Anti-tau antibodies for the treatment of AD and tauopathy

Abstract

The present disclosure provides a human tau protein (Tau) binding protein comprising an antigen-binding domain of an antibody, said antigen-binding domain specifically binds to an epitope of tau that causes inhibition of tau aggregate seeding and can be used to inhibit the proliferation of tau aggregates in vivo. Also disclosed herein are nucleic acids encoding such a tau binding protein, expression vectors, and recombinant viruses, as well as compositions comprising such a tau binding protein. Additionally, methods for the use of such compositions in the diagnosis, prevention, treatment, and prognosis of tauopathy, e.g., Alzheimer's disease, frontotemporal dementia, Pick's disease, and progressive supranuclear palsy are disclosed. Genetically modified cells for the expression of the disclosed tau binding protein and their use for the production of the tau binding protein are also disclosed.

Description

Technology Field

[0001] This specification generally relates to the field of therapeutics. More specifically, this specification relates to tau epitopes, tau binding proteins (antibodies) and compositions targeting these epitopes, and related methods for the production of tau binding proteins and screening of tau binding proteins, as well as methods for the diagnosis, prevention, and treatment of tau-related conditions, e.g., tauopathy. Background Technology

[0002] Most neurodegenerative disorders are characterized by the progressive pathological aggregation of specific proteins. Alzheimer's disease belongs to a class of neurodegenerative disorders known as tauopathy, which is induced by the aggregation of the microtubule-associated protein "Tau." The pathophysiology of tauopathy involves the prion-like proliferation and replication of tau protein aggregates, known as "seeds," across various brain regions, leading to progressive synaptic dysfunction and loss, brain inflammation, severe memory impairment, and ultimately massive neuronal loss in the hippocampus and cortex. Attempts to halt or reverse this process in preclinical models and human patients have included immunotherapy against tau, but to date, these have demonstrated very limited or no efficacy.

[0003] Therefore, there is a continuous and urgent need to identify therapeutic agents that effectively target tau and effectively treat tauopathy, such as Alzheimer's disease.

[0004] In producing the present invention, the inventors intended to produce a reagent (e.g., a protein comprising an antibody and its antigen-binding domain) that binds to human tau protein in monomeric and aggregated forms and inhibits its ability to promote tau protein aggregation, which is a key pathological process in tauopathy. The inventors produced an exemplary antibody that binds to human monomeric and aggregated tau and, surprisingly, inhibits the ability of tau to promote further tau aggregation or “seed.” The inventors also performed epitope mapping and discovered that an exemplary antibody designated as “RNJ1” binds to tau at the N-terminus of the protein. Furthermore, the inventors demonstrated that administration of this antibody successfully improved symptoms in a preclinical model of tauopathy. For convenience, the “tau protein” referred to herein will be referred to as “tau.” Accordingly, “tau-binding proteins,” e.g., antibodies that bind to tau protein, will be referred to as “tau-binding proteins.”

[0005] Based on the foregoing, the inventor has produced a class of proteins comprising an antigen-binding domain of an antibody, and it will be obvious to those skilled in the art that said antigen-binding domain can bind to or specifically bind to human tau protein and inhibit tau protein seeding. The inventor has also produced a method for treating tauopathy.

[0006] Therefore, in one embodiment, the variable region of the immunoglobulin heavy chain (V H ) and immunoglobulin light chain variable region (V L An isolated tau-binding protein comprising ) is provided herein, said isolated tau-binding protein is V H and V L It specifically binds to the epitope of human tau protein through the antigen-binding site formed by, and the amino acid sequence of the epitope is Sequence No. 1 It corresponds to. V in the related modality HDomain, for example, single-domain antibodies (sdAbs), also known as "nanobodies" ® A tau-binding protein comprising only "in some embodiments K of the antigen-binding site is provided herein. In some embodiments, K of the antigen-binding site D is approximately 5.0x10 -7 It is M or less. In some embodiments, the isolated tau-binding protein binds to the epitope when the human tau protein is in either a monomeric or aggregated form. In some embodiments, the isolated tau-binding protein inhibits human tau protein seeding of tau protein aggregates. In some embodiments, the isolated tau-binding protein is such that the tau seed concentration is 200 ng / mL and the tau-binding protein concentration is about 300 nM. In a test tube It inhibits human tau protein seeding of tau protein aggregates by at least about 30%. In some embodiments, the isolated tau-binding protein has a higher affinity for aggregated tau than the tau antibody "HJ8.5" and / or the tau antibody "RNF5". In some embodiments, the tau-binding protein, In vivo When administered, it inhibits the diffusion of tau aggregates in the brain.

[0007] In some embodiments of the isolated tau-binding protein disclosed herein:

[0008] ( i ) V H The amino acid sequence of is:

[0009] (a) having up to 5 amino acid substitutions Sequence No. 2 ,

[0010] (b) having up to 6 amino acid substitutions Sequence No. 3 , and

[0011] (c) having up to 6 amino acid substitutions Sequence No. 4 Includes,

[0012] Sequence No. 2 It includes HC-CDR1 corresponding to residuals 31-35 according to the Kavat numbering system, and Sequence No. 3It includes HC-CDR2 corresponding to remaining 50-65 according to the Kavat numbering system, and Sequence No. 4 ...includes and / or includes HC-CDR3 corresponding to residuals 95-102 according to the Kavat numbering system;

[0013] ( ii ) V L The amino acid sequence of is:

[0014] (a) having up to 4 amino acid substitutions Sequence number 5 ,

[0015] (b) having up to 4 amino acid substitutions Sequence number 6 , and

[0016] (c) having up to 5 amino acid substitutions Sequence number 7 Including; Sequence number 5 It includes LC-CDR1 corresponding to residuals 24-34 according to the kavat numbering system, and Sequence number 6 It includes LC-CDR2 corresponding to residuals 50-56 according to the Kavat numbering system, and Sequence number 7 It includes LC-CDR3 corresponding to residuals 89-97 according to the Kavat numbering system.

[0017] In some embodiments of the isolated tau-binding protein disclosed herein:

[0018] ( i ) V H The amino acid sequence of is:

[0019] (a) having up to 5 amino acid substitutions Sequence No. 2 ,

[0020] (b) having up to 6 amino acid substitutions Sequence number 8 , and

[0021] (c) having up to 6 amino acid substitutions Sequence No. 4 Includes, Sequence No. 2 It includes HC-CDR1 corresponding to residual positions 31-35 according to the kavat numbering system, and Sequence number 8 It includes HC-CDR2 corresponding to residual positions 50-65 according to the Kavat numbering system, and Sequence No. 4 ...includes and / or includes HC-CDR3 corresponding to residual positions 95-102 according to the kavat numbering system;

[0022] ( ii ) V L The amino acid sequence of is:

[0023] (a) having up to 4 amino acid substitutions Sequence number 5 ,

[0024] (b) having up to 4 amino acid substitutions Sequence number 9 , and

[0025] (c) having up to 5 amino acid substitutions Sequence number 10 Includes; Sequence number 5 It includes LC-CDR1 corresponding to residual positions 24-34 according to the kavat numbering system, and Sequence number 9 It includes an LC-CDR2 corresponding to residual positions 50-56 according to the kavat numbering system, and Sequence number 10 It includes LC-CDR3 corresponding to residue positions 89-97 according to the Kavat numbering system. In some embodiments of any of the aforementioned tau-binding proteins disclosed herein, the tau-binding protein is V in (i). H and V in (ii) L Includes

[0026] In some embodiments of the recently mentioned isolated tau-binding protein having amino acid substitutions within a CDR, the isolated tau-binding protein comprises at least one amino acid substitution within (i) one CDR; (ii) two CDRs; (iii) three CDRs; (iv) four CDRs; (v) five CDRs; or (vi) six CDRs.

[0027] In some embodiments of any of the aforementioned isolated tau-binding proteins disclosed herein, V H The amino acid sequence of is Sequence number 11 or Sequence number 13 The amino acid sequence of is at least 80% identical to V L silver Sequence number 12 or Sequence number 14 It is at least 80% identical to the amino acid sequence of.

[0028] In some embodiments of any of the isolated tau-binding proteins disclosed herein, six or fewer amino acid substitutions are made in each of the following within the isolated tau-binding protein:

[0029] (i) at residual locations according to the kavat numbering scheme selected from the group consisting of locations 30-33, 35, 50, 52A, 53, 54, 56, 58, 95, 96, 97, 98, 99, and 101 Sequence number 11 or Sequence number 13 V corresponding to H ; or

[0030] (ii) at residual locations according to the kavat numbering scheme selected from the group consisting of positions 30, 31, 32, 34, 49, 50, 53, 55, 91-94, and 96 Sequence number 12 or Sequence number 14 V corresponding to L .

[0031] In some embodiments, the isolated tau-binding protein comprises one or fewer amino acid substitutions in each CDR according to the sequence mentioned above. In some embodiments, the isolated tau-binding protein is V H One or fewer amino acid substitutions within any of the CDRs and any V L It includes one or fewer amino acid substitutions within any of the CDRs.

[0032] V in some embodiments H The amino acid sequence of is ( i ) Sequence No. 2 ; ( ii ) Sequence No. 3 or Sequence number 8 ; and ( iii ) Sequence No. 4 Includes. In some embodiments, V L The amino acid sequence of is ( i ) Sequence number 5 ; ( ii ) Sequence number 6 or Sequence number 9 ; and ( iii ) Sequence number 7 or Sequence number 10 Includes

[0033] In some embodiments of isolated tau-binding protein, V H Is Sequence number 11 or Sequence number 13 Includes the amino acid sequence of, and V L silver Sequence number 12 or Sequence number 14 It includes the amino acid sequence of.

[0034] V in some embodiments H and V L It is within a single polypeptide chain. V H and V L In some embodiments within this single polypeptide chain, the isolated tau-binding protein is: (i) a single-chain Fv fragment (scFv); (ii) a dimeric scFv (di-scFv); or (iii) at least one of (i) and / or (iii) connected to Fc or a heavy chain constant domain CH2 and / or CH3.

[0035] V in another embodiment H and V L It is within a separate polypeptide chain. V H and V L In some embodiments within this distinct polypeptide chain, the isolated tau-binding protein is: (i) a divalent antibody; (ii) Fab´ (iii) a diabody; (iv) a tribody; or (iv) a tetrabody.

[0036] In any of the embodiments of the isolated tau-binding proteins mentioned above, the isolated tau-binding protein further comprises a functional moiety selected from the group consisting of (i) a detection moiety; (ii) a stabilization moiety; (iii) a transfer moiety; and (iv) a combination thereof. In some embodiments, the isolated tau-binding protein comprises both a transfer moiety and a detection moiety. In some embodiments, the isolated tau-binding protein is detectably labeled (e.g., labeled with a fluorescent dye or nanoparticle). In some embodiments, the functional moiety is V H or V L It is encoded as a fusion protein containing the amino acid sequence of.

[0037] In a related embodiment, an amino acid sequence encoding any of the isolated tau-binding proteins mentioned above or its V H or V L Isolated nucleic acids encoding are provided herein. In some embodiments, the isolated nucleic acid is a modified mRNA. In some embodiments where the isolated nucleic acid is a modified mRNA, the modified mRNA is self-amplified mRNA (sa-mRNA) or circular RNA (circ-RNA).

[0038] Any of the isolated tau-binding proteins mentioned above in other related embodiments, or its V H or V L A non-viral expression vector comprising an isolated nucleic acid encoding an amino acid sequence encoding [something] is provided herein.

[0039] In a further related aspect, an isolated cell comprising an isolated nucleic acid encoding an amino acid sequence encoding any of the isolated tau-binding proteins mentioned above is provided herein. In some embodiments, the genetically modified cell is a mammalian cell, a yeast cell, a bacterial cell, an insect cell, a plant cell, or a filamentous fungal cell. In some embodiments, the genetically modified cell is a bacterial cell or a yeast cell. In some embodiments, the genetically modified cell is a rodent cell or a human cell. In some embodiments, the genetically modified cell is a human cell. In some embodiments, the genetically modified cell is a mouse cell, a rat cell, or a hamster cell. In some embodiments, the isolated cell expresses the encoded protein.

[0040] In other related embodiments, a recombinant virus for mammalian expression of any of the isolated tau-binding proteins disclosed herein is provided. In some embodiments, the recombinant virus is a virus type selected from the group consisting of adeno-associated virus (AAV), adenovirus (AdV), lentivirus, herpes simplex virus (HSV), baculovirus, and anellovirus.

[0041] In additional embodiments, a pharmaceutical composition is provided herein comprising any of the isolated tau-binding protein, isolated nucleic acid, nonviral expression vector, genetically modified cell, or recombinant virus disclosed herein, and a pharmaceutically acceptable carrier. In some preferred embodiments, the pharmaceutical composition comprises the isolated tau-binding protein.

[0042] In some embodiments, the pharmaceutical composition also contains lipid shell-type microbubbles or lipid shell-type nanobubbles. In some embodiments, the lipid shell-type microbubbles comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000 (DSPE-PEG2000).

[0043] In another related embodiment, a method for producing an isolated tau-binding protein disclosed herein is provided, the method comprising the steps of: culturing a cell comprising a nucleic acid encoding any of the proteins corresponding to the isolated tau-binding protein disclosed herein to express said protein; and subsequently isolating said expressed tau-binding protein.

[0044] In a further related embodiment, a method for preventing or treating tauopathy in a subject in need is provided herein, said method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition disclosed herein to said subject.

[0045] In another related embodiment, a method for preventing or inhibiting the spread of tau protein aggregates within the central nervous system (CNS) of a subject identified as needing said method is provided herein, said method comprising the step of administering an effective amount of the pharmaceutical composition disclosed herein to said subject.

[0046] In some embodiments, the aforementioned method for prevention, treatment, or inhibition also includes a scanning ultrasound transient permeation step of the blood-brain barrier in the subject.

[0047] In some embodiments, the route of administration is intravenous, intrathecal, intra-arterial, intracerebral, intraventricular, intranasal, intramuscular, or subcutaneous.

[0048] In a related embodiment, any of the compositions disclosed herein (e.g., pharmaceutical compositions) are used in the manufacture of a drug for the prevention or treatment of tauopathy. In another related embodiment, any of the isolated tau-binding proteins disclosed herein are used in the manufacture of a drug for the diagnosis or prognosis of tauopathy.

[0049] In additional related embodiments, compositions disclosed herein (e.g., pharmaceutical compositions) are provided for (i) preventing or treating tauopathy; (ii) preventing or inhibiting the spread of tau protein aggregates in subjects requiring it; and (iii) diagnosing or prognosing tauopathy.

[0050] In another embodiment, a method for determining the level of extracellular tau protein or extracellular tau protein aggregates within a subject is provided herein, said method:

[0051] (i) the isolated tau-binding protein disclosed herein in a biological sample from said subject under conditions allowing specific binding of the isolated tau-binding protein to the tau protein present in the biological sample ex vivo A step of contacting and determining the level of tau protein bound to the isolated tau binding protein; or

[0052] (ii) administer the isolated tau-binding protein disclosed herein (e.g., detectably labeled isolated tau-binding protein) to the subject, and In vivo The method includes the step of detecting the isolated tau-binding protein bound to the aggregated tau protein at one or more locations.

[0053] In some embodiments of the method for detecting the extracellular tau protein or tau protein aggregates just mentioned, ex vivoThe biological sample in contact is a biological sample comprising cerebrospinal fluid (CSF), plasma, serum, urine, rhinorrhea, tear fluid, or postmortem brain extract. In some embodiments, this method identifies that the subject from whom the biological sample was obtained suffers from tauopathy, or that within the biological sample or for one or more threshold levels or ranges In vivo It also includes a step of determining the progression of tauopathy based on the level of aggregated tau protein.

[0054] In some embodiments of any of the methods for prevention, treatment, inhibition, diagnosis, or prognosis, the tauopathy is selected from the group consisting of Alzheimer's disease, frontotemporal dementia, Pick's disease, chronic traumatic encephalopathy, progressive supranuclear palsy, Parkinson's disease, Parkinson's disease-related disorders, primary age-related tauopathy, silicomeric granulosa disease, and spherocytic tauopathy. In some embodiments, the tauopathy is Alzheimer's disease.

[0055] In some preferred embodiments, the object in the method mentioned above is a human object.

[0056] In another aspect, a method for selecting an immunoglobulin that binds to a human tau protein is provided herein, said method:

[0057] (i) Heavy chain variable region (V H ), immunoglobulin light chain variable region (V L A step of providing a library of immunoglobulins comprising ), or both;

[0058] (ii) The amino acid sequences of multiple immunoglobulin proteins Sequence No. 1 A step of contacting an isolated tau protein, a fragment thereof, or a peptide thereof containing an epitope of the human tau protein corresponding to;

[0059] (iii) identifying an immunoglobulin from a plurality of immunoglobulins that specifically binds to an isolated tau protein, a fragment thereof, or a peptide thereof; and

[0060] (iv) binding of immunoglobulin to isolated tau protein, its fragment, or its peptide Sequence No. 1 The method includes the step of selecting the immunoglobulin only when it is competitively inhibited by a peptide consisting of the amino acid sequence of

[0061] In some embodiments of the screening method just mentioned:

[0062] (i) The amino acid sequence of VH is:

[0063] (a) having up to 5 amino acid substitutions Sequence No. 2 ,

[0064] (b) having up to 6 amino acid substitutions Sequence No. 3 or Sequence number 8 , and

[0065] (c) having up to 6 amino acid substitutions Sequence No. 4 Includes,

[0066] Sequence No. 2 It includes HC-CDR1 corresponding to residuals 31-35 according to the Kavat numbering system, and Sequence No. 3 or Sequence number 8 It includes HC-CDR2 corresponding to residuals 50-65 according to the Kavat numbering system, and Sequence No. 4 ...includes and / or includes HC-CDR3 corresponding to residuals 95-102 according to the Kavat numbering system;

[0067] (ii) The amino acid sequence of VL is:

[0068] (a) having up to 4 amino acid substitutions Sequence number 5 ,

[0069] (b) having up to 4 amino acid substitutions Sequence number 6 or Sequence number 9 , and

[0070] (c) having up to 5 amino acid substitutions Sequence number 7 or Sequence number 10 Includes; Sequence number 5 It includes LC-CDR1 corresponding to residuals 24-34 according to the kavat numbering system, and Sequence number 6 It includes LC-CDR2s corresponding to residuals 50-56 according to the Kavat numbering system, and Sequence number 7 It includes LC-CDR3 corresponding to residuals 89-97 according to the Kavat numbering system.

[0071] In some embodiments, immunoglobulin is V H and V L Includes both. In some embodiments, V H and / or V L It includes at least one amino acid substitution within (i) 1 CDR; (ii) 2 CDRs; (iii) 3 CDRs; (iv) 4 CDRs; (v) 5 CDRs; or (vi) 6 CDRs. In some embodiments, the library to be used in the method is a phage display library.

[0072] In the relevant mode, V H and / or V L A variable domain expression library comprising a plurality of expression vectors encoding is provided herein:

[0073] (i) V H The amino acid sequence of is:

[0074] (a) having up to 5 amino acid substitutions Sequence No. 2 ,

[0075] (b) having up to 6 amino acid substitutions Sequence No. 3 , and

[0076] (c) having up to 6 amino acid substitutions Sequence No. 4 Includes,

[0077] Sequence No. 2... comprises HC-CDR1 corresponding to residues 31-35 according to the Kavat numbering system, and sequence number 3 comprises HC-CDR2 corresponding to residues 50-65 according to the Kavat numbering system, and Sequence No. 4 ...includes and / or includes HC-CDR3 corresponding to residuals 95-102 according to the Kavat numbering system;

[0078] (ii) V L The amino acid sequence of is:

[0079] (a) having up to 4 amino acid substitutions Sequence number 5 ,

[0080] (b) having up to 4 amino acid substitutions Sequence number 6 , and

[0081] (c) having up to 5 amino acid substitutions Sequence number 7 Includes; Sequence number 5 It includes LC-CDR1 corresponding to residuals 24-34 according to the kavat numbering system, and Sequence number 6 It includes LC-CDR2s corresponding to residuals 50-56 according to the Kavat numbering system, and Sequence number 7 It includes LC-CDR3 corresponding to residuals 89-97 according to the Kavat numbering system.

[0082] In another mode, Sequence No. 1 A non-naturally occurring immunogenic polypeptide or peptide comprising an amino acid sequence corresponding to is provided herein.

[0083] In a further embodiment, an immunogenic peptide having a length of about 12 to about 80 amino acids is provided herein, said immunogenic peptide having an amino acid sequence of the amino acid sequence of said immunogenic peptide Sequence No. 1 Includes

[0084] Sequence list core (Sequence provided in Appendix 1)

[0085] Sequence No. 1 : sapient ( Homo sapiens ) Amino acid sequence of the tau epitope (AA:9-20)

[0086] Sequence No. 2 : Antibody RNJ1 V H Amino acid sequence of the CDR1 region of the chain

[0087] Sequence No. 3 : Antibody RNJ1 V H Amino acid sequence of the CDR2 region of the chain

[0088] Sequence No. 4 : Antibody RNJ1 V H Amino acid sequence of the CDR3 region of the chain

[0089] Sequence number 5 : Antibody RNJ1 V L Amino acid sequence of the CDR1 region of the chain

[0090] Sequence number 6 : Antibody RNJ1 V L Amino acid sequence of the CDR2 region of the chain

[0091] Sequence number 7 : Antibody RNJ1 V L Amino acid sequence of the CDR3 region of the chain

[0092] Sequence number 8 : Antibody RNJ1 V H Exemplary amino acid sequence of the fully humanized CDR2 region of the chain

[0093] Sequence number 9 : Antibody RNJ1 V L Exemplary amino acid sequence of the fully humanized CDR2 region of the chain

[0094] Sequence number 10 : Antibody RNJ1 V L Exemplary amino acid sequence of the fully humanized CDR2 region of the chain

[0095] Sequence number 11 : Antibody RNJ1 V H amino acid sequence of the chain

[0096] Sequence number 12 : Antibody RNJ1 V L amino acid sequence of the chain

[0097] Sequence number 13: V of antibody RNJ1 containing a fully humanized CDR2 region and a fully humanized CDR3 region H Exemplary amino acid sequence of the chain

[0098] Sequence number 14 : V of antibody RNJ1 containing a fully humanized CDR2 region L Exemplary amino acid sequence of the chain

[0099] Sequence number 15 : Antibody RNJ1 V H Exemplary amino acid sequence of the partially degenerated humanized CDR2 region of the chain

[0100] Sequence number 16 : Antibody RNJ1 V H Exemplary amino acid sequence of a partially degenerated humanized CDR3 region of the chain

[0101] Sequence number 17 : Antibody RNJ1 V L Exemplary amino acid sequence of the partially degenerated humanized CDR2 region of the chain

[0102] Sequence number 18 : Antibody RNJ1 V L Exemplary amino acid sequence of a partially degenerated humanized CDR3 region of the chain

[0103] Sequence number 19 : Positive clone forward primer

[0104] Sequence number 20 : Positive clone reverse primer

[0105] Sequence number 21 : Trimmed tau aa 1-44

[0106] Sequence number 22 : Trimmed tau aa 1-22

[0107] Sequence number 23 : HJ8.5 truncated tau linkage site aa 23-44

[0108] Sequence number 24 : HJ8.5 reported tau epitope aa 25-30

[0109] Sequence number 25: NTer1 aa 1-10

[0110] Sequence number 26 : NTer2 aa 3-12

[0111] Sequence number 27 : NTer3 aa 5-14

[0112] Sequence number 28 : NTer4 aa 7-16

[0113] Sequence number 29 : NTer5 aa 9-18

[0114] Sequence number 30 : NTer6 aa 11-20

[0115] Sequence number 31 : NTer7 aa 13-22

[0116] Sequence number 32 : RNJ1 tau binding site aa 9-22

[0117] Sequence number 33 : RNJ1 V H Whole-length sequence-human framework variant (IGHV3a)

[0118] Sequence number 34 : RNJ1 V H Whole-length sequence-human framework variant (IGHV3b)

[0119] Sequence number 35 : RNJ1 V H Whole-length sequence-human framework variant (IGHV3c)

[0120] Sequence number 36 : RNJ1 V H Whole-field sequence-human framework variant (IGHV3d)

[0121] Sequence number 37 : RNJ1 V L Whole-length sequence-human framework variant (IGKV1a)

[0122] Sequence number 38 : RNJ1 V L Whole-length sequence-human framework variant (IGKV6a)

[0123] Sequence number 39 : RNJ1 V LWhole-length sequence-human framework variant (IGKV2a)

[0124] Sequence number 40 : RNJ1 V L Whole-length sequence-human framework variant (IGKV4a)

[0125] Sequence number 41 : RNJ1 V L Whole-length sequence-human framework variant (IGKV3a)

[0126] Sequence number 42 : RNJ1 V H Exemplary nucleotide human codon optimized sequence

[0127] Sequence number 43 : RNJ1 V L Exemplary nucleotide human codon optimized sequence

[0128] Sequence number 44 : RNJ1 V H Exemplary nucleotide Chinese hamster codon optimized sequence

[0129] Sequence number 45 : RNJ1 V L Exemplary nucleotide Chinese hamster codon optimized sequence

[0130] Sequence number 46 : Humanized RNJ1 V H Exemplary nucleotide human codon optimized sequence

[0131] Sequence number 47 : Humanized RNJ1 V L Exemplary nucleotide human codon optimized sequence

[0132] Sequence number 48 : Humanized Tau Protein-F Subtype (Uniprot ID P10636-8) Brief explanation of the drawing

