HK1245810B - Tau-binding antibodies - Google Patents

Description

TAU-binding antibodies

Field of the Invention

The present invention relates in particular to therapeutic and diagnostic Tau-binding antibodies and binding fragments thereof, methods for preparing such antibodies, and their use for treating and/or diagnosing tauopathies, such as Alzheimer's disease; amyotrophic lateral sclerosis/Parkinson's disease-dementia complex; argyrophilic grain disease; chronic traumatic encephalopathy; corticobasal degeneration; diffuse neurofibrillary tangles with calcifications; Down syndrome; British familial dementia; Danish familial dementia; frontotemporal dementia on chromosome 17 associated with MAPT mutations and Parkinson's disease; Gerstmann-Strauss syndrome. Tausler-Scheinker disease; Guadeloupe Parkinson's disease; myotonic dystrophy; neurodegeneration with brain iron accumulation; Niemann-Pick disease type C; non-Guam motor neuron disease with neurofibrillary tangles; Pick disease; postencephalitic Parkinson's disease; prion protein cerebral amyloid angiopathy; progressive subcortical gliosis; progressive supranuclear palsy; SLC9A6-related mental retardation; subacute sclerosing panencephalitis; tangle-only dementia; white matter tauopathy with globular glial inclusions (Clavaguera et al., Brain Pathology 23 (2013) 342-349). The present invention also relates to methods of treating human subjects suffering from or suspected of being susceptible to the above-mentioned tauopathy (particularly, tauopathy such as Alzheimer's disease and progressive supranuclear palsy).

Alzheimer's disease (AD) and progressive supranuclear palsy (PSP) are neurodegenerative diseases with high unmet medical needs, high costs to the health system, and a heavy burden on affected families. AD clinical symptoms include decreased memory, cognition, reasoning, judgment, and emotional stability, and ultimately death. PSP includes severe and progressive gait control and balance problems, falls, vertical eye movement disorders, cognitive problems, depression, expressionlessness, and mild dementia. Late-stage symptoms include blurred vision, uncontrollable eye movements, slurred speech, difficulty swallowing, and death.

For more than a decade, improved strategies have targeted the amyloid-β peptide through various mechanisms. In contrast, much less progress has been made in addressing intracellular tau pathology, the second hallmark of AD. Neurofibrillary inclusions, or tangles, containing hyperphosphorylated tau aggregates define AD pathology and several other tauopathies, including PSP.

In such diseases, there is a strong correlation between symptom progression and the level and distribution of Tau aggregates within the nerve. In AD neurons, Tau tangles first appear in the entorhinal cortex, from which they spread to the hippocampus and neocortex. The tangles observed in AD neurons are composed of hyperphosphorylated insoluble Tau aggregates. It has been proposed that the direct toxic effects of pathological Tau substances and/or axonal transmission loss (due to the sequestration of functional Tau into hyperphosphorylated aggregated forms, which are no longer able to support axonal transmission) lead to the disease.

Tau is a highly soluble cytoplasmic microtubule-binding protein in its non-pathological state. Due to alternative splicing, it appears in the human central nervous system (CNS) in 6 major isoforms ranging in length from 352 to 441 amino acids. These isoforms may have zero, one or two N-terminal insertion sequences (ON, 1N, 2N), and three or four C-terminal "repeat" sequences (3R or 4R). These 30 to 32 amino acid C-terminal repeats R1, R2, R3 and R4 together constitute the Tau microtubule binding region (MTBR). In fact, the main role of Tau is believed to be to assemble and stabilize axonal microtubules. Microtubules form tracks for axonal transport and cytoskeletal components for cell growth (Clavaguera et al., Brain Pathology 23 (2013) 342-349). Three Tau isoforms have been shown to contain three microtubule binding regions (MTBRs):

- isoform 4, also known as 3RON, NCBI reference sequence NP_058525.1 (352 amino acids),

- Isoform 7, also known as 3R1N, NCBI reference sequence NP_001190180.1 (381 amino acids)

- Isoform 8, also known as 3R2N, NCBI reference sequence NP_001190181.1 (410 amino acids).

The other three tau isoforms contain four MTBRs:

- Isoform 2, also known as 4R2N, NCBI reference sequence NP_005901.2 (441 amino acids),

- isoform 3, also known as 4RON, NCBI reference sequence NP_058518.1 (383 amino acids), and

Subtype 5, also known as 4R1N, NCBI reference sequence NP_001116539.1 (412 amino acids).

Tau contains 85 potential serine (S), threonine (T), and tyrosine (Y) phosphorylation sites. Many phosphorylated residues on Tau are found in its proline-rich domain (flanking the microtubule-binding domain). All six Tau isoforms are present in the normal adult human brain, and Tau phosphorylation is relatively reduced during this stage (Noble et al., 2013 Front Neurol. 2013; 4: 83). In various tauopathies, the deposited Tau in pathological lesions is always highly phosphorylated. Phosphorylated serine 202 and phosphothreonine 205 have been detected in Tau aggregates in brain samples and cerebrospinal fluid obtained from patients with PSP and AD (Buée et al., Brain Research Reviews 33 (2000) 95-130; Wray et al., J Neurochem. 2008 Jun 1; 105(6): 2343-52; Hanger et al., J Biol Chem. 2007 Aug 10; 282(32): 23645-54; Maccioni et al., Neurobiol Aging. 2006 Feb; 27(2): 237-44).

The only symptomatic treatments currently available for these diseases have little or no efficacy. There is currently no therapy that can be used to slow or ideally halt the progression of the disease. Therefore, there is still a need in the prior art for new compounds and compositions suitable for treating tauopathies.

OBJECTIVES AND SUMMARY OF THE INVENTION

It is an object of the present invention, in particular, to provide a medicament for the treatment or diagnosis of a tauopathy, such as Alzheimer's disease (AD) or progressive supranuclear palsy (PSP). Furthermore, it is an object of the present invention, in particular, to provide a method for the treatment or diagnosis of a tauopathy, such as Alzheimer's disease (AD) or progressive supranuclear palsy (PSP).

These and other objects, which will become apparent from the following description, are achieved by the subject matter of the independent claims. Certain specific aspects and embodiments thereof encompassed by the present invention form the subject matter of the dependent claims. Further aspects and embodiments thereof encompassed by the present invention can be derived from the following description.

In a first aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises:

a light chain variable region comprising a CDR1 selected from SEQ ID No.: 1 or a sequence at least 90% identical thereto, a CDR2 selected from SEQ ID No.: 2 or a sequence at least 90% identical thereto, and a CDR3 selected from SEQ ID No.: 3 or a sequence at least 90% identical thereto; and/or

A heavy chain variable region comprising a CDR1 selected from SEQ ID No.: 4 or a sequence at least 90% identical thereto, a CDR2 selected from SEQ ID No.: 5 or a sequence at least 90% identical thereto, and a CDR3 selected from SEQ ID No.: 6 or a sequence at least 90% identical thereto.

In a second aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises:

A light chain variable region comprising SEQ ID No.: 7 or a sequence at least 80% identical thereto, and/or

A heavy chain variable region comprising SEQ ID No.: 8 or a sequence at least 80% identical thereto.

In a third aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises:

A light chain variable region comprising SEQ ID No.: 9 or a sequence at least 80% identical thereto, and/or

A heavy chain variable region comprising SEQ ID No.: 10 or a sequence at least 80% identical thereto.

In a fourth aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises:

A light chain variable region comprising SEQ ID No.: 13 or a sequence at least 80% identical thereto, and/or

A heavy chain variable region comprising SEQ ID No.: 16 or a sequence at least 80% identical thereto.

In a fifth aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment binds to a phosphorylated Tau fragment comprising amino acids 197 to 206 of SEQ ID NO:55.

As an embodiment of the first and fifth aspects, the antibodies or binding fragments thereof provided by the present disclosure may be chimeric, humanized or fully human antibodies or binding fragments thereof.

As an embodiment of the second or third aspect, the antibody or binding fragment thereof provided by the present disclosure may be a chimeric antibody or binding fragment thereof.

As an embodiment of the fourth aspect, the antibody or binding fragment thereof provided by the present disclosure may be a humanized antibody or binding fragment thereof.

In a sixth aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof competes for binding to Tau with the Tau-binding antibody or binding fragment thereof of any one of the first to fourth aspects and embodiments thereof.

In a seventh aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to substantially the same Tau epitope as the Tau-binding antibody or binding fragment thereof of any one of the first to fifth aspects and embodiments thereof.

As an embodiment of the sixth and seventh aspects, the present disclosure provides a monoclonal antibody or a binding fragment thereof, which is a humanized antibody or a binding fragment thereof.

The antibodies and binding fragments thereof of the first to seventh aspects and embodiments thereof are capable of binding to a soluble form of human Tau, paired helical filaments (PHF) of human Tau, or binding to a soluble form of human Tau and paired helical filaments (PHF) of human Tau, which comprises a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID NO: 55.

In an eighth aspect, the present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence, such as a DNA sequence encoding the heavy and/or light chain of the antibody or binding fragment of the first to seventh aspects and embodiments thereof.

In a ninth aspect, the present disclosure provides cloning or expression vectors comprising these aforementioned nucleic acid molecules.

In a tenth aspect, the present disclosure provides host cells comprising these aforementioned nucleic acid molecules, cloning vectors or expression vectors.

In an eleventh aspect, the present disclosure provides methods of making the antibodies and binding fragments thereof of the first to seventh aspects and embodiments thereof.

The twelfth aspect of the present disclosure is use of the antibodies and binding fragments thereof according to the first to seventh aspects and embodiments thereof for treating tauopathies (particularly, such as AD and PSP).

Another aspect of the present disclosure is the use of the antibodies and binding fragments thereof of the first to seventh aspects and embodiments thereof for diagnosing Tauopathies, in particular, such as AD and PSP.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Binding of AB1 having the rabbit VL sequence of SEQ ID No.: 7 (VL_AB1) and the rabbit VH sequence of SEQ ID No.: 8 (VH_AB1) to the biotinylated T197 peptide relative to the binding to the biotinylated peptides T174, T211, T230 and T396 in the ELISA analysis of experiment 2.3.

Figure 2: Graph illustrating the cell aggregation analysis of Experiment 3.1.

Figure 3: Efficacy of Tau binding antibodies having a light chain of SEQ ID No.: 17 and a heavy chain of SEQ ID No.: 20 (A) and Tau binding antibodies having a light chain of SEQ ID No.: 17 and a heavy chain of SEQ ID No.: 21 (B), or negative control IgG4 antibody A33 (C) in a cellular Tau aggregation assay using human Tau pathological fibrils recovered from human PSP patients (PSP-PHF8) as seeds.

Figure 4: Western blot showing the binding properties of Tau-binding antibody AB1 having a light chain of SEQ ID No.: 9 and a heavy chain of SEQ ID No.: 10, against Tau-containing lysates obtained from human AD or PSP.

Figure 5: A) Depicts the donor VL (VL_AB1) of AB1 of SEQ ID No.: 7, wherein CDR1 (SEQ ID No.: 1), CDR2 (SEQ ID No.: 2), and CDR3 (SEQ ID No.: 53) are underlined. B) Depicts the VL sequence of the human acceptor region IGKV1-39 of SEQ ID No.: 44, wherein the acceptor CDR1, CDR2, and CDR3 are underlined. C) Depicts the CDR-grafted sequence gVL4_AB1 of SEQ ID No.: 11, wherein CDR1 (SEQ ID No.: 1), CDR2 (SEQ ID No.: 2), and CDR3 (SEQ ID No.: 53) are underlined. D) depicts the CDR-grafted sequence gVL9_AB1 of SEQ No.: 12, wherein CDR 1 (SEQ ID No.: 1), CDR 2 (SEQ ID No.: 2) and CDR 3 (SEQ ID No.: 54) are underlined; compared to VL_AB1, CDR3 contains the A91 mutation.

Figure 6: A) Depicts the donor VH (VH_AB1) of AB1 of SEQ ID No. 8, with CDR1 (SEQ ID No. 4), CDR2 (SEQ ID No. 5), and CDR3 (SEQ ID No. 48) underlined. B) Depicts the VH sequence of the human acceptor region IGHV4-39 of SEQ ID No. 45, with acceptor CDR1, CDR2, and CDR3 underlined. C) Depicts the CDR-grafted sequence gVH41_AB1 of SEQ No. 14, with CDR1 (SEQ ID No. 4), CDR2 (SEQ ID No. 5), and CDR3 (SEQ ID No. 49) underlined. Donor residues are shown in italics and bold: K71 and V78. Mutations in the framework are highlighted (E1). Compared to VH_AB1, CDR3 contains an N100Q substitution. D) Depicts the CDR-grafted sequence of gVH49_AB1 of SEQ ID No.: 15, with CDR 1 (SEQ ID No.: 4), CDR 2 (SEQ ID No.: 5), and CDR 3 (SEQ ID No.: 50) underlined. Donor residues are italicized and bold (K71 and V78). Mutations in the framework are highlighted (E1). Compared to VH_AB1, CDR3 contains an N100A substitution.

Figure 7: Efficacy of a Tau binding antibody having a light chain of SEQ ID No.: 9 and a heavy chain of SEQ ID No.: 10, and the literature-described Tau binding antibody AT8 that binds to an epitope comprising phosphorylated residues 202 and 205 of SEQ ID NO: 55, or the negative control antibody 101.4 in a cellular Tau aggregation assay using human Tau pathological fibrils recovered from human AD patients as seeds.

Figure 8: Efficacy of a Tau binding antibody having a light chain of SEQ ID NO: 17 and a heavy chain of SEQ ID NO: 20 in a cellular Tau aggregation assay using human Tau pathological fibrils recovered from human AD patients or human PSP patients or human FTD patients as seeds.

Detailed Description of the Invention

The disclosure illustratively described below may suitably be practiced in the absence of any component or components, limitation or limitations not specifically disclosed herein.

The present disclosure will be described in terms of particular aspects and embodiments thereof and with reference to certain figures and examples but the invention is not limited thereto.

Unless otherwise indicated, technical terms are used according to their ordinary meanings. Where specific meanings are assigned to certain terms, the definitions of the terms are provided below in the context in which the terms are used.

If the term "comprising" is used in the present description and claims, it does not exclude other components. For the purpose of this disclosure, the term "consisting of" is considered a preferred embodiment of the term "comprising of". If a group is defined below as comprising at least a certain number of embodiments, this is also understood to disclose a group that preferably consists only of these embodiments.

For the purposes of this disclosure, the term “obtained” is considered a preferred embodiment of the term “obtainable.” If hereinafter, for example, an antibody is defined as being obtainable from a particular source, this should also be understood as disclosing that the antibody was obtained from this source.

When referring to a singular noun, if the indefinite or definite article is used, for example "a", "an" or "the", this includes the plural noun, unless specifically stated otherwise. The term "about" or "approximately" indicates an interval of accuracy that a person skilled in the art will understand still ensures the technical effect of the relevant feature. The term generally indicates a deviation of ±10% from the specified numerical value, and preferably a deviation of ±5%.

It should be understood that any reference to a Tau-binding antibody or binding fragment thereof as a preferred embodiment of the various aspects encompasses a monoclonal Tau-binding antibody or binding fragment thereof.

In various aspects, the present disclosure refers to antibodies and binding fragments thereof comprising CDRs and variable regions of corresponding light and/or heavy chain regions. Antibodies or binding fragments thereof comprising only light chain variable regions or heavy chain variable regions may be useful, for example, in manufacturing methods, or for example, in screening for variable regions that can effectively associate with the corresponding other variable regions. However, it should be understood that whenever reference is made to antibodies and binding fragments thereof comprising CDRs and variable regions of corresponding light and/or heavy chain regions, it is always considered to be a preferred embodiment antibody and binding fragment thereof comprising CDRs and variable regions of corresponding light and heavy chain regions.

As used herein, the terms "treatment," "treating," and similar terms refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic, in the sense of completely or partially preventing a disease or its symptoms, and/or therapeutic, in the sense of partially or completely curing a disease and/or adverse effects attributable to the disease. Therapy thus encompasses any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the development of the disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its progression; and (c) alleviating the disease, i.e., causing regression of the disease.

Referring to a Tau-binding antibody or a binding fragment thereof as a "therapeutically active agent" refers to the use of the Tau-binding antibody or a binding fragment thereof for treating a disease.

A "therapeutically effective amount" refers to an amount of a tau-binding antibody or binding fragment thereof that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The therapeutically effective amount will vary depending on the tau-binding antibody or binding fragment thereof, the disease and its severity, and the age, weight, etc., of the subject to be treated.

Referring to a Tau-binding antibody or a binding fragment thereof as a "diagnostically active agent" refers to the use of the Tau-binding antibody or a binding fragment thereof for diagnosing a disease.

A "diagnostically effective amount" refers to an amount of a tau binding antibody or binding fragment thereof that, when used in a diagnostic test performed on a biological sample, is sufficient to allow identification of a disease or to monitor diseased tissue as a means of monitoring the efficacy of a therapeutic intervention.

This application is based, in part, on the identification of an antibody (designated AB1) that binds to human tau. As is customary in the art, tau residue numbering herein refers to tau isoform 2 of SEQ ID No.: 55 (NCBI Reference Sequence: NP_005901.2). As demonstrated below, AB1 was isolated from an immunized rabbit and recognizes a phosphorylated tau region within amino acids 197 to 206 of SEQ ID No.: 55.

The examples demonstrate that AB1 is able to bind to paired helical filaments (PHFs) of human Tau (see Example 2.4) and that AB1 is able to detect intraneuronal neurofibrillary tangles (NFTs), extraneuronal NFTs, neuritic plaque-like structures, and neurofibrillary filaments in frozen sections of human samples (see Example 3.2). It seems reasonable to assume that this behavior is mediated, at least in part, by the complementarity determining regions (CDRs) in the light chain variable region (VL) and heavy chain variable region (VH) of AB1.

