WO2025253337A2 - Protéines de liaison à tau monospécifiques et bispécifiques et compositions associées - Google Patents

Protéines de liaison à tau monospécifiques et bispécifiques et compositions associées

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Publication number
WO2025253337A2
WO2025253337A2 PCT/IB2025/055816 IB2025055816W WO2025253337A2 WO 2025253337 A2 WO2025253337 A2 WO 2025253337A2 IB 2025055816 W IB2025055816 W IB 2025055816W WO 2025253337 A2 WO2025253337 A2 WO 2025253337A2
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Prior art keywords
tau
binding protein
seq
bispecific
tfr
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WO2025253337A3 (fr
Inventor
Philippe Bertrand
Béatrice Cameron
Tarik Dabdoubi
Catherine Devaud
Simon DUJARDIN
Stéphanie EYQUEM
Dominique Lesuisse
Mireille Meunier
Nadine MICHOT
Souad Naimi
Fabienne Soubrier
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Sanofi SA
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Sanofi SA
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Publication of WO2025253337A2 publication Critical patent/WO2025253337A2/fr
Publication of WO2025253337A3 publication Critical patent/WO2025253337A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • 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
    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • Tau is a microtubule-associated protein abundant in central nervous system (CNS) neurons. Tau functions to stabilize the microtubule network in axons, thereby regulating axonal transport. Truncation of tau can result in conversion to pathological conformations that may form insoluble aggregates.
  • the microtubule binding region (MTBR) of tau is a core component for tau assembly and formation of seeding species.
  • tau protein aggregates are a pathological hallmark of around 20 neurodegenerative pathologies known as tauopathies.
  • the tauopathies are structurally and neuropathologically diverse. Across at least four tauopathies (Alzheimer’s disease, progressive supranuclear palsy, argyrophilic grain disease, and Pick’s disease), the deposition of tau aggregates progresses in a stereotypical manner in the brain, following synaptic connections to actively spread from neuron to neuron through the brain. In a manner similar to prion propagation, misfolding of the tau protein leads to trans-synaptic transfer of aggregates and seeding of pathology.
  • the present disclosure provides anti-tau binding domains, as well as bispecific binding proteins that bind to tau and a target in the central nervous system (CNS), such as an endothelial cell receptor (ECR) of the blood-brain barrier (BBB).
  • CNS central nervous system
  • ECR endothelial cell receptor
  • BBB blood-brain barrier
  • the ECR may be, for example, transferrin receptor 1 (TfR).
  • the present disclosure provides a tau-binding protein comprising an anti-tau binding domain that comprises: a) a heavy chain variable region (VH) comprising heavy chain complementaritydetermining regions (HCDR) 1-3 set forth in SEQ ID NOs: 1, 2, and 3, respectively; and a light chain variable region (VL) comprising light chain CDR (LCDR) 1-3 set forth in SEQ ID NOs: 4, 5, and 6, respectively; or b) a VH comprising HCDR1-3 set forth in SEQ ID NOs: 9, 10, and 11, respectively; and a VL comprising LCDR1-3 set forth in SEQ ID NOs: 12, 13, and 14, respectively.
  • VH heavy chain variable region
  • HCDR heavy chain complementaritydetermining regions
  • VL light chain variable region
  • LCDR light chain CDR
  • the VH and the VL of the tau-binding protein are at least 90% identical to:
  • the VH and the VL of the tau-binding protein comprise: SEQ ID NOs: 7 and 8, respectively;
  • the tau-binding protein herein may have at least one property selected from a) binds to full-length tau monomers with an ECso of 0.1-0.4 nM as determined by ELISA; b) binds to full-length tau fibrils with an ECso of 0.1-0.4 nM as determined by ELISA; c) binds to tau microtubule binding region (MTBR) monomers; d) binds to tau MTBR fibrils; e) binds to tau pathological forms in progressive supranuclear palsy, Alzheimer’s disease, Pick’s disease, or any combination thereof; f) inhibits tau seeding and aggregation as determined by homogeneous time resolved fluorescence (HTRF); g) inhibits aggregation of endogenous full-length tau induced by aggregated wild-type tau MTBR in vitro.
  • HTRF homogeneous time resolved fluorescence
  • h reduces tau pathology in the hippocampus, cortex, or both in THY-Tau22 mice; i) inhibits tau seeding and aggregation as determined by in cell fluorescence resonance energy transfer (FRET); j) inhibits uptake of tau aggregates into neurons; k) decreases neurofilament light (NFL) in the cerebrospinal fluid of THY-Tau22 mice; l) reverses cognitive deficits in THY-Tau22 mice; m) improves motor deficits in THY-Tau22 mice; or n) any combination of a)-m).
  • FRET cell fluorescence resonance energy transfer
  • the tau-binding protein may have at least properties a)-h), or all of properties a)- m).
  • the tau-binding protein herein is a monoclonal antibody (“anti-tau antibody”) (e.g., of human isotype subclass IgGl, IgG2, IgG3, or IgG4) or an antigen-binding fragment thereof (e.g., comprising a Fab, Fab’, F(ab’)2, or scFv).
  • anti-tau antibody comprises a) a human IgGl constant region; b) a human kappa light chain constant region, optionally comprising SEQ ID NO: 30; or c) both a) and b).
  • the anti-tau antibody comprises a human IgGl heavy chain constant region that comprises SEQ ID NO: 27.
  • the human IgGl heavy chain constant region comprises mutations selected from i) L234A and L235A, ii) H435R and Y436F, and iii) both i) and ii), wherein the mutation positions are according to Eu numbering.
  • the anti-tau antibody comprises a first heavy chain constant region that comprises the mutation of i), and a second heavy chain constant region that comprises the mutations of i) and ii).
  • the tau-binding protein herein may be fused to a cellpenetrating peptide that binds a central nervous system (CNS) target (e.g., TfR).
  • the tau-binding protein herein may comprise an Fc region with one chain modified to bind a CNS target (e.g., TfR), and is optionally a bivalent anti-tau antibody or antigen-binding fragment thereof.
  • the present disclosure also provides a bispecific binding protein comprising a) a tau-binding protein herein or an anti-tau binding domain thereof, and b) a binding domain specific for another, distinct target protein, which may be a CNS target (e.g., TfR).
  • a CNS target e.g., TfR
  • the bispecific binding protein is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe-N-bindyl
  • the bispecific binding protein herein comprises two heavy chains and two light chains, wherein one pair of heavy and light chains forms one arm, and the other pair of heavy and light chains forms another arm, of the bispecific binding protein.
  • one arm of the bispecific binding protein comprises a first anti-tau binding domain and an anti-CNS target binding domain
  • the other arm comprises a second anti-tau binding domain, wherein the first and second anti-tau binding domains are of a tau-binding protein herein.
  • the first and second anti-tau binding domains may have the same HCDR 1-3 and same LCDR 1-3, or may be the same.
  • one arm of the bispecific binding protein comprises a first heavy chain comprising a VH of the anti-CNS target binding domain and a VH of the first anti-tau binding domain and a first light chain comprising a VL of the first anti-tau binding domain and a VL of the anti-CNS target binding domain; and the other arm comprises a second heavy chain comprising a VH of the anti-tau domain and a second light chain comprising a VL of the second anti-tau domain.
  • the VH of the anti-CNS target binding domain is N-terminal to the VH of the first anti-tau binding domain
  • the VL of the first anti-tau binding domain is N-terminal to the VL of the anti-CNS target binding domain
  • one arm of the bispecific binding protein comprises first and second anti-tau binding domains, and the other arm comprises an anti-CNS target binding domain, wherein the first and second anti-tau binding domains are of a tau-binding protein herein.
  • the first and second anti-tau binding domains may have the same HCDR 1-3 and same LCDR 1-3, or may be the same.
  • one arm of the bispecific binding protein comprises a first heavy chain comprising a VH of the first anti-tau binding domain and a VH of the second anti-tau binding domain and a first light chain comprising a VL of the second anti-tau binding domain and a VL of the first anti-tau binding domain; and the other arm comprises a second heavy chain comprising a VH of the anti-CNS target binding domain and a second light chain comprising a VL of the anti-CNS target binding domain.
  • the VH of the first anti-tau binding domain is N-terminal to the VH of the second anti-tau binding domain
  • the VL of the second anti-tau binding domain is N-terminal to the VL of the first anti-tau binding domain
  • the CNS target is an endothelial cell receptor (ECR) of the blood brain barrier.
  • ECR may be, e.g., a transferrin receptor, insulin receptor, low-density lipoprotein receptor, or folate receptor.
  • the ECR is transferrin receptor 1 (TfR), and the bispecific binding protein has an anti-TfR binding domain.
  • the anti-TfR binding domain competes for binding with, or binds to the same epitope as, an anti-TfR antibody comprising VH and VL as set forth in SEQ ID NOs: 25 and 26.
  • the anti-TfR binding domain comprises a VH comprising HCDR1-3 set forth in SEQ ID NOs: 19, 20 and 21, respectively; and a VL comprising LCDR1-3 set forth in SEQ ID NOs: 22, 23, and 24, respectively.
  • the VH and the VL of the anti-TfR binding domain are at least 90% identical to, or comprise:
  • the bispecific binding protein may have one or more of the following properties: a) binds to human tau with an ECso of 5-35 pM as determined by ELISA; b) binds to human TfR with an ECso of 1-50 nM as determined by FACS; c) binds to cynomolgus TfR with an ECso of 1-250 nM as determined by FACS; d) binds to human TfR with a KD of 1-50 nM as determined by SPR; e) binds to cynomolgus TfR with a KD of 1-200 nM as determined by SPR; f) binds to human TfR with a KD of 10-40 nM as determined by OctetTM; g) binds to cynomolgus TfR with a KD of 50-200 nM as determined by OctetTM; h) has improved brain penetrance in h
  • the bispecific binding protein herein comprises an Fc region.
  • the bispecific binding protein is a bispecific antibody.
  • the Fc region is of an antibody of human isotype subclass IgGl, IgG2, IgG3, or IgG4.
  • the bispecific binding protein may comprise, e.g., a) a human IgGl heavy chain constant region; b) a human kappa light chain constant region, optionally comprising SEQ ID NO: 30; or c) both a) and b).
  • the bispecific antibody comprises a first heavy chain constant region that comprises one or more knob mutations, optionally wherein the knob mutations comprise S354C and T366W; and a second heavy chain constant region that comprises one or more hole mutations, optionally wherein the hole mutations comprise Y349C, T366S, L368A, and Y407V, wherein the mutation positions are according to Eu numbering.
  • the human IgGl heavy chain constant region or the bispecific antibody comprises mutations selected from i) L234A and L235A, ii) H435R and Y436F, and iii) both i) and ii), wherein the mutation positions are according to Eu numbering.
  • the bispecific antibody comprises a first heavy chain constant region that comprises the mutation of i), and a second heavy chain constant region that comprises the mutations of i) and ii).
  • the first heavy chain constant region further comprises knob mutations of S354C and T366W
  • the second heavy chain constant region further comprises hole mutations of Y349C, T366S, L368A, and Y407V (Eu numbering).
  • the human IgGl constant region may comprise, e.g., any one of SEQ ID NOs: 27-29.
  • the bispecific binding protein comprises two heavy chain constant regions that both comprise SEQ ID NO: 27; or a first heavy chain constant region that comprises SEQ ID NO: 28 and a second heavy chain constant region that comprises SEQ ID NO: 29.
  • the present disclosure provides a bispecific binding protein that binds to tau and TfR, wherein the bispecific binding protein is bivalent for tau and monovalent for TfR, comprising a first heavy chain that comprises SEQ ID NO: 41, a second heavy chain that comprises SEQ ID NO: 43, a first light chain that comprises SEQ ID NO: 42, and a second light chain that comprises SEQ ID NO: 44.
  • the present disclosure provides a bispecific binding protein that binds to tau and TfR, wherein the bispecific binding protein is bivalent for tau and monovalent for TfR, comprising a first heavy chain that comprises SEQ ID NO: 37, a second heavy chain that comprises SEQ ID NO: 39, a first light chain that comprises SEQ ID NO: 38, and a second light chain that comprises SEQ ID NO: 40.
  • the present disclosure provides a bispecific binding protein that binds to tau and TfR, wherein the bispecific binding protein is bivalent for tau and monovalent for TfR, comprising a first heavy chain that comprises SEQ ID NO: 31, a second heavy chain that comprises SEQ ID NO: 33, a first light chain that comprises SEQ ID NO: 32, and a second light chain that comprises SEQ ID NO: 34.
  • the present disclosure provides a bispecific binding protein that binds to tau and TfR, wherein the bispecific binding protein is bivalent for tau and monovalent for TfR, comprising a first heavy chain that comprises SEQ ID NO: 35, a second heavy chain that comprises SEQ ID NO: 33, a first light chain that comprises SEQ ID NO: 36, and a second light chain that comprises SEQ ID NO: 34.
  • the present disclosure provides a bispecific binding protein comprising two heavy chains and two light chains, wherein one pair of heavy and light chains forms one arm, and the other pair of heavy and light chains forms another arm, of the bispecific binding protein, wherein one arm comprises a first anti-tau binding domain and an anti-CNS target binding domain (e.g., an anti-TfR binding domain), and the other arm comprises a second anti-tau binding domain.
  • one arm comprises a first anti-tau binding domain and an anti-CNS target binding domain (e.g., an anti-TfR binding domain)
  • the other arm comprises a second anti-tau binding domain.
