Assay for evaluating tau propagation

Abstract

The present disclosure relates to assays for evaluating tau propagation activity, in particular in vivo assays for evaluating tau propagation activity. The disclosure further provides methods to evaluate compounds for their effect on tau propagation, and anti-tau compounds such as antibodies which reduce tau propagation in the assay of the present disclosure and can be used to treat Alzheimer's disease in a human patient.

Description

ASSAY FOR EVALUATING TAU PROPAGATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 490,715, filed March 16, 2023, the entire contents of which is incorporated herein by reference in its entirety. FIELD

[0002] The present disclosure relates to assays for evaluating tau propagation activity, in particular in vivo assays for evaluating tau propagation activity, as well as methods to evaluate compounds for their effect on tau propagation, and compounds identified through such analyses. BACKGROUND

[0003] Tau is a neuronal microtubule-associated protein found predominantly in axons which functions to promote tubulin polymerization and stabilize microtubules. There are six tau isoforms expressed in human adult brain, which are produced by alternative mRNA splicing of transcripts from the MAPT gene. These isoforms contain either three or four microtubule-binding repeats (3R or 4R tau, respectively), and 0-2 N-terminal inserts (0N, 1N, or 2N tau).

[0004] The physiological functions of tau are highly regulated by a range of posttranslational modifications, including phosphorylation, acetylation, glycosylation, isomerization, nitration, SUMOylation, and ubiquitination. The alteration of these modifications can affect tau functions and potentially lead to pathological conditions. Hyperphosphorylated tau is a major component of neurofibrillary tangles (NFTs), one of the characteristic pathological features of Alzheimer’s disease (AD). Tau deposits in the brain correlate with memory decline, confirming the importance of tau pathology in AD. See Braak et al., “Neuropathological staging of Alzheimer- related changes,” Acta Neuropathol.1991; 82:239-59; Nelson et al., “Correlation of Alzheimer Disease Neuropathologic changes with Cognitive Status: A Review of the Literature,” J. Neuropathol. Exp. Neurol.2012; 72:362-81.

[0005] Physiological and pathological tau have become potential targets for AD therapies. Several therapeutic approaches have been proposed, including inhibition of protein kinases, inhibition of tau aggregation, and active and passive immunotherapies. Numerous anti-tauantibodies and vaccines have been evaluated in preclinical studies, and at least eleven anti-tau antibodies have reached the clinic. See, e.g., Guo et al., “Tau-targeting therapy in Alzheimer’s disease: critical advances and future opportunities”, Ageing Neur Dis 2022;2:11; Panza et al., “The Challenges of anti-tau therapeutics in Alzheimer Disease”, Nature Reviews Neurology 18, 577-78 (2022); Jadhav et al., “A Walk Through Tau Therapeutic Strategies,” Acta Neuropathica Communications, 22 (2019).

[0006] In Alzheimer’s disease, tau pathology spreads from one area of the brain to another in a stereotypical pattern along a neural network. NFTs appear in the entorhinal cortex and spread to anatomically connected regions across the entire cerebral cortex via the hippocampal areas. See Braak, supra. The progression of cognitive impairment correlates well with the tau pathology, as assessed by hallmarks such as aggregation or hyperphosphorylation. See Braak et al., “Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunohistochemistry,” Acta Neuropathol.2006; 112:389-404; Braak et al., “Neuropathological staging of Alzheimer-related changes,” Acta Neuropathol.1991;82:239-59. It is hypothesized that disease progression in AD may be caused by the interneuronal transfer of pathological tau protein, a phenomenon known as tau propagation.

[0007] Various in vivo assays have been reported as being directed to tau propagation. See, e.g., Vogels et al, “Propagation of tau pathology: integrating insights from postmortem and in vivo studies”, Biological Psychiatry, 87(9), 808-818 (May 2020). Such assays involve injection of lysate into a particular brain region and observing aggregated tau in different brain regions. However, such assays do not distinguish tau propagation from tau spreading, wherein tau may be picked up by the synapse at the site of injection, and carried to the cell body of the neuron by retrograde transport. The term “propagation” is sometimes used in literature to describe distant tau seeding-induced pathology, but the terms are not synonymous.

[0008] There remains a need in the art for in vivo assays capable of specifically evaluating tau propagation to aid in the development of novel therapies for treating Alzheimer’s disease. SUMMARY

[0009] The present disclosure provides an assay for evaluating the effect of an anti-tau compound on tau propagation. In embodiments, the assay involves injecting a lysate derived from brain tissue of patients having Alzheimer’s disease into the olfactory bulb of an hTaumouse. In embodiments, the assay further comprises administering an anti-tau compound to the mouse after injection of the lysate. In embodiments, aggregated tau is quantified from at least one region selected from the group consisting of the piriform cortex, the medial thalamus, the hippocampus, and the entorhinal cortex. Then, the amount of aggregated tau achieved after administration of the anti-tau compound is compared with the amount of aggregated tau achieved after administration of a reference compound.

[0010] In embodiments, the anti-tau antibody may be injected one day, two days, three days, four days, five days, six days, or seven days after injection of the brain lysate. In embodiments, the anti-tau antibody is injected one day after injection of the lysate. In embodiments, the anti-tau antibody is injected about three days after injection of the lysate. In embodiments, the anti-tau antibody is injected about seven days after injection of the lysate.

[0011] In embodiments, the anti-tau compound is an anti-tau antibody. In embodiments, the anti-tau antibody is administered at a dose of from about 1 to about 100 mg / kg, such as from about 1 to about 60 mg / kg, or from about 1 to about 30 mg / kg.

[0012] In embodiments, the anti-tau compound (e.g., anti-tau antibody) is administered at a once-weekly or twice-weekly dose. In embodiments, the weekly dose begins one day after injection of the lysate, or three days after injection of the lysate, or seven days after injection of the lysate. In embodiments, the anti-tau compound is administered as a once-weekly or twice- weekly dose for at least about 4 weeks, or at least about 5 weeks, or at least about 6 weeks, or at least about 7 weeks, or at least about 8 weeks.