Against this background, the present disclosure provides Tau-binding antibodies or binding fragments thereof comprising the CDRs or specificity-determining residues of the VL region of AB1 (SEQ ID No.: 7) and/or the CDRs of the VH region of AB1 (SEQ ID No.: 8).

The residues in the variable domains of antibodies are conventionally numbered according to the system devised by Kabat et al. This system is described in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al. (supra)"). This numbering system is used in this specification unless otherwise indicated.

Kabat residue designations do not always correspond directly to the linear numbering of amino acid residues. Due to shortening of or insertion into structural components (framework or complementarity determining regions (CDRs)) of the basic variable domain structure, the actual linear amino acid sequence may contain fewer amino acids than the strict Kabat numbering or contain other amino acids. For a given antibody, the correct Kabat numbering of the residues can be determined by aligning the homologous residues in the antibody sequence with the "standard" Kabat numbering sequence. However, according to Chothia (Chothia, C. and Lesk, A.M.J.Mol.Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32.

Thus, the CDR1, CDR2, and CDR3 in the VL of AB1 were identified as corresponding to SEQ ID NOs.: 1, 2, and 53, respectively. Thus, the CDR1, CDR2, and CDR3 in the VH of AB1 were identified as corresponding to SEQ ID NOs.: 4, 5, and 48, respectively. One skilled in the art can readily test the effects of amino acid substitutions, additions, and/or deletions on the CDRs, for example, by using the methods described in the Examples. In the initially identified CDR3 (CDRH3) of the VH (i.e., SEQ ID NO.: 48), a potential asparagine deamidation site was identified and modified by replacing the asparagine residue with glutamine, alanine, aspartic acid, or serine. This resulted in the CDRH3 sequences of SEQ ID NOs.: 49, 50, 51, and 52, respectively. For brevity, the three CDRH3 sequences (i.e., SEQ ID NOs.: 48, 49, 50, 51, and 52) are combined into SEQ ID NO.: 6. Similarly, in the CDR3 of VL (CDRL3), a potential glutamine deamidation site was identified and modified by replacing the adjacent glycine with an alanine. This resulted in the sequence SEQ ID NO: 54. For brevity, the two sequences of CDRL3 (i.e., SEQ ID NOs: 53 and 54) are combined into SEQ ID NO: 3.

It will be appreciated that the CDRs can be further modified, such as by substitutions, additions, and/or deletions, without substantially altering, for example, binding properties (compared to AB1). This can be achieved, for example, by replacing amino acids in the CDRs with similar amino acids. As used herein, "similarity" means that at any particular position in the compared sequences, the type of amino acid residue is similar between the sequences. For example, leucine can be substituted for isoleucine or valine. Other amino acids that are commonly substituted for each other include, but are not limited to:

- phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains);

- lysine, arginine, and histidine (amino acids with basic side chains);

- Aspartic acid and glutamic acid (amino acids with acidic side chains);

- Asparagine and glutamine (amino acids with amide side chains); and

- Cysteine and methionine (amino acids with sulfur-containing side chains).

Against this background, the present disclosure provides, in one aspect, an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises:

a light chain variable region comprising a CDR1 selected from SEQ ID No.: 1 or a sequence at least 90% identical thereto, a CDR2 selected from SEQ ID No.: 2 or a sequence at least 90% identical thereto, and a CDR3 selected from SEQ ID No.: 3 or a sequence at least 90% identical thereto; and/or

A heavy chain variable region comprising a CDR1 selected from SEQ ID No.: 4 or a sequence at least 90% identical thereto, a CDR2 selected from SEQ ID No.: 5 or a sequence at least 90% identical thereto, and/or a CDR3 selected from SEQ ID No.: 6 or a sequence at least 90% identical thereto.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises:

a light chain comprising a CDR1 selected from SEQ ID No.: 1 or a sequence at least 90% identical thereto, a CDR2 selected from SEQ ID No.: 2 or a sequence at least 90% identical thereto, and a CDR3 selected from SEQ ID No.: 3 or a sequence at least 90% identical thereto; and

Heavy chain.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises:

light chain; and

A heavy chain variable region comprising a CDR1 selected from SEQ ID No.: 4 or a sequence at least 90% identical thereto, a CDR2 selected from SEQ ID No.: 5 or a sequence at least 90% identical thereto, and/or a CDR3 selected from SEQ ID No.: 6 or a sequence at least 90% identical thereto.

As used herein, "identity" means that at any particular position in the aligned sequences, the amino acid residues between the sequences are the same. The degree of identity can be readily calculated using, for example, BLAST ^™ software available from NCBI (Altschul, SF et al., 1990, J. Mol. Biol. 215: 403-410; Gish, W & States, DJ 1993, Nature Genet. 3: 266-272. Madden, TL et al., 1996, Meth. Enzymol. 266: 131-141; Altschul, SF et al., 1997, Nucleic Acids Res. 25: 3389-3402; Zhang, Jk Madden, TL 1997, Genome Res. 7: 649-656).

The identity of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3 to SEQ ID NO.: 1, 2, 3, 4, 5 and 6, respectively, can be at least 90%, but can also be higher, such as at least 95%, 96%, 97%, 98% or 99%, with higher identities being preferred. Different positions of identity can be selected based on similarity considerations.

In this context, the present disclosure specifically contemplates Tau binding antibodies or binding fragments thereof comprising a VL having CDRL1, CDRL2, and CDRL3 of SEQ ID Nos: 1, 2, and 3, respectively, and a VH having CDRH1, CDRH2, and CDRH3 of SEQ ID Nos: 4, 5, and 6, respectively. The present disclosure also contemplates Tau-binding antibodies or binding fragments thereof comprising: VLs having CDRL1, CDRL2, and CDRL3 of SEQ ID Nos.: 1, 2, 53, respectively, and VHs having CDRH1, CDRH2, and CDRH3 of SEQ ID Nos.: 4, 5, and 6, respectively; Tau-binding antibodies or binding fragments thereof comprising: VLs having CDRL1, CDRL2, and CDRL3 of SEQ ID Nos.: 1, 2, 54, respectively, and VHs having CDRH1, CDRH2, and CDRH3 of SEQ ID Nos.: 4, 5, and 6, respectively; Tau-binding antibodies or binding fragments thereof comprising: VLs having CDRL1, CDRL2, and CDRL3 of SEQ ID Nos.: 1, 2, 3, respectively, and VHs having CDRH1, CDRH2, and CDRH3 of SEQ ID Nos.: 4, 5, and 48, respectively; Tau-binding antibodies or binding fragments thereof comprising: VLs having CDRL1, CDRL2, and CDRL3 of SEQ ID Nos.: 1, 2, 3, respectively, and VHs having CDRH1, CDRH2, and CDRH3 of SEQ ID Nos.: 4, 5, and 48, respectively; No.: 1, 2, 3 of CDRL1, CDRL2 and CDRL3 VL and SEQ ID No: 4, 5 and 49, respectively, CDRH1, CDRH2 and CDRH3 VH; comprising the following Tau binding antibodies or binding fragments thereof: having SEQ ID No.: 1, 2, 3 of CDRL1, CDRL2 and CDRL3 VL and SEQ ID No: 4, 5 and 50, respectively, CDRH1, CDRH2 and CDRH3 VH; comprising the following Tau binding antibodies or binding fragments thereof: having SEQ ID No.: 1, 2, 3 of CDRL1, CDRL2 and CDRL3 VL and SEQ ID No: 4, 5 and 50, respectively, CDRH1, CDRH2 and CDRH3 VH; comprising the following Tau binding antibodies or binding fragments thereof: having SEQ ID No.: 1, 2, 3 of CDRL1, CDRL2 and CDRL3 VL and SEQ ID No: 4, 5 and 50, respectively, CDRH1, CDRH2 and CDRH3 VH; comprising the following Tau binding antibodies or binding fragments thereof: having SEQ ID No.: 1, 2, 3 of CDRL1, CDRL2 and CDRL3 VL and SEQ ID No: No.: 4, 5 and 51 CDRH1, CDRH2 and CDRH3 VH; and a Tau binding antibody or binding fragment thereof comprising the following: VL having CDRL1, CDRL2 and CDRL3 of SEQ ID No.: 1, 2, 3, respectively, and VH having CDRH1, CDRH2 and CDRH3 of SEQ ID No.: 4, 5 and 52, respectively. The invention also provides a method for preparing a Tau-binding antibody or a binding fragment thereof, comprising a VL having CDRL1, CDRL2 and CDRL3 of SEQ ID No.: 1, 2, 53, respectively, and a VH having CDRH1, CDRH2 and CDRH3 of SEQ ID No.: 4, 5 and 48, respectively; a method for preparing a Tau-binding antibody or a binding fragment thereof, comprising a VL having CDRL1, CDRL2 and CDRL3 of SEQ ID No.: 1, 2, 53, respectively, and a VH having CDRH1, CDRH2 and CDRH3 of SEQ ID No.: 4, 5 and 49, respectively; a method for preparing a Tau-binding antibody or a binding fragment thereof, comprising a VL having CDRL1, CDRL2 and CDRL3 of SEQ ID No.: 1, 2, 53, respectively, and a VH having CDRH1, CDRH2 and CDRH3 of SEQ ID No.: 4, 5 and 50 ...H having CDRH1, CDRH2 and CDRH3 of SEQ ID No.: 4, No.: 1, 2, 53 of CDRL1, CDRL2 and CDRL3 VL and having SEQ ID No: 4, 5 and 51, respectively, CDRH1, CDRH2 and CDRH3 VH; and comprising the following Tau binding antibodies or binding fragments thereof: having SEQ ID No.: 1, 2, 53 of CDRL1, CDRL2 and CDRL3 VL and having SEQ ID No: 4, 5 and 52, respectively, CDRH1, CDRH2 and CDRH3 VH; comprising the following Tau binding antibodies or binding fragments thereof: having SEQ ID No.: 1, 2, 54 of CDRL1, CDRL2 and CDRL3 VL and having SEQ ID No: 4, 5 and 48, respectively, CDRH1, CDRH2 and CDRH3 VH; comprising the following Tau binding antibodies or binding fragments thereof: having SEQ ID No.: 1, 2, 54 of CDRL1, CDRL2 and CDRL3 VL and having SEQ ID No: 4, 5 and 48, respectively, CDRH1, CDRH2 and CDRH3 VH; comprising the following Tau binding antibodies or binding fragments thereof: having SEQ ID No.: 1, 2, 54 of CDRL1, CDRL2 and CDRL3 VL and having SEQ ID No: No.: 4, 5 and 49 CDRH1, CDRH2 and CDRH3 VH; comprising the following Tau binding antibodies or binding fragments thereof: VL having CDRL1, CDRL2 and CDRL3 of SEQ ID No.: 1, 2, 54, respectively, and VH having CDRH1, CDRH2 and CDRH3 of SEQ ID No.: 4, 5 and 50, respectively; comprising the following Tau binding antibodies or binding fragments thereof: VL having CDRL1, CDRL2 and CDRL3 of SEQ ID No.: 1, 2, 54, respectively, and VH having CDRH1, CDRH2 and CDRH3 of SEQ ID No.: 4, 5 and 51, respectively; and comprising the following Tau binding antibodies or binding fragments thereof: VL having CDRL1, CDRL2 and CDRL3 of SEQ ID No.: 1, 2, 54, respectively, and VH having CDRH1, CDRH2 and CDRH3 of SEQ ID No.: 4, 5 and 52, respectively.

The Tau-binding antibodies or binding fragments thereof encompassed by the first aspect may comprise these CDRs embedded in framework regions of different origins. Thus, the CDRs may be contained within the native framework regions of AB1 (i.e., the rabbit VL region of SEQ ID No.: 7 and the rabbit VH region of SEQ ID No.: 8). However, the CDRs may also be embedded in framework regions of different species origin, such as mouse or human framework regions. Depending on the origin of the framework regions and constant regions that may be combined with such framework regions, chimeric, murinized, or humanized Tau-binding antibodies or binding fragments thereof may be obtained.

Chimeric Tau-binding antibodies or binding fragments thereof will comprise CDRs within framework regions of non-human origin combined with constant regions from a different species (such as murine or human origin). Murinized Tau-binding antibodies or binding fragments thereof will comprise CDRs within framework regions of murine origin combined with constant regions of murine origin. Humanized Tau-binding antibodies or binding fragments thereof will comprise CDRs within framework regions of human origin combined with constant regions of human origin.

Against this background, the present disclosure provides, in another aspect, an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises:

A light chain variable region comprising SEQ ID No.: 7 or a sequence at least 80% identical thereto, and/or

A heavy chain variable region comprising SEQ ID No.: 8 or a sequence at least 80% identical thereto.

The VL and VH sequences can be at least 80% identical to SEQ ID Nos. 7 and 8, respectively, but can also be higher, such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, with higher identities being preferred. Different positions of identity can be selected based on similarity considerations. It will be appreciated that greater flexibility in identity can exist in the framework regions relative to the CDRs.

In this context, the present invention specifically contemplates a Tau binding antibody or binding fragment thereof comprising a VL of SEQ ID No.: 7 and a VH of SEQ ID No.: 8.

The present disclosure particularly encompasses humanized Tau binding antibodies or binding fragments thereof.

To this end, the CDRs can be grafted onto human framework regions. It will be appreciated that identification of such humanized CDR-grafted Tau-binding antibodies or binding fragments thereof can be achieved using methods well established in the art. When grafting CDRs or specificity-determining residues, any appropriate acceptor human variable region framework sequence relevant to the class/type of donor antibody from which the CDRs are derived can be used (see, e.g., Boss et al., U.S. Patent No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M.S. et al., WO 86/01533; Neuberger, M.S. et al., European Patent No. 0,194,276 B1; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, E.A. et al., European Patent Application No. 0,519,596 A1).

In addition, in the antibody variable region of the CDR transplantation of the present disclosure, the framework region does not need to have a sequence identical to that of the receptor antibody. Thus, CDRs with or without framework changes can be transplanted. Introducing framework changes based on the comparison between the donor variable region framework region and the receptor framework region can allow, for example, antibody affinity to be maintained, otherwise, as a result of humanization, antibody affinity may be reduced. For example, abnormal residues can be changed to residues that are more frequently present in the receptor chain class or type. Alternatively, the selected residues in the receptor framework region can be changed so that they correspond to the residues found in the same position in the donor antibody (see Riechmann et al., 1998, Nature, 332, 323-324). Such changes should be reduced to the minimum necessary to restore the affinity of the donor antibody. The scheme outlined by Adair et al. (1991) (Humanised antibodies. WO91/09967) can be used to select residues for change. In the CDR-grafted antibodies of the present disclosure, the acceptor heavy and light chains need not be derived from the same antibody and, if desired, may comprise composite chains having framework regions derived from different chains.

Examples of human acceptor frameworks that can be used in the present disclosure are KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (Kabat et al., supra). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain, and EU, LAY, and POM can be used for both the heavy and light chains. Alternatively, human germline sequences can be used; these sequences are available at: (http://vbase.mrc-ce.cam.ac.uk/, or http://www.imgt.org). The present invention specifically contemplates the use of the human V region IGKV1-39 + JK4 J region SEQ ID No.: 44 (IMGT, http://www.imgt.org/) as an acceptor framework for the light chain CDRs and the human V region IGHV4-39 + JH4 J region SEQ ID No.: 45 (IMGT, http://www.imgt.org/) as an acceptor framework for the heavy chain CDRs. In SEQ ID No.:45, for example, positions 1, 73, and 80 are suitable for residue changes in the framework regions. The glutamine residue at position 1 can be changed to glutamic acid. The valine residue at position 73 can be changed to lysine. The phenylalanine at position 80 can be changed to valine. Other positions for residue changes in the framework regions in SEQ ID No.:45 may be positions 39 and/or 75. For example, the isoleucine residue at position 39 of SEQ ID No.:45 can be changed to valine. The threonine residue at position 75 can be changed to serine. Positions for residue changes in the framework regions in SEQ ID No.:44 may be positions 2 and/or 63. For example, the isoleucine residue at position 2 of SEQ ID No.:44 can be changed to valine. The serine residue at position 63 of SEQ ID No.:44 can be changed to lysine.

Against this background, the present disclosure provides, in another aspect, an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises: a light chain variable region comprising SEQ ID No.: 9 or a sequence at least 80% identical thereto, and/or a heavy chain variable region comprising SEQ ID No.: 10 or a sequence at least 80% identical thereto.

In another aspect, the present disclosure further provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises: a light chain variable region comprising SEQ ID No.: 13 or a sequence at least 80% identical thereto, and/or a heavy chain variable region comprising SEQ ID No.: 16 or a sequence at least 80% identical thereto.

Such an isolated Tau-binding antibody or binding fragment thereof may comprise: a light chain variable region comprising SEQ ID No.: 13 or a sequence at least 80% identical thereto, and/or a heavy chain variable region comprising SEQ ID No.: 14, 15 or a sequence at least 80% identical thereto.

In addition, such an isolated Tau-binding antibody or binding fragment thereof may comprise: a light chain variable region comprising SEQ ID No.: 11, 12, or a sequence at least 80% identical thereto, and/or a heavy chain variable region comprising SEQ ID No.: 16, or a sequence at least 80% identical thereto.

Such an isolated Tau-binding antibody or binding fragment thereof may comprise: a light chain variable region comprising SEQ ID No.: 11 or a sequence at least 80% identical thereto, and/or a heavy chain variable region comprising SEQ ID No.: 14, 15 or a sequence at least 80% identical thereto.

Such an isolated Tau-binding antibody or binding fragment thereof may comprise: a light chain variable region comprising SEQ ID No.: 12 or a sequence at least 80% identical thereto, and/or a heavy chain variable region comprising SEQ ID No.: 14, 15 or a sequence at least 80% identical thereto.

The VL and VH sequences can be at least 80% identical to SEQ ID Nos. 13 and 16, respectively, but can also be higher, such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, with higher identities being preferred. Different positions of identity can be selected based on similarity considerations. It should be understood that greater flexibility in identity can exist in the framework regions relative to the CDRs.

