Monospecific and bispecific tau binding proteins and compositions thereof

Abstract

The present disclosure provides binding proteins that target tau, as well as bispecific binding proteins that target tau and a central nervous system protein (e.g., transferrin receptor 1). Also provided is the use of these binding proteins to treat tauopathies.

Description

MONOSPECIFIC AND BISPECIFIC TAU BINDING PROTEINS ANDCOMPOSITIONS THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to EP Patent Application No. 24305886.4, filed June 5, 2024, and EP Patent Application No. 24306578.6, filed September 27, 2024. The disclosures of those priority applications are incorporated by reference herein in their entirety.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on May 27, 2025, is named “122548.WO036.xml” and is 141,766 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTION

[0003] 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.

[0004] 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.

[0005] In view of the role of tau in neurodegenerative disease, there remains a need for tau-targeted therapies for treatment of such diseases (e.g., tauopathies), and for delivery of the therapies to the brain.SUMMARY OF THE INVENTION

[0006] 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). The ECR may be, for example, transferrin receptor 1 (TfR).

[0007] In some embodiments, 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.

[0008] In certain embodiments, the VH and the VL of the tau-binding protein are at least 90% identical to:SEQ ID NOs: 7 and 8, respectively;SEQ ID NOs: 15 and 16, respectively;SEQ ID NOs: 73 and 75, respectively; orSEQ ID NOs: 74 and 76, respectively.

[0009] In certain embodiments, the VH and the VL of the tau-binding protein comprise: SEQ ID NOs: 7 and 8, respectively;SEQ ID NOs: 15 and 16, respectively;SEQ ID NOs: 73 and 75, respectively; orSEQ ID NOs: 74 and 76, respectively.

[0010] 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.: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).For example, the tau-binding protein may have at least properties a)-h), or all of properties a)- m).

[0011] In some embodiments, 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). In certain embodiments, the 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).

[0012] In some embodiments, the anti-tau antibody comprises a human IgGl heavy chain constant region that comprises SEQ ID NO: 27. In some embodiments, 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. In certain embodiments, 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).

[0013] In some embodiments, the tau-binding protein herein may be fused to a cellpenetrating peptide that binds a central nervous system (CNS) target (e.g., TfR). In someembodiments, 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.

[0014] 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).In some embodiments, the bispecific binding protein is- bivalent for tau and monovalent for the CNS target; monovalent for tau and monovalent for the CNS target; monovalent for tau and bivalent for the CNS target; or- bivalent for tau and bivalent for the CNS target.

[0015] In some embodiments, 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.

[0016] In some embodiments, one arm of the bispecific binding protein comprises a first anti-tau binding domain and an anti-CNS target binding domain, and 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.

[0017] In certain embodiments, 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. In particular embodiments, on the first heavy chain, the VH of the anti-CNS target binding domain is N-terminal to the VH of the first anti-tau binding domain, and on the first light chain, the VL of the first anti-tau binding domain is N-terminal to the VL of the anti-CNS target binding domain.

[0018] In some embodiments, 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 proteinherein. 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.

[0019] In certain embodiments, 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. In particular embodiments, on the first heavy chain, the VH of the first anti-tau binding domain is N-terminal to the VH of the second anti-tau binding domain, and on the first light chain, the VL of the second anti-tau binding domain is N-terminal to the VL of the first anti-tau binding domain.

[0020] In some embodiments of the bispecific binding protein herein, the CNS target is an endothelial cell receptor (ECR) of the blood brain barrier. The ECR may be, e.g., a transferrin receptor, insulin receptor, low-density lipoprotein receptor, or folate receptor. In particular embodiments, the ECR is transferrin receptor 1 (TfR), and the bispecific binding protein has an anti-TfR binding domain.

[0021] In some embodiments, 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. In some embodiments, 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. In certain embodiments, the VH and the VL of the anti-TfR binding domain are at least 90% identical to, or comprise:SEQ ID NOs: 25 and 26, respectively; orSEQ ID NOs: 77 and 78, respectively.

[0022] 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 Octet™; g) binds to cynomolgus TfR with a KD of 50-200 nM as determined by Octet™;h) has improved brain penetrance in hTfR knock-in mice by at least 3 -fold over the monospecific anti-tau binding domain; i) has increased brain and CSF exposure by at least 5-fold in cynomolgus monkeys over the monospecific anti-tau binding domain; j) reduces tau seeding in vitro., k) inhibits uptake of tau aggregates into neurons; l) reduces AT8+cells in vivo in the cortex of hTfR / THY-Tau22 mice; or m) any combination of a)-l).

[0023] In some embodiments, the bispecific binding protein herein comprises an Fc region. In certain embodiments, the bispecific binding protein is a bispecific antibody. In particular embodiments, 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).

[0024] In some embodiments, 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.

[0025] In some embodiments, 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. In certain embodiments, 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). In particular embodiments, the first heavy chain constant region further comprises knob mutations of S354C and T366W, and 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. In someembodiments, 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.

[0026] In some embodiments, 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.

[0027] In some embodiments, 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.

[0028] In some embodiments, 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.

[0029] In some embodiments, 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.

[0030] In some embodiments, 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. In certain embodiments, on the first heavy chain, 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, and on the first light chain, 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 andsame LCDR1-3, and in some embodiments are the same. In particular embodiments, 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, respectively.In certain embodiments, 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.In particular embodiments, 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.

[0031] The present disclosure also provides pharmaceutical composition comprising a tau- binding protein or bispecific binding protein herein and a pharmaceutically acceptable excipient.

[0032] Further, the present disclosure provides isolated nucleic acid molecule(s) encoding a tau-binding protein or bispecific binding protein herein. In some embodiments, the nucleic acid molecule(s) are expression constructs. Also provided are 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.

[0033] 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.

[0034] In some embodiments, 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). In particular embodiments, the primary tauopathy is progressive supranuclear palsy (PSP).

[0035] In some embodiments, 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. In particular embodiments, the secondary tauopathy is Alzheimer’s disease.

[0036] Other features, objectives, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way ofillustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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).

[0042] 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.

[0043] 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.

[0044] 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.

[0045] FIG. 9 is a schematic showing the different positions of the anti-TfR domain within various bispecific anti -tau / anti-TfR antibody constructs.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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).

[0051] 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.

[0052] 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.

[0053] 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.17B)

[0054] 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).

[0055] 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).

[0056] 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.

[0057] FIG. 21 is a pair of bar graphs showing the levels of tau-PFF-pHrodo™ inside iPSC-derived neurons following incubation with increasing amounts of the indicatedantibodies. “Blank” refers to no tau-PFF-pHrodo™ added, “Control” refers to tau-PFF- pHrodo™ without addition of antibodies.

[0058] FIG. 22 is a bar graph showing the levels of tau-PFF-pHrodo™ inside iPSC- derived neurons following incubation with the indicated antibodies.

[0059] FIG. 23 is a set of bar graphs showing the levels of tau-PFF-pHrodo™ inside iPSC-derived neurons following incubation with increasing amounts of the indicated antibodies. “Blank” refers to no tau-PFF-pHrodo™ added, “Control” refers to tau-PFF- pHrodo™ without addition of antibodies.

[0060] 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.

[0061] 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.

[0062] FIG. 26 is a schematic showing the formats of the indicated bispecific antibodies.DETAILED DESCRIPTION OF THE INVENTION

[0063] 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. Without wishing to be bound by theory, it is contemplated that by binding extracellular tau, 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.

[0064] 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)). The domain that binds to the CNS target may facilitate transport of the anti-tau binding domain across the BBB.

[0065] Unless otherwise indicated, 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 SequenceNo. NP 001116538.2 (SEQ ID NO: 17). The MTBR of tau has a sequence of SEQ ID NO: 46. Unless otherwise indicated, TfR herein refers to human TfR (human TfRl). A human TfR polypeptide sequence is available under UniProt Accession No. P02786 (SEQ ID NO: 18).I. Binding Proteins

[0066] The present disclosure provides tau-binding domains / proteins, such as antibodies or antigen-binding fragments thereof. The term “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). The 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). 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.

[0067] The precise amino acid sequence boundaries of a given CDR or FR can be defined by several well-known systems, including those described by Kabat et al., 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (“Kabat” system); Al- Lazikani et al., J Mol Biol. (1997) 273:927-48) (“Chothia” system); MacCallum et al., J Mol Biol. (1996) 262:732-45 (“contact” system); Lefranc et al., Dev Comp Immunol. (2003) 27(l):55-77 (“IMGT” system); Honegger and Pliickthun, J Mol Biol. (2001) 309(3):657-70 (“Aho” system); and Whitelegg and Rees, Protein Eng. (2000) 13(12):819-24 (“AbM” system). The boundaries of a given CDR or FR may vary depending on the system used. For example, the Kabat system is based on sequence alignments, while 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 Chothiasystem. 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.