[0013] In embodiments, the first dose of the anti-tau antibody is administered at a dose of from about 1 to about 100 mg / kg, such as from about 1 to about 50 mg / kg, or from about 1 to about 30 mg / kg, and subsequent doses are administered at the same dose as the first dose. In embodiments, a first dose of the anti-tau antibody is administered at a bolus dose of from about 40 to about 100 mg / kg, and subsequent doses are administered at a dose of from about 1 to about 40 mg / kg.

[0014] In embodiments, aggregated tau is quantified in the mouse brain at least about one day, or at least about two days, or at least about three days, or at least about four days, or at least about five days, or at least about six days, or at least about seven days, after the last dose of the anti-tau compound is administered. In embodiments, the mouse brain is collected at least aboutone day, or at least about two days, or at least about three days, or at least about four days, or at least about five days, or at least about six days, or at least about seven days after the last dose of the anti-tau compound. In embodiments, the mouse brain is collected about 7 days after the last dose of the anti-tau compound. In embodiments, the aggregated tau in the mouse brain is quantified by counting cell bodies that are immunoreactive (IR) to the AT100 anti-tau antibody (Thermo Fisher, Waltham, MA USA), i.e., AT100 IR cell bodies, in the olfactory bulb, the piriform cortex, the entorhinal cortex, the hippocampus, and / or the medial thalamus.

[0015] In embodiments, aggregated tau is quantified in the olfactory bulb and at least one first synapse brain region selected from the group consisting of the piriform cortex and the entorhinal cortex. In embodiments, aggregated tau is quantified in the olfactory bulb and at least one second synapse brain region selected from the group consisting of the hippocampus and the thalamus. In embodiments, aggregated tau is quantified in the olfactory bulb, at least one first synaptically connected brain region selected from the group consisting of the piriform cortex and the entorhinal cortex, and at least one second synaptically connected brain region selected from the group consisting of the hippocampus and the thalamus.

[0016] In embodiments, the assay described in the present disclosure involves injection of a lysate derived from the brain tissue of patients having Alzheimer’s disease. In embodiments, the lysate has a human tau protein concentration of from about 750 to about 2500 nM. In embodiments, the lysate has a tau concentration of at least about 750 nM. In embodiments, the lysate has a tau concentration of at least about 800 nM.

[0017] In embodiments, the present disclosure provides a method of identifying a compound for use in treating Alzheimer’s disease, wherein the method comprises evaluating an anti-tau compound for its ability to reduce tau propagation in an in vivo tau propagation assay. In embodiments, the method comprises injecting a lysate derived from brain tissue of patient(s) having Alzheimer’s disease into the olfactory bulb of an hTau mouse, and administering the anti- tau compound to the mouse after injection of the lysate. Aggregated tau is quantified in the olfactory bulb and at least one region selected from the group consisting of the piriform cortex, the medial thalamus, the hippocampus, and the entorhinal cortex. In embodiments, the amount of aggregated tau after administration of the anti-tau compound may then be compared with theamount of aggregated tau achieved with a reference compound. In embodiments, the anti-tau compound is an anti-tau antibody.

[0018] In embodiments, the present disclosure provides an anti-tau compound which reduces tau propagation in a tau propagation assay as described in the present disclosure. For example, in embodiments, the present disclosure provides an anti-tau compound which reduces tau propagation in an in vivo tau propagation assay, the assay comprising: injecting a lysate derived from brain tissue of a patient(s) having Alzheimer’s disease into the olfactory bulb of an hTau mouse; administering an anti-tau compound to the mouse after injection of the lysate; and quantifying aggregated tau in the olfactory bulb and at least one region selected from the group consisting of the piriform cortex, the medial thalamus, the hippocampus, and the entorhinal cortex.

[0019] In embodiments, the anti-tau compound which reduces tau propagation in the tau propagation assay is used to treat Alzheimer’s disease in a human patient. In embodiments, the anti-tau compound which reduces tau propagation in the tau propagation assay is an anti-tau antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 provides a schematic of an in vivo tau propagation model for Alzheimer’s disease. Lysate purified from brain tissue of AD patients is used to seed tau into the olfactory bulb of hTau mice. Evidence of tau propagation is observed by the presence of neurons containing aggregated tau in brain regions that are synaptically connected to the olfactory bulb, including the piriform cortex and entorhinal cortex, which are first synapse brain regions. The piriform cortex sends projections into the medial thalamus (second synapse region), and the entorhinal cortex neurons project to the CA1 of the hippocampal formation (second synapse region).

[0021] Figure 2A and Figure 2B illustrate that seeding purified human tau derived from the brain tissue of AD patients in the olfactory bulb of female hTau mice yields a tau concentration- dependent increase in tau aggregations in multiple brain structures. In particular, Figure 2A shows tau aggregates in the olfactory bulb, piriform cortex, medial thalamus, and CA1 hippocampus two months after injection of AD brain lysates containing decreasing amounts of tau into the olfactory bulb of hTau mice. Figure 2B illustrates that the concentration of tau in theinjected lysate correlates with the mean number of neurons containing tau aggregates in the various brain regions, indicating that tau concentration in the donor samples facilitates subsequent tau seeding and propagation in hTau mice.

[0022] Figure 3 illustrates aggregated tau expression as measured by AT100 IR across pathways following seeding to the olfactory bulb in the assay according to the present disclosure. When purified human tau derived from brain tissue of AD patients was seeded into the olfactory bulb of female hTau mice, synaptically connected pathway propagation was observed; neurons containing AT100 IR were observed in a time and region specific pattern across synaptically connected pathways in the brain.

[0023] Figure 4 illustrates the effects of olfactory deprivation on reducing propagation of human tau in hTau mice. Olfactory deprivation induced by insertion of a nose plug results in anatomical and morphological changes to the olfactory bulb, including reduction in olfactory bulb size, modulation of olfactory bulb neurochemical expression, and effects on sensory and synaptic physiology. See, e.g., Cummings et al., “Olfactory Bulb Recovery After Early Sensory Deprivation,” J. Neurosci.1997 Oct 1;17(19): 7433-7440. In the present study, olfactory deprivation at day 0 post-tau seeding dramatically reduced seeding as well as propagation to the first synapse brain regions (piriform cortex or entorhinal cortex) and eliminated propagation to the second synapse brain regions (thalamus or hippocampus). Olfactory deprivation at day 7 partially eliminated propagation to the first and second synapse brain regions, and olfactory deprivation at day 14 or day 21 did not affect tau propagation.