In this context, the present application specifically contemplates a Tau-binding antibody or binding fragment thereof comprising a VL of SEQ ID No.: 11 and a VH of SEQ ID No.: 14; a Tau-binding antibody or binding fragment thereof comprising a VL of SEQ ID No.: 11 and a VH of SEQ ID No.: 15; a Tau-binding antibody or binding fragment thereof comprising a VL of SEQ ID No.: 12 and a VH of SEQ ID No.: 14; and a Tau-binding antibody or binding fragment thereof comprising a VL of SEQ ID No.: 12 and a VH of SEQ ID No.: 15.

The humanized CDR-grafted Tau binding antibody or its binding fragment may comprise a constant region of human origin. Antibodies or immunoglobulins are divided into the following categories depending on the constant region amino acid sequence of their heavy chains: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (subtypes), such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. In particular, when the antibody molecule is intended for therapeutic use and antibody effector functions are required, human IgG constant region domains, especially IgG1 and IgG3 isotypes, can be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required, IgG2 and IgG4 isotypes can be used. The present invention particularly contemplates humanized antibodies of IgG1 and IgG4 subtypes.

It will be appreciated that sequence modifications of these constant region domains may also be used. For example, one or more amino acid substitutions, additions, and/or deletions may also be made in the antibody constant domains, such as one or two amino acids, without significantly altering the antibody's ability to bind to Tau. IgG4 molecules in which the serine at position 241 has been changed to a proline, as described in Angal et al., Molecular Immunology, 1993, 30(1), 105-108, may also be used.

Antibody effector functions include ADCC and CDC. ADCC refers to antibody-dependent cellular cytotoxicity. To determine whether an antibody is capable of mediating ADCC in principle, ADCC can be measured, for example, by so-called Cr ⁵¹ , Eu, and S ³⁵ release assays. Target cells containing the antigen of interest (i.e., Tau) can be labeled with these compounds. After binding of the therapeutic antibody, the cells are washed and effector cells expressing Fc receptors (such as FcγRIII) are co-incubated with the antibody-labeled target cells and the lysis of the target cells can be monitored based on the release of the label. Another method is to use the so-called aCella TOX ^™ assay. CDC refers to complement-dependent cellular cytotoxicity. To determine whether an antibody is capable of mediating CDC in principle, CDC can be measured in vitro as described, for example, in Delobel A et al., Methods Mol Biol. (2013); 988: 115-43 or Current Protocols in Immunology, Chapter 13 Complement (Print ISSN: 1934-3671).

Against this background, the present disclosure provides, in another aspect, an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises: a light chain comprising SEQ ID No.: 19 or a sequence at least 70% identical thereto, and/or a heavy chain comprising SEQ ID No.: 22 or a sequence at least 70% identical thereto.

Such an isolated Tau-binding antibody or binding fragment thereof may comprise: a light chain comprising SEQ ID No.: 19 or a sequence at least 70% identical thereto, and/or a heavy chain comprising SEQ ID No.: 20, 21, or a sequence at least 80% identical thereto.

Such an isolated Tau-binding antibody or binding fragment thereof may comprise: a light chain comprising SEQ ID No.: 17, 18, or a sequence at least 70% identical thereto, and/or a heavy chain comprising SEQ ID No.: 22, or a sequence at least 80% identical thereto.

Such an isolated Tau-binding antibody or binding fragment thereof may comprise: a light chain comprising SEQ ID No.: 17, 18, or a sequence at least 70% identical thereto, and/or a heavy chain comprising SEQ ID No.: 20, 21, or a sequence at least 80% identical thereto.

The light and heavy chains can be at least 70% identical to SEQ ID Nos.: 19 and 22, respectively, but can also be higher, such as at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, with higher identities being preferred. Different positions of identity can be selected based on similarity considerations. It should be understood that there can be more flexibility in the framework regions relative to the CDRs with respect to identity, and even more flexibility in the constant regions.

In this context, the present application specifically contemplates a Tau-binding antibody or binding fragment thereof comprising a light chain of SEQ ID No.: 17 and a heavy chain of SEQ ID No.: 20, a Tau-binding antibody or binding fragment thereof comprising a light chain of SEQ ID No.: 17 and a heavy chain of SEQ ID No.: 21, a Tau-binding antibody or binding fragment thereof comprising a light chain of SEQ ID No.: 18 and a heavy chain of SEQ ID No.: 20, and a Tau-binding antibody or binding fragment thereof comprising a light chain of SEQ ID No.: 18 and a heavy chain of SEQ ID No.: 21.

In addition, the present invention provides, in another aspect, an isolated Tau-binding antibody or a binding fragment thereof, wherein the Tau-binding antibody or a binding fragment thereof comprises: a light chain comprising SEQ ID No.: 19 or a sequence at least 70% identical thereto, and/or a heavy chain comprising SEQ ID No.: 25 or a sequence at least 70% identical thereto.

Such an isolated Tau-binding antibody or binding fragment thereof may comprise: a light chain comprising SEQ ID No.: 19 or a sequence at least 70% identical thereto, and/or a heavy chain comprising SEQ ID No.: 23 or SEQ ID No.: 24 or a sequence at least 70% identical thereto.

Such an isolated Tau-binding antibody or binding fragment thereof may comprise: a light chain comprising SEQ ID No.: 19 or a sequence at least 70% identical thereto, and/or a heavy chain comprising SEQ ID No.: 23 or SEQ ID No.: 24 or a sequence at least 80% identical thereto.

Such an isolated Tau-binding antibody or binding fragment thereof may comprise: a light chain comprising SEQ ID No.: 17, 18, or a sequence at least 70% identical thereto, and/or a heavy chain comprising SEQ ID No.: 23 or SEQ ID No.: 24, or a sequence at least 80% identical thereto.

In this context, the present application specifically contemplates a Tau-binding antibody or binding fragment thereof comprising a light chain of SEQ ID No.: 17 and a heavy chain of SEQ ID No.: 23, a Tau-binding antibody or binding fragment thereof comprising a light chain of SEQ ID No.: 17 and a heavy chain of SEQ ID No.: 24, a Tau-binding antibody or binding fragment thereof comprising a light chain of SEQ ID No.: 18 and a heavy chain of SEQ ID No.: 23, and a Tau-binding antibody or binding fragment thereof comprising a light chain of SEQ ID No.: 18 and a heavy chain of SEQ ID No.: 24.

The light chain and heavy chain can be at least 70% identical to SEQ ID No.: 19 and SEQ ID No.: 23 or 24, respectively, but can also be higher, such as at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, with higher identities being preferred. Different positions of identity can be selected based on similarity considerations. It should be understood that there can be more flexibility in the framework regions relative to the CDRs with respect to identity, and even more flexibility in the constant regions.

The present disclosure also provides specific regions or epitopes of human Tau that are bound by the antibodies or binding fragments thereof provided by the present disclosure, specifically, antibodies or binding fragments thereof comprising any one of the following: CDR-H1 (SEQ ID No.: 4), CDR-H2 (SEQ ID No.: 5), CDR-H3 (SEQ ID No.: 6), CDR-L1 (SEQ ID No.: 1), CDR-L2 (SEQ ID No.: 2), or CDR-L3 (SEQ ID No.: 3), for example, an antibody comprising the VL of SEQ ID No.: 7 and the VL of SEQ ID No.: 8.

The present disclosure further provides specific regions or epitopes of human Tau, specifically, a phosphorylated Tau region within amino acids 197-206 of SEQ ID No.: 55, which is bound by the antibodies or binding fragments thereof provided in the present disclosure, specifically, antibodies or binding fragments thereof comprising the VL of SEQ ID No.: 7 and the VH of SEQ ID No.: 8.

The Tau region within 197 to 206 of SEQ ID No.: 55 contains four potential phosphorylation sites, corresponding to serine residues at positions 198 (S198), 199 (S199), 202 (S202) and a threonine residue at position 205 (T205).

The term "phosphorylated Tau region within amino acids 197-206 of SEQ ID No.: 55" refers to a Tau region within amino acids 197 to 206 of SEQ ID No.: 55 that contains at least one phosphorylated residue selected from S198, S199, S202, and T205. As known to those skilled in the art, a phosphorylated residue may also be referred to as, for example, Ser(PO ₃ H ₂ ) or Thr(PO ₃ H ₂ ).

Binding of a tau-binding antibody to this specific region or epitope of tau can be identified by any suitable epitope mapping method known in the art in combination with any of the antibodies provided herein. Examples of such methods include screening peptides of varying lengths derived from SEQ ID No.: 55 for binding to a tau-binding antibody or binding fragment thereof of the present disclosure using the smallest fragment of a sequence containing the epitope recognized by the tau-binding antibody or binding fragment thereof that specifically binds to the antibody. If different isoforms of tau exist in the central nervous system, it will be understood that any such isoforms can be used in the methods detailed herein. In a specific example, the longest isoform of tau, isoform 2 as defined in SEQ ID No.: 55, can be used. The tau peptide of SEQ ID No.: 55 can be produced recombinantly, synthetically, or by proteolytic digestion of a tau polypeptide. Peptides that bind to the antibody can be identified by, for example, Western blotting or mass spectrometry. In another example, NMR spectroscopy or X-ray crystallography can be used to identify the epitope bound by a tau-binding antibody or binding fragment thereof. Once identified, epitope fragments that bind to the antibodies of the present disclosure can be used as immunogens, if necessary, to obtain other antibodies that bind to the same epitope. In addition, epitope fragments that bind to the antibodies of the present disclosure can be used to obtain proteins that bind to the same epitope and, if necessary, at least inhibit Tau aggregation, such as proteins or polypeptide compounds comprising more than 10 amino acids based on protein backbones derived from, for example, lipocalin ("anticalcin"), fibronectin ("adnectins, trinectins"), kunez domains, C-type lectins, transferrin, γ-crystallins, cysteine-nots, ankyrin repeats ("DARPins"), or protein A ("affine") and antibodies), as known in the art (Tomlinson, 2004; Mosavi et al., 2004; Gill and Damle, 2006; Nilsson and Tolmachev, 2007; Binz et al., 2004). In addition, molecules that bind to the same epitope include other organic molecules, including peptides and cyclic peptides comprising no more than 10 amino acids, and peptidomimetics. Peptide mimetics are compounds based on amino acid sequences found at protein-protein interaction sites and are known in the art (Sillerud and Larson, 2005).

Against this background, the present disclosure provides, in another aspect, an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.:55.

Such antibodies may be or may be used to obtain chimeric, murinized, humanized or fully human monoclonal antibodies.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least one phosphorylated residue selected from S198, S199, S202, and T205.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least one phosphorylated residue selected from S198 and S199.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least one phosphorylated residue selected from S202 and T205.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least one phosphorylated residue comprising S198.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least one phosphorylated residue comprising S199.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least one phosphorylated residue comprising S202.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least one phosphorylated residue comprising T205.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least two phosphorylated residues selected from S198, S199, S202, and T205.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least two phosphorylated residues comprising S198 and S199.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least two phosphorylated residues comprising S199 and S202.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least two phosphorylated residues comprising S202 and T205.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least two phosphorylated residues comprising S198 and T205.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least two phosphorylated residues comprising S198 and S202.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least two phosphorylated residues comprising S199 and T205.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least three phosphorylated residues selected from S198, S199, S202, and T205.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least three phosphorylated residues comprising S198 and S199.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least three phosphorylated residues comprising S199 and S202.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least three phosphorylated residues comprising S202 and T205.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least three phosphorylated residues comprising S198 and T205.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least three phosphorylated residues comprising S198 and S202.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises at least three phosphorylated residues comprising S199 and T205.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55, wherein the phosphorylated Tau region comprises the following four phosphorylated residues S198, S199, S202, and T205.

Such antibodies may be or may be used to obtain chimeric, murinized, humanized or fully human monoclonal antibodies.

In another aspect, the present disclosure provides an isolated neutralizing Tau binding antibody or binding fragment thereof, wherein the neutralizing Tau binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 35.

Such antibodies may be or may be used to obtain chimeric, murinized, humanized or fully human monoclonal antibodies.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof binds to substantially the same Tau epitope as the Tau-binding antibody or binding fragment thereof described above. Binding to the epitope can be determined as described for epitope mapping, using, for example, a Tau-binding antibody or binding fragment thereof comprising a VL of SEQ ID No.: 7 and a VH of SEQ ID No.: 8 as a reference.

Such antibodies may be or may be used to obtain chimeric, murinized, humanized or fully human monoclonal antibodies.

In another aspect, the present disclosure provides an isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof competes with the above-mentioned Tau-binding antibody for binding to Tau.

In this context, the present invention specifically encompasses isolated Tau-binding antibodies or binding fragments thereof, wherein the Tau-binding antibodies or binding fragments thereof compete for Tau binding with a Tau-binding antibody or binding fragment thereof comprising the VL of SEQ ID No.: 7 and the VH of SEQ ID No.: 8.

Such antibodies may be or may be used to obtain chimeric, murinized, humanized or fully human monoclonal antibodies.

Competition for Tau binding can be determined by reducing Tau binding by at least about 50%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, or about 100% in the presence of a reference antibody or binding fragment thereof that may comprise a VL of SEQ ID No.: 7 and a VH of SEQ ID No.: 8, or a VL of SEQ ID No.: 9 and a VH of SEQ ID No.: 10. Binding can be measured using surface plasmon resonance (using an instrument), various fluorescence detection techniques (e.g., fluorescence correlation spectroscopy, fluorescence cross-correlation, fluorescence lifetime measurement, etc.), or various types of radioimmunoassays or other assays for tracking antibodies bound to a target molecule.

The term "Tau-binding antibody or binding fragment thereof" means an antibody or binding fragment thereof that specifically binds to Tau via its variable region, i.e., binds to the Tau antigen with a greater affinity than other antigens that are not Tau homologs. A "Tau-binding antibody or binding fragment thereof" binds to Tau via its variable region with an affinity that is at least two-fold, at least five-fold, at least 10, 20, 100, 10 ³ , 10 ⁴ , 10 ⁵ , or at least 10 ⁶ fold greater than other antigens that are not Tau homologs. It should be understood that Tau-binding antibodies and binding fragments thereof may also interact with other proteins (e.g., Staphylococcus aureus protein A or other antibodies in ELISA techniques) via interactions with sequences outside the variable region of the Tau-binding antibody or binding fragment thereof. The term "Tau-binding antibody or binding fragment thereof" is not intended to encompass such latter binding properties that are mediated by sequences outside the variable region of the Tau-binding antibody or binding fragment thereof, and in particular, by the constant region of the Tau-binding antibody or binding fragment thereof. Screening assays for determining antibody binding specificity are well known and routinely performed in the art. Tau-binding antibodies or binding fragments thereof may have an equilibrium dissociation constant ( _KD ) in the nanomolar concentration range with respect to the affinity with which the antibody (or its binding fragment) binds to its antigen. Thus, the _KD may be less than about 1* ^10-6 , for example, less than about 5* ^10-7 , such as about 2* ^10-7 or less than 2* ^10-7 , and may be measured using, for example, surface plasmon resonance and BIAcore devices, as described in the Examples.

As described above, the present disclosure provides Tau-binding antibodies or binding fragments thereof. A full-length antibody comprises a constant domain and a variable region. The constant region may not necessarily be present in its full-length form in an antigen-binding fragment of an antibody. However, it should be understood that whenever the present application contemplates the use of antibody-mediated ADCC and/or CDC, the binding fragment must comprise a constant region that is still long enough to mediate ADCC and/or CDC.

As described above, the present invention also relates to human Tau binding antibodies or binding fragments thereof, which can be produced as an alternative to humanization. For example, transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies without producing endogenous murine antibodies after immunization are known in the art. For example, homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice has been described, resulting in complete inhibition of endogenous antibody production. Transferring the human germline immunoglobulin gene array into such germline mutant mice will produce human antibodies specific for a particular antigen (after immunizing the transgenic animal carrying the human germline immunoglobulin gene with the antigen). Techniques for producing such transgenic animals and techniques for isolating and producing human antibodies from such transgenic animals are known in the art (Lonberg, 2005; Green, 1999; Kellermann and Green, 2002; Nicholson et al., 1999). Alternatively, in a transgenic animal (e.g., mouse), only the immunoglobulin genes encoding the variable regions of the mouse antibodies are replaced with corresponding human variable immunoglobulin gene sequences. The mouse germline immunoglobulin genes encoding antibody constant regions remain unchanged. In this way, the antibody effector function and therefore B cell development in the immune system of transgenic mice are substantially unchanged, so that antibody response can be improved after antigen attack in vivo. Once the gene encoding the specific antibody of interest has been separated from such transgenic animals, the gene encoding the constant region can be replaced with human constant region genes to obtain fully human antibodies. Other methods for obtaining the antibody fragment of human antibodies in vitro are based on display technology, such as phage display or ribosome display technology, wherein a recombinant DNA library produced at least in part by artificial means or from the immunoglobulin variable (V) domain gene pool of a donor is used. Phage and ribosome display technology for producing human antibodies are well known in the art (Winter et al., 1994; Hoogenboom, 2002; Kretzschmar and von Ruden, 2002; Groves and Osbourn, 2005; Dufner et al., 2006).

Human antibodies can also be produced from isolated human B cells that are immunized ex vivo with the antigen of interest and subsequently fused to produce hybridomas, which can then be screened for production of the best human antibodies (Grasso et al., 2004; Li et al., 2006).

As used herein, the term "neutralizing Tau binding antibody" refers to an antibody that binds to Tau and inhibits at least one biological activity of Tau. In a specific embodiment, as used herein, "neutralizing Tau binding antibody" refers to an antibody that binds to Tau and inhibits Tau aggregation in an in vitro assay, such as the in vitro assay described in Experiment 3.1 below.