  • the VH of the anti-CNS target binding domain (e.g., anti-TfR binding domain) is N- terminal to the VH of the first anti-tau binding domain
  • the VL of the first anti-tau binding domain is N-terminal to the VL of the anti-CNS target binding domain.
  • the first and second anti-tau binding domains may have the same HCDR1-3 and same LCDR1-3, and in some embodiments are the same.
  • the first anti-tau binding domain, the second anti-tau binding domain, or both bind specifically to tau fibrils and/or bind to the MTBR region of tau.
  • the first anti-tau binding domain, the second anti-tau binding domain, or both may comprise, e.g., a) a VH and a VL comprising HCDR1-3 and LCDR1-3 as set forth in i) SEQ ID NOs: 1-6, respectively; or ii) SEQ ID NOs: 9-14, respectively; b) the VH and VL of a), wherein the VH and VL are at least 90% identical to i) SEQ ID NOs: 7 and 8, respectively; ii) SEQ ID NOs: 15 and 16, respectively; iii) SEQ ID NOs: 73 and 75, respectively; or iv) SEQ ID NOs: 74 and 76, respectively; or c) a VH and VL that comprise i) SEQ ID NOs: 7 and 8, respectively; ii) SEQ ID NOs: 15 and 16, respectively; iii) SEQ ID NOs: 73 and 75, respectively; or iv) SEQ ID NOs: 74 and 76
  • the first anti-tau binding domain, the second anti-tau binding domain, or both may comprise, e.g., a) a VH and a VL comprising HCDR1-3 and LCDR1-3 as set forth in SEQ ID NOs: 1-6, respectively; b) the VH and VL of a), wherein the VH and VL are at least 90% identical to i) SEQ ID NOs: 7 and 8, respectively; ii) SEQ ID NOs: 73 and 75, respectively; or iii) SEQ ID NOs: 74 and 76, respectively; or c) a VH and VL that comprise i) SEQ ID NOs: 7 and 8, respectively; ii) SEQ ID NOs: 73 and 75, respectively; or iii) SEQ ID NOs: 74 and 76, respectively.
  • the anti-CNS target binding domain is an anti-TfR binding domain that binds to human TfR (hTR) with a KD of 1-50 nM (e.g., 1-30 nM or 1-20 nM) as determined by SPR, and/or binds to cynomolgus TfR (cTfR) with a KD of 1-200 nM (e.g., 30-
  • the present disclosure also provides pharmaceutical composition comprising a tau- binding protein or bispecific binding protein herein and a pharmaceutically acceptable excipient.
  • the present disclosure provides isolated nucleic acid molecule(s) encoding a tau-binding protein or bispecific binding protein herein.
  • the nucleic acid molecule(s) are expression constructs.
  • a host cell comprising the isolated nucleic acid molecule(s), optionally wherein the host cell is a mammalian cell, and a method of producing a tau-binding protein or a bispecific binding protein herein, comprising: culturing the host cell under conditions that allow expression of the tau-binding protein or bispecific binding protein, and isolating the tau-binding protein or bispecific binding protein from the cell culture.
  • the present disclosure also provides a method of treating a tauopathy in a subject (e.g., a human subject in need thereof), comprising administering to the subject a therapeutically effective amount of a tau-binding protein or bispecific binding protein herein. Also provided are use of a tau-binding protein or bispecific binding protein herein for the manufacture of a medicament for treating a tauopathy in a subject (e.g., a human subject in need thereof), and a tau-binding protein or bispecific binding protein herein for use in treating a tauopathy in a subject (e.g., a human subject in need thereof). Further, the present disclosure provides a tau-binding protein or bispecific binding protein herein for use as a medicament.
  • the tauopathy is a primary tauopathy, e.g., selected from progressive supranuclear palsy (PSP), frontotemporal lobar degeneration with MAPT mutations, argyrophilic grain disease, corticobasal degeneration, Pick’s disease, globular glial tauopathy, aging-related tau astrogliopathy (ARTAG), and primary age-related tauopathy (PART).
  • the primary tauopathy is progressive supranuclear palsy (PSP).
  • the tauopathy is a secondary tauopathy, e.g., selected from Alzheimer’s disease, chronic traumatic encephalopathy, anti-IgLON5-related tauopathy, Down syndrome, Niemann-Pick disease type C, and myotonic dystrophy type 1 and 2.
  • the secondary tauopathy is Alzheimer’s disease.
  • FIGs. 1A-1C are a set of photographs showing the specific cellular and regional staining of the 22-MTBR antibody in various tauopathies via immunohistochemical staining.
  • FIG. 2 is a plot showing the change in tau pathology in THY-Tau22 mice after treatment with the indicated antibodies, as measured by HTRF.
  • FIG. 3 is a bar graph showing the number of AT8 + cells in the hippocampus of THY-Tau22 mice following treatment with the indicated antibodies. Each symbol represents a separate animal.
  • FIG. 4 is box and whisker plot showing neurofilament light concentration (a measure of neuroprotective effect) in the cerebrospinal fluid of THY-Tau22 mice after treatment with antibody 22-MTBR.
  • FIGs. 5A and 5B are bar graphs indicating the ability of mice (wild-type or THY- Tau22) to perform an object recognition task when treated with the indicated antibodies (FIG. 5A) or with antibody 22-MTBR at the indicated concentrations (FIG. 5B).
  • FIG. 6 is a bar graph showing the fraction of THY-Tau22 mice exhibiting high, low, or no deficits in motor functions upon treatment with 22-MTBR.
  • FIG. 7 is a set of bar graphs showing the levels of hyperphosphorylated tau (as measured using antibody AT8) in the indicated brain regions of THY-Tau22 mice following administration of the tau microtubule binding region (MTBR) and treatment with control (PBS) or antibody 523 -MTBR.
  • MTBR tau microtubule binding region
  • PBS treatment with control
  • 523 -MTBR antibody 523 -MTBR
  • FIG. 8 is a set of protein structures showing the binding of antibodies 22-MTBR (left) and 523 -MTBR (right) to a region of the tau protein MTBR (“peptide,” sequence shown below the structure).
  • IGSLDNITHV SEQ ID NO: 95.
  • THVPGGGNKK SEQ ID NO: 96.
  • Underlined amino acids in SEQ ID NO: 95 have a confirmed direct interaction with 22- MTBR.
  • Underlined amino acids in SEQ ID NO: 96 have a confirmed direct interaction with 523 -MTBR.
  • FIG. 9 is a schematic showing the different positions of the anti-TfR domain within various bispecific anti -tau/ anti-TfR antibody constructs.
  • FIG. 10 is a set of line graphs indicating the concentration of the indicated antibodies in the brains of mice over time following antibody treatment.
  • FIG. 11 is a line graph showing the concentration of the indicated antibodies in the brains of THY-Tau22 mice over time following intraperitoneal (IP) antibody administration.
  • FIGs. 12A and 12B are box and whisker plots showing the number of AT8 + (FIG. 12A) and Gallyas + (FIG. 12B) cells in brain samples of THY-Tau22 mice following treatment with the indicated antibodies.
  • FIG. 13 is a line graph showing the concentration of the indicated monospecific (“Tau monospecific”) and bispecific (“aTfR-Tau”) antibodies in the brains of THY- Tau22/human TfR knock in mice over time following the final dose of antibody treatment.
  • Anti -TfR/anti -tau bispecific antibodies 22-53 lv25-4 or 523-53 lv25-l.
  • Anti-tau monospecific antibodies 22-MTBR or 523-MTBR.
  • FIG. 14A is a line graph showing the total concentration and the concentration of free antibody in the cerebrospinal fluid of cynomolgus monkeys over time following treatment with the indicated antibodies.
  • FIG. 14B is a bar graph showing the total concentration of bispecific antibody 22-53 lv25-3 in various tissues of the cynomolgus monkeys (monospecific 22-MTBR was below the lower limit of quantification (LLOQ) of 0.22 nmol/kg).
  • LLOQ lower limit of quantification
  • FIG. 15 is a bar graph showing the frequency of FRET spots in HEK293 cells transfected with MTBR-mTurquoise-2A-MTBR-mVenus following incubation with the indicated antibodies.
  • FIG. 16 is a bar graph showing the fraction of AT8 + cells in the cortex of humanized TfR-knock in/THY-Tau22 mice following treatment with the indicated antibodies.
  • FIGs. 17A and 17B are a pair of line graphs showing association and dissociation of antibody 22-MTBR to tau monomers (FIG. 17A) or tau pre-formed fibrils (PFFs) (FIG.
  • FIGs. 18A and 18B are a pair of line graphs showing association and dissociation of bispecific antibody 22-53 lv25-3 to tau monomers (FIG. 18A) or tau PFFs (FIG. 18B).
  • FIGs. 19A and 19B are a pair of line graphs showing association and dissociation of antibody HT7 to tau monomers (FIG. 19A) or tau PFFs (FIG. 19B).
  • FIG. 20 is a set of line graphs showing association and dissociation of the indicated antibodies to the transferrin receptor in the presence or absence of tau PFFs.
  • FIG. 21 is a pair of bar graphs showing the levels of tau-PFF-pHrodoTM inside iPSC-derived neurons following incubation with increasing amounts of the indicated antibodies. “Blank” refers to no tau-PFF-pHrodoTM added, “Control” refers to tau-PFF- pHrodoTM without addition of antibodies.
  • FIG. 22 is a bar graph showing the levels of tau-PFF-pHrodoTM inside iPSC- derived neurons following incubation with the indicated antibodies.
  • FIG. 23 is a set of bar graphs showing the levels of tau-PFF-pHrodoTM inside iPSC-derived neurons following incubation with increasing amounts of the indicated antibodies. “Blank” refers to no tau-PFF-pHrodoTM added, “Control” refers to tau-PFF- pHrodoTM without addition of antibodies.
  • FIG. 24 is a pair of bar graphs showing the levels of hyperphosphorylated tau (as measured using antibody AT8) in the ipsilateral and contralateral hippocampi of THY-Tau22 mice following inoculation with tau PFFs in the presence or absence of the indicated antibodies.
  • FIG. 25 is a set of photographs showing hyperphosphorylated tau pathology (as measured using antibody AT8) in the hippocampi of THY-Tau22 mice following inoculation with tau PFFs in the presence or absence of the indicated antibodies.
  • FIG. 26 is a schematic showing the formats of the indicated bispecific antibodies.
  • the present disclosure provides isolated binding domains/binding proteins, such as antibodies and antigen-binding fragments thereof, that bind human tau. These tau-binding proteins bind to an epitope in the microtubule binding region (MTBR) of tau, allowing them to recognize various tau forms across different tauopathies.
  • MTBR microtubule binding region
  • the tau-binding proteins herein inhibit propagation of misfolded tau through extracellular spaces or along axonal paths, halting or slowing tau pathology (e.g., tauopathy) progression and cognitive decline.
  • the present disclosure also provides multispecific (e.g., bispecific) binding proteins that pair an anti-tau binding domain with a domain that binds to a CNS target (e.g., an epithelial cell receptor (ECR) of the BBB, such as transferrin receptor 1 (TfR)).
  • a CNS target e.g., an epithelial cell receptor (ECR) of the BBB, such as transferrin receptor 1 (TfR)
  • ECR epithelial cell receptor
  • TfR transferrin receptor 1
  • tau herein refers to human tau.
  • a human tau polypeptide sequence is available under UniProt Accession No. P10636-8 (SEQ ID NO: 45).
  • a full-length human tau polypeptide sequence is available under NCBI Reference Sequence No. NP 001116538.2 (SEQ ID NO: 17).
  • the MTBR of tau has a sequence of SEQ ID NO: 46.
  • TfR herein refers to human TfR (human TfRl).
  • a human TfR polypeptide sequence is available under UniProt Accession No. P02786 (SEQ ID NO: 18).
  • the present disclosure provides tau-binding domains/proteins, such as antibodies or antigen-binding fragments thereof.
  • antibody herein includes monospecific and multispecific (e.g., bispecific) antibodies.
  • Antibody” (Ab) or “immunoglobulin” (Ig), as used herein, may refer to a tetramer comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region or domain (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable region or domain (VL) and a light chain constant region (CL).
  • VH and VL domains can be subdivided further into regions of hypervariability, termed “complementarity-determining regions” (CDRs), interspersed with regions that are more conserved, termed “framework regions” (FRs).
  • CDRs complementarity-determining regions
  • FRs frame regions
  • Each VH and VL is composed of three CDRs (HCDR herein designates a CDR from the heavy chain; and LCDR herein designates a CDR from the light chain) and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the boundaries of a given CDR or FR may vary depending on the system used.
  • the Kabat system is based on sequence alignments
  • the Chothia system is based on structural information. Numbering for both the Kabat and Chothia systems is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a.” The two systems place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering.
  • the contact system is based on analysis of complex crystal structures and is similar in many respects to the Chothia system.
  • the CDRs of the antibodies described herein can be defined, e.g., by a system selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.
  • the antibodies provided herein may be of any immunoglobulin isotype, such as IgG (e.g., IgGl, IgG2, IgG3, or IgG4).
  • the antibodies preferably comprise a human IgG (e.g., IgGl) constant region.
  • the IgG constant region may comprise mutations that improve the therapeutic potential of the antibody, such as mutations that reduce or eliminate effector functions of the antibody (see, e.g., Wang et al., Protein Cell (2016) 9(l):63-73).
  • the antibody may comprise a human IgGl constant region with the mutation(s) L235E, L234A/L235A (“LALA” mutations), or L234A/L235A/G237A (“LALAGA” mutations); M252Y/S254T/T256E (“YTE” mutations); and/or S298N/T299A/Y300S (“NNAS” mutations); in any combination.