[0024] Figure 5 illustrates the impact of treatment interval with antibody PHF-1 post-seeding effect on tau propagation. PHF-1 is an anti-tau antibody previously shown to reduce tau pathology in P301L and P301S mice following passive immunization. See Chai et al., Passive immunization with anti-tau antibodies in two transgenic models, J Bio Chem 2012;286:34457- 34467. PHF-1 has been described in PCT Application No. WO 9620218, the disclosure of which is incorporated by reference herein in its entirety. At the first synapse, the treatments within one day of seeding were effective at reducing tau aggregation in the piriform cortex and entorhinal cortex. PHF-1 treatment significantly reduced aggregated tau in second synapse regions, the medial thalamus, and the CA1 hippocampus, even when dosing started seven days post-seeding.

[0025] Figure 6 illustrates the post-seeding effect of 1 mg, 3 mg, 10 mg, and 30 mg / kg / week PHF-1 antibody on tau propagation in the assay according to the present disclosure. Weekly treatment with PHF-1 at 10 and 30 mg / kg significantly reduced tau aggregation in both the first (piriform cortex and entorhinal cortex) and second (thalamus and hippocampus) synapses. DETAILED DESCRIPTION

[0026] In embodiments, the present disclosure provides assays for assessing tau propagation, in particular in vivo assays for assessing tau propagation.

[0027] In embodiments, the assay comprises injecting a lysate derived from brain tissue of patients having Alzheimer’s disease into the olfactory bulb of a mouse. In embodiments, the mouse is an hTau mouse. In embodiments, the assay comprises evaluating tau propagation over time in the mouse brain, in particular by quantifying tau aggregates in synaptically connected regions of the brain. For example, in embodiments, tau aggregates may be quantified over time in at least one first synapse brain region (e.g., piriform cortex or entorhinal cortex). In embodiments, tau aggregates may be evaluated in at least one second synapse brain region (e.g., thalamus or hippocampus). In embodiments, tau aggregates may be evaluated over time at the site of tau seeding, in the olfactory bulb. In embodiments, tau aggregates may be evaluated in both first synapse (e.g., piriform cortex and entorhinal cortex) and second synapse (e.g., thalamus and hippocampus) brain regions. Accordingly, in embodiments, the assay of the present disclosure may be used to evaluate tau progression along synaptically connected pathways over time.

[0028] In embodiments, propagation to the first synapse (e.g., piriform cortex or entorhinal cortex) may be observed after about 4 weeks post-injection of the AD lysate. In embodiments, propagation to the second synapse (e.g., medial thalamus or CA1 hippocampus) may be observed after about 6 weeks post-injection of the AD lysate, or after about 8 weeks post-injection, or after about 12 weeks post-injection. In embodiments, propagation to the second synapse is first observed after about 6 weeks post-injection of the AD lysate, and becomes more robust about 8 to 12 weeks post-injection.

[0029] In embodiments, the assay of the present disclosure is carried out in mice which do not express murine tau. In embodiments, the assay is carried out in mice which do not express aggregated tau, even with aging. In embodiments, the assay is carried out in mice which do notexpress mutated human tau. In embodiments, the assay is carried out in mice which express both the 3R and 4R tau isoforms. In embodiments, the assay is carried out in mice which express all six isoforms of the human MAPT gene (including both 3R and 4R forms). For example, in embodiments, the assay is conducted in hTau mice. hTau mice express the human tau isoforms, but no endogenous mouse tau is detected. See, e.g., Andorfer et al., “Hyperphosphorylation and aggregation of tau in mice expressing normal human tau isoforms,” J. Neurochemistry, 2003, 86, 582-590; Phillips et al., “Olfactory and Visuospatial Learning and Memory Performance in Two Strains of Alzheimer’s Disease Model Mice—A Longitudinal Study,” PLoS One, 2001; 6(5): e195657, the disclosures of each of which are incorporated by reference herein in their entireties.

[0030] In embodiments, the assay of the present disclosure can be used to evaluate tau progressing along synaptically connected pathways in hTau mice. In embodiments, the assay comprises injecting a lysate derived from brain tissue of a patient or patients having Alzheimer’s disease into the olfactory bulb of an hTau mouse, and observing aggregated tau in neurons outside the olfactory bulb after injection of the AD lysate. For example, in embodiments, propagation to the first synapse brain region (e.g., piriform cortex, entorhinal cortex) may be observed after about 4 weeks. In embodiments, propagation to second synapse brain regions (e.g., CA1 hippocampus and medial thalamus) may be observed after about 6 weeks, and is more robust by about 8 weeks and about 12 weeks post-injection of the AD brain lysate.

[0031] In embodiments, the present disclosure provides a method for evaluating the effect of an anti-tau compound on tau propagation, the method comprising injecting a lysate derived from brain tissue of patients having Alzheimer’s disease into the olfactory bulb of an hTau mouse. In embodiments, the method further comprises administering an anti-tau compound to the mouse after injection of the lysate. In embodiments, the anti-tau compound is an anti-tau antibody. In embodiments, aggregated tau is quantified in the olfactory bulb. In embodiments, aggregated tau is quantified in synaptically connected brain regions. For example, in embodiments, aggregated tau may be quantified in at least one region selected from the group consisting of the piriform cortex, the medial thalamus, the hippocampus, and the entorhinal cortex. In embodiments, aggregated tau may be quantified in at least one first synapse brain region selected from the group consisting of the piriform cortex and the entorhinal cortex. In embodiments, aggregated tau may be quantified at a second synapse brain region selected from the group consisting of the medial thalamus and the CA1 hippocampus. In embodiments, the method may comprisecomparing the amount of aggregated tau after administration of the anti-tau compound with the amount of aggregated tau achieved with a reference compound. In embodiments, the reference compound is an IgG antibody which does not bind tau.

