are outside the STA core and thus fall within the fuzzy coat, corroborating our recent results that these biomarkers may not be directly associated with tau-PET in humans. This clarifies why their levels start to plateau in advanced- stage AD (that is, dementia) where tau pathology is more severe and tau-PET is a better predictor of AD pathology. It is reasonable to hypothesize that the fuzzy coat
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66A6790.DOCX Attorney Docket No. 06527-2501 181 contents have less propensity to aggregate and are thus released or secreted into biofluids in the early stages of disease when NFT formation is minimal and most of the available tau species are in the soluble form. However, as STAs mature into fibrils and tangles, fewer free-floating tau fragments bearing these N-terminal to mid-region p- tau markers are available for release into biofluids because of sequestration into NFTs. Characterization of the STA core, p-tau262 and p-tau356 will thus be important in directing the development of tau aggregation-relevant biomarkers and therapies.
[00244] The polymerization of tau into STAs, and further aggregation into fibrils and NFTs, occurs through the MTBR, specifically the R2 and R3 repeat domains or even shorter hexapeptide motifs within these domains, via [3-pleated sheet conformation59- 61 . The aggregation process does not require phosphorylation per se as tau proteins and peptides produced recombinantly or by chemical synthesis can polymerize into fibrils in vitro. In normal full-length tau, the MTBR is masked by the N-terminal and C- terminal projection domains; therefore, because of a cysteine residue each in R2 and R3, it can form only dimers but not oligomers63-65. Abnormal phosphorylation at serine-262, serine-356 and related serine and threonine sites unmasks the MTBR and promotes self-aggregation. While tau species lacking portions of the N-terminal and C-terminal domains through truncation can also self-aggregate, with pathologically relevant truncation at various sites reported, STAs in the AD brain consisted of nearfull-length peptides. These tau protein species contain the MTBR together with N- terminal, mid-region or C-terminal sequences, in agreement with previous studies.
[00245] MS showed, beside N-terminal forms, abundance of tau species containing not only defined peptides within the MTBR but also in the mid-region in brain TBS- soluble fractions, supporting previous results in human biospecimen and cell models. However, in the CSF, the mid-region peptides — but not the MTBR forms — remain abundant, supporting approved assays currently used in specialized clinics and recently developed mid-region-tau-targeting CSF assays. Furthermore, in agreement with earlier investigations that suggested that tau forms bearing MTBR fragments were lacking in the CSF, we demonstrated in this study that their levels are indeed low among tau-PET- young adults and cognitively normal older adults. However, STA levels increased with disease severity, being higher in tau-PET+ MCI and AD dementia groups. Taking a ratio against a mid-region-targeting nonphosphorylated tau (that is, t-tau) improved this association but the direction was reversed. Importantly, our findings extend previous results in cell models showing that MTBR-containing tau
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66A6790.DOCX Attorney Docket No. 06527-2501 181 becomes available extracellularly only with neuronal death, suggesting that cellular compromise is critical for MTBR tau release.
[00246] Histopathological analysis indicated that p-tau262 and p-tau356 are early indicators of tau pathology in hippocampal pyramidal neurons; hyperphosphorylation promotes tau self-aggregation, with p-tau262 and p-tau356 being critical to initiate this pathological process. Furthermore, p-tau262 and p-tau356 have been reported in AD brain extracts from several studies; however, their relevance to NFT formation has remained unclear. We have more directly shown that p-tau262-immunoreactive and p- tau356-immunoreactive hippocampal neurons in both low and high Braak stages are not colabeled with a marker of tau fibrils (X-34) and are therefore truly representative of pre-tangles. This agrees with our observations that p-tau262 and p-tau356 immunoreactivity occurred not only in cases with advanced Braak-staged NFT pathology but also in very early cases including at Braak stage II.