  • the IgG constant region may comprise mutations that improve the serum half-life of the antibody, such as the M428L and/or N434S mutations.
  • the IgG constant region may comprise mutations that improve manufacturing and yield of the antibody, such as H435R and Y436F mutations, which reduce binding to protein A and thus are advantageous for antibody purification.
  • the IgG constant region may also comprise knob-in-hole mutations (see, e.g., the descriptions herein). Human constant regions with mutation(s) as described above are still considered “human” constant regions herein. Unless otherwise indicated, all residue numbers in IgG constant regions are Eu numbers.
  • an IgG heavy chain constant region in combination with a light chain constant region, may additionally or alternatively comprise CR3/NN3 charge-pair mutations that facilitate specific heavy and light chain pairing (CR3: T187E mutation in the heavy chain constant region and N137K/S114A mutations in the light chain constant region; NN3: K213E and K218D mutations in the heavy chain constant region and E123K and D122K mutations in the light chain constant region).
  • the monospecific or multispecific antibody herein comprises a human IgGl constant region comprising mutation(s) selected from a) L234A and L235A, b) H435R and Y436F, and c) both a) and b).
  • the monospecific or multispecific antibody herein comprises a first human IgGl constant region comprising L234A and L235A mutations and a second human IgGl constant region comprising L234A, L235A, H435R, and Y436F mutations.
  • the first and second human IgGl constant regions herein may also comprise knob-in-hole mutations, e.g., as described herein.
  • Exemplary knob mutations may comprise S354C and/or T336W.
  • Exemplary hole mutations may comprise Y349C, T366S, L368A, Y407V, or any combination thereof.
  • one of the human IgGl constant regions may comprise knob mutations of S354C and T336W
  • the other human IgGl constant region e.g., the second constant region, with mutations as described above
  • the monospecific or multispecific antibody herein comprises a human IgGl constant region comprising any one of SEQ ID NOs: 27-29.
  • the monospecific or multispecific antibody may comprise, e.g., two human IgGl constant regions both comprising SEQ ID NO: 27, or a first human IgGl constant region comprising SEQ ID NO: 28 and a second human IgGl constant region comprising SEQ ID NO: 29.
  • the binding proteins herein are antigen-binding fragments of full (tetrameric) antibodies.
  • the term “antigen-binding fragment” or “antigen-binding portion” herein encompasses genetically engineered and/or otherwise modified forms of immunoglobulins that do not have the conventional full-length tetrameric structure.
  • the term encompasses intrabodies, peptibodies, diabodies, triabodies, tetrabodies, Fv, Fab, Fab’, Fab’ - SH, F(ab’)2, single-chain antibody molecules (e.g., scFv or sFv), tandem di-scFv, and tandem tri-scFv.
  • the present tau-binding proteins bind specifically to human tau. “Specifically” herein indicates that binding proteins bind to their target with an affinity described herein or higher.
  • KD target binding affinity
  • SPR surface plasmon resonance
  • BLI bio-layer interferometry
  • Flow cytometry assay e.g., FACS
  • FACS Flow cytometry assay
  • the binding proteins of the present disclosure are tau- binding proteins comprising anti-tau binding domains, such as anti-tau antibodies or antigenbinding fragments thereof.
  • the anti-tau binding domain competes for binding with, or binds to the same epitope as, an anti-tau antibody comprising VH and VL that comprise SEQ ID NOs: 7 and 8, respectively. In some embodiments, the anti-tau binding domain competes for binding with, or binds to the same epitope as, an anti-tau antibody comprising VH and VL that comprise SEQ ID NOs: 15 and 16, respectively.
  • the anti-tau binding domain comprises HCDR1-3 and LCDR1-3 set forth in
  • the anti-tau binding domain herein comprises the HCDR1-3 in a VH comprising SEQ ID NO: 7 and the LCDR1-3 in a VL comprising SEQ ID NO: 8, or the H-CDR1-3 in a VH comprising SEQ ID NO: 15 and the LCDR1-3 in a VL comprising SEQ ID NO: 16.
  • the assignment of CDR regions may be in accordance with any method known in the art, such as IMGT® , Kabat, Chothia, Martin, Contact, or AHo definitions, or any combination of any of these definitions (Kabat plus Chothia, for example). Examples of CDR definitions under different methods are shown below for the VH and VL of exemplified anti-tau antibodies 22-MTBR and 523-MTBR:
  • HCDR1-3 sequences of SEQ ID NOs: 1, 2, and 3, respectively may be replaced in any embodiment described herein by SEQ ID NOs: 47, 48, and 49, respectively;
  • LCDR1-3 sequences of SEQ ID NOs: 4, 5, and 6, respectively may be replaced in any embodiment described herein by SEQ ID NOs: 55, 56, and 6, respectively; or
  • HCDR1-3 sequences of SEQ ID NOs: 9, 10, and 11, respectively may be replaced in any embodiment described herein by
  • LCDR1-3 sequences of SEQ ID NOs: 12, 13, and 14, respectively may be replaced in any embodiment described herein by SEQ ID NOs: 68, 69, and 14, respectively; or SEQ ID NOs: 70, 71 and 72, respectively.
  • HCDR1 may be defined by the Kabat method
  • HCDR2 may be defined by the IMGT® method, etc.
  • These methods or combinations of methods may be used to define the CDRs in the VH or VL of any binding domain herein.
  • the anti-tau binding domain comprises a VH and/or a VL at least 80% (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NOs: 7 and 8, respectively (optionally wherein any differences from the reference sequence(s) do not occur in the CDRs).
  • the anti-tau binding domain comprises a VH and/or a VL at least 80% (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NOs: 15 and 16, respectively (optionally wherein any differences from the reference sequence(s) do not occur in the CDRs).
  • the anti-tau binding domain herein comprises a VH and a VL with charge mutations to facilitate correct VH/VL pairing, such as in a multispecific (e.g. bispecific) context.
  • charge mutations may appear in, e.g., VH/VL pairs wherein the VH domain comprises a Q39K mutation and the VL comprises a Q37E mutation, or wherein the VH domain comprises a Q39E mutation and the VL comprises a Q37K mutation.
  • the anti-tau binding domain comprises a VH at least 80% (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 73 or 74, and/or a VL at least 80% (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 75 or 76 (optionally wherein any differences from the reference sequence(s) do not occur in the CDRs).
  • the anti-tau binding domain comprises a VH selected from SEQ ID NOs: 7, 73, and 74 and a VL selected from any one of SEQ ID NOs: 8, 75, and 76.
  • the anti-tau binding domain comprises a VH and/or a VL at least 80% (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NOs: 73 and 75, respectively (optionally wherein any differences from the reference sequence(s) do not occur in the CDRs).
  • the anti-tau binding domain comprises a VH and/or a VL at least 80% (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NOs: 74 and 76, respectively (optionally wherein any differences from the reference sequence(s) do not occur in the CDRs).
  • the anti-tau binding domain comprises a VH and a VL set forth in SEQ ID NOs: 7 and 8, respectively.
  • the anti-tau binding domain comprises a VH and a VL set forth in SEQ ID NOs: 73 and 75, respectively.
  • the anti-tau binding domain comprises a VH and a VL set forth in SEQ ID NOs: 74 and 76, respectively.
  • the anti-tau binding domain comprises a VH and a VL set forth in SEQ ID NOs: 15 and 16, respectively.
  • the present disclosure provides an anti-tau antibody comprising an anti-tau binding domain herein, or an antigen-binding fragment of the antibody.
  • the anti-tau antibody may comprise any heavy and light chain constant regions described herein.
  • the anti-tau antibody comprises a human IgGl heavy chain constant region, optionally with mutations as described herein.
  • the human IgGl heavy chain constant region may comprise any one of SEQ ID NOs: 27-29.
  • the antibody comprises two heavy chain constant regions both comprising SEQ ID NO: 27, or said sequence with “LALA” mutations; additionally or alternatively, one heavy chain constant region may have “RF” mutations.
  • the antibody comprises a first heavy chain constant region comprising SEQ ID NO: 28 and a second heavy chain constant region comprising SEQ ID NO: 29.
  • the anti-tau antibody comprises a human kappa or lambda light chain constant region.
  • the anti-tau antibody comprises one or two human kappa light chain constant regions, e.g., comprising SEQ ID NO: 30.
  • an anti-tau antibody herein may comprise an HC comprising SEQ ID NOs: 7 and 27, or sequences at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to said sequences, and an LC comprising SEQ ID NOs: 8 and 30, or sequences at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to said sequences; an HC comprising SEQ ID NOs: 73 and 27, or sequences at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to said sequences, and an LC comprising SEQ ID NOs: 75 and 30, or sequences at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to said sequences; an HC comprising
  • one HC of the anti-tau antibody may comprise “RF” mutations.
  • Percent (%) sequence identity or homology with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways using available computer software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In some embodiments, the query sequence has at least 70% (e.g., at least 75, 80, 85, 90, or 95%) of the length of the reference sequence. For purposes herein, sequence homology or identity may be identified by BLAST, a bioinformatics program available at the server of the United States National Center for Biotechnology Information, using default parameters.
  • the tau-binding proteins herein bind to full-length tau monomers, full-length tau fibrils, monomers of the tau MTBR, and/or fibrils of the tau MTBR (e.g., to all of said tau forms).
  • the tau-binding proteins bind to tau monomers and/or tau fibrils, e.g., with an ECso of no more than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 nM (e.g., no more than 0.3 nM) as determined by ELISA.
  • the tau-binding proteins bind to tau monomers and/or tau fibrils, e.g., with an ECso of 0.01-10 nM, e.g., 0.1-0.4 nM, as determined by ELISA.
  • the tau- binding proteins may bind to tau monomers and/or fibrils with an EC50 of 0.1-0.25 nM or 0.25-0.35 nM.
  • the tau-binding proteins bind to tau monomers and/or tau fibrils, e.g., with an EC50 as determined in Example 1.
  • the tau-binding proteins herein bind to both tau monomers and tau fibrils with ECsosZECso ranges as described above.
  • the tau-binding proteins herein bind to an epitope of tau comprising the sequence SLDNITHVPG (SEQ ID NO: 79).
  • a tau-binding protein herein may bind the sequence QSKIGSLDNITHVPG (SEQ ID NO: 80), and may optionally further bind the sequence APVPMPDLKNVKSKI (SEQ ID NO: 81) and may optionally further bind the sequence NVQSKCGSKDNIKHV (SEQ ID NO: 82) and/or SKVTSKCGSLGNIHH (SEQ ID NO: 83).
  • a tau-binding protein herein may bind the sequence SLDNITHVPGGGNKK (SEQ ID NO: 84), and may optionally further bind the sequence APVPMPDLKNVKSKI (SEQ ID NO: 81) and may optionally further bind the sequence SKVTSKCGSLGNIHH (SEQ ID NO: 83), QSKIGSLDNITHVPG (SEQ ID NO: 80), and/or THVPGGGNKKIETHK (SEQ ID NO: 85), in any combination.
  • the tau-binding proteins herein bind to tau pathological forms in progressive supranuclear palsy, Alzheimer’s disease, Pick’s disease, or any combination thereof.
  • the tau-binding proteins herein inhibit tau seeding and aggregation, e.g., with an ICso of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nM (e.g., no more than 4 nM) as determined by homogeneous time resolved fluorescence (HTRF®).
  • the tau-binding proteins herein inhibit tau seeding and aggregation with an IC50 of 1-5 nM, e.g., 2-4 nM.
  • the tau-binding proteins herein inhibit the aggregation of endogenous full-length tau induced by aggregated wild-type tau MTBR (determined, for example, in a U20S cell assay as described in Example 1), e.g., with an IC50 of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nM, e.g., no more than 4 nm).
  • the tau- binding proteins herein inhibit the aggregation of endogenous full-length tau induced by aggregated wild-type tau MTBR with an IC50 of 1-5 nM, e.g., 2-4 nM.
  • the tau-binding proteins herein inhibit tau seeding and aggregation in vitro in a fluorescence resonance energy transfer (FRET) assay (e.g., as described in Example 3).
  • FRET fluorescence resonance energy transfer
  • the tau-binding proteins herein inhibit uptake of tau aggregates (such as tau PFFs) into neurons (e.g., as described in Example 6).
  • the tau-binding proteins herein reduce hippocampal and/or cortical tau pathology, and/or decrease neurofilament light (NFL) in cerebrospinal fluid, in a mouse model for tau aggregation (e.g., in a THY-Tau22 mouse model assay as described in Example 1).
  • the tau-binding proteins herein reverse cognitive deficits and/or improve motor deficits in a mouse model for tau aggregation (e.g., in a THY-Tau22 mouse model assay as described in Example 1).
  • the tau-binding protein herein has one or more of the following properties: a) binds to full-length tau monomers with an ECso of 0.1-0.4 nM as determined by ELISA; b) binds to full-length tau fibrils with an ECso of 0.1-0.4 nM as determined by ELISA; c) binds to tau microtubule binding region (MTBR) monomers; d) binds to tau MTBR fibrils; e) binds to tau pathological forms in progressive supranuclear palsy, Alzheimer’s disease, Pick’s disease, or any combination thereof; f) inhibits tau seeding and aggregation as determined by homogeneous time resolved fluorescence (HTRF); g) inhibits aggregation of endogenous full-length tau induced by aggregated wild-type tau MTBR in vitro.