[0068] 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. In some embodiments, 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 (2018) 9(l):63-73). For example, 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. In some embodiments, the IgG constant region may comprise mutations that improve the serum half-life of the antibody, such as the M428L and / or N434S mutations. In some embodiments, 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.

[0069] In any embodiments of constant regions herein, 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).

[0070] In certain embodiments, 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).

[0071] In certain embodiments, the monospecific or multispecific antibody herein comprises a first human IgGl constant region comprising L234A and L235A mutations and asecond human IgGl constant region comprising L234A, L235A, H435R, and Y436F mutations.

[0072] In certain embodiments, e.g., of a multispecific antibody herein, 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. For example, one of the human IgGl constant regions (e.g., the first constant region, with mutations as described above) may comprise knob mutations of S354C and T336W, and the other human IgGl constant region (e.g., the second constant region, with mutations as described above) may comprise hole mutations of Y349C, T366S, L368A, and Y407V.

[0073] In particular embodiments, 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.

[0074] In some embodiments, 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.

[0075] 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. Several techniques can be used to characterize target binding affinity (KD), such as surface plasmon resonance (SPR, using, e.g., BIAcore™) or bio-layer interferometry (BLI, using, e.g., Octet™ from ForteBio). Flow cytometry assay (e.g., FACS) using cells expressing membrane-bound targets can also be used to determine ECso or ICso values of the binding proteins; these values are indicative of the binding to the targets in their native conformation.A. Tau-Binding Proteins

[0076] In some embodiments, 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.

[0077] 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: 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.

[0078] In some embodiments, the anti-tau binding domain comprises HCDR1-3 and LCDR1-3 set forth inSEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; orSEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively.

[0079] In some embodiments, 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:SEQ: SEQ ID NO:SEQ: SEQ ID NO:

[0080] Thus, for example, the 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;SEQ ID NOs: 50, 51, and 49, respectively; orSEQ ID NOs: 52, 53, and 54, respectively.

[0081] Similarly, the 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; orSEQ ID NOs: 57, 58, and 59, respectively.

[0082] Further, the HCDR1-3 sequences of SEQ ID NOs: 9, 10, and 11, respectively, may be replaced in any embodiment described herein bySEQ ID NOs: 60, 61, and 62, respectively; SEQ ID NOs: 63, 64, and 62, respectively; orSEQ ID NOs: 65, 66 and 67, respectively.

[0083] Similarly, the 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.

[0084] Also contemplated is a set of CDRs specified according to any combination of the methods for defining CDRs shown above (e.g., 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.

[0085] In certain embodiments, 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).

[0086] In certain embodiments, 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).

[0087] In some embodiments, 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. In anti-tau VH and VL sequences herein, such 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. In certain embodiments, 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). In some embodiments, 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.

[0088] In certain embodiments, 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).

[0089] In certain embodiments, 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).

[0090] In certain embodiments, the anti-tau binding domain comprises a VH and a VL set forth in SEQ ID NOs: 7 and 8, respectively.

[0091] In certain embodiments, the anti-tau binding domain comprises a VH and a VL set forth in SEQ ID NOs: 73 and 75, respectively.

[0092] In certain embodiments, the anti-tau binding domain comprises a VH and a VL set forth in SEQ ID NOs: 74 and 76, respectively.

[0093] In certain embodiments, the anti-tau binding domain comprises a VH and a VL set forth in SEQ ID NOs: 15 and 16, respectively.

[0094] 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. In some embodiments, the anti-tau antibody comprises a human IgGl heavy chain constant region, optionally with mutations as described herein. For example, the human IgGl heavy chain constant region may comprise any one of SEQ ID NOs: 27-29. In some embodiments, 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. In some embodiments, 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. In some embodiments, the anti-tau antibody comprises a human kappa or lambda light chain constant region. In certain embodiments, the anti-tau antibody comprises one or two human kappa light chain constant regions, e.g., comprising SEQ ID NO: 30.

[0095] In some embodiments, 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 SEQ ID NOs: 74 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: 76 and 30, or sequences at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to said sequences; or an HC comprising SEQ ID NOs: 15 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: 16and 30, or sequences at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to said sequences.Also contemplated are the above HC and LC combinations wherein SEQ ID NO: 27 in the HC is modified by “LALA” mutations. Additionally or alternatively, in some embodiments, one HC of the anti-tau antibody may comprise “RF” mutations.

[0096] 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.

[0097] In some embodiments, 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). In certain embodiments, 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. In certain embodiments, 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. For example, 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. In particular embodiments, the tau-binding proteins bind to tau monomers and / or tau fibrils, e.g., with an EC50 as determined in Example 1. In some embodiments, the tau-binding proteins herein bind to both tau monomers and tau fibrils with ECsosZECso ranges as described above.

[0098] In some embodiments, the tau-binding proteins herein bind to an epitope of tau comprising the sequence SLDNITHVPG (SEQ ID NO: 79). For example, in certain embodiments, 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). In certain embodiments, 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.

[0099] In some embodiments, the tau-binding proteins herein bind to tau pathological forms in progressive supranuclear palsy, Alzheimer’s disease, Pick’s disease, or any combination thereof.

[0100] In some embodiments, 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®). In certain embodiments, the tau-binding proteins herein inhibit tau seeding and aggregation with an IC50 of 1-5 nM, e.g., 2-4 nM.

[0101] In some embodiments, 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). In certain embodiments, 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.

[0102] In some embodiments, 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).

[0103] In some embodiments, the tau-binding proteins herein inhibit uptake of tau aggregates (such as tau PFFs) into neurons (e.g., as described in Example 6).

[0104] In some embodiments, 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).

[0105] In some embodiments, 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).

[0106] In certain embodiments, 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.: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).

[0107] In some embodiments, the tau-binding protein has properties a)-m), or a)-i) and k)- m) (e.g., 22-MTBR). In some embodiments, the bispecific antibody has at least properties a)- h) (e g., 523-MTBR).

[0108] It is contemplated that 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).

[0109] In some embodiments, 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. In some embodiments of a brain-targeted tau-binding protein herein, 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. In some embodiments, 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.

[0110] In some embodiments, 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). In certain embodiments, 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). In particular embodiments, 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). In some embodiments, 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. [OHl] In some embodiments, a brain-targeted tau-binding protein herein may be or comprise a multispecific, in particular a bispecific, binding protein, as described below.II. Multispecific Binding Proteins

[0112] The present disclosure also provides tau-binding proteins that are multispecific, e.g., bispecific. In some embodiments, the present disclosure provides a multispecific binding protein (e.g., a bispecific antibody) comprising to 1) a domain that binds to tau (e.g., the MTBR of tau) (“anti-tau binding domain”), and 2) a central nervous system (CNS) target,such as a brain receptor. In certain embodiments, 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. In particular embodiments, the ECR is transferrin 1 (TfR).

[0113] In some embodiments, the anti-tau binding domain of the multispecific binding protein (e.g., bispecific antibody) may be, e.g., an anti-tau binding domain as described herein.

[0114] In some embodiments, 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”). In certain embodiments, the anti-TfR binding domain may be an anti-TfR antibody or an antigenbinding fragment thereof disclosed in PCT Patent Application PCT / IB2023 / 062266. In some embodiments, 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.

[0115] Sequence identifiers (SEQ ID NOs:) 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:*: with charge mutations**: amino acid sequence of YTS

[0116] In some embodiments, 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.

[0117] In some embodiments, 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.

[0118] In some embodiments, 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. For example, 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:SEQ ID NOs: 25 and 26, respectively; orSEQ ID NOs: 77 and 78, respectively.

[0119] In some embodiments, 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 inSEQ ID NOs: 25 and 26, respectively; orSEQ ID NOs: 77 and 78, respectively.

[0120] 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.

[0121] In some embodiments, a multispecific binding protein herein may comprise an anti-tau binding domain comprisingHCDR1-3 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.

[0122] In some embodiments, 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, or- 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 comprisinga 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.

[0123] In some embodiments, 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.

[0124] In embodiments in which the multispecific binding protein is bivalent for tau, it may comprise two anti-tau binding domains, which may have the same VH and VL pair or different VH and VL pairs. For example, in certain embodiments, 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.In particular embodiments, 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.

[0125] Where 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). For example, 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).

[0126] In embodiments where the multispecific antibody has two different heavy chains, to promote heterodimerization of the two heavy chains during manufacturing, 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. For example, 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.