As used herein, the term 'antibody' refers essentially to an intact (complete, full-length) antibody, i.e., an assembly comprising two heavy chains and two light chains. Antibodies may comprise other binding domains, such as the molecules DVD-Ig disclosed in WO 2007/024715, or the so-called (FabFv)2Fc described in WO 2011/030107. Thus, antibodies as used herein include bivalent, trivalent, or tetravalent full-length antibodies.

Antibody binding fragments include single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab') ₂ , Fv, Fab-Fv, Fab-dsFv, Fab-scFv, Fab-scFc, disulfide-stabilized Fab-scFv, single domain antibodies (e.g., VH or VL or VHH), scFv, scFv-scFc, dsscFv, dsscFv-scFc, bivalent, trivalent or tetravalent antibodies, diabodies, triabodies, triabodies, tetrabodies; domain antibodies (dAbs), such as sdAbs; VHH and VNAR fragments, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and manufacturing these antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009/040562 and its disulfide-stabilized Fab-dsFv was first disclosed in WO2010/035012. The disulfide-stabilized form of Fab-scFv is described in WO2013/068571. Antibody formats including scFc formats were first described in WO2008/012543. Other antibody fragments used in the present disclosure include Fab and Fab' fragments described in International Patent Applications WO2005/003169, WO2005/003170, and WO2005/003171.

Multivalent antibodies may comprise multiple specificities, such as bispecifics, or may be monospecific (see, for example, WO92/22583 and WO05/113605). An example of the latter is a Tri-Fab (or TFM) as described in WO92/22583.

In one embodiment, a Fab fragment is provided.

In one embodiment, a Fab' fragment is provided.

A typical Fab' molecule comprises a heavy and light chain pair, wherein the heavy chain comprises a variable region VH, a constant domain CH1 and a native or modified hinge region and the light chain comprises a variable region VL and a constant domain CL.

In one embodiment, a dimer of a Fab' according to the present disclosure is provided to generate F(ab')2, for example dimerization can occur via a hinge.

In one embodiment, an antibody or binding fragment thereof comprises a binding domain. The binding domain will typically comprise six CDRs, three from the heavy chain and three from the light chain. In one embodiment, the CDRs are located in a framework and together form a variable region. Thus, in one embodiment, an antibody or binding fragment comprises a binding domain specific for an antigen comprising a light chain variable region and a heavy chain variable region.

It should be understood that the affinity of the Tau binding antibodies or binding fragments thereof provided herein can be altered using suitable methods known in the art. The present disclosure therefore also relates to variants of the antibody molecules of the present disclosure that have improved affinity for Tau. Such variants can be obtained by a variety of affinity maturation schemes, including mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), using E. coli mutant strains (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996), and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998). These affinity maturation methods are discussed in Vaughan et al. (supra).

Tau-binding antibodies and binding fragments thereof may also encompass, for example, any of the light chain or heavy chain amino acid sequences specifically mentioned above with one or more conservative substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative substitutions). Positions in the amino acid sequence can be identified as candidates for conservative substitutions, and synthetic and naturally occurring amino acids can be selected to achieve conservative substitutions for any particular amino acid. Factors considered for selecting conservative substitutions include the context in which any particular amino acid substitution is made, the hydrophobicity or polarity of the side chain, the general size of the side chain, and the pK value of the side chain having acidic or basic characteristics under physiological conditions. For example, substitutions of lysine, arginine, and histidine for each other are generally suitable. As is known in the art, this is because all three amino acids have basic side chains, yet the pK values of the side chains of lysine and arginine are closer to each other (approximately 10 and 12) than those of histidine (approximately 6). Similarly, substitution of glycine, alanine, valine, leucine, and isoleucine for one another is generally suitable, provided that glycine does not frequently substitute for other members of the group. Other amino acid groups that are suitable for frequent substitution for one another include, but are not limited to, the group consisting of glutamic acid and aspartic acid; the group consisting of phenylalanine, tyrosine, and tryptophan; and the group consisting of serine, threonine, and optionally tyrosine.

As referred to in the context of the present disclosure, Tau-binding antibodies and binding fragments thereof may encompass derivatives of the exemplary antibodies, fragments, and sequences disclosed herein. "Derivatives" include Tau-binding antibodies and binding fragments thereof that have been chemically modified. Examples of chemical modifications include covalent attachment of one or more polymers, such as water-soluble polymers, N-linked or O-linked carbohydrates, sugars, phosphates, and/or other such molecules, such as detectable labels, such as fluorophores.

If necessary, the Tau binding antibodies or binding fragments thereof used in the present disclosure can therefore be bound to one or more effector molecules. It should be understood that the effector molecule may comprise a single effector molecule or two or more such molecules that are so connected to form a single portion that can be connected to the antibody of the present invention. In the case where it is necessary to obtain an antibody fragment connected to an effector molecule, this can be prepared by a standard chemical or recombinant DNA procedure in which the antibody fragment is directly or via a coupling agent connected to the effector molecule. The techniques for binding such effector molecules to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd edition, Robinson et al., ed., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62: 119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). These techniques for binding effector molecules may include site-specific binding or non-site-specific or random binding. Specific chemical procedures include, for example, those described in WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476, and WO 03/031581. Alternatively, where the effector molecule is a protein or polypeptide, recombinant DNA procedures, such as those described in WO 86/01533 and EP 0392745, can be used to achieve linkage. Alternatively, specific linkage sites can be engineered into the antibodies or antigen-binding fragments thereof of the present disclosure, such as those described in WO 2008/038024. In addition, coupling agents can be used to link the effector molecule to the antibodies or antigen-binding fragments thereof of the present disclosure, such as those described in WO 2005/113605. It will be appreciated by those skilled in the art that the above possibilities can be used individually or in combination.

As used herein, the term effector molecule includes, for example, drugs, toxins, biologically active proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof (e.g., DNA, RNA and fragments thereof), radionuclides (particularly radioiodine), radioisotopes, chelated metals, nanoparticles, and reporter groups, such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy. As used herein, effector molecules also include therapeutic agents, such as chemotherapeutic agents, therapeutic polypeptides, nanoparticles, liposomes, or therapeutic nucleic acids.

Other effector molecules may include chelated radionuclides such as ¹¹¹ In and ⁹⁰ Y, Lu ¹⁷⁷ , bismuth ²¹³ , californium ²⁵² , iridium ¹⁹² , and tungsten ¹⁸⁸ /rhenium ¹⁸⁸ ; or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids, and suramin.

Other effector molecules include proteins, peptides, and enzymes. Enzymes of interest include, but are not limited to, proteases, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides, and peptides of interest include, but are not limited to, immunoglobulins; toxins such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as insulin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, or tissue plasminogen activator; thrombotic agents or anti-angiogenic agents, such as angiostatin or endostatin; or biological response modifiers, such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte Macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factors and immunoglobulins, or other protein or polypeptide compounds comprising more than 10 amino acids based on a protein backbone derived from, for example, lipocalins ("anticalins"), fibronectins ("Adnectins," terinary ligands), quinone domains, C-type lectins, transferrin, gamma-crystallins, cysteine knots, ankyrin repeats ("DARPins"), Fyn SH3 domains ("fynomers") or protein A ("affibrin"), as known in the art (Tomlinson, 2004; Mosavi et al., 2004; Gill and Damle, 2006; Nilsson and Tolmachev, 2007; Binz et al., 2004; Silacci et al., 2014).

Other effector molecules include peptides and proteins that enhance or promote blood-brain barrier permeation. For example, WO2010/043047, WO2010/063122, WO2010/063123, or WO2011/041897 describe peptides or polypeptides that can act as carriers capable of transporting therapeutic molecules across the blood-brain barrier and methods of conjugating them to therapeutic molecules. In the context of blood-brain barrier permeation, peptides and proteins of interest include, but are not limited to, those that bind to blood-brain barrier receptors such as transferrin receptor, glucose receptor, insulin receptor, insulin-like growth factor receptor, low-density lipoprotein receptor-related protein 8, low-density lipoprotein receptor-related protein 1, and heparin-binding epidermal growth factor-like growth factor. Alternatively, the effector molecule is an antibody fragment that specifically binds to one of the above-mentioned blood-brain barrier receptors, such as a domain antibody, a camelid antibody, or a shark-derived antibody (VNAR).

Other effector molecules may include detectable substances suitable for use in, for example, diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radionuclides, positron-emitting metals (such as those used in positron emission tomography or single photon emission computerized tomography), and non-radioactive paramagnetic metal ions. For metal ions that can be bound to antibodies suitable for use in diagnostics, see generally U.S. Patent No. 4,741,900. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radionuclides include ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ¹¹¹ In, ⁹⁹ Tc, ⁸⁹ Zr, ⁹⁰ Y, ⁶⁴ Cu, ⁶⁸ Ga, and ¹⁸ F. Particular types of effector molecules suitable for diagnostics as detectable substances include electron-deficient tetrazines and trans-cyclooctenes (TCOs), as described in Wyffels et al., 2014, Nuclear Medicine and biology 41 (2014): 513-523, which can be administered to a Tau-binding antibody of the present disclosure linked to a tetrazine and allowed to achieve maximum uptake and adequate clearance from non-target sites, followed by subsequent administration of a TCO or optimized TCO analog labeled with a suitable radionuclide, such that the TCO is covalently bound to the tetrazine on the Tau-binding antibody of the present disclosure, and detection thereof is achieved by, for example, positron emission tomography or single photon emission computerized tomography.

In one embodiment, a Tau-binding Fab, Fab', or scFv is provided that is linked to a radionuclide or to a tetrazine. Linkage to the radionuclide or to the tetrazine can be achieved by linkage via any available amino acid side chain or terminal amino acid functional group (e.g., any free amine, imine, thiol, hydroxyl, or carboxyl group) located in the antibody fragment. Such amino acids may be naturally present in the antibody fragment or may be engineered into the fragment using recombinant DNA methods (see, e.g., US 5,219,996; US 5,667,425; WO98/25971, WO2008/038024). In one embodiment, the Tau-binding antibody or binding fragment thereof of the present disclosure is a modified Fab fragment, wherein the modification is the addition of one or more amino acids to the C-terminus of its heavy chain to enable attachment of an effector molecule. Suitably, the other amino acids form a modified hinge region containing one or more cysteine residues that can be linked to the effector molecule. In one embodiment, if the radionuclide is a metal ion, such as ¹¹¹ In, ⁹⁹ Tc, ⁸⁹ Zr, ⁹⁰ Y, ⁶⁴ Cu, or ⁶⁸ Ga, it can be bound by a macrocyclic chelator, such as described by Turner et al. (Br. J. Cancer, 1994, 70: 35-41; Comparative biodistribution of indium-111-labeled macrocycle chimeric B72.3 antibody conjugates in tumor-bearing mice), which is in turn covalently linked to the aforementioned amino acid side chains or terminal amino acid functional groups of the antibody or antibody fragment. In another embodiment, the macrocyclic chelator that binds the radionuclide can be an effector molecule as described in WO05/113605, which is part of a cross-linker that links two or more anti-Tau antibodies or fragments thereof.

In another example, the effector molecule can extend the in vivo half-life of the antibody, and/or reduce the immunogenicity of the antibody and/or enhance the transport of the antibody across the epithelial barrier to the immune system. Examples of suitable such effector molecules include polymers, albumin and albumin binding proteins or albumin binding compounds, such as those described in WO05/117984.

Where the effector molecule is a polymer, it may generally be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide (e.g., a homopolysaccharide or a heteropolysaccharide).

Specific substituents that may optionally be present on the above synthetic polymers include one or more hydroxyl, methyl or methoxy groups.

Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, especially optionally substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) or derivatives thereof.

Particular naturally occurring polymers include lactose, amylose, polydextrose, glycogen or derivatives thereof.

In one embodiment, the effector molecule is albumin or a fragment thereof, such as human serum albumin or a fragment thereof.

The polymer size can vary as desired, but the average molecular weight is typically in the range of 500 to 50,000 Da, for example 5,000 to 40,000 Da, such as 20,000 to 40,000 Da. The polymer size can be selected based on, among other things, the intended use of the product, such as its ability to be localized in certain tissues (such as the brain) or to extend its circulatory half-life (for a review, see Chapman, 2002, Advanced Drug Discovery Reviews, 54, 531-545). Thus, for example, it may be desirable for the product to leave the circulation and penetrate into tissues.

Suitable polymers include polyalkylene polymers such as poly(ethylene glycol) or especially methoxypoly(ethylene glycol) or derivatives thereof, and especially having a molecular weight in the range of about 15,000 Da to about 40,000 Da.

In one embodiment, the antibody used in the present disclosure is linked to a poly(ethylene glycol) (PEG) moiety. In a specific example, the antibody is a Tau binding antibody or a binding fragment thereof and the PEG molecule can be linked via any available amino acid side chain or terminal amino acid functional group located in the antibody fragment, such as any free amino group, imino group, sulfhydryl group, hydroxyl group, or carboxyl group. Such amino acids may be naturally present in the antibody fragment or may be engineered into the fragment using recombinant DNA methods (see, for example, US 5,219,996; US 5,667,425; WO98/25971, WO2008/038024). In one example, the Tau binding antibody or binding fragment thereof of the present disclosure is a modified Fab fragment, wherein the modification is the addition of one or more amino acids to the C-terminus of its heavy chain to enable the connection of an effector molecule. Suitably, the other amino acids form a modified hinge region containing one or more cysteine residues that can be linked to the effector molecule. Multiple sites can be used to connect two or more PEG molecules.

PEG molecules are suitably covalently linked via the sulfhydryl group of at least one cysteine residue located in the antibody fragment. Each polymer molecule connected to the modified antibody fragment can be covalently linked to the sulfur atom of the cysteine residue located in the fragment. The covalent bond is generally a disulfide bond or especially a sulfur-carbon bond. In the case where a sulfhydryl group is used as a connection point, an appropriately activated effector molecule can be used, for example, a sulfhydryl selective derivative, such as maleimide and cysteine derivatives. Activated polymers can be used as starting materials to prepare polymer-modified antibody fragments as described above. Activated polymers can be any polymer containing a thiol reactive group, such as an α-halocarboxylic acid or ester, for example, iodoacetamide, imide (e.g., maleimide), vinyl sulfone or disulfide. Such starting materials are commercially available (e.g., purchased from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or can be prepared from commercially available starting materials using conventional chemical procedures. Specific PEG molecules include 2OK methoxy-PEG-amine (available from Nektar, formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (available from Nektar, formerly Shearwater).

In another aspect, the present disclosure provides nucleic acid molecules comprising nucleic acid sequences encoding Tau-binding antibodies and binding fragments thereof; nucleic acid molecules comprising nucleic acid sequences encoding the variable light and/or heavy chains thereof; and nucleic acid molecules comprising nucleic acid sequences encoding the CDR1, CDR2 and/or CDR3 of the variable light and/or heavy chains thereof.

For example, the VL of AB1 (SEQ ID No.: 7) can be encoded by SEQ ID No.: 26. The VH of AB1 (SEQ ID No.: 8) can be encoded by SEQ ID No.: 27.

The humanized VL of SEQ ID No.: 11 can be encoded by SEQ ID No.: 30. The humanized VL of SEQ ID No.: 12 can be encoded by SEQ ID No.: 31. The humanized VH of SEQ ID No.: 14 can be encoded by SEQ ID No.: 32 and the humanized VH of SEQ ID No.: 15 can be encoded by SEQ ID No.: 33.

The humanized light chain of SEQ ID No.: 17 can be encoded by SEQ ID No.: 34. The humanized light chain of SEQ ID No.: 18 can be encoded by SEQ ID No.: 35. The humanized heavy chain of SEQ ID No.: 20 can be encoded by SEQ ID No.: 36 and the humanized heavy chain of SEQ ID No.: 21 can be encoded by SEQ ID No.: 37. The humanized heavy chain of SEQ ID No.: 23 can be encoded by SEQ ID No.: 38 and the humanized heavy chain of SEQ ID No.: 24 can be encoded by SEQ ID No.: 39.

Tau-binding antibodies and binding fragments thereof can be encoded by a single nucleic acid (e.g., a single nucleic acid comprising nucleotide sequences encoding the light and heavy chain polypeptides of the antibody), or by two or more separate nucleic acids (each encoding a different portion of the antibody or antibody fragment). In this regard, the present disclosure provides one or more nucleic acids encoding any of the aforementioned antibodies or binding fragments. Nucleic acid molecules can be DNA, cDNA, RNA, and analogs thereof.

For example, the DNA sequence encoding part or all of the antibody heavy and light chains can be synthesized as needed from the determined DNA sequence or based on the corresponding amino acid sequence. The DNA encoding the receptor framework sequence is widely available to those skilled in the art and is easily synthesized based on its known amino acid sequence.

The DNA sequence encoding the antibody molecules of the present disclosure can be prepared using standard molecular biology techniques. The desired DNA sequence can be synthesized in whole or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques can be used where appropriate.

Preferably, the encoding nucleic acid sequence is operably linked to an expression control sequence, thereby allowing expression in prokaryotic or eukaryotic cells. The expression of the polynucleotide comprises transcribing the polynucleotide into translatable mRNA. Regulatory elements ensuring that expression is achieved in eukaryotic cells (preferably mammalian cells) are well known to those skilled in the art. It generally comprises a regulatory sequence ensuring transcription initiation and optionally comprises a poly A signal ensuring transcription termination and transcript stabilization. Other regulatory elements may include transcription and translation enhancers, and/or naturally associated or heterogeneous promoter regions.

Thus, the present disclosure provides, in another aspect, cloning or expression vectors comprising such nucleic acid sequences encoding Tau-binding antibodies and binding fragments thereof.

A "vector" is any molecule or composition capable of carrying a nucleic acid sequence into a suitable host cell, e.g., where synthesis of the encoded polypeptide can occur. Typically and preferably, a vector is a nucleic acid that has been engineered using recombinant DNA techniques known in the art to incorporate a desired nucleic acid sequence, e.g., a nucleic acid of the present disclosure. An expression vector typically contains one or more of the following components (if not already provided by a nucleic acid molecule): a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

Vectors are generally selected to function in the host cell in which they are to be used that is compatible with the host cell machinery, such that gene amplification and/or gene expression can occur.