  • HTRF homogeneous time resolved fluorescence
  • h reduces tau pathology in the hippocampus, cortex, or both in THY-Tau22 mice; i) inhibits tau seeding and aggregation as determined by in cell fluorescence resonance energy transfer (FRET); j) inhibits uptake of tau aggregates into neurons; k) decreases neurofilament light (NFL) in the cerebrospinal fluid of THY-Tau22 mice; l) reverses cognitive deficits in THY-Tau22 mice; m) improves motor deficits in THY-Tau22 mice; or n) any combination of a)-m).
  • FRET cell fluorescence resonance energy transfer
  • the tau-binding protein has properties a)-m), or a)-i) and k)- m) (e.g., 22-MTBR).
  • the bispecific antibody has at least properties a)- h) (e g., 523-MTBR).
  • anti-tau binding domains herein may be for delivery to the CNS, e.g., to the brain.
  • the anti-tau binding domains herein thus may form part of a brain- targeted tau-binding protein, such as a binding protein comprising a moiety that facilitates transport across the BBB (e.g., one or more cell-penetrating peptides, an Fc region modified to bind to a CNS target, or a second binding domain that binds to an endothelial cell receptor of the BBB).
  • a brain-targeted tau-binding protein herein may comprise an anti-tau binding domain herein associated with a cell-penetrating peptide.
  • Cell-penetrating peptides are short peptides that can penetrate biological membranes, facilitating delivery of associated cargos. Where cell-penetrating peptides are targeted to the CNS, they can promote transport of a given cargo across the BBB and into the brain.
  • an anti-tau binding domain herein may be linked to a cell-penetrating peptide that binds to a CNS target (e.g., TfR or another endothelial cell receptor of the BBB, such as those described herein).
  • the cell-penetrating peptide may be, e.g., a peptide described in Kang et al., Drug Delivery (2022) 29(l):2375-85 (incorporated herein by reference in its entirety), such as the T7 peptide.
  • the anti- tau binding domain of the brain-targeted tau-binding protein may be or form part of, e.g., a bivalent antibody, a monovalent antibody, Fab, Fab’, F(ab’)2, or scFv.
  • a brain-targeted tau-binding protein herein may comprise an anti-tau binding domain herein associated with a moiety that facilitates receptor-mediated transcytosis (RMT) at the BBB, e.g., an Fc region or a fragment thereof wherein one or both chains of the Fc region, preferably one chain, are engineered to bind to an endothelial cell receptor of the BBB (e.g., TfR or another ECR, such as those described herein).
  • the Fc region is derived from a human IgGl constant region, and may comprise KIH mutations and/or mutations to reduce or eliminate effector function (e.g., “LALA” mutations).
  • the Fc region may bind to TfR, and may be, e.g., a BBB transport vehicle (TV) as described in Kariolis et al., Sci TranslMed. (2020) 12(545):eaayl359 or Arguello et al., J Exp Med. (2022) 219(3):e20211057 (incorporated herein by reference in their entirety).
  • the anti-tau binding domain of the brain-targeted tau-binding protein may be or form part of, e.g., a bivalent anti-tau antibody or an antigen-binding fragment thereof comprising the Fc region as defined herein.
  • a brain-targeted tau-binding protein herein may be or comprise a multispecific, in particular a bispecific, binding protein, as described below.
  • the present disclosure also provides tau-binding proteins that are multispecific, e.g., bispecific.
  • a multispecific binding protein e.g., a bispecific antibody
  • the brain receptor is an endothelial cell receptor of the blood-brain barrier (BBB), for example, a transferrin receptor, insulin receptor, low-density lipoprotein receptor, folate receptor, etc.
  • the domain of the multispecific binding protein that binds to the ECR of the BBB may act as a shuttle to transport the anti-tau binding domain across the BBB.
  • the ECR is transferrin 1 (TfR).
  • the anti-tau binding domain of the multispecific binding protein may be, e.g., an anti-tau binding domain as described herein.
  • the domain of the multispecific binding protein (e.g., bispecific antibody) that binds to the CNS target (“anti-CNS target domain”) binds to an ECR (“anti-ECR binding domain”) such as TfR (“anti-TfR binding domain”).
  • anti-TfR binding domain may be an anti-TfR antibody or an antigenbinding fragment thereof disclosed in PCT Patent Application PCT/IB2023/062266.
  • the anti-TfR binding domain binds to human TfR (hTR) with a KD of 1-50 nM (e.g., 1-30 nM or 1-20 nM) as determined by SPR. In some embodiments, the anti-TfR binding domain binds to cynomolgus TfR (cTfR) with a KD of 1-200 nM (e.g., 30-170 nM or 50-150 nM) as determined by SPR.
  • hTR human TfR
  • cTfR cynomolgus TfR
  • Sequence identifiers for exemplary anti-tau and anti-TfR binding domains, which may be used in the multispecific binding proteins (e.g., bispecific antibodies) herein, are shown in the table below:
  • the anti-TfR binding domain competes for binding with or binds to the same epitope of TfR as an anti-TfR antibody comprising VH and VL sequences set forth in SEQ ID NOs: 25 and 26, respectively.
  • the anti-TfR binding domain comprises HCDR1-3 and LCDR1-3 set forth in SEQ ID NOs: 19, 20, 21, 22, 23, and 24, respectively.
  • the anti-TfR binding domain comprises a VH at least 80% (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 25 or 77 and a VL at least 80% (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 26 or 78.
  • the anti-TfR binding domain may comprise a VH and a VL at least 80% (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to:
  • the anti-TfR binding domain comprises a VH of SEQ ID NO: 25 or 77 and a VL of SEQ ID NO: 26 or 78. In certain embodiments, the anti-TfR binding domain comprises a VH and a VL set forth in
  • any combination of an anti-tau binding domain and an anti-TfR binding domain described herein is contemplated for the multispecific binding proteins (e.g., bispecific antibodies) herein.
  • a multispecific binding protein herein may comprise an anti-tau binding domain comprising
  • HCDR1-3 and LCDR1-3 set forth in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; or - HCDR1-3 and LCDR1-3 set forth in SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively; and an anti-TfR binding domain comprising HCDR1-3 and LCDR1-3 set forth in SEQ ID NOs: 19, 20, 21, 22, 23, and 24, respectively.
  • a multispecific binding protein herein may comprise an anti-tau binding domain comprising a VH and a VL set forth in SEQ ID NOs: 7 and 8, respectively, optionally wherein
  • the VH has a Q39K mutation and the VL has a Q37E mutation
  • the VH has a Q39E mutation and the VL has a Q37K mutation; or a VH and a VL set forth in SEQ ID NOs: 15 and 16, respectively; and an anti-TfR binding domain comprising a VH and a VL set forth in SEQ ID NOs: 25 and 26, respectively, optionally wherein the VH has a Q39K mutation and the VL has a Q38E mutation.
  • a multispecific binding protein herein may comprise an anti-tau binding domain comprising a VH and a VL set forth in SEQ ID NOs: 7 and 8, respectively; a VH and a VL set forth in SEQ ID NOs: 73 and 75, respectively; a VH and a VL set forth in SEQ ID NOs: 74 and 76, respectively; or a VH and a VL set forth in SEQ ID NOs: 15 and 16, respectively; and an anti-TfR binding domain comprising a VH and a VL set forth in SEQ ID NOs: 25 and 26, respectively; or a VH and a VL set forth in SEQ ID NOs: 77 and 78, respectively.
  • the multispecific binding protein may comprise two anti-tau binding domains, which may have the same VH and VL pair or different VH and VL pairs.
  • the multispecific binding protein may comprise a first anti-tau binding domain comprising a VH and a VL set forth in SEQ ID NOs: 73 and 75, respectively; and a second anti-tau binding domain comprising a VH and a VL set forth in SEQ ID NOs: 74 and 76, respectively.
  • the multispecific binding protein may also comprise an anti-TfR binding domain comprising a VH and a VL set forth in SEQ ID NOs: 77 and 78, respectively.
  • the multispecific binding protein is a multispecific antibody (e.g., a bispecific antibody)
  • it may be of any immunoglobulin isotype, such as human IgG (e.g., IgGl, IgG2, IgG3, or IgG4).
  • the multispecific antibodies herein may comprise a human IgGl constant region, e.g., with mutations to improve the clinical potential of the antibody (such as mutations that reduce or eliminate effector functions, improve the serum half-life of the antibody, or improve manufacturing and yield of the antibody, as described herein, in any combination).
  • mutations may be introduced to the heavy chains to physically (e.g., by steric hinderance, “knobs” into “holes”) or biochemically (e.g., by electrostatic interactions) deter coupling of heavy chains of the same type.
  • knobs-in-holes (KIH) mutations can be introduced to create a “knob” heavy chain and a “hole” heavy chain that preferentially pair with each other.
  • Exemplary KIH mutations comprise S354C and T366W in one heavy chain and Y349C/T366S/L368A/Y407V in the other heavy chain. See also WO 2009/089004 and U.S. Pat. 8,642,745; and Brinkmann and Kontermann, MAbs. (2017) 9(2): 182-212.
  • a multispecific antibody herein is of human IgGl isotype subclass and comprises two different heavy chain constant regions comprising: a) KIH mutations, (e.g., S354C and T336W knob mutations or Y349C, T366S, L368A, and Y407V hole mutations), b) L234A and L235A (“LALA”) mutations, c) H435R and Y436F (“RF”) mutations, or d) any combination of a)-c).
  • KIH mutations e.g., S354C and T336W knob mutations or Y349C, T366S, L368A, and Y407V hole mutations
  • LALA L234A and L235A
  • RF H435R and Y436F
  • the multispecific antibody comprises a first heavy chain constant region with a) (e.g., knob) mutations and b) mutations, and a second heavy chain constant region with a) (e.g., hole) mutations, b) mutations, and c) mutations.
  • the multispecific antibody comprises a knob heavy chain constant region comprising SEQ ID NO: 29 and a hole heavy chain constant region comprising SEQ ID NO: 28.
  • a multispecific antibody herein comprises a human kappa or lambda light chain constant region.
  • the multispecific antibody comprises a kappa light chain constant region of SEQ ID NO: 30.
  • a multispecific binding protein herein may be monovalent or bivalent for tau and monovalent or bivalent for the CNS target (e.g., TfR), in any combination.
  • the multispecific binding protein may be bivalent for tau and monovalent for the CNS target, monovalent for tau and bivalent for the CNS target, monovalent for both tau and the CNS target, or bivalent for both tau and the CNS target.
  • the anti -tau binding domain, the anti-CNS target binding domain, or both may be full antibodies, antigenbinding fragments (e.g., Fab or scFv), or any combination thereof.
  • the anti-tau and anti-CNS target portions of the multispecific binding protein, or regions thereof may be functionally linked, e.g., by noncovalent association, chemical coupling, protein fusion, etc.
  • the portions or regions thereof are genetically linked (peptide bond) without a peptide linker.
  • the portions or regions thereof are linked through a peptide linker.
  • the peptide linker may, for example, predominantly include the following amino acid residues: Gly, Ser, Ala, or Thr.
  • the peptide linker may have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain their respective desired activity.
  • the linker is 1 to 50 (e.g., 1 to 30, 1 to 20, 1 to 10 or 1 to 5) amino acids in length.
  • Useful linkers include glycine-serine polymers, such as, for example, (GS)n, (GSGGS)n (SEQ ID NO: 87), (GGGGS)n (SEQ ID NO: 88), and (GGGS)n (SEQ ID NO: 89), where n is an integer of at least one (an example is GGGGSGGGGS (SEQ ID NO: 86)); glycine-alanine polymers; alanine-serine polymers; XTEN linkers; and other flexible linkers.
  • the linker is GGGG (SEQ ID NO: 90) or SGSGGGG (SEQ ID NO: 91).
  • Additional exemplary linkers for linking antibody fragments or single-chain variable fragments can include AAEPKSS (SEQ ID NO: 92), AAEPKSSDKTHTCPPCP (SEQ ID NO: 93), or GGGGDKTHTCPPCP (SEQ ID NO: 94).
  • the multispecific binding protein may comprise sets of peptide linkers to link components of binding domains (e.g., VH and/or VL domains) and/or to link such components to constant regions.
  • binding domains e.g., VH and/or VL domains
  • linker sets might comprise four linkers: two to connect a first light chain variable domain (VL1), a second light chain variable domain (VL2), and a light chain constant region (CL), and two to connect a first heavy chain variable domain (VH1), a second heavy chain variable domain (VH2), and a heavy chain constant region (CH).
  • VL1 first light chain variable domain
  • VL2 second light chain variable domain
  • CL light chain constant region
  • VH1 first heavy chain variable domain
  • VH2 second heavy chain variable domain
  • CH heavy chain constant region
  • the variable domains, constant regions, and linkers may be in the following conformation:
  • linker sets include 5-5-5-5 linkers (wherein linkers 1-4 have the amino acid sequences of DKTHT, DKTHT, DKTHT, and DKTHT, respectively), 7-5- 1-2 linkers (wherein linkers 1-4 have the amino acid sequences of GQPKAAP, TKGPS, S, and RT, respectively), and 10-10-0-0 linkers (wherein linkers 1 and 2 have the amino acid sequences of GGGGSGGGGS and GGGGSGGGGS, and there are no linkers 3 and 4). [0132] Exemplary bispecific antibody formats are discussed below.
  • the CNS target is a brain receptor, such as an endothelial cell receptor of the BBB (e.g., insulin receptor, low-density lipoprotein receptor, folate receptor, etc.).
  • the CNS target is TfR.
  • the bispecific antibody is bivalent for tau and monovalent for the CNS target.