[0032] In embodiments, the anti-tau compound may be administered at least about 1 day post lysate injection. In embodiments, the anti-tau compound is administered at least about 2 days post lysate injection. In embodiments, the anti-tau compound may be administered at least about 3 days post lysate injection. In embodiments, the anti-tau compound may be administered at least about 4 days post lysate injection. In embodiments, the anti-tau compound may be administered at least about 5 days post lysate injection. In embodiments, the anti-tau compound may be administered at least about 6 days post lysate injection. In embodiments, the anti-tau compound may be administered at least about 7 days post lysate injection.

[0033] In embodiments, the anti-tau compound is an anti-tau antibody. In embodiments, the anti-tau antibody may be administered at a dose of from about 1 to about 100 mg / kg, such as from about 20 to about 90 mg / kg, or about 30 to about 80 mg / kg, or about 40 to about 60 mg / kg, or from about 1 to about 60 mg / kg, or from about 1 to about 50 mg / kg, or from about 1 to about 40 mg / kg, or from about 1 to about 30 mg / kg. In embodiments, the anti-tau antibody may be administered at about 1 mg / kg, or about 5 mg / kg, or about 10 mg / kg, or about 20 mg / kg, or about 30 mg / kg, or about 40 mg / kg, or about 50 mg / kg, or about 60 mg / kg, or about 70 mg / kg, or about 80 mg / kg, or about 90 mg / kg, or about 100 mg / kg. In embodiments, the anti-tau antibody may be administered about 40 mg / kg. In embodiments, the anti-tau antibody may be administered at 80 mg / kg. In embodiments, the anti-tau antibody is administered as a once weekly dose. In embodiments, the anti-tau antibody may be administered as a twice weekly dose. In embodiments, the anti-tau antibody may be administered at a weekly dose of from about 1 to about 100 mg / kg, such as from about 20 to about 90 mg / kg, or about 30 to about 80 mg / kg, or about 40 to about 60 mg / kg, or from about 1 to about 60 mg / kg, or from about 1 to about 50 mg / kg, or from about 1 to about 40 mg / kg, or from about 1 to about 30 mg / kg. In embodiments, the anti-tau antibody may be administered a twice-weekly dose of from about 20 to about 100 mg / kg, such as from about 30 to about 90 mg / kg, or about 40 to about 80 mg / kg. In embodiments, the anti-tau antibody may be administered at 40 mg / kg / week. In embodiments, the anti-tau antibody may be administered at 80 mg / kg / week. In embodiments, the anti-tau antibody may be administered at a twice weekly dose of about 40 mg / kg.

[0034] In embodiments, subsequent doses of the anti-tau antibody may be administered at the same dose as the first dose. In embodiments, the anti-tau compound may be injected as a bolus dose at least one week after injection of the lysate, and subsequent doses of the anti-tau antibody are administered at a lower dose than the first dose. For example, in embodiments, the anti-tau compound may be an antibody administered at a dose of about 1 to 100 mg / kg. In embodiments, the initial dose may be followed by additional doses of the anti-tau compound on a weekly or biweekly basis. In embodiments, the weekly doses may be the same dose as the initial dose. In other embodiments, the initial dose may be higher than the subsequent doses; for example, in embodiments, a first dose of the anti-tau antibody is administered at a bolus dose of from about 40 to about 100 mg / kg, and subsequent doses are administered at a dose of from about 1 to about 40 mg / kg.

[0035] In embodiments, aggregated tau is quantified in the mouse brain at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, after the last dose of the anti-tau compound is administered. In embodiments, the mouse brain is collected about 7 days after the last dose of the anti-tau compound. For example, in embodiments, a mouse may be injected with a first dose of an anti-tau compound at about 7 days post-injection of the AD brain lysate, followed by weekly doses of the anti-tau compound at weeks 2-8, and the mouse brain is collected about 7 days following the dose at week 8.

[0036] In embodiments, aggregated tau is quantified in the olfactory bulb. In embodiments, aggregated tau is quantified in at least one synaptically connected region selected from the group consisting of the piriform cortex, the entorhinal cortex, the medial thalamus, and the hippocampus. For example, in embodiments, aggregated tau is quantified in at least one first synaptically connected region selected from the group consisting of the piriform cortex and the entorhinal cortex. In embodiments, aggregated tau is quantified in at least one second synaptically connected region selected from the group consisting of the hippocampus and the thalamus. In embodiments, aggregated tau is quantified in the olfactory bulb, at least one first synaptically connected region selected from the group consisting of the piriform cortex and the entorhinal cortex, and at least one second synaptically connected region selected from the group consisting of the hippocampus and the thalamus. In embodiments, aggregated tau is quantified in the piriform cortex. In embodiments, aggregated tau is quantified in the thalamus. Inembodiments, aggregated tau is quantified in the entorhinal cortex. In embodiments, aggregated tau is quantified in the hippocampus. In embodiments, aggregated tau is quantified in the olfactory bulb, the piriform cortex, and the thalamus. In embodiments, aggregated tau is quantified in the olfactory bulb, the entorhinal cortex, and the hippocampus.

[0037] In embodiments, aggregated tau is quantified by AT100 immunoreactivity quantification. AT100 is an anti-tau antibody which recognizes tau protein phosphorylated at Thr212 and Ser214. In embodiments, aggregated tau is quantified by counting cell bodies that are immunoreactive to the AT100 anti-tau antibody (i.e., AT100 IR cell bodies).

[0038] In embodiments, the methods of the present disclosure involve injection of an AD brain lysate. In embodiments, the lysate of the present disclosure is a sarkosyl-insoluble AD lysate. Sarkosyl extraction of tau has been described, for example, in Guo et al., Seeding of Normal Tau by Pathological Tau Conformers Drives Pathogenesis of Alzheimer-like Tangles, Journal Biol. Chem. Vol.286, Issue 17, 15317-15331 (2011), and Guo et al., Unique Pathological Tau Conformers from Alzheimer’s brains transmit tau pathology in nontransgenic mice, J. Exp. Med. 2016 Nov 14;213(12):2635-2654, the disclosures of which are incorporated herein by reference in their entireties.