The present disclosure therefore provides, in another aspect, a host cell comprising a cloning or expression vector as described above and/or a nucleic acid sequence encoding a Tau-binding antibody and binding fragment thereof as described above.

The host cell can be any cell type that can be transformed with a nucleic acid or vector to produce a Tau-binding antibody or binding fragment thereof encoded therefrom. Host cells containing nucleic acids or vectors can be used to produce Tau-binding antibodies or binding fragments thereof, or portions thereof (e.g., heavy chain sequences or light chain sequences encoded by nucleic acids or vectors). After the nucleic acid or vector is introduced into the cell, the cell is cultured under conditions suitable for expression of the encoded sequence. The antibody, antigen-binding fragment, or a portion of the antibody can then be isolated from the cell.

The host cell can be a prokaryotic host cell (such as E. coli) or a eukaryotic host cell (such as a yeast cell, an insect cell, or a vertebrate cell). When cultured under appropriate conditions, the host cell expresses the antibody or its binding fragment, which can then be collected from the culture medium (if the host cell secretes the antibody or its binding fragment into the culture medium) or directly from the host cell producing the antibody or its binding fragment (if the antibody or its binding fragment is not secreted). The selection of an appropriate host cell will depend on a variety of factors, such as the desired expression amount, polypeptide modifications required or necessary for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule. The choice of host cell depends in part on whether the antibody or its binding fragment is post-transcriptionally modified (e.g., glycosylated and/or phosphorylated). If so, yeast, insect, or mammalian host cells are preferred.

Suitable mammalian host cells include CHO, myeloma, or hybridoma cells. Types of Chinese hamster ovary (CHO) cells suitable for use in the present disclosure may include CHO and CHO-K1 cells, including dhfr-CHO, such as CHO-DG44 cells and CHODXB11 cells that can be used in conjunction with the DHFR selective marker, or CHOKI-SV cells that can be used in conjunction with the glutamine synthetase selective marker. Many are available from the American Type Culture Collection (ATCC), Manassas, Va. Examples include mammalian cells such as Chinese hamster ovary cells (CHO) (ATCC No. CCL61), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), 3T3 cells (ATCC No. CCL92), or PER.C6 cells. Other cell types for expressing antibodies include lymphocytic cell lines, such as NSO myeloma cells and SP2 cells, COS cells.

Another aspect of the present disclosure provides a method of producing a Tau-binding antibody or a binding fragment thereof, comprising culturing a host cell containing, e.g., a vector, under conditions suitable for expressing the Tau-binding antibody or the binding fragment thereof from, e.g., DNA encoding the Tau-binding antibody or the binding fragment thereof and isolating the antibody molecule.

Tau-binding antibodies or binding fragments thereof may comprise only a heavy or light chain polypeptide, in which case only the heavy or light chain polypeptide encoding sequence needs to be transfected into the host cells. To produce a product containing both heavy and light chains, the cell line can be transfected with two vectors, the first encoding the light chain polypeptide and the second encoding the heavy chain polypeptide. Alternatively, a single vector can be used, comprising sequences encoding both the light and heavy chain polypeptides.

Tau binding antibodies or binding fragments thereof (antibodies and fragments according to the present disclosure) are expressed in high amounts in host cells. Therefore, the properties of the antibodies and/or fragments are conducive to commercial processing.

Thus, a method is provided for culturing host cells and expressing a Tau-binding antibody or a binding fragment thereof, isolating the latter and optionally purifying it to provide an isolated Tau-binding antibody or a binding fragment thereof. In one embodiment, the method further comprises the step of conjugating an effector molecule to the isolated antibody or fragment (e.g., to a PEG polymer, particularly as described herein).

Tau-binding antibodies or binding fragments thereof can be formulated into compositions, particularly pharmaceutical or diagnostic compositions. Pharmaceutical compositions comprise a therapeutically or prophylactically effective amount of a tau-binding antibody or binding fragment thereof in admixture with a suitable carrier (e.g., a pharmaceutically acceptable agent). Diagnostic compositions comprise a diagnostically effective amount of a tau-binding antibody or binding fragment thereof in admixture with a suitable carrier (e.g., a diagnostically acceptable agent).

Pharmaceutically acceptable agents used in the pharmaceutical compositions of the present invention include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavorings and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffers, antimicrobial agents and surfactants.

The compositions may be in liquid form or in lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and/or bulking agents (see, e.g., U.S. Patents 6,685,940, 6,566,329 and 6,372,716).

The composition may be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into animals by any route available to those skilled in the art, such as intra-articular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional routes. Parenteral formulations are typically sterile, pyrogen-free, isotonic aqueous solutions optionally containing a pharmaceutically acceptable preservative.

Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Aqueous vehicles include water, alcoholic solutions/aqueous solutions, emulsions or suspensions, including physiological saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose (Ringer's dextrose), dextrose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (such as those based on Ringer's dextrose), and analogs thereof. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, inert gases and analogs thereof, may also be present. See generally Remington's Pharmaceutical Science, 16th edition, Mack, 1980, which is incorporated herein by reference.

The pharmaceutical compositions described herein can be formulated for controlled or sustained delivery in a manner that provides local concentrations of the product in a specific local environment and/or increases stability or half-life (e.g., bolus, reservoir action). Compositions can include antibodies, binding fragments, nucleic acids, or vectors of the present disclosure with formulations of particulate formulations of polymeric compounds such as polylactic acid, polyglycolic acid, and the like, and agents such as biodegradable matrices, injectable microspheres, microencapsulated particles, microcapsules, bioerodible particle beads, liposomes, and implantable delivery devices that provide controlled or sustained release of the active agent, followed by reservoir injection delivery.

Alternatively or additionally, the composition can be administered locally by implanting a membrane, sponge, or other appropriate material into the affected area that has absorbed or encapsulated the antibodies, binding fragments, nucleic acids, or vectors of the disclosure. Where an implant device is used, the device can be implanted in any suitable tissue or organ, and the antibodies, binding fragments, nucleic acids, or vectors of the disclosure can be delivered directly via the device, by bolus injection, or by continuous administration or by using a catheter for continuous infusion.

Pharmaceutical compositions comprising tau-binding antibodies or binding fragments thereof can be formulated for inhalation, such as in dry powder form. Inhalation solutions can also be formulated in liquefied propellants for aerosol delivery. In yet another formulation, the solution can be nebulized.

One aspect of the present disclosure relates to the use of Tau-binding antibodies and binding fragments thereof as therapeutic agents for treating disease.

Another aspect of the present disclosure relates to the use of Tau binding antibodies and their binding fragments for treating Tau diseases. Tau diseases containing Tau inclusions have been described (Clavaguera et al., Brain Pathology 23 (2013) 342-349) including Alzheimer's disease (AD); amyotrophic lateral sclerosis/Parkinson's disease-dementia syndrome; argyrophilic grain disease; chronic traumatic encephalopathy; corticobasal degeneration; diffuse neurofibrillary tangles with calcifications; Down syndrome; British familial dementia; Danish familial dementia; frontotemporal dementia and Parkinson's disease associated with chromosome 17 caused by MAPT mutations; Gusmann-Stausler-Scheinker disease ; Guadeloupe Parkinsonism; myotonic dystrophy; neurodegeneration with brain iron accumulation; Niemann-Pick disease type C; non-Guam motor neuron disease with neurofibrillary tangles; Pick disease; postencephalitic Parkinsonism; prion protein cerebral amyloid angiopathy; progressive subcortical gliosis; progressive supranuclear palsy (PSP); SLC9A6-related mental retardation; subacute sclerosing panencephalitis; tangle-only dementia; and white matter tauopathy with globular glial inclusions.

Another aspect of the present disclosure therefore relates to the use of Tau binding antibodies and binding fragments thereof for treating Alzheimer's disease and/or progressive supranuclear palsy.

Accordingly, the present invention also relates to methods of treating tauopathies (particularly, Alzheimer's disease and/or progressive supranuclear palsy) by administering to a subject in need thereof a therapeutically active amount of a tau-binding antibody or binding fragment thereof.

The present invention also relates to the use of a Tau-binding antibody or a binding fragment thereof for the manufacture of a medicament for treating Tauopathy (particularly, Alzheimer's disease and/or progressive supranuclear palsy).

In another aspect of the present disclosure, a Tau-binding antibody or binding fragment thereof can be used alone or in combination with other agents for treatment. For example, a Tau-binding antibody or binding fragment thereof can be co-administered with at least one other therapeutic agent. In certain aspects, the other therapeutic agent is a therapeutic agent that is effective for treating the same or a different condition when treated with the Tau-binding antibody or binding fragment thereof. Exemplary additional therapeutic agents include, but are not limited to, cholinesterase inhibitors (such as donepezil, galantamine, rivastigmine, and tacrine), NMDA receptor antagonists (such as memantine), amyloid beta peptide aggregation inhibitors, antioxidants, gamma secretase modulators, nerve growth factor (NGF) mimetics or NGF gene therapy, PPARy agonists, HMS-CoA reductase inhibitors (statins), ampaquines, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulin, muscarinic receptor agonists, nicotinic receptor modulators, active or passive amyloid beta peptide immunization, phosphodiesterase inhibitors, serotonin receptor antagonists, and anti-amyloid beta peptide antibodies or other anti-Tau antibodies. Other exemplary neurological drugs can be selected from growth hormones or neurotrophic factors; examples include, but are not limited to, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-4/5, fibroblast growth factor (FGF)-2 and other FGFs, neurotrophin (NT)-3, erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF)-α, TGF-β, vascular endothelial growth factor (VEGF), interleukin-1 receptor antagonist (IL-1ra), ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), neurturin, platelet-derived growth factor (PDGF), and glial-derived neurotrophic factor (GDNF). ), heregulin, neuregulin, artemin, persephin, interleukins, glial cell line-derived neurotrophic factor (GFR), granulocyte colony-stimulating factor (CSF), granulocyte-macrophage-CSF, axon guidance factors (netrins), cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF), midkine, pleiotrophin, bone morphogenetic protein (BMP), axon guidance factors, saponins, semaphorins and stem cell factor (SCF). In certain embodiments, at least one other therapeutic agent is selected that can alleviate one or more side effects of neurological drugs. Such combination therapies mentioned above encompass both combined administration (wherein two or more therapeutic agents are included in the same or separate formulations) and separate administration, in which case the administration of the Tau binding antibody or its binding fragment may be performed before, simultaneously and/or after the administration of the other therapeutic agents and/or adjuvants. Tau-binding antibodies or binding fragments thereof may also be used in combination with other interventional therapies, such as, but not limited to, radiation therapy, behavioral therapy, or other therapies known in the art and suitable for treating or preventing neurological disorders.

Another aspect of the present disclosure relates to the use of Tau binding antibodies and binding fragments thereof as diagnostic agents.

One aspect of the present disclosure also relates to the use of Tau-binding antibodies and binding fragments thereof for the diagnosis of tauopathies, in particular, Alzheimer's disease and/or progressive supranuclear palsy.

The term "biological sample" refers to a sample obtained from a human or animal subject and is used for diagnosis or monitoring analysis. ...

Diagnostic tests are preferably performed on biological samples that have not come into contact with the human or animal body. Such diagnostic tests are also called in vitro tests.

In vitro diagnostic tests can rely on methods for detecting Tau in a biological sample obtained from a subject in vitro, the methods comprising the following steps: i) contacting the biological sample with a Tau binding antibody or its binding fragment as described herein; and ii) detecting the binding of a Tau binding antibody or its binding fragment to Tau as described herein. By comparing the detected Tau levels with a suitable control, the presence or possibility of tauopathy (such as Alzheimer's disease and/or progressive supranuclear palsy) can then be diagnosed. Therefore, such detection methods can be used to determine whether a subject suffers from tauopathy (including determining the stage (severity) of tauopathy) or is at risk of developing tauopathy.

The present disclosure therefore provides a method for in vitro diagnosis of a tauopathy (such as Alzheimer's disease and/or progressive supranuclear palsy) in a subject, the method comprising the following steps: i) assessing the level or state of Tau in a biological sample obtained from the subject by using a Tau-binding antibody or binding fragment thereof as described herein; and ii) comparing the level or state of Tau to a reference value, a standard value, or a normal control value indicative of the level or state of Tau in a normal control subject. A significant difference between the level and/or state of Tau polypeptide in the biological sample and the normal control value indicates that the subject suffers from a tauopathy, such as Alzheimer's disease and/or progressive supranuclear palsy.

In light of the various aspects and embodiments described herein, the present disclosure encompasses, inter alia:

1. An isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises

a light chain variable region comprising a CDR1 selected from SEQ ID No.: 1 or a sequence at least 90% identical thereto, a CDR2 selected from SEQ ID No.: 2 or a sequence at least 90% identical thereto, and a CDR3 selected from SEQ ID No.: 3 or a sequence at least 90% identical thereto; and/or

A heavy chain variable region comprising a CDR1 selected from SEQ ID No.: 4 or a sequence at least 90% identical thereto, a CDR2 selected from SEQ ID No.: 5 or a sequence at least 90% identical thereto, and/or a CDR3 selected from SEQ ID No.: 6 or a sequence at least 90% identical thereto.

2. The Tau binding antibody or binding fragment thereof according to embodiment 1, wherein the Tau binding antibody or binding fragment thereof comprises

a light chain variable region comprising a CDR1 selected from SEQ ID No.: 1, a CDR2 selected from SEQ ID No.: 2, and a CDR3 selected from SEQ ID No.: 3; and

A heavy chain variable region comprising a CDR1 selected from SEQ ID No.: 4, a CDR2 selected from SEQ ID No.: 5 and/or a CDR3 selected from SEQ ID No.: 6.

3. The Tau-binding antibody or binding fragment thereof according to embodiment 1 or 2, wherein X1 in SEQ ID No.: ₃ is A.

4. The Tau-binding antibody or binding fragment thereof according to embodiment 1 or 2, wherein X1 in SEQ ID No.: ₃ is G.

5. The Tau-binding antibody or binding fragment thereof according to embodiment 1 or 2, wherein X2 in SEQ ID No.: ₆ is A.

6. The Tau binding antibody or binding fragment thereof according to embodiment 1 or 2, wherein X2 in SEQ ID No.: ₆ is Q.

7. The Tau binding antibody or binding fragment thereof according to embodiment 1 or 2, wherein X2 in SEQ ID No.: ₆ is N.

8. The Tau binding antibody or binding fragment thereof according to embodiment 1 or 2, wherein X2 in SEQ ID No.: ₆ is D.

9. The Tau binding antibody or binding fragment thereof according to embodiment 1 or 2, wherein X2 in SEQ ID No.: ₆ is S.

10. The Tau-binding antibody or binding fragment thereof of any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, or 9, wherein the Tau-binding antibody or binding fragment thereof is a monoclonal antibody.

11. The Tau-binding antibody or binding fragment thereof of embodiment 10, wherein the Tau-binding antibody or binding fragment thereof is a chimeric, humanized, or fully human antibody.

12. The Tau-binding antibody or binding fragment thereof according to embodiment 11, wherein the Tau-binding antibody or binding fragment thereof is a humanized antibody of the IgG1 or IgG4 subtype.

13. The Tau binding antibody or binding fragment thereof of any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, wherein the Tau binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197-206 of SEQ ID No.: 55.

14. The Tau-binding antibody or binding fragment thereof of any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, wherein the Tau-binding antibody or binding fragment binds to a soluble form of human Tau, paired helical filaments (PHFs) of human Tau, or both a soluble form of human Tau and paired helical filaments (PHFs) of human Tau.

15. An isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises

A light chain variable region comprising SEQ ID No.: 7 or a sequence at least 80% identical thereto, and/or

A heavy chain variable region comprising SEQ ID No.: 8 or a sequence at least 80% identical thereto.

16. The Tau binding antibody or binding fragment thereof of embodiment 15, wherein the Tau binding antibody or binding fragment thereof comprises

A light chain variable region comprising SEQ ID NO: 7, and

The heavy chain variable region comprises SEQ ID No.: 8.

17. The Tau binding antibody or binding fragment thereof according to embodiment 15 or 16, wherein X1 in SEQ ID No.: ₇ is A.

18. The Tau binding antibody or binding fragment thereof according to embodiment 15 or 16, wherein Xi in SEQ ID No.: ₇ is G.

19. The Tau binding antibody or binding fragment thereof according to embodiment 15 or 16, wherein X2 in SEQ ID No.: ₈ is A.

20. The Tau binding antibody or binding fragment thereof of embodiment 15 or 16, wherein X2 in SEQ ID No.: ₈ is Q.

21. The Tau binding antibody or binding fragment thereof of embodiment 15 or 16, wherein X2 in SEQ ID No.: ₈ is N.

22. The Tau binding antibody or binding fragment thereof of embodiment 15 or 16, wherein X2 in SEQ ID No.: ₈ is D.

23. The Tau binding antibody or binding fragment thereof of embodiment 15 or 16, wherein X2 in SEQ ID No.: ₈ is S.

24. The Tau-binding antibody or binding fragment thereof of any one of embodiments 15, 16, 17, 18, 19, 20, 21, 22, or 23, wherein the Tau-binding antibody or binding fragment thereof is a monoclonal antibody.

25. The Tau-binding antibody or binding fragment thereof of embodiment 24, wherein the Tau-binding antibody or binding fragment thereof is a chimeric antibody.

26. The Tau binding antibody or binding fragment thereof of any one of embodiments 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, wherein the Tau binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197 to 206 of SEQ ID No.: 55.

27. The Tau-binding antibody or binding fragment thereof of any one of embodiments 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, wherein the Tau-binding antibody or binding fragment binds to a soluble form of human Tau, paired helical filaments (PHFs) of human Tau, or both a soluble form of human Tau and paired helical filaments (PHFs) of human Tau.