  • the bispecific antibody may comprise a first arm that comprises a first anti-tau binding domain and an anti-CNS target binding domain (e.g., an anti-TfR binding domain as described herein), and a second arm that comprises a second anti-tau binding domain.
  • the HCDR1-3 and LCDR1-3 of the first and second anti-tau binding domains are the same.
  • the first and second anti-tau binding domains are the same except for having different pairs of charge mutations (e.g., charge mutations described herein).
  • the first and second anti-tau binding domains are the same.
  • the bispecific antibody comprises
  • a first heavy chain comprising the VH of a first anti-tau binding domain described herein and the VH of an anti-CNS target binding domain;
  • the VH of the anti-CNS target binding domain is N- terminal to the VH of the first anti-tau binding domain
  • the VL of the first anti-tau binding domain is N-terminal to the VL of the anti-CNS target binding domain.
  • the VH of the first anti-tau binding domain is N-terminal to the VH of the anti- CNS target binding domain
  • the VL of the anti-CNS target binding domain is N-terminal to the VL of the first anti-tau binding domain.
  • the N-terminal VL may be connected to the C-terminal VL via a first linker, and the C-terminal VL may be connected to the light chain constant region via a second linker.
  • the first and second linkers may be, e.g., linkers as described herein. In certain embodiments, the first and second linkers are the same, and have the sequence GGGGSGGGGS (SEQ ID NO: 86).
  • the N-terminal VH may be connected to the C-terminal VH via a third linker, and the C-terminal VH may be connected to the heavy chain constant region via a fourth linker.
  • the first, second, third, and fourth linkers may, in particular embodiments, be 5-5-5- 5, 7-5-1-2, or 10-10-0-0 linker sets.
  • the first and second heavy chains may comprise KIH mutations as described herein (e.g., knob mutations of S354C and T336W and hole mutations of Y349C, T366S, L368A, and Y407V), optionally wherein the first heavy chain comprises the knob mutations and the second heavy chain comprises the hole mutations.
  • the first and/or second heavy chains e.g., both heavy chains
  • the first and/or second heavy chains e.g., both heavy chains
  • one heavy chain e.g., the heavy chain with hole mutations
  • the bispecific antibody may comprise a first heavy chain amino acid sequence comprising SEQ ID NO: 41 and a first light chain amino acid sequence comprising SEQ ID NO: 42, and a second heavy chain amino acid sequence comprising SEQ ID NO: 43 and a second light chain amino acid sequence comprising SEQ ID NO: 44; or a first heavy chain amino acid sequence comprising SEQ ID NO: 37 and a first light chain amino acid sequence comprising SEQ ID NO: 38, and a second heavy chain amino acid sequence comprising SEQ ID NO: 39 and a second light chain amino acid sequence comprising SEQ ID NO: 40.
  • the first heavy chain may pair with the first light chain
  • the second heavy chain may pair with the second light chain
  • the bispecific antibody may comprise a first arm that comprises first and second anti-tau binding domains and a second arm that comprises an anti- CNS target binding domain (e.g., an anti-TfR binding domain as described herein).
  • the HCDR1-3 and LCDR1-3 of the first and second anti-tau binding domains are the same.
  • the first and second anti-tau binding domains are the same except for having different pairs of charge mutations (e.g., charge mutations described herein).
  • the first and second anti-tau binding domains are the same.
  • the bispecific antibody comprises
  • the VH of the first anti-tau binding domain is N-terminal to the VH of the second anti-tau binding domain
  • the VL of the second anti-tau binding domain is N-terminal to the VL of the first anti-tau binding domain, or vice-versa for the first and second anti-tau binding domains.
  • the N-terminal VL may be connected to the C-terminal VL via a first linker
  • the C-terminal VL may be connected to the light chain constant region via a second linker.
  • the first and second linkers may be, e.g., linkers as described herein.
  • the first and second linkers are the same, and have the sequence GGGGSGGGGS (SEQ ID NO: 86).
  • the N- terminal VH may be connected to the C-terminal VH via a third linker, and the C-terminal VH may be connected to the heavy chain constant region via a fourth linker.
  • the first, second, third, and fourth linkers may, in particular embodiments, be 5-5-5-5, 7-5- 1-2, or 10- 10-0-0 linker sets.
  • the first and second heavy chains may comprise KIH mutations as described herein (e.g., knob mutations of S354C and T336W and hole mutations of Y349C, T366S, L368A, and Y407V), optionally wherein the first heavy chain comprises the knob mutations and the second heavy chain comprises the hole mutations.
  • the first and/or second heavy chains e.g., both heavy chains
  • the first and/or second heavy chains e.g., both heavy chains
  • one heavy chain e.g., the heavy chain with hole mutations
  • the bispecific antibody may comprise a first heavy chain amino acid sequence comprising SEQ ID NO: 31 and a first light chain amino acid sequence comprising SEQ ID NO: 32, and a second heavy chain amino acid sequence comprising SEQ ID NO: 33 and a second light chain amino acid sequence comprising SEQ ID NO: 34; or a first heavy chain amino acid sequence comprising SEQ ID NO: 35 and a first light chain amino acid sequence comprising SEQ ID NO: 36, and a second heavy chain amino acid sequence comprising SEQ ID NO: 33 and a second light chain amino acid sequence comprising SEQ ID NO: 34.
  • the first heavy chain may pair with the first light chain
  • the second heavy chain may pair with the second light chain
  • the multispecific binding protein (e.g., bispecific antibody herein) binds to human tau with an ECso of no more than 50, 40, 35, 30, 25, 20, 15, 10, or 5 pM (e.g., no more than 35 pM) as determined by ELISA. In certain embodiments, the multispecific binding protein binds to human tau with an ECso of 1-40 pM (e.g., 5-35 pM, or 10-15 pM) as determined by ELISA.
  • the multispecific binding protein herein binds to human TfR (hTfR) with an ECso of no more than 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1 nM (e.g., no more than 25 nM) as determined by FACS.
  • the multispecific binding protein binds to hTfR with an ECso of 1-50 nM (e.g., 3-40 nM, or 5-25 nM) as determined by FACS.
  • the multispecific binding protein herein binds to hTfR with a KD of no more than 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nM (e.g., no more than 30 nM) as determined by SPR. In certain embodiments, the multispecific binding protein binds to hTfR with a KD of 1-50 nM (e.g., 5- 30 nM) as determined by SPR.
  • the multispecific binding protein herein binds to hTfR with a KD of no more than 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nM (e.g., no more than 30 nM) as determined by OctetTM.
  • the multispecific binding protein binds to hTfR with a KD of 10-40 nM (e.g., 15-30 nM) as determined by OctetTM.
  • the multispecific binding protein herein binds to cynomolgus TfR (cTfR) with an ECso of no more than 250, 200, 175, 150, 125, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nM (e.g., no more than 170 nM) as determined by FACS.
  • the multispecific binding protein binds to cTfR with an ECso of 1-250 nM (e.g., 15-150 nM) as determined by FACS.
  • the multispecific binding protein binds to cTfR with an ECso of 1-20 nM as determined by FACS.
  • the multispecific binding protein herein binds to cTfR with a KD of no more than 250, 200, 175, 150, 125, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nM (e.g., no more than 175 nM) as determined by SPR.
  • the multispecific binding protein binds to cTfR with a KD of 1-200 nM (e.g., 10-200 nM, or 10-175 nM) as determined by SPR.
  • the multispecific binding protein herein binds to cTfR with a KD of no more than 250, 200, 175, 150, 125, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nM (e.g., no more than 125 nM) as determined by OctetTM.
  • the multispecific binding protein binds to cTfR with a KD of 50-200 nM (e.g., 100-150 nM, or 100-125 nM) as determined by OctetTM.
  • the multispecific binding protein herein binds to tau, hTfR, and/or cTfR with binding affinities (i.e., an ECso or KD) as determined in Example 3.
  • the multispecific binding protein herein e.g., a bispecific antibody herein
  • has improved brain penetrance over the monospecific anti-tau antibody e.g., by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12- fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold (such as by at least 3-fold).
  • the multispecific binding protein herein e.g., a bispecific antibody herein
  • the multispecific binding protein achieves brain exposure that is at least 5- to 20-fold (e.g., 3- to 16-fold, or 7- to 16-fold) higher than that of the monospecific anti-tau antibody.
  • the multispecific binding protein herein e.g., a bispecific antibody herein
  • reduces tau seeding e.g., as determined by the assay described in Example 1 or 3.
  • the multispecific binding protein herein e.g., a bispecific antibody herein
  • inhibits uptake of tau aggregates into neurons e.g., as determined by the assay described in Example 6).
  • the multispecific binding protein herein reduces the number of cells positive for antibody AT8 (“AT8 + cells”) in the cortex of hTfR knock-in mice (e.g., as determined by the assay described in Example 3). AT8 binds to hyperphosphorylated tau.
  • a multispecific binding protein with any combination of the above properties is also contemplated.
  • the multispecific binding protein herein has one or more of the following properties: a) binds to human tau with an EC50 of 5-35 pM as determined by ELISA; b) binds to human TfR with an EC50 of no more than 1-50 nM as determined by FACS; c) binds to cynomolgus TfR with an ECso of no more than 1-250 nM as determined by FACS; d) binds to human TfR with a KD of 1-50 nM as determined by SPR; e) binds to cynomolgus TfR with a KD of 1-200 nM as determined by SPR; f) binds to human TfR with a KD of 10-40 nM as determined by OctetTM; g) binds to cynomolgus TfR with a KD of 50-200 nM
  • the multispecific binding protein has all of properties a)-l). In some embodiments, the multispecific binding protein has at least properties a)-h).
  • the present disclosure provides a bispecific binding protein comprising two heavy chains and two light chains, wherein one pair of heavy and light chains forms one arm, and the other pair of heavy and light chains forms another arm, of the bispecific binding protein, wherein one arm comprises a first anti-tau binding domain and an anti-CNS target binding domain (e.g., an anti-TfR binding domain), and the other arm comprises a second anti-tau binding domain.
  • one arm comprises a first anti-tau binding domain and an anti-CNS target binding domain (e.g., an anti-TfR binding domain)
  • the other arm comprises a second anti-tau binding domain.
  • the VH of the anti-CNS target binding domain (e.g., anti-TfR binding domain) is N- terminal to the VH of the first anti-tau binding domain
  • the VL of the first anti-tau binding domain is N-terminal to the VL of the anti-CNS target binding domain.
  • the first and second anti-tau binding domains may have the same HCDR1-3 and same LCDR1-3, and in some embodiments are the same.
  • the first anti-tau binding domain, the second anti-tau binding domain, or both bind specifically to tau fibrils and/or bindsto the MTBR region of tau.
  • the anti-tau binding domain(s) may have one or more of properties a)-e) below, in any combination (e.g., all of a)-e)): a) binds to full-length tau monomers with an ECso of 0.1-0.4 nM as determined by ELISA; b) binds to full-length tau fibrils with an ECso of 0.1-0.4 nM as determined by ELISA; c) binds to tau microtubule binding region (MTBR) monomers; d) binds to tau MTBR fibrils; and e) binds to tau pathological forms in progressive supranuclear palsy, Alzheimer’s disease, Pick’s disease, or any combination thereof.
  • properties a)-e) below, in any combination (e.g., all of a)-e)): a) binds to full-length tau monomers with an ECso of 0.1-0.4 nM as determined by ELISA; b) bind
  • the first and/or second anti-tau domain may be, e.g., an anti-tau domain described herein.
  • the anti-tau domain may comprise VH and VL domains comprising HCDR1-3 and LCDR1-3 that comprise the amino acid sequences of SEQ ID NOs: 1-6, respectively, or SEQ ID NOs: 9-14, respectively, optionally wherein the VH and VL domains comprise amino acid sequences that are or are at least 90% identical to SEQ ID NOs: 7 and 8, 15 and 16, 73 and 75, or 74 and 76, respectively.
  • the anti-tau domain may comprise HCDR1-3 and LCDR1-3 comprising the amino acid sequences of SEQ ID NOs: 1-6, respectively, or VH and VL domains comprising the amino acid sequences of SEQ ID NOs: 7 and 8, 73 and 75, or 74 and 76, respectively.
  • the anti-CNS target binding domain is an anti-TfR binding domain that binds to human TfR (hTR) with a KD of 1-50 nM (e.g., 1-30 nM or 1-20 nM) as determined by SPR, and/or binds to cynomolgus TfR (cTfR) with a KD of 1-200 nM (e.g., 30-170 nM or 50-150 nM) as determined by SPR.
  • hTR human TfR
  • cTfR cynomolgus TfR
  • the anti-TfR binding domain may have one or more of properties a)-f) below, in any combination (e.g., all of a)-f)): a) binds to human TfR with an ECso of 1-50 nM as determined by FACS; b) binds to cynomolgus TfR with an ECso of 1-250 nM as determined by FACS; c) binds to human TfR with a KD of 1-50 nM as determined by SPR; d) binds to cynomolgus TfR with a KD of 1-200 nM as determined by SPR; e) binds to human TfR with a KD of 10-40 nM as determined by OctetTM; f) binds to cynomolgus TfR with a KD of 50-200 nM as determined by OctetTM;
  • the anti-TfR binding domain may be, e.g., an anti-TfR binding described herein.
  • the binding proteins may be produced recombinantly using isolated nucleic acid molecules such as expression constructs encoding each chain of the proteins.