[00247] Mechanistically, p-tau262 decreases tau binding to microtubules. The anti- p-tau262 antibody labeled clusters of vesicles strongly resembling those reported to also express markers of granulovacuolar degeneration bodies in pre-tangles, which probably precede fibrillar tau aggregation in classic NFTs. The p-tau356 site is shared by both the STA and fibril core regions and might thus be an indicator of a biochemical change from soluble to insoluble aggregation. Notably, Augustinak et al. reported that a p-tau262 antibody preferentially detected pre-NFTs while another antibody against both p-tau262 and p-tau356 was indiscriminate for pre-NFTs, intracellular NFTs and extracellular NFTs. However, a marker of tau fibrils was not used to unequivocally identify NFTs versus pre-NFTs in that study.
[00248] The estimated core of AD-type tau fibrils differs from those in PSP, PiD and CBD brains. Our results, supported by others, show that these differences extend to AD-type versus non-AD STAs. Moreover, both the soluble and insoluble tau forms are surrounded by N-terminal and C-terminal fuzzy coat structures. However, the faster aggregation and higher neurotoxicity of STA versus fibril core peptides suggest unique biochemical properties. The presence of the second MTBR repeat region containing the 275VQIINK280 aggregation-promoting hexapeptide motif and the p-tau262 site in the STA core but not the fibril core might, at least partly, explain these results.
[00249] The STA core peptide addresses a critical need in drug development, that is, the discovery of druggable therapeutic target(s) for early-stage tau aggregates in AD. As recently shown for A|3 leading to approved therapies, targeting smaller, early-
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66A6790.DOCX Attorney Docket No. 06527-2501 181 stage soluble, pre-tangle tau assemblies might have higher chances to provide clinically meaningful outcomes than insoluble fibrils. Moreover, assessments of p- tau262-immunoreactive and p-tau356-immunoreactive tau species have the potential to identify individuals with prefibrillar tau pathology who may not (yet) be tau-PET+. Future development of biofluid-based biomarker assays for these p-tau sites should enable identification of living individuals with this profile for mechanistic studies and inclusion in therapeutic trials for preventing tau pathology in AD.
[00250] Together, we identified a core peptide of STAs in AD brains, revealed aggregation-relevant phosphorylation sites and translated these findings to develop an accessible CSF biomarker of AD-type tau pathology that will pave the way for the quantification of early-stage soluble (prefibrillar) tau assemblies in CSF and the development of therapies against these soluble pathological entities that may not be detectable using tau-PET.
Example 3
Methods
[00251] The utility of the pSer262 tau-directed antibody in early detection of p-tau neuropathological change and staging of the progression of p-tau lesions in the brain during the course of Alzheimer’s disease (AD) was evaluated by IHC-based staining of fixed tissue sections of the middle temporal gyrus from the same cases used in the analysis of the hippocampus (see FIG. 2 for IHC-based staining of the hippocampus). The pSer262 directed antibody and IHC methods used for the single-label chromogenbased IHC staining of middle temporal gyrus sections were the same as those used in the analysis of the hippocampus (see above). We also examined the degree to which the pSer262 and pSer356 antibodies labeled non-AD tau pathologies using the same methods described above.