[0039] Tau propagation in the assay of the present disclosure correlates with the tau concentration in the AD lysate; that is, tau concentration in donor samples used to prepare the lysate facilitates subsequent tau aggregation in hTau mice. In embodiments, the lysate of the present disclosure has a concentration of at least about 250 nM, or at least about 350 nM, or at least about 450 nM, or at least about 550 nM, or at least about 650 nM. In embodiments, the lysate of the present disclosure has a tau concentration of at least about 750 nM, such as at least about 760 nM, or at least about 770 nM, or at least about 780 nM, or at least about 790 nM, or at least about 800 nM, or at least about 850 nM, or at least about 900 nM, or at least about 950 nM, or at least about 1000 nM, or at least about 1100 nM, or at least about 1200 nM, or at least about 1300 nM, or at least about 1400 nM, or at least about 1500 nM, or at least about 1600 nM, or at least about 1700 nM, or at least about 1800 nM, or at least about 1900 nM, or at least about 2000 nM. In embodiments, the lysate has a tau concentration of from about 750 to about 2500 nM, such as from about 780 to about 2000 nM, or about 800 to about 2000 nM. In embodiments, thelysate has a tau concentration of at least about 800 nM. In embodiments, the tau in the AD lysate is primarily pathological tau protein, i.e., nonsoluble tau.

[0040] In embodiments, the present disclosure further provides an anti-tau compound, wherein the compound is effective to reduce tau propagation in a tau propagation assay as described in the present disclosure. In embodiments, the anti-tau compound is an anti-tau antibody. In embodiments, the anti-tau compound is used to treat Alzheimer’s disease in a human patient. In embodiments, when evaluated in the tau propagation assay as described in the present disclosure, the anti-tau compound reduces tau aggregation observed in at least one first synaptically connected brain region (e.g., piriform cortex, entorhinal cortex). In embodiments, when evaluated in the tau propagation assay of the present disclosure, the anti-tau compound reduces tau aggregates observed in at least one second synaptically connected brain region (e.g., hippocampus, thalamus). In embodiments, when evaluated in the tau propagation assay as described in the present disclosure, the anti-tau compound reduces tau aggregates observed in at least one first synaptically connected brain region selected from the group consisting of the piriform cortex and the entorhinal cortex, and at least one second synaptically connected brain region selected from the group consisting of the hippocampus and the thalamus. In embodiments, reduction in tau aggregates observed with the anti-tau compound is compared to a control compound, such as an IgG antibody which does not bind tau.

[0041] In embodiments, the anti-tau compound of the present disclosure reduces tau aggregates in at least one first synaptically connected brain region (e.g., entorhinal cortex, piriform cortex) by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, as compared to IgG control. In embodiments, the anti-tau compound of the present disclosure reduces tau aggregates in at least one second synaptically connected brain region (e.g., hippocampus, thalamus) by at least about 10%, or at least about 20%, or at least about 30%, or at least about 35%, or at least about 40%, as compared to IgG control. In embodiments, tau aggregates are quantified by measuring AT100 cell bodies.

[0042] It is noted that, as used in this specification and the intended claims, the singular form “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes a single compound as well as one or more of the same or different compounds; reference to “a pharmaceutically acceptable carrier” means asingle pharmaceutically acceptable carrier as well as one or more pharmaceutically acceptable carriers, and the like. Abbreviations EGTA Ethyleneglycol- bis(β-aminoethyl)-N,N,Nʹ,Nʹ-tetraacetic acid FRET Fluorescence resonance energy transfer PBS Phosphate-buffered saline PBST Phosphate-buffered saline containing Tween® 20 TBS TRIS-buffered saline TRIS 2-Amino-2-(hydroxymethyl)-1,3-propanediol EXAMPLES

[0043] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims. Materials and Methods Preparation of human sarkosyl insoluble tau AD lysate from donor samples

[0044] Donor brain tissue was weighed and homogenized three times in TBS lysate buffer (50 mM TRIS, 150 mM NaCl, 20 mM NaF, 1 mM NaVO4, 0.5 mM MgSO4 (pH 7.4) containing protease and phosphatase inhibitors using a Precellys Evolution homogenizer (Bertin Technologies, Bretonneux, FR). Following homogenization, lysates were centrifuged at 27,000 x g for 20 minutes at 4 ℃. The supernatant was saved and the resulting pellet was resuspended in 2X salt / sucrose buffer (1.6 M NaCl, 20% sucrose, 20 mM TRIS HCl, 2 mM EGTA) and sonicated on ice. The sonicated samples were then centrifuged at 27,000 x g for 20 minutes at 4 ℃. The two supernatants were combined and sarkosyl detergent was added at 1.5% (w / v). To the supernatants, an equal volume of 2X salt / sucrose buffer was added. The supernatants were then shaken at ambient conditions for 1.5 hours. Following shaking, the samples were centrifuged at250,000 x g for 90 minutes at 4 ℃. The resulting pellet was resuspended in PBS, sonicated on ice and centrifuged at 100,000 x g for 60 minutes at 4 ℃. The resulting pellet was resuspended in PBS, sonicated and then centrifuged at 10,000 x g for 30 minutes at 4 ℃. The resulting supernatant was the sarkosyl insoluble tau AD lysate used to seed tau.

[0045] Aggregated tau species were quantified using a homogenous time-resolved fluorescence (HTRF)-based tau aggregation assay kit (PerkinElmer - Cisbio, Waltham, MA). The manufacturer’s protocol was followed to ensure the appropriate dilution for the donor and receptor fluorophore-tagged antibodies. Assay reactions (20 μL) were carried out in 384 microplate (Greiner Bio-One, Kremsmünster, AT), where samples were incubated with the fluorophore-tagged antibodies for 24 hours at room temperature. The fluorescent signal was detected using Cytation 5 (Agilent, Santa Clara, CA) by excitation at 340 nm and emission at 620 nm and 665 nm. The relative FRET rate for each sample was determined by calculating the ratio of the two fluorescence intensities (665 / 620). Data were plotted in GraphPad Prism (Dotmatics, Boston, MA) and a One-way ANOVA was used to compare statistical significance between human AD and control brain lysates.