  • Biomolecules e.g., nucleic acid or polypeptide molecules
  • isolated or purified are those that (1) have been separated away from the biomolecules (e.g., nucleic acids of the genomic DNA or cellular RNA, or polypeptides, of their source of origin; and/or (2) do not occur in nature.
  • the encoding sequences for each polypeptide chain may be cloned into a single vector or cloned into separate vectors.
  • Mammalian cell lines available as hosts for expression include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines.
  • ATCC American Type Culture Collection
  • cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells, and yeast cell lines. Cell lines may be selected based on their expression levels.
  • the binding proteins may be isolated and purified from the host cell culture using well known methods, such as centrifugation, ultracentrifugation, protein A, protein G, protein A/G, or protein L purification, and/or ion exchange chromatography.
  • the present disclosure also provides pharmaceutical compositions comprising the binding proteins (e.g., monospecific or multispecific binding proteins) herein.
  • the pharmaceutical compositions may comprise one or more pharmaceutically acceptable excipients, carriers, or diluents.
  • pharmaceutically acceptable with reference to a carrier,” “excipient,” or “diluent” includes appropriate solvents, dispersion media, antibacterial and antifungal agents, isotonic agents, and the like.
  • the pharmaceutical composition is a sterile aqueous solution, and may comprise a buffer; a surfactant; a polyol; an antioxidant; and/or a chelating agent.
  • the pharmaceutical composition is provided in a lyophilized form and is reconstituted before administration.
  • lyophilized antibody formulations may comprise a bulking agent.
  • the pharmaceutical composition may be administered to patients by parenteral administration (e.g., by injection or infusion).
  • parenteral administration e.g., by injection or infusion
  • the pharmaceutical composition may be administered by an intravenous, intracerebral, intracranial, or spinal route.
  • the monospecific and bispecific binding proteins herein are useful in treating a human patient with, or at risk of developing, tauopathy.
  • the binding proteins or pharmaceutical compositions may be used to treat a primary tauopathy (e.g., selected from progressive supranuclear palsy (PSP), frontotemporal lobar degeneration with MAPT mutations, argyrophilic grain disease, corticobasal degeneration, Pick’s disease, globular glial tauopathy, aging-related tau astrogliopathy (ARTAG), and primary age-related tauopathy (PART)) or a secondary tauopathy (e.g., selected from Alzheimer’s disease, chronic traumatic encephalopathy, anti- IgLON5 -related tauopathy, Down syndrome, Niemann-Pick disease type C, and myotonic dystrophy type 1 and 2).
  • the primary tauopathy is PSP.
  • the secondary tauopathy is Alzheimer’s disease.
  • the terms “treat,” “treatment,” and “treating” refers to a deliberate intervention to a physiological disease state resulting in the reduction in severity of a disease or condition; the reduction in the duration of a disease or condition; the amelioration or elimination of one or more symptoms associated with a disease or condition; or the provision of beneficial effects to a subject with a disease or condition. Treatment does not require curing the underlying disease or condition.
  • a pharmaceutical composition herein may be provided to the patient at a dosage strength and a frequency determined as appropriate by a health care provider.
  • Therapeutically effective amounts are those sufficient to ameliorate one or more symptoms associated with the disease or affliction to be treated.
  • a “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dosage” of the binding protein herein protects a subject against the onset of a disease or promotes disease regression or stabilization as evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention or delay of impairment or disability due to the disease affliction.
  • the present disclosure also provides the use of the present binding proteins for the manufacture of a medicament, e.g., for treating a tauopathy (such as a tauopathy described herein).
  • the medicament is capable of crossing the BBB.
  • the present disclosure provides the present binding proteins for use as a medicament, e.g., for treating a tauopathy (such as a tauopathy described herein).
  • the present disclosure also provides the use of the present binding proteins (e.g., monospecific tau-binding proteins) for diagnostic processes (e.g., in vitro or ex vivo).
  • the binding proteins can be used to detect and/or measure the level of tau in a biological sample from a patient (e.g., a tissue sample such as a brain sample, or a fluid sample such as a blood, plasma, or CSF sample).
  • a biological sample e.g., a tissue sample such as a brain sample, or a fluid sample such as a blood, plasma, or CSF sample.
  • Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays, radioimmunoassays, and immunohistochemistry.
  • kits e.g., diagnostic kits comprising the binding proteins described herein.
  • the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.
  • mice Using the classical method as described by Wennerberg et al., supra, 6-8 weeks old female BALB/c mice (S082342; Charles River Labs, Bar Harbor, ME) each received three rounds of immunization over a course of 60 days. Antigens were administered intraperitoneally to ventral sites of mice. Three days after the last injection, mice were sacrificed and spleens were isolated aseptically and washed with fresh RPMI medium.
  • Lymphocytes were released from the spleens and the single-cell suspension was washed twice with RPMI medium before being fused with P3X63-AG8.653 myeloma cells using polyethylene glycol. After fusion, the cell mixture was incubated in an incubator at 37°C for 16-24 hours. The resulting cell preparation was transferred into selective semi-solid medium and aseptically plated out into 100 mm Petri plates and incubated at 37°C. Ten days after initiation of selection, the plates were examined for hybridoma growth, and visible colonies were picked up and placed into 96-well plates containing 200 pL of growth medium. The 96-well plates were kept in an incubator at 37°C for 2 to 4 days.
  • Antibodies were also screened on monomeric tau MTBR (VA2-13-023) and aggregated a-synuclein (VA2-13-073) in the same conditions described previously. Binding Kinetics of Anti-Tau Antibodies by SPR
  • lyophilized a-synuclein (Millipore, AG938) was dissolved in 500 pL of 10 mM Tris-HCl buffer pH 7.4. The sample was incubated and shaken (750 rpm) for 7 days at 37°C. After the incubation, the sample was centrifuged at 100,000 g for 1 hour at 12°C. The supernatant was discarded. The pellet was washed twice with 500 pL of PBS IX then resuspended in 200 pL of PBS IX. Aliquots were stored at -80°C.
  • Variants were expressed in polyclonal stable CHO-9E4 cell lines generated by PiggyBacTM transposon technology.
  • Cells were transfected using a MaxCyte electrotransfection system and then selected in cell selection and amplification medium containing 25 pM of MSX. After 8 to 10 days, cells were scaled up for production in rProtein production medium containing 25 pM of MSX for 14 to 20 days. Cell viability was controlled to be between 50 and 70%. After clarification, the culture supernatant was harvested and led in a two-step purification process including affinity chromatography (ProtA) and Preparative Size exclusion chromatography (Prep SEC).
  • ProtA affinity chromatography
  • Prep SEC Preparative Size exclusion chromatography
  • the ProtA was performed using a Mab Select SuRe (MSS) resin packed in column (5 mL up to 50 mL) connected to AKTApureTM 150 (Cytiva) placed in a cooled cabinet at a flow rate of 30-100 cm/h.
  • the binding was performed in DPBS-1X pH 7.5, for equilibration (20CV) and wash (20CV) steps and the elution in acetic acid glacial 0.1 N pH 2.8 (5CV).
  • the elution fractions of interest were grouped together.
  • the pool was neutralized by adding a 1 M Tris buffer solution pH 8.3 ( ⁇ 4% of pool volume) to adjust the pH value to 6-7. The pool was then filtered through 0.22 pm.
  • Protein concentration is determined by UV (Lunatic device, Unchained Labs) using the calculated epsilon absorbance of mAb at 280 nm.
  • the Prep SEC was performed using a SuperdexTM 200 resin packed in column (CV of 320 mL up to 6.6 L) connected AKTApureTM 150 (Cytiva) placed in a cooled cabinet at a 30 bm/h flow rate in a buffer consisting of 10 mM Histidine-HCl pH 6.0, 150 mM NaCl for isocratic step (1.5CV). At the end of the step, the elution fractions including the mAb monomeric antibodies were pooled (HMWs/LMWs were discarded).
  • the mAb concentration of the pool was determined by UV (Lunatic device, Unchained Labs) using the calculated epsilon absorbance of 280 nm.
  • UV Liquiatic device, Unchained Labs
  • epsilon absorbance 280 nm.
  • ultrafiltration step centrifugal or tangential flow filtration (TFF)
  • the mAb was concentrated to reach values > 10 mg/mL before being filtered through 0.22 pm.
  • the final batch was aliquoted and stored at +4°C.
  • SEC HPLC Size Exclusion Chromatography-High Pressure Liquid Chromatography
  • MS mass spectrometry
  • DLS Thermal Stability and Dynamic Light Scattering
  • SEC HPLC was performed using a 1260 PDA HPLC system from Agilent and ChemStation software (Agilent) at a flow rate of 300 pL/min (90cm/h) in a S200 Increase GL 5-150, 5 x 150 mm column (3 mL, GE Healthcare) at room temperature. Samples were diluted to 1 mg/mL before analysis and IX D-PBS was used as a buffer. Detection was performed at 280 nm, and 30 pL were injected.
  • Mass spectrometry was performed using a Q-ExactiveTM Plus with Acquity UPLC system (Thermo Fisher) with GeneData expressionist software.
  • Buffer A was H2O, 0.1% formic acid
  • Buffer B was acetonitrile, 0.1% formic acid.
  • the temperature of the column was 80°C when the sample temperature was 4-8°C. Samples were diluted to 1 mg/mL before analysis and 1 pL was injected. The mass of the reduced molecules was measured.
  • Thermal stability was assessed using a PrometheusTM NT 48 (Nanotemper Technology). Thermal stability parameters: Tonset, T ms and T agg were determined simultaneously in the same run. The experiments were repeated twice, and the provided values are the averaged parameters with error bar being less than 0.05°C. Standard nanoDSF NT capillaries were filled with 10 pL of protein solutions at the provided concentration. For simple thermal stability study, a linear gradient of temperature was applied at the range of 20°C to 95°C at 1°C heating rate/min, in duplicate. Data were analyzed with PR Stability Analysis software.
  • DLS Dynamic light scattering
  • Monomeric Tau MTBR (1.46 mg/mL) was diluted at 5 g/mL in coating buffer (phosphate buffered saline (Gibco Cat. No. 14190-094), 0.05 M sodium bicarbonate buffer pH 9.8, NaHCOs (Sigma S5761)), then 25 pL were added per well to polystyrene microplates (384 well polystyrene microplate Thermo Scientific Nunc Cat No. 464718). Plates were centrifuged at 300 rpm for 1 min, then incubated overnight at 4°C or one hour at room temperature. The coating protein was removed from the well.
  • coating buffer phosphate buffered saline (Gibco Cat. No. 14190-094), 0.05 M sodium bicarbonate buffer pH 9.8, NaHCOs (Sigma S5761)
  • washing buffer phosphate buffered saline (Gibco 14190-094), 0.05% Tween® 20 (Sigma P1379)
  • 50 pL of blocking buffer phosphate buffered saline (Gibco 14190-094), 0.05% Tween® 20 (Sigma P1379), 1 % BSA (Sigma A3803)
  • the plates were covered and incubated at 37°C for two hours. The blocking buffer was aspirated, and the wells were washed two times with 50 pL washing buffer.
  • the mAbs were diluted in blocking buffer at appropriate dilutions, with a starting concentration of 0.74 pg/mL, followed by 1/3 fold dilutions (33.33 pL diluted into 66.66 pL) blocking buffer to 1.25xl0' 15 pg/mL, then 25 pL were added per well in duplicates. Plates were centrifuged at 500 rpm for 1 min, incubated at 37°C for 1 hour or at 4°C overnight, then washed 5 times with 50 pL of washing buffer per well. Goat anti-human IgG Fc secondary antibody HRP (Invitrogen 1 mg/mL (goshu EcHRP affinity) Cat. No.
  • A18817, Lot 58-15-070717 was diluted at 1/2000, then 25 pL were added per well.
  • the plates were centrifuged at 500 rpm for 1 min, incubated at 37°C for 2 hours in darkness, then washed 5 times with 50 pL washing buffer.
  • KPL TMB microwell peroxidase substrate system was prepared according to manufacturer's instructions (mix equal volumes of TMB peroxidase substrate (Cat. No. 5120-00048) and peroxidase substrate solution (Cat. No. B 5120-0037)), then 25 pL were added to each well.
  • the plates were incubated in the dark at room temperature for 10 minutes.
  • the reaction was stopped by addition of 25 pL of stop solution to each well.
  • the plates were read with Tecan Infinite M200 with the MagellanTM software at 450 mm within 30 minutes after adding stop solution.
  • U2OS cells stably overexpressing the full-length human tau (441 amino acids 2N4R) with G272V and P301S mutations were cultured in DMEM + GlutaMAXTM (Gibco 31966-021), 10% heat inactivated Foetal Bovine Serum and 50 mg/mL hygromycin.
  • Cells were plated at 10,000 cells per well in a 96 well plate. 24 hours after plating, cells were incubated with wild-type human MTBR at 0.71 pg/mL ( ⁇ 50 nM) with or without incubation with various concentrations of the anti-tau-MTBR antibodies. 48 hours after treatment, cells were washed with PBS, and lysed using the HTRF lysis buffer (Cisbio). Tau aggregation was then quantified using the Cisbio HTFR kit according to manufacturer’s instructions (Cisbio).
  • Shape object recognition consisted of three phases. On days 1 and 2 (habituation phase) animals were placed into the open fields and allowed twice a day to freely explore for 10 min the open-field arena in the absence of objects. Locomotor activity was recorded. On Day 3, mice were placed again for 10 min into the open-field arena containing, this time, two identical objects (familiarization phase). After a retention interval of 20 minutes, the mouse is placed back into the open-field arena in the presence of a familiar object and a new one. The test lasts 10 min. Object recognition is distinguished by more time spent interacting with the novel object. Object exploration was considered whenever the mouse interacted with the object by smelling or touching the object.