[0127] In some embodiments, 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).In certain embodiments, 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. In particular embodiments, for example, 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.

[0128] In some embodiments, a multispecific antibody herein comprises a human kappa or lambda light chain constant region. In particular embodiments, the multispecific antibody comprises a kappa light chain constant region of SEQ ID NO: 30.

[0129] Any multispecific binding protein format (e.g., multispecific antibody format) is contemplated for the multispecific binding proteins of the present invention. A multispecific binding protein herein (e.g., a bispecific antibody) may be monovalent or bivalent for tau and monovalent or bivalent for the CNS target (e.g., TfR), in any combination. For example, 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. In some embodiments, 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.

[0130] The anti-tau and anti-CNS target portions of the multispecific binding protein, or regions thereof (e.g., VH and / or VL), may be functionally linked, e.g., by noncovalent association, chemical coupling, protein fusion, etc. In some embodiments, the portions or regions thereof are genetically linked (peptide bond) without a peptide linker. In someembodiments, 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. In some embodiments, 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. In some embodiments, 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).

[0131] In some embodiments, 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. For example, where a subpart of the multispecific binding protein (e.g., an antibody arm) comprises two binding domains, such 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). The variable domains, constant regions, and linkers may be in the following conformation:VL2 - Linker 1 - VL1 - Linker 2 - CL VH1 - Linker 3 - VH2 - Linker 4 - CHExamples of 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. In some embodiments, 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.). In particular embodiments, the CNS target is TfR.

[0133] In some embodiments, the bispecific antibody is bivalent for tau and monovalent for the CNS target. For example, in certain embodiments, 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. In some embodiments, the HCDR1-3 and LCDR1-3 of the first and second anti-tau binding domains are the same. In some embodiments, 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). In some embodiments, the first and second anti-tau binding domains are the same.

[0134] In certain embodiments, 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;- a first light chain comprising the VL of the first anti-tau binding domain and the VL of the anti-CNS target binding domain;- a second heavy chain comprising the VH of a second anti-tau binding domain described herein; and- a second light chain comprising the VL of the second anti-tau binding domain.

[0135] In some embodiments, the VH of the anti-CNS target binding domain is N- terminal to the VH of the first anti-tau binding domain, and the VL of the first anti-tau binding domain is N-terminal to the VL of the anti-CNS target binding domain. In some embodiments, the VH of the first anti-tau binding domain is N-terminal to the VH of the anti- CNS target binding domain, and the VL of the anti-CNS target binding domain is N-terminal to the VL of the first anti-tau binding domain. In certain embodiments, 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). In certain embodiments, 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 fourthlinker. 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.

[0136] 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. In certain embodiments, the first and / or second heavy chains (e.g., both heavy chains) further comprise LALA mutations. Additionally or alternatively, one heavy chain (e.g., the heavy chain with hole mutations) may further comprise RF mutations.

[0137] In particular embodiments, 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, and the second heavy chain may pair with the second light chain.

[0138] In certain embodiments, 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). In some embodiments, the HCDR1-3 and LCDR1-3 of the first and second anti-tau binding domains are the same. In some embodiments, 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). In some embodiments, the first and second anti-tau binding domains are the same. In certain embodiments, the bispecific antibody comprises- a first heavy chain comprising the VH of a first anti-tau binding domain described herein and the VH of a second anti-tau binding domain described herein;- a first light chain comprising the VL of the first anti-tau binding domain and the VL of the second anti-tau binding domain;- a second heavy chain comprising the VH of an anti-CNS target binding domain; and- a second light chain comprising the VL of the anti-CNS target binding domain.

[0139] In some embodiments, the VH of the first anti-tau binding domain is N-terminal to the VH of the second anti-tau binding domain, and 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. In certain embodiments, 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). In certain embodiments, 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.

[0140] 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. In certain embodiments, the first and / or second heavy chains (e.g., both heavy chains) further comprise LALA mutations. Additionally or alternatively, one heavy chain (e.g., the heavy chain with hole mutations) may further comprise RF mutations.

[0141] In particular embodiments, 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, and the second heavy chain may pair with the second light chain.

[0142] In some embodiments, 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 5pM (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.

[0143] Additionally or alternatively, in some embodiments, the multispecific binding protein herein (e.g., a bispecific antibody 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. In certain embodiments, 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.

[0144] In some embodiments, the multispecific binding protein herein (e.g., a bispecific antibody 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.

[0145] In some embodiments, the multispecific binding protein herein (e.g., a bispecific antibody 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 Octet™. In certain embodiments, the multispecific binding protein binds to hTfR with a KD of 10-40 nM (e.g., 15-30 nM) as determined by Octet™.

[0146] Additionally or alternatively, in some embodiments, the multispecific binding protein herein (e.g., a bispecific antibody 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. In certain embodiments, the multispecific binding protein binds to cTfR with an ECso of 1-250 nM (e.g., 15-150 nM) as determined by FACS. In certain embodiments, the multispecific binding protein binds to cTfR with an ECso of 1-20 nM as determined by FACS.

[0147] In some embodiments, the multispecific binding protein herein (e.g., a bispecific antibody 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. In certain embodiments, 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.

[0148] In some embodiments, the multispecific binding protein herein (e.g., a bispecific antibody 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 Octet™. Incertain embodiments, 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 Octet™.

[0149] In particular embodiments, the multispecific binding protein herein (e.g., a bispecific antibody herein) binds to tau, hTfR, and / or cTfR with binding affinities (i.e., an ECso or KD) as determined in Example 3.

[0150] In some embodiments, 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).

[0151] In some embodiments, the multispecific binding protein herein (e.g., a bispecific antibody herein) achieves brain and / or CSF exposure that is at least 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold (e.g., at least 5-fold) higher than that of the monospecific anti-tau antibody. In certain embodiments, 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.

[0152] In some embodiments, 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).

[0153] In some embodiments, 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).

[0154] In some embodiments, the multispecific binding protein herein (e.g., a bispecific antibody 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.

[0155] A multispecific binding protein with any combination of the above properties is also contemplated.

[0156] In certain embodiments, the multispecific binding protein (e.g., bispecific antibody) 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 Octet™; g) binds to cynomolgus TfR with a KD of 50-200 nM as determined by Octet™; h) has improved brain penetrance in hTfR knock-in mice by at least 3 -fold over the monospecific anti-tau binding domain; i) has increased brain and CSF exposure by at least 5-fold in cynomolgus monkeys over the monospecific anti-tau binding domain; j) reduces tau seeding in vitro., k) inhibits uptake of tau aggregates into neurons; l) reduces AT8+cells in vivo in the cortex of hTfR knock-in / THY-Tau22 mice; or m) any combination of a)-l).In some embodiments, the multispecific binding protein has all of properties a)-l). In some embodiments, the multispecific binding protein has at least properties a)-h).

[0157] In some embodiments, 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. In certain embodiments, on the first heavy chain, 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, and on the first light chain, 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. In some embodiments, 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. For example, 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.The first and / or second anti-tau domain may be, e.g., an anti-tau domain described herein.For example, 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. In particular embodiments, 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. In some embodiments, 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. For example, 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 Octet™; f) binds to cynomolgus TfR with a KD of 50-200 nM as determined by Octet™;The anti-TfR binding domain may be, e.g., an anti-TfR binding described herein.II. Making of Binding Proteins

[0158] The binding proteins (e.g., monospecific or bispecific binding proteins) described herein 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) referred to herein as “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.

[0159] Methods of producing proteins such as antibodies are well known. The present binding proteins such as antibodies may be produced in, e.g., mammalian host cells, using appropriate expression constructs. 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. Other 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.III. Pharmaceutical Compositions and Uses

[0160] 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. As used herein, “pharmaceutically acceptable” with reference to a carrier,” “excipient,” or “diluent” includes appropriate solvents, dispersion media, antibacterial and antifungal agents, isotonic agents, and the like. In some embodiments, the pharmaceutical composition is a sterile aqueous solution, and may comprise a buffer; a surfactant; a polyol; an antioxidant; and / or a chelating agent. In some embodiments, the pharmaceutical composition is provided in a lyophilized form and is reconstituted before administration. In certain embodiments, lyophilized antibody formulations may comprise a bulking agent.

[0161] The pharmaceutical composition may be administered to patients by parenteral administration (e.g., by injection or infusion). For example, the pharmaceutical composition may be administered by an intravenous, intracerebral, intracranial, or spinal route.

[0162] The monospecific and bispecific binding proteins herein (e.g., in the pharmaceutical compositions herein) are useful in treating a human patient with, or at risk ofdeveloping, tauopathy. For example, 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). In particular embodiments, the primary tauopathy is PSP. In particular embodiments, the secondary tauopathy is Alzheimer’s disease.

