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WO2025198916A1 - Anticorps anti-tau - Google Patents

Anticorps anti-tau

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Publication number
WO2025198916A1
WO2025198916A1 PCT/US2025/019566 US2025019566W WO2025198916A1 WO 2025198916 A1 WO2025198916 A1 WO 2025198916A1 US 2025019566 W US2025019566 W US 2025019566W WO 2025198916 A1 WO2025198916 A1 WO 2025198916A1
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Prior art keywords
tau
seq
amino acid
set forth
cdr
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Inventor
Xavier Jean Michel LANGLOIS
Tammy D. SMITH
Kiran YANAMANDRA
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AbbVie Inc
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AbbVie Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • ABV21639USO1 ANTI-TAU ANTIBODIES REFERENCE TO A SEQUENCE LISTING [0000]
  • This application includes a Sequence Listing submitted electronically as an xml file named ABV21639USO1_ST26.xml, created on February 14, 2025, with a size of 19,848 bytes. The Sequence Listing is incorporated herein by reference.
  • tau 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.
  • the deposition of abnormal tau protein in the brain is associated with a group of neurodegenerative disorders known collectively as “tauopathies,” which includes Alzheimer’s disease. [0002] Alzheimer’s disease represents the most common cause of dementia in the elderly population; in 2020, approximately 50 million people worldwide suffered from dementia, with 60 to 70% of dementia cases attributed to AD. By 2050, the number of dementia cases is expected to increase to more than 150 million worldwide.
  • AD Alzheimer’s disease
  • Alzheimer’s disease tau pathology spreads from one area of the brain to another in a stereotypical pattern along a neural network in a “prion-like” manner. 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 anti-tau antibody zagotenemab was discontinued after its failure in a phase II study in Alzheimer’s disease. See Muller, “Anti-tau antibody failures stack up,” Nature Reviews Drug Discovery 20, 888 (2021). ABV21639USO1 [0006] There remains a need for novel anti-tau antibodies for use in treatment of neurodegenerative diseases like Alzheimer’s Disease. SUMMARY [0007] The present disclosure provides antibodies that specifically bind to human tau, in particular antibodies that selectively bind to hyperphosphorylated tau T231 (pT231).
  • the present disclosure provides a full-length anti-tau antibody comprising a heavy chain variable region comprising a CDR-H1, a CDR-H2, and a CDR-H3, and a light chain variable region comprising a CDR-L1, a CDR-L2, and a CDR-L3, and wherein: vH CDR-H1 has the amino acid sequence set forth as SEQ ID NO: 3; vH CDR-H2 has the amino acid sequence set forth as SEQ ID NO: 4; vH CDR-H3 has the amino acid sequence set forth as SEQ ID NO: 5; vL CDR-L1 has the amino acid sequence set forth as SEQ ID NO: 8; vL CDR-L2 has the amino acid sequence set forth as SEQ ID NO: 9; and vL CDR-L3 has the amino acid sequence set forth as SEQ ID NO: 10.
  • the full-length anti-tau antibody comprises a heavy chain variable region having the amino acid sequence set forth as SEQ ID NO: 2 and a light chain variable region having the amino acid sequence set forth as SEQ ID NO: 7.
  • the present disclosure provides a full-length humanized monoclonal IgG1 anti-tau antibody comprising two heavy chains and two light chains, wherein said heavy chains each comprise the amino acid sequence set forth as SEQ ID NO: 1, and said light chains each comprise the amino acid sequence set forth as SEQ ID NO: 6.
  • the present disclosure provides a full-length humanized monoclonal IgG 1 anti-tau antibody comprising two heavy chains and two light chains, wherein said heavy chains each comprise the amino acid sequence set forth as SEQ ID NO: 16, and said light chains each comprise the amino acid sequence set forth as SEQ ID NO: 6.
  • the present disclosure provides a full-length humanized monoclonal IgG 1 anti-tau antibody comprising two heavy chains and two light chains, wherein said heavy chains each comprise the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 16, and said light chains each comprise the amino acid sequence set forth as SEQ ID NO: 6.
  • compositions comprising an anti-tau antibody of the present disclosure and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of treating Alzheimer’s Disease, the method comprising administering an antibody according to the present disclosure to a patient in need thereof.
  • the present disclosure provides a method of treating Alzheimer’s Disease, the method comprising administering a composition comprising an antibody of the present disclosure and a pharmaceutically acceptable carrier to a patient in need thereof.