[0046] Total tau levels were measured using the V-PLEX Human total tau kit (Meso Scale Discovery, Rockville, MD) in a sandwich immunoassay. Precoated plates were blocked using 150μL of Diluent 35 to each well, sealed with an adhesive plate sealer and incubated for 1 hour at ambient conditions on a shaker. A stock of loading dye was prepared using 9 parts of 4x laemmli buffer (Bio-Rad, Hercules, CA) and 1 part β-mercaptoethanol (Bio-Rad, Hercules, CA). For sample preparation, we prepared a 1:5 dilution of the sample using the loading dye and PBS, following which samples were vortexed and spun down using a bench-top microcentrifuge. Samples were then heated at 95 °C for 5 minutes on a heating block and then placed on ice. For total tau measurement, samples were diluted to 1:3000 in diluent 35 and run in duplicates. The tau calibrator (Meso Scale Discovery) was diluted to 28,800 pg / mL as the highest concentration. A three-fold dilution series of the calibrator was performed until 39.5 pg / mL and a blank was also included for background signal deduction. After 1 hour of blocking, the plates were washed three times with 150 μL of 0.05% PBST per well. The calibrator and unknowns were added at 50 μL per replicate and the plates were sealed with an adhesive plate sealer and incubated for 1 hour on a shaker at ambient conditions. After 1 hour, the plates were washed three times with 150 μL of 0.05% PBST per well. The detection antibody SULFO-Tag was diluted from 50x to1x (in Diluent 35) and 25 μL of 1x antibody was added to each well. The plates were sealed with an adhesive plate sealer and incubated for 1 hour at ambient conditions on a shaker. After one hour, the plates were washed again with 150 μL of 0.05% PBST per well. The read buffer was diluted from 4x to 1x and 150μL of diluted buffer was added to each well. The plates were incubated for 7 minutes with the read buffer and the plates were read on the Meso Sector S600 (Meso Scale Discovery). Animals and experimental design

[0047] Female hTau mice (B6.Cg-Mapt Tg(MAPT)8cPdav / J, stock #005491) that were homozygous for the targeted allele and hemizygous for the transgene were used for these studies. The mice were bred and maintained at Jackson laboratories (Bar Harbor, ME, USA). The mice were provided at about 3 months of age and were housed five / cage with 12:12 hour dark / light cycle with ad libitum access to food and water.

[0048] At 4.5-5 months of age, mice were prepped for aseptic stereotaxic surgery and 2 μL of sarkosyl-insoluble AD lysate was injected into the olfactory bulb using a Hamilton syringe. After recovery from surgery, mice were returned to their home cages.

[0049] All experiments were performed in full compliance with the Principles of Laboratory Animal Care (NIH publication No.86-23) and AbbVie’s Institutional Animal Care and Use Committee (IACUC). Animal studies were conducted in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) accredited facility where veterinary care and oversight was provided to ensure appropriate animal care. Tissue processing and immunohistochemistry

[0050] At the appropriate time point, mice were euthanized with sodium pentobarbital, perfused with phosphate buffered saline (PBS), and the brains were quickly removed. The entire brain was drop-fixed in 10% formalin for 48 hours before being switched to 70% ethanol:water. Brains were stored in 70% ethanol until all brains from the study were collected to allow all samples to be processed for paraffin embedding at the same time (Leica, Buffalo Grove, IL, USA).

[0051] After processing, three brains from different treatment groups or time points were embedded together into paraffin blocks (Sakura Finetek, Torrance, CA, USA) in a horizontalplane. Every 5th, 5 µm paraffin sections was collected through the entire brain and mounted onto glass slides. Twenty-five sections per block were stained for AT100 immunoreactivity using the BOND RX stainer with the Refine Detection kit (Leica). Briefly, the slides were deparaffinized, rehydrated, and placed on the BOND RX where they underwent a series of pretreatments including peroxide block, 5% donkey serum blocking (Leica) and were then incubated overnight in the AT100 mouse monoclonal antibody to tau (Thermo Fisher, Waltham, MA, USA) at a concentration of 0.006 µg / mL.

[0052] Slides were then returned to the BOND RX where they underwent a series of washes before being incubated in Biotin-SP-conjugated F(ab')2 donkey anti mouse IgG(H+L) secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA, USA) at a concentration of 2 µg / mL for 20 minutes. After additional washing steps, the slides were incubated for 20 minutes in Streptavidin / Horseradish Peroxidase (Leica) and the immunoreactivity was visualized using diaminobenzidine (DAB; Leica) and counterstained with hematoxylin (Leica). AT100 immunoreactivity quantification

[0053] AT100 IR cells were counted in matched sections through the entire extent of the olfactory bulb (8 – 10 slides / brain), piriform cortex (6 – 8 sections / brain), entorhinal cortex (8 – 10 sections / brain), CA1 hippocampus (10-12 sections / brain) and medial thalamus (8 – 10 sections / brain) by an observer blind to the treatment or time point. Cell counts for each region were totaled to give one number / brain region / mouse. Example 1. Propagation model of tau protein following seeding in the olfactory bulb

[0054] Using lysate purified from brain tissue of AD patients, a model of tau propagation was developed (Figure 1) in hTau mice where lysate was used to seed tau into the olfactory bulb. Evidence of tau propagation would be subsequently determined by presence of neurons containing aggregated tau as measured by AT100 IR in brain regions that are synaptically connected to the olfactory bulb. Brain regions analyzed included the piriform cortex and entorhinal cortex which are first synapse brain regions. The piriform cortex sends projections to the medial thalamus (second synapse region) and the entorhinal cortex neurons project to the CA1 of the hippocampal formation (second synapse region).Example 2. The effect of h tau protein dose on evidence of tau aggregation (AT100 IR) following seeding of the olfactory bulb

[0055] When purified human tau derived from brain tissue from various AD patients was injected into the olfactory bulb of female hTau mice, the level of tau aggregation was correlated with the concentration of tau present in the lysate (Figure 2A and 2B). In this study, individual preparations of AD donor brain tissue were prepared. In addition, lysates from multiple donors were mixed and amount of insoluble tau present in the samples determined.