[0163] As used herein, 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.

[0164] 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.

[0165] 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). In particular embodiments, the medicament is capable of crossing the BBB. Further, 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).

[0166] 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). For example, 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 fluidsample 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. The present disclosure further encompasses kits (e.g., diagnostic kits) comprising the binding proteins described herein.

[0167] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used 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.

[0168] According to the present disclosure, back-references in the dependent claims are meant as short-hand writing for a direct and unambiguous disclosure of each and every combination of claims that is indicated by the back-reference. Further, headers herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any manner.

[0169] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.EXAMPLESExample 1: Generation and Validation of Anti-hTau-MTBR AntibodiesMaterials and MethodsImmunization and Hybridoma Generation

[0170] Immunizations, fusion and screening were performed using P3X63-Ag8.653 myeloma cells with fibril tau MTBR (VA213048) as described in Wennerberg et al., Am J Pathol, (1993). 143(4), 1050-54.

[0171] 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.

[0172] 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.Screening and In Vitro Characterization of Murine Anti-Tau MTBR Antibodies

[0173] Primary screening for anti-tau MTBR IgG production was performed by Enzyme- Linked Immunosorbent Assay (ELISA) using fibril tau MTBR (VA213048) as the capturing antigen. Plates were coated with fibril tau MTBR at 0.5 pg / well in PBS and 100 pL / well of anti-tau MTBR antibodies were added to the plate. The plate was incubated at 37°C for one hour and washed five times with PBS containing 0.05% Tween-20 (PBS-T). Then, 100 pL of a 1 :50,000 dilution of rabbit anti-mouse IgG conjugated with horseradish peroxidase (Sigma; Cat. No. A9044) was added to each well. Following incubation at 37°C for one hour in darkness, plates were washed with PBS-T five times. Antibody binding was visualized by adding TMB-H2O2 buffer and read at a wavelength of 450 nm.

[0174] 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

[0175] Kinetic constants of the anti-tau antibodies were measured by surface plasmon resonance (SPR) on a Biacore™ T200 instrument. Anti-huFc antibody was covalently immobilized on CM5 Sensor Chips (Cytiva) following provider’s instructions. About 10,000 RU of anti-huFc was obtained on each flowcell. All samples were diluted in running buffer (1XHBS-EP+, Cytiva). Anti-tau antibodies were captured at 0.5 pg / ml on the anti-huFc surface. Serial concentrations of tau MTBR (VA214191) starting from 200 nM were injected over the anti-tau antibody surface for 3 min at 30 pL / min using single-cycle kinetics injection. Dissociation was monitored for 5 min. Surfaces were regenerated with one 30 sec pulse of 3M MgCh at 20 pL / min. Biacore™ T200 Evaluation software was used for analysis. Sensorgrams were double referenced with reference surface (bulk and weak nonspecific binding subtraction) and blank (drift removal) and curves were fitted with a 1 : 1 binding model and RI fixed to 0.Preparation of q-Synuclein Fibrils

[0176] 1 mg of 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. The determination of fibril concentration was performed using the Bradford test and fibrils were characterized by a Thioflavin T (ThT) fluorescence assay with the following conditions: 50 pL of pure fibril sample or diluted at i or 1 / 10 in PBS IX + 5 pL of ThT 10 pM final. After 30 min of incubation at room temperature, fluorescence was measured on a Tecan device (Saphire) with kExcitation=455nm and kEmission=485nmProduction and Purification of Humanized Antibody Variants

[0177] Variants were expressed in polyclonal stable CHO-9E4 cell lines generated by PiggyBac™ 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). The ProtA wasperformed using a Mab Select SuRe (MSS) resin packed in column (5 mL up to 50 mL) connected to AKTApure™ 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). At the end of the step, the elution fractions of interest were grouped together. Immediately afterwards, 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 Superdex™ 200 resin packed in column (CV of 320 mL up to 6.6 L) connected AKTApure™ 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. By 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.

[0178] Quality control of the antibodies was performed by Size Exclusion Chromatography-High Pressure Liquid Chromatography (SEC HPLC), mass spectrometry (MS), and Thermal Stability and Dynamic Light Scattering (DLS). 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.

[0179] Mass spectrometry was performed using a Q-Exactive™ Plus with Acquity UPLC system (Thermo Fisher) with GeneData expressionist software. Column MabPAC RP 4 pm, 2.1 x 100 mm (0.346 mL) (Thermo Scientific) was used at a flow rate of 300 pL / min (520 cm / h). Buffer A was H2O, 0.1% formic acid, and 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.

[0180] Thermal stability was assessed using a Prometheus™ NT 48 (Nanotemper Technology). Thermal stability parameters: Tonset, Tmsand Taggwere 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.

[0181] Dynamic light scattering (DLS) was performed to estimate particle size distribution and to document the aggregation state of the solution. Samples were processed in their own buffer without any dilution. A 2 pL aliquot of each sample was added to Stunner plates (Unchained Labs). Measures were performed at 20°C. Three independent replicates were performed. Autocorrelation function drew correlograms that were further transformed into histograms giving the size and hydrodynamic diameter distributions of the different populations. The number and sharpness of peaks indicated whether the sample is heterogeneous or not. UV concentration and hydrodynamic diameter were also measured in a single experiment.ELISA for Tau Binding

[0182] 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. 30 pL of washing buffer (phosphate buffered saline (Gibco 14190-094), 0.05% Tween® 20 (Sigma P1379)) were added per well, incubated 15-30 seconds, and aspirated (two times). 50 pL of blocking buffer (phosphate buffered saline (Gibco 14190-094), 0.05% Tween® 20 (Sigma P1379), 1 % BSA (Sigma A3803)) were added to each well. 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 1.25xl0'15pg / 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 with50 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 Magellan™ software at 450 mm within 30 minutes after adding stop solution.U2OS Seeding Assay

[0183] U2OS cells stably overexpressing the full-length human tau (441 amino acids 2N4R) with G272V and P301S mutations were cultured in DMEM + GlutaMAX™ (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).Immunohi stochemi stry

[0184] Immunohistochemistry of human derived tissue and immunocytochemistry were performed using fully automated immunohistology on a DISCOVERY Ultra system (Ventana, Roche) using the 22-MTBR and 523-MTBR antibodies and the OmniMap anti-Rb HRP (RUO) (Roche). Single marker DAB staining was accomplished using a BenchMark Ultra system and the ultraView Universal DAB Detection Kit, according to the manufacturer’s recommendations. The DISCOVERY Universal Procedure was used to create a protocol. For iHC, paraffin embedded slides were deparaffinized by warming the slide to 70°C for 3 cycles each 8 min long. Antigen retrieval was performed by applying Cell Conditioning 1 (VMSI Cat. No. 950-124) and warming the slide to 94°C for 64 min.Staining of each antibody was performed by incubation with primary antibody, washing in reaction buffer, incubation with secondary antibody conjugated to either peroxidase, andwashing with reaction buffer. Slides were then counterstained with diluted Hematoxylin II for 4 min and washed with reaction buffer. Slides were then manually dehydrated through an ethanol series (2 x 80% ethanol, 1 min each, 2 x 90% ethanol, 1 min each, 3 x 100% ethanol, 1 min each, 3 x xylene, 1 min each), at ambient temperature, and mounted in Cytoseal™ XYL mounting medium (ThermoFisher).Object Recognition Assay

[0185] 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. Climbing onto the object (unless the mouse smells the object it has climbed on) or chewing the object does not qualify as exploration. Animals that do not explore each object for more than 1 sec or explore nothing during the first six minutes were excluded from analysis. Behavior was analyzed by an experimenter blind to the treatment and group conditions.Extension Reflex

[0186] Animals were suspended by the tail and kept at a height of 40 cm above the table top for a duration of 30 s, during which the presence of hindlimb clasping behavior was observed.Epitope Mapping

[0187] For epitope mapping, dot blot and ELISA were used. Overlapping recombinant peptides spanning the whole sequence of the tau MTBR (15 amino acid length - peptides every 6 amino acids starting with SRLQTAPVPMPDLKN (SEQ ID NO: 100) and ending with GGNKKIETHKLTFRE (SEQ ID NO: 101) were generated. For dotblots, 10 pg of each peptide was deposited on a PVDF 0.22 pm membrane and air-dried for a few minutes. 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). For 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. 30 pL of washing buffer (phosphate buffered saline (Gibco 14190-094), 0.05% Tween® 20 (Sigma P1379)) were added per well, incubated 15-30 seconds, and aspirated (two times). 50 pL of blocking buffer (phosphate buffered saline (Gibco 14190-094), 0.05% Tween® 20 (Sigma P1379), 1 % BSA (Sigma A3803)) were added to each well. 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 1.25xl0'5pg / 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 Magellan™ software at 450 mm within 30 minutes after adding stop solution.Results