Results
[00252] FIG. 12 depicts pSer262 tau antibody-based IHC analyses of p-tau lesions in middle temporal gyrus based on Braak NFT Stages. In this experiment, tissue sections were obtained from the middle temporal gyrus from cases neuropathologically determined to be Braak Stage I (A), II (not shown), III (not shown), IV (B), V (C), and VI (D). We observed positive pSer262 tau immunostaining in middle temporal gyrus sections in cases from each of the six Braak Stages. However, the morphological appearance of tau pathology structures labeled with pSer262 IHC differed across Braak Stages. In Braak Stages I and II, pSer262 immunosignal was
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66A6790.DOCX Attorney Docket No. 06527-2501 181 mostly seen in small (approximately 10 pm) circumscribed clusters of loosely associated granule-like structures (A, a, b). Less frequently, granule clusters exhibited a more condensed arrangement on a background of confluent staining (c, d). The latter pattern was observed most frequently in Braak Stages III and IV, with dense intracellular and occasional neuritic staining (B). Clusters of pSer262 labeled granules were sparse in the Braak Stage V and rare in the Braak Stage VI (C, D). In Braak Stage V, many of the pSer262 immunolabeled neurons showed extension of staining from cell soma into apical dendrite and were surrounded by scattered neuropil threads and small clusters of short, thickened processes resembling dystrophic neurites of neuritic plaques (C, e, f). In the Braak Stage VI, pSer262 immunosignal was robust, showing abundantly in numerous classic NFT, dense network of neuropil threads, and frequent plaque-like clusters of dystrophic neurites (D, g, h). Scale bars: 100 pm (A- D), 10 pm (a-d), 20 pm (e-h).
[00253] FIG. 13 depicts AT8 (pSer202/pThr205; A, a, b), pSer262 tau (B, c), and pSer356 (C, d) antibody-based IHC studies of CA1 hippocampus and adjacent fiber tracts in a case of AD (Braak Stage III) with a common non-AD related tau copathology termed aging-related tau astrogliopathy (ART AG), which consists of p-tau accumulation in glial cells (primarily astroglia) at the pial surface, near the ependyma of the ventricles, in the white matter, and in the gray matter and appears to be associated with aging rather than specific known disease processes. We observed intense AT8 immunolabeling of astroglia near the ependyma of the ventricle (A, a) in addition to AD-specific NFT in hippocampus CA1 (A, b). In contrast, pSer262 tau and pSer356 tau immunolabeling was not observed in areas with dense AT8- immunolabeled astroglia, while infrequent pyramidal cells with the appearance of pretangles and NFT were clearly labeled in CA1 hippocampus with the pSer262 tau antibody (B, c) and the pSer356 tau antibody (C, d). Scale bars: 400 pm (A-C), 200 pm (a-d).
[00254] FIG. 14 depicts AT8 (pSer202/pThr205; A, a, b), pSer262 tau (B, c), and pSer356 (C, d) antibody-based IHC studies of cortical white matter in a case of Alzheimer’s disease (Braak Stage III) with ART AG co-pathology. We observed clusters of AT8 immunoreactive astroglia (A, a) and occasional NFT (A, b). The clusters of AT8 immunoreactive astroglia were not labeled with pSer262 tau (B) or pSer356 tau (C) antibodies, though rare small neurons were immunolabeled with
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66A6790.DOCX Attorney Docket No. 06527-2501 181 antibody pSer262 tau (B, c) and antibody pSer356 (C, d). Scale bars: 400 pm (A-C), 50 pm (a-d).