[0056] The lysates were injected into the olfactory bulb of hTau mice. Two months after tau seeding, neuron containing aggregated tau visualized by AT100 IR were quantified in the olfactory bulb, piriform cortex, entorhinal cortex, medial thalamus, and CA1 hippocampus (Figure 2A). Analysis showed that the concentration of tau in the AD lysate was predictive of the level of tau pathology with coefficients of variation between 0.67 and 0.814 (Figure 2B). The tau aggregates from the mixture samples correlated with the results from individual samples, confirming that the level of tau seeding and propagation in the model system was dependent on the concentration of insoluble tau present in the lysate injected into the olfactory bulb. This finding led to standardization of mixed AD lysate used for subsequent studies to be approximately 800 nM human tau protein. Example 3. Timing of synaptically connected pathway transmission of tau aggregates following seeding of the olfactory bulb

[0057] When purified human tau derived from brain tissue of AD patients was seeded onto the olfactory bulb of female hTau mice, synaptically connected pathway propagation was observed (Figure 3). Within four weeks post seeding, tau aggregates were observed in mice outside of the olfactory bulb with propagation to the first synapse (piriform cortex). Propagation of tau aggregates to the second synapse (thalamus) was first observed at six weeks post seeding and was more robust by 8 and 12 weeks post-seeding. Following the observation of tau aggregates in the olfactory bulb, tau aggregation was observed after six weeks in the entorhinal cortex and at eight weeks after seeding in the CA1 hippocampus. Example 4. Effect of olfactory deprivation on propagation of tau aggregates following seeding of the olfactory bulb

[0058] hTau mice were injected with purified AD brain lysate into the olfactory bulb and nose plugs were inserted either on the day of surgery (D0), or at 7, 14, or 21 days after surgery. Four months post tau seeding, aggregated tau was assessed by AT100 IR antibody in brain regions associated with human tau propagation.

[0059] Within the olfactory bulb, olfactory deprivation by nostril plugging reduced tau aggregation particularly if deprivation occurred at day 0 and partially if deprivation occurred within the first two weeks after seeding (Figure 4). Olfactory deprivation at day 0 dramatically reduced propagation to the first synapse brain regions (piriform cortex or entorhinal cortex) and eliminated propagation to the medial thalamus or hippocampus, the sites of the second synapse. Olfactory deprivation at day 7 partially eliminated propagation to the first and second synapses, while olfactory deprivation at 14 or 21 days post-tau seeding did not affect tau propagation as compared to the sham control (Figure 4). Example 5. Efficacy of anti-tau antibody treatment timing on tau propagation

[0060] To assess whether an anti-tau antibody could prevent tau propagation, the antibody PHF- 1 was used for a dosing study. Four and a half-month-old female hTau mice were injected with purified AD brain lysate into the olfactory bulb and intraperitoneal (IP) dosing with antibody started either 1, 3 or 7 days later (Figure 5). In the 80 mg / kg / week doses, the injections were made twice a week (40 mg / kg, ip), while the 40 mg / kg / week were administered once weekly.

[0061] Two months after tau seeding, neurons containing tau aggregates, as measured by AT100 IR, were counted in the olfactory bulb as well as first and second synapse brain regions. In the olfactory bulb, PHF-1 treatment did not affect tau pathology regardless of when dosing started post-seeding. At the first synapse, dosing with PHF1 significantly reduced numbers of AT100 IR neurons when dosing started 1 day post-seeding, but not 3 or 7 days. However, PHF-1 treatment significantly reduced aggregated tau in second synapse brain regions, the medial thalamus and the CA1 hippocampus, even when dosing started seven days post-seeding. These data suggest that anti-tau antibody treatment can alter propagation without affecting tau seeding. Example 6. Efficacy of PHF-1 antibody treatment dose on tau propagation

[0062] A study was performed to determine if the PHF-1 tau antibody could dose-dependently affect tau propagation and subsequent aggregation. Four and a half-month-old female hTau micewere injected with purified AD brain lysate into the olfactory bulb, and intraperitoneal dosing with PHF-1 or control antibody started 7 days post seeding at doses ranging from 1 to 30 mg / kg / week, with IgG serving as a control. The mice were sacrificed 2 months post seeding, and analysis of AT100 IR tau aggregates showed that weekly treatment with PHF-1 at 10 and 30 mg / kg significantly reduced tau aggregation in both the first (piriform cortex and entorhinal cortex) and second (thalamus and hippocampus) synapses (Figure 6). This result suggests not a dose-dependent response, but a threshold response to PHF-1 treatment, above which significant control of tau aggregation is observed.

Claims

CLAIMS 1. An assay for evaluating the effect of an anti-tau compound on tau propagation, the assay comprising: injecting a lysate derived from brain tissue of patients having Alzheimer’s disease into the olfactory bulb of an hTau mouse; administering an anti-tau compound to the mouse after the injection of the lysate; quantifying aggregated tau in the olfactory bulb and at least one region selected from the group consisting of the piriform cortex, the medial thalamus, the hippocampus, and the entorhinal cortex; and comparing the amount of aggregated tau after administration of the anti-tau compound with the amount of aggregated tau achieved with a reference compound.

2. The assay according to claim 1, wherein the anti-tau compound is injected at least about one day after the injection of the lysate.

3. The assay according to claim 1, wherein the anti-tau compound is an anti-tau antibody administered at a dose of about 1 to about 100 mg / kg.

4. The assay according to claim 1, wherein the anti-tau compound is administered as a once- weekly or twice-weekly dose for at least about 4 weeks.

5. The assay according to claim 4, wherein the anti-tau compound is administered for at least about 6 weeks.

6. The assay according to claim 4, wherein the anti-tau compound is administered for at least about 8 weeks.

7. The assay according to claim 1, wherein the aggregated tau is quantified about 7 days after the last dose of the anti-tau compound.

8. The assay according to claim 1, wherein aggregated tau is quantified by counting AT100 IR cell bodies in at least one region selected from the group consisting of the piriform cortex, entorhinal cortex, hippocampus, and medial thalamus.

9. The assay according to claim 1, wherein aggregated tau is quantified in at least one first synaptically connected region selected from the group consisting of the piriform cortex and the entorhinal cortex.