[0188] To generate antibodies against the microtubule binding region (MTBR) of tau protein (Tau-MTBR), recombinant aggregated human MTBR was produced and used for mouse immunization and hybridoma cell production. The resulting anti-tau-MTBR cloneswere tested for their ability to bind fibrils and monomers of full-length tau as well as tau MTBR fibrils and monomers ELISA (Table 1). Two clones — 22-MTBR and 523-MTBR — demonstrated the ability to bind to all four tau species. Additionally, the measured affinities of the 1 :1 interaction with monomers for these species were in agreement with previous data. In comparison, commercial antibodies MCI and PHF1 that comprise epitopes outside of the tau MTBR were unable to recognize tau monomers or MTBR species.Table 1: Tau Species Binding Activity of Anti-Tau-MTBR AntibodiesFL: full-length

[0189] 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.Table 2: EC50 Values of Anti-Tau-MTBR Antibodies via ELISA Assay| ] monomer, ] fibrils, ] a-Synuclein Fibril| | ELISA | ELISA | Binding, ELISA ECso || 22-MTBR | 0.16 nM | 0.18 nM | No binding || 523-MTBR | 0.30 nM | 0.29 nM | No binding |

[0190] Additionally, the selected anti-tau-MTBR antibodies were tested for their ability to recognize pathological tau. Alzheimer’s disease (AD) and control human brain tissue as well as brain sections from a mouse model of tauopathy (THY-Tau22) and 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. Different cell lines overexpressing tau and their non-overexpressing counterparts (SHSY-Tau, SHSY-WT, U2OS-Tau, U2OS-WT, and iPSC cells) were stained with 22-MTBR and 523-MTBR. From immunocytochemistry analysis, both antibodies showed binding to human tau in overexpressing cell lines and iPSC cells while staining of non-overexpressing cells showed minimal binding. To note, 22-MTBR showed better binding than 523-MTBR suggesting a distinct profile of tau recognition. Additionally, immunohistochemical staining of cells with 22-MTBR or 523-MTBR showed specific cellular and regional staining of various tauopathies including Progressive Supranuclear Palsy (PSP), Alzheimer’s disease (AD), and Pick’s Disease (FIGs. 1A-1C). This suggests that 22-MTBR and 523-MTBR are able to recognize various tau isoforms and can be used to treat various tauopathies.

[0191] Furthermore, 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).

[0192] 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

[0193] To determine the binding kinetics of the selected antibodies, 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.

[0194] To further determine the effectiveness of anti-tau-MTBR treatment, 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. Following treatment, the 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). For 22-MTBR, the reduction in hippocampal tau pathology was also observed by immunohistochemistry (AT8+cell number) with a 30% reduction in tau pathology progression (FIG. 3).

[0195] The 22-MTBR antibody was further evaluated for neuroprotective effect via measurement of neurofilament light (NFL) in the cerebrospinal fluid. 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.

[0196] Both antibodies were effective at reversing cognitive deficits in a THY-Tau22 model. To evaluate the effectiveness of these antibodies as a curative treatment in vivo, male THY-Tau22 mice were administered 22-MTBR and 523-MTBR antibodies at a dose of 20 mg / kg via intraperitoneal injection. Four days after the antibody treatment, an object recognition test was performed to assess the effect of the antibody on tau-related cognitive deficit. Both 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). In a follow-up experiment, various concentrations of 22-MTBR were intraperitoneally injected in THY-Tau22 mice weekly for 14 weeks. Following this treatment, the 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).

[0197] To further test the antibody treatment efficacy, 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. Following MTBR injection, the 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).

[0199] Finally, to further characterize the anti-tau-MTBR antibodies, epitope mapping was conducted for 22-MTBR and 523-MTBR using dot blots and ELISA with tiling peptides from the MTBR of the tau protein. The dot blot confirmed strong interaction of 22-MTBR with the peptide QSKIGSLDNITHVPG (SEQ ID NO: 80) within the fourth microtubulebinding region repeat sequence (FIG. 8, left). This interaction was confirmed with ELISA, which also found weak / moderate interactions with the sequences in the “inter-repeats” of the microtubule-binding region (APVPMPDLKNVKSKI; SEQ ID NO: 81) and the second and third microtubule-binding region repeat sequences (NVQSKCGSKDNIKHV (SEQ ID NO: 82) and SKVTSKCGSLGNIHH (SEQ ID NO: 83), respectively). ELISA analysis indicated strong interaction of 523-MTBR with the peptide sequence SLDNITHVPGGGNKK (SEQ ID NO: 84) in the fourth microtubule-binding region repeat sequence, and found weak / moderate interactions with the inter-repeats region (APVPMPDLKNVKSKI (SEQ ID NO: 81)), and the third and fourth microtubule-binding region repeat sequences (SKVTSKCGSLGNIHH (SEQ ID NO: 83) and QSKIGSLDNITHVPG (SEQ ID NO: 80), respectively) (FIG. 8, right).Example 2: Generation and Validation of Mouse Bispecific Anti-TfR / Anti-Tau-MTBR AntibodiesMaterials and MethodsBinding Kinetics of Anti-TfR Antibodies by SPR

[0200] Kinetic constants of the anti-TfR antibodies were measured by SPR on Bruker MASS-2 instrument with HBS-EP+ as running buffer and sample diluent (Cytiva Life Sciences, #BR100826). HCA sensorchips (Bruker) were immobilized with anti -His antibody (His capture kit, Cytiva Life Sciences) following provider’s instructions. About 10,000 RU of anti-His was obtained. His-tagged TfR proteins (internal productions; 5 pg / ml) werecaptured for 1 min at 10 pL / min. Concentration series of anti-TfR antibodies were then injected for 4 min at 30 pL / min over TfR surfaces and dissociation was monitored for 5 min. Surfaces were regenerated with one 1 min pulse of 10 mM Glycine-HCl pH 1.5. Bruker Sierra Analyzer software was used for analysis. Sensorgrams were double referenced with reference surface (bulk and weak non-specific binding subtraction) and blank (drift removal) and curves were fitted with a 1 : 1 binding model.Octet™ Analysis of Binding to Human and Cynomolgus TfR

[0201] For biolayer interferometry (BLI) assays, human and cynomolgus monkey transferrin receptor (TfR) were produced in Expi293 cells and biotinylated on an N-terminal AviTag using BirA. The human TfR amino acid sequence used is found in SEQ ID NO: 97. The cynomolgus TfR amino acid sequence used is found in SEQ ID NO: 98.

[0202] To measure the binding affinity of antibodies to TfR, we performed Octet™ binding assays and utilized the monovalent anti-TfR antibody (53 lv25) as a control. The Octet™ HTX system was used with streptavidin coated biosensors (Sartorius Cat. No. 18- 5019) bound to recombinant biotinylated human or cynomolgus TfR loaded at 4 pg / mL for 120 seconds. Antibodies were diluted 5-fold in lx HBSP buffer (Cytiva Cat. No. BR100671) to generate working stock; the monovalent antibody was already at an appropriate working stock concentration. 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, ka(1 / Ms), kaerror, 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 Octet™ version number for software: Octet™ Analysis Studio 12.2. For data correction and preprocessing, the following software options were chosen:Align Y axis: Align data to average of baseline stepInter-step correction: Align data to Baseline stepFiltering: check box for Savitzky-Golay filtering

[0203] For kinetic analysis fitting parameters, the following settings were used:Step to analyze: Association and dissociation Binding model: 1 : 1 Fitting type: Global Group by : Sample ID second Group by: loading sample IDRmax values: sensorFACS-Based Assay to Determine Binding of Antibodies to hTfR

[0204] 300.19 huTFRC-expressing cells (clone 5650-24; # AG-953) or 300.19CynoTFRC-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® easyCyte™ 8HT Flow Cytometry System. Apparent KD and ECso values were estimated using BIOST@T-BINDING and BIOST@T-SPEED software, respectively.AT8 Immunohistology

[0205] 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. Slides were then washed with water and dehydrated using ethanol and xylene (2 x80% ethanol, 1 min each, 2 x 90% ethanol, 1 min each, 3 x 100% ethanol, 1 min each, 3x xylene, 1 min each), at ambient temperature, and mounted in Cytoseal™ XYL (ThermoFisher).In Vivo Studies

[0206] 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.NFL ELISA

[0207] 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.HEK293T FRET Assay for Tau Aggregation

[0208] To assess if bispecific anti-tau / anti-TfR antibodies can engage seeding competent tau species (tau pre-formed fibrils (PFFs) that induce tau aggregation), immunoprecipitation of tau-PFFs was performed and the supernatant was applied to an HEK293T tau aggregation FRET (Fluorescence Resonance Energy Transfer) assay. A plasmid construct (SEQ ID NO: 99) was generated with sequences for mVenus tagged tau-MTBR and mTurquois-MTBR separated by a 2A self-cleaving sequence.