28. An isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises

A light chain variable region comprising SEQ ID No.: 9 or a sequence at least 80% identical thereto, and/or

A heavy chain variable region comprising SEQ ID No.: 10 or a sequence at least 80% identical thereto.

29. An isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises

A light chain variable region comprising SEQ ID No.: 13 or a sequence at least 80% identical thereto, and/or

A heavy chain variable region comprising SEQ ID No.: 16 or a sequence at least 80% identical thereto.

30. The Tau binding antibody or binding fragment thereof of embodiment 29, wherein the Tau binding antibody or binding fragment thereof comprises

A light chain variable region comprising SEQ ID NO: 13, and

The heavy chain variable region comprises SEQ ID No.: 16.

31. The Tau binding antibody or binding fragment thereof of embodiment 29 or 30, wherein X1 in SEQ ID No.: ₁₃ is A.

32. The Tau binding antibody or binding fragment thereof of embodiment 29 or 30, wherein X1 in SEQ ID No.: ₁₃ is G.

33. The Tau binding antibody or binding fragment thereof of embodiment 29 or 30, wherein _X2 in SEQ ID No.: 16 is A.

34. The Tau binding antibody or binding fragment thereof of embodiment 29 or 30, wherein X2 in SEQ ID No.: ₁₆ is Q.

35. The Tau binding antibody or binding fragment thereof of embodiment 29 or 30, wherein _X2 in SEQ ID No.: 16 is N.

36. The Tau binding antibody or binding fragment thereof of embodiment 29 or 30, wherein _X2 in SEQ ID No.: 16 is D.

37. The Tau binding antibody or binding fragment thereof of embodiment 29 or 30, wherein X2 in SEQ ID No.: ₁₆ is S.

38. The Tau-binding antibody or binding fragment thereof of embodiment 29, wherein the heavy chain variable region comprises SEQ ID No.: 14 or 15.

39. The Tau-binding antibody or binding fragment thereof of embodiment 29, wherein the light chain variable region comprises SEQ ID No.: 11 or 12.

40. The Tau-binding antibody or binding fragment thereof of any one of embodiments 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39, wherein the Tau-binding antibody or binding fragment thereof is a monoclonal antibody.

41. The Tau-binding antibody or binding fragment thereof of embodiment 40, wherein the Tau-binding antibody or binding fragment thereof is a humanized antibody.

42. The Tau-binding antibody or binding fragment thereof of embodiment 41, wherein the Tau-binding antibody or binding fragment thereof has an IgG1 or IgG4 subtype.

43. The Tau-binding antibody or binding fragment thereof of any one of embodiments 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42, wherein the Tau-binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197-206 of SEQ ID No.: 55.

44. The Tau-binding antibody or binding fragment thereof of any one of embodiments 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43, wherein the Tau-binding antibody or binding fragment binds to a soluble form of human Tau, paired helical filaments (PHFs) of human Tau, or both a soluble form of human Tau and paired helical filaments (PHFs) of human Tau.

45. An isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof comprises

A light chain comprising SEQ ID No.: 19 or a sequence at least 70% identical thereto, and/or

A heavy chain comprising SEQ ID No.: 22 or a sequence at least 70% identical thereto.

46. The Tau binding antibody or binding fragment thereof of embodiment 45, wherein the Tau binding antibody or binding fragment thereof comprises

comprising a light chain of SEQ ID No.: 19, and

Comprising a heavy chain of SEQ ID No.: 22.

47. The Tau binding antibody or binding fragment thereof of embodiment 45 or 46, wherein _X1 in SEQ ID No.: 19 is A.

48. The Tau binding antibody or binding fragment thereof of embodiment 45 or 46, wherein Xi in SEQ ID No.: ₁₉ is G.

49. The Tau binding antibody or binding fragment thereof of embodiment 45 or 46, wherein X2 in SEQ ID No.: ₂₂ is A.

50. The Tau binding antibody or binding fragment thereof of embodiment 45 or 46, wherein X2 in SEQ ID No.: ₂₂ is Q.

51. The Tau binding antibody or binding fragment thereof of embodiment 45 or 46, wherein X2 in SEQ ID No.: ₂₂ is N.

52. The Tau binding antibody or binding fragment thereof of embodiment 45 or 46, wherein X2 in SEQ ID No.: ₂₂ is D.

53. The Tau binding antibody or binding fragment thereof of embodiment 45 or 46, wherein X2 in SEQ ID No.: ₂₂ is S.

54. The Tau binding antibody or binding fragment thereof of embodiment 45, wherein the heavy chain variable region comprises SEQ ID No.: 14 or 15.

55. The Tau binding antibody or binding fragment thereof of embodiment 45, wherein the light chain variable region comprises SEQ ID No.: 11 or 12.

56. The Tau-binding antibody or binding fragment thereof of any one of embodiments 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55, wherein the Tau-binding antibody or binding fragment thereof is a monoclonal humanized antibody.

57. The Tau binding antibody or binding fragment thereof of embodiment 56, wherein the Tau binding antibody or binding fragment thereof has an IgG1 or IgG4 subtype.

58. The Tau binding antibody or binding fragment thereof of any one of embodiments 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, or 57, wherein the Tau binding antibody or binding fragment thereof binds to the phosphorylated Tau region contained within amino acids 197-206 of SEQ ID No.: 55.

59. The Tau-binding antibody or binding fragment thereof of any one of embodiments 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, wherein the Tau-binding antibody or binding fragment binds to a soluble form of human Tau, paired helical filaments (PHFs) of human Tau, or both a soluble form of human Tau and paired helical filaments (PHFs) of human Tau.

60. An isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment binds to the phosphorylated Tau region contained within amino acids 197-206 of SEQ ID No.: 55.

61. The Tau-binding antibody or binding fragment thereof of embodiment 60, wherein the Tau-binding antibody or binding fragment thereof is a monoclonal antibody.

62. The Tau-binding antibody or binding fragment thereof of embodiment 60 or 61, wherein the Tau-binding antibody or binding fragment thereof is a chimeric, humanized, or fully human antibody.

63. The Tau binding antibody or binding fragment thereof of embodiment 62, wherein the Tau binding antibody or binding fragment thereof is a monoclonal humanized antibody or binding fragment thereof of the IgG1 or IgG4 subtype.

64. The Tau-binding antibody or binding fragment thereof of any one of embodiments 60, 61, 62, or 63, wherein the Tau-binding antibody or binding fragment binds to a soluble form of human Tau, paired helical filaments (PHFs) of human Tau, or both a soluble form of human Tau and paired helical filaments (PHFs) of human Tau.

65. An isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof competes for Tau binding with the Tau-binding antibody or binding fragment thereof of any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64.

66. The Tau-binding antibody or binding fragment thereof of embodiment 65, wherein the Tau-binding antibody or binding fragment thereof competes for Tau binding with a Tau-binding antibody or binding fragment comprising: a light chain variable region comprising SEQ ID NO: 11 or 12, and a heavy chain variable region comprising SEQ ID No.: 14 or 15.

67. An isolated Tau-binding antibody or binding fragment thereof, wherein the Tau-binding antibody or binding fragment thereof competes for binding to substantially the same Tau epitope as the Tau-binding antibody or binding fragment thereof of any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67.

68. The Tau-binding antibody or binding fragment thereof of embodiment 67, wherein the Tau-binding antibody or binding fragment thereof binds to substantially the same Tau epitope as a Tau-binding antibody or binding fragment comprising: a light chain variable region comprising SEQ ID NO: 11 or 12, and a heavy chain variable region comprising SEQ ID No.: 14 or 15.

69. The isolated Tau-binding antibody or binding fragment thereof of any one of embodiments 65, 66, 67 or 68, wherein the Tau-binding antibody or binding fragment thereof is a monoclonal antibody.

70. The Tau-binding antibody or binding fragment thereof of embodiment 69, wherein the Tau-binding antibody or binding fragment thereof is a chimeric, humanized, or fully human antibody.

71. The Tau-binding antibody or binding fragment thereof of embodiment 70, wherein the Tau-binding antibody or binding fragment thereof is a humanized antibody of IgG1 or IgG4 subtype.

72. The Tau binding antibody or binding fragment thereof of any one of embodiments 65, 66, 67, 68, 69, 70, or 71, wherein the Tau binding antibody or binding fragment thereof binds to a phosphorylated Tau region within amino acids 197-206 of SEQ ID No.: 55.

73. The Tau-binding antibody or binding fragment thereof of any one of embodiments 65, 66, 67, 68, 69, 70, 71 or 72, wherein the Tau-binding antibody or binding fragment binds to a soluble form of human Tau, paired helical filaments (PHFs) of human Tau, or both a soluble form of human Tau and paired helical filaments (PHFs) of human Tau.

74. As in embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73, wherein the Tau binding antibody or binding fragment thereof is a Fab, Fab', F(ab') ₂ , Fd and Fv, scFv, Fab-Fv, Fab-scFv, Fab-dsFv, Fab-scFc, scFv-scFc, dsscFv, dsscFv-scFc, a diabody, a triabody, a tetrabody, a linear antibody, or a VHH-containing antibody.

75. An isolated nucleic acid molecule encoding the protein of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74, or a light chain and/or heavy chain of a Tau-binding antibody or binding fragment thereof.

76. A cloning or expression vector comprising one or more nucleic acid sequences according to embodiment 75.

77. A host cell comprising one or more nucleic acid sequences according to embodiment 75 or one or more cloning or expression vectors according to embodiment 76.

78. The host cell according to embodiment 77, which is not a human embryonic stem cell.

79. A method of manufacturing a method as described in embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 74, or a method for producing a Tau-binding antibody or binding fragment thereof, the method comprising at least the steps of: a) culturing the host cell of embodiment 77 or 78, and b) isolating the Tau-binding antibody or binding fragment thereof.

80. An isolated Tau-binding antibody or binding fragment thereof according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 74 for use as a therapeutically active agent.

81. The isolated tau-binding antibody or binding fragment thereof of any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 74 for use in treating a tauopathy.

82. An isolated Tau binding antibody or binding fragment thereof for use in embodiment 81, wherein the tauopathy is Alzheimer's disease.

83. An isolated Tau binding antibody or binding fragment thereof for use in embodiment 81, wherein the tauopathy is progressive supranuclear palsy.

84. A method of treating tauopathy, comprising administering to a subject in need thereof an agent as described in embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 6, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 74, or a Tau binding antibody or binding fragment thereof.

85. The method of embodiment 84, wherein the tauopathy is Alzheimer's disease.

86. The method of embodiment 85, wherein the tauopathy is progressive supranuclear palsy.

87. The isolated Tau-binding antibody or binding fragment thereof of any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 74 for use as a diagnostic agent.

88. An isolated tau-binding antibody or binding fragment thereof according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 74 for use in diagnosing a tauopathy.

89. An isolated Tau binding antibody or binding fragment thereof for use in embodiment 88, wherein the tauopathy is Alzheimer's disease.

90. An isolated Tau binding antibody or binding fragment thereof for use in embodiment 88, wherein the tauopathy is progressive supranuclear palsy.

The present invention will now be described with reference to some embodiments, which, however, should not be construed as limiting.

experiment

Experiment 1 - Generation of Tau-binding Antibodies

1.1 Tau peptide design and production

Peptides and immunogens were supplied by Peptide Protein Research Ltd., Bishop's Waltham, UK, and synthesized by Fmoc solid-phase peptide chemistry according to the method of Atherton and Sheppard (Reference: Atherton, E.; Sheppard, RC (1989). Solid Phase Peptide Synthesis: A Practical Approach. Oxford, England: IRL Press). Peptides containing phosphorylated serine (pSer), phosphorylated threonine (pThr), 3-nitrotyrosine (nTyr), and N-ε-acetyl lysine (aLys) were synthesized using the Fmoc-protected precursors Fmoc-Ser(PO(OBzl)OH)-OH, Fmoc-Thr(PO(OBzl)OH)-OH, Fmoc-Tyr(3- _NO2 )-OH, and Fmoc-Lys(Ac)-OH.

Peptides were designed and used to generate Tau-binding antibodies that recognize all Tau isoforms that are post-translationally modified with phosphorylated serine (pSer), phosphorylated threonine (pThr), and/or nitrosotyrosine (nTyr); they represent residues 197 to 206, as aligned with Tau isoform 2 (SEQ ID NO: 55, Uniprot code: P10636-8, NCBI reference number: NP_005901.2): N-Acetyl-nTyr pSer pSer Pro Cys* pSer Pro Gly pThr Pro-Amide

(The peptide is called T197 and is defined in SEQ ID NO: 56).

The N- and C-termini of the peptides are capped with acetyl and amide groups, respectively, and cys* indicates a cysteine side chain sulfhydryl group, which serves as a point of attachment to a carrier protein or biotin for preparation of immunogens or analytical reagents, respectively, as described in detail below. The analytical reagent used to monitor antiserum titers was prepared by reacting an equal mass of maleimido-PEG-biotin with the peptide. Three different immunogens were prepared by reacting the peptides with the following carrier proteins: keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and ovalbumin (OVA), in which the ε-aminolysine side chains had been substituted with maleimido groups.

1.2 Immunization

Two female New Zealand white rabbits (>2 kg) were subcutaneously immunized using a syringe with 500 μg of a total peptide mixture (T197 and T211) emulsified in an equal volume of complete Freund's adjuvant (CFA) obtained by vigorous mixing. Peptides were designed to bind to KLH, OVA, and BSA and were immunized alternately. Rabbits were given two booster injections at 21-day intervals using incomplete Freund's adjuvant (IFA) and bled from the ears on day 14 after immunization. 14 days after the last booster immunization, single cell suspensions of spleen, bone marrow, and peripheral blood mononuclear cells were prepared and frozen at -80°C in 10% DMSO/FCS.

1.3B cell culture

B cell cultures were prepared using a method similar to that described by Zubler et al. (1985). Briefly, PBMC-derived B cells from immunized rabbits were cultured at a density of approximately 3,000 cells per well in barcoded 96-well tissue culture plates at 37°C in a 5% _CO2 atmosphere for seven days. The plates contained 200 μl of RPMI 1640 medium (Gibco BRL) per well, supplemented with 10% FCS (PAA Laboratories Ltd.), 2% HEPES (Sigma Aldrich), 1% L-glutamine (Gibco BRL), 1% penicillin/streptomycin solution (Gibco BRL), 0.1% β-mercaptoethanol (Gibco BRL), 3% activated spleen cell culture supernatant, and gamma-irradiated mutant EL4 murine thymoma cells (5 x ¹⁰⁴ /well). A total of approximately 1.2 x ¹⁰⁷ B cell samples were obtained.

1.4 Initial Screening

B cell culture supernatants were assayed for the presence of T197 peptide-specific antibodies using a fluorescence-based homogeneous binding assay using Superavidin ^™ beads (Bangs Laboratories) coated with biotinylated T197 peptide as the target antigen source. The screen involved transferring 10 μl of supernatant from a barcoded 96-well tissue culture plate to a barcoded 384-well black-walled assay plate containing T197 immobilized on beads (10 μl/well) using a Matrix Platemate liquid handler. Binding was visualized using a goat anti-rabbit IgG Fcγ-specific Cy-5 conjugate (Jackson). Plates were read on an Applied Biosystems 8200 Cellular Detection System.

1.5 Secondary Screening

After the initial screening, positive supernatants were pooled onto barcoded 96-well master plates using an Aviso Onyx sorting robot, and the B cells in the cell culture plates were frozen at -80°C. The master plates were then screened against the T197 peptide and streptavidin alone in an ELISA assay. This was done to determine the peptide specificity of each well and to exclude false-positive wells showing nonspecific binding to Superavidin beads. The ELISA assay involved capturing biotinylated T197 onto 384-well Maxisorp plates (ThermoScientific/Nunc) coated with _{carbonate coating buffer (dH2O + 0.16% Na2CO3 + 0.3} % _NaHCO3 ) containing streptavidin. The plates were blocked with 1% w/v PEG/PBS and then incubated with 10 ul of B cell culture supernatant per well (diluted 1:1 in blocking buffer). HRP-conjugated goat anti-rabbit IgG fc secondary antibody (Stratech Scientific Ltd/Jackson ImmunoResearch) was added to the plate, followed by visualization of binding using TMB substrate (3,3',5,5'-tetramethylbenzidine, obtained from EMD Millipore; 10 μl/well). Optical density was measured at 630 nM using a BioTek Synergy2 microplate reader. Initial binding analysis identified 880 successful results and after ELISA screening, 406 of them showed specific binding to T197. B cell supernatants that exhibited specificity for T197 were selected for further Biacore analysis to identify those with the best affinity.

1.6 Variable region restoration

In order to allow the recovery of antibody variable region genes from the selected wells of interest, a deconvolution step must be performed to identify antigen-specific B cells in designated wells containing heterogeneous B cell populations. This is achieved using the fluorescent focus method (Clargo et al., 2014). Briefly, immunoglobulin-secreting B cells obtained from positive wells are mixed with streptavidin beads (New England Biolabs) coated with biotinylated T197 peptide and a goat anti-rabbit Fcγ fragment-specific FITC conjugate (Jackson) at a final dilution of 1:1200. After static incubation at 37°C for 1 hour, antigen-specific B cells can be identified due to the presence of a fluorescent halo around the B cells. Eppendorf micromanipulators are then used to pick a plurality of these individual B cell clones identified using an Olympus microscope and deposited into PCR tubes.

The antibody variable region genes were recovered from single cells using heavy chain and light chain variable region specific primers by reverse transcription (RT)-PCR. Two rounds of PCR were performed on an Aviso Onyx liquid handling robot. Nested 2 ° PCR introduced restriction enzyme sites at 3 ' and 5 ' ends, thereby allowing the variable region to be cloned into rabbit IgG (VH) or rabbit κ (VL) mammalian expression vectors. The anti-T197 antibody gene obtained from 31 different wells was successfully cloned into the expression vector. Fectin293 (Invitrogen) was used to co-transfect heavy chain and light chain constructs into HEK-293 cells and recombinant antibodies were expressed in 30 ml volumes in 125 ml Erlenmeyer flasks. After 5-7 days of expression, supernatants were collected and purified using affinity chromatography.