[00255] FIG. 15 depicts pSer356 tau immunoreactivity compared to immunoreactivity seen with antibody clone AT8 (pSer202/pThr205) in middle frontal cortex from a case neuropathologically diagnosed with the 4R tau tauopathy, corticobasal degeneration (CBD), and a case neuropathologically diagnosed with the primarily 3R tau tauopathy, Pick’s disease (PiD). In frontal cortex sections from the CBD case, we observed numerous AT8 immunolabeled astrocytic plaques (one of the defining lesions of the disease) throughout the gray matter (A). In contrast, pSer262 immunoreactivity was absent (not shown), while pSer356 immunoreactivity was present in clusters of small puncta arranged in patterns resembling the very much more robust AT8-immunolabeled astrocytic plaques (B). Rare, small pSer356 immunoreactive neurons were also observed (C, dashed box area in B). In frontal cortex sections from the PiD case, AT8-immunlabeling was observed in numerous, laminarly arranged Pick bodies (one of the defining lesions of the disease) on a background of dense neuropil threads (D). In contrast, pSer262 immunoreactivity was confined to rare, lightly labeled neurons (not shown), while robust pSer356 immunoreactivity was observed in Pick bodies but not in neuropil threads (E, F, the latter is the boxed area in E). Scale bars: 100 pm (A, B, D, E), 25 pm (C, F). Discussion
[00256] As described fully above, cases of AD have been stratified into Braak NFT Stages that reflect the neuropathological severity of the disease, providing order and classification to p-tau neurofibrillary pathology, specifically NFT, a major lesion of AD, and “based chiefly on the topographical expansion of the lesions”. In short, six Braak Stages characterized by differential regional involvement with NFT can be viewed as three groups, Braak Stages l-ll where NFT are restricted to the allocortex (transentorhinal and entorhinal cortex) and rarely, CA1 hippocampus, Braak Stages lll-IV characterized by expansion of NFT into the hippocampus and limbic cortex, and Braak Stages V-VI characterized by presence of NFT in neocortical association areas. As described in Section [0074], Galiyas silver impregnation technique and/or IHC with antibody clone AT8 (pSer202/pThr205 tau) have been used traditionally in neuropathological evaluation of Alzheimer’s disease NFT pathology neither of which have fluid of PET biomarker equivalents, precluding pre-mortem classification of individuals into Braak Stages. This prevents identification of individuals at the early
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66A6790.DOCX Attorney Docket No. 06527-2501 181 stages of the disease and is clinically relevant from a therapeutic standpoint as early identification of the disease is critical for proper timing of therapeutic intervention. We identified two novel antibodies, one directed to the tau phospho-epitope pSer262 and the other to pSer356, that label early p-tau accumulation in form of granules within pyramidal neurons of the hippocampus. The absence of, or only partial co-labeling with antibody clone AT8 and lack of fibrillar tau aggregates reactive to amyloid binding dyes such as X-34 indicate that these two epitopes are highly likely to mark the earliest stage of tau neuropathological development, supporting their use as early biomarkers of the p-tau neuropathological process in AD. Our histopathological studies of middle temporal gyrus described above provide further evidence for this claim. The middle temporal gyrus is a neocortical association area that integrates higher order sensory information with the brain default mode network for modulation of future, goal directed behavior. According to Braak Stages, and confirmed in many studies, IHC with antibody clone AT8 reveals NFT pathology in the middle temporal gyrus to be limited to cases considered Braak stage V-VI though they can appear sparsely in Braak Stage IV. Thus, unlike what was described for antibody clone AT8, we observed pSer262 tau immunoreactivity, indicative of accumulation of tau phosphorylated at Ser262 tau, at all Braak Stages. Furthermore, we observed that the type of structure immunolabeled with the pSer262 tau antibody evolved across Braak Stages, from lower (i.e., Braak Stages l-ll) to middle (i.e., Braak Stages 11 l-l V) to higher (i.e., Braak Stage V-VI). These data indicate that detection of tau phosphorylated at Ser262 represents an early stage of NFT development in the neocortex even at the earliest Braak Stages. We also observed that antibodies targeting pSer262 and pSer356 are not potent markers of ARTAG, a common aging-related tau pathology in glia, supporting the specificity of these antibodies for Alzheimer’s disease, a disease of the aged. Additionally, we observed that the pSer262 tau antibody did not label hallmark 4R-rich tau lesions of CBD or the 3R-rich tau lesions of Pick’s disease, while the pSer356 tau antibody showed only partial labeling of the former and prominent labeling of the latter. These observations indicate that the pSer262 tau antibody is highly specific for AD tau lesions from the earliest to late stages of their development.
[00257] Having described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof. References incorporated herein by reference
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66A6790.DOCX Attorney Docket No. 06527-2501181 are incorporated for their technical disclosure and only to the extent that they are consistent with the present disclosure.