  • BRIEF DESCRIPTION OF THE DRAWINGS [0016] Figure 1 illustrates the in vitro specificity of the anti-tau antibody AbA for phosphorylated pT231 peptide.
  • Figure 2 illustrates the in vitro binding of the anti-tau antibody AbA to both phospho T231 tau and tau aggregates derived from the brain tissue of AD patients in a dose-dependent manner.
  • Figure 3 illustrates the in vivo tau seeding model according to the present disclosure.
  • Figure 4 provides a summary of the protocol for the in vivo tau seeding model and illustrates the effect of an anti-tau antibody of the present disclosure on hippocampal seeding in the rTG4510 mouse seeding model.
  • Figure 5A provides a schematic of an in vivo tau propagation model for Tau pathology.
  • Figure 5B shows photomicrographs of aggregated tau (AT100 IR) containing neurons in the first and second synapse brain regions following seeding in the olfactory bulb in the tau propagation assay according to the present disclosure. Following seeding with AD lysate, AT100 IR containing neurons increases in both a time and region dependent manner indicating propagation of pathology along synaptically connected pathways.
  • Figure 6A illustrates tau aggregates (AT100 IR cells) in the medial thalamus, and CA1 hippocampus 2 months after seeding with AD lysates containing various concentrations of tau into the olfactory bulb.
  • Figure 6B illustrates that the concentration of tau in the injected lysate correlates with the mean number of neurons containing tau aggregates, indicating that tau concentration in the donor samples facilitates subsequent tau seeding and propagation in hTau mice.
  • Figure 7A illustrates the study design of an hTau propagation efficacy study with anti-tau antibody chAbA.
  • Figure 7B illustrates that weekly intraperitoneal (i.p.) dosing with chAbA significantly reduced numbers of neurons containing AT100 IR tau aggregates as compared to control IgG treatment in both the medial thalamus and CA1 hippocampus of female hTau mice 2 months after injection of tau lysate into the olfactory bulb.
  • FIG 8 illustrates that weekly intraperitoneal (i.p.) dosing chAbA significantly reduced numbers of neurons containing aggregated tau (AT100 IR) as compared to IgG treated hTau mice as well as mice dosed with N-terminal Pan-Tau antibodies HJ8.5 (mouse version of Tilavonemab), Semorinemab or IPN002.
  • the three Pan-Tau antibodies did not affect numbers of AT100 IR neurons in any brain region analyzed.
  • Figure 9A illustrates that weekly intraperitoneal (i.p.) dosing with chAbA for both 2 and 8 months significantly reduced the percentage of neurons containing aggregated tau as measured by AT100 IR in the CA1 hippocampus and medial thalamus as compared to IgG treated mice.
  • Figure 9B illustrates that weekly intraperitoneal (i.p.) dosing with chAbA resulted in a 54% and 87% reduction in spreading of tau pathology in the hippocampus and thalamus, respectively, between 2 and 8 months post-seeding.
  • DETAILED DESCRIPTION [0029] Described herein are embodiments of anti-tau antibodies, in particular antibodies directed to pathological species of human tau.
  • AbA is a humanized monoclonal IgG1 antibody that binds to human tau.
  • AbA is a humanized monoclonal IgG1 antibody that binds human tau peptide that is phosphorylated at threonine 231 (pT231).
  • an antibody of the present disclosure comprises variable regions and CDRs (complementarity determining regions) identified according to rules developed in the art and/or by aligning sequences against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, N.Y., 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, N.J., 2000.
  • CDRs may be identified in accordance with one of the schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5 th Ed.), U.S. Dept.
  • anti-tau antibody AbA comprises CDRs as recited in Table 1.