  • the membranes were then washed three times for 10 min with PBS containing 0.05% Tween® 20 (PBS-T) and then incubated in PBS-T with 3% bovine serum albumin for one hour at room temperature. Membranes were then incubated overnight at 4°C with 22-MTBR and 523- MTBR antibodies at 1/1000 in PBS-T with 1% bovine serum albumin. Membranes were then washed three times for 10 min with PBS containing 0.05% Tween® 20 (PBS-T) and incubated with anti-human IgG (Li-Cor) for one hour at room temperature.
  • Membranes were then washed three times for 10 min with PBS-T and the membrane was imaged using an Odyssey infrared imaging system (Li-Cor).
  • Li-Cor Odyssey infrared imaging system
  • ELISA either 1 pg or 10 pg of peptides were adsorbed on polystyrene microplates (384 well polystyrene microplate Thermo Scientific Nunc Cat. No. 464718). Plates were centrifuged at 300 rpm for 1 min, then incubated overnight at 4°C or 1 hour at room temperature. The coating protein was removed from the well.
  • washing buffer phosphate buffered saline (Gibco 14190-094), 0.05% Tween® 20 (Sigma P1379)
  • 50 pL of blocking buffer phosphate buffered saline (Gibco 14190-094), 0.05% Tween® 20 (Sigma P1379), 1 % BSA (Sigma A3803)
  • the plates were covered and incubated at 37°C for 2 hours. The blocking buffer was aspirated, and the wells were washed two times with 50 pL washing buffer.
  • the antibodies 22-MTBR and 523 -MTBR were diluted in blocking buffer at appropriate dilutions, with a starting concentration of 0.74 pg/mL, followed by 1/3 fold dilutions (33.33 pL diluted into 66.66 pL) blocking buffer to 1.25xl0' 5 pg/mL, then 25 pL were added per well in duplicates. Plates were centrifuged at 500 rpm for 1 min, incubated at 37°C for 1 hour or at 4°C overnight, then washed 5 times with 50 pL of washing buffer per well. Goat anti-human IgG Fc secondary antibody HRP (Invitrogen 1 mg/mL (goshu EcHRP affinity) Cat. No.
  • A18817, Lot 58-15-070717 was diluted at 1/2000, then 25 pL were added per well.
  • the plates were centrifuged at 500 rpm for 1 min, incubated at 37°C for 2 hours in darkness, then washed 5 times with 50 pL washing buffer.
  • KPL TMB microwell peroxidase substrate system was prepared according to manufacturer's instructions (mix equal volumes of TMB peroxidase substrate (Cat. No. 5120-00048) and peroxidase substrate solution (Cat. No. B 5120-0037)), then 25 pL were added to each well.
  • the plates were incubated in the dark at room temperature for 10 minutes.
  • the reaction was stopped by addition of 25 pL of stop solution to each well.
  • the plates were read with Tecan Infinite M200 with the MagellanTM software at 450 mm within 30 minutes after adding stop solution.
  • Antibodies 22-MTBR and 523-MTBR were screened via ELISA assay for binding to full length tau monomers or fibrils as well as aggregated a-synuclein (Table 2). Both antibodies demonstrated high affinity for both tau monomers and fibrils with no binding observed to a-synuclein fibrils.
  • AD Alzheimer’s disease
  • THY-Tau22 mouse model of tauopathy
  • wild-type mice were immunohistochemically stained with either 0.1 pg/mL of 22-MTBR or 1 pg/mL of 523- MTBR (secondary antibody: mouse IgG2b and mouse IgGl, respectively). Both antibodies displayed good binding to tau pathology from AD and THY-Tau22 mice.
  • the selected antibodies were characterized by an in vitro intracellular seeding assay using U2OS cells.
  • the cells stably express mutated human full length tau proteins that develop into intracellular tau aggregates when incubated with exogenous recombinant aggregated human MTBR.
  • This “seeding” with aggregated human MTBR induces the aggregation of endogenous full-length human tau protein which can be detected by homogenous time resolved fluorescence (HTRF).
  • HTRF time resolved fluorescence
  • the cells were seeded with wild-type human MTBR at 0.71 pg/mL ( ⁇ 50 nM) for 48 hours and the change in fluorescence measured after incubation with various concentrations of the anti-tau-MTBR antibodies. Both 22-MTBR and 523-MTBR inhibited endogenous tau aggregation induced by aggregated WT MTBR seeding in vitro (Table 3).
  • Table 3 Aggregation Inhibition of Anti-Tau-MTBR Antibodies via HTRF Assay
  • 22-MTBR and 523-MTBR were tested using surface plasmon resonance in the presence of either full-length tau monomers and fibrils, or tau MTBR monomers and fibrils. Both antibodies displayed fast on- and off-rates, and the affinities for both antibodies were in the same range of 100-300 nM, although the kinetics of 22-MTBR were slightly slower.
  • 22-MTBR and 523-MTBR were administered via intraperitoneal injection to six-month old male THY- Tau22 mice weekly for three months at a dose of 20 mg/kg.
  • tau aggregation in the hippocampus was measured relative to the baseline tau pathology in THY- Tau22 mice via HTRF (FIG. 2).
  • Treatment with 22-MTBR prevented the increase of tau aggregation in the hippocampus, with 84% less tau pathology progression compared to a control antibody (anti -DM4 antibody).
  • anti -DM4 antibody anti-DM4 antibody
  • the reduction in hippocampal tau pathology was also observed by immunohistochemistry (AT8 + cell number) with a 30% reduction in tau pathology progression (FIG. 3).
  • the 22-MTBR antibody was further evaluated for neuroprotective effect via measurement of neurofilament light (NFL) in the cerebrospinal fluid.
  • NNL neurofilament light
  • THY-Tau22 mice were intraperitoneally injected weekly with 22-MTBR and the concentration of NFL measured after nine months of treatment at various antibody concentrations (0, 2, 6, and 20 mg/kg) (FIG. 4). Following treatment, 38% neuroprotection was observed, indicating that the antibody was effective in a THY-Tau22 model.
  • Both antibodies were effective at reversing cognitive deficits in a THY-Tau22 model.
  • male THY-Tau22 mice were administered 22-MTBR and 523-MTBR antibodies at a dose of 20 mg/kg via intraperitoneal injection.
  • an object recognition test was performed to assess the effect of the antibody on tau-related cognitive deficit.
  • 22-MTBR and 532-MTBR rescued cognitive deficits in THY-Tau22 mice when compared to WT mice and THY-Tau22 mice administered a control antibody (anti-DM4 antibody) (FIG. 5A).
  • mice that received dosages of > 6 mg/kg 22-MTBR showed reversal of cognitive deficits comparable to wild-type mice (FIG. 5B) (control: anti-TNP antibody).
  • THY-Tau22 mice were administered various concentrations of 22-MTBR for three months over a test period of six to nine months of age. Following administration, the extension reflex of the mice was evaluated as a measure of tau aggregate-associated motor deficits. Mice were given a score of normal, low deficit, or high deficit depending on their ability to be fully stretched when hung by the tail (FIG. 6). Dosages of 22-MTBR > 6 mg/kg improved motor deficits in mice. [0198] To further investigate the efficacy of anti-tau-MTBR antibodies, an in vivo seeding experiment was conducted by intrahippocampally injecting THY-Tau22 mice with recombinant aggregated human MTBR.
  • mice were intraperitoneally administered 10 mg/kg doses of 523-MTBR weekly for three weeks, and the levels of AT8, an antibody against hyperphosphorylated tau, were measured and visualized by immunohistochemical staining of the brain tissue.
  • Treatment with 523-MTBR resulted in significant reduction in measured tau levels, and reduced hippocampal and cortical tau pathology (FIG. 7).
  • Antibody serial dilutions were then performed in a 96-well plate (Greiner bio-one microplate, Black, Cat. No. 655900) generating concentrations of 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.63 nM, 7.81 nM, and 3.91 nM. Additional wells contained buffer-only as negative controls. During the assay, plates were maintained at 25°C and shaken at 1000 rpm. Baseline measures of probes were taken both before and after the sensors were loaded with transferrin receptor. Probes were then dipped in antibody or control wells and the association step proceeded for 90 seconds. Then probes were moved to buffer- only wells and antibody dissociation was measured for 180 seconds.
  • Additional reference assays were performed to measure background levels from the streptavidin biosensors to control for any non-specific interactions between probes and antib ody/analyte. From the binding and dissociation curves, the KD (M), KD error, k a (1/Ms), k a error, kdis (1/s), and kdis error were determined.
  • the window of interest used for association was 0 to 90 seconds.
  • the window of interest used for dissociation was 0 to 60 seconds. Analysis was performed using OctetTM version number for software: OctetTM Analysis Studio 12.2. For data correction and preprocessing, the following software options were chosen:
  • Align Y axis Align data to average of baseline step
  • Inter-step correction Align data to Baseline step
  • CynoTFRC-expressing cells were seeded at 70,000 cells/well on 96-well plates (BD-Falcon; #353910). 100 pL/well of antibodies (antibody concentration range: 3-fold serial dilutions starting at 455 nM up to 12 points) were incubated for 20 min at 4°C. Plates were washed 3 times with PBS 1% BSA. 100 pL/well of goat anti-human IgG conjugated with Alexa488 (Jackson ImmunoResearch; # 109-545-098) were incubated for 20 min at 4°C. Plates were washed 3 times with PBS 1% BSA.
  • Antibody binding was evaluated after centrifugation and resuspension of cells by adding 200 pL/well PBS 1% BSA and read using the Guava® easyCyteTM 8HT Flow Cytometry System. Apparent KD and ECso values were estimated using BIOST@T-BINDING and BIOST@T-SPEED software, respectively.
  • Histologies for AT8 binding to hyperphosphorylated tau were performed on floating sections of either 20 or 30 pm. Sections were washed three times with PBS containing 0.15% Triton X-100 (PBS-T - Sigma) and then incubated for one hour in PBS containing 0.3% of H2O2 and 50% methanol. Sections were washed three times with PBS-T and then incubated in PBS, 3% BSA for one hour. Sections were then incubated at room temperature for 24 hours with biotinylated AT8 primary antibody (1/100 in PBS-T, Invitrogen).
  • Sections were washed three times with PBS-T and incubated for one hour in ABC Vectastain (1/400 in PBS) under gentle agitation. Sections were washed 1 time in PBS- T for 10 min followed by 2 washes in PBS for 10 min. Sections were incubated in 3,3’- diaminobenzidine tetrahydrochloride (DAB - Sigma) at 0.5 mg/mL and 0.003% H2O2 in PBS until stained and then quickly washed twice in PBS. Sections were mounted on slides and air-dried.
  • DAB - Sigma 3,3’- diaminobenzidine tetrahydrochloride
  • mice For in vivo studies, THY-Tau22 mice, THY-Tau22 mice crossed with hTfR-KI mice, or cynomolgus macaques were used. Compounds were diluted in sterile PBS or 10 mM histidine pH 6, 150 mM NaCl and injected either intraperitoneally or intravenously by an experimenter blind to treatment. For repeated injection studies of humanized compounds, mice were immunotolerized by the injection of anti-CD4 antibodies (GK1.5 - BioXcell) according to manufacturer’s instructions. At different time points, animals were sacrificed after anesthesia and CSF / blood collected.
  • CSF was snap frozen on dry ice and blood centrifugated at 5,000 rpm for 10 min, 4°C and plasma collected, aliquoted and snap frozen on dry ice. Animals were transcardially perfused with ice-cold PBS and then tissues were collected for further analysis.
  • Neurofilament light chain and total human tau concentrations were determined using the Neurology 4-Plex B (N4PB) kit (SIMOA - Quanterix) according to the manufacturer’s instructions.
  • Cleavage generates two tau-MTBR fragments that when aggregated, give off a FRET signal based on the proximity of the mVenus and mTurquoise2 tags.
  • a concentrated LipofectamineTM 2000 (Invitrogen Cat. No. 52758) stock of 80 pL/mL was made in Opti-MEMTM (Gibco Cat. No. 31985-070) and concentrated stock of 20 pg/mL plasmid was made, then combined at a 1 : 1 volume ratio and incubated at room temperature for 5 minutes. The volume was then increased to lOx using Opti-MEMTM.
  • This transfection solution was then applied at 100 pL/well to HEK293T cells that were plated at 15,000/well in media (DMEM containing 10% serum and lx Penicillin Streptomycin) in 96-well Cell- Carrier ultra plates coated with Poly-D-lysine.
  • media DMEM containing 10% serum and lx Penicillin Streptomycin
  • Full-length tau-PFFs (StressMarq Cat# SPR-480) were prepared by sonication in Diagenode Bioruptor® bath sonicator at 4°C for 5-cycles (1-cycle: 30-seconds on, 30-seconds off). After sonication, 800 ng of tau was diluted to 100 pL in PBS. This solution was added to the antibody-bead complexes, mixed by pipetting, then incubated on a rotator for 10 minutes at room temperature.
  • HEK293T cells were lifted with trypsin and replated in maintenance medium (DMEM, Gibco Cat. No. 11995-065; 10% fetal bovine serum, Gibco Cat. No. A56695; lx Penicillin/Streptomycin, Gibco Cat. No. 15140-122) at 15,000 cells/well in 96-well cell carrier ultra plates (FisherScientific Cat. No. 50-209-9831) coated with poly-D-lysine (Sigma Cat. No. P7886-100MG).