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66A6790.DOCX

Claims

Attorney Docket No. 06527-2501181 THE INVENTION CLAIMED IS
1 . A method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample, comprising: obtaining a sample from a patient suspected of having or at risk of developing a tauopathy; incubating the sample with a composition comprising a first binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein; and detecting binding between the first binding reagent and the tau protein, wherein detecting binding between the first binding reagent and the tau protein indicates the presence of a pre-stage NFT in the patient sample.
2. The method of claim 1 , wherein the first binding reagent is an antibody against Ser262 or Ser356.
3. The method of claim 1 , wherein the first binding reagent is an antibody against phosphorylated Ser262 (pSer262) or phosphorylated Ser356 (pSer356).
4. The method of claim 1 , wherein the composition comprises the first binding reagent bound to a substrate.
5. The method of claim 4, wherein the substrate is a bead.
6. The method of claim 5, wherein the bead is a magnetic bead.
7. The method of claim 4, wherein detecting binding between the first binding reagent and the tau protein comprises a step of eluting the first binding reagent and tau protein from the substrate, thereby generating a free protein-first binding reagent complex, and performing an immunoblotting assay or protein separation and immunodetection assay on the free protein-first binding reagent complex.
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8. The method of claim 1 , further comprising staining the sample with a dye, optionally wherein the dye has the formula C24H18O6.
9. The method of claim 8, further comprising localizing binding of the first binding reagent to the tau protein based on localization of the binding reagent relative to the stain.
10. The method of claim 1 , further comprising incubating the sample with a second binding reagent, the second binding reagent specific to Ser202/Thr205.
1 1. The method of claim 10, wherein the second binding reagent is specific to phosphorylated Ser202 (pSer202) and phosphorylated Thr205 (pThr205), optionally wherein the second binding reagent is a monoclonal antibody, optionally wherein the second binding reagent is a monoclonal antibody derived from clone AT8.
12. The method of claim 10, further comprising detecting binding between the second binding reagent and the tau protein, wherein detecting binding between the second binding reagent and the tau protein indicates the presence a mature NFT in the patient sample.
13. The method of claim 12, wherein detecting binding between the first binding reagent and the tau protein and an absence of binding between the second binding reagent and the tau protein indicates the presence of a pre-NFT in the patient sample.
14. The method of claim 1 , wherein the tau protein is a human tau protein from a human patient sample.
15. The method of claim 14, wherein the human patient sample is a sample from the patient’s central nervous system, optionally a tissue sample, optionally from hippocampus, entorhinal cortex, or basal forebrain tissue.
16. The method of claim 14, wherein the human patient sample is a sample from the patient’s blood.
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17. The method of claim 15 or claim 16, wherein the sample is a from a living patient.
18. The method of claim 17, further comprising treating the patient for early-stage AD if the sample comprises a pre-NFT.
19. The method of claim 1 , wherein the first binding reagent is a diluted binding reagent.
20. The method of claim 19, wherein the first binding reagent is diluted 1 :250
21. The method of claim 1 , wherein the first binding reagent comprises an IgG antibody.
22. A method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample, comprising: incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a composition comprising a first-binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein; and detecting binding between the first binding reagent and the tau protein, wherein detecting binding between the first binding reagent and the tau protein indicates the presence of a pre-stage NFT in the patient sample.
23. A method of identifying a pre-stage neurofibrillary tangle (NFT) in a patient sample, comprising: incubating a sample obtained from a patient suspected of having or at risk of developing a tauopathy with a composition comprising a first-binding reagent and a second binding reagent, wherein the first binding reagent is specific to Ser262 and/or Ser356 of a tau protein and the second binding reagent is specific to Ser202/Thr205 of the tau protein; and
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66A6790.DOCX Attorney Docket No. 06527-2501 181 detecting binding between the first binding reagent and the tau protein and between the second binding reagent and the tau protein, wherein: detecting binding between the first binding reagent and the tau protein and an absence of binding between the second binding reagent and the tau protein indicates the presence of a pre-NFT in the patient sample.
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