  • CDRs (Kabat) of anti-tau antibody AbA CDR Identity SEQ ID NO Sequence Location Sequence Heavy Chain, CDR-H1 SEQ ID NO: 3 Residues 31-35 of DYYMN SEQ ID NO: 1 Heavy Chain, CDR-H2 SEQ ID NO: 4 Residues 50-66 of VFYPHLGYTIYNQKFKG SEQ ID NO: 1 Heavy Chain, CDR-H3 SEQ ID NO: 5 Residues 99-109 of PYYYGSSSLDY SEQ ID NO: 1 Light Chain, CDR-L1 SEQ ID NO: 8 Residues 24-39 of RSSQDLVESDADTYLH SEQ ID NO: 6 Light Chain, CDR-L2 SEQ ID NO: 9 Residues 55-61 of KVSNRFS SEQ ID NO: 6 Light Chain, CDR-L3 SEQ ID NO: Residues 94-102 of SQSTHVPFT 10 SEQ ID NO:
  • the anti-tau antibody of the present disclosure is an IgG isotype. In embodiments, the anti-tau antibody of the present disclosure is an IgG1 isotype or an IgG2 isotype or an IgG3 isotype or an IgG4 isotype. In embodiments, the anti-tau antibody of the present disclosure is an IgG1 isotype. In embodiments, the anti-tau antibody of the present disclosure is an IgG2 isotype. [0035] In embodiments, the anti-tau antibody of the present disclosure is a humanized antibody.
  • an anti-tau antibody of the present disclosure comprises a heavy chain variable region having the amino acid sequence set forth as SEQ ID NO: 2 (vH CDRs underlined, and shown as SEQ ID NOs: 3, 4, and 5 respectively, in order of appearance): EVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMNWVRQAPGQGLEWIGVFYPH LGYTIYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCASPYYYGSSSLDYW GQGTLVTVSS (SEQ ID NO: 2) ABV21639USO1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 7 (vL-CDRs underlined, and shown as SEQ ID NOs: 8, 9, and 10 respectively, in order of appearance): DVQMTQSPSSVSASVGDRVTITCRSSQDLVESDADTYLHWYQQKPGKAPKLLIYKV SNRFSGVPSRF
  • an anti-tau antibody of the present disclosure comprises a heavy chain having the amino acid sequence set forth as SEQ ID NO: 1 (variable region is bold; constant region is italicized; CDRs are underlined and shown as SEQ ID NOs: 3, 4, and 5 respectively, in order of appearance): EVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMNWVRQAPGQGLEWIGVFYPH LGYTIYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCASPYYYGSSSLDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
  • an anti-tau antibody of the present disclosure comprises a light chain having amino acid SEQ ID NO: 6 (variable region is bold; constant region is italicized; CDRs are underlined and shown as SEQ ID NOs: 8, 9, and 10 respectively, in order of appearance): DVQMTQSPSSVSASVGDRVTITCRSSQDLVESDADTYLHWYQQKPGKAPKLLIYKV SNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQSTHVPFTFGQGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 6).
  • an anti-tau antibody of the present disclosure comprises two heavy chains each having the amino acid sequence set forth as SEQ ID NO: 1 and two light chains each having the amino acid sequence set forth as SEQ ID NO: 6. [0041] In embodiments, an anti-tau antibody of the present disclosure comprises two heavy chains each having the amino acid sequence set forth as SEQ ID NO: 16 and two light chains each having the amino acid sequence set forth as SEQ ID NO: 6. [0042] In embodiments, an anti-tau antibody of the present disclosure comprises two heavy chains each having the amino acid sequence set forth as SEQ ID NO:1 or SEQ ID NO: 16 and two light chains each having the amino acid sequence set forth as SEQ ID NO: 6.
  • the present disclosure provides one or more nucleic acids encoding any of the polypeptides as described herein.
  • the present disclosure provides a polynucleotide encoding a polypeptide of a heavy chain as described herein, such as a polynucleotide encoding a polypeptide of a heavy chain as set forth in SEQ ID NO: 1 or SEQ ID NO: 16.
  • the present disclosure provides a polynucleotide encoding a polypeptide of a light chain as described herein, such as a polynucleotide encoding a polypeptide of a light chain as set forth in SEQ ID NO: 6.
  • the present disclosure provides a polynucleotide encoding (1) a polypeptide of a heavy chain as set forth in SEQ ID NO: 1 or SEQ ID NO: 16, and (2) a polypeptide of a light chain as set forth in SEQ ID NO: 6.
  • Compositions [0044]
  • the antibodies of this disclosure may be provided as a composition suitable for administration to a subject.
  • the antibody composition is a ABV21639USO1 pharmaceutical composition, including an antibody of this disclosure and a pharmaceutically acceptable carrier.
  • Methods of Use Provided herein are embodiments of methods for treating Alzheimer’s disease (AD) comprising administering to a subject in need thereof a therapeutically effective amount of an anti-tau antibody disclosed herein.
  • the term “subject”, as used herein, refers to a human.
  • the terms “human,” “patient,” and “subject” are used interchangeably herein.