[0209] Cleavage generates two tau-MTBR fragments that when aggregated, give off a FRET signal based on the proximity of the mVenus and mTurquoise2 tags. For transfection, a concentrated Lipofectamine™ 2000 (Invitrogen Cat. No. 52758) stock of 80 pL / mL was made in Opti-MEM™ (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-MEM™. This transfection solution was then applied at 100 pL / well to HEK293T cells that were plated at 15,000 / well inmedia (DMEM containing 10% serum and lx Penicillin Streptomycin) in 96-well Cell- Carrier ultra plates coated with Poly-D-lysine.

[0210] For immunoprecipitation, 750 pg of Protein-G Dynabeads™ (Invitrogen Cat. No. 100070) were mixed, added to 1.5 mL tubes, and washed with PBS in a magnetic field. Dynabeads™ were then incubated with 5 pg of antibodies in PBS on a rotator for 10 minutes at room temperature. The supernatant was removed while the bead-antibody complexes were immobilized by a magnetic field. Beads were washed once with 100 pL of PBS. 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.

[0211] One day prior to the immunoprecipitation, 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). Approximately 6 hours after transfection of HEK293T cells with the FRET biosensor, 10 pL of supernatant from the tau immunoprecipitation was added to 89 pL of Opti-MEM™ and 1 pL of Lipofectamine™, then the 100 pL mixture was added on top of the 100 pL media that was used for the transfection.Pharmacokinetic Assessment of Total Antibodies and Unbound Antibodies

[0212] Streptavidin coated MSD GOLD 96-well SMALLSPOT® plates (Meso Scale Discovery) were blocked with Superblock™ T20 (Thermo Scientific) for 1 hour at room temperature (RT) and washed three times using PBS / 0.05% Tween® 20 on an automated plate washer. The capture tool, biotinylated rat anti-human kappa mAb (2.0 pg / mL; Synabs; Cat. No. Lo-hK-3) or biotinylated recombinant tau MTBR (1.0 pg / mL), was applied and incubated for 1 hour at RT, while shaking at 600 rpm. 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.Results

[0213] To enhance the efficacy of the anti-tau-MTBR antibody, trivalent cross-over dual variable (TrioCODV) antibodies were designed to incorporate a wild-type anti-mouse TfR antibody 8D3 to act as brain shuttle with the anti-tau 22-MTBR antibody. Three different positions of the anti-TfR domain within the antibody structure were tested: position 1, wherein the anti-TfR domain is in the “inner” position; position 2, wherein the anti-TfR domain is in on the Fab arm; or position 3, wherein the anti-TfR domain is in the “outer” position (FIG. 9). These antibodies were designed with a mouse IgGl-KIH (Knob Into Hole) Fc region; some variants were also designed with mutations to the light chain (Y92A) or heavy chain (Y52A) of the anti-TfR arm. Additionally, a set of bispecific Duobody CODVs were designed using a mouse IgGl-cFAE (controlled Fab Arm Exchange) Fc region with the anti-TfR 8D3 and anti-tau 22-MTBR antibody arms. All antibodies were expressed in stable CHO cells, purified, and evaluated for their affinity to tau and TfR, their cellular activity via tau seeding in U2OS cells, and their brain exposure (Table 4).Table 4: Production and In Vitro Evaluation of Bispecific Mouse Anti-TfR / Anti-Tau AntibodiesPos: position of TfR-8D3 binding domain (see FIG. 9)

[0214] 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.

[0215] These studies indicated that the position of the TfR paratope had little influence on the in vitro TfR affinity, and the construct with mitigated TfR affinity displayed better brain exposure. Thus, 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

[0216] To demonstrate the in vivo efficacy of the TrioCODV 22-8D3-4 anti-TfR / anti-Tau- MTBR antibody, THY-Tau22 mice were injected with either the TrioCODV 22-8D3-4 antibody (twice at a dose of 42 nmol / kg) or the monospecific 22-MTBR antibody (twice at a dose of either 42 nmol / kg or 10 nmol / kg). Following administration of the antibodies, 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.Example 3: Generation and Validation of Human Bispecific Anti-TfR / Anti-Tau-MTBR Materials and Methods

[0217] Assays were performed as described in the above Examples.Results

[0218] To enhance the efficacy of an anti-tau-MTBR antibody in humans, 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. In some constructs 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). Additionally, 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. Additionally, different linkers were utilized in the design: 10-10-0-0 classical linker or 7-5-1-2 linker. These constructs were expressed and purified for analysis of their affinity for tau and TfR (Table 6). The constructs with the best parameters -- 22-53 lv25-3, 22-53 lv25-4, and 523-53 lv25-l — were utilized for pharmacokinetic studies.Table 6: Production and In Vitro Evaluation of Bispecific Human Anti-TfR / Anti-Tau Antibodies* vs ECso value of 22-MTBR-hIgGl-LALA (mAb control)_ECso= 6pM

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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 IDNO: 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.

[0223] The three selected variants — 22-53 lv25-3, 22-53 lv25-4, and 523-53 lv25-l — were evaluated for their ability to bind tau and human or cynomolgus TfR by ELISA, fluorescence-activated cell sorting (FACS), surface plasmon resonance (SPR), or Octet™ assays (Table 7). Antibody 22-53 lv25-3 demonstrated better affinity for both tau and TfR.Table 7: Pharmacokinetic Evaluation of Anti-TfR / Anti-Tau Antibodies* vs ECso value of 22-MTBR-hIgGl-LALA (mAb control )_ECso= 6pM

[0224] The brain exposure of the anti -TfR / anti -tau antibodies was also evaluated following antibody treatment. 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.

[0225] 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 profilein 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. In these analyses, the bispecific 523-MTBR antibody with the TfR component on the Fab (523-53 lv25-l) showed better brain exposure. Additionally, 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.Table 8: Brain Pharmacokinetic Profile of Anti-TfR / Anti-Tau AntibodiesTable 9: Plasma Pharmacokinetic Profile of Anti-TfR / Anti-Tau Antibodies

[0226] The brain and plasma exposure at different dose levels was also examined by intraperitoneally injecting THY-Tau22 / humanized TfR-knock in mice with 10, 42, or 200 nmol / kg of either the bispecific anti-TfR / anti-tau antibody 22-53 lv25-3, or the monospecific 22-MTBR antibody 22-MTBR. Samples were collected several days after administration and the pharmacokinetic profile evaluated (Plasma: Table 10; Brain: Table 11). The lower limit of quantification (LLOQ) for 22-53 lv25-3 was 0.241 nmol / L in plasma, and the lower limit of quantification for 22-MTBR was 0.285 nmol / L in plasma. Across the dose levels tested, both 22-53 lv25-3 and 22-MTBR exhibited plasma exposure increases that appeared in proportion with the dose increases. However, 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.Table 10: Plasma Pharmacokinetic Profile of Anti-TfR / Anti-Tau AntibodyTable 11: Brain Pharmacokinetic Profile of Anti-TfR / Anti-Tau Antibody* due to values below the LLOQ

[0227] 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). 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. Additionally, 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.

[0228] To test the efficacy of the bispecific anti-TfR / anti-tau antibody in human cells, 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). Incubation of PFFs with either bispecific 22-53 lv25-3 or monospecific 22-MTBR resulted ina reduction in FRET spots, a measure of tau aggregation, indicating that the bispecific antibody effectively recognizes tau seeding competent species in vitro.