10. The assay according to claim 1 or 9, wherein aggregated tau is quantified in at least one second synaptically connected brain region selected from the group consisting of the hippocampus and the thalamus.

11. The assay according to claim 1, wherein aggregated tau is quantified in the olfactory bulb, at least one first synaptically connected brain region selected from the group consisting of the piriform cortex and the entorhinal cortex, and at least one second synaptically connected brain region selected from the group consisting of the hippocampus and the thalamus.

12. The assay according to claim 1, wherein the lysate has a tau protein concentration of at least about 750 nM.

13. The assay according to claim 1, wherein the lysate has a tau protein concentration of at least about 800 nM.

14. The assay according to claim 1, wherein the lysate is a Sarkosyl-insoluble AD brain lysate.

15. A method of identifying a compound for use in treating Alzheimer’s disease, the method comprising evaluating an anti-tau compound for its ability to reduce tau propagation in an in vivo tau propagation assay comprising:injecting a lysate derived from brain tissue of patients having Alzheimer’s disease into the olfactory bulb of an hTau mouse; administering an anti-tau compound to the mouse after the injection of the lysate; quantifying aggregated tau in the olfactory bulb and at least one region selected from the group consisting of the piriform cortex, the medial thalamus, the hippocampus, and the entorhinal cortex; and comparing the amount of aggregated tau after administration of the anti-tau compound with the amount of aggregated tau achieved with a reference compound.

16. The method according to claim 15, wherein the anti-tau compound is an anti-tau antibody.

17. The method according to claim 15, wherein in the tau propagation assay, the anti-tau compound is injected at least one day after the injection of the lysate.

18. The method according to claim 16, wherein in the tau propagation assay, the anti-tau antibody is administered at a dose of about 1 to about 100 mg / kg.

19. The method according to claim 15, wherein in the tau propagation assay, the anti-tau compound is administered as a once-weekly or twice-weekly dose for at least about 4 weeks.

20. The method according to claim 19, wherein the anti-tau compound is administered for at least about 6 weeks.

21. The method according to claim 19, wherein the anti-tau compound is administered for at least about 8 weeks.

22. The method according to claim 15, wherein in the tau propagation assay, the aggregated tau is quantified at least about 7 days after the last dose of the anti-tau compound.

23. The method according to claim 15, wherein aggregated tau is quantified by counting AT100 IR cell bodies in at least one region selected from the group consisting of the piriform cortex, entorhinal cortex, hippocampus, and medial thalamus.

24. The method according to claim 15, wherein aggregated tau is quantified in the olfactory bulb and at least one first synaptically connected region selected from the group consisting of the piriform cortex and the entorhinal cortex.

25. The method according to claim 15, wherein aggregated tau is quantified in at least one second synaptically connected brain region selected from the group consisting of the hippocampus and the thalamus.

26. The method according to claim 15, wherein aggregated tau is quantified in the olfactory bulb, at least one first synaptically connected brain region selected from the group consisting of the piriform cortex and the entorhinal cortex, and at least one second synaptically connected brain region selected from the group consisting of the hippocampus and the thalamus.

27. The method according to claim 15, wherein the lysate has a tau protein concentration of at least about 750 nM.

28. The method according to claim 15, wherein the lysate has a tau protein concentration of at least about 800 nM.

29. The method according to claim 15, wherein the lysate is a Sarkosyl-insoluble AD brain lysate.

30. An anti-tau compound, wherein the anti-tau compound reduces tau propagation in an in vivo tau propagation assay, the assay comprising: injecting a lysate derived from brain tissue of patients having Alzheimer’s disease into the olfactory bulb of an hTau mouse;administering an anti-tau compound to the mouse after the injection of the lysate; quantifying aggregated tau in the olfactory bulb and at least one region selected from the group consisting of the piriform cortex, the medial thalamus, the hippocampus, and the entorhinal cortex; and comparing the amount of aggregated tau after administration of the anti-tau compound with the amount of aggregated tau achieved with a reference compound.

31. The compound according to claim 1, wherein in the tau propagation assay, the anti-tau compound is injected at least one day after injection of the lysate.

32. The compound according to claim 30, wherein the anti-tau compound is an anti-tau antibody.

33. The compound according to claim 32, wherein in the tau propagation assay, the anti-tau antibody is administered at a dose of from about 1 to about 100 mg / kg.

34. The compound according to claim 30, wherein in tau propagation assay, the anti-tau compound is administered at a once-weekly or twice-weekly dose for at least about 4 weeks.

35. The compound according to claim 34, wherein the anti-tau compound is administered for at least about 6 weeks.

36. The compound according to claim 34, wherein the anti-tau compound is administered for at least about 8 weeks.

37. The compound according to claim 30, wherein in the tau propagation assay, the aggregated tau is quantified about 7 days after the last dose of the anti-tau compound.

38. The compound according to claim 30, wherein in the tau propagation assay, aggregated tau is quantified by counting AT100 IR cell bodies in at least one region selected from the groupconsisting of the piriform cortex, the entorhinal cortex, the hippocampus, and the medial thalamus.

39. The compound according to claim 30, wherein in the tau propagation assay, aggregated tau is quantified in at least one first synaptically connected region selected from the group consisting of the piriform cortex and the entorhinal cortex.

40. The compound according to claims 30 and 39, wherein in the tau propagation assay, aggregated tau is quantified in at least one second synaptically connected brain region selected from the group consisting of the hippocampus and the thalamus.

41. The compound according to claim 30, wherein in the tau propagation assay, aggregated tau is quantified in the olfactory bulb, at least one first synaptically connected brain region selected from the group consisting of the piriform cortex and the entorhinal cortex, and at least one second synaptically connected brain region selected from the group consisting of the hippocampus and the thalamus.

42. The compound according to claim 30, wherein in the tau propagation assay, the lysate has a tau protein concentration of at least about 750 nM.

43. The compound according to claim 30, wherein in the tau propagation assay, the lysate has a tau protein concentration of at least about 800 nm.

44. The compound according to claim 30, wherein in the tau propagation assay, the lysate is a Sarkoskyl-insoluble AD brain lysate.

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