  • Assays for Assessing Tau Seeding and Propagation [0047] In embodiments, disclosed herein are assays for assessing tau propagation, in particular in vivo assays for assessing tau propagation. [0048] In embodiments, the activity of an antibody of the present disclosure may be evaluated in a tau seeding assay.
  • the assay comprises injecting a lysate derived from brain tissue of patients having Alzheimer’s disease stereotactically into the hippocampus (2.5 ⁇ l, coordinates: AP: -1,9mm; ML: -1,5mm; DV: -1,6) of rTG4510 mice (Santacruz K et al, Tau suppression in a neurodegenerative mouse model improves memory function. Science.2005 Jul 15;309(5733):476-81. doi: 10.1126/science.1113694. PMID: 16020737; PMCID: PMC1574647).
  • the assay comprises evaluating tau seeding in the mouse brain, in particular by quantifying the percent area of the CA1 hippocampus covered by tau aggregates.
  • tau aggregates may be quantified in the CA1 hippocampus.
  • the assay of the present disclosure may be used to evaluate tau seeding in the CA1 hippocampus.
  • the present disclosure provides assays for assessing tau propagation, in particular in vivo assays for assessing tau propagation.
  • the assay comprises injecting a lysate derived from brain tissue of patients having Alzheimer’s disease into the olfactory bulb of a hTau mouse; in embodiments, the mouse is an hTau mouse.
  • 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.
  • tau aggregates ABV21639USO1 may be quantified over time at the site of tau seeding, in the olfactory bulb.
  • tau aggregates may be evaluated in both first and second synapse brain region (e.g., piriform cortex and entorhinal cortex; medial thalamus and CA1 hippocampus). Accordingly, in embodiments, the assay of the present disclosure may be used to evaluate tau progression along synaptically connected pathways over time.
  • propagation to the first synapse may be observed after about 4 weeks post-injection of the AD lysate.
  • propagation to the second synapse e.g., thalamus or hippocampus
  • propagation to the second synapse 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.
  • propagation to the second synapse e.g., medial thalamus or CA1 hippocampus
  • propagation to the second synapse is first observed after about 6 weeks post-injection of the AD lysate, and becomes more robust after about 8 - 12 weeks post- injection.
  • 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 not express 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.
  • the assay of the present disclosure can be used to evaluate tau progressing along synaptically connected pathways in hTau mice.
  • 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 ABV21639USO1 in neurons outside the olfactory bulb after injection of the AD lysate.
  • a lysate derived from brain tissue of a patient or patients having Alzheimer’s disease into the olfactory bulb of an hTau mouse
  • aggregated tau ABV21639USO1 in neurons outside the olfactory bulb after injection of the AD lysate.
  • propagation to the first synapse brain region e.g., piriform cortex, entorhinal cortex
  • propagation to second synapse brain regions 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.
  • 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.
  • the method further comprises administering an anti-tau compound to the mouse after injection of the lysate.
  • the anti-tau compound is an anti- tau antibody.
  • 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.
  • the method may comprise comparing the amount of aggregated tau after administration of the anti-tau compound with the amount of aggregated tau achieved with a reference compound.
  • the reference compound is an IgG antibody which does not bind tau.
  • 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.
  • the anti-tau compound may be administered at least about 6 days post lysate injection. In ABV21639USO1 embodiments, the anti-tau compound may be administered at least about 7 days post lysate injection.
  • 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.
  • 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.
  • aggregated tau is quantified in the olfactory bulb.
  • 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.
  • 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.
  • aggregated tau is quantified in at least one second synaptically connected region selected from the group consisting of the hippocampus and the thalamus.
  • 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.
  • aggregated tau is quantified in the piriform cortex.
  • aggregated tau is quantified by counting cell bodies that are immunoreactive to the AT100 anti-tau antibody (i.e., AT100 IR cell bodies).
  • ABV21639USO1 the methods of the present disclosure involve injection of an AD brain lysate.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, the lysate has a tau concentration of at least about 800 nM.
  • the tau in the AD lysate is primarily pathological tau protein, i.e., nonsoluble tau.
  • Figure 5A provides a schematic of an in vivo tau propagation model for tau pathology according to the present disclosure. Lysate purified from brain tissue of AD patients is used to seed tau into the olfactory bulb of female hTau mice, which express all six isoforms of normal human tau.