  • maintenance medium Gibco Cat. No. 11995-065
  • 10% fetal bovine serum Gibco Cat. No. A56695
  • lx Penicillin/Streptomycin Gibco Cat. No. 15140-122
  • the calibration curve and QC samples were incubated for 30 minutes at RT while shaking at 600 rpm. Prior to loading to the MSD plate, all samples were diluted in assay diluent (PBS/0.1% casein). For the sampling step, the diluted samples were applied onto the plate and incubated for 1 hour at RT, while shaking at 600 rpm. As a detection step, 2.0 pg/mL mouse anti-human Fc-Sulfo was applied and incubated for 1 hour at room temperature, covered from light, while shaking at 600 rpm. The ECL signal was obtained via readout using the Sector Imager QuickPlex SQ 120 (Mesoscale Discovery), within 10 minutes after applying MSD GOLD read buffer A.
  • TrioCODV trivalent cross-over dual variable
  • the mouse TrioCODVs and Duobody COD Vs were tested for brain exposure in THY-Tau22 or WT mice after intraperitoneal administration of 14 nmol/kg of antibody. Samples were taken at several timepoints following antibody administration and concentration of antibody measured (FIG. 10).
  • the Duobody CODVs showed up to 21-fold improved brain exposure compared to the monospecific 22-MTBR antibody. Additionally, the bispecific 22-8D3-5 mutant demonstrated up to 68-fold improved brain exposure in WT mice compared to monospecific 22-MTBR.
  • the 22-8D3-4 construct was used for further in vivo evaluation in comparison with monospecific 22-MTBR.
  • the antibodies were administered intraperitoneally to THY-Tau22 mice at a dose of 14 nmol/kg and the concentration measured over time (FIG. 11).
  • the 22-8D3-4 construct demonstrated approximately 15-fold increased brain penetration compared to 22-MTBR (Table 5).
  • Table 5 Characterization of TrioCODV Anti-TfR/Anti-Tau-MTBR Antibody
  • the number of AT8 + (Tau + ) or Gallyas + (i.e., positive for tau aggregates detected by the Galiyas silver impregnation method) cells in brain samples (CAI region) were measured to determine the impact of the antibodies on tau pathology progression (FIGs. 12A and 12B).
  • the TrioCODV 22-8D3-4 significantly decreased the progression of tau pathology.
  • TrioCODV antibodies were designed to incorporate either of two humanized anti-tau-MTBR antibody variants (22-MTBR or 523-MTBR) with a humanized anti-TfR shuttle antibody (531V25). These antibodies were designed with various Fc regions.
  • the Fc region comprised a human IgGl Knob into Hole (KIH) Fc region with LALA mutations, deletion of the terminal lysine, and RF mutations to abolish Protein A binding (hlgGl-LALA-KIH-RF- dK, where dK represents deletion of the terminal lysine).
  • the Fc region in some constructs comprised one of two backbones with optimized sequences by additional mutations for improving correct pairing and thermal stability of multi-specific antibodies: charge mutations CM1 on the CODV arm (with two binding domains) (VL Q44E; VH Q44K in IMGT numbering) and CM2 on the Fab arm (with one binding domain) (VL Q44K; VH Q44E in IMGT numbering), or charge mutations CM1-CM3 on the CODV arm (CM3: light chain kappa constant region mutation S176R, heavy chain CHI mutation L145E, in Eu numbering) and CM2-CR3-NN3 on the Fab arm.
  • 22-53 lv25-3 comprises a first heavy chain that comprises SEQ ID NO: 41, a second heavy chain that comprises SEQ ID NO: 43, a first light chain that comprises SEQ ID NO: 42, and a second light chain that comprises SEQ ID NO: 44.
  • 22-53 lv25-l comprises a first heavy chain that comprises SEQ ID NO: 37, a second heavy chain that comprises SEQ ID NO: 39, a first light chain that comprises SEQ ID NO: 38, and a second light chain that comprises SEQ ID NO: 40.
  • 22-53 lv25-2 is a version of 22-53 lv25-l that uses a different linker, as described above.
  • 22-53 lv25-4 comprises a first heavy chain that comprises SEQ ID NO: 31, a second heavy chain that comprises SEQ ID NO: 33, a first light chain that comprises SEQ ID NO: 32, and a second light chain that comprises SEQ ID NO: 34.
  • 523-53 lv25-l comprises a first heavy chain that comprises SEQ ID NO: 35, a second heavy chain that comprises SEQ ID NO: 33, a first light chain that comprises SEQ ID NO: 36, and a second light chain that comprises SEQ ID NO: 34.
  • 523-53 lv25-2 is a version of 523-53 lv25-l with charge mutations CM1-CM3 on the CODV arm and CM2-CR3-NN3 on the Fab arm.
  • THY-Tau22/human TfR knock in mice were administered either 22-53 lv25-4 or 523-53 lv25-l, or the monospecific antibodies 22-MTBR or 523- MTBR, at a dose of 42 nmol/kg twice a week for three months.
  • the brain antibody concentration was measured at various timepoints following the final antibody dose (FIG. 13).
  • the anti-TfR/anti-tau antibodies both showed improved brain penetrance compared to the non-TfR (monospecific) control antibodies.
  • the anti-TfR/anti-tau antibodies were also pharmacokinetically evaluated in vivo using humanized mice.
  • THY-Tau22/human TfR knock in mice were intravenously injected with 7.2 mg/kg (42 nmol/kg) of antibody and evaluated based on the pharmacokinetic profile in the brain (Table 8) when compared with the monospecific 22-MTBR antibody.
  • the brain exposure was increased by 7- to 16-fold upon treatment with the bispecific antibodies compared with 22-MTBR.
  • the bispecific 523-MTBR antibody with the TfR component on the Fab (523-53 lv25-l) showed better brain exposure.
  • the plasma pharmacokinetic profiles of these antibodies were analyzed (Table 9), with the bispecific TrioCODV antibodies showing reduced plasma concentration compared to monospecific 22-MTBR.
  • both 22-53 lv25-3 and 22-MTBR exhibited plasma exposure increases that appeared in proportion with the dose increases.
  • the plasma exposure of 22-53 lv25-3 was approximately 5-fold lower than that of 22-MTBR that lacks a TfR recognition sequence.
  • the bispecific antibody 22-53 lv25-3 exhibited brain exposure that was from 3- to 16-fold higher than that of monospecific antibody 22-MTBR, depending on the dose.
  • the bispecific anti-TfR/anti-tau antibody 22-53 lv25-3 was further tested in cynomolgus monkeys through intravenous injection of antibody at a dose of 7 mg/kg.
  • the total concentration of antibody and the concentration of free antibody were measured in the cerebrospinal fluid (CSF) of the monkeys at 6 and 24 hours post injection (FIG. 14A).
  • CSF cerebrospinal fluid
  • the total CSF concentration was approximately two-fold higher than the free antibody concentration, suggesting binding of 22-53 lv25-3 to the tau protein.
  • the brain and CSF exposure of 22-53 lv25-3 were approximately five-fold higher than that of monospecific antibody 22-MTBR.
  • FIG. 14B shows the concentration of total forms of 22- 53 lv25-3 in various brain tissues.
  • HEK293 cells were transfected with MTBR-mTurquoise-2A-MTBR-mVenus to fluorescently image tau aggregation in cells. Six hours after transfection, the cells were seeded with tau protein via propagation using preformed fibrils (PFF) and after 24 hours the cells were imaged for fluorescence resonance energy transfer (FRET) activity (FIG. 15).
  • PFF preformed fibrils
  • FRET fluorescence resonance energy transfer
  • mice were immunotolerized by the injection of anti-CD4 antibodies.
  • anti-CD4 antibodies One week following anti-CD4 injection, the mice were injected twice weekly for three months with anti-TfR/anti-tau antibodies at a dose of 42 nmol/kg.
  • samples were collected and the fraction of AT8 + (Tau + ) cells was measured in the cortex (FIG. 16), showing significant reduction in AT8 positive staining with 22-53 lv25-3 and 22-53 lv25-4 and not with 523- 531v25-l.
  • Biotinylated HT7 antibody (Invitrogen, Cat. No. MN1000B, lot# YB3804864). Antibodies were immobilized on biosensors at concentrations ranging from 10 to 17 pg/mL. Ligand loading was performed for 300 seconds, followed by a baseline step in buffer for 60 seconds. A series of two-fold dilutions of tau proteins (monomers and PFFs) was prepared, with the highest concentration at 1000 nM. Association was measured for 120 seconds, followed by dissociation in buffer for 180 seconds. Reference sensors with no ligand were used to correct for non-specific binding. Data processing and analysis were performed using Octet® Data Analysis software (Sartorius).
  • P reprocessing steps included reference sensor subtraction, alignment to the baseline step, and Savitzky-Golay filtering.
  • Kinetic parameters were determined using a 1 : 1 binding model with global fitting. The window of interest for association was set to 0-90 seconds and for dissociation to 0-60 seconds. Results
  • BLI was performed to assess antibody binding to human TfR, but in the presence or absence of human tau monomer or tau fibrils.
  • Human transferrin receptor (TfR) was produced in Expi293 cells and biotinylated on an N-terminal AviTagTM using BirA. The human TfR amino acid sequence used is found in SEQ ID NO: 97.
  • To measure the binding affinity of antibodies to TfR we performed OctetTM binding assays and utilized the monovalent anti-TfR antibody (53 lv25) as a control.
  • the OctetTM HTX system was used with streptavidin coated biosensors (Sartorius Cat. No.
  • Additional wells contained buffer-only as negative controls. During the assay, plates were maintained at 25°C and shaken at 1000 rpm. Baseline measures of probes were taken both before and after the sensors were loaded with transferrin receptor. Probes were then dipped in antibody, antibody with tau, or control wells, and the association step proceeded for 90 seconds. Then probes were moved to buffer-only wells and antibody dissociation was measured for 180 seconds. Additional reference assays were performed to measure background levels from the streptavidin biosensors to control for any non-specific interactions between probes and antib ody/analyte.
  • iPSC induced pluripotent stem cell
  • iCell® GlutaNeurons Cell® GlutaNeurons, Cellular Dynamics International
  • PFFs human full-length wild-type tau pre-formed fibrils
  • the labeled tau-PFFs were used at a final concentration of 5 pg/mL for all experiments. Antibodies were serially diluted to achieve concentrations ranging from 0.78 nM to 800 nM. Antibodies were pre-incubated with pHrodoTM-labeled tau-PFFs (5 pg/mL) for 30 minutes at room temperature before addition to GlutaNeurons. The internalization of tau-PFFs was monitored using an Incucyte® SX5 live-cell imaging system, which captured fluorescent images at regular intervals. Quantitative analysis of tau-PFF internalization was performed by measuring pHrodoTM fluorescence intensity normalized to cell confluence.
  • 523- 531v25-la is a version of 523-53 lv25-l comprising a heavy chain sequence with mutations Q39K, L148E, T259D, and T310Q as compared to SEQ ID NO: 33; a light chain sequence with mutations Q38E and S176R as compared to SEQ ID NO: 34; a heavy chain sequence with mutations Q39E, Q152E, L254R, T365D, and T416Q as compared to SEQ ID NO: 35; and a light chain sequence with mutations N32S, N33Q, N35Q, Q43K, N154S, N155Q, N157Q, Q165K, and S313E as compared to SEQ ID NO: 36.
  • Injections consisted of either 1.15 pL of tau aggregates or 1.15 pL of the tau aggregates co-incubated with TfR-Tau antibodies for 30 min at 37°C, which were injected unilaterally at a flow rate of 0.5 pL per min. All injected animals were monitored throughout the surgery and post-surgery, until full recovery. After surgery, the skin was sutured. One hour before the surgery, a subcutaneous injection of long-release buprenorphine was administered. Two weeks post-injection, mice were euthanized with CO2 and transcardially perfused with cold PBS until the organs were clear of blood. The brains were immediately removed, fixed for 48 h in 10% formalin 4% PF A, and placed in PBS until sectioned.
  • tau PFFs were co-incubated with MTBR-22, 22-53 lv25-3, and 22-53 lv25-4 and then injected into the CAI region of the left hippocampus of young ThyTau22 mice. Two weeks post-injection, the mice were sacrificed and their brains collected and stained for tau pathology using the AT8 hyperphosphorylated tau antibody.
  • tau pathology in the ipsilateral hippocampus is indicative of seeding of pathology (uptake of aggregates inside neurons and recruitment of hyperphosphorylated tau inside aggregates).
  • Presence of tau pathology in the contralateral hippocampus (non-injected side) is indicative of spreading of tau pathology from the ipsilateral side through neuron-to-neuron connections.
  • Examples 6 and 7 demonstrate that two bispecific antibodies (22-53 lv25-3 and 22- 53 lv25-4) with the same anti-tau and anti-TfR binding domains, but different formats (FIG. 26), can exhibit markedly different properties.
  • the data highlights the importance of not only the identity of the anti-tau binding domain, but also the format, of the bispecific binding molecule.

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Abstract

La présente invention concerne des protéines de liaison qui ciblent tau, ainsi que des protéines de liaison bispécifiques qui ciblent tau et une protéine du système nerveux central (p. ex. le récepteur 1 de la transferrine). L'invention concerne également l'utilisation de ces protéines de liaison pour traiter des tauopathies.
PCT/IB2025/055816 2024-06-05 2025-06-05 Protéines de liaison à tau monospécifiques et bispécifiques et compositions associées Pending WO2025253337A2 (fr)

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