[0133] Fig. 1: Isolation of human tau binder after panning of Tomlinson I + J library. (a) Three consecutive pannings were performed on whole-length human tau (hTau-441). ELISAs of polyclonal phages from each panning, and (b)It shows the enhancement of positive binders for hTau-441 from each library as the phage colony-forming unit (cfu) count from each panning session. I Library (c) and J library (d) As an ELISA of scFv isolated following three pannings from hTau-441, it was tested against skim milk powder in PBS (MPBS) as a control. (e) ELISA of isolated scFv clones RNJ1 and RNJ15 against recombinant hTau-441 and comparison with the tau monoclonal antibody Tau5. (f) As shown by Western blotting, scFv clones RNJ1 and RNJ15 show specific binding to hTau-441 but not to the control protein BSA. Fig. 2: Antibody RNJ1 binds to the amino-terminal epitope of tau and has a stronger reactivity to aggregated tau than amino-terminal antibodies HJ8.5 and RNF5. (a) Schematic diagram depicting epitope mapping of novel antibody RNJ1 and a tau fragment expressed as a fusion protein with a maltose-binding protein for benchmarking antibody HJ8.5. (b) Epitope mapping of HJ8.5 by indirect ELISA using tau fusion protein as antigen. (c) Epitope mapping of RNJ1 by indirect ELISA using tau fusion protein. (d) High-resolution mapping using 10-amino acid (aa) long overlapping peptides of 22 amino-terminal aa of tau conjugated to BSA (center panel). (e) Indirect ELISA epitope mapping using BSA-conjugated non-phosphorylated and phosphorylated peptides (right panel). (f) Determination of RNJ1 and HJ8.5 concentrations yielding EC50, the maximum half-binding for monomeric recombinant human 2N4R tau (hTau-441), by indirect ELISA. (g)Surface plasmon resonance (SPR) sensograms of RNJ1 and HJ8.5 (analytes) binding to monomeric recombinant hTau-441. Sensograms shown for antibody injection at 1.0, 3.0, 9.1, 27.3, and 82.0 nM. Both sensograms were fitted to the binding model. The experimental curve for RNJ1 is shown in red, for HJ8.5 in blue, and the model fit in gray. (h) Binding curves of RNJ1, HJ8.5, and RNF5 from indirect ELISA using sarcosyl-insoluble rTg4510 transformation and human AD tauside as antigens. (i) SPR sensigrams of RNJ1, HJ8.5, and RNF5 (analytes) binding to sarcosyl-insoluble tauside derived from rTg4510 mice. In bc, and e, n=4 descriptive replicates. In d, f, and h, n=3 descriptive replicates. Data are shown as mean ± SEM. Fig. 3: Binding of RNJ1 to the amino-terminus of tau determined by surface plasmon resonance. Peptides from a synthetic library spanning human tau amino acids (aa) 1–22 were first conjugated to BSA and then used as immobilized ligands on an SPR sensor chip. Subsequently, RNJ1 was used as an analyte to detect binding to different peptides. Fig. 4: RNJ1 inhibits the seeding activity of sarcosyl-insoluble tau in two distinct tau biosensor cells. (a) Representative epifluorescence microscopy images of tau RD P301S FRET HEK293 biosensor cells 48 h after exposure to rTg4510 transgenic mouse-derived tau seeds in the presence or absence of 300 nM RNJ1, HJ8.5, or RNF5. All samples were treated with the same amount of tau seeds. (b) Representative FRET-based flow cytometry scatter plot of HEK293 biosensor cells treated with rTg4510 tau seed with or without antibodies. The percentage of FRET-positive cells is shown as mean ± SEM. No FRET signal was detected in biosensor cells without tau seed exposure. The scale bar represents 100 μm. (c)HEK293 biosensor cells treated with rTg4510 seed and tau antibody. A percentage reduction in the integrated FRET intensity value from flow cytometric analysis is shown compared to the antibody-free control value. (d) Complementary tau seed neutralization assay performed on a novel tau RD P301S SH-SY5Y FRET biosensor system. (e) Seeding neutralization assay in HEK293 cells performed as in c, but using AD tau seed. In b and c, n=4 descriptive replicates for each concentration, 50,000 cells per replicate were analyzed. In d and e, n=3 descriptive replicates for each concentration, 50,000 cells per replicate were analyzed. Statistical analysis was performed using binary ANOVA with Holm-Sidak's multiple comparison correction. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Fig. 5: RNJ1 treatment improves motor function in female K3 mice. (a) Experimental design of the longitudinal treatment study: Baseline behavioral and motor functions were determined in 4-week-old K369I tau mutant K3 mice with tau pathology and WT litter controls to assign mice to experimental groups with equivalent mean rotarod performance. The experimental groups were vehicle-treated WT mice (WTVeh) and K3 mice treated with vehicle (K3Veh), RNJ1 (K3RNJ1), HJ8.5 (K3HJ8.5), or antibody combinations (K3R+H). Treatments were performed weekly, and rotarod performance was evaluated every 4 weeks for 14 weeks. (b) The average rotor drop latency (top panel) for all study groups during the treatment period (n=12 mice per group) was compared by calculating the area under the corresponding curve (AUC) (bottom panel). (c)Mean fall latency from the rotarod (top panel) and corresponding AUC (bottom panel), shown separately for male and female mice in the K3Veh group. Female K3 mice demonstrated improved motor function and balance compared to K3 males (n=6 mice per group). (d) Group-based sex-stratified mean fall latency (n=6 mice per group) (top panel) and corresponding AUC (bottom panel) during the treatment period. (e.g.). Female K3 RNJ1 mice showed improved motor performance compared to female K3Veh mice. (e) Average weight per group throughout the study. (f) Mean foreleg grip strength per group at baseline time point and end of treatment. (bf): Data are expressed as mean ± SEM. Statistical comparisons of rotarod AUC and grip strength in the treatment groups were performed using one-way ANOVA with Holm-Sidak's multiple comparison correction, or unpaired t-tests were used to compare rotarod AUC based on sex at c. Comparisons of rotarod drop latency times according to time point were performed using two-way ANOVA with Holm-Sidak's multiple comparison correction. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Fig. 6: Total and phospho-tau levels are reduced in RNJ1-treated K3 mice. (a) Immunoblot of forebrain lysates from female K3 mice probed for total tau (DakoTau) and S202 / T205 phosphorylated tau (AT8). (b) Quantification of a using total protein staining (Revert™ 700) for normalization is expressed as the fold change relative to the K3Veh control group (n=6 per group). (c) Representative (20x) image of AT8 immunoreactivity (green) with DAPI counterstaining (blue) for nuclei in the hippocampus and motor cortex of female mice. Scale bars represent 400 μm (top) and 100 μm (bottom). (d)Quantification of integrated fluorescence intensity within the hippocampus and cortex in c. K3Veh, n=6; K3RNJ1, n=6; K3HJ8.5, n=6; K3R+H, n=5 (1 mouse excluded due to tissue damage). (e) Rotarod performance across treatment studies (Rotarod AUC) shows a moderate inverse correlation with AT8 immunoreactivity for the hippocampus (left panel) (Pearson correlation r = -0.62, p = 0.0018) and cortex (right panel) (Pearson correlation r = -0.60, p = 0.0025). (f) Mass spectrometry quantification of protein abundance for K369I hTau transgene and endogenous mouse tau (n=4 female mice per group). (g) The level of hTau K369I showed a strong inverse correlation with performance in the rotarod test (Pearson correlation r = -0.81, p = 0.0013), whereas the level of endogenous mouse tau showed only a weak inverse correlation (Pearson correlation r = -0.32, p = 0.32). (h) Heatmap of tau phosphopeptides detected by quantitative mass spectrometry. Phosphopeptides with fold change achieving p < 0.05 are highlighted with an asterisk. Data within the heatmap are displayed as mean log2 (fold change) for K3Veh. All other data are displayed as mean ± SEM. Statistical comparisons were performed using one-way ANOVA with Holm-Sidak's post-hoc multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Fig. 7: Quantitative mass spectrometry analysis shows the disregard of proteins involved in metabolic pathways and microtubule-based processes in the K3 model of tauopathy. (a) As a volcano plot of proteins with different abundances (DA) in K3Veh relative to WTVeh brain, it shows 342 DA proteins (165 proteins with lower abundance and 177 proteins with higher abundance) out of a total of 6,623 proteins (6,281 with no significant change) detected in this study. (b)A Venn diagram depicting that only a small subset of the 342 proteins have recently been reported as tau interactors via proximity-labeled MS (6). (c) Volcano plot showing a protein subset in a reported tau interactor. (d) Overpresentation analysis (ORA) for reduced proteins in K3Veh mice revealed that gene ontology (GO) terms and reactome pathways associated with metabolic processes were highly enhanced, particularly for proteins annotated with the terms 'mitochondrial matrix' (GO cellular component) and 'pyruvate metabolism and citric acid (TCA) cycle' (reactome pathway). GO ORA for proteins with higher abundance in K3Veh mice demonstrates potent overpresentation of microtubule-based processes with highly enhanced axonal transport. (e) As a gene-concept network showing DA proteins in comparison between K3Veh and WTVeh associated with the most highly enhanced GO term, the latter is indicated as a gray node with connections to proteins having different abundances in K3Veh mice. Fig. 8: Functional protein-protein interaction network of proteins with different abundances in K3Veh for WTVeh mice. Functional STRING networks using a confidence cutoff of 0.7 for all proteins with different abundances (DA) in K3Veh in WTVeh mice. Clusters formed by more than three proteins are indicated. Protein circles are color-coded as linear continuous mappings of their t-statistics in comparisons between K3Veh and WTVeh mice. Line widths reflect the confidence of the association. Dashed rectangles highlight clusters where the majority of proteins are associated with the listed biological processes. Fig. 9: Proteomic changes throughout the brain in response to antibody treatment. (a) Principal Component Analysis (PCA) shows three clusters for PC2 and PC3, and the K3RNJ1 cluster moves toward WTVeh. (b)Volcano plot for K3RNJ1 mice showing DA proteins (p < 0.05 after spurious detection correction), with 186 decreased and 137 increased proteins compared to K3Veh (left panel). The K3HJ8.5 group shows higher similarity to K3Veh, with only 5 proteins identified as DA (right panel). (c) Analysis of therapeutic effects focusing on the set of 342 DA proteins in K3Veh compared to WTVeh (pie chart and Fig. 5a). (d) The heatmap shows the multiple change in protein in antibody-treated mice compared to K3Veh, the top heatmap shows reduced DA protein in K3Veh compared to WTVeh, and the bottom heatmap shows increased DA protein, showing the conversion to WTVeh by the antibody. (e) Scatter plot showing the direction of change in the abundance of 342 DA proteins in WTVeh and antibody-treated mice relative to K3Veh (Top: RNJ1, Bottom: HJ8.5). The majority of the 342 proteins (approx. 75.1% for RNJ1; approximately 72.5% for HJ8.5) fall into Quadrants I and III, with Quadrant I showing higher proteins in WTVeh and antibody-treated mice relative to K3Veh, and Quadrant III showing lower proteins in WTVeh and antibody-treated mice relative to K3Veh, repeating the transition of the treatment groups relative to WT. Proteins with significant fold change (DA) are indicated by closed circles, and those with insignificant (NS) are indicated by open circles. The number of proteins in each quadrant is shown. (f)The Venn diagram shows the number of significantly altered (DA) crossover and non-crossover proteins in the comparisons of K3Veh vs. WTVeh and K3RNJ1 vs. K3Veh. The heatmap shows the row z-scores of log2-transformed protein abundance in the overlapping portions, showing significant similarities in this subset for the WTVeh and K3RNJ1 groups, and highlights proteins that are significantly affected in the K3 model and recovered to WT levels by treatment. Gene symbols in bold and underlined indicate the corresponding DA proteins in K3HJ8.5 compared to K3Veh. Columns (individual mice) are clustered by correlation (top). Taken together, the proteomic analysis suggests that tau antibody treatment initiates the restoration of homeostasis—a surrogate for achieving therapeutic outcomes. Fig. 10: Heatmap of proteins with all different abundances in K3Veh for the WTVeh group. (a) Heatmap of all proteins increased in K3Veh compared to WTVeh, showing row z-score values ​​for treatment groups WTVeh, K3RNJ1, K3HJ8.5, and K3Veh. (b) Corresponding heatmaps of all proteins reduced in K3Veh relative to WTVeh. Both heatmaps are shown as row z-scores of log2-transformed protein abundance values. Columns and rows were clustered by correlation. Fig. 11: Analysis of the effect of antibody treatment on altered proteins in K3Veh mice. (a) Heatmap of proteins with different abundances (DA) in WTVeh and K3RNJ1 compared to K3Veh (see Fig. 6f), showing the recovery of wild-type protein levels for K3RNJ1 for 45 out of 48 proteins. The heatmap shows the row z-scores of log2-transformed protein abundance values. (b) As an analysis of the overpresentation of DA proteins in the overlap shown in Panel A, enhanced terms for proteins with increased abundance in WTVeh and K3RNJ1 mice are shown in red, and proteins with decreased abundance in WTVeh and K3RNJ1 are shown in blue. (c)Functional PPI of proteins from Panel A. Node size is linearly encoded by connectivity. Node colors describe the direction of fold change in protein abundance ratios in WTVeh and K3RNJ1 relative to K3Veh. Green labels show proteins annotated with the GO term 'presynapse'. Purple labels show proteins annotated with both the GO terms 'presynapse' and 'synaptic vesicle'. Fig. 12: Changes in the brain-wide phosphoproteome in response to antibody treatment. (a) Principal component analysis (PCA) of the phosphoroteome identifies three clusters, WTVeh, K3RNJ1 and K3Veh and one formed by K3HJ8.5. (b) The volcano plot shows different abundances (DA) of phosphopeptides (p < 0.05 after correction for false discovery) for comparisons of K3Veh vs. WTVeh, K3RNJ1 vs. K3Veh, and K3HJ8.5 vs. K3Veh. 541 phosphopeptides are DA between K3Veh and WTVeh mice, and 653 are DA between K3RNJ1 and K3Veh mice. The therapeutic effect analysis of the phosphoproteome focused on the 541 DA phosphopeptides in K3Veh compared to WTVeh. (c)The scatter plot (left: RNJ1, right: HJ8.5) shows the direction of change in the abundance of 541 DA phosphopeptides in mice treated with WTVeh and antibodies against K3Veh. The majority of the 541 DA phosphopeptides (approximately 82.1% for RNJ1; approximately 75.4% for HJ8.5) fall into Quadrants I and III; Quadrant I shows phosphopeptides with higher abundance in mice treated with WTVeh and antibodies against K3Veh, while Quadrant III shows those with lower abundance in mice treated with WTVeh and antibodies against K3Veh, indicating a shift in the treatment group relative to WT. Phosphopeptides with significant fold changes are represented by closed circles, and those without significant changes are represented by open circles. The number of phosphopeptides in each quadrant and their corresponding percentages of the total DA phosphopeptides are shown. (d) Venn diagrams showing the number of crossover and non-crossover DA phosphopeptides in both K3Veh vs. WTVeh and K3RNJ1 vs. K3Veh comparisons. The heatmap shows the row z-scores of the phosphopeptide abundance ratios log2-transformed in the overlap, and by showing significant similarities in this subset for WTVeh and K3RNJ1, it demonstrates that phosphopeptides significantly affected by treatment in the K3 model have recovered to WT levels. (e) Protein-protein interaction (PPI) network of proteins containing DA phosphopeptides shown in Panel C. Node size and color are linearly related to the degree of connectivity of nodes within the network. (f) SynGO analysis of proteins containing DA phosphopeptides shown in Panel C demonstrates the enhancement of presynaptic and postsynaptic proteins. The number of proteins in the analyzed set annotated in each category is shown. (g)S2448-phosphorylated mTOR and T188-phosphorylated MAPK1 show higher abundance in K3RNJ1 and WTVeh mice compared to K3Veh, whereas total protein abundance does not show a significant difference. Data in Panel G are shown as mean ± SEM. Statistical comparisons of protein abundance were performed using a generalized linear model with Bayesian reduction for group comparisons. P-values ​​were derived from adjusted t-tests and corrected for multiple hypotheses using the Benjaminy-Hochberg (FDR) method (54). *p <0.05, **p <0.01, ***p <0.001. Fig. 13: 3D computational model of the variable domain of the RNJ1 tau antibody Cartoon illustrating the computationally predicted 3D structure of the RNJ1 tau antibody variable domain (based on ABodyBuilder2 modeling). The top structure is V H It corresponds to the domain, and the lower structure is V L Corresponds to domains. In each domain, the darker loop corresponds to the predicted CDR region. PDB-format predicted atomic coordinates for the structure are provided in Appendix 2 of Australian provisional patent 2023903449 filed on October 27, 2023, the whole of which is incorporated herein by reference. Fig. 14: 3D computational model of a peptide containing an N-terminal tau epitope to which mAb RNJ-1 binds. The tau N-terminal epitope to which mAb RNJ-1 binds ( Sequence No. 1 A cartoon illustrating the computationally predicted 3D structure of a peptide composed of ). PDB-format predicted atomic coordinates for the structure are provided in Appendix 2 of Australian provisional patent 2023903449 filed on October 27, 2023, the whole of which is incorporated herein by reference. Specific details for implementing the invention

[0134] general details

[0135] Throughout this specification, unless otherwise specifically stated or otherwise required by the context, a designation for a single step, composition of a material, group of steps, or group of material compositions shall be deemed to encompass one and plural (i.e., one or more) such step, composition of a material, group of steps, or group of material compositions.

[0136] Those skilled in the art will understand that modifications and variations other than those specifically described in this disclosure are possible. This disclosure should be understood to include all such modifications and variations. This disclosure also includes all steps, features, compositions, and compounds mentioned or indicated herein, individually or collectively, and any and any combination of any two or more of said steps or features.

[0137] The scope of this disclosure is not limited by the specific examples described herein for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0138] Any example of the present disclosure thereof, unless otherwise specifically stated, any other example of the present disclosure thereof Applied by analogy It should be considered as such.

[0139] Any example of the present disclosure relating to tau-binding proteins is to tau protein-binding antibodies Applied by analogy It should be considered as such.

[0140] Unless otherwise specifically defined, all technical and scientific terms used herein shall be deemed to have the same meaning as generally understood by those skilled in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, biochemistry, and bioinformatics).

[0141] Unless otherwise indicated, the recombinant proteins, cell cultures, and immunological techniques utilized in this disclosure are standard procedures well known to those skilled in the art. Such techniques are based on materials, e.g., J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook except Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Vol. 1 and 2, IRL Press (1991), DM Glover and BD Hames (editors), DNA Cloning: A Practical Approach, Vol. 1–4, IRL Press (1995 and 1996), and F.M. Ausubel except (Editor), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (Editor), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan except (Editor) It has been described and explained throughout the literature of Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0142] The description and definition of the variable region and parts thereof, immunoglobulins, antibodies, and fragments thereof are Kabat Sequences of Proteins of Immunological Interest , National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork. except , J Mol. Biol . 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901 -917, 1987, Chothia except Nature 342, 877-883, 1989 and / or Al-Lazikani except , J Mol Biol 273, This can be further clarified by the discussion in 927-948, 1997.

[0143] The term "and / or," for example, "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be considered to provide explicit support for either of the two meanings or for one of the two meanings.

[0144] Throughout this specification, the word “comprising,” or modifications, such as “comprising” or “comprising,” will be understood to imply the inclusion of the specified elements, integers, or steps, or a group of elements, integers, or steps, but not the exclusion of any other elements, integers, or steps, or a group of elements, integers, or steps.

[0145] As used herein, the term "derived from" should be deemed to indicate that a specified integer may be obtained from a specific source, even if not necessarily directly from that source.

[0146] For example, the designation of the scope of residues in this application shall be understood as comprehensive. For example, the designation of "region comprising amino acids 56 to 65" shall be understood in a comprehensive manner, and in other words , the above region includes the sequence of amino acids numbered 56, 57, 58, 59, 60, 61, 62, 63, 64, and 65 in the specified sequence.

[0147] Selected definition

[0148] For the purpose of nomenclature alone and without limitation, an exemplary sequence of the human microtubule-associated protein tau is presented as P10636-8 in the Uniprot database (and Sequence number 48 As presented). Sequences of tau proteins from other species may be determined using the sequences provided herein and / or publicly available databases, or standard techniques (e.g., Ausubel except , (Editor), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook except It can be determined using (as described in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

[0149] The designation for human microtubule-associated protein tau may be abbreviated as human tau protein or simply "tau protein" or "tau". The designation for tau protein is provided herein Sequence No. 1 It includes the original subtype of human tau protein or its mammalian paralog, comprising an N-terminal amino acid sequence corresponding to. In some exemplary embodiments, the human tau protein is the 2N4R subtype (441 amino acids - Uniprot ID P10636-8), also referred to as subtype "tau-F", and its sequence is Sequence number 48 It is provided as.

[0150] The terms “isolated protein” or “isolated polypeptide” refer to a protein or polypeptide that is not associated with any naturally-associated component in its original state due to its origin or source of origin; and is substantially free of any other protein from the same source. The protein may be substantially free of the naturally associated component or substantially purified by isolation using protein purification techniques known in the art. “Substantially purified” means that the protein is substantially free of contaminants, for example, that it is free of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of contaminants.

[0151] The term "recombinant" should be understood to refer to the product of artificial genetic recombination. Therefore, in the context of recombinant proteins containing antibody-antigen binding domains, this term does not encompass antibodies that occur naturally within the subject's body, which are the product of natural recombination occurring during B cell maturation. However, if such antibodies are isolated, they are considered isolated proteins containing antibody-antigen binding domains. Similarly, if nucleic acids encoding a protein are isolated and expressed using recombinant means, the resulting protein is a recombinant protein containing an antibody-antigen binding domain. Recombinant proteins also encompass proteins expressed by artificial recombinant means, for example, when present within the cell, tissue, or subject in which they are expressed.

[0152] The term "protein" should be understood to include a single polypeptide chain, that is, a series of consecutive amino acids connected by peptide bonds, or a series of polypeptide chains connected to one another covalently or non-covalently (i.e., polypeptide complexes). For example, a series of polypeptide chains may be covalently connected using suitable chemicals or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0153] The term "polypeptide" or "polypeptide chain" will be understood from the preceding paragraph to mean a series of consecutive amino acids connected by peptide bonds.

[0154] As used herein, the term "antigen-binding domain" refers to a region of an antibody capable of specifically binding to an antigen, i.e., V H or V L or V H and V L It should be understood to mean an Fv that includes both. The antigen-binding domain does not need to be in the context of a whole antibody and may be, for example, isolated (e.g., a domain antibody) or in other forms as described herein, such as a scFv.

[0155] For the purposes of this disclosure, the term "antibody" refers to one or several closely related antigens (e.g., at least) due to the antigen-binding domain contained within the Fv. Sequence No. 1It includes proteins capable of specifically binding to peptides, polypeptides, or proteins containing [specific components]. This term includes 4-chain antibodies (e.g., two light chains and two heavy chains), and recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-transplant antibodies, primateized antibodies, deimmunization antibodies, synthetic humanized antibodies, anti-antibodies, and bispecific antibodies). Antibodies generally contain a constant domain, which may be arranged into a constant region, a constant fragment, or a crystallizable fragment (Fc). Exemplary forms of antibodies include a 4-chain structure as their basic unit. Full-length antibodies contain two covalently linked heavy chains (~50 to 70 kD) and two light chains (~23 kDa each). The light chains generally contain a variable region (if present) and a constant domain, and are either the κ light chain or the λ light chain in mammals. Heavy chains generally contain one or two constant domain(s) connected to additional constant domain(s) by a variable region and a hinge region. Mammalian heavy chains are one of the following types: α, δ, ε, γ, or μ. Each light chain is also covalently linked to one of the heavy chains. For example, two heavy chains and heavy and light chains are held together by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds can vary between different types of antibodies. Each chain has an N-terminal variable region (V H or V L and each has a length of approximately 110 amino acids) and one or more constant domains at the C-terminus. The constant domain of the light chain (C, which is approximately 110 amino acids in length) L ) is the first constant domain of the heavy chain (C, which has a length of 330 to 440 amino acids). H 1) is aligned with and disulfide-bonded. The variable region of the light chain is aligned with the variable region of the heavy chain. The antibody heavy chain has two or more additional C H Domain (e.g., C H 2, CH It may include 3 etc.) and C H 1 and C H A hinge region may be included between the two constant domains. The antibody may be an immunoglobulin type, class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass selected from IgG, IgE, IgM, IgD, IgA, and IgY. In some preferred embodiments, the antibody is a human antibody. In some embodiments, the antibody is a murine (mouse or rat) antibody or a primate (e.g., human) antibody. In some embodiments, the antibody heavy chain is missing a C-terminal lysine residue. In some embodiments, the antibody is humanized, synthetically humanized, chimeric, CDR-transplanted, or deimmunized.

[0156] The terms "whole antibody," "whole antibody," or "total antibody" are used interchangeably to refer to a substantially whole form of an antibody, as opposed to an antigen-binding fragment of the antibody. Specifically, total antibodies include those having a heavy chain and a light chain containing an Fc region. The constant domain may be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or an amino acid sequence variant thereof.

[0157] As used herein, "variable region" refers to a portion of the light chain and / or heavy chain of an antibody as defined herein that is capable of specifically binding to an antigen, and a complementarity determining region (CDR), in other words Includes the amino acid sequences of , CDR1, CDR2, and CDR3, and the framework region (FR). For example, the variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) along with three CDRs. V H refers to the variable region of the heavy chain. V L It refers to the variable region of the light chain.

[0158] As used herein, the term "complementary determining region" (synonymously CDR, i.e., CDR1, CDR2, and CDR3) refers to amino acid residues in an antibody variable region whose presence is a major contributor to specific antigen binding. Each variable region domain (V H or V L ) typically has three CDRs identified as CDR1, CDR2, and CDR3. In some embodiments, the amino acid positions assigned to CDR and FR are of the kavat. Sequences of Proteins of Immunological Interest , National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the “Kavat Numbering System”). In other embodiments, the amino acid positions assigned to CDR and FR are defined according to the Enhanced Chothia Numbering Scheme (bioinf.org.uk / abs / info.html). According to the Kavat Numbering System, V H FR and CDR are generally, but not exclusively, located as follows: residues 1 to 30 (FR1), 31 to 35 (CDR1), 36 to 49 (FR2), 50 to 65 (CDR2), 66 to 94 (FR3), 95 to 102 (CDR3), and 103 to 113 (FR4). According to the kavat numbering system, V L FR and CDR are located as follows: residues 1 to 23 (FR1), 24 to 34 (CDR1), 35 to 49 (FR2), 50 to 56 (CDR2), 57 to 88 (FR3), 89 to 97 (CDR3), and 98 to 107 (FR4). The present disclosure is not limited to FR and CDR as defined by the Kavat numbering system, but includes the standard numbering system or Chothia and Lesk J. Mol. Biol. 196 : 901-917, 1987; Chothia et al., Nature 342 : 877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273: 927-948, System of 1997; Honnegher and Pl kthun J. Mol. Biol. 309 : 657-670, 2001 numbering system; or Giudicelli except , Nucleic Acids Res. Includes all numbering systems including the IMGT system discussed in 25: 206-211 1997. In one example, CDR is defined according to the Kavat numbering system. Optionally, heavy chain CDR2 according to the Kavat numbering system does not include the five C-terminal amino acids listed herein, or one or more of these amino acids are substituted with other naturally occurring amino acids. In this regard, Padlan except , FASEB J., 9 : 133-139, 1995 established that the five C-terminal amino acids of heavy chain CDR2 generally do not participate in antigen binding.

[0159] The Framework Area (FR) is a variable area residue other than the CDR residue.

[0160] As used herein, the term "Fv" is V H and V L A complex that is associated and has an antigen-binding domain, in other words , is considered to mean any protein capable of specifically binding to an antigen, regardless of whether it consists of multiple polypeptides or a single polypeptide. V forming the antigen-binding domain H and V L It may be within a single polypeptide chain or different polypeptide chains. Additionally, the Fv of the present disclosure (as well as any protein of the present disclosure) may have multiple antigen-binding domains that may or may not bind to the same antigen. The term should be understood to encompass proteins corresponding to such fragments produced using recombinant means as well as fragments directly derived from antibodies. In some examples, V His a heavy chain invariant domain (C H Not connected to )1 or V L The light chain invariant domain (C L It is not linked to ). An exemplary Fv containing a polypeptide or protein is a Fab fragment, Fab' fragment, F(ab') fragment, scFv, diabody, triabody, tetrabody, or higher-order complex, or an invariant region or a domain thereof, e.g., C H 2 or C H 3. Any of the aforementioned connected to the domain, e.g., a minibody. The "Fab fragment" consists of a monovalent antigen-binding fragment of immunoglobulin and may be produced by digesting the whole antibody with the enzyme papain to produce a fragment consisting of an intact light chain and a portion of the heavy chain, or may be produced using recombinant means. The "Fab' fragment" of the antibody may be obtained by treating the whole antibody with pepsin and subsequently reducing it, and the intact light chain and V H and mass-produces a molecule consisting of a portion of a heavy chain containing a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. Fab' fragments can also be produced by recombinant means. The "F(ab')2 fragment" of the antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds and can be obtained by treating the entire antibody molecule with the enzyme pepsin without subsequent reduction. The "Fab2" fragment is, for example, produced by leucine zipper or C H It is a recombinant fragment containing two Fab fragments linked using three domains. "Single-chain Fv" or "scFv" is a recombinant molecule containing a variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0161] nanobody ® "Single-domain antibodies" (sdAbs), also known as monomolecular variable antibody domains, typically VH It consists of domains. In some embodiments, the nanobody is based on a camelid antibody. In other embodiments, the nanobody comprises one or more CDRs derived from mouse or human antibodies implanted on a nanobody scaffold.

[0162] As used herein, in relation to the interaction between a human tau-binding protein or its antigen-binding domain and an antigen, the term “binds” means that the interaction depends on the presence of a specific structure on the antigen (e.g., an epitope or epitope). For example, antibodies recognize and bind to specific protein structures rather than the protein as a whole. If an antibody binds to epitope “A,” the presence of a molecule containing epitope “A” (or free, unlabeled “A”) in a reaction containing labeled “A” and protein will reduce the amount of labeled “A” bound to the antibody.