  • Example 1 Anti-Tau Antibody Generation
  • the murine anti-tau antibody Ab1 was identified from CD-1 mice immunized with a mixture of six phospho-peptides derived from the human tau sequence: N-terminus and C- terminus KLH conjugated pT231; N-terminus and C-terminus KLH conjugated pS235; N- terminus and C-terminus KLH conjugated pT231, pS235.
  • Immune libraries were constructed from the tissues of immunized mice and screened using a phage panning strategy. The phage output was evaluated for binding by ELISA to pT231 peptide, pS235 peptides, and pT231 / pS235 peptides.
  • the murine antibody Ab1 was identified, which has a heavy chain sequence set forth as SEQ ID NO: 11 and a light chain sequence set forth as SEQ ID NO: 12, demonstrated specificity to the phosphorylated T231 (pT231) peptide but not to the non-phosphorylated T231 peptide.
  • Ab1 was subsequently humanized and subjected to CDR engineering to eliminate potential liabilities and potential deamidation sites. This yielded the humanized antibody AbA, which has a heavy chain sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 16 and a light chain sequence set forth as SEQ ID NO: 6.
  • AbA maintained specificity to the phosphorylated T231 peptide, while also demonstrating higher affinity to phosphorylated T231 peptide than the murine Ab1 parent antibody.
  • chAbA is a mouse IgG2a/k antibody having a heavy chain ABV21639USO1 sequence set forth as SEQ ID NO: 17 and a light chain sequence set forth as SEQ ID NO: 18.
  • chAbA maintained specificity to the phosphorylated T231 peptide.
  • Example 2 Anti-Tau Antibody AbA shows specificity for phosphorylated (pT231) peptide
  • the peptide was biotinylated, phosphorylated (or not phosphorylated), and interacted with bound antibody in an ELISA format.
  • ELISA was performed using Protein G 96 well plates (Thermo Fisher Scientific, Waltham, MA, USA) washed 1X in 200 ⁇ L of phosphate-buffered saline containing 0.05% Polysorbate 20 (PBS-T) using a BioTek ELx405 plate washer (Agilent, Santa Clara, CA, USA). The plates were then incubated with AbA or a control hu IgG1-QL at 1 ⁇ g/mL concentration in 0.1% bovine serum albumin (BSA) in PBS-T for 2 hours under ambient conditions on an orbital shaker at 120 RPM.
  • BSA bovine serum albumin
  • HT7 mAb was used as the detection antibody.
  • HT7 mAb was used as both the capture and detection antibody.
  • Antibody labeled beads and biotinylated detection antibodies were used according to the manufacturer’s protocol (Quanterix ® , Billerica, MA, USA).
  • Recombinant full-length P301L tau (SEQ ID NO: 14; Spillantini MG, et al. Tau pathology in two Dutch families with mutations in the microtubule-binding region of tau (Am J Pathol.1998 Nov;153(5):1359-63.) was purified by the method of Barghorn et al.
  • the hyperphosphorylated tau from SF9 cells was purchased as cell paste containing the recombinant protein from GenScript (GenScript ProBio, Piscataway, NJ, USA) and purified by the method of Tepper (supra). Full-length P301L tau aggregates and SF9 cell derived hyperphosphorylated tau were used as calibrators and included in each run to generate a standard curve.
  • FIG. 1 illustrates the in vitro binding of the anti-tau antibody AbA to both phosphorylated tau pT231 and tau aggregates derived from the brain tissue of AD patients in a dose-dependent manner.
  • Example 5 Effect of an anti-tau antibody in an in vivo model of tau seeding
  • a model of tau seeding was developed in transgenic rTG4510 mice where lysate was used to directly seed tau into the hippocampus ( Figure 3). Briefly, stereotactic intrahippocampal injection of protein lysate generated from brain tissue in AD patients in rTG4510 mice ( Figure 3A) results in the expression of aggregated tau (AT100 IR) in the CA1 region of the hippocampus ( Figure 3B).
  • Transgenic rTG4510 mice were used for the hippocampal seeding studies (Santacruz 2005; 10.1126/science.1113694).
  • mice The original breeder mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) under the license from Mayo Clinic (Rochester, MN, USA). Mice were bred at Charles River Laboratories (Sulzfeld, DE). Animal health and comfort were veterinary controlled. Mice were group housed in temperature- and humidity- controlled rooms with a 12:12 hour dark/light cycle with ad libitum access to water and food.