Experiment 2 - Further screening of identified antibodies

2.1 Tau production in E. coli

Genes encoding different Tau isoforms were synthetically generated and codon-optimized for expression in E. coli. Standard molecular biology techniques were used to subclone into a modified pET32 vector engineered to produce Tau with an N-terminal 6His-TEV tag.

E. coli BL 21 (DE3) cells were transformed with the above vectors, and proteins were expressed using standard techniques.

E. coli cells were then recovered by centrifugation, lysed, and Tau protein was captured from the soluble fraction by affinity chromatography using NiNTA (Qiagen). The 6His tag was removed using TEV protease, followed by a second NiNTA chromatography step. Depending on the application, the purified Tau was buffer exchanged in an appropriate buffer. Samples generated for immunization were endotoxin-free using Proteus NoEndo ^™ columns (Vivaproducts).

Production of isotope-labeled Tau for nuclear magnetic resonance (NMR) studies: Protein expression was performed as described above, but minimal medium was used to incorporate ¹⁵ N, ¹³ C, and ² H into the protein. E. coli cell pellets were lysed and Tau protein was purified using a NiNTA (Qiagen) affinity chromatography step, the 6His tag was removed using TEV protease, and then the Tau protein was purified by gel filtration using a Superdex 200 unit (GE-Healthcare).

2.2 Production of Tau in HEK293

The wild-type DNA sequence was used to synthetically generate the gene encoding Tau isoform 2. It was subcloned into the expression vector pMV-10HisTEV (containing a CMV promoter), which was engineered to produce Tau with an N-terminal 10His-TEV tag, using standard molecular biology techniques.

The resulting vector was transfected using the Expi293 ^™ Expression System (Invitrogen) according to the manufacturer's protocol. This system uses Expi293F human cells derived from the HEK293 cell line.

Tau protein accumulates in the culture medium and is recovered using immobilized metal affinity chromatography (Ni Sepharose Excel) (GE Healthcare). The 10His tag is then removed using TEV protease, followed by reapplication to a Ni Sepharose column and collection of the cleaved Tau flow-through. The purified Tau is buffer exchanged into an appropriate buffer, depending on the application.

2.3 Preparation of PHF Tau fibrils using human brain samples

Paired helical filament (PHF)-Tau protein was purified from brain samples obtained from donors with Alzheimer's disease (AD), progressive supranuclear palsy (PSP), or frontotemporal dementia (FTD) according to the protocol published by Ksiezak-Reding and Wall (Neurobiology of Aging 15, 11-19, 1994). The previously described PHF-Tau enriched fraction 8 (equivalent to crude PHF-Tau before sucrose gradient centrifugation in this reference) and fraction 11 (equivalent to fraction A2, SDS-soluble PHF as described in this reference) were recovered and used for BIAcore analysis and cell analysis in Experiment 3.

2.4ELISA screening

The purified antibodies were then further screened by ELISA and Biacore to confirm the activity of the recombinant antibodies and select the highest affinity and most specific antibodies. The ELISA analysis again involved capturing biotinylated T197 onto 384-well Maxisorp plates (ThermoScientific/Nunc) coated with streptavidin-containing carbonate coating buffer ( _dH2O + 0.16% _Na2CO3 + 0.3% _NaHCO3 ). Separate plates were also coated with different Tau peptides mapped to alternative regions of the Tau molecule to examine the specificity of binding to only the T197 sequence. The plates were blocked with ₁ % w/v PEG/PBS and then incubated with several dilutions of the purified transient supernatant. An HRP-conjugated goat anti-rabbit IgGfc secondary antibody (Stratech Scientific Ltd/Jackson ImmunoResearch) was added to the plate, and binding was subsequently visualized using TMB substrate (3,3',5,5'-tetramethylbenzidine, obtained from EMD Millipore; 10 μl/well). Optical density was measured at 630 nM using a BioTek Synergy 2 microplate reader. Data for the selected antibody (AB1 with rabbit VL of SEQ ID No.: 7 and rabbit VH of SEQ ID No.: 8) are shown in Figure 1. As can be seen, AB1 exhibited highly selective binding only to T197, but not to the four peptides corresponding to other regions of the Tau molecule:

-T174N-acetyl-C*K pT P P A P K pT P P amide (SEQ ID No.: 62);

-T211N-acetyl-R pT P pS L P pT P C*amide (SEQ ID No.: 63);

-T230N-acetyl-R pT P P K pS P pS SC*amide (SEQ ID No.: 57); and

-T396N-acetyl-C*pS P V V pS G D pT pS amide (SEQ ID No.: 64);

*Position where biotin is bound.

2.5 BIAcore screening

Selected rabbit anti-Tau IgG antibody clones were transiently expressed, purified, and analyzed using the SPR Biacore T200 platform. Antibodies were first captured on a CM5 sensor chip using an immobilized goat F(ab')2 anti-rabbit Fcγ reagent. Flow cells 2, 3, and 4 showed immobilization levels between 5600-6100 RU, while blocked flow cell 1 (polydextrose only) served as a reference. Purified IgG was diluted to 0.5 μg/ml in HBS-EP+ buffer obtained from GE Healthcare and captured on the chip at a flow rate of 10 μl/min. After each capture step, analytes with peptide/streptavidin complexes, buffer controls, and dephosphorylated peptide/streptavidin complexes were injected for 180 seconds. Peptide dephosphorylation was performed using 10 μl of a 100 μM peptide solution in HBS-EP+ containing 3 mM EDTA and 1% DMSO. The solution was diluted 1:10 in HBS-N buffer (GE Healthcare) containing 0.3 mM EDTA and saturated with 2 μl of 0.5 M _MgCl₂ in EDTA. 2 μl of a 1:10 dilution of calf intestinal alkaline phosphatase (NEB, catalog number M0290S) in HBS-N was added and incubated at 37°C for one hour, followed by storage at 4°C. The phosphatase reaction was inhibited by adding 1 μl of 0.5 M EDTA to the mixture. This solution was used to prepare a dephosphorylated peptide/streptavidin complex. For the rabbit antibody having the VL region of SEQ ID No. 7 and the VH region of SEQ ID No. 8, peptide T230, obtained from the Tau region 230-238 having the amino acid sequence SEQ ID No. 57, was used as a negative control. The data were fit with a bivalent analyte model.

Table 1 shows the affinity values measured for this antibody when binding to T197 and T230 phosphorylated and dephosphorylated peptide/streptavidin complexes.

Table 1:

Selected monoclonal Fab fragments (mFabs) were prepared from a murinized mAB1 antibody with a light chain of SEQ ID No. 58 and a heavy chain of SEQ ID No. 59 using the Pierce Ficin lysis kit (Cat. No. 44980, Thermo Scientific) according to the manufacturer's protocol. The concentration of the Fab stock solution was determined using absorbance at 280 nm in Biacore analysis. Insoluble Tau protein preparations obtained from Alzheimer's patients (AD-PHF, fraction 11), HEK-derived Tau isoform-2 monomers (amino acids 1-441), and isoform-2 monomers expressed in E. coli were amine-immobilized onto a CM5 chip, and binding of the anti-Tau mFabs was measured using a Biacore T200 instrument. Immobilization was performed using HBS-EP buffer from GE Healthcare, except that AD-PHF used 10 mM acetic acid (pH 3.0). HBS-EP+ buffer was supplemented with 300 mM NaCl and 1.25% CM-Dextran (Sigma) and used as the assay buffer. While using flow cell (Fc)1 as a reference, the following RU values were obtained for Fc2-4: 44 RU for 5 μg/ml E. coli Tau, 56 RU for 5 μg/ml HEK Tau, and 500 RU for a 1:20 dilution of the AD-PHF material. Regeneration was performed using two 60-second cycles of 10 mM glycine (pH 1.7). A flow rate of 10 μl/min was used for immobilization and regeneration, while a flow rate of 30 μl/min was used for analyte binding. For AD-PHF, multiple manual injections were used to achieve 500 RU, including EDC/NHS and EtoA capping. A 90 μl analyte injection was used for either 180 seconds or 300 seconds for dissociation, with five priming cycles and 12 cycles for each mFab sample or buffer control. Eleven 1:3 dilutions of a 600 nM solution of each mFab were used with buffer.

Table 2. Binding of mFabs from mAbs and control antibody 101.4 to monomeric Tau isoform 2 expressed in E. coli, mammalian HEK293 cells, and isolated Tau PHF fibrils from Alzheimer's patients.

2.6 Epitope mapping by BIAcore

Using a Biacore 3000 instrument and HBS-EP (GE Healthcare) as the operating buffer, recombinant Tau isoform-2 monomers (amino acids 1-441) expressed and purified from HEK cells and insoluble Tau protein (AD-PHF) isolated from the brain of an Alzheimer's disease patient in the form of a paired helical filament preparation were immobilized onto a CM5 chip via amines. After activating the carboxymethyl polydextrose surface of these flow cells and the reference flow cell by injecting 70 μl of a fresh mixture of 50 mM N-hydroxysuccinimide and 200 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide at a flow rate of 10 μl/min, the former were amine-coupled to flow cells 2 and 4, respectively. Tau monomers were immobilized by injecting 160 μl of 50 μg/ml 10 mM acetate pH 5.0 buffer, while AD-PHFs were immobilized by injecting twenty 160 μl aliquots of 2 μg/ml in 10 mM acetic acid, pH 3.0. The test and control flow cell surfaces were deactivated using a pulse of 50 μl of 1 M ethanolamine.HCl, pH 8.5.

Epitope mapping was performed by pre-incubating solutions of a tau-binding humanized antibody (L17H20) with a light chain of SEQ ID NO: 17 and a heavy chain of SEQ ID NO: 20 with test peptides or buffer controls (prepared in running buffer at 200 nM and 5000 nM, respectively). These were tested in a series of sensing profile cycles at a constant flow rate of 10 μl/min by injecting 50 μl onto a reference cell, tau monomer, and AD-PHF flow cell. Response units of antibody binding were recorded as the difference between the test and reference flow cells at a reporter point taken 15 seconds after the end of each injection. At the end of each cycle, the chip was regenerated with two 20 μl injections of phosphate-buffered saline containing 1.5 M guanidine.

Table 3: Abbreviations: nY is nitrotyrosine, pS is phospho-serine; pT is phosphothreonine; N.S. is not significant at the 95% confidence level.

Antibody reactivity to the indicated test peptides is shown as the percent inhibition level of antibody binding to immobilized tau monomers or binding to AD-PHF relative to the average antibody binding value calculated from control cycles.

Test peptides were prepared and analyzed based on a peptide containing residues 196 to 206 of the Tau protein (SEQ ID NO: 65), and the effects of post-translational modifications of phosphorylated serine, phosphorylated threonine, and/or nitrosotyrosine were analyzed as defined in Table 3.

Based on this analysis, it was concluded that the minimal epitope was the region of phosphorylated Tau defined by amino acids 201 to 206 of SEQ ID NO:55 (corresponding to the motif Gly pSer Pro Gly pThr Pro), and that this epitope necessarily required the presence of phosphorylated threonine at position 205 of SEQ ID NO:55 and phosphorylated serine at position 202 of SEQ ID NO:55.

Experiment 3 - Further characterization of identified antibodies

3.1 Cell analysis

Preparation of crude soluble and insoluble fractions using Tau transgenic mice to induce Tau aggregation

In these experiments, transgenic mice expressing human Tau P301S (Allen et al., 2002 J. Neurosci. 22(21):9340-51) and human Tau P301L (Lewis et al., 2000 Nat Genet. (4):402-5.; J et al., 2001 J Biol Chem. 276(1):529-34) were used.

Crude soluble and insoluble fractions were prepared from the brains of P301S and P301L Tau transgenic mice by differential centrifugation. Briefly, brain tissue obtained from P301S (spinal cord and brainstem) and P301L (midbrain and brainstem) Tau transgenic mice was homogenized in ice-cold TBS (Fisher Scientific) in a 1.5 ml microcentrifuge tube using a handheld homogenizer Pellet Pestle Motor (Kontes) on ice. The homogenate (H) was then centrifuged at 4,000 g for 10 minutes at 4°C to remove tissue debris. The resulting supernatant (S0) was centrifuged at 20,000 g for 20 minutes at 4°C to provide a supernatant corresponding to the crude soluble fraction (S1). The remaining pellet (P1) was resuspended in 1 ml of 1% sarkosyl solution prepared in TBS, incubated at room temperature for 1 hour, and then centrifuged at 100,000 g for 1 hour at 4°C. The supernatant (S2) was discarded. The pellet (P2) was washed with 5 ml of ice-cold TBS and then resuspended in TBS to provide a crude insoluble fraction (P2').

Preparation of HEK-293-F cells expressing human Tau with the P301S mutation

HEK-293-F cells (Life Technologies) were transfected with pcDNA3.1(+) vector expressing human Tau isoform 2 with the P301S mutation using 293fectin (Life Technologies) according to the manufacturer's instructions. Aliquots of the transfected cells were stored in liquid nitrogen.

Inducing Tau aggregation

Figure 2 illustrates the different steps of a cell aggregation assay used to characterize the activity of a Tau therapeutic antibody. On day 1, HEK-293-F cells expressing human Tau isoform 2 with a P301S mutation (P301S-tau) were thawed at 37°C and diluted in 293 expression medium (Life Technologies) containing 10% fetal bovine serum and 1% penicillin-streptomycin (FFBS). Cells were counted using an automated cell counter (Vi-CELL XR, Beckman Coulter) and then plated at a density of 25,000 viable cells per well in poly-D-lysine pre-coated 96-well plates (Greiner Bio-One). Cells were maintained at 37°C in 5% _CO2 . On the same day, sonicated human insoluble Tau from patients with Alzheimer's disease (AD-PHF, fraction 8), progressive supranuclear palsy (PSP-PHF, fraction 8), or frontotemporal dementia (FTD-PHF) or brain fractions from P301S or P301L transgenic mice (used as seeds for inducing Tau aggregation) were incubated with or without anti-Tau antibodies in FFBS medium overnight at 4°C with gentle agitation. For AD and PSP samples, AD-PHF (fraction 8) was used at 80 ng/μl and 60 ng/μl, respectively; soluble brain fractions from transgenic mice P301S and P301L were used at 0.1 μg/μl and 1.2 μg/μl, respectively. On the second day, seeds or seed/antibody mixtures were applied to the cells for 24 hours. On day 3, the culture medium was replaced with fresh FFBS medium containing the antibody, and the cells were maintained in culture for another 24 hours. On day 4, Tau aggregation was measured using a Tau aggregation assay kit (Cisbio) based on homogeneous time-lapse fluorescence energy transfer (HTRF) according to the manufacturer's instructions. Fluorescence was measured using a SpectraMax Paradigm (Molecular Devices). Aggregation is reported as a percentage of aggregation relative to the control (-), which corresponds to the maximum aggregation response induced by exogenous fibrils or fractions in the absence of the antibody.

The effects of AB1 and other Tau-binding antibodies from the prior art on induced Tau aggregation were tested. The prior art antibodies are IPN002 from WO2014/028777A2, PT3 from WO2013/096380A2, mAb2.10.3 from WO2010/142423A2, and HJ8.5 from WO 2014/008404.

The results of this analysis are summarized in Table 3 and Figure 3.

Table 4 summarizes the potency (IC 50 ) and maximal efficacy (I max , 300 nM) of AB1 having a murinized VL of SEQ ID NO: 9 and a murinized VH of SEQ ID No.: 10 (VL9VH10), a Tau-binding antibody having a light chain of SEQ ID No.: 17 and a heavy chain of SEQ ID No.: 23 (L17H23), a Tau-binding antibody having a light chain of SEQ ID No.: 17 and a heavy chain of SEQ ID No.: 24 (L17H24), a Tau-binding antibody having a light chain of SEQ ID No.: 17 and a heavy chain of SEQ ID No.: 20 (L17H20), a Tau-binding antibody having a light chain of SEQ ID No.: 17 and a heavy chain of SEQ ID _No .: ₂₁ (L17H21), and a series of competing antibodies against Tau seeds obtained from various brain extracts. While Figure 3 shows the efficacy of a Tau-binding antibody having a light chain of SEQ ID No.: 17 and a heavy chain of SEQ ID No.: 20 (L17H20) and a Tau-binding antibody having a light chain of SEQ ID No.: 17 and a heavy chain of SEQ ID No.: 21 (L17H21) in a cell aggregation assay using human Tau pathological fibrils obtained from human PSP patients.

Table 4

ND: Not determined.

(*) Maximum efficacy at 100 nM

In the cell assay, further experiments were performed using Tau seeds obtained from P301L (n=2), AD (n=3), and PSP (n=3) to determine the activity of AB1 (VL9VH10) with a murine VL of SEQ ID NO: 9 and a murine VH of SEQ ID No.: 10, resulting in final _IC50 values of 15nM, 27nM, and 70nM, respectively; and _Imax values of 79%, 68%, and 57%, respectively.

3.2 Histological analysis

ABl having a rabbit VL of SEQ ID NO:7 and a rabbit VH of SEQ ID No.:8, humanized ABl having a VL of SEQ ID No.:11 and a VH of SEQ ID No.:14 (VL11VH14) or a VL of SEQ ID No.:11 and a VH of SEQ ID No.:15 (VL11VH15), and prior art antibodies IPN002, PT3, and Mab2.10.3 were analyzed, and optimal concentrations were determined using frozen sections of human hippocampus obtained from a donor with Alzheimer's disease, which had previously been shown to contain pathological Tau structures using AT8 immunostaining (such as described in Braak and Braak, 1995, Neurobiol Aging; 16(3):271-8; and Porzig et al., 2007 Biochem Biophys Res Commun; 358(2):644-9). With the exception of 101.4 (negative control antibody), AB1 and all prior art antibodies exhibited specific and concentration-dependent immunoreactivity. Based on these data, a single, optimal concentration of antibody was selected and used to screen a panel of six human brain samples. Three samples were from donors with Alzheimer's disease or from elderly donors exhibiting high levels of Tau pathology (positive Tau pathology detected using AT8 immunostaining), and three samples were from donors without Tau pathology (negative Tau pathology detected using AT8 immunostaining).