[0229] To assess the efficacy of a bispecific anti-TfR / anti-tau antibody as a curative treatment in vivo, six-month old humanized TfR-knock in / THY-Tau22 mice were immunotolerized by the injection of 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. Following treatment, 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.Example 4: Biolayer Interferometry Assay of Tau Monomers and FibrilsMaterials and methods

[0230] Human recombinant tau-441 (2N4R) wild-type protein monomers, human tau-441 (2N4R) P301S mutant protein monomers, and human tau pre-formed fibrils (PFFs) were obtained from StressMarq Biosciences. Biolayer interferometry binding kinetics were measured using an Octet® HTX system (Sartorius) at room temperature. Assays were performed in HBSP buffer (10 mM HEPES, 150 mM NaCl, 0.05% P20, pH 7.4). Sartorius Octet® AHC Biosensors (Cat. No. 18-5060, lot 2307007611) were used for internal antibodies, while Sartorius Octet® SA Biosensors (Cat. No. 18-5019, lot 2308004611) were used for 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

[0231] The binding kinetics of 22-53 lv25-3 and 22-MTBR to tau monomers and fibrils were assessed using biolayer interferometry. A high affinity commercially available antibody (HT7) was used as positive control in the experiment. Both 22-MTBR and 22-53 lv25-3 exhibited similar binding profiles to tau monomers and aggregates. Specifically, both antibodies showed binding to monomers but quick dissociation (FIGs. 17A, 17B, 18A, and 18B) HT7, on the other hand, also bound tau monomer but had a slower dissociation rate (FIGs. 19A and 19B) When the binding of the three antibodies to tau fibrils was assessed, 22-MTBR, 22-53 lv25-3, and HT7 bound tau-PFFs had minimal detectable off-rates. This is indicative of 22-MTBR and 22-53 lv25-3 being selective for tau fibrillar species.Example 5: Biolayer Interferometry (BLI) Assay of Transferrin Receptor in the Presence of Tau ProteinsMaterials and methods

[0232] 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 AviTag™ 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 Octet™ binding assays and utilized the monovalent anti-TfR antibody (53 lv25) as a control. The Octet™ HTX system was used with streptavidin coated biosensors (Sartorius Cat. No. 18-5019) bound to recombinant biotinylated human TfR loaded at 4 pg / mL for 120 seconds. Antibodies were diluted 10-fold in lx HBSP buffer (Cytiva Cat. No. BR100671) to generate working stock; the monovalent antibody was already at an appropriate working stock concentration. Human recombinant Tau-441 (2N4R) wild-type protein monomers, and human Tau pre-formed fibrils (PFFs) were obtained from StressMarq Biosciences. Tau PFFs were thawed then moved to Bioruptor® sonication tubes and PFFs were sonicated at 4 degrees for 5 cycles (1 cycle is 30 seconds on, 30 seconds off); monomers were not sonicated. After sonication, antibodies were preincubated with tau PFFs or monomers on a room temperature rotator for 30 minutes prior to addition to the assay.

[0233] 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 werethen 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.Results

[0234] The binding kinetics of 22-53 lv25-3 and 22-53 lv25-4 and of 53 lv25 (mono-arm version) to the transferrin receptor were assessed by biolayer interferometry and evidenced profile differences when bound or unbound to tau PFFs. Specifically striking was the significant decrease in the off-rate of 22-53 lv25-4 in the presence of tau PFF aggregates, indicating a stronger binding to TfR in the presence of tau. This stronger binding in the presence of tau was not observed with either 22-53 lv25-3 or the monospecific 53 lv25 recognizing the human transferrin receptor, which retained stable off rates (FIG. 20). This result indicates that the format of the TfR-Tau bispecific antibody governs the biophysical response of the antibody to binding of both targets.Example 6: Tau Aggregate Uptake Assay in iPSC-Derived NeuronsMaterials and Methods

[0235] To assess antibody-mediated inhibition of tau-PFF internalization, human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (iCell® GlutaNeurons, Cellular Dynamics International) were cultured according to the manufacturer's instructions. Cells were seeded at a density of 35,000 cells per well in CellCarrier™ Ultra plates and maintained under standard culture conditions. Human full-length wild-type tau pre-formed fibrils (PFFs) were labeled with pHrodo™, a pH-sensitive fluorescent dye that increases in fluorescence intensity in acidic environments, using the pHrodo™ iFL Microscale Protein Labeling Kit according to the manufacturer's protocol (Invitrogen). 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 pHrodo™-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 pHrodo™ fluorescence intensity normalized to cell confluence.Results

[0236] Tau preformed fibrils (PFFs) were labelled with pHRodo™, a pH sensitive dye, to monitor internalization of tau aggregates into the endolysosomal system. Increasing concentrations of antibodies were incubated with the pHRodo™-PFFs (5 pg / mL) and incubated with iPSC-derived neurons. Both 22-52 lv25-3 and 22-MTBR antibodies inhibited uptake of tau PFF aggregates into neurons in a dose dependent manner (FIG. 21).

[0237] Monospecific tau antibodies, including 22-MTBR, inhibited tau uptake into neurons, and while the bispecific 22-52 lv25-3 also exhibited this property, it was not shared by all TfR-Tau bispecific antibodies. Indeed 22-52 lv25-4 exhibited a significant enhancement of tau PFF uptake into neurons at 200 nM (FIG. 22).

[0238] Dose-dependent enhancement of tau PFF uptake was also seen with the 523- MTBR anti-tau antibody in a TrioCODV format, using 523-53 lv25-la (FIG. 23). 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.Example 7: Acute In Vivo Seeding AssayMaterials and Methods

[0239] Two-month-old ThyTau22 x hTfRKI mice were deeply anesthetized with 2% (vol / vol) isoflurane, immobilized in a stereotaxic frame and tau preformed fibrils were injected at the following coordinates from the bregma suture (anteroposterior, -2.5 mm; mediolateral, -2 mm; dorsoventral, -2 mm from the dura) with a Hamilton syringe under aseptic conditions. 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 wasadministered. 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. Coronal sections (30 pm thick) were sliced using a freezing microtome for immunohistochemical analysis. AT8 immunohistology was performed as described in Example 2. Histological analysis was performed using Halo analysis software. Brain regions were identified using the Allen Brain Atlas as an anatomical reference. To minimize bias, the analyst was blinded to all treatment groups. Quantification included measurement of AT8- positive staining across different brain regions. Data were analyzed using GraphPad Prism software. Comparisons between treatment groups were performed to assess differences in tau pathology burden as measured by AT8 immunoreactivity.Results

[0240] To understand the functional effect of the different properties of 22-53 lv25-3 and 22-53 lv25-4 (22-53 lv25-4 having increased uptake of tau aggregates into neurons and decreased off-rate for TfR binding when bound to tau), 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. The presence of tau pathology in the ipsilateral hippocampus (injected side) 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.

[0241] In both the ipsilateral and contralateral hippocampi, tau pathology was drastically increased when tau PFFs were pre-incubated with 22-53 lv25-4 compared to tau PFFs incubated with the vehicle (FIGs. 24 and 25). For both MTBR-22 and 22-53 lv25-3, no increased pathology was observed (FIGs. 24 and 25). This result indicates that the different properties of 22-53 lv25-4 and 22-53 lv25-3 have a clear functional consequence, with 22- 53 lv25-4 acutely enhancing tau pathology seeding and spreading in the brain of this tauopathy mouse model.

[0242] 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.SEQUENCES

[0244] Sequences described in the present disclosure are summarized in the table below (SEQ: SEQ ID NO).

Claims

CLAIMS1. 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.

2. The tau-binding protein of claim 1, wherein the VH and the VL are at least 90% identical to:SEQ ID NOs: 7 and 8, respectively;SEQ ID NOs: 15 and 16, respectively;SEQ ID NOs: 73 and 75, respectively; orSEQ ID NOs: 74 and 76, respectively.

3. The tau-binding protein of claim 1 or 2, wherein the VH and the VL comprise:SEQ ID NOs: 7 and 8, respectively;SEQ ID NOs: 15 and 16, respectively;SEQ ID NOs: 73 and 75, respectively; orSEQ ID NOs: 74 and 76, respectively.

4. The tau-binding protein of any one of claims 1-3, wherein the tau-binding protein has 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 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).

5. The tau-binding protein of claim 4, wherein the tau-binding protein has all of properties a)-m), or at least properties a)-h).

6. The tau-binding protein of any one of claims 1-5, wherein the tau-binding protein is a monoclonal antibody or an antigen-binding fragment thereof.

7. The tau-binding protein of claim 6, wherein the tau-binding protein is an antigenbinding fragment comprising a Fab, Fab’, F(ab’)2, or scFv.

8. The tau-binding protein of claim 6, wherein the tau-binding protein is an anti-tau antibody of human isotype subclass IgGl, IgG2, IgG3, or IgG4.

9. The tau-binding protein of claim 8, wherein the anti-tau antibody comprises 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).

10. The tau-binding protein of claim 8 or 9, wherein the human IgGl heavy chain constant region comprises SEQ ID NO: 27.

11. The tau-binding protein of claim 8 or 9, wherein 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.

12. The tau-binding protein of claim 11, comprising 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).

13. The tau-binding protein of any one of claims 1-12, wherein the tau-binding protein is fused to a cell-penetrating peptide that binds a central nervous system (CNS) target.

14. The tau-binding protein of any one of claims 1-12, wherein the tau-binding protein comprises an Fc region with one chain modified to bind a CNS target.

15. The tau-binding protein of claim 14, wherein the tau-binding protein is a bivalent anti- tau antibody or antigen-binding fragment thereof.

16. The tau-binding protein of any one of claims 13-15, wherein the CNS target is transferrin receptor 1 (TfR).

17. A bispecific binding protein comprising a) a tau-binding protein of any one of claims 1-12 or an anti-tau binding domain thereof, andb) a binding domain specific for another, distinct target protein.

18. The bispecific binding protein of claim 17, wherein the distinct target protein is a CNS target.

19. The bispecific binding protein of claim 18, wherein the bispecific binding protein is bivalent for tau and monovalent for the CNS target; monovalent for tau and monovalent for the CNS target; monovalent for tau and bivalent for the CNS target; or bivalent for tau and bivalent for the CNS target.

20. The bispecific binding protein of any one of claims 17-19, wherein the bispecific binding protein 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 bi specific binding protein.

21. The bispecific binding protein of claim 20, wherein one arm comprises a first anti -tau binding domain and an anti-CNS target binding domain, and 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 of any one of claims 1-12, optionally wherein the first and second anti-tau binding domains have the same HCDR1-3 and same LCDR1- 3, further optionally wherein the first and second anti-tau binding domains are the same.

22. The bispecific binding protein of claim 21, wherein one arm 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.

23. The bispecific binding protein of claim 22, whereinon the first heavy chain, the VH of the anti-CNS target binding domain is N-terminal to the VH of the first anti-tau binding domain, and on the first light chain, the VL of the first anti-tau binding domain is N-terminal to the VL of the anti-CNS target binding domain.

24. The bispecific binding protein of claim 20, wherein one arm 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 of any one of claims 1-12, optionally wherein the first and second anti-tau binding domains have the same HCDR1-3 and same LCDR1-3, further optionally wherein the first and second anti-tau binding domains are the same.

25. The bispecific binding protein of claim 24, wherein one arm 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.

26. The bispecific binding protein of claim 25, wherein on the first heavy chain, the VH of the first anti-tau binding domain is N-terminal to the VH of the second anti-tau binding domain, and on the first light chain, the VL of the second anti-tau binding domain is N-terminal to the VL of the first anti-tau binding domain.

27. The bispecific binding protein of any one of claims 18-26, wherein the CNS target is an endothelial cell receptor (ECR) of the blood brain barrier.

28. The bispecific binding protein of claim 27, wherein the ECR is a transferrin receptor, insulin receptor, low-density lipoprotein receptor, or folate receptor.

29. The bispecific binding protein of claim 27 or 28, wherein the ECR is transferrin receptor 1 (TfR), and the binding domain of b) is an anti-TfR binding domain.

30. The bispecific binding protein of claim 29, wherein 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.

31. The bispecific binding protein of claim 29 or 30, wherein 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.

32. The bispecific binding protein of any one of claims 29-31, wherein the VH and the VL of the anti-TfR binding domain are at least 90% identical to:SEQ ID NOs: 25 and 26, respectively; or SEQ ID NOs: 77 and 78, respectively.

33. The bispecific binding protein of any one of claims 29-32, wherein the VH and the VL of the anti-TfR binding domain comprise:SEQ ID NOs: 25 and 26, respectively; or SEQ ID NOs: 77 and 78, respectively.

34. The bispecific binding protein of any one of claims 29-33, wherein the bispecific binding protein has at least one property selected from: 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 Octet™; g) binds to cynomolgus TfR with a KD of 50-200 nM as determined by Octet™; h) has improved brain penetrance in hTfR knock-in mice by at least 3 -fold over the monospecific anti-tau binding domain;i) has increased brain and CSF exposure by at least 5-fold in cynomolgus monkeys over the monospecific anti-tau binding domain; j) reduces tau seeding in vitro., k) inhibits uptake of tau aggregates into neurons; l) reduces AT8+cells in vivo in the cortex of hTfR / THY-Tau22 mice; or m) any combination of a)-l).

35. The bispecific binding protein of any one of claims 17-34, wherein the bispecific binding protein comprises an Fc region, optionally wherein the bispecific binding protein is a bispecific antibody.

36. The bispecific binding protein of claim 35, wherein the Fc region is of human isotype subclass IgGl, IgG2, IgG3, or IgG4.

37. The bispecific binding protein of claim 36, comprising 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).

38. The bispecific binding protein of claim 36 or 37, comprising 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 (Eu numbering).

39. The bispecific binding protein of claim 37 or 38, wherein 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.

40. The bispecific binding protein of claim 39, comprising 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).

41. The bispecific binding protein of claim 40, wherein the first heavy chain constant region further comprises knob mutations of S354C and T366W, and the second heavy chain constant region further comprises hole mutations of Y349C, T366S, L368A, and Y407V (Eu numbering).

42. The bispecific binding protein of claim 37, wherein the human IgGl heavy chain constant region comprises any one of SEQ ID NOs: 27-29.

43. The bispecific binding protein of claim 42, comprising 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.

44. 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.

45. 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.

46. 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 thatcomprises 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.

47. 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.

48. 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, and the other arm comprises a second anti-tau binding domain, and wherein on the first heavy chain, the VH of the anti-CNS target binding domain is N-terminal to the VH of the first anti-tau binding domain, and on the first light chain, the VL of the first anti-tau binding domain is N-terminal to the VL of the anti-CNS target binding domain, optionally wherein the CNS target is TfR.

49. The bispecific binding protein of claim 48, wherein the first and second anti-tau binding domains have the same HCDR1-3 and same LCDR1-3, optionally wherein the first and second anti-tau binding domains are the same.

50. The bispecific binding protein of claim 48 or 49, wherein the first anti-tau binding domain, the second anti-tau binding domain, or both a) bind specifically to tau fibrils, b) bind to the MTBR region of tau, or c) a) and b).

51. The bispecific binding protein of any one of claims 48-50, wherein the first anti-tau binding domain, the second anti-tau binding domain, or both comprise 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, respectively.

52. The bispecific binding protein of any one of claims 48-51, wherein the anti-CNS target binding domain is an anti-TfR binding domain that a) binds to human TfR (hTR) with a KD of 1-50 nM as determined by SPR, b) binds to cynomolgus TfR (cTfR) with a KD of 1-200 as determined by SPR, or c) a) and b).

53. A pharmaceutical composition comprising the tau-binding protein of any one of claims 1-16 or the bispecific binding protein of any one of claims 17-52 and a pharmaceutically acceptable excipient.

54. Isolated nucleic acid molecule(s) encoding the tau-binding protein of any one of claims 1-16 or the bispecific binding protein of any one of claims 17-52.

55. The isolated nucleic acid molecule(s) of claim 54, wherein the nucleic acid molecule(s) are expression constructs.

56. A host cell comprising the isolated nucleic acid molecule(s) of claim 54 or 55, optionally wherein the host cell is a mammalian cell.

57. A method of producing a tau-binding protein or a bispecific binding protein, comprising: culturing the host cell of claim 56 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.

58. A method of treating a tauopathy in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of the tau-binding protein of any one of claims 1-16 or the bispecific binding protein of any one of claims 17-52.

59. Use of the tau-binding protein of any one of claims 1-16 or the bispecific binding protein of any one of claims 17-52 for the manufacture of a medicament for treating a tauopathy in a human subject in need thereof.

60. The tau-binding protein of any one of claims 1-16 or the bispecific binding protein of any one of claims 17-52, for use as a medicament.

61. The tau-binding protein of any one of claims 1-16 or the bispecific binding protein of any one of claims 17-52, for use in treating a tauopathy in a human subject in need thereof.

62. The method of claim 58, the use of claim 59, or the tau-binding protein or bispecific binding protein for use of claim 61, wherein the tauopathy is a primary tauopathy.

63. The method, use, or tau-binding protein or bispecific binding protein for use of claim 62, wherein the primary tauopathy is 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).

64. The method, use, or tau-binding protein or bispecific binding protein for use of claim 62, wherein the primary tauopathy is progressive supranuclear palsy (PSP).

65. The method of claim 58, the use of claim 59, or the tau-binding protein or bispecific binding protein for use of claim 61, wherein the tauopathy is a secondary tauopathy.

66. The method, use, or tau-binding protein or bispecific binding protein for use of claim 65, wherein the secondary tauopathy is selected from Alzheimer’s disease, chronic traumatic encephalopathy, anti -IgL0N5 -related tauopathy, Down syndrome, Niemann-Pick disease type C, and myotonic dystrophy type 1 and 2.

67. The method, use, or tau-binding protein or bispecific binding protein for use of claim 65, wherein the secondary tauopathy is Alzheimer’s disease.