  • chAbA represents a chimeric version of AbA on a mouse IgG backbone as described above.
  • mice were prepared for aseptic stereotactic surgery and were unilaterally injected with protein lysate derived from human AD brain (AD lysate, prepared as described above in Example 2) into the hippocampus (2.5 ⁇ L, coordinates: AP: -1,9mm; ML: -1,5mm; DV: -1,6) using a Hamilton syringe (Hamilton, Reno, NV, USA). After recovery from surgery, mice were returned to their home cages. [0078] Three weeks later, mice were euthanized, perfused with phosphate buffered saline (PBS), and the brains were quickly removed.
  • PBS phosphate buffered saline
  • the trimmed forebrain was drop-fixed in 10% formalin for 24 hours before being switched to 70% ethanol:water.
  • 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 ABV21639USO1 from the study were collected to allow all samples to be processed for paraffin embedding at the same time (Leica Biosystems, Wetzlar, DE). [0079] After processing, four brains were embedded together into paraffin blocks in coronal plane (Medite, Burgdorf, DE). Brains were randomized based on the test group and paraffin blocks containing 4 randomized brains were generated.
  • the threshold was set so that the positive brown DAB stain of the AT100 immunoreactivity was recognized by the software and any background/non-specific staining was excluded from the analysis.
  • the software measured the percentage of the area of interest (CA1 region) containing the positive immunoreactivity for AT100.
  • the AT100 IR area values for each animal were then plotted and analyzed in GraphPad Prism (Dotmatics, Boston, MA, USA) [0081]
  • Figure 4 summarizes the protocol for antibody pre-dosing, seed injection, and tissue and serum collection, and presents the AT100 IR results illustrating the effect of the anti-tau antibody chAbA on hippocampal seeding in the rTG4510 seeding model.
  • Example 6 In Vivo Tau Propagation Assay Preparation of human sarkosyl insoluble tau AD lysate from donor samples [0082] Donor brain tissue was weighed and homogenized three times in TBS lysate buffer (50 mM TRIS, 150 mM NaCl, 20 mM NaF, 1 mM Na3VO4, 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 °C.
  • TBS lysate buffer 50 mM TRIS, 150 mM NaCl, 20 mM NaF, 1 mM Na3VO4, 0.5 mM MgSO4 (pH 7.4) containing protease and phosphatase inhibitors using a Precellys Evolution homogenizer (Ber
  • the resulting supernatant was the sarkosyl insoluble tau AD lysate used to seed tau.
  • Aggregated tau species were quantified using a homogenous time-resolved fluorescence (HTRF)-based tau aggregation assay kit (PerkinElmer - Cisbio, Waltham, MA, USA). The manufacturer’s protocol was followed to ensure the appropriate dilution for the donor and receptor fluorophore-tagged antibodies. Assay reactions (20 ⁇ L) were carried out in 384 microplate (Greiner Bio-One, Kremsmünster, AT), where samples were incubated with the fluorophore-tagged antibodies for 24 hours at ambient temperature.
  • HTRF time-resolved fluorescence
  • the fluorescent signal was detected using Cytation 5 (Agilent, Santa Clara, CA, USA) 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 ABV21639USO1 plotted in GraphPad Prism (Dotmatics) and a One-way ANOVA was used to compare statistical significance between human AD and control brain lysates.
  • Total tau levels were measured using the R-PLEX Human total tau kit (Meso Scale Discovery, Rockville, MD) in a sandwich immunoassay.
  • Biotinylated capture antibody was diluted in coating diluent (MSD Diluent 100) 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 ⁇ -mercaptoethanol (Bio-Rad).
  • 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. Samples were diluted to 1:80,000 in MSD Diluent 101 and assayed in duplicates.
  • R-plex human tau calibrator was prepared as a 7-point calibration curve starting at 5000 pg/mL with a four-fold dilution series down to 1.2pg/mL, as recommended by the manufacturer. A blank was also included for background signal deduction.
  • plates were washed with 1x MSD Tris wash buffer at 150 ⁇ L/well.
  • the calibrator and unknowns were added at 25 ⁇ L/replicate, plates were sealed with an adhesive plate sealer and incubated for 1 hour at ambient conditions on a shaker. After 1 hour, plates were washed with 150 ⁇ L/well with 1X MSD Tris wash buffer.