AB1, VL11VH14, VL11VH15, PT3, and Mab2.10.3 showed similar immunostaining patterns in AT8-positive samples. Specific immunostaining for neurofibrillary tangles (NFTs within neurons), cytoplasmic Tau, neuritic plaque-like structures, and neurofibrillary filaments was observed in the hippocampus and temporal cortex of positive Tau pathology samples. However, much less immunostaining was detected in AT8-negative tissues. This result suggests that these antibodies preferentially recognize pathological Tau compared to non-pathological Tau.

IPN002 provided similar signals in both positive and negative Tau pathology samples.

3.3 Western blotting

Western blotting using chemiluminescence readout: Lysates prepared from human AD and PSP were loaded onto a 10% polyacrylamide gel (20 μg protein per lane). Proteins were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and electrotransferred to a PVDF (polyvinylidene fluoride) membrane. The membrane was blocked in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH adjusted to pH 7.6 with HCl) containing 4% BSA (bovine serum albumin). The membrane was incubated overnight at 4°C with primary antibody or non-immune IgG control antibody, rinsed with TBST, incubated with a secondary antibody (mouse anti-biotin) for 1 hour, rinsed with TBST, incubated with a tertiary antibody (anti-mouse IgG-peroxidase) for 1 hour, rinsed with TBST, and developed using ECL (enhanced chemiluminescence).

mABl with the murinized VL of SEQ ID NO: 9 and the murinized VH of SEQ ID NO.: 10 binds to pathological Tau from human AD samples, but binds weakly to PSP. See FIG4 .

Experiment 4 - Humanization of identified antibodies

AB1 having a VL of SEQ ID No.: 7 and a VH of SEQ ID No.: 8 was humanized by grafting CDRs from a rabbit antibody V region onto a human germline antibody V region framework. To restore antibody activity, many framework residues from the rabbit V region were also retained in the humanized sequence. These residues can be selected using the scheme outlined by Adair et al. (1991) (Humanized antibodies. WO 91/09967). An alignment of the rabbit antibody (donor) V region sequence with the human germline (acceptor) V region sequence and the designed humanized sequence are shown in Figures 5 and 6. The CDRs grafted from the donor to the acceptor sequence were defined as those of Kabat (Kabat et al., 1987), except that the combined Chothia/Kabat definition was used for CDR-H1 (see Adair et al., 1991 Humanized antibodies. WO 91/09967).

The human V region IGKV1-39 and JK4J region (IMGT, http://www.imgt.org/) were selected as acceptors for the light chain CDRs of antibody AB1. The light chain framework residues in the grafts gL4 and gL9 were all derived from human germline genes. The gL9 graft was used to mutate CDRL3 to modify a potential deamidation site. The human V region IGHV4-39 and JH4J region (IMGT, http://www.imgt.org/) were selected as acceptors for the heavy chain CDRs of antibody AB1. Common to many rabbit antibodies, the VH gene of antibody AB1 is shorter than the selected human acceptor. When aligned with the human acceptor sequence, framework 1 in the VH region of antibody AB1 lacks the N-terminal residues retained in the humanized antibody ( Figure 6 ). Framework 3 in the AB1 rabbit VH region also lacks two residues (75 and 76) located in the loop between the D and E strands of the β sheet: in grafts gH41 and gH49, the gaps are filled with the corresponding residues from selected human acceptor sequences (lysine 75, K75; asparagine 76, N76) (Figure 6). The heavy chain framework residues in grafts gH41 and gH49 are all derived from human germline genes, but one or more of the residues are selected from the group containing residues 71 and 78 (Kabat numbering), in which the donor residues lysine (K71) and valine (V78), respectively, are retained. Retention of residues K71 and V78 is required to achieve full potency of the humanized antibody. The glutamine residue at position 1 of the human framework is replaced with glutamic acid (E1) to provide for expression and purification of homogeneous products: conversion of the N-terminal glutamine of antibodies and antibody fragments to pyroglutamic acid has been widely reported. CDRH3 was mutated using grafts gH41 and gH49 to modify potential deamidation sites.

Genes encoding many variant heavy and light chain V region sequences of various antibodies were designed and constructed by automated synthesis methods of DNA2.0 Inc. Additional variants of the heavy and light chain V regions were generated by modifying the VH and VK genes by oligonucleotide site-directed mutagenesis (in some cases, mutations within the CDRs were included to modify potential deamidation sites). For transient expression, the humanized light chain V region genes were cloned into the UCB human light chain expression vector pMhCK, which contains DNA encoding the human kappa chain constant region (Km3 allotype). The humanized heavy chain V region genes were cloned into the UCB human gamma-4 heavy chain expression vector pMhγ4P FL, which contains DNA encoding the human gamma-4 heavy chain constant region with the hinge stabilizing mutation S241P (Angal et al., Mol Immunol. 1993, 30(1): 105-8). Alternatively, humanized VH genes were cloned into the UCB γ-1 heavy chain expression vector pMhγ1FL, which contains DNA encoding the human γ-1 constant region (G1m17, 1 allotype). To evaluate the monovalent binding kinetics of humanized antibodies, humanized VH genes were also cloned into the UCB human Fab-HIS expression vector pMhFab10HIS, which contains DNA encoding the human γ-1 CH1-hinge domain with a C-terminal tag of ten histidine residues. The histidine tag facilitates affinity chromatography purification of the expressed Fab. The resulting heavy and light chain vectors were co-transfected into HEK293 suspension cells using 293Fectin (12347-019, Invitrogen), and humanized recombinant antibodies were expressed as human IgG4P, IgG1, or Fab-HIS formats.

Variant humanized antibody chains and combinations thereof are expressed and evaluated for their potency (relative to the parent antibody), their biophysical properties and suitability for downstream processing.

To stably express humanized recombinant antibodies in mammalian cells, the humanized light chain V region gene is ligated to a DNA sequence encoding a human C-κ constant region (Km3 allotype) to generate adjacent light chain genes. The humanized heavy chain gene is ligated to DNA encoding a human γ-4β heavy chain constant region or a human γ-1 heavy chain constant region (G1m17, 1 allotype) to generate adjacent heavy chain genes. The heavy and light chain genes are cloned into a mammalian expression vector.

Sequence Listing

<110> UCB BIOPHARMA sprl

<120> TAU binding antibodies

<130> PF0031-WO-PCT

<160> 65

<170> PatentIn version 3.5

<210> 1

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> CDR-L1

<400> 1

Gln Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 2

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CDR-L2

<400> 2

Ala Ala Ser Tyr Leu Ala Ser

1 5

<210> 3

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> CDR-L3

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Xaa can be A or G

<400> 3

Gln Gln Xaa Tyr Thr Arg Thr Asp Ile Asp Asn Thr

1 5 10

<210> 4

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CDR-H1

<400> 4

Gly Ile Asp Leu Ser Thr Trp Arg Met Asn

1 5 10

<210> 5

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> CDR-H2

<400> 5

Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Tyr Ala Asn Trp Ala Lys Gly

1 5 10 15

<210> 6

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> CDR-H3

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> Xaa can be A, Q, N, D or S

<400> 6

Leu Gly Ala Asn Asn Xaa Gly Tyr Pro Leu Asp Leu

1 5 10

<210> 7

<211> 110

<212> PRT

<213> Rabbit

<220>

<221> MISC_FEATURE

<222> (91)..(91)

<223> Xaa can be A or G

<400> 7

Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val Gly

1 5 10 15

Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Tyr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys

65 70 75 80

Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Xaa Tyr Thr Arg Thr Asp

85 90 95

Ile Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Val Val Glu

100 105 110

<210> 8

<211> 117

<212> PRT

<213> Rabbit

<220>

<221> MISC_FEATURE

<222> (100)..(100)

<223> Xaa can be A, Q, N, D or S

<400> 8

Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Trp Arg

20 25 30

Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly

35 40 45

Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Tyr Ala Asn Trp Ala Lys Gly

50 55 60

Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Val Thr

65 70 75 80

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Leu Gly

85 90 95

Ala Asn Asn Xaa Gly Tyr Pro Leu Asp Leu Trp Gly Pro Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 9

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> Murinization

<400> 9

Asp Val Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Gln Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Tyr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Asn Ile His Pro Val Glu Glu

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Arg Thr Asp

85 90 95

Ile Asp Asn Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 10

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> Murinization

<400> 10

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Trp

20 25 30

Arg Met Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Phe Ser Ile Ser Lys Asp Ser Thr Gln Val Phe Leu Lys Met

65 70 75 80

Asn Ser Leu Gln Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Leu

85 90 95

Gly Ala Asn Asn Asn Gly Tyr Pro Leu Asp Leu Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 11

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 11

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Tyr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Arg Thr Asp

85 90 95

Ile Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 12

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Tyr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Thr Arg Thr Asp

85 90 95

Ile Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 13

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<220>

<221> MISC_FEATURE

<222> (91)..(91)

<223> Xaa can be A or G

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Tyr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Xaa Tyr Thr Arg Thr Asp

85 90 95

Ile Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 14

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 14

Glu Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Trp

20 25 30

Arg Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Leu Gly Ala Asn Asn Gln Gly Tyr Pro Leu Asp Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 15

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 15

Glu Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Trp

20 25 30

Arg Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Leu Gly Ala Asn Asn Ala Gly Tyr Pro Leu Asp Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 16

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<220>

<221> MISC_FEATURE

<222> (103)..(103)

<223> Xaa can be A, Q, N, D or S

<400> 16

Glu Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Trp

20 25 30

Arg Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Leu Gly Ala Asn Asn Xaa Gly Tyr Pro Leu Asp Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 17

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 17

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Tyr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Arg Thr Asp

85 90 95

Ile Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105 110

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

115 120 125

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

130 135 140

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155 160

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

165 170 175

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

180 185 190

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 18

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 18

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Tyr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Thr Arg Thr Asp

85 90 95

Ile Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105 110

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

115 120 125

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

130 135 140

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155 160

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

165 170 175

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

180 185 190

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 19

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<220>

<221> MISC_FEATURE

<222> (91)..(91)

<223> Xaa can be A or G

<400> 19

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Tyr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Xaa Tyr Thr Arg Thr Asp

85 90 95

Ile Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105 110

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

115 120 125

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

130 135 140

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155 160

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

165 170 175

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

180 185 190

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 20

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 20

Glu Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Trp

20 25 30

Arg Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Leu Gly Ala Asn Asn Gln Gly Tyr Pro Leu Asp Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 21

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 21

Glu Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Trp

20 25 30

Arg Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Leu Gly Ala Asn Asn Ala Gly Tyr Pro Leu Asp Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 22

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<220>

<221> MISC_FEATURE

<222> (103)..(103)

<223> Xaa can be A, Q, N, D or S

<400> 22

Glu Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Trp

20 25 30

Arg Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Leu Gly Ala Asn Asn Xaa Gly Tyr Pro Leu Asp Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 23

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 23

Glu Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Trp

20 25 30

Arg Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Leu Gly Ala Asn Asn Gln Gly Tyr Pro Leu Asp Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 24

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 24

Glu Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Trp

20 25 30

Arg Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Leu Gly Ala Asn Asn Ala Gly Tyr Pro Leu Asp Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 25

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<220>

<221> MISC_FEATURE

<222> (103)..(103)

<223> Xaa can be A, Q, N, D or S

<400> 25

Glu Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Trp

20 25 30

Arg Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Leu Gly Ala Asn Asn Xaa Gly Tyr Pro Leu Asp Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 26

<211> 330

<212> DNA

<213> Rabbit

<400> 26

gatgttgtga tgacccagac tccagcctcc gtggaggcag ctgtgggagg cacagtcacc 60

atcaagtgcc aggccagtca gagcgttagt agttatacttag cctggtatca gcagaaacca 120

gggcagcctc ccaagctcct gatctatgct gcatcctatc tggcatccgg ggtcccatca 180

cggttcaaag gcagtggatc tgggacagag ttcactctca ccatcagcga cctggagtgt 240

gccgatgctg ccacttatta ctgtcaacag ggttatacta ggactgatat tgataatact 300

ttcggcggag ggaccaaggt ggtggtcgaa 330

<210> 27

<211> 351

<212> DNA

<213> Rabbit

<400> 27

cagtcgctgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60

tgcacagtct ctggaatcga cctcagtact tggagaatga actgggtccg ccaggctcca 120

gggaaggggc tggaatggat cggaatcatt ggtactggtg gtagaacata ctacgcgaac 180

tgggcaaaag gccgattcac catctccaaa acctcgacca cggtggatct gaaggtcacc 240

agtccgacaa ccgaggacac ggccacctat ttctgtgcca gattgggtgc taataataat 300

ggttatcctt tggacttgtg gggcccgggg accctggtca ccgtctcgag t 351

<210> 28

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> Murinization

<400> 28

gatgtcgtga tgactcagtc acccgcaagc ctggctgtgt cgctgggaca gcgggctact 60

atctcgtgcc aagcgtccca gtctgtctcc tcgtacttgg cctggtatca gcagaagcca 120

ggacagccgc cgaaactcct catctacgcc gcctcatacc ttgcgtccgg agtgccttcg 180

agattcaaag gaagcggaag cggcactgag tttaccctga acatccaccc ggtggaagaa 240

gaggacgcag ccacgtacta ctgtcaacaa gggtacaccc gcaccgatat tgacaatacc 300

ttcggtggcg ggactaagct ggaaatcaag 330

<210> 29

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Murinization

<400> 29

caagtgcagc tgaaagaatc aggaccggga ctggtggcac caagccagtc cctgtcgatc 60

acctgtactg tgtccggaat cgacctctcc acctggcgca tgaattgggt ccggcagcct 120

cccggaaagg gcctcgaatg gattggcatc atcggtactg ggggcagaac ttactacgcc 180

aattgggcga aggggcgctt ctcaatctcg aaagactcga cccaagtgtt cttgaagatg 240

aacagccttc agaccgagga taccgctacc tacttttgcg cgaggctggg agccaacaac 300

aacggttacc cgctcgatct gtggggagcc ggaacgactg tgactgtctc gagc 354

<210> 30

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> Humanization

<400> 30

gatatccaaa tgacccagtc cccatcctcc ctgtccgctt ctgtcggtga ccgcgtgacc 60

attacttgtc aagccagcca gtcggtgtcc tcatacctcg cctggtatca gcagaagccg 120

ggaaaggcgc ccaaactttt gatctacgcc gcctcgtacc tggcatccgg cgtgccgtca 180

agattcagcg ggtcgggctc cggaactgac ttcaccctga ccatcagcag cctgcagcct 240

gaggactttg ccacttacta ctgccagcag ggatacacccc ggaccgatat tgacaacacc 300

ttcggcgggg gaactaaggt cgaaatcaag 330

<210> 31

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> Humanization

<400> 31

gatatccaaa tgacccagtc cccatcctcc ctgtccgctt ctgtcggtga ccgcgtgacc 60

attacttgtc aagccagcca gtcggtgtcc tcatacctcg cctggtatca gcagaagccg 120

ggaaaggcgc ccaaactttt gatctacgcc gcctcgtacc tggcatccgg cgtgccgtca 180

agattcagcg ggtcgggctc cggaactgac ttcaccctga ccatcagcag cctgcagcct 240

gaggactttg ccacttacta ctgccagcag gcctacaccc ggaccgatat tgacaacacc 300

ttcggcgggg gaactaaggt cgaaatcaag 330

<210> 32

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Humanization

<400> 32

gagctccaac tgcaagaatc cggccctggt ctggtcaagc cctccgaaac cctgagcctc 60

acctgtactg tgtccggggat cgacttgagc acctggagga tgaactggat tcgccagcca 120

ccgggaaagg gactggagtg gatcggcatt atcggtactg ggggacggac ctactacgcc 180

aattgggcaa agggcagagt gacgatttca aaggacacct cgaagaacca ggtgtccctg 240

aaactgtcct ccgtgactgc ggctgacacc gccgtgtact attgcgcccg gctggggagcc 300

aacaaccagg gctacccgct ggatctttgg ggacagggaa ccctcgtgac tgtctcgagt 360

<210> 33

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Humanization

<400> 33

gagctccaac tgcaagaatc cggccctggt ctggtcaagc cctccgaaac cctgagcctc 60

acctgtactg tgtccggggat cgacttgagc acctggagga tgaactggat tcgccagcca 120

ccgggaaagg gactggagtg gatcggcatt atcggtactg ggggacggac ctactacgcc 180

aattgggcaa agggcagagt gacgatttca aaggacacct cgaagaacca ggtgtccctg 240

aaactgtcct ccgtgactgc ggctgacacc gccgtgtact attgcgcccg gctggggagcc 300

aacaacgccg gctacccgct ggatctttgg ggacagggaa ccctcgtgac tgtctcgagt 360

<210> 34

<211> 651

<212> DNA

<213> Artificial sequence

<220>

<223> Humanization

<400> 34

gatatccaaa tgacccagtc cccatcctcc ctgtccgctt ctgtcggtga ccgcgtgacc 60

attacttgtc aagccagcca gtcggtgtcc tcatacctcg cctggtatca gcagaagccg 120

ggaaaggcgc ccaaactttt gatctacgcc gcctcgtacc tggcatccgg cgtgccgtca 180

agattcagcg ggtcgggctc cggaactgac ttcaccctga ccatcagcag cctgcagcct 240

gaggactttg ccacttacta ctgccagcag ggatacacccc ggaccgatat tgacaacacc 300

ttcggcgggg gaactaaggt cgaaatcaag cggaccgtgg ccgctccctc cgtgttcatc 360

ttcccaccct