[0163] As used herein, the terms “specifically bind” or “specifically bind” should be construed to mean that the human tau binding protein of the present disclosure reacts or associates with a specific antigen more frequently, more rapidly, for a longer duration, and / or with greater affinity than alternative antigens. For example, the human tau binding protein is a human tau protein in monomeric or aggregated form (e.g., human tau protein monomer, aggregate, Sequence No. 1 It binds to another polypeptide or peptide containing the amino acid sequence presented in ). The designation "bind" provides explicit support for the term "specific binding". The same applies to the opposite case. .

[0164] As used herein, the terms “tau protein aggregate,” “tau aggregate,” or “aggregated form of tau” encompass any of tau oligomers, tau fibrils, tau fibers, coupled helical filaments, or linear filaments.

[0165] As used herein, the term “not detectably bound” should be understood to mean that the human tau binding protein, e.g., antibody, binds to the candidate antigen at a level of less than 10%, 8%, 6%, or 5% of the background. The background may be the level of the binding signal detected in the absence of the protein and / or in the presence of a negative control protein (e.g., an isomorphic control antibody) and / or the binding level detected in the presence of a negative control antigen. The binding level is detected using a biosensor assay (e.g., Biacore) in which the antigen (e.g., polypeptide) is immobilized and contacts the human tau binding protein.

[0166] As used herein, the term “does not bind significantly” should be understood to mean that the binding level of the human tau-binding protein polypeptide of the present disclosure is not statistically significantly higher than the level of the binding signal detected in the background, e.g., in the absence of the human tau-binding protein and / or in the presence of a negative control protein (e.g., an isomorphic control antibody) and / or the binding level detected in the presence of a negative control polypeptide. For example, the binding level is detected using a biosensor assay (e.g., Biacore) in which an antigen (e.g., polypeptide) is immobilized and contacted with the human tau-binding protein. Sequence No. 1 Dissociation constant (Kd), binding constant (Ka), and / or affinity constant (K) of an immobilized tau binding protein for a tau polypeptide or peptide containing an epitope corresponding to D ). "Kd" or "Ka" or "K" for tau binding protein D " is measured in one example by a radiolabeled or fluorescently labeled tau binding assay. In the case of "Kd", this assay is performed with labeled tau protein or peptide in the presence of a titration series of unlabeled tau protein or peptide ( Sequence No. 1Equilibrate the tau-binding protein to the minimum concentration of (including ). After washing to remove unbound tau polypeptide or peptide, the amount of labeling is determined, which represents the Kd of the protein.

[0167] According to other examples, Kd, ​​Ka, or K D is a surface plasmon resonance assay, for example, immobilized tau protein or Sequence No. 1 Its region including, or is measured using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized tau binding protein.

[0168] In some examples, the isolated tau-binding protein inhibits human tau protein seeding of tau protein aggregates. In some embodiments, the isolated tau-binding protein is such that the tau seed concentration is 200 ng / mL and the tau-binding protein concentration is about 300 nM. In a test tube It inhibits human tau protein seeding of tau protein aggregates by at least about 30%. In some embodiments, the isolated tau binding protein has a higher affinity for aggregated tau than the tau antibody "HJ8.5" and / or the tau antibody "RNF5".

[0169] In some examples, the tau binding protein is K similar to the antibody RNJ1 disclosed herein. D or improved K D (i.e., K D It has a value lower than.

[0170] tau-binding proteins of the present disclosure In vivo Half-life is also used in pharmacokinetic studies, for example, Kim et al., Eur J of Immunol 24It can be measured according to the method described in :542, 1994. According to this method, a radiolabeled tau-binding protein is injected intravenously into a mouse, and its plasma concentration is measured periodically as a function of time, for example, from 3 minutes to 72 hours after injection. The clearance curve thus obtained must be bipolar, namely alpha and beta phases. of the protein In vivo To determine the half-life, the clearance rate in the beta phase is calculated and compared to that of the wild-type or unmodified protein.

[0171] As used herein, phrases referring to "reduced binding" or "lower levels of binding" in relation to an antigen refer to human tau binding protein, for example, an antibody to a control epitope or antigen (e.g., Sequence No. 1 Antigen (e.g., as described herein) with an affinity at least about 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, or 200 times lower than ) Sequence No. 1 It will be understood to mean binding to an antigen containing a mutation in the epitope sequence present in.

[0172] Human tau binding protein or antibody K of protein or antibody against other polypeptides D Dissociation constant less than (K D If it binds to the polypeptide in question via ), it may be considered to "preferentially bind" to the polypeptide in question. In some embodiments, the human tau binding protein or antibody K of the protein or antibody against another polypeptide. D Affinity that is at least about 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, or 200 times higher than (i.e., K D When binding to a polypeptide with ), it is considered to bind preferentially to that polypeptide.

[0173] For clarity and as will be apparent to those skilled in the art based on the subject matter exemplified herein, the designation "affinity" in this specification refers to the K of human tau binding protein or antibody. D It is a designation for.

[0174] For clarity and as will be apparent to those skilled in the art based on the description herein, the designation “at least affinity of the drug” is to be understood to mean that the affinity is equal to or higher than the value mentioned (i.e., the value mentioned as affinity is lower), that is, the affinity of 2 nM is greater than the affinity of 3 nM. In other words, this term may be “an affinity of X or less,” where X is the value mentioned herein.

[0175] "At least the IC of the medicine 50 "is IC 50 This mentioned value is equal to or less than (i.e., IC 50 It will be understood to mean that the value mentioned as is lower), that is, an IC of 2μg / ml. 50 The IC of 1 μg / ml 50 It is larger than. In other words, this term refers to "ICs less than or equal to X". 50 It may be, and X is the value mentioned in the present invention.

[0176] As used herein, the term “epitope” (synonym “antigenic determinant”) should be understood to mean a region of the human tau protein to which the human tau binding protein binds, comprising the antigen-binding domain of the antibody. This term is not necessarily limited to specific residues or structures to which the human tau binding protein contacts. For example, this term includes a region spanning 5 to 10 (or more) or 2 to 5 or 1 to 3 amino acids to which the human tau binding protein contacts.

[0177] The term "competitively inhibits" means that the test human tau binding protein (or its antigen-binding domain) of the present disclosure is a reference antibody or human tau protein of the human tau protein binding protein, for example Sequence No. 1 It should be understood that this means reducing or preventing binding to [the antibody]. This may be because the human tau protein binding protein (or antigen-binding domain) and the antibody bind to the same or overlapping epitopes. From the foregoing, it will be apparent that the human tau protein binding protein does not need to completely inhibit the binding of the antibody, but rather only needs to reduce binding by a statistically significant amount, for example, at least about 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 95%. For example, the human tau protein binding protein reduces the binding of the antibody by at least about 30%, for example, at least about 50%, for example, at least about 70%, for example, at least about 75%, more preferably at least about 80% or 85%, for example, at least about 90%. Methods for determining competitive inhibition of binding are known in the art and / or described herein.

[0178] As used herein, the term “neutralize” should be construed to mean that the human tau protein binding protein can block, reduce, or prevent human tau protein aggregate seeding. In some embodiments, neutralizing activity also includes the ability of the human tau binding protein to inhibit or reduce the diffusion of tau aggregates, such as tau oligomers and / or fibers, in the central nervous system (e.g., between different brain regions) of a subject to whom the human tau binding protein has been administered.

[0179] As used herein, the term "tauopathy" refers to a class of neurodegenerative disorders associated with the aggregation of tau proteins.

[0180] As used herein, the terms “preventing,” “preventing,” or “prevention” include administering the human tau-binding protein disclosed herein to thereby stop or interfere with the development of at least one symptom or biomarker of tauopathy. The terms also encompass treatment for subjects in remission to prevent or interfere with relapse. Prevention does not need to be the complete suppression of the onset of the targeted condition, but may include a delay in the onset of the condition or a delay in specific symptoms associated with the onset or progression of the condition, e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 80%, 90%, or other delays in the onset of the condition, which, based on a clinical evaluation of the subject, would be otherwise observed in subjects at risk in the absence of treatment / prevention therapy.

[0181] As used herein, the terms “treating,” “treat,” or “treatment” include administering the human tau protein-binding protein described herein to reduce or eliminate at least one symptom of the disease or condition specified thereby.

[0182] As used herein, the term “object” refers to any animal including humans, e.g., mammals. Exemplary objects include, but are not limited to, humans and non-human primates. For example, an object is a human.

[0183] antibodies

[0184] In some embodiments, the human tau protein binding protein as disclosed herein is an antibody.

[0185] Methods for generating antibodies are known in the art and / or described in Harlow and Lane (Editor) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in these methods, human tau protein or its regions (e.g., N-terminal fragment or Sequence No. 1 The peptide containing the sequence presented in (i.e., the peptide) or cells expressing and presenting it (i.e., immunogens) are optionally formulated with any suitable or desired carrier, ajuvant, or pharmaceutically acceptable excipient and administered to non-human animals, e.g., mice, chickens, rats, rabbits, guinea pigs, dogs, horses, cattle, goats, or pigs.

[0186] Therefore also Sequence No. 1 It includes an amino acid sequence according to, but tau ( Sequence number 48 An amino acid sequence of more than 50 to 100 consecutive amino acids from ), e.g. 97 amino acids, 90 amino acids, 85 amino acids, 80 amino acids, 75 amino acids, 70 amino acids, 65 amino acids, 60 amino acids, 55 amino acids, or Sequence number 48 A non-naturally occurring immunogenic peptide or polypeptide is disclosed herein that does not contain 50 to 100 or fewer consecutive amino acids of a different number thereof. In other embodiments, a length of 12 to about 80 amino acids, e.g. 14, 15, 17, 18, 20, 22, 25, 27, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or a different number of amino acids of a length of 12 to about 80 amino acids. Sequence No. 1 An immunogenic peptide comprising an amino acid corresponding to is disclosed herein. In some embodiments, the amino acid sequence of the immunogenic peptide is Sequence number 1 It consists of amino acids corresponding to .

[0187] The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes. Alternatively, the peptide is V as described herein. H -V L It can be used to screen expression display libraries (e.g., phage display libraries) that express antigen-binding domains. In another embodiment, the peptide is used to screen single-domain antibody libraries (also known as "nanobody" libraries).

[0188] The production of polyclonal antibodies can be monitored by sampling blood from immunized animals at various points after immunization. If necessary to achieve the desired antibody titer, one or more additional immunizations may be administered. The boosting process is repeated until a suitable titer is achieved. Once the desired level of immunogenicity is obtained, blood is drawn from the immunized animals to isolate and store serum, or the animals are used to generate monoclonal antibodies (mAbs).

[0189] Monoclonal antibodies are one exemplary form of the antibodies considered by the present disclosure. The terms “monoclonal antibody” or “mAb” refer to a homogeneous population of antibodies capable of binding to the same antigen(s), e.g., the same epitope within the antigen. The term is not intended to be limited in relation to the source of the antibodies or the manner in which they are manufactured.

[0190] Any one or more of the many known techniques for the production of mAbs may be used, for example, US4196265 or The above This is the procedure exemplified in Harlow and Lane (1988).

[0191] For example, suitable animals are immunized with immunogens under conditions sufficient to stimulate antibody-producing cells. Rodents, such as rabbits, mice, and rats, are exemplary animals. Genetically engineered to express human antibodies, for example, mice and mice that do not express antibodies may also be used to produce the antibodies of the present disclosure (e.g., as described in WO2002 / 066630).

[0192] After immunization, somatic cells capable of producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generation protocol. These cells may be obtained from biopsies of the spleen, tonsils, or lymph nodes, or from peripheral blood samples. B cells from the immunized animal are then fused with immortal myeloma cells, typically derived from the same species as the animal immunized with the immunogen.

[0193] Hybrids of nucleotides in tissue culture medium De Novo It is amplified by culture in a selective medium containing agents that block synthesis. Exemplary agents are aminopterin, methotrexate, and azacerin.

[0194] The amplified hybridoma undergoes functional selection for antibody specificity and / or titer by, for example, flow cytometry and / or immunoassays (e.g., radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, etc.).

[0195] Alternatively, ABL-MYC technology (NeoClone, Madison, WI 53713, USA) is used to produce Mab-secreting cell lines (e.g., Largaespadia). except , J. Immunol. Methods. 197 : As described in 85-95, 1996).

[0196] Antibodies may also be produced or isolated by screening a display library, e.g., a phage display library, as described in, for example, US6300064 and / or US5885793.

[0197] For example, the grip display library is Sequence No. 1 It is screened for polypeptides or peptides containing.

[0198] In additional examples, human tau protein, N-terminal domain, or Sequence No. 1 A peptide containing [the peptide] is used as a "bait" antigen and is contacted with an RNJ1 antibody as disclosed herein. A phage display library is then contacted with the bait antigen, and a phage expressing a protein capable of competing with RNJ1 for binding is subsequently selected.

[0199] In some embodiments, the antibodies disclosed in this disclosure are synthetic. Such synthetic antibodies include, but are not limited to, chimeric antibodies, humanizing antibodies, human antibodies, synthetic humanizing antibodies, primateizing antibodies, or deimmunizing antibodies. In some preferred embodiments, the antibodies are human antibodies.

[0200] Deimmunized, chimeric, CDR-transplanted, humanized, synthetic humanized, primate-ized, human, and complex human tau-binding protein

[0201] In some preferred embodiments, the tau protein-binding protein of the present disclosure is a human protein. In other embodiments, the tau protein-binding protein of the present disclosure may be a CDR-transplanted protein comprising a CDR from an antibody of a non-human species (e.g., mouse or rat or non-human primate) that is transplanted onto or inserted into the FR from a human antibody, or a CDR-transplanted protein comprising a CDR derived from an antibody of one type of antibody (e.g., one type of human antibody) that is transplanted onto or inserted into the FR from another type of antibody (e.g., another type of human antibody). The term also encompasses a complex human tau-binding protein comprising, for example, one or more CDR-transplanted variable regions and one or more, for example, a human variable region, a chimeric variable region, a synthetic humanized variable region, or a primateized variable region.

[0202] In some embodiments, the tau-binding protein of the present disclosure is a humanized protein.

[0203] The term “humanized protein” should be understood to refer to a protein comprising a human-like variable region, comprising a CDR derived from an antibody of a non-human species (e.g., mouse, rat, rabbit, or non-human primate) that is transplanted onto or inserted into the FR from a human antibody (antibodies of this type belong to the class of “CDR-transplanted antibodies”). Humanized human tau protein-binding proteins also comprise proteins in which one or more residues of the human protein are modified by one or more amino acid substitutions or one or more FR residues of the human protein are replaced by the corresponding non-human residues. Humanized proteins may also comprise residues not found in either the human antibody or the non-human antibody. Any additional region of the protein (e.g., the Fc region) is generally human. Humanization may be performed using methods known in the art, e.g., US5225539, US6054297, US7566771, US5585089. The expert can also combine calculation methods, such as human antibody sequence databases, such as the Observed Antibody Space (OAS available at opig.stats.ox.ac.uk / webapps / oas / ) with Prihoda except (2022), mAbs , the "BioPhi" platform described in (doi: 10.1080 / 19420862.2021.2020203); or Mlokosiewicz et al. (2022), Bioinformatics As described regarding the "AbDiver" platform in , 38(9):2628-2630, the corresponding human V H and V LWe will recognize various methods for evaluating the "humanity" of variable region sequences by comparison with the human natural antibody repertoire to determine residues proposed for substitution based on positional frequencies in framework and CDR sequences. Additionally, a computational method ("CUMAb") for selecting the optimal human framework for the transplantation of non-human CDRs while maintaining antigen affinity based on energy-based ranking is Tennenhouse except (2023), Nature Biomedical Engineering , (doi: 10.1038 / s41551-023-01079-1) is described. The term “humanized protein” also encompasses super-humanized proteins as described, for example, in US7732578. This term also encompasses complex proteins comprising, for example, one or more humanized variable regions and one or more, for example, human variable regions, chimeric variable regions, synthetic humanized variable regions or primateized variable regions.

[0204] In some preferred embodiments, the human tau-binding protein disclosed herein is of human origin. As used herein in relation to the human tau-binding protein, “human protein” or “human origin” refers to a protein having a variable antibody region found in humans. In embodiments, where the antibody also includes a constant antibody region, such constant antibody region is also found in humans, e.g., from human germlines or somatic cells, or from a library generated using such region. “Human” protein is an amino acid residue not encoded by a human sequence, e.g. In a test tubeIt may include mutations introduced by random or site-specific mutations (particularly mutations involving substitutions or mutations in a small number of residues of the protein, e.g., 1, 2, 3, 4, or 5 residues of the protein). Such “human proteins” do not necessarily need to be produced as a result of a human immune response, but rather may be produced using recombinant means (e.g., phage display library screening) and / or by transgenic animals (e.g., mice) containing nucleic acids encoding human antibody constant and / or variable regions, or by guided selection (e.g., as described in US5565332). The term also encompasses the affinity-mature forms of such antibodies. For the purposes of the present disclosure, human proteins will also be considered to include FRs comprising a FR from a human antibody or a FR comprising a sequence from a consensus sequence of a human FR, and one or more of the CDRs corresponding to those disclosed herein as part of the tau-binding protein, antibody “RNJ1”, as well as proteins that are amino acid-substituted and / or humanized variants as disclosed herein.

[0205] Accordingly, in some exemplary embodiments, the human tau binding protein V comprises the following set of CDR sequences H and V L It is an antibody containing a region:

[0206] (i) Sequence No. 2 (CDR1), Sequence No. 3 (CDR2), and Sequence No. 4 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 6 (CDR2), and Sequence number 7 V containing the amino acid sequence presented in (CDR3) L ;

[0207] (ii) Sequence No. 2 (CDR1), Sequence number 8(CDR2), and Sequence No. 4 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 6 (CDR2), and Sequence number 7 V containing the amino acid sequence presented in (CDR3) L ;

[0208] (iii) Sequence No. 2 (CDR1), Sequence No. 3 (CDR2), and Sequence No. 4 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 9 (CDR2), and Sequence number 7 V containing the amino acid sequence presented in (CDR3) L ;

[0209] (iv) Sequence No. 2 (CDR1), Sequence No. 3 (CDR2), and Sequence No. 4 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 6 (CDR2), and Sequence number 10 V containing the amino acid sequence presented in (CDR3) L ;

[0210] (v) Sequence No. 2 (CDR1), Sequence No. 3 (CDR2), and Sequence No. 4 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 9 (CDR2), and Sequence number 10 V containing the amino acid sequence presented in (CDR3) L

[0211] (vi) Sequence No. 2 (CDR1), Sequence number 8 (CDR2), and Sequence No. 4 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 9(CDR2), and Sequence number 7 V containing the amino acid sequence presented in (CDR3) L ;;

[0212] (vii) Sequence No. 2 (CDR1), Sequence number 8 (CDR2), and Sequence No. 4 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 6 (CDR2), and Sequence number 10 V containing the amino acid sequence presented in (CDR3) L ;

[0213] (viii) Sequence No. 2 (CDR1), Sequence number 8 (CDR2), and Sequence No. 4 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 9 (CDR2), and Sequence number 10 V containing the amino acid sequence presented in (CDR3) L ;

[0214] (ix) Sequence No. 2 (CDR1), Sequence No. 3 (CDR2), and Sequence No. 4 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 17(CDR2), and Sequence number 7 V containing the amino acid sequence presented in (CDR3) L ;

[0215] (x) Sequence No. 2 (CDR1), Sequence No. 3 (CDR2), and Sequence No. 4 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 6 (CDR2), and Sequence number 18 V containing the amino acid sequence presented in (CDR3) L ;

[0216] (xi) Sequence No. 2 (CDR1), Sequence No. 3 (CDR2), and Sequence No. 4 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 17 (CDR2), and Sequence number 18 V containing the amino acid sequence presented in (CDR3) L ;

[0217] (xii) Sequence No. 2 (CDR1), Sequence number 15 (CDR2), and Sequence No. 4 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 6 (CDR2), and Sequence number 7 V containing the amino acid sequence presented in (CDR3) L ;

[0218] (xiii) Sequence No. 2 (CDR1), Sequence No. 3 (CDR2), and Sequence number 16 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 6 (CDR2), and Sequence number 7 V containing the amino acid sequence presented in (CDR3) L ;

[0219] (xiv) Sequence No. 2 (CDR1), Sequence number 15 (CDR2), and Sequence number 16 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 6 (CDR2), and Sequence number 7 V containing the amino acid sequence presented in (CDR3) L ;

[0220] (xv) Sequence No. 2 (CDR1), Sequence number 15(CDR2), and Sequence number 16 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 17 (CDR2), and Sequence number 7 V containing the amino acid sequence presented in (CDR3) L ;

[0221] (xvi) Sequence No. 2 (CDR1), Sequence number 15 (CDR2), and Sequence number 16 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 6 (CDR2), and Sequence number 18 V containing the amino acid sequence presented in (CDR3) L ;

[0222] (xvii) Sequence No. 2 (CDR1), Sequence number 15 (CDR2), and Sequence number 16 V containing the amino acid sequence presented in (CDR3) H ; and Sequence number 5 (CDR1), Sequence number 17 (CDR2), and Sequence number 18 V containing the amino acid sequence presented in (CDR3) L .

[0223] Optionally, the above V H It is connected to a heavy chain constant region, for example, an IgG4 heavy chain constant region or a stabilized IgG4 constant region. In one example, the heavy chain constant region lacks a C-terminal lysine residue.

[0224] Optionally, the above V L It is connected to the light chain invariant region.

[0225] The tau-binding protein of the present disclosure may be a synthetic humanized protein. The term "synthetic humanized protein" refers to a protein prepared by the method described in WO2007 / 019620. The synthetic humanized tau-binding protein comprises a variable region of an antibody, wherein the variable region comprises a FR derived from a New World primate antibody variable region and a CDR derived from a non-New World primate antibody variable region. For example, the synthetic humanized tau-binding protein comprises a variable region of an antibody, wherein the variable region comprises a FR derived from a New World primate antibody variable region and a CDR derived from a mouse or rat antibody. In some embodiments, the synthetic humanized tau-binding protein is a synthetic humanized tau protein-binding antibody in which one or both of the variable regions are synthetic humanized tau protein-binding antibodies. The term also encompasses a complex protein comprising, for example, one or more synthetic humanized variable regions and one or more, for example, a human variable region or a humanized variable region or a chimeric variable region.

[0226] In some embodiments, the tau-binding protein disclosed herein is a primate-forming protein. The "primate-forming protein" comprises variable regions derived from antibodies produced after immunization of a non-human primate (e.g., crab-eating monkey). Optionally, the variable region of the non-human primate antibody is linked to a human constant region to produce a primate-forming antibody. An exemplary method for producing a primate-forming antibody is described in US6113898. The term also encompasses a complex protein comprising, for example, one or more primate-forming variable regions and one or more, for example, a human variable region or a humanized variable region or a chimeric variable region.

[0227] In some embodiments, the tau-binding protein disclosed herein is a chimeric protein. The term "chimeric protein" refers to a protein in which the antigen-binding domain is derived from a specific species (e.g., rodents, e.g., mouse or rat) or belongs to a specific class or subclass of antibodies, while the remainder of the protein is derived from a different species (e.g., human or non-human primates) or belongs to a different class or subclass of antibodies. In some embodiments, the chimeric protein is a V derived from a non-human antibody (e.g., a rodent antibody). H and / or V L It is a chimeric antibody comprising [specific region], wherein the remaining region of the antibody is derived from a human antibody. The production of such chimeric proteins is known in the art and can be achieved by standard means (e.g., as described in US6331415; US5807715; US4816567 and US4816397). The term also encompasses complex proteins comprising, for example, one or more chimeric variable regions and one or more, for example, human variable regions or humanized variable regions or chimeric variable regions.

[0228] This disclosure also considers deimmunized tau-binding proteins by a process, for example, as described in WO2000 / 34317 and WO2004 / 108158. Deimmunized antibodies and proteins have one or more epitopes, for example, B cell epitopes or T cell epitopes, removed (i.e., mutated) so as to reduce the likelihood that a subject will elicit an immune response to the antibody or protein. In some embodiments, deimmunization is performed by a computational approach, such as Prihoda, which assesses humanization and presents residue mutations to increase the humanization of a given antibody. Other than the aboveIt is facilitated by what is described. For example, the tau-binding protein disclosed herein is analyzed to identify one or more B or T cell epitopes, and one or more amino acid residues within the epitopes are mutated to thereby reduce the immunogenicity of the tau-binding protein.

[0229] From the aforementioned disclosure, to those skilled in the art, a "complex" protein is one form of V H (e.g., human) and other forms of V L It will be apparent that it includes (e.g., humanization). The present disclosure is V H and V L It explicitly encompasses all combinations of forms.

[0230] Protein containing antibody binding domain

[0231] Short chain Fv(scFv)

[0232] In some embodiments, the tau protein binding is scFv. The skilled person knows that scFv is V within a single polypeptide chain. H and V L Includes the region and V H and V L scFv forms a suitable structure for antigen binding by including polypeptide linkers between them (i.e., V of a single polypeptide chain). H and V L You will recognize that these can be linked together to form Fv. For example, linkers contain more than 12 amino acid residues, and (Gly4Ser)3 is one of the preferred linkers for scFv.

[0233] The present disclosure also includes a single cysteine ​​residue V H FR and V L Consider disulfide-stabilized Fv (or diFv or dsFv), which is introduced into FR and has cysteine ​​residues connected by disulfide bonds to produce a stable Fv.

[0234] In some embodiments, the tau binding protein is a dimeric scFv, that is, a protein comprising two scFv molecules connected by non-covalent or covalent bonds, for example, by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, the two scFvs are connected by a peptide linker of sufficient length to allow the two scFvs to be formed and bind to an antigen, as described, for example, in US20060263367.

[0235] Diabadi, Triabadi, Tetrabadi

[0236] In some embodiments, the tau-binding protein of the present disclosure is a diabody, triabody, tetrabody, or higher-order protein complex, such as that described in WO98 / 044001 and / or WO94 / 007921, or comprises such.

[0237] For example, a diabody is a protein containing two associated polypeptide chains, each polypeptide chain having structure V L -XV H or V H -XV L Includes, and V L is the variable region of the antibody light chain, and V H is the variable region of the antibody heavy chain, and X is V within a single polypeptide chain. H and V L A linker containing or lacking residues insufficient to cause this association (or form Fv), wherein V of one polypeptide chain H is V of a different polypeptide chain L It binds to form an antigen-binding domain, that is, forms an Fv molecule capable of specifically binding to one or more antigens. V L and V H can be the same in each polypeptide chain, or V H and V LEach polypeptide chain is different and can form a dispecific diabody (i.e., containing two Fvs with different specificities).

[0238] Single-domain antibody

[0239] In some examples, the tau-binding protein of the present disclosure is a single-domain antibody ("sdAb" is the term "domain antibody" or "dAb" or "nanobody") ® (used interchangeably with ") or includes. A single-domain antibody is a single polypeptide chain comprising all or part of the heavy chain variable region of an antibody. In a specific example, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, US6248516).

[0240] Tau binding protein and sequence variants

[0241] In addition, it includes one or more mutations in one or more CDRs disclosed herein, and Sequence number 1 A tau-binding protein is disclosed herein for binding to an epitope of a human tau protein, which is the amino acid sequence of the epitope corresponding to . In some embodiments, K of the tau-binding protein for said epitope d is approximately 5 x 10 -7 It is less than M. Sequence No. 1 The dissociation constant (Kd), binding constant (Ka), and / or affinity constant (KD) of an immobilized tau-binding protein for a tau polypeptide or peptide containing an epitope corresponding to. The "Kd" or "Ka" or "KD" for the tau-binding protein is, in one example, measured by a radiolabeled or fluorescently labeled tau binding assay. For "Kd", the assay is performed on the labeled tau protein or peptide (in the presence of a titration series of unlabeled tau protein or peptide). Sequence No. 1Equilibrate the tau-binding protein to the minimum concentration of (including). After washing to remove unbound tau polypeptides or peptides, the amount of labeling is determined and represents the Kd of the protein.

[0242] According to another example, K D Or Ka is a surface plasmon resonance assay, for example, immobilized tau protein or Sequence No. 1 Its region including, or is measured using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized tau binding protein.

[0243] In some examples, the tau-binding protein is K, which is more similar to the antibody RNJ1 disclosed herein. D or improved K D (i.e., lower K D It has a value.

[0244] Accordingly, in some embodiments of the tau-binding protein disclosed herein:

[0245] ( i ) V H The amino acid sequence of is:

[0246] (a) having up to 5 amino acid substitutions, e.g., 0, 1, 2, 3, 4, or 5 amino acid substitutions Sequence No. 2 ,

[0247] (b) having up to 6 amino acid substitutions, e.g., 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions Sequence No. 3 , and

[0248] (c) having up to 6 amino acid substitutions, e.g., 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions Sequence No. 4 Includes,

[0249] Sequence No. 2 It includes HC-CDR1 corresponding to residuals 31-35 according to the Kavat numbering system, and Sequence No. 3It includes HC-CDR2 corresponding to remaining 50-65 according to the Kavat numbering system, and Sequence No. 4 ...includes and / or includes HC-CDR3 corresponding to residuals 95-102 according to the Kavat numbering system;

[0250] ( ii ) V L The amino acid sequence of is:

[0251] (a) having up to 4 amino acid substitutions, e.g., 0, 1, 2, 3, or 4 amino acid substitutions Sequence number 5 ,

[0252] (b) having up to 4 amino acid substitutions, e.g., 0, 1, 2, 3, or 4 amino acid substitutions Sequence number 6 , and

[0253] (c) having up to 5 amino acid substitutions, e.g., 0, 1, 2, 3, 4, or 5 amino acid substitutions Sequence number 7 Includes; Sequence number 5 It includes LC-CDR1 corresponding to residuals 24-34 according to the kavat numbering system, and Sequence number 6 It includes LC-CDR2 corresponding to residuals 50-56 according to the Kavat numbering system, and Sequence number 7 It includes LC-CDR3 corresponding to residuals 89-97 according to the Kavat numbering system.

[0254] Those skilled in the field will understand that the humanization of antibodies is useful in reducing the likelihood of an immune response to antibodies administered to human patients. Humanization can be extended to various parts of the antibody, including the constant region, the variable domain framework region, and, in some cases, the CDR.

[0255] In some embodiments, tau binding proteins are Sequence No. 2 HC CDR1 including, Sequence number 5LC CDR1 comprising... and at least one of HC CDR2, HC CDR3, LC CDR2, or LC CDR3 comprising variant sequences as summarized in Table 1 below, wherein the standard RNJ1 antibody start sequence is: HC CDR1( Sequence No. 2 ), HC CDR2( Sequence No. 3 ), HC CDR3( Sequence No. 4 ) LC CDR1( Sequence number 5 ), LC CDR2( Sequence number 6 ), LC CDR3( Sequence number 7 )am.

[0256]

[0257] In some embodiments, the tau binding protein comprises one or more variant CDRs:

[0258] ( i ) V H The amino acid sequence of is:

[0259] (a) having up to 5 amino acid substitutions, e.g., 0, 1, 2, 3, 4, or 5 amino acid substitutions Sequence No. 2 ,

[0260] (b) having up to 6 amino acid substitutions, e.g., 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions Sequence number 8 , and

[0261] (c) having up to 6 amino acid substitutions, e.g., 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions Sequence No. 4 Includes,

[0262] Sequence No. 2 It includes HC-CDR1 corresponding to residuals 31-35 according to the Kavat numbering system, and Sequence number 8 It includes HC-CDR2 corresponding to remaining 50-65 according to the Kavat numbering system, and Sequence No. 4 ...includes and / or includes HC-CDR3 corresponding to residuals 95-102 according to the Kavat numbering system;

[0263] ( ii) V L The amino acid sequence of is:

[0264] (a) having up to 4 amino acid substitutions, e.g., 0, 1, 2, 3, or 4 amino acid substitutions Sequence number 5 ,

[0265] (b) having up to 4 amino acid substitutions, e.g., 0, 1, 2, 3, or 4 amino acid substitutions Sequence number 9 , and

[0266] (c) having up to 5 amino acid substitutions, e.g., 0, 1, 2, 3, 4, or 5 amino acid substitutions Sequence number 10 Including; Sequence number 5 It includes LC-CDR1 corresponding to residuals 24-34 according to the kavat numbering system, and Sequence number 9 It includes LC-CDR2 corresponding to residuals 50-56 according to the Kavat numbering system, and Sequence number 10 It includes LC-CDR3 corresponding to residuals 89-97 according to the Kavat numbering system.

[0267] In some embodiments, the tau binding protein includes (i) and (ii).

[0268] In some embodiments, the tau binding protein comprises at least one of the maximum number of substitutions mentioned above in (i) 1 CDR; (ii) 2 CDRs; (iii) 3 CDRs; (iv) 4 CDRs; (v) 5 CDRs; or (vi) 6 CDRs.

[0269] In some embodiments of the tau-binding protein, the number of substitutions in either the sequence-specified heavy chain or light chain CDR does not exceed the specified maximum value (e.g., 6 substitutions for HC CDR2), and V H The identity of the entire amino acid sequence of V disclosed herein is H At least 80% identical in amino acid sequence, and V L The identity of the entire amino acid sequence of V disclosed herein is LAt least 80% identical with respect to the amino acid sequence, for example, V disclosed herein H Amino acid sequence (V H (in the case of comparison with amino acid sequences) and V disclosed herein L Amino acid sequence (V L (in the case of comparison with amino acid sequences) at least 80% to 100% identical, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or other percentage identical, and said tau-binding protein specifically binds to tau protein as disclosed herein. For clarity, V H and V L The amino acid sequences of do not need to have the same percent amino acid sequence identity with respect to their respective reference sequences, for example, tau-binding protein V H The amino acid sequence of is V disclosed herein H It can be 90% identical to the reference sequence; V of the same tau-binding protein L V disclosed herein L It can be 97% identical to the reference sequence.

[0270] In some embodiments, V for the tau binding protein disclosed herein H The amino acid sequence of is Sequence number 11 or Sequence number 13 At least 80% identical to the amino acid sequence of and V L The amino acid sequence of is Sequence number 12 or Sequence number 14 The amino acid sequences are at least 80% identical, for example, each V H and V L It is 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or other percentage identity with respect to the amino acid sequence, which is at least 80% to 100%. In some preferred embodiments of the tau-binding protein disclosed herein, V H The amino acid sequence of is Sequence number 11At least 80% identical to the amino acid sequence of and V L The amino acid sequence of is Sequence number 12 It is at least 80% identical to the amino acid sequence of. In some preferred embodiments, V H The amino acid sequence of is Sequence number 11 Includes the amino acid sequence of and V L The amino acid sequence of is Sequence number 12 It includes the amino acid sequence of, and such V H and V L The amino acid sequence corresponds to that of the disclosed "RNJ1" antibody. The numbering according to the kavat numbering system is V disclosed herein. H and V L The amino acid sequence is provided and is provided in Tables 2 and 3 of the present invention.

[0271] In some cases, structural information is obtained from the experimentally solved crystal structures of antibodies, synonymous antigens, and antibody-antigen complexes. More recently, it has become possible to use computational tools to generate 3D structural models of antibody variable domains and variable domain-antigen complexes, and to infer probable CDR-antigen contact residues. For example, Abanades except (2023), Communications Biology 6, 575(doi.org / 10.1038 / s42003-023-04927-7), Desta except (2023), Nature Protocols, 18:1814-1840; and Ambrosetti except , 2020, Bioinformatics , 36(20):5107-5108; and Ambrosetti except (2020), Structure , 28(1):119-129 Refer to .

[0272] Sequence variation

[0273] Those skilled in the art know that, in antibodies, e.g., tau-binding proteins, one or more amino acid substitutions within the antibody's CDR may, in principle, alter the binding properties (e.g., affinity, specificity, or immunogenicity) of the antigen-binding domain containing the altered CDR, and furthermore, that the number of theoretically possible amino acid substitutions is very large. However, those skilled in the art likewise know that the possibility of substantial functional alteration of the antigen-binding domain can be minimized in practice by a combination of (i) general and individual antibody-specific heuristics for selecting substitutions and / or (ii) sequence and structure-based computational tools to avoid substitutions that may impair key tau-binding protein properties.

[0274] Accordingly, in some embodiments, where any CDR substitution is to be performed, the CDR substitution is performed based on the generally expected mean binding free energy (△△G) contribution of each CDR, and the CDR with the lower binding energy contribution should generally be the first to which the amino acid substitution is introduced, that is, the preferred order of CDR selection for substitution from lower △△G to higher △△G is ( 1 ) V L CDR2 < ( 2 ) V L CDR1 < ( 3 ) V L CDR3 V H CDR1 < ( 4 ) V H CDR2 ≤ ( 5 ) V H It is CDR3. For example, Robin et al. (2014), Journal of Molecular Biology , 426:3729-3743 Refer to Therefore, for example, V, which generally contributes little free energy to paratop-antigen binding. LAmino acid substitutions in CDR2 have generally been found to contribute substantially to the free binding energy, V H It is less likely to affect the affinity of antibodies for antigens than substitutions in CDR2. Additionally, it has been observed that certain amino acids and amino acid types are significantly overpresented in CDR, while conversely, some amino acids are underpresented. Furthermore, it has been found that most of the amino acids overpresented in CDR make a disproportionate contribution to the binding free energy for synonymous antigens. This suggests that when performing substitutions in CDR, substitutions at positions occupied by amino acids known to be one of the most presented / overpresented in CDR and contributing to the highest binding free energy should generally be avoided. Therefore, in some embodiments, substitutions of the following "specific" group of amino acids are not "prohibited" but should be avoided as much as possible:

[0275] Tyr, Trp, Phe, His, Asn, Asp, and Pro

[0276] Where it is not possible or required to avoid the substitution of one of the above amino acids, the preferred proposed substitution is as follows, based on the free energy of the binding contribution (including ordered preference) and structural / functional conservation in preferences ordered from left to right:

[0277] Tyr → Trp, Phe, Leu, Ile, Val, or Met

[0278] Trp → Tyr, Phe, Leu, Ile, Met, or Val

[0279] Phe → Tyr, Trp, Leu, Ile, Met, or Val

[0280] His → Arg, Asn, Lys, Thr

[0281] Asn → Asp, His, Ser, Thr

[0282] Asp → Asn, His, Ser, Thr

[0283] Pro → Val, Thr, Gly, His, Ala

[0284] In some embodiments, where the amino acid to be substituted does not belong to the specific group of amino acids (i.e., Y, T, F, H, N, D, and P), the substitution is based on the similarity of physicochemical properties, for example, "Grantham's distance" related to characteristics of composition, polarity, and molecular volume (Grantham, 1974, Science Based on , doi: 10.1126 / science.185.4154.862), it must be as conservative as possible, the shorter the distance, the more conservative the substitution, and the original amino acid of any pair o Substituted amino acids from s Distance to " D os " is calculated as follows:

[0285] D os = [ α ( c o - c s ) 2 + β( p o - p s ) 2 + γ ( v o - v s ) 2 ] 1 / 2

[0286] Table 2 below shows the Graham distances calculated for each amino acid, and the shorter the distance between amino acid pairs, the more similar they are considered to be overall.

[0287] Table 2: Grantham distance between amino acids

[0288]

[0289] An alternative method for determining conservative amino acid substitutions is, for example, Yampolsky et al. (2005). Genetics It is known in the art as described in , 170(4):1459-1472.

[0290] Furthermore, it is recognized in the art that specific variable domain positions (mostly CDR positions) are more frequently found to contribute to the binding free energy for antigens. In fact, it has been found that the 30 highest contributing residue positions alone contribute approximately 80% of the total binding free energy ( The above (Robin et al.), therefore, it is most likely to be sensitive, in particular, to non-conservative substitutions or substitutions that deviate from the aforementioned preferred substitutions. In the Kavat numbering system, more sensitive CDR (or CDR-proximate) positions are frequently found in the following:

[0291] V H Location (Kavat): (CDR1) 27, 32, 33, and 35; (CDR2) 52, 52A, 53, 54, 56, and 58; and (CDR3) 95, 96, S97, 98, and 99; and

[0292] V L Location (Kavat): (CDR1) 32; (CDR2) 49 and 50; (CDR3) 91, 92, 94, and 96

[0293] Tau-binding proteins disclosed herein (e.g., V H Sequence number 11 and V L Sequence number 12 Referring to the amino acid sequence of ), the amino acids corresponding to the aforementioned "sensitive" CDR positions are as follows:

[0294] V H Location (Kavat):(CDR1) F27, Y32, M33, and S35; (CDR2) G52, W52A, N53, G54, S56, and A58; and (CDR3) R95, G96, S97, P98, and F99; and

[0295] V L Location (Kavat): (CDR1) Y32; (CDR2) Y49 and R50; (CDR3) G91, M92, R94, and I96.

[0296] Guidance on preferred substitution sites in some embodiments is, for example, Ambrosetti et al., (2020), Bioinformatics As described in , 36(20):5107-5108, it is based on the computational prediction of paratop / contact residues for each CDR using "Prediction of AntiBody contacts v2 (proABC-2). Therefore, the corresponding (tau) antigen epitope ( Sequence No. 1 The predicted contact residues for the tau-binding protein disclosed herein to contact ) are as follows:

[0297] V H ( Sequence number 11 ): Kabat locations 30-33, 35, 50, 52, 52A, 53, 54, 56-58; 94-99, and 101; and

[0298] V L ( Sequence number 12 ): Kabat locations 30-32, 34, 49, 50, 51, 53-56, and 91-96.

[0299] Accordingly, in some embodiments of the tau-binding protein disclosed herein, the total number of substitutions including the predicted contact sites is as follows:

[0300] (i) V H Is Sequence number 11 or Sequence number 13Comprising an amino acid sequence corresponding to, and comprising six or fewer amino acid substitutions at residue positions according to a Kavat numbering scheme selected from the group consisting of positions 30-33, 35, 50, 52, 52A, 53, 54, 56-58; 94-99, and 101, e.g., 0, 1, 2, 3, 4, 5, or six substitutions;

[0301] (ii) V L silver Sequence number 12 or Sequence number 14 It includes an amino acid sequence corresponding to, and includes six or fewer amino acids, for example, 0, 1, 2, 3, 4, 5, or six substitutions, at residue positions according to a Kavat numbering scheme selected from the group consisting of positions 30, 31, 32, 34, 49, 50, 53, 55, 91-94, and 96.

[0302] In another embodiment, a CDR-containing sequence (e.g., Sequence number 2-4 V within ) H (for example, Sequence number 11 or Sequence number 13 My); or CDR-containing sequence (e.g., Sequence number 5-7 V within ) L (for example, Sequence number 12 or Sequence number 14 Tau-binding proteins containing amino acid substitutions within) are likely at position V, which is not predicted to be an antigen contact site. H 23-26, 28, 29, 34, 36, 37, 51, 59-65, 93, 101, and 102; and V L These substitutions are present only in 24-29, 33, 51, 52, 89, 90, 95, and 97.

[0303] In another embodiment, where substitutions must be made at both predicted contact and non-contact locations, where possible, in order to reduce the likelihood of interfering with antigen binding, the substitutions are preferably made from the substitution predicted to be least likely to be a contact residue to the substitution predicted to be most likely to be a contact residue. The following sequence is also described above and The above Consider the bond free energies generally contributed by amino acids Y, T, F, H, N, D, and P, as described by Robin et al.

[0304] Accordingly, the disclosed herein Sequence No. 2 (CDR2), Sequence No. 3 (CDR3), or Sequence No. 4 V containing any of (CDR3) H Domain and / or Sequence number 5 (CDR1), Sequence number 6 (CDR2), or Sequence number 7 V containing any of (CDR3) L In some embodiments of a tau-binding protein containing a domain, the order of preferred substitutions from most preferred to least preferred (based on the Kavat numbering system) is as follows:

[0305] V H

[0306] Sequence No. 2 -Include CDR1: A23 = A24 > G26 > S25 > T28 > V37 > S35 > A33 > S30 = S31 >W36 > F27 = F29 >Y32;

[0307] Sequence No. 3 - Including CDR2: G65 > K64 = V63 > A60 > G50 > I51 > D61 > G52 > Y59 > G54 > T55 > S56 > N53 > W52(A); and

[0308] Sequence No. 4- Included CDR3: A93 > K94 > G96 > R95 > S97 = P98 > Y102 > D101 > F99 V L

[0309] Sequence number 5 -Include CDR1: A25 > R24 = S26 = Q27 > S28 > I29 > S30 > S31 > L33 > N34 > Y32;

[0310] Sequence number 6 - Including CDR2: G65 > K64 = V63 > S62 > A60 > I51 > G50 > D61 > G52 > Y59 > A58 > T55 > G54 > S56 > N53 > W52(A); and

[0311] Sequence number 7 - Included CDR3: A93 > K94 > G96 > R95 > S97 > P98 > Y102 > D101 > F99

[0312] In some preferred embodiments, where CDR substitution is to be performed, the order of selection of CDRs for substitution follows the order mentioned above (based on the contribution of CDR bond free energy), i.e., ( 1 ) V L CDR2 < ( 2 ) V L CDR1 < ( 3 ) V L CDR3 ≡ V H CDR1 < ( 4 ) V H CDR2 ≤ ( 5 ) V H It is CDR3.

[0313] V H or V L In addition to the aforementioned guidelines for generating a tau-binding protein comprising a CDR position substitution that minimizes the possibility of functional alteration in functional characteristics (e.g., antigen affinity), a tau-binding protein, e.g., an RNJ1 mAb, and the tau antigen to which it binds, e.g., Sequence number 1 3D structural modeling of peptides composed of corresponding amino acids is useful for computationally modeling the 3D structure of antibody-antigen complexes, and is particularly useful for determining the possible effects of any given substitution at paratop / contact residues on antigen binding. Therefore, Abanades et al. (2023), Communications Biology RNJ1 antibody (V), modeled based on the ABodyBuilder2 platform as described at 6:575 H As a hierarchy Sequence number 11 and V L As a hierarchy Sequence number 12 cast A 3D computational model of a tau-binding protein corresponding to (including) is provided herein. The cartoon model of FIG. 13 and Appendix 2 of Australian provisional patent application 2023903449 filed October 27, 2023, which is incorporated herein by reference in its entirety. Refer to.

[0314] Also, the tau N-terminal epitope to which mAb RNJ-1 binds ( Sequence No. 1 A 3D structure of a peptide composed of ) is provided, which is Lamiable et al. (2016), Nucleic Acids Research It was modeled based on the PEPFOLD3 platform described in , doi: 10.1093 / nar / gkw329. The cartoon model of FIG. 14 and the Australian provisional patent application 2023903449 filed on October 27, 2023, the whole of which is incorporated herein by reference. Refer to. Methods for "docking" 3D structural binding partners including antibody-antigen complexes are known in the art, for example, Ambrosetti et al. (2023), Methods in Molecular Biology It uses the "HADDOCK" platform as described in , 2252:267-282. Also, Ambrosetti except (2020), Structure , 28(1):119-129; Brenke except (2012), Bioinformatics, 28:2608-2614; Desta except (2023), Nature Protocols , 18:1814-1840; and Davila except (2022), Bioinformatics Advances , doi.org / 10.1093 / bioadv / vbac015 Refer to Other platforms, such as the graph-based signature model "mmCSM-AB2," can be used to computationally predict the effect of point mutations on antibody affinity (Myung et al., 2020). Nucleic Acids Research , doi: 10.1093 / nar / gkaa389). Also, Ascher et al. (2023), Methods in Molecular Biology , 2252:375-397; and Sheng et al. (2023), Frontiers in Immunology , doi: 10.3389 / fimmu.2023.1190416 Refer to .

[0315] Therefore, a skilled user can [use] tau-binding protein V without substantial loss of affinity or specificity H or V L Multiple sequence variants of the sequence can be provided without great effort. As referred to herein, "substantial loss" with respect to affinity or specificity as used herein refers to a reduction of about 10% to about 95% in affinity or specificity for a reference tau-binding protein, e.g., 15%, 18%, 20%, 25%, 30%, 35%, 37%, 40%, 42%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or the reference tau-binding protein disclosed herein, e.g., V, respectively. H and V L As an amino acid sequence Sequence number 11 and 12 It refers to a different percentage reduction in the affinity or specificity of the tau-binding protein for sequence variants including .

[0316] An exemplary method for producing a sequence-altered form of a tau-binding protein includes the following:

[0317] - DNA(Thie except , Methods Mol. Biol. 525 : 309-322, 2009) or RNA(Kopsidas except , Immunol. Lett . 107 :163-168, 2006; Kopsidas et al., BMC Biotechnology, 7 : 18, 2007; and mutagenicity of WO1999 / 058661);

[0318] - Introduction of nucleic acids encoding polypeptides into mutant cells, e.g., XL-1Red, XL-mutS, and XL-mutS-Kanr bacterial cells (Stratagene);

[0319] - DNA shuffling, for example, Stemmer, Nature 370 : As disclosed in 389-91, 1994; and

[0320] - Site-specific mutagenicity, e.g. Dieffenbach (edit) and Dveksler (edit) ( In : As described in PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).

[0321] An exemplary method for determining the biological activity of the sequence variant tau-binding protein of the present disclosure will be apparent to those skilled in the art and / or is described herein, for example, antigen binding. For example, methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation, and therapeutic efficacy are described herein.

[0322] Those skilled in the art, for example, Dondelinger et al. (2018), Frontiers in ImmunologyIt is acknowledged that numerous antibody variable domain and CDR numbering schemes have been developed in the art, as reviewed in 9:2278. These include Kavat, Chotia, IMGT, Martin, and North-AHO. In this document, unless otherwise specified, CDRs are defined according to the Kavat numbering scheme. However, in some cases, sequence numbers as mentioned herein contain additional residues not included in the Kavat-defined CDR, which may nevertheless be considered part of the CDR according to other antibody numbering schemes, e.g., North-AHO.

[0323] V against tau-binding protein RNJ1 antibody based on the Kavat numbering system H ( Sequence number 11 ) and V L ( Sequence number 12 The amino acid residue positions for ) are shown in Tables 3 and 4 below.

[0324] Table 3 - RNJ1 V H Kavart numbering position for (sequence number 11)

[0325]

[0326] Underlined positions 23-37 correspond to sequence number 2; bold underlined positions 31-35 are Kavart-defined V H Corresponds to CDR1; bold underlined positions 50-65 are Sequence No. 3 and Kavart-defined V H Corresponds to CDR2; underlined positions 93-102 correspond to sequence number 4, and bold underlined positions 95-102 are Kavart-defined V H It corresponds to CDR3.

[0327] Table 4 - RNJ1 V L Kavart numbering position for (sequence number 12)

[0328]

[0329] The boldly underlined positions 24-34 are Sequence number 5 and Kabat-defined V LCorresponds to CDR1; underlined positions 49-56 are Sequence number 6 This corresponds to, and the bold underlined positions 50-56 are Kabat-defined V L Corresponds to CDR2; the boldly underlined positions 89-97 are Sequence number 7 and Kabat-defined V L It corresponds to CDR3.

[0330] In some embodiments, the tau-binding protein comprises one or fewer amino acid substitutions in each CDR. In other embodiments, the tau-binding protein V H One or fewer permutations in any of the CDRs and V L It includes one amino acid substitution in any one of the CDRs.

[0331] In some embodiments of the tau binding protein, V H The amino acid sequence of is:

[0332] (i) Sequence No. 2 CDR1-containing amino acid sequence corresponding to;

[0333] (ii) Sequence No. 3 or Sequence number 8 CDR2-containing amino acid sequence corresponding to; and

[0334] (iii) Sequence number 4 It includes a CDR3-containing amino acid sequence corresponding to.

[0335] In some embodiments of the tau binding protein, V L The amino acid sequence of is:

[0336] (i) Sequence number 5 CDR1-containing amino acid sequence corresponding to;

[0337] (ii) Sequence number 6 or In sequence number 9 Corresponding CDR2-containing amino acid sequence; and

[0338] (iii) Sequence number 7 or Sequence number 10It includes a CDR3-containing amino acid sequence corresponding to.

[0339] In some embodiments, V H The amino acid sequence of is:

[0340] (i) Sequence number 2 CDR1-containing amino acid sequence corresponding to;

[0341] (ii) Sequence No. 3 CDR2-containing amino acid sequence corresponding to; and

[0342] (iii) Sequence No. 4 It includes a CDR3-containing amino acid sequence corresponding to.

[0343] In some embodiments, V L The amino acid sequence of is:

[0344] (i) Sequence number 5 CDR1-containing amino acid sequence corresponding to;

[0345] (ii) Sequence number 6 CDR2-containing amino acid sequence corresponding to; and

[0346] (iii) Sequence number 7 It includes a CDR3-containing amino acid sequence corresponding to.

[0347] In some embodiments, V H The amino acid sequence of is:

[0348] (i) Sequence No. 2 CDR1-containing amino acid sequence corresponding to;

[0349] (ii) Sequence No. 3 CDR2-containing amino acid sequence corresponding to; and

[0350] (iii) Sequence number 4 It includes a CDR3-containing amino acid sequence corresponding to; V L The amino acid sequence of is:

[0351] (i) Sequence number 5 CDR1-containing amino acid sequence corresponding to;

[0352] (ii) Sequence number 6 CDR2-containing amino acid sequence corresponding to; and

[0353] (iii) Sequence number 7 It includes a CDR3-containing amino acid sequence corresponding to.

[0354] In some embodiments, V H The amino acid sequence of is Sequence number 11 or Sequence number 13 It includes or consists of the amino acid sequence corresponding to.

[0355] In some embodiments, V L The amino acid sequence of is Sequence number 12 or Sequence number 14 It includes or consists of an amino acid sequence corresponding to .

[0356] In some embodiments, V H The amino acid sequence of is Sequence number 11 or Sequence number 13 Includes or consists of an amino acid sequence corresponding to;

[0357] V L The amino acid sequence of is Sequence number 12 or Sequence number 14 It includes or consists of the amino acid sequence corresponding to.

[0358] V in some embodiments H The amino acid sequence of is Sequence number 11 Includes or is composed of an amino acid sequence corresponding to;

[0359] V L The amino acid sequence of is Sequence number 12 It includes or consists of the amino acid sequence corresponding to.

[0360] Those skilled in the art will recognize that tau-binding proteins, for example, tau antibodies, may be provided in various antibody forms or structures comprising any of the variable domain amino acid sequences disclosed herein. Accordingly, in some embodiments, the tau protein is V within a single polypeptide chain H and VL It includes all domains. Suitable examples of such single polypeptide tau-binding proteins are (i) short-chain F v (ii) a fragment (scFv), (ii) a dimeric scFv (di-scFv), or (iii) at least one of (i) and / or (ii) is an Fc or a heavy chain invariant domain (C H )2 and / or C H Includes, but is not limited to, connected to 3.

[0361] In some embodiments, one or more CDR V derived from tau binding protein H Domains are used to generate single-domain antibodies, also known as "nanobodies." A method for generating nanobodies by transplanting a CDR from a mammalian antibody onto a framework derived from the variable domain of a heavy chain-only antibody (VHH) is, for example, Wagner et al. (2018). International Journal of Molecular Sciences It is known in the art as exemplified in , 19(11), 3444. Also, Vincke et al. (2009), Journal of Biological Chemistry , 284(5):3273-3284 Refer to .

[0362] In other embodiments, the tau binding protein is: (i) a divalent antibody, (ii) Fab'; (iii) a diabadi; (iv); a triabadi; or (v) a tetrabadi.

[0363] In some embodiments, any of the tau-binding proteins disclosed herein further comprise (i) a detection moiety; (ii) a stabilization moiety; (iii) a delivery moiety; and (iv) a functional moiety selected from any combination thereof.

[0364] In some embodiments, the tau-binding protein of the present disclosure is provided as a conjugate with a functional moiety. For example, the functional moiety is selected from the group consisting of radioisotopes, detectable labels, therapeutic compounds, peptides, proteins, compounds that increase the half-life of the tau-binding protein in a target, and combinations thereof.

[0365] The above functional moiety may be directly or indirectly bound to a tau-binding protein (e.g., in the case of indirect binding, it may include a linker). Examples of detectable moiety include radioisotopes (e.g., carbon 11 or fluorine 18), detectable labels (e.g., near-infrared (NIR) fluorescent dyes, e.g., BTC1070, BTC980, BTC982), or NIR quantum dots. For example, Gil except (2021), iScience , 24(3):102189 Refer to. In some embodiments, the tau-binding protein is labeled detectably, for example, by the conjugation of a detectable moiety.

[0366] In some embodiments, the functional moiety is encoded as a fusion protein with the tau-binding protein disclosed herein (e.g., as an N-terminal or C-terminal fusion protein). Thus, in some embodiments, the detectable moiety is a reporter protein fused to the tau-binding protein. In some embodiments, the reporter protein is a fluorescent protein or an enzyme. In some embodiments, where the reporter protein is a fluorescent protein, the fluorescent protein is selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), mCerulean3, and mScarlet3. In other embodiments, where the reporter protein is an enzyme, the enzyme is selected from the group consisting of β-galactosidase (LacZ), luciferase, β-lactamase, and HaloTag.

[0367] Examples of stabilizing moiety are polyethylene glycol (PEG), (Q311R / M428L-substituted) Fc domain (Ko et al., 2022, Experimental Molecular Medicine , 54:1850-1861), and unstructured Pro-Ala-Ser (PAS) polypeptide chains (Schlapschy et al., 2013, Protein Engineering, Design and Selection Examples of transport moiety include, but are not limited to, , 26(8):489-501). Examples of transport moiety are those that facilitate the transport of cell-permeable peptides, particularly across the blood-brain barrier (e.g., Blades et al., 2023, Labeled Compounds and Radiopharmaceuticals , reviewed at DOI 10.1002 / jlcr.4023), polypeptides targeting receptors on the blood-brain barrier (e.g., transferrin receptor TfR or insulin receptor) to facilitate receptor-mediated transcytosis, such as scFv or nanobodies (fused to tau-binding proteins), but not limited thereto—e.g., Kouhi et al. (2021), International Journal of Molecular Sciences , 22(12):6331 Refer to . In another embodiment, the targeting moiety is Edavettal except (2022), Med As described in , 3(12):860-882 and international patent application PCT / IB2021 / 052889, it is a polypeptide comprising a transcytosis-capable module (TEM) of the form anti-scFv (e.g., TfR-scFv or CD98-scFv).

[0368] invariant region

[0369] In some embodiments, the tau-binding protein and / or antibody disclosed herein comprises an invariant region of the antibody. This comprises an antigen-binding fragment of the antibody fused to Fc.

[0370] Sequences of constant regions useful for producing the protein of the present disclosure may be obtained from a number of sources. In some preferred embodiments, the constant region of the protein or a portion thereof is derived from a human antibody. The constant region or a portion thereof may be derived from any class of antibodies including IgM, IgG, IgD, IgA, and IgE, and any antibody isoforms including IgG1, IgG2, IgG3, and IgG4. In some embodiments, the constant region is a human isoform IgG4 or a stabilized IgG4 constant region. In other embodiments, the constant region is an IgG1 constant region, which can facilitate the uptake of tau or tau aggregate-binding antibody complexes into microglia, thereby facilitating the removal of extracellular aggregated forms of tau from the brain. In other embodiments, the constant region within the tau-binding protein (e.g., a tau antibody) is an IgG2b constant region.

[0371] In some embodiments, the Fc region of the constant region has a reduced ability to induce effector function compared to, for example, the natural or wild-type human IgG1 or IgG3 Fc region. In the context of this disclosure, "effector function" refers to such biological activity mediated by a cell or protein binding to the Fc region of an antibody (the original sequence Fc region or the amino acid sequence variant Fc region) that results in the activation of the Fc receptor. Examples of effector functions induced by antibodies include antibody-dependent phagocytosis (ADCP) in the relevant portion. Methods for evaluating the level of effector function of an Fc region-containing protein are known in the art.

[0372] In some embodiments, where the tau-binding protein comprises an Fc region, said Fc region is an IgG4 Fc or IgG1 region (i.e., derived from an IgG4 or IgG1 constant region), for example, a human IgG4 Fc or IgG1 Fc region. Sequences for IgG Fc regions are available in publicly accessible databases (e.g., available from the National Center for Biotechnology Information).

[0373] In some embodiments, where the IgG4 Fc region is utilized, the invariant region is a stabilized IgG4 invariant region. The term "stabilized IgG4 invariant region" will be understood to mean an IgG4 invariant region modified to reduce the tendency to undergo Fab arm exchange or Fab arm exchange, or the formation of half antibodies or the tendency to form half antibodies. "Fab arm exchange" refers to a type of protein modification for human IgG4 in which the IgG4 heavy chain and attached light chain (half molecule) are swapped with a heavy-light chain pair from another IgG4 molecule. Thus, the IgG4 molecule can acquire two different Fab arms that recognize two distinct antigens (resulting in a heterospecific molecule). Fab arm exchange is In vivo It occurs naturally and is produced by purified blood cells or reducing agents, such as reduced glutathione. In a test tube It can be induced. "Half antibodies" are formed when IgG4 antibodies dissociate to form two molecules containing a single heavy chain and a single light chain, respectively.

[0374] In some embodiments, the stabilized IgG4 constant region contains proline at position 241 of the hinge region according to the Kabat system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991). This position is in the EU numbering system (Kabat except , Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 2001 and Edelman et al., Proc. Natl. Acad. USA , 63 It corresponds to position 228 of the hinge region according to *[ ], 78-85, 1969]). In human IgG4, this residue is typically serine. Following the substitution of serine with proline, the IgG4 hinge region contains the sequence CPPC. In this regard, those skilled in the art will recognize that the "hinge region" is a proline-rich portion of the antibody heavy chain constant region that connects the Fc and Fab regions, which confer mobility to the two Fab arms of the antibody. The hinge region contains cysteine ​​residues involved in the inter-heavy chain disulfide bond. This is generally defined as extending from Glu226 to Pro243 in human IgG1 according to the Kavat numbering system. The hinge regions of other IgG isoforms can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain disulfide (SS) bond at the same location (see, for example, WO2010 / 080538).

[0375] Further examples of stabilized IgG4 antibodies include antibodies in which arginine at position 409 of the heavy chain constant region of human IgG4 (according to the EU numbering system) is substituted with lysine, threonine, methionine, or leucine (e.g., as described in WO2006 / 033386). The Fc region of the constant region may additionally or alternatively include a residue selected from the group consisting of alanine, valine, glycine, isoleucine, and leucine at the position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region contains proline at position 241 ( in other words Includes , CPPC sequence) (as described above).

[0376] In another example, the Fc region is a region modified to have reduced effector function, namely the "non-immunostimulating Fc region." For example, the Fc region is an IgG1 Fc region containing substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In another example, the Fc region is an IgG1 Fc region containing one or more of the following changes E233P, L234V, L235A and a deletion of G236 and / or one or more of the following changes A327G, A330S, and P331S (Armour et al., Eur J Immunol. 29 :2613-2624, 1999; Shields et al., J Biol Chem. 276(9) (6591-604, 2001). Additional examples of non-immunostimulatory Fc regions are, for example, Dall'Acqua et al., J Immunol. 177 : 1129-1138 2006; and / or Hezareh J Virol ;75 : Explained in 12161-12168, 2001).

[0377] In some embodiments, the Fc region is a chimeric Fc region, for example, at least one C from an IgG4 antibody. H At least one C from the 2 domains and IgG1 antibody HIt comprises three domains, and the Fc region comprises substitutions at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409, and 427 (EU numbering) (e.g., as described in WO2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0378] Exemplary tau-binding protein

[0379] Some exemplary variable-region containing tau-binding proteins disclosed herein are described in Table 5.

[0380] Table 5 : Exemplary tau-binding protein V H -V L Amino acid sequence of combination Fv

[0381]

[0382] Nucleic acid encoding tau-binding protein, expression vector, recombinant virus, and nucleic acid library

[0383] Also, any tau binding protein or its V disclosed herein H or V L A nucleic acid encoding the amino acid sequence of is provided herein. In some embodiments, the nucleic acid is throughout its entire length Sequence number like 11, 13, and 33-36 At least 80% identity with any one of the, for example, Sequence numbers 11, 13, and 33-36 Tau-binding protein V having 85%, 87%, 90%, 92%, 95%, 97%, or other percentage identity from at least 80% to 100% identity with any one of the above. H It encodes an amino acid sequence. In some embodiments, the encoding nucleic acid over its entire length Sequence numbers 12, 14 , and 37-41At least 80% identity with any one of the, for example, Sequence numbers 12, 14 , and 37-41 Tau-binding protein V having 85%, 87%, 90%, 92%, 95%, 97%, or other percentage identity from at least 80% to 100% identity with any one of the above. L Encodes an amino acid sequence.

[0384] In another example, the nucleic acid of the present disclosure is presented herein and is a tau-binding protein, its V H or V L It comprises a sequence that is at least about 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to the sequence encoding. The present disclosure also includes the tau-binding protein of the present disclosure and its V, which differ from the sequence exemplified herein as a result of the degeneracy of the genetic code. H and V L It includes nucleic acids that encode.

[0385] Percentage identity of nucleic acids or polypeptides is GAP with a gap creation penalty of 5 and a gap extension penalty of 0.3 (Needleman and Wunsch. Mol. Biol. 48 (, 443-453, 1970) It is determined by analysis (GCG program). The query sequence is at least 50 residues long, and the GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues long, and the GAP analysis aligns the two sequences over a region of at least 100 residues. For example, the two sequences are aligned over their entire length.

[0386] This disclosure also covers the tau-binding protein, V described herein. H or V LConsider a nucleic acid that is hybridized under strict hybridization conditions to a nucleic acid encoding [the nucleic acid]. “Moderate strictness” is defined herein as hybridization and / or washing performed in 2 x SSC buffer, 0.1% (w / v) SDS, or equivalent conditions at a temperature in the range of 45°C to 65°C. “High strictness” is defined herein as hybridization and / or washing performed in 0.1 x SSC buffer, 0.1% (w / v) SDS, or lower salt concentrations, and at a temperature of at least 65°C, or equivalent conditions. Designations for a specific level of strictness herein encompass equivalent conditions using washing / hybridization solutions other than SSC known in the art. For example, methods for calculating the temperature at which the strands of a double-stranded nucleic acid dissociate (also known as the melting temperature, or Tm) are known in the art. A temperature similar to (e.g., within 5°C or within 10°C) or equal to the Tm of the nucleic acid is considered high strictness. Intermediate strictness is considered to be that the calculated Tm of nucleic acid is within 10°C to 20°C or 10°C to 15°C.

[0387] In some embodiments, the isolated nucleic acid provided herein is DNA. In other embodiments, the isolated nucleic acid is mRNA, for example, modified mRNA. In some embodiments, modified mRNA (also referred to as “chemically modified mRNA” or “mmRNA”) refers to synthetic RNA produced in vitro, in which at least one type of nucleotide, for example, a proportion of cytosine (e.g., 10%, 30%, 50%, or 100%) is chemically modified to increase its stability in cells or otherwise in vivo. For example, in some cases, the modified cytosine is 5-methylcytosine. Such polynucleotides are particularly useful for delivery / transfection into cells in vivo, especially when combined with a transfection agent / transfer agent. In some cases, the chemically modified mRNA is a chemically modified mRNA in which the majority (e.g., all) of the cytosines are 5-methylcytosine and the majority (e.g., all) of the uracils are pseudouracils. The synthesis and use of such modified RNA are described, for example, in WO 2011 / 130624. In some embodiments, the encoding nucleic acid is mRNA or In the case of mmRNA, the mRNA is self-amplifying mRNA (sa-mRNA), which may be useful for increasing the expression of the encoded tau-binding protein or its variable domain. In other embodiments, the encoding nucleic acid is circular RNA (circ-RNA). For example, Chen et al. (2023), Nature Biotechnology , 41:262-272 Refer to .

[0388] In addition, nonviral expression vectors encoding the tau-binding protein disclosed herein are disclosed herein. Suitable examples of nonviral expression vectors include, but are not limited to, mammalian expression vectors, yeast expression vectors, insect cell expression vectors, bacterial expression vectors, and plant expression vectors. In some embodiments, the nonviral expression vector is a mammalian expression vector. In other embodiments, the nonviral expression vector is a yeast expression vector. Molecular cloning techniques for achieving these purposes are known in the art, and, for example, Ausubel except , (Editor), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook except , described in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and In a test tube The amplification method is suitable for the construction of non-viral expression vectors. Methods for producing recombinant antibodies are also known in the art, for example, US4816567 or US5530101 Refer to Also, Frenzel et al., (2013), Frontiers in Immunology , 4:217 Refer to .

[0389] After isolation, the nucleic acid is operably linked and inserted into a promoter within an expression construct or expression vector for further cloning (DNA amplification) or for expression in a cell-free system or cell.

[0390] As used herein, the term “promoter” should be taken in the broadest possible context and includes transcriptional regulatory sequences of genomic genes containing TATA boxes or initiator elements required for precise transcription initiation, with or without additional regulatory elements (e.g., upstream activation sequences, transcription factor binding sites, enhancers, and silencers) that alter nucleic acid expression in a tissue-specific manner, for example, in response to development and / or external stimuli. In this context, the term “promoter” is also used to describe recombinant, synthetic, or fusion nucleic acids or derivatives that confer, activate, or enhance the expression of operably linked nucleic acids. An exemplary promoter may contain additional copies of one or more specific regulatory elements to further enhance and / or improve the expression of said nucleic acids or to modify spatial expression and / or temporal expression.

[0391] As used herein, the term "operably linked" means positioning a promoter on a nucleic acid so that the expression of the nucleic acid is controlled by the promoter.

[0392] Many vectors are available for intracellular expression. Vector components generally include, but are not limited to, one or more of a signal sequence, a sequence encoding a protein (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. A person skilled in the art will recognize a sequence suitable for protein expression. Exemplary signal sequences include prokaryotic secretory signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretory signals (e.g., invertase leader, factor α leader, or acid phosphatase leader), or mammalian secretory signals (e.g., herpes simplex gD signal).

[0393] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-α promoter (EF1), micronucleus RNA promoter (U1a and U1b), α-myosin heavy chain promoter, monkey virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), adenovirus major late promoter, β-actin promoter; and hybrid regulatory elements comprising the CMV enhancer / β-actin promoter or immunoglobulin promoter or an active fragment thereof.

[0394] Yeast cells, for example, Pichia pastoris ( Pichia pastoris) , Saccharomyces cerevisiae( Saccharomyces cerevisiae ) and S. Pombe( S. pombe) A typical promoter suitable for expression in yeast cells selected from the group including ADH1 Promoter, GAL1 Promoter, GAL4 Promoter, CUP1 Promoter, PHO5 Promoter, nmt Promoter, RPR1 promoter, or TEF1 Includes, but is not limited to, promoters.

[0395] In other embodiments, a viral vector for the expression of a tau-binding protein disclosed herein is disclosed herein. As used herein, a “viral expression vector” is a DNA construct capable of inducing the expression of one or more polynucleotides in a host cell (e.g., mammalian sensory hair cell) and comprises a recombinant viral vector or a viral sequence that enables replication of the vector and / or packaging into a viral capsid so as to produce a recombinant virus as a viral genome or part thereof. In some embodiments, the recombinant viral expression vector is a recombinant adeno-associated virus (AAV) vector, a recombinant adenovirus (AdV) vector, or a recombinant lentivirus (LV) vector. Methods for designing AAV, AdV, and LV vectors are known in the art, for example, regarding AAV, Li et al. (2020), Nature Reviews Genetics , 21:255-272 and Gray except (2011), Methods Mol Biol , 807:25-46; regarding AdV, Sallard et al. (2023) Viruses , 15(1):204; and regarding LV, Merten et al. (2016), Molecular Therapy - Methods & Clinical Development , 3, 16017. Also, Gene Therapy Protocols Methods in Mol. Biol., Vols. 1 & 2, Joseph LeDoux (ed.), 2008 Refer to .

[0396] In some embodiments, the expression vector (non-viral or viral) disclosed herein comprises at least two proteins, for example, tau binding protein V H and tau binding protein V L It includes at least one polycistronic expression cassette encoding. The polycistronic expression cassette refers to an expression cassette ("polycistronic expression cassette") encoding polycistronic mRNA, which, upon translation, produces independent polypeptides containing different amino acid sequences or functionalities, for example, tau-binding protein VH Domain-containing polypeptide and tau-binding protein V L A domain-containing polypeptide is generated. In some embodiments, the polycistronic expression cassette encodes a "polyprotein" comprising multiple polypeptide sequences that are encoded and separated by a picornavirus, e.g., foot-and-mouth disease virus (FMDV) virus 2A peptide sequence. The 2A peptide sequence acts co-translatingly by preventing the normal formation of a peptide bond between the conserved glycine and the final proline, causing the ribosome to skip to the next codon and cleave between the nascent peptides Gly and Pro. After cleaving, the short 2A peptide remains fused to the C-terminus of the 'upstream' protein, while the proline is added to the N-terminus of the 'downstream' protein. This enables the nascent polypeptide sequence to be cleaved into distinct polypeptides during translation. For example, Trichas et al. (2008) BMC Biology , 6, 40 and Liu et al. (2017) Nature Scientific Reports See , 7, 2193, doi.org / 10.1038 / s41598-017-02460-2.

[0397] In another embodiment, the polycistronic expression cassette may incorporate one or more internal ribosome entry sites (IRES) sequences between open reading frames incorporated within the polycistronic expression cassette. IRES sequences and their uses are known in the art, for example, Martinez-Sales, (1999), Current Opinion in Biotechnology Examples are provided in , 10, 458-464.

[0398] Additionally, a recombinant virus containing any one of the recombinant virus expression vectors disclosed herein is disclosed herein, thereby the recombinant virus can be used to transfect and express the tau-binding protein disclosed herein in a host cell genetically modified by transduction by the recombinant virus. In some embodiments, the recombinant virus is an adeno-associated virus (AAV), an adenovirus (AdV), a lentivirus (LV), a baculovirus (including silkworm baculovirus), and anellovirus. Methods for packaging and purifying the recombinant virus and for quantifying the same are, for example, for AAV, Mietzsch et al. (2021), Journal of Virology , 95(19) doi.org / 10.1128 / jvi.00773-21; regarding AdV, Sayedahmed et al. (2019) Methods in Molecular Biology , 1937:155-175; regarding LV, Brown et al. (2020) STAR Protocols , doi.org / 10.1016 / j.xpro.2020.100152; regarding silkworm baculovirus, Ebihara et al. (2021), Molecular Biotechnology ..., 63, 1223-1234; and anelloviruses are known in the art as described in International Patent Application Publication WO2023064781. Additionally, services for packaging and purifying recombinant viruses are commercially available from a number of companies, e.g., VectorBuilder, Charles River Laboratories, and ThermoFisher Scientific.

[0399] The expert is tau binding protein V H and / or V L It will be understood that when a nucleic acid encoding a specific species is to be expressed in a host cell derived from a specific species, the expression of the encoded polypeptide can be enhanced by the use of species-fit codon optimization. For example, the tau-binding protein V disclosed herein H or VL If this is to be expressed in human cells, an exemplary human codon-optimized nucleotide sequence for a tau-binding protein is, for example, Sequence number 42 or 46 (V H in the case of) or Sequence number 43 or 47 (V L Includes any one of the cases of).

[0400] In another embodiment, for example, for the production of a tau-binding antibody in Chinese hamster ovary (CHO) cells, the use of codons is optimized for expression in Chinese hamster host cells. An exemplary Chinese hamster codon-optimized nucleotide sequence for a tau-binding protein is, for example, Sequence number 44 (V H (in the case of) and Sequence number 45 (V L Includes the case of). Codon usage frequencies are known in the art and applications for codon optimization are publicly available, for example, LeRoy et al. (2023), bioRxiv , doi.org / 10.1101 / 2023.05.22.541759 exists. Also, the Optipyzer.com web server Refer to .

[0401] Also V H and / or V L A tau-binding protein variable domain expression library comprising a plurality of expression vectors encoding:

[0402] (i) The above V H The amino acid sequence of is:

[0403] (a) having up to 5 amino acid substitutions Sequence No. 2 ,

[0404] (b) having up to 6 amino acid substitutions Sequence No. 3 , and

[0405] (c) having up to 6 amino acid substitutions Sequence No. 4 Includes,

[0406] Sequence No. 2 It includes HC-CDR1 corresponding to residuals 31-35 according to the Kavat numbering system, and Sequence No. 3 It includes HC-CDR2 corresponding to residuals 50-65 according to the Kavat numbering system, and Sequence No. 4 ...includes and / or includes HC-CDR3 corresponding to residuals 95-102 according to the Kavat numbering system;

[0407] (ii) The above V L The amino acid sequence of is:

[0408] (a) having up to 4 amino acid substitutions Sequence number 5 ,

[0409] (b) having up to 4 amino acid substitutions Sequence number 6 , and

[0410] (c) having up to 5 amino acid substitutions Sequence number 7 Includes; Sequence number 5 It includes LC-CDR1 corresponding to residuals 24-34 according to the kavat numbering system, and Sequence number 6 It includes LC-CDR2s corresponding to residuals 50-56 according to the Kavat numbering system, and Sequence number 7 It includes LC-CDR3 corresponding to residuals 89-97 according to the Kavat numbering system.

[0411] Such libraries are useful for identifying tau-binding proteins having improved characteristics, e.g., higher antigen-binding affinity, increased solubility, or lower potential immunogenicity in humans. In some embodiments, the variable domain expression library is V H or V L It includes only the domain, and this Sequence No. 1 It is useful for the development of single-domain antibodies against tau epitopes defined by, for example, nanobodies. In some preferred embodiments, the variable domain expression library is to be co-expressed V to facilitate the assembly of the tau-binding protein variable domain. H and VL It includes all domains. In some preferred embodiments, the variable domain expression library is a phage display expression vector library, which facilitates the rapid expression screening and identification of tau-binding protein variable domains. A method for constructing and screening human Fab and scFab phage display libraries is, for example, Lai et al., (2023), Methods Mol Biol , 2702:39-58; and Rader (2012), Methods Mol Biol As exemplified in , 901:81-99, it is known in the art. In other embodiments, the variable domain expression library is, for example, Chang et al., (2023), Antibody Therapeutics It is designed to be expressed as a mammalian cell display library as described in , 6(3):157-169.

[0412] In addition, a method for selecting an immunoglobulin that binds to human tau protein is provided herein, said method:

[0413] (i) Heavy chain variable region (V H ), immunoglobulin light chain variable region (V L A step of providing an immunoglobulin library comprising ), or both;

[0414] (ii) the above plurality of immunoglobulin proteins is isolated from tau protein, its fragment, or amino acid sequence Sequence No. 1 A step of contacting with his peptide containing an epitope of the human tau protein corresponding to;

[0415] (iii) identifying an immunoglobulin that specifically binds to the isolated tau protein, its fragment, or its peptide from the plurality of immunoglobulins; and

[0416] (iv) binding of the immunoglobulin to the isolated tau protein, its fragment, or its peptide Sequence No. 1by a peptide composed of the amino acid sequence of or Sequence number 11 V containing the amino acid sequence corresponding to H and Sequence number 12 V containing the amino acid sequence corresponding to L The step of selecting the immunoglobulin only when it is competitively inhibited by a tau binding protein including

[0417] A screening method for identifying scFvs of interest from a scFv phage display library is, for example, Singh et al. (2022), Bio Protocol It is well established, as exemplified in , 12(12):e4450. In addition, skilled individuals can see that scFv or other types of immunoglobulin have a specific epitope of interest, e.g., an amino acid sequence Sequence No. 1 We are well aware of competitive binding assays for determining whether to bind to the tau protein epitope disclosed herein corresponding to [the example].

[0418] In some embodiments, the tau-binding protein disclosed herein (e.g., tau scFv or antibody) is conjugated to a detectable label, e.g., a fluorescent label or a radioactive label. The labeled antibody and the candidate tau-binding protein are mixed and Sequence No. 1An immobilized peptide or polypeptide containing a tau epitope corresponding to is contacted with the labeled tau antibody under conditions permissible for binding to the immobilized peptide or polypeptide containing SEQ ID NO. 1. Following a washing step to reduce any non-specific binding, the level of the labeled antibody is determined and compared with the level determined when the labeled antibody binds to the immobilized peptide in the absence of the candidate tau binding protein. If the level of the labeled antibody bound in the presence of the test tau binding protein is reduced compared to the case in the absence of the candidate tau binding protein, the candidate tau binding protein is considered to competitively inhibit the binding of the tau binding protein disclosed herein. Sequence No. 1 There is a possibility that it binds to a tau epitope corresponding to. In another embodiment, the candidate tau-binding protein is detectably labeled, and in a glass solution, the amino acid sequence is Sequence No. 1 In the presence or absence of a competitor peptide composed of Sequence No. 1 It binds to an immobilized tau protein or a fragment thereof containing... If the level of the bound candidate tau binding protein is reduced in the presence of the competitor peptide compared to the absence of the competitor peptide, the binding of the candidate tau binding protein to the tau protein is considered to be competitively inhibited by said competitor peptide. Sequence No. 1 There is a possibility that it specifically binds to the tau epitope corresponding to.

[0419] More generally, skilled individuals [are familiar with] tau binding protein-tau antigen binding kinetics, for example, Hearty et al. (2012), Methods Mol. Biol You will recognize that it can be evaluated by surface plasmon resonance as described in , 907:411-442.

[0420] Recombinant expression of genetically modified cells and tau-binding protein

[0421] Also disclosed herein is a genetically modified cell comprising any of an isolated nucleic acid or an expression vector encoding a tau-binding protein as disclosed herein. In some embodiments, the genetically modified cell is a mammalian cell, a yeast cell, a bacterial cell, an insect cell, a plant cell, or a filamentous fungal cell. In some preferred embodiments, the modified mammalian cell is a rodent cell or a human cell. Accordingly, also disclosed herein is a method for producing the tau-binding protein disclosed herein by culturing the genetically modified cell comprising an expression vector for the recombinant expression of a tau-binding protein for the recombinant expression of such protein, and then purifying / isolating the expressed protein. In some embodiments, the tau-binding protein disclosed herein was produced by culturing a hybridoma under conditions sufficient to produce the protein, as described herein and / or as known in the art.

[0422] Genetically modified host cells, for example E. coli, Yeast cells, insect cells, or mammalian cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce protein may be used to express and produce the tau-protein disclosed herein. Exemplary cells used to express the tau-binding protein are CHO cells, myeloma cells, or HEK cells. In other embodiments, the genetically modified host cell is a filamentous fungal cell, for example, Aspergillus oryzae ( Aspergillus oryzae ) originates from, for example, Huyn et al. (2020), Fungal Biology and Biotechnology As described in , doi.org / 10.1186 / s40694-020-00098-w, it can be particularly useful for the recombinant expression of antibodies at a low cost on a large scale.

[0423] Means for introducing isolated nucleic acids or expression constructs containing them into cells for expression are known to those skilled in the art. The techniques used for a given cell depend on known successful techniques. Means for introducing recombinant DNA into cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes, such as using Lipofectamine (Gibco, MD, USA) and / or Cellpectin (Gibco, MD, USA), PEG-mediated DNA absorption, electroporation and microparticle bombardment, such as using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA), among others.

[0424] Mammalian host cells used to produce proteins can be cultured in various media depending on the type of cell used. Commercially available media, such as Hem F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco Modified Eagle Medium (DMEM) (Sigma), are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein, for example, yeast or filamentous fungi, are known in the art.

[0425] Purification of tau binding protein

[0426] Methods for purifying binding proteins, e.g., antibodies, are known in the art and / or described herein.

[0427] In some embodiments where tau-binding proteins are secreted into the culture medium, the supernatant from such expression system may first be concentrated using a commercially available protein concentration filter, e.g., an Amicon or Millipore Pelicon ultrafiltration unit. A protease inhibitor, e.g. PMSF, may be included in any of the aforementioned steps to inhibit protein degradation, and an antibiotic may be included to prevent the growth of incidental contaminants. Alternatively, or additionally, the supernatant may be filtered and / or separated from the protein-expressing cells, e.g., using continuous centrifugation.

[0428] Tau-binding proteins prepared from cells may be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing. Such methods are known in the art and are described, for example, in WO99 / 57134 or in Ed Harlow and David Lane (editor) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Fishman and Berg, (2019), Cold Spring Harbor Protocols , also see doi:10.1101 / pdb.top099101.

[0429] Those skilled in the art will also recognize that proteins may be modified to include tags, e.g., poly-histidine tags, e.g., hexahistidine tags, or influenza virus hemagglutinin (HA) tags, or monkey virus 5 (V5) tags, or FLAG tags, or glutathione S-transferase (GST) tags, to facilitate purification or detection. The resulting proteins are subsequently purified using methods known in the art, such as affinity purification. For example, proteins containing a hexahistidine tag are purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds to a hexahistidine tag immobilized on a solid or semi-solid support, washing the sample to remove unbound proteins, and subsequently eluting the bound proteins. Alternatively, or additionally, ligands or antibodies that bind to the tag are used in the affinity purification method.

[0430] composition

[0431] Additionally, compositions are disclosed herein, comprising: (i) a tau-binding protein disclosed herein; (ii) an isolated nucleic acid encoding such a tau-binding protein (e.g., mRNA, mmRNA, or circ-RNA); (iii) a nonviral expression vector; (iv) a genetically modified cell as disclosed herein; or (v) a recombinant virus as disclosed herein; and a pharmaceutically acceptable carrier. In some preferred embodiments, the pharmaceutical composition for use in the therapeutic methods disclosed herein contains the tau-binding protein disclosed herein. The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” in this application refer to any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Such carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable carriers / excipients can be found in Remington's Pharmaceutical Sciences, Gennaro, AR, ed., 20th edition, 2000: Williams and Wilkins, PA, USA. Exemplary pharmaceutically acceptable carriers may include salts, e.g., mineral acid salts, e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids, e.g., acetate, propionate, malonate, benzoate, etc. For example, the compositions described herein may be provided in liquid form and may be formulated as saline-based aqueous solutions of physiological pH with or without a detergent, e.g., 0.01-1% polysorbate-80, or a carbohydrate additive, e.g., mannitol, sorbitol, or trehalose. Commonly used buffers include histidine, acetate, phosphate, or citrate. The infusion solution may contain 0 to 10% dextrose.

[0432] In some embodiments, the pharmaceutical composition disclosed herein further comprises one or more agents to facilitate the delivery of an activator, e.g., a tau-binding protein or a nucleic acid encoding a tau-binding protein, across the blood-brain barrier (BBB). Accordingly, in some embodiments, the pharmaceutical composition comprises lipid-shelled microbubbles or lipid-shelled nanobubbles. In some embodiments, the lipid-shelled microbubbles comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000 (DSPE-PEG2000). Regarding the generation, therapeutic formulation, and use of lipid-shelled microbubbles or lipid-shelled nanobubbles, particularly for the delivery of biotherapeutics across the BBB, e.g., Omata et al. (2020), Advanced Drug Delivery Reviews , 154-155:236-244; Yang et al. (2022); Interdisciplinary Materials It is known in the art as , doi.org / 10.1002 / idm2.12050; and as exemplified in international patent publications WO 2021 / 108861 and WO 2020 / 077380.

[0433] In some embodiments, the pharmaceutical composition disclosed herein is formulated for intravenous, intra-arterial, intrathecal, intraventricular, intracerebral, intranasal, intraperitoneal, intramuscular, or subcutaneous administration routes.

[0434] Formulation of (i) a tau-binding protein or other of the activator (ii) to (v) into a form suitable for administration to a subject (e.g., a pharmaceutical composition) is known in the art and includes, for example, methods as described in Remington's Pharmaceutical Sciences, Academic Press; 23rd edition (2020); and US Pharmacopeia: The National Formulary (2023). Rockville, MD: United States Pharmacopeial Convention, Inc.

[0435] The pharmaceutical compositions of the present disclosure are particularly useful for parenteral administration, e.g., intravenous, intra-arterial administration, or administration to the CNS (e.g., intrathecal route) or to the brain (e.g., intraventricular route). The compositions for administration commonly comprise a solution of tau-binding protein dissolved in a pharmaceutically acceptable carrier, e.g., an aqueous carrier. Various aqueous carriers, e.g., buffered saline, etc., may be used. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, e.g., pH adjustment and buffers, toxicity modifiers, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. In these formulations, the concentration of the tau-binding protein of the present disclosure may vary widely and will be selected primarily based on liquid volume, viscosity, body weight, level of intracerebral absorption, etc., depending on the specific mode of administration selected and the specific patient's needs (e.g., stage of disease). Exemplary carriers include water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. In some cases, non-aqueous vehicles, such as mixed oils and ethyl oleate, may also be used. The vehicle may contain small amounts of additives, such as buffers and preservatives, to improve isotonicity and chemical stability. Those skilled in the art will understand that the carrier for the pharmaceutical compositions disclosed herein will be selected based on the active agent to which it is formulated, and may differ from one another depending on the extent to which a specific active agent (e.g., genetically modified cells versus mmRNA; or recombinant viruses versus non-viral expression vectors) may require a specific stabilizer. Notwithstanding any specific differences in these formulations, each formulation is to be taken into account the selected route of administration. In vivo It is physiologically compatible for administration.

[0436] Various preclinical models for evaluating the therapeutic efficacy of the pharmaceutical compositions disclosed herein (e.g., those comprising the tau binding protein disclosed herein or a recombinant virus for the expression of the tau binding protein) are known in the art. Numerous animal models for tauopathy are known, for example, Ittner et al. (2008), Proc. Natl. Acad. Sci A “K3” transgenic mouse model expressing human tau carrying the frontotemporal dementia (FTD)-associated tau K369I mutation as described in USA, 105(41):15997-16002; a dual transgenic lycogenic mouse expressing human tau combined with a 5xFAD mouse as a model of Alzheimer's disease (Barendrecht et al., 2023, Alzheimer's Research & Therapy Useful tests for evaluating validity include spatial learning and memory tests (e.g., Morris water maze), motor behavior tests (e.g., using the rotarod test), and biochemical and immunocytochemical evaluations of tau hyperphosphorylation.

[0437] How to use

[0438] Also disclosed herein is a method for preventing or treating tauopathy in a subject in need, said method comprising the step of administering to said subject a therapeutically effective amount of a pharmaceutical composition comprising (i) a tau-binding protein disclosed herein; (ii) an isolated nucleic acid encoding such a tau-binding protein (e.g., mmRNA or circ-RNA); (iii) a nonviral expression vector (e.g., a plasmid construct); (iv) a genetically modified cell as disclosed herein; or (v) any of a recombinant virus as disclosed herein. Also disclosed herein is a method for preventing or inhibiting the spread of tau protein aggregates in the central nervous system (CNS) of a subject identified as in need, said method comprising the step of administering to said subject a prophylactic amount of said pharmaceutical composition. In some embodiments, said subject is a rodent, e.g., a mouse, e.g., a genetically modified mouse or rat model of tauopathy. In other embodiments, said subject to be treated is a non-human primate. In some preferred embodiments, said subject to be treated is a human subject.

[0439] Those skilled in the art will understand that any of the therapeutic agents and related compositions disclosed herein (e.g., the tau-binding proteins disclosed herein) may be used in the manufacture of agents for the prevention or treatment of tauopathy. In other embodiments, the composition is used in the manufacture of agents for the diagnosis or prognosis of tauopathy. In other embodiments, the composition is intended for use in the manufacture of agents for preventing or inhibiting the spread of tau protein aggregates in the central nervous system (e.g., brain) of a subject requiring such agents. In some preferred embodiments, the pharmaceutical composition to be administered comprises the tau-binding proteins disclosed herein. In other embodiments, the pharmaceutical composition comprises a nucleic acid encoding the tau-binding proteins disclosed herein and is in a form operable to be expressed in cells typically found in the mammalian brain (e.g., neurons, astrocytes, and / or microglia).

[0440] In some embodiments of the method, where the route of administration of the pharmaceutical composition is a systemic administration route (e.g., parenteral administration route), particularly intravenous or intra-arterial administration, the method also includes a step of temporarily enhancing BBB permeability before, during, or after administration of the pharmaceutical composition or activator. In some embodiments, the temporary BBB permeability includes a scanning ultrasound temporary permeability step. In some preferred embodiments, the scanning ultrasound temporary permeability is used in combination with lipid-shelled microbubbles or nanobubbles, for example, DSPE-PEG2000 microbubbles pre-formulated in the pharmaceutical composition to be administered or administered individually. Suitable methods for scanning ultrasound temporary BBB permeability are described, for example, in International Patent Application Publication WO 2021 / 108861.

[0441] In an embodiment combining scanning ultrasound penetration with the use of lipid-shelled microbubbles or lipid-shelled nanobubbles, penetration is achieved by mechanical interaction between the microbubbles or nanobubbles and the blood vessel wall as pulsed focused ultrasound is applied, which results in compression and rarefaction cycles of the microbubbles. This induces the transient disruption of tight junctions and the absorption of blood-derived factors by the brain. Microbubbles within a target volume are "acoustically activated" by what is known as acoustic cavitation. In this process, the microbubbles expand and contract over several cycles in response to negative pressure rarefaction and compression.

[0442] In some embodiments, microbubbles or nanobubbles are provided as a continuous infusion. In other embodiments, they are provided as a single bolus dose. In some preferred embodiments, a continuous infusion of microbubbles pre-formulated with an activator, such as the tau-binding protein disclosed herein, is provided individually or during the period of acoustic energy application. In some embodiments, the microbubble agent is administered intravenously via systemic circulation. The delivery of acoustic energy may include the delivery of acoustic energy from an acoustic energy source, such as a focused ultrasound transducer, to deliver transcranial ultrasound. For example, Kee et al., (2023), Scientific Reports , 6963; doi.org / 10.1038 / s41598-023-33671-5 Refer to Various ultrasound parameters can be manipulated to increase the permeability of the blood-brain barrier, and these include pressure amplitude, ultrasound frequency, burst length, pulse repetition frequency, focal spot size, and focal depth.

[0443] Increased permeability of the blood-brain barrier can be determined by any suitable imaging method. Preferably, the imaging method is MRI, optical imaging method, positron emission tomography (PET), computed tomography (CT) or computed axial tomography (CAT) or ultrasound.

[0444] tauopathy

[0445] The method described above is useful for the prevention and / or treatment of a wide range of tauopathy based on their common etiology, which is underpinned by the seeding, aggregation, and proliferation of pathological forms of tau aggregates ("seeds") throughout the brain and CNS by prion-like mechanisms. Accordingly, in some embodiments, the subject to be treated suffers from a tauopathy selected from the group consisting of Alzheimer's disease, frontotemporal dementia, Pick's disease, chronic traumatic encephalopathy, progressive supranuclear palsy, Parkinson's disease, Parkinson's disease-related disorders, primary age-related tauopathy, silicomeric granulosa disease, and spherocytic tauopathy. In some embodiments, the subject to be treated suffers from Alzheimer's disease or frontotemporal dementia.

[0446] Diagnostic criteria and prognostic approaches for various tauopathy are, for example, Riley et al. (2022), Journal of Neuroimaging As reviewed at , doi.org / 10.1111 / jon.13001, this is known in the art. Generally, the diagnosis of tauopathy will utilize a combination of behavioral / psychological measurements, neuroimaging, and molecular biomarker assays. In particular, brain tau-PET imaging using ligands that specifically target tau protein is particularly useful for the diagnosis and prognosis of tauopathy. For example, Petersen et al., (2022), Current Opinion in Neurology , 35(2):230-239 Refer toNeuroimaging methods, such as tau-PET imaging, are particularly useful for evaluating the sustained response and progression of a subject during and after any of the prophylactic and therapeutic regimens disclosed herein.

[0447] Combination therapy

[0448] In some embodiments, the tau-binding protein of the present disclosure is administered in combination with other therapeutic agents useful for treating the pathological conditions described herein as a combined or additional therapeutic step or as an additional component of a therapeutic formulation.

[0449] For example, other therapeutic agents are antibodies against amyloid-β fibrils, for example, the humanized IgG1 lecanemab "Leqembi" (Eisai) (CAS 1260393-98-3), and the monoclonal antibody donanemab "Kisunla" (Eli Lilly) (CAS 1931944-80-7), approved in the United States for the treatment of Alzheimer's disease. Alternatively, or additionally, other compounds are immunosuppressants. In other embodiments, other therapeutic agents for use in combination with the compositions disclosed herein are cholinesterase inhibitors, for example, donepezil (Aricept), galantamine (Razadyne), or rivastigmine (Exelon). Alternatively, or additionally, additional therapeutic agents are, for example, Jahreis et al., (2023), Alzheimer's & Dementia These are serotonin receptor 7 (5-HT7R) inverse agonists, such as amisulpride, which have been shown to reduce tau hyperphosphorylation as described in doi.org / 10.1002 / alz.13090. Alternatively, or in addition, other therapeutic agents are inhibitors of RIPK3, which have recently been shown to play a role in neuronal necroptosis in Alzheimer's disease (Balusu et al., 2023). Science, 381(6663):1176-1182). Alternatively, or additionally, another therapeutic agent is the tau aggregation inhibitor 5-(1,3-benzodioxol-5-yl)-3-(3-bromophenyl)-1H-pyrazole (anle138b).

[0450] In another embodiment, additional therapeutic agents are Mummery et al., (2023), Nature Medicine As described in , 29:1437-1447), antisense oligonucleotides (ASOs) for tau mRNA, e.g., BIIB080 (IONIS Pharmaceuticals), are mycophenolate mofetil. Alternatively, or in addition, other therapeutic agents are non-steroidal anti-inflammatory drugs (NSAIDs) such as tolfenamic acid, which can reduce total tau levels (Chang et al., 2018, Current Alzheimer Research , 15:549-562).

[0451] Dosage and time of administration

[0452] The appropriate dosage of the tau-binding protein of the present disclosure or a preparation encoding such tau-binding protein (e.g., a recombinant virus) will vary depending on the specific tau-binding protein, the condition to be treated, and / or the subject being treated. Determining the appropriate dosage is within the capacity of a skilled physician and is accomplished, for example, by starting with a suboptimal dosage and gradually changing the dosage to determine the optimal or useful dosage. Alternatively, to determine an appropriate dosage for treatment / prevention, cell culture assays or animal study data are used, and the appropriate dosage is the ED of the active compound with little to no toxicity. 50It is a range of circulating concentrations including. The dosage may vary within this range depending on the adopted administration form and the route of administration utilized. The therapeutically or prophylactically effective dose can be initially estimated from cell culture assays, particularly utilizing human cell 2D or 3D culture models (e.g., patient hiPSC-derived models). The dose is IC as determined from cell culture. 50 It can be formulated in animal models to achieve a range of circulating plasma concentrations including (i.e., the concentration or amount of the compound that achieves semi-maximal suppression of symptoms). This information can be used to more accurately determine a useful dose in humans. Levels in plasma can be measured, for example, by high-performance liquid chromatography.

[0453] In some preferred embodiments, the method of the present disclosure comprises the step of administering a prophylactic or therapeutically effective amount of the protein described herein.

[0454] In the context of the methods disclosed herein, the term “therapeutic effective dose” refers to an amount of therapeutic agent (e.g., tau-binding protein or an expression vector encoding tau-binding protein or a recombinant virus) that, when administered to a subject requiring treatment, improves and / or improves the subject’s prognosis and / or condition, or reduces or suppresses one or more symptoms of the clinical conditions described herein to a level below that observed and accepted as a clinical diagnosis or clinical characteristic of said condition. The amount to be administered to the subject will vary depending on the specific characteristics of the condition to be treated, the type and stage of the condition being treated, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and body weight. A person skilled in the art will be able to determine an appropriate dose based on these and other factors. Accordingly, this term should not be interpreted as limiting the present disclosure to a specific amount, e.g., of protein(s), nucleic acid, or recombinant virus; rather, the present disclosure encompasses any amount of tau-binding protein, encoding nucleic acid or vector, or genetically modified cell that secretes the recombinant tau-binding protein, sufficient to achieve the results mentioned in the subject.

[0455] As used herein, the term “prophylactic effective dose” should be understood to mean an amount of protein sufficient to prevent, suppress, or delay the onset of one or more detectable symptoms of a clinical condition. Those skilled in the art will recognize that such amount may vary, for example, depending on the specific tau-binding protein(s) administered and / or the specific subject and / or the type, severity, or level of the condition and / or predisposition (genetic or otherwise) to the condition. Accordingly, this term should not be interpreted as limiting the present disclosure to specific amounts of the disclosed tau-binding protein and related compositions (e.g., encoding nucleic acids); rather, the present disclosure encompasses any amount of such preparations to achieve a clinically significant prophylactic effect in the subject.

[0456] In some embodiments, the route of administration used in the treatment, prevention, diagnosis, or prognosis method disclosed herein is intravenous, intra-arterial, intrathecal, intracerebral, intraventricular, intranasal, intramuscular, or subcutaneous.

[0457] In some embodiments, when the tau-binding protein disclosed herein is administered systemically, a suitable dose is about 1 mg / kg to about 100 mg / kg of the individual's body weight per day, e.g., 2.5 mg / kg, 5.0 mg / kg, 10 mg / kg, 15 mg / kg, 22 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, or other dose ranges of about 1 mg / kg to about 100 mg / kg. In some embodiments, when the route of administration is intrathecal, intracerebral, or intracerebrovascular, the dose is in a lower range of about 0.1 mg / kg to about 20 mg / kg to reflect the higher relative dose reaching the brain due to the more direct route of administration. In the case of repeated administration over several days or longer, treatment may continue until the desired symptom suppression is achieved, depending on the severity of the tauopathy-related disease or disorder to be treated.

[0458] In some examples, the tau-binding protein is administered systemically at an initial (or loading) dose between about 1 mg / kg and about 50 mg / kg, e.g., about 1 mg / kg to about 10 mg / kg, or about 1 mg / kg, about 2 mg / kg, or 5 mg / kg. The tau-binding protein may then be administered at a lower maintenance dose of 10% to about 50% of the initial ("loading") dose, e.g., 12%, 15%, 20%, 25%, 30%, 35%, 40%, or another lower maintenance dose of about 10% to about 50% of the initial dose. The maintenance dose may be administered every 7 to 30 days, e.g., every 10 to 15 days, e.g., every 10 or 11 or 12 or 13 or 14 or 15 days.

[0459] In some embodiments, where the pharmaceutical composition comprises a recombinant virus for expressing a tau-binding protein as disclosed herein, for example, the dose by intravenous administration is about 1 x 10⁻⁶ 12 Viral genome / kg (VG / kg) to about 2 x 10⁶ 14 VG / kg, for example, 1.5 x 10 12 VG / kg, 1.7 x 10 12 VG / kg, 2 x 10 12 VG / kg, 2.5 x 10 12 VG / kg, x 10 13 VG / kg, 3 x 10 12 VG / kg, 4 x 10 12 VG / kg, 5, x 10 12 VG / kg, 7 x 10 12 VG / kg, 8 x 10 12 VG / kg, 9 x 10 12 VG / kg, 1 x 10 13 VG / kg, 2 x 10 13 VG / kg, 3 x 10 13 VG / kg, 4 x 10 13 VG / kg, 5 x 10 13 VG / kg, 6 x 1013 VG / kg, 7 x 10 13 VG / kg, 8 x 10 13 VG / kg, 9 x 10 13 VG / kg, 1 x 10 14 VG / kg, 1.5 x 10 14 VG / kg, or about 1 x 10⁻⁶ 12 VG / kg to about 2 x 10 14 There may be different dose ranges of VG / kg. In some preferred embodiments, the recombinant virus in the pharmaceutical composition is a recombinant AAV for the expression of scFv encoding a tau binding protein.

[0460] In some embodiments, the dose is administered at periodic intervals, for example, every week, every two weeks, every three weeks, once a month, once every two months, or at other intervals from about every week to about two months. In some embodiments, a treatment period lasting from about two months to about 24 months covering one of the above administration intervals is, for example, 3 months, 4 months, 5 months, 6 months, 8 months, 9 months, 10 months, 12 months, 16 months, 20 months, 22 months, or other treatment periods from about two months to about 24 months.

[0461] For subjects who do not respond adequately to treatment after the treatment period, a second treatment period may be initiated and / or an increased dosage regimen may be used.

[0462] In some embodiments where the subject experiences an adverse reaction, the initial (or loading) dose may be divided over several days or consecutive days within a week.

[0463] The administration of a pharmaceutical composition for providing a tau-binding protein according to the method of the present disclosure may be continuous or intermittent, for example, depending on the physiological condition of the recipient, whether the purpose of administration is prophylactic or therapeutic, and other factors known to a skilled practitioner. In some embodiments, the administration of the tau-binding protein is essentially continuous for a predetermined period of time. In other embodiments, the administration is provided in a series of intervald doses, for example, during or after the onset of tauopathy.

[0464] Tau and Tau Aggregate Detection Test

[0465] Also disclosed herein is a method for determining the level of tau or extracellular tau aggregates in a subject, said method comprising, under conditions causing specific binding of the tau binding protein to the tau protein present in the biological sample, the biological sample from said subject and the tau binding protein disclosed herein ex vivo The method includes the step of contacting (e.g., by a third-party provider) and determining the level of tau protein bound to the tau binding protein. Suitable examples of biological samples in which the level of tau or tau aggregates is quantified include, but are not limited to, cerebrospinal fluid (CSF), plasma, serum, urine, rhinorrhea, tear fluid, or postmortem brain extract.

[0466] The following assay may be performed using the tau-binding proteins of the present disclosure, for example, tau-binding proteins conjugated to detectable labels as discussed herein. Detection of tau or tau aggregates using the assays described herein is particularly useful for diagnosing tauopathy or predicting its prognosis.

[0467] Immunoassays are a typical assay format for diagnosing pathological conditions in subjects or detecting tau and tau aggregates in samples. The present disclosure considers any form of immunoassay including Western blotting, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), competitive assay, radioimmunoassay, lateral flow immunoassay, penetrating immunoassay, electrochemiluminescence assay, turbidity measurement-based assay, turbidity measurement-based assay, and fluorescence-activated cell sorting (FACS)-based assay.

[0468] One suitable form of immunoassay is, for example, ELISA or FLISA.

[0469] In some embodiments, this assay involves immobilizing the tau-binding protein of the present disclosure on a solid matrix, e.g., polystyrene or polycarbonate microwells or dipsticks, membranes, or glass supports (e.g., glass slides). The test sample is then brought into direct contact with the tau-binding protein to bind or capture tau or tau aggregates within the sample. After washing to remove any unbound proteins within the sample, the tau-binding protein binding to tau at individual epitopes is brought into direct contact with the captured tau or tau aggregates. These "detection proteins" are generally labeled with a detectable reporter molecule, e.g., an enzyme (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), or β-galactosidase) in the case of ELISA, or a fluorescent dye in the case of FLISA. Alternatively, a second labeled protein binding to the detection protein may be used. After washing to remove any unbound proteins, the detectable reporter molecule is detected in the case of ELISA by the addition of a substrate, e.g., hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indole-beta-D-galatopyranoside (x-gal). Those skilled in the art will know that the immobilized (captured) protein and the detection protein described above may be used in the opposite manner, namely, the detectably labeled tau-binding protein of the present disclosure ( Sequence No. 1 It will be understood that this is used to detect tau or tau aggregates captured by immobilized tau-binding proteins that bind to an alternative epitope.

[0470] The level of tau antigen in a sample is determined by using a standard curve generated with known amounts of markers or by comparison with a control sample.

[0471] The test described above is easily modified to use chemiluminescence or electrochemiluminescence as the basis for detection.

[0472] As will be obvious to those skilled in the art, it will be apparent that other detection methods based on immunosorbent assays are useful for carrying out the present disclosure. For example, there are immunosorbent methods based on the above description that use a radiolabel for detection, use a gold label (e.g., colloidal gold) for detection, use a liposome encapsulating NAD+ for detection, or use an acridinium-binding immunosorbent assay.

[0473] In some examples of the present disclosure, the level of the tau binding protein-tau antigen complex is measured by a surface plasmon resonance detector, such as that described in US7205159 (e.g., BIAcore TM , GE Healthcare, Piscataway, NJ), through-flow device; micro or nano immunoassay device (e.g., as described in US20030124619); lateral flow device (e.g., as described in US20040228761 or US20040265926); fluorescence polarization immunoassay (FPIA, e.g., as described in US4593089 or US4751190); or immunoturbidity assay (e.g., as described in US5571728 or US6248597) is determined using.

[0474] In another embodiment, the detection of tau or tau aggregates is In vivo Accordingly, the detectably labeled tau-binding protein of the present disclosure is administered to a subject to be diagnosed or to be predicted for prognosis, and the administered detectably labeled tau-binding protein bound to extracellular tau or aggregated tau is detected at one or more locations of the subject (e.g., within the brain).

[0475] In some embodiments, tau or tau aggregates In vivoDetection is performed by administering a suitable radiolabeled tau-binding protein of the present disclosure to a subject and subsequently performing PET imaging to detect tau or tau aggregate-associated labels. In some embodiments, the tau-binding protein is N-succinimidyl-4-[ 18 It is radiolabeled with [F]fluorobenzoate. In some embodiments, the tau-binding protein is, for example, Banka et al., (2023), Frontiers in Nuclear Medicine As described in , doi 10.3389 / fnume.2023.1173693, a fusion antibody containing a domain that facilitates transport across the BBB, for example, can be provided as a fusion of a tau binding protein and a transferrin receptor (TfR) antibody.

[0476] In some embodiments, the above-mentioned ex vivo or In vivo The detection method is within or in biological samples or In vivo Aggregated tau protein levels are useful for identifying a subject as having tauopathy or determining the progression of tauopathy for one or more threshold levels or ranges (e.g., by a party different from the party performing the detection assay). Accordingly, such methods or associated compositions disclosed herein may be used to diagnose or predict the prognosis of tauopathy selected from the group consisting of Alzheimer's disease, frontotemporal dementia, Pick's disease, chronic traumatic encephalopathy, progressive supranuclear palsy, Parkinson's disease, Parkinson's disease-related disorders, primary age-related tauopathy, silicoaffin granulosa disease, and spherocytic tauopathy. In some embodiments, the tauopathy is Alzheimer's disease. In a preferred embodiment, the subject is a human subject.

[0477] Diagnostic and Prognostic Thresholds

[0478] As is obvious to those skilled in the art, some of the examples described herein require some degree of quantification to determine the levels of tau or tau aggregates. Such quantification for reference threshold levels can be determined by including a suitable control sample in the assay of the present disclosure.

[0479] In some embodiments, a suitable control sample is a sample derived from a healthy subject or a normal subject. In the context of the invention, the term “healthy subject” is considered to mean an individual known not to suffer from tauopathy, e.g., Alzheimer’s disease, or to have an above-average risk of developing it.

[0480] The term "normal subject" is considered to mean an individual having normal levels of extracellular tau or tau aggregates compared to a population of healthy individuals of matching age, preferably.

[0481] The present disclosure also considers a control sample as a data set obtained from normal and / or healthy subjects or a population of normal and / or healthy subjects.

[0482] In some embodiments, the diagnostic or prognostic method of the present disclosure additionally includes or determines the level of tau or tau aggregates in a control sample, for example, using the method described herein.

[0483] In some embodiments, the levels of tau or tau aggregates in the subject or subject sample being evaluated and the reference control subject or subject sample are determined at approximately or substantially the same time.

[0484] In some preferred embodiments, where the level of tau or tau aggregates in a biological sample is to be determined, the sample from the subject and the control sample are tested using the same method to enable a meaningful comparison of the results.

[0485] Kit

[0486] The present disclosure further comprises a kit containing one or more of the following:

[0487] (i) isolated tau binding protein of the present disclosure, expression construct(s), or recombinant virus encoding the same;

[0488] (ii) cells of the present disclosure; or

[0489] (iii) Pharmaceutical composition of the present disclosure.

[0490] In the case of a kit for detecting tau or tau aggregates, the kit may further include, for example, a detection means connected to a tau binding protein of the present disclosure.

[0491] For kits intended for therapeutic or preventive use, the kit may additionally include a pharmaceutically acceptable carrier.

[0492] Optionally, the kit of the present disclosure is packaged with instructions for use in the method described herein according to any example.

[0493] The present disclosure includes the following non-limiting embodiments.

[0494] Examples

[0495] Example 1: Materials and Method

[0496] Preparation of recombinant human tau and library panning

[0497] Recombinant whole-length human tau-441 (hTau-441) was prepared as previously described (Liu et al. 2016). Purified recombinant hTau-441 was panned against the human single-dip scFv library Tomlinson I + J. Immunotubes (Nunc) were coated with 20 μg / ml hTau-441 in PBS and left overnight at 4°C. The next day, the tubes were washed three times with PBS and then blocked with 2% skim milk powder (MPBS) in PBS for 2 hours at room temperature. The library (1012 phage) in skim milk powder (MPBS) in PBS was added to the coated tubes and incubated at room temperature for 1 hour while rotating, followed by leaving for 1 hour. Unbound phages were removed by washing 10 times with PBS-T (0.1% Tween 20), and subsequently, bound phages were eluted by spinning with 500 μl of 1 mg / ml trypsin in PBS at room temperature for 10 minutes. Cultured in 2YT (16 g tryptone, 10 g yeast extract, and 5 g NaCl in 1 L of distilled water) E. coli XL1-Blue Cell (Agilent)([OD] 600= 0.4) was infected with 250 μl of eluted phage and incubated in a 37°C water bath for 30 minutes. To measure phage titer, 10 μl of infected XL1-Blue cells were serially diluted, and 10 μl of each dilution was plated onto TYE plates containing 100 μg / ml ampicillin and 1% glucose (15 g Bactoagar, 8 g NaCl, 10 g tryptone, 5 g yeast extract in 1 L of distilled water). The remaining infected E. coli were plated onto TYE plates containing 100 μg / ml ampicillin and 1% glucose. All plates were incubated overnight at 37°C. To amplify the phages, bacteria on the plate were collected by cell scraping within 2 ml of 2YT, and 50 μl of the resuspended bacteria were used to inoculate 50 ml of 2YT containing 100 μg / ml ampicillin and 1% glucose. The culture [OD] 600 Incubation was performed until the value reached 0.4. KM13 helper phage (5 x 10¹⁰) was added to 10 ml of culture and incubated in a 37°C water bath for 30 minutes. Subsequently, the culture was centrifuged at 3,000 xg for 10 minutes, and the pellet was resuspended in 50 ml of 2YT to which 100 μg / ml ampicillin, 50 μg / ml kanamycin, and 0.1% glucose were added. The culture was incubated overnight at 30°C with shaking at 200 rpm. The phage was purified from the overnight culture by centrifugation at 3,000 rpm for 15 minutes, and then incubated on ice with PEG / NaCl (20% polyethylene glycol 6000, 2.5 M NaCl) for 1 hour. The samples were centrifuged at 3,300 xg for 30 minutes, and the pellet was resuspended in 2 ml of PBS prepared for the next panning. A total of three pannings were performed. Polyclonal phages binding to hTau-441 were determined by indirect ELISA.

[0498] scFv preparation for monoclonal ELISA

[0499] HB2151 E. coli Cell ([OD] 600 10 μl of phages eluted from the last selection was inoculated into (= 0.4) and incubated in a 37°C water bath for 30 minutes. Serial dilutions were performed with 10 μl of infected HB2151 cells, and 10 μl of each dilution was dropped onto TYE plates containing 100 μg / ml ampicillin and 1% glucose and incubated overnight at 37°C. From the plates incubated overnight, 20 colonies were selected for each library and inoculated into 2YT plates containing 100 μg / ml ampicillin and 1% glucose in 96-well plates and incubated overnight at 37°C and 250 rpm. Subsequently, the cultures incubated overnight were used to inoculate fresh 2YT plates containing 100 μg / ml ampicillin and 0.1% glucose, and 0D 600 Incubation was performed until = 0.9. scFv expression was induced with IPTG at a final concentration of 1 mM and incubated overnight at 30°C with shaking at 250 rpm. Plates were centrifuged at 1,800 xg for 10 minutes, and the supernatant was collected. Subsequently, scFv binding to recombinant hTau-441 was tested on the supernatant by ELISA. Positive clones were sequenced using the following primers: Forward 5'- CGA CCC GCC ACC GCC GCT G-3'( Sequence number 19 ) and reverse 5'- CTA TGC GGC CCC ATT CA-3'( Sequence number 20 ).

[0500] Refining and Western Blotting of scFv

[0501] RNJ1 and RNJ15 scFvs were expressed in HB2151 cells and purified from periplasm as previously described (Nisbet et al., 2017). Western blotting was performed to further verify scFv binding to tau. Recombinant hTau-441 (80 ng) was electrophoresed on a 10% Tris-glycine gel (Thermo Fisher) and then transferred to a nylon membrane. The membrane was blocked in 5% skim milk powder in TBS (M-TBST) containing 0.1% Tween 20, and then incubated overnight at 4°C with purified scFv (1:20) in 2.5% M-TBST. Subsequently, scFv binding was detected for 1 hour using an anti-Myc-HRP conjugate antibody (Cell Signaling Technologies) in 2.5% M-TBST. The blots were developed using chemiluminescence.

[0502] production of mouse IgG

[0503] The variable domain of RNJ1, a clone that showed the best binding to hTau-441, was individually cloned into a mammalian expression vector containing the mouse IgG1 heavy chain and kappa light chain constant regions using InFusion Cloning (National Biologics Facility, Queensland node). RNJ1 IgG was expressed in ExpiCHO cells and purified using Protein G (National Biologics Facility, Queensland node).

[0504] Example 2: Isolation of Tau Antibody RNJ1 from Human Phage Display Library and Epitope Mapping

[0505] To isolate tau-specific antibodies, a library of Tomlinson I + J human synthetic single-fold short-chain variable fragments (scFvs) was panned multiple times against full-length human recombinant tau (hTau-441) to obtain enrichment of RNJ1-positive scFvs (Figs. 1a-d). These scFvs bound to recombinant hTau-411 (the longest human brain tau subtype) by ELISA and Western blot analysis (Figs. 1d,e). To further characterize RNJ1 and determine its preclinical efficacy, its variable heavy chain and variable light chain were reformatted into a mouse IgG1 backbone containing a kappa light chain.

[0506] Using sequentially cleaved recombinant hTau-441 (Fig. 2a), the RNJ1 epitope was mapped by indirect ELISA, and the epitope of the clinically tested tau antibody HJ8.5 (Yanamandra et al. 2015) was identified, cloned, and purified for benchmarking purposes. Both RNJ1 and HJ8.5 exclusively bound to the tau form containing the first 44 amino acids (aa); however, while RNJ1 had a binding site within aa 1-22, HJ8.5 recognized a variant containing aa 23-44, consistent with the reported epitope (aa 25-30) (Fig. 2b, c) (Yanamandra et al. 2015).

[0507] Next, to map the RNJ1 epitope with higher resolution, a library of overlapping synthetic peptides spanning aa 1–22 with peptide lengths of 10 aa and offsets of 2 aa was used (Fig. 2d). The peptides were conjugated to bovine serum albumin (BSA) and used as antigens in an indirect ELISA. Maximum binding was detected for the peptides spanning aa 9–18 and 11–20, while residual binding was observed for aa 13–22. Similarly, surface plasmon resonance (SPR) using BSA-conjugated peptides as ligands and RNJ1 as an analyte showed maximum binding for aa 9–18 and 11–20, partial binding for aa 13–22, and almost no detectable signal for the remaining peptides (Fig. 3). In summary, these results show that RNJ1 binds to the tau segment spanning aa 9-22.

[0508] In addition, considering that tyrosine 18 (Y18) is a substrate of Fyn and that phosphorylation of this epitope is found in tauopathy and animal models of tauopathy (Bhaskar et al. 2010; Li et al. 2017), RNJ1 reactivity was tested for synthetic peptides aa 9-18, 11-20, and 13-22 phosphorylated at Y18. However, no binding was detected, suggesting that RNJ1 binds to the segment of aa 9-22 that is not phosphorylated at Y18 (Fig. 2e).

[0509] Example 3: RNJ1 exhibits stronger reactivity to aggregated tau than HJ8.5 and RNF5.

[0510] Considering that epitope mapping reveals adjacent epitopes for RNJ1 (aa 9-22) and HJ8.5 (aa 25-30) within the amino-terminal domain of tau, we investigated whether these differences affect the binding profiles for monomeric and aggregated tau. The semi-maximum binding concentration (EC50) was estimated via indirect ELISA using recombinant hTau-441 in monomeric form as an antigen, and the EC50 of HJ8.5 (0.09 nM, 95% CI: 0.077-0.093 nM) was approximately 5 times lower than that of RNJ1 (0.51 nM, 95% CI: 0.46-0.57 nM) (Fig. 2f). Binding kinetic constants were measured in a multicycle kinetic SPR assay using monomer hTau-441 as the ligand and two antibodies at various concentrations as analytes. Based on the SPR sensogram, the antibodies showed good agreement with the divalent analyte model, and HJ8.5 exhibited a higher affinity (equilibrium dissociation constant, KD = 1.8 nM) for monomer tau than RNJ1 (KD = 19.15 nM) (Fig. 2g).

[0511] To evaluate binding to aggregated tau, ELISA plates were coated with sarcosyl-insoluble tau obtained from P301L mutant tau-transformed rTg4510 mice (19) and human AD brain lysates, and RNF5, a third amino-terminal antibody exhibiting specificity for aa 35-44 as previously described, was tested in addition to RNJ1 and HJ8.5 (20). Both HJ8.5 and RNF5 reached binding saturation at low antibody concentrations, whereas RNJ1 did not reach saturation within the tested concentration range and consequently showed a significantly higher maximum response to sarcosyl-insoluble tau from both mouse and human brains (Fig. 1h). To gain further insight into the binding characteristics of antibodies to aggregated tau, SPR was performed using sarcosyl-insoluble rTg4510 tau as the ligand and the antibody as the analyte. Although the structural complexity of aggregated tau excludes deriving binding kinetic constants with high reliability in SPR, it was evident from the sensogram that RNJ1 generated a much higher binding signal than HJ8.5 and RNF5 at equivalent antibody concentrations (Fig. 1i), which reflects the higher reactivity of RNJ1 to aggregated tau determined by indirect ELISA (Fig. 1h). Taken together, the differential binding profile of RNJ1 to aggregated tau determined by ELISA and SPR suggested that RNJ1 may have a higher ability to neutralize template aggregation induced by proteopathic tau seeds.

[0512] Example 4: RNJ1 is In a test tube It demonstrates remarkable efficiency in neutralizing protein-pathogenic tauside.

[0513] In AD, pathogenic tau possesses seeding ability (Dujardin et al. 2020), and since RNJ1 showed increased responsiveness to aggregated tau compared to HJ8.5 and RNF5, we investigated whether RNJ1 possesses a higher ability to neutralize tau seeds in a biosensor system. To this end, we adopted the widely used tau RD P301S FRET HEK293 (human embryonic kidney) cells, which co-express the microtubule-binding repeat domain (RD) of the P301S mutant tau fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). In this system, the internalization of exogenous tau seeds induces the aggregation of recombinant RD tau expressed by the cells, generating a Foerster resonance energy transfer (FRET) signal that can be measured by microscopy or flow cytometry (Holmes et al. 2014).

[0514] To evaluate the effect of antibody treatment on tau inclusion body formation, HEK293 biosensor cells were treated with sarcosyl-insoluble rTg4510 tau seeds in the presence or absence of antibodies (Fig. 4a), and FRET signals were measured by flow cytometry. Antibodies were tested at concentrations ranging from 10 to 300 nM using 200 ng / ml of tau seeds. When comparing the effects of antibodies on tau-seeded agglutination, RNJ1 treatment was most effective in reducing the mean integrated fluorescence intensity (IFI) of the FRET signal at all tested concentrations, reducing the IFI to approximately 30% of the 'antibody-free' control at the highest antibody concentration of 300 nM, compared to approximately 42% for HJ8.5 and 70% for RNF5 (Figs. 4a-c).

[0515] Neutralization of tau seeding was evaluated in a more neuronal-like model using a complementary FRET-biosensor system established in human SH-SY5Y neuroblastoma cells. Once again, RNJ1 demonstrated superior neutralizing ability, reducing the IFI of the FRET signal at the highest antibody concentration tested (300 nM) to ~29% of the 'no antibody' control group, compared to ~45% and ~55% for HJ8.5 and RNF5, respectively (Fig. 2d). The mean IFI was consistently lower for RNJ1 than for HJ8.5 across all tested concentrations, but the difference achieved statistical significance only at 100 nM. Notably, RNJ1 was significantly superior to RNF5 across all four tested concentrations.

[0516] We subsequently investigated whether antibodies could neutralize AD brain-derived tau seeds. The HEK293 biosensor was treated with sarcosyl-insoluble AD tau (Fig. 4e). Treatment with 100 nM RNJ1 reduced seeding intensity to nearly 60% of the seeding detected in the 'antibody-free' control, whereas inhibition by an equivalent dose of HJ8.5 achieved only a reduction of 88%. Treatment with RNJ1 achieved significantly higher seeding inhibition against HJ8.5 at both 20 nM and 100 nM concentrations. Taken together, these results demonstrate that RNJ1 exhibits a superior ability to neutralize tau seeds obtained from both tauopathy mouse models and human AD brain tissue. Importantly, since the three tested antibodies bind to amino-terminal epitopes that are lacking in the tau RD constructs expressed in biosensor cells, the observed seeding inhibition appears to occur by binding to exogenous tau seeds rather than the RD tau expressed by biosensor cells.

[0517] Example 5: RNJ1 treatment improves motor function in K3 mouse model of tauopathy

[0518] We established that HJ8.5 and RNJ1 differ in binding profiles and neutralizing abilities for seed-eligible tau, and subsequently compared their efficacy alone and together in a longitudinal study using the K369I frontotemporal dementia-mutant tau transgenic K3 tauopathy mouse model (Ittner et al. 2008). K3 mice exhibit distinct motor dysfunction with tau pathology and an early onset age of 4 weeks, which manifests as a lack of motor ability and coordination, for example, as evaluated in the rotarod test (Pandit et al. 2019).

[0519] For treatment group assignment, 48 K3 mice and 12 age-matched wild-type (WT) littermates (equal sex ratio) were subjected to a rotarod baseline test at 4 weeks of age and subsequently assigned to five treatment groups (Fig. 5a) based on equivalent mean performance: K3 mice treated with RNJ1 (K3RNJ1), HJ8.5 (K3HJ8.5), antibody combination (K3R+H), and vehicle (K3Veh), and vehicle-treated WT mice (WTVeh). Mice were administered 14 weekly intraperitoneal antibody doses starting at 5 weeks of age at 50 mg / kg for the single treatment and 25 mg / kg for the combination. Rotarod performance was tracked longitudinally every 4 weeks, and a final test was performed at the end of treatment. K3 mice in all groups showed significantly impaired performance in the rotarod test compared to WTVeh, and WTVeh mice reached a maximum fall latency (180s) in almost all test sessions (Fig. 5b, top). K3RNJ1 mice showed a higher average fall latency than all other K3 groups in all test sessions after the start of treatment, but this difference did not reach statistical significance (Fig. 5b, top). To evaluate the overall performance of the groups during the treatment process, the area under the curve (AUC) of fall latency was also calculated. Notably, K3RNJ1 mice showed a 27 percent increase in average AUC compared to K3Veh, but this did not reach statistical significance (p = 0.0911) (Fig. 5b, bottom). K3HJ8.5 and K3R+H mice did not show improvement compared to K3Veh throughout the treatment or at the time of completion (Fig. 5b). Surprisingly, K3Veh females performed better than K3Veh males, showing significant differences in the second and third rotarod tests (at 8 and 12 weeks of age) (Fig. 5c, top), and the AUC was significantly higher for K3Veh females (Fig. 5c, bottom).Therefore, to increase the sensitivity of behavior readings, the treatment groups were stratified based on gender (Fig. 5d).

[0520] Sex-stratified analysis of the treatment groups showed that K3RNJ1 females showed significant improvement in rotarod performance, with the second and third tests (8 and 12 weeks of age) showing a trend of improvement, reaching statistical significance in the fourth test (16 weeks of age) (Fig. 5d, top left). This improvement was also reflected in a statistically significant increase in the AUC of fall latency time (Fig. 5d, bottom left). Conversely, the treated males showed no improvement (Fig. 5d, top right and bottom right). Weight (Fig. 5e) and mean grip strength (Fig. 5f) differed significantly between the WT and all K3 groups, with no treatment effect. Taken together, these findings demonstrate that RNJ1 effectively improved motor function in female K3 mice.

[0521] Example 6: RNJ1 treatment causes a slight reduction in total tau levels in the cerebral homogenate of female K3 mice.

[0522] Considering that RNJ1 treatment improved rotarod performance in female K3 mice, biochemical and histopathological changes induced by antibody treatment were evaluated in the female cohort. Tau accumulates and undergoes hyperphosphorylation in disease (Noble et al. 2013). Immunoreactivity to total (mouse and human) tau and tau phosphorylated at pathological AT8 epitopes (pS202 / pT205) was evaluated by Western blot analysis using whole-brain lysates (Fig. 6a). This showed a slight but statistically significant mean reduction of ~12% of total tau in the K3RNJ1 group compared to K3Veh (p = 0.0102) (Fig. 6a, b), and the K3R+H group also showed a statistically significant reduction of 8.5% of total tau (p = 0.049). The levels of AT8 showed greater variability, and K3RNJ1 mice showed a reduction of ~27%; however, this reduction did not reach statistical significance (p = 0.1069) (Fig. 6a, b).

[0523] Next, AT8 responsiveness was evaluated by immunofluorescence in the motor cortex and hippocampus (Fig. 6c), which are brain regions characterized by tau deposition in K3 mice (Bajracharya et al. 2022; Pandit et al. 2019). Mossy fibers in dentate granule cells distributing neurons to spiracle cells and CA3 pyramidal cells showed particularly distinct responsiveness (Fig. 6c). The integrated density of AT8 immunofluorescence in the hippocampus was reduced in some K3RNJ1 mice, but this reduction was not statistically significant (Figs. 6c, 6d). In the motor cortex, AT8 immunoreactivity showed mainly diffuse neural network staining and additional intense responsiveness in the cell bodies of neuronal subsets (Fig. 6c). The integrated density of AT8 immunofluorescence in the motor cortex showed a high degree of variability within the treated cancers, and the differences between groups were not statistically significant (Figs. 6c, 6d). However, when examining the combined AT8 fluorescence intensity for the AUC of rotarod fall latency for individual K3 mice within all treatment groups, moderate correlations with rotarod performance (AUC) were found in both the hippocampus (Pearson correlation r = -0.62, p = 0.0018) and the cortex (Pearson correlation r = -0.60, p = 0.0025) (Fig. 4e). Taken together, these findings demonstrated a slight reduction in tau pathology in the forebrain lysates of RNJ1-treated K3 mice, which was not observed in other treatment groups.

[0524] Example 7: Quantitative proteomics and phosphoproteomics show a reduction in the abundance and phosphorylation of human tau in response to RNJ1 treatment.

[0525] Quantitative proteomic and phosphoproteomic mass spectrometry (MS) analysis was performed on whole-brain lysates of treated female mice as an unbiased approach to gain insight into the differences between K3 and WT mice as well as the effects of antibody treatment. To this end, the TMTpro 18-flex labeling approach was adopted for the quantitative analysis of both trypsin peptides and phosphopeptides by tandem mass spectrometry enhanced by the "TiSH" method (Engholm-Keller et al. 2016). Considering the sample size limitations of the labeling method, MS analysis was focused on the WTVeh, K3Veh, K3RNJ1, and K3HJ8.5 groups, while combination groups (K3R+H) that did not show synergistic effects in behavioral improvement or tau pathology as determined by Western blotting were excluded.

[0526] Human tau showed a 17.7% reduction in average abundance in K3RNJ1 compared to K3Veh (p = 0.0559), while K3HJ8.5 showed a 9.1% reduction (p = 0.2124). Changes in endogenous mouse tau levels were more subtle, with the K3RNJ1 group showing a 6.7% reduction in average abundance (p = 0.1960) (Fig. 6f). Surprisingly, human tau levels showed a strong inverse correlation with rotarod performance (Pearson correlation r = -0.81, p = 0.0013), which was particularly pronounced in the K3RNJ1 group (Fig. 6g). In contrast, mouse tau levels showed only a weak inverse correlation that was not statistically significant (Pearson correlation r = -0.32, p = 0.32). This, along with Western blot analysis, suggests that the reduction of human tau may contribute to therapeutic efficacy in K3 mice.

[0527] Next, quantitative phosphopeptide MS data were analyzed for changes in phosphorylation at individual tau phosphorylation sites. A total of 41 tau phosphopeptides were detected, containing 27 unique phosphorylation sites within tau (including known disease-related epitopes AT270, AT8, AT100, AT180, 12E8, PHF1, and PG5) (Fig. 6h). Antibody treatment affected phosphorylation at these sites to varying degrees. Four phosphopeptides—pS202, part of the AT8 epitope; pS404 and pS400 / pS404, part of the PHF1 epitope; and pS409, part of the PG5 epitope—achieved a statistically significant reduction in abundance in K3RNJ1 compared to K3Veh, whereas pS262 and pS356 (which together form the 12E8 epitope) were reduced in K3HJ8.5 mice. Taken together, immunoblotting, histopathological, and quantitative mass spectrometry analyses showed that RNJ1-treated female K3 mice had reduced levels of total tau and hyperphosphorylated tau, and that this reduction correlated with rotarod performance.

[0528] Example 8: Quantitative proteomics demonstrates the disregard of proteins involved in metabolic and microtubule-related processes in K3 mice.

[0529] To elucidate how RNJ1 treatment affects proteins with which tau interacts directly or indirectly, we described how the brain-wide proteome of K3 mice differs from that of wild-type mice. Of the 6,623 proteins detected in the analysis, 342 proteins showed differential abundance (DA, defined as a fold change in protein levels achieving p<0.05 after correction for false discovery rate (FDR)) in K3Veh mice compared to WTVeh mice, of which 165 were decreased and 177 were increased (Fig. 7a). Interestingly, only about 10% of these DA proteins have been reported to interact with tau via proximate labeling MS (Tracy et al., 2022) and primarily encoded cytoskeletal proteins. This distribution suggests that the majority of the 342 DA proteins may be indirectly affected (Fig. 7b, c).

[0530] These DA proteins were applied to gene ontology (GO) and reactome pathway overpresentation analysis (ORA) to identify functional domains potentially affected by these DA proteins in K3 mice. By applying ORA to the set of proteins with lower abundance in K3 mice, several metabolic processes were found to be strongly overpresented (Fig. 7d, left), and the most significantly enhanced terms in the reactome pathways were 'pyruvate metabolism and the citric acid (TCA) circuit'. Conversely, in the set of proteins with higher abundance, the overpresented terms were primarily associated with microtubule-related processes, including proteins that play a role in axonal transport (Fig. 7d, right). As shown in the gene concept network of proteins annotated with enhanced terms in this comparative analysis, several kinesin, tubulin subtypes, and dinactin subunits were upregulated, similar to Stasmin 1, a key regulator of microtubule dynamics (Fig. 7e) (Lyu et al., 2020; Yadav et al., 2016). Furthermore, in addition to identifying small clusters of DA proteins involved in neurite growth and clathrin-dependent endocytosis, STRING analysis revealed a protein-protein interaction (PPI) network of DA proteins involved in metabolic and microtubule-related processes (Fig. 8). The pronounced decrease in proteins involved in metabolic processes, along with the increase in microtubule-related proteins, reflects phenotypic dysfunction in mitochondrial function and axonal transport previously reported by the present and other groups in different tauopathy models (David et al., 2005; Cummins et al., 2019; DuBoff et al., 2012). This analysis emphasizes the concept that proteome alterations in K3 mice accurately model dysfunction across various functional domains.

[0531] Example 9: Antibody treatment restores deregulated protein levels in K3 mice

[0532] We subsequently investigated whether antibody treatment affects the proteome (and consequently its functionality) to restore disregulated protein levels and associated pathways in K3 mice in this model (Fig. 9a). First, principal component analysis (PCA) showed that K3RNJ1 mice clustered separately from other K3 groups (K3Veh and K3HJ8.5) (Fig. 9a). Furthermore, compared to K3Veh, only 5 protein levels (3 elevated and 2 decreased) in the K3HJ8.5 group showed DA, whereas RNJ1 treatment induced DA changes in 323 proteins (186 elevated and 137 decreased) in the K3Veh group (Fig. 9b). Taken together, these two analyses indicate substantial differences in proteome levels in K3RNJ1 compared to K3Veh mice.

[0533] Considering that this protein subset characterizes the K3 phenotype at the proteome level, the treatment effects specifically related to 342 DA proteins in K3Veh compared to WTVeh were subsequently considered (Fig. 6c). In the first instance, since these changes may be subtle at the individual level but can nevertheless lead to coordinated responses, treatment on this 342-protein subset was analyzed without considering statistical significance. Antibody treatment increased the abundance of the majority of downregulated proteins (Fig. 9d, top; Fig. 10a) and decreased the abundance of the majority of upregulated proteins in K3Veh compared to WTVeh (Fig. 9d, bottom; Fig. 10b), suggesting that antibody treatment shifts the levels of deregulated proteins in K3 to those in WT. To further understand this coordinated shift, the relationship between these 342 protein levels in K3RNJ1 and WTVeh mice compared to K3Veh mice was investigated (Fig. 9e, top). When the fold change value was greater than or less than 1, respectively, antibody treatment was first considered to increase or decrease protein levels in K3Veh mice. Of the 165 proteins with reduced abundance in K3Veh mice compared to WTVeh, 131 (~79.4%) shifted to higher values ​​in K3RNJ1 mice (Fig. 9e, top, Quadrant I). Similarly, of the 177 proteins with higher levels in K3Veh (Quadrant III), 126 (~71.2%) shifted to lower abundance in K3RNJ1. Combined, 257 (~75.1%) of the 342 DA proteins (K3Veh vs. WTVeh) approached the abundance of WT levels after RNJ1 treatment. The same analysis showed a similar response in K3HJ8.5 mice, and approximately 72.5% of these DA proteins (K3Veh vs. WTVeh) also showed changes in the same direction as WTVeh (see Quadrants I and III) (Fig. 9e, bottom).In addition, changes in protein levels in antibody-treated mice against K3Veh mice and WTVeh mice were positively correlated (Pearson correlation r=0.42, p<0.0001 for RNJ1, Fig. 6e, top; Pearson correlation r=0.45, p<0.0001 for HJ8.5, Fig. 9e, bottom).

[0534] Subsequently, the analysis was narrowed down to this subset of 342 proteins that met the criteria for becoming DA upon antibody treatment compared to K3Veh. In the K3RNJ1 group, 48 proteins met this criterion (top of Fig. 9e and closed circles in Fig. 9f), while in the K3HJ8.5 group, only 3 met it (bottom of Fig. 9e and closed circles in Fig. 9f), the latter also included among the 48 proteins identified in the K3RNJ1 group. Interestingly, all 45 of these 48 proteins in the RNJ1 group and all 3 in the HJ8.5 group converted to WT levels due to treatment. A positive correlation was also found between the changes in protein levels of K3RNJ1 and WTVeh mice relative to K3Veh mice (Pearson correlation r=0.89, p<0.0001, closed circles at the top of Fig. 9e). In summary, this analysis reinforces the concept that K3 protein levels are restored to WT after tau antibody treatment.

[0535] In a subset of 45 overlapping DA proteins, clathrin light chain A (Clta) and clathrin coat assembly protein AP180 (Snap91), proteins involved in synaptic vesicle endocytosis, showed reduced abundance in both RNJ1-treated K3Veh and WTVeh mice. To further infer functional domains altered in K3 mice and potentially restored by RNJ1 treatment, ORA was performed on the overlapping DA proteins, and surprisingly, the highest enhancement for the GO terms 'presynapse' and 'endocytosis' was found for proteins with lower abundance in WTVeh and K3RNJ1 for K3Veh (Fig. 11a,b). Additionally, functional PPI analysis revealed a network of 34 proteins within this subset, of which 6 proteins were annotated with the GO term 'presynapse' and 3 with the GO term 'synaptic vesicle' (Fig. 11c). In summary, intensive protemic analysis indicates that the two antibody treatments initiated a response to restore protein levels within the protein subset characterizing the K3 phenotype.

[0536] Example 10: Differential analysis of the phosphoproteome also shows a transition to wild-type levels in RNJ1-treated K3 mice.

[0537] To understand the additional effects of antibody treatment, differential quantitative analysis of the phosphoproteome was performed. Consistent with the proteome analysis (Fig. 9a), PCA of the phosphoproteome showed three clusters: first formed by K3RNJ1 mice, then by WTVeh, and third by both K3HJ8.5 and K3Veh, indicating that K3 mice possess an altered phosphoproteome which is consequently affected by RNJ1 treatment (Fig. 12a). 541 DA phosphopeptides were initially identified in K3Veh mice compared to WTVeh, with 283 phosphopeptides showing lower levels and 258 showing higher abundance in K3Veh (Fig. 12b, left). Surprisingly, 635 phosphopeptides were DA in K3RNJ1 compared to K3Veh, of which 333 showed lower levels and 320 showed higher abundance, reflecting changes in the phosphoproteome following RNJ1 treatment consistent with PCA analysis (Fig. 12b, center). In contrast, no DA phosphopeptides were detected in K3HJ8.5 compared to K3Veh (Fig. 12b, right).

[0538] Similar to the proteomic analysis (Fig. 9c), 541 phosphopeptides with DA between K3Veh and WTVeh were used to determine how antibody treatment affected this subset. When the fold change value was greater than or less than 1, respectively, antibody treatment was initially considered to increase or decrease phosphopeptide levels in K3Veh mice (open and closed circles in Fig. 9c). Of the 258 phosphopeptides with higher abundance in K3Veh compared to WTVeh, 205 (~79.4%) were converted to lower abundance in the K3RNJ1 group (Fig. 12c, left, quadrant III). Similarly, of the 283 phosphopeptides with lower abundance in K3Veh compared to WTVeh, 239 (~84.5%) (quadrant I) had higher abundance in K3RNJ1. In particular, the abundance of 444 (~82.1%) of the total 541 phosphopeptides changed for WTVeh, and the degree of this conversion in the phosphoproteome was much more significant than in the proteomic analysis mentioned above (Fig. 9d). Although less pronounced, a similar conversion was observed in K3HJ8.5 mice, with 183 phosphopeptides decreasing and 217 increasing for both K3HJ8.5 and WTVeh compared to the K3Veh group (Quadrants I and III), meaning that 408 (~75.4%) of the total 541 phosphopeptides were converted for WT (Fig. 12c, right).

[0539] Next, we investigated the statistically distinct overlap of phosphopeptides in both WTVeh and K3RNJ1 for K3Veh and found that it contained 101 phosphopeptides derived from 90 proteins. Surprisingly, similar to the proteomic findings mentioned above, the levels of 99 of these 101 phosphopeptides were restored in K3RNJ1 mice (Fig. 12d). This set of 99 phosphopeptides originated from a total of 89 proteins, and applying this to the PPI analysis revealed a network formed by 41 proteins, with numerous functional associations between these proteins being affected in the K3 model and restored to WT levels upon RNJ1 treatment (Fig. 12e). In addition, analysis of 89 proteins with phosphopeptides restored to WTVeh levels in K3RNJ1 mice using the SynGO database (Koopmans et al. 2019) showed that 28 of these were proteins found in synapses, and that the highest enhancement and protein number were found in postsynaptic proteins (Bhaskar et al. 2010) (Fig. 12f).

[0540] When key proteins within the PPI network were investigated, surprisingly, phosphorylation of mTOR at serine 2448 and MAPK1 at threonine 188 (pT188) was increased in K3RNJ1 mice, and both phosphopeptides were also upregulated in WTVeh compared to K3Veh (Fig. 12g). These instances of phosphorylation may indicate the upregulation of mTOR and MAPK / ERK signaling pathways involved in numerous neuronal functions, including synaptic plasticity (Eng et al. 2016; Tang et al. 2002).

[0541] Taken together, these findings suggest that proteomic and phosphoproteomic changes collectively transform defects in signaling pathways and, consequently, can contribute to improved functional outcomes in response to tau-targeted antibody therapies.

[0542] References within the examples

[0543] Bajracharya et al. (2022) J Control Release 349:634-648.

[0544] Bhaskar et al (2010) Neuropathol Appl Neurobiol 36:6 ​​462-77.

[0545] Cummins et al (2019) J EMBO 1;38(3): e99360.

[0546] David et al. (2005) J Biol Chem 280, 23802-23814.

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[0564] Those skilled in the art will understand that numerous modifications and / or variations may be made without departing from the spirit or scope of the invention as extensively described with respect to the invention as shown in specific embodiments. Accordingly, the embodiments shall be considered exemplary in all respects and not restrictive.

[0565] This application claims priority from Australian provisional application No. 2023903449, titled “Tau-binding proteins and related compositions and methods,” filed on October 27, 2023, the entirety of which is incorporated herein by reference.

[0566] All publications cited herein are incorporated herein by reference in their entirety. Where URLs or other such identifiers or addresses are referenced, it should be understood that such identifiers may change and specific information on the Internet may appear and disappear, but equivalent information can be found by searching the Internet. References to this demonstrate the availability and public distribution of such information.

[0567] Any discussion of documents, acts, materials, devices, articles, etc., contained herein is solely for the purpose of providing background to the present invention. This shall not be construed as an acknowledgment that any or all of these matters constitute part of the basis of the prior art or were common knowledge in the field related to the present invention prior to the priority date of each claim of this application.

[0568] Appendix 1: Order

[0569] Sequence No. 1

[0570] N-terminal human tau epitope (residues 9-20) for mAb RNJ1

[0571] EVMEDHAGTYGL

[0572] Sequence No. 2

[0573] RNJ1 V H -CDR1-containing sequence

[0574] AASGFTFSSYAMSWV

[0575] Sequence No. 3

[0576] RNJ1 V H -CDR2-containing sequence

[0577] GIGWNGTSTAYADSVKG

[0578] Sequence No. 4

[0579] RNJ1 V H -CDR3-containing sequence

[0580] AKRGSPFDY

[0581] Sequence number 5

[0582] RNJ1 V L -CDR1-containing sequence

[0583] RASQSISSYLN

[0584] Sequence number 6

[0585] RNJ1 V L -CDR2-containing sequence

[0586] YRASALQS

[0587] Sequence number 7

[0588] RNJ1 V L -CDR3-containing sequence

[0589] QQGMRRPIT

[0590] Sequence number 8

[0591] RNJ1 VH-CDR2-containing sequence (humanized)

[0592] AISGSGGSTYYADSVKG

[0593] Sequence number 9

[0594] RNJ1 VL-CDR2-containing sequence (humanized)

[0595] YAASSLQS

[0596] Sequence number 10

[0597] RNJ1 VL-CDR3-containing sequence (humanized)

[0598] QQSYSTPWT

[0599] Sequence number 11

[0600] RNJ1 V H Battlefield Order

[0601] MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIGWNGTSTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRGSPFDYWGQGTLVTVSS

[0602] Sequence number 12

[0603] RNJ1 V L Battlefield Order

[0604] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGMRRPITFGQGTKVEIK

[0605] Sequence number 13

[0606] RNJ1 V H Battlefield hierarchy - humanized

[0607] MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRGSPFDYWGQGTLVTVSS

[0608] Sequence number 14

[0609] RNJ1 V L Battlefield hierarchy - humanized

[0610] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIK

[0611] Sequence number 15

[0612] RNJ1 V H -CDR2-Humanized (Degenerate location exists)

[0613]

[0614] Sequence number 16

[0615] RNJ1 V H -CDR3-Humanized (Degenerate location exists)

[0616]

[0617] Sequence number 17

[0618] RNJ1 V L -CDR2-Humanized (Degenerate location exists)

[0619]

[0620] Sequence number 18

[0621] RNJ1 V L -CDR3-Humanized (Degenerate location exists)

[0622]

[0623] Sequence number 19

[0624] Positive clone forward primer

[0625] CGACCCGCCACCGCC GCTG

[0626] Sequence number 20

[0627] Positive clone reverse primer

[0628] CTATGCGGCCCCATTCA

[0629] Sequence number 21

[0630] Truncate tau aa 1-44

[0631] MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLK

[0632] Sequence number 22

[0633] RNJ1 truncated tau junction site aa 1-22

[0634] MAEPRQEFEVMEDHAGTYGLGD

[0635] Sequence number 23

[0636] HJ8.5 truncated tau linkage site aa 23-44

[0637] RKDQGGYTMHQDQEGDTDAGLK

[0638] Sequence number 24

[0639] HJ8.5 reported tau epitope aa 25-30

[0640] DQGGYT

[0641] Sequence number 25

[0642] NTer1 aa 1-10

[0643] MAEPRQEFEV

[0644] Sequence number 26

[0645] NTer2 aa 3-12

[0646] EPRQEFEVME

[0647] Sequence number 27

[0648] NTer3 aa 5-14

[0649] RQEFEVMEDH

[0650] Sequence number 28

[0651] NTer4 aa 7-16

[0652] EFEVMEDHAG

[0653] Sequence number 29

[0654] NTer5 aa 9-18

[0655] EVMEDHAGTY

[0656] Sequence number 30

[0657] NTer6 aa 11-20

[0658] MEDHAGTYGL

[0659] Sequence number 31

[0660] NTer7 aa 13-22

[0661] DHAGTYGLGD

[0662] Sequence number 32

[0663] RNJ1 tau binding site aa 9-22

[0664] EVMEDHAGTYGLGD

[0665] Sequence number 33

[0666] RNJ1 V H Whole-length sequence-human framework variant (IGHV3a)

[0667] MAEVQLVESGGGLVQPGGSLRLSCSASGFTFSSYAMSWVRQAPGKGLEWVSGIGWNGTSTAYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCAKRGSPFDYWGRGTLVTVSS

[0668] Sequence number 34

[0669] RNJ1 V H Whole-length sequence-human framework variant (IGHV3b)

[0670] MAEVQLVESGGGLVQPGGSLRLSCSASGFTFSSYAMSWVRQAPGKGLEWVSGIGWNGTSTAYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCAKRGSPFDYWGQGTLVTVSS

[0671] Sequence number 35

[0672] RNJ1 V H Whole-length sequence-human framework variant (IGHV3c)

[0673] MAEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIGWNGTSTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRGSPFDYWGQGTMVTVSS

[0674] Sequence number 36

[0675] RNJ1 V H Whole-field sequence-human framework variant (IGHV3d)

[0676] MAEVQLVESGGGLVQPGGSLRLSCSASGFTFSSYAMSWVRQAPGKGLEWVSGIGWNGTSTAYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCAKRGSPFDYWGQGTMVTVSS

[0677] Sequence number 37

[0678] RNJ1 V L Whole-length sequence-human framework variant (IGKV1a)

[0679] AIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGMRRPITFGQGTKVEIK

[0680] Sequence number 38

[0681] RNJ1 V L Whole-length sequence-human framework variant (IGKV6a)

[0682] EIVLTQSPDFQSVTPKEKVTITCRASQSISSYLNWYQQKPDQSPKLLIYRASALQSGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQGMRRPITFGQGTKVEIK

[0683] Sequence number 39

[0684] RNJ1 V L Whole-length sequence-human framework variant (IGKV2a)

[0685] DIVMTQTPLSLSVTPGQPASISCRASQSISSYLNWYLQKPGQPPQLLIYRASALQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQGMRRPITFGQGTKLEIK

[0686] Sequence number 40

[0687] RNJ1 V L Whole-length sequence-human framework variant (IGKV4a)

[0688] DIVMTQSPDSLAVSLGERATINCRASQSISSYLNWYQQKPGQPPKLLIYRASALQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGMRRPITFGPGTKVDIK

[0689] Sequence number 41

[0690] RNJ1 V L Whole-length sequence-human framework variant (IGKV3a)

[0691] EIVLTQSPATLSLSPGERATLSCRASQSISSYLNWYQQKPGQAPRLLIYRASALQTGIPARFSGSGSGTDFTLTISSLEPEDFAVYCQQGMRRPITFGQGTKVEIK

[0692] Sequence number 42

[0693] RNJ1 V H Example nucleotide human codon-optimized sequence

[0694] ATGGCGGAAGTACAACTCCTAGAGTCAGGCGGCGGATTAGTACAACCAGGAGGCTCCCTCCGACTATCCTGGCTGCATCCGGCTTCACTTTTTCCAGCTACGCCATGTCCTGGGTCCGTCAAGCGCCCGGCAAAGGGCTAGAATGGGTGTCAGGCATAGGATGGAACGGCACCTCCA CAGCATACGCCGACTCCGTTAAAGGACGCTTCACCATCTCACGCGACAACTCCAAAAACACCCTATACCTACAAATGAACTCACTCCGTGCAGAAGACACAGCTGTCTACTACTGCGCCAAACGCGGATCACCATTCGACTATTGGGGCCAAGGAACCCTTGTAACTGTATCATCCTGA

[0695] Sequence number 43

[0696] RNJ1 V L Example nucleotide human codon-optimized sequence

[0697] GACATTCAAATGACACAATCCCCCTCTTCCCTCTCAGCATCAGTCGGCGACCGCGTAACCATCACTTGTCGAGCGTCACAATCAATCTCCTCATATCTCAACTGGTATCAACAAAAACCTGGCAAAGCCCCAAAACTCCTCATATATCGTGCCTCCGCCCTA CAATCCGGGGTTCCAAGCCGCTTTAGCGGCTCCGGATCAGGAACAGATTTCACACTCACAATATCCTCTCTACAACCCGAGGATTTTGCTACATACTATTGCCAACAAGGAATGCGACGCCCCATAACATTTGGACAAGGCACCAAAGTAGAAATAAAATGA

[0698] Sequence number 44

[0699] RNJ1 V H Example nucleotide Chinese hamster codon-optimized sequence

[0700] ATGGCGGAGGTTCAGTTGCTGGAGTCAGGCGGCGGTCTGGTGCAACCTGGAGGGTCATTGAGGCTGAGTTGTGCTGCCAGTGGATTCACTTTTTCCAGCTACGCAATGAGTTGGGTGAGGCAAGCCCCAGGAAAGGGACTGGAGTGGGGTGAGCGGCATCGGGTGGAACGGCACCTCTA CAGCATACGCTGACTCCGTGAAGGGCCGATTCACAATCTCCCGAGACAATTCTAAGAATACCCTCTACCTGCAGATGAACAGCCTGCGAGCGGAAGATACTGCAGTCTACTATTGCGCGAAACGCGGCAGTCCCTTTGATTACTGGGGGCAGGGTACACTTGTTACTGTTTCATCATGA

[0701] Sequence number 45

[0702] RNJ1 V L Example nucleotide Chinese hamster codon-optimized sequence

[0703] GACATCCAGATGACTCAGTCACCATCTTCTCTGAGTGCATCTGTGGGTGATCGGGTAACAATCACCTGCCGGGCGAGCCAGTCCATCTCTTCTTACTTGAATTGGTACCAGCAGAAGCCAGGTAAGGCACCAAAACTCCTGATCTACCGGGCTAGCGCTCTT CAGAGCGGTGTCCCCTCACGCTTCTCAGGTTCAGGGTCCGGCACTGACTTTACACTGACCATTTCTTCTCTTCAGCCAGAGGACTTCGCGACATACTACTGTCAACAAGGTATGCGACGCCCTATCACATTTGGACAGGGCACCAAGGTAGAAATAAAGTGA

[0704] Sequence number 46

[0705] Humanization RNJ1 V H Example nucleotide human codon-optimized sequence

[0706] ATGGCGGAGGTTCAGCTGCTCGAATCAGGCGGTGGACTTGTGCAACCAGGCGGAAGTCTGCGGCTCTCCTGCGCAGCGAGTGGGTTTACATTCAGCTCCTATGCAATGTCCTGGGTCCGGCAGGCCCCTGGCAAGGGCCTGGAATGGGTGAGCGCGATCAGTGGGTCAGGGGGTTCTA CTTATTATGCTGATTCTGTGAAAGGGCGGTTCACCATCTCCCGCGACAACAGCAAGAACACACTCTACTTGCAGATGAACTCCCTGAGAGCCGAGGATACGGCCGTGTATTATTGTGCGAAGAGGGGATCCCCTTTCGATTACTGGGGACAGGGTACTCTGGTCACTGTCAGTTCCTGA

[0707] Sequence number 47

[0708] Humanization RNJ1 V L Example nucleotide human codon-optimized sequence

[0709] GACATTCAGATGACCCAGTCCCCCAGTTCTCTGAGCGCTTCCGTGGGTGACAGGGTTACTATCACTTGTCGCGCCAGCCAGAGCATTAGTTCTTACTTGAACTGGTATCAACAGAAGCCCGGAAAGGCCCCTAAACTGCTGATCTACGCAGCCAGCAGTCTCCAGAGCGGCGTTCCATCCAGATTCAGCGGCAGCGGGTCAGGCACAGATTTTACGTTGACCATCTCCTCCCTGCAGCCTGAAGATTTTGCCACATATTACTGTCAGCAGAGCTACAGTACCCCCTGGACCTTCGGCCAAGGCACAAAGGTGGAGATTAAGTAA

[0710] Sequence number 48

[0711] 인간 타우 단백질-F 아형(Uniprot ID P10636-8)

[0712] MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

Claims

Claim 1 Immunoglobulin heavy chain variable region (V H ) and immunoglobulin light chain variable region (V L As an isolated tau-binding protein comprising ), said isolated tau-binding protein is V H and V L Through the antigen binding site formed by, Sequence No. 1 Isolated tau-binding protein that specifically binds to the epitope of human tau protein, which is the amino acid sequence of the epitope corresponding to. Claim 2 In claim 1, the isolated tau-binding protein is an isolated tau-binding protein that inhibits human tau protein seeding. Claim 3 In paragraph 1 or 2, ( i ) V H The amino acid sequence of is: (a) having up to 5 amino acid substitutions Sequence No. 2 , (b) having up to 6 amino acid substitutions Sequence No. 3 , and (c) having up to 6 amino acid substitutions Sequence No. 4 Includes, Sequence No. 2 It includes HC-CDR1 corresponding to residuals 31-35 according to the Kavat numbering system, and Sequence No. 3 It includes HC-CDR2 corresponding to residuals 50-65 according to the Kavat numbering system, and Sequence No. 4 ...includes and / or includes HC-CDR3 corresponding to residuals 95-102 according to the Kavat numbering system;( ii ) V L The amino acid sequence of is: (a) having up to 4 amino acid substitutions Sequence number 5 , (b) having up to 4 amino acid substitutions Sequence number 6 , and (c) having up to 5 amino acid substitutions Sequence number 7 Including; Sequence number 5 It includes LC-CDR1 corresponding to residuals 24-34 according to the kavat numbering system, and Sequence number 6 It includes LC-CDR2s corresponding to residuals 50-56 according to the kavat numbering system, and Sequence number 7 Isolated tau-binding protein containing LC-CDR3 corresponding to residues 89-97 according to the Kavat numbering system. Claim 4 In paragraph 1 or 2, ( i ) V H The amino acid sequence of is: (a) having up to 5 amino acid substitutions Sequence No. 2 , (b) having up to 6 amino acid substitutions Sequence number 8 , and (c) having up to 6 amino acid substitutions Sequence number 4 Includes, Sequence No. 2 It includes HC-CDR1 corresponding to residual positions 31-35 according to the kavat numbering system, and Sequence number 8 It includes HC-CDR2 corresponding to residual positions 50-65 according to the kavat numbering system, and Sequence number 4 ...includes and / or includes HC-CDR3 corresponding to residual positions 95-102 according to the kavat numbering system;( ii ) V L The amino acid sequence of is: (a) having up to 4 amino acid substitutions Sequence number 5 , (b) having up to 4 amino acid substitutions Sequence number 9 , and (c) having up to 5 amino acid substitutions Sequence number 10 Includes; Sequence number 5 It includes LC-CDR1 corresponding to residual positions 24-34 according to the kavat numbering system, and Sequence number 9 It includes an LC-CDR2 corresponding to residual positions 50-56 according to the kavat numbering system, and Sequence number 10 Isolated tau-binding protein containing LC-CDR3 corresponding to residue positions 89-97 according to the Kavat numbering system. Claim 5 In any one of paragraphs 1 through 4, V H The amino acid sequence of is Sequence number 11 or Sequence number 13 The amino acid sequence of is at least 80% identical to V L The amino acid sequence of is Sequence number 12 or Sequence number 14 An isolated tau-binding protein that is at least 80% identical to the amino acid sequence of. In any one of claims 1 to 5, (i) at residue positions according to a kavat numbering scheme selected from the group consisting of positions 30-33, 35, 50, 52, 52A, 53, 54, 56-58; 94-99, and 101 Sequence number 11 or Sequence number like 13 V corresponding to H ; or (ii) at residual positions according to the kavat numbering scheme selected from the group consisting of positions 30-32, 34, 49, 50, 51, 53-56, and 91-96 Sequence number 12 or Sequence number 14 V corresponding to L Isolated tau-binding proteins, each having six or fewer substitutions. Claim 6 An isolated tau-binding protein comprising one or fewer amino acid substitutions in each CDR in any one of claims 3 to 5. Claim 7 In Paragraph 6, V H One or fewer amino acid substitutions in any of the CDRs and V L Isolated tau-binding protein containing one amino acid substitution in any of the CDRs. Claim 8 In any one of paragraphs 1 through 7, V H The amino acid sequence of is:( i ) Sequence No. 2 ;( ii ) Sequence No. 3 or Sequence number 8 ; and( iii ) Sequence No. 4 Isolated tau-binding protein containing Claim 9 In any one of paragraphs 1 through 7, V L The amino acid sequence of is:( i ) Sequence number 5 ;( ii ) Sequence number 6 or Sequence number 9 ; and( iii ) Sequence number 7 or Sequence number 10 Isolated tau-binding protein containing Claim 10 In any one of paragraphs 1 through 7, V H The amino acid sequence of is Sequence number 11 or Sequence number 13 Includes the amino acid sequence of, and V L The amino acid sequence of is Sequence number 12 or Sequence number 14 Isolated tau-binding protein containing the amino acid sequence of Claim 11 In any one of paragraphs 1 through 10, V H and V L is an isolated tau-binding protein within a single polypeptide chain. Claim 12 In any one of paragraphs 1 through 10, V L and V H is an isolated tau-binding protein located within a separate polypeptide chain. Claim 13 An isolated tau-binding protein according to any one of claims 1 to 12, further comprising (i) a detection moiety; (ii) a stabilization moiety; (iii) a delivery moiety; and (iv) a functional moiety selected from the group consisting of combinations thereof. Claim 14 In paragraph 13, the above functional moiety is V H or V L Isolated tau-binding protein encoded as a fusion protein containing the amino acid sequence of Claim 15 Isolated tau-binding protein or its V according to any one of claims 1 to 14 H or V L Isolated nucleic acid encoding the amino acid sequence of Claim 16 In paragraph 15, the isolated nucleic acid is an isolated nucleic acid that is a modified mRNA. Claim 17 A non-viral expression vector containing nucleic acid according to paragraph 15. Claim 18 A genetically modified cell comprising a nucleic acid isolated according to paragraph 15 or 16, or a non-viral expression vector according to paragraph 17, wherein the genetically modified cell expresses a protein. Claim 19 A pharmaceutical composition comprising an isolated tau-binding protein according to any one of claims 1 to 14, an isolated nucleic acid according to claim 15 or 16, a non-viral expression vector according to claim 17, or a genetically modified cell according to claim 18. Claim 20 A method for producing an isolated tau-binding protein according to any one of claims 1 to 12, comprising the step of culturing a genetically modified cell according to claim 16 to express a protein encoded by the non-viral expression vector and isolating the expressed tau-binding protein. Claim 21 A method for preventing or treating tauopathy in a subject in need, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition according to claim 19 to the subject. Claim 22 A method for preventing or inhibiting the spread of tau protein aggregates in the central nervous system (CNS) of a subject identified as needing, comprising the step of administering a prophylactic effective amount of a pharmaceutical composition according to claim 19 to said subject. Claim 23 A method according to claim 21 or 22, wherein the tauopathy is selected from the group consisting of Alzheimer's disease, frontotemporal dementia, Pick's disease, chronic traumatic encephalopathy, progressive supranuclear palsy, Parkinson's disease, Parkinson's disease-related disorders, primary age-related tauopathy, silicomeric granulosa disease, and spherocytic tauopathy. Claim 24 In paragraph 23, the above tauopathy is an Alzheimer's disease, method. Claim 25 A method for selecting an immunoglobulin that binds to human tau protein, wherein the method comprises: (i) a heavy chain variable region (V H ), immunoglobulin light chain variable region (V L (ii) providing a library of immunoglobulins comprising ), or both; (ii) a plurality of immunoglobulin proteins, wherein the amino acid sequences are Sequence No. 1 A step of contacting an isolated tau protein, a fragment thereof, or a peptide thereof comprising an epitope of a human tau protein corresponding to; (iii) a step of identifying from the plurality of immunoglobulins an immunoglobulin that specifically binds to the isolated tau protein, the fragment thereof, or the peptide thereof; and (iv) the binding of the immunoglobulin to the isolated tau protein, the fragment thereof, or the peptide thereof Sequence number 1 by a peptide composed of the amino acid sequence of or Sequence number 11 V containing the amino acid sequence corresponding to H and Sequence number 12 V containing the amino acid sequence corresponding to L A method comprising the step of selecting the immunoglobulin only when it is competitively inhibited by a tau-binding protein including Claim 26 V H and V L A variable domain expression library comprising a plurality of expression vectors encoding, ( i ) The above V H The amino acid sequence of is: (a) having up to 5 amino acid substitutions Sequence No. 2 , (b) having up to 6 amino acid substitutions Sequence No. 3 , and (c) having up to 6 amino acid substitutions Sequence No. 4 Includes, Sequence No. 2 It includes HC-CDR1 corresponding to residuals 31-35 according to the Kavat numbering system, and Sequence No. 3 It includes HC-CDR2 corresponding to residuals 50-65 according to the Kavat numbering system, and Sequence No. 4 ...includes and / or includes HC-CDR3 corresponding to residuals 95-102 according to the Kavat numbering system;( ii ) The above V L The amino acid sequence of is: (a) having up to 4 amino acid substitutions Sequence number 5 , (b) having up to 4 amino acid substitutions Sequence number 6 , and (c) having up to 5 amino acid substitutions Sequence number 7 Including; Sequence number 5 It includes LC-CDR1 corresponding to residuals 24-34 according to the kavat numbering system, and Sequence number 6 It includes LC-CDR2s corresponding to residuals 50-56 according to the kavat numbering system, and Sequence number 7 A variable domain expression library containing LC-CDR3 corresponding to residues 89-97 according to the Kavat numbering system. Claim 27 amino acid sequence Sequence No. 1 Immunogenic peptide composed of

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