  • the 100x SULFO-Tag Detection antibody (Meso Scale Discovery) was diluted to 1x using MSD Diluent 101 and added to the plate at 50 ⁇ L/well. After 1 hour of incubation, plates were washed with 1X MSD Tris wash buffer. In the last step, MSD Read Buffer T (4x) was diluted to 2X working solution in deionized water and added to the plates at 150 ⁇ L/well. Plates were immediately read on the Meso Sector S600 (Meso Scale Discovery).
  • ABV21639USO1 [0086] At 4.5-5 months of age, female hTau mice were prepped for aseptic stereotaxic surgery and 2 ⁇ L of sarkosyl-insoluble AD lysate was injected into the olfactory bulb using a Hamilton syringe (Hamilton). After recovery from surgery, mice were returned to their home cages. Animals were dosed weekly i.p. starting 7 days after the seed injections (see Figure 7A).All experiments were performed in full compliance with the Principles of Laboratory Animal Care (NIH publication No.86-23). Experiments performed in the U.S. were performed in full compliance with AbbVie’s Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • PBS phosphate buffered saline
  • paraffin blocks (Sakura Finetek, Torrance, CA, USA) in a horizontal plane. Every 5 th , 5 ⁇ m paraffin sections was collected through the entire brain and mounted onto glass slides. Twenty-five sections (every 5 th section) per block were stained for AT100 immunoreactivity using the BOND RX stainer with the Refine Detection kit (Leica).
  • 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 Scientific, Waltham, MA, USA) at a concentration of 0.006 ⁇ g/mL.
  • AT100 immunoreactivity quantification [0090] 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 (9 – 11 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.
  • ABV21639USO1 Table 2 Cortical Tissues from AD donors with various concentrations of tau [0092] The lysates were injected into the olfactory bulb of hTau mice. Two months after tau seeding, neurons containing aggregated tau were visualized by AT100 IR and quantified in the olfactory bulb, piriform cortex, entorhinal cortex, medial thalamus, and CA1 hippocampus ( Figure 6A). Analysis showed that the concentration of tau in the AD lysate correlated with the level of tau pathology with coefficients of variation between 0.67 and 0.814 ( Figure 6B).
  • mice Four and a half-month-old female hTau mice were injected with purified AD brain lysate into the olfactory bulb; intraperitoneal (i.p.) dosing with chAbA antibody started 7 days later at doses ABV21639USO1 X, Y, and Z (where X ⁇ Y ⁇ Z). Weekly dosing continued for 8 weeks post-seeding with AD lysate.
  • chAbA antibody intraperitoneal (i.p.) dosing with chAbA antibody started 7 days later at doses ABV21639USO1 X, Y, and Z (where X ⁇ Y ⁇ Z). Weekly dosing continued for 8 weeks post-seeding with AD lysate.

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Abstract

La présente divulgation concerne des anticorps anti-tau, y compris des compositions et des méthodes d'utilisation de tels anticorps pour traiter la maladie d'Alzheimer.
PCT/US2025/019566 2024-03-16 2025-03-12 Anticorps anti-tau Pending WO2025198916A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150175682A1 (en) * 2012-04-05 2015-06-25 Ac Immune S.A. Humanized tau antibody
US20190112364A1 (en) * 2017-10-16 2019-04-18 Eisai R&D Management Co., Ltd. Anti-tau antibodies and uses thereof
US20220275067A1 (en) * 2019-02-08 2022-09-01 Prothena Biosciences Limited Antibodies recognizing tau
US20230049732A1 (en) * 2019-12-30 2023-02-16 University Of Louisville Research Foundation, Inc. Virus-like particle binding agents, related compositions, and related methods
US20240084005A1 (en) * 2021-09-16 2024-03-14 Allakos Inc. Anti-siglec-6 antibodies and methods of use thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150175682A1 (en) * 2012-04-05 2015-06-25 Ac Immune S.A. Humanized tau antibody
US20190112364A1 (en) * 2017-10-16 2019-04-18 Eisai R&D Management Co., Ltd. Anti-tau antibodies and uses thereof
US20220275067A1 (en) * 2019-02-08 2022-09-01 Prothena Biosciences Limited Antibodies recognizing tau
US20230049732A1 (en) * 2019-12-30 2023-02-16 University Of Louisville Research Foundation, Inc. Virus-like particle binding agents, related compositions, and related methods
US20240084005A1 (en) * 2021-09-16 2024-03-14 Allakos Inc. Anti-siglec-6 antibodies and methods of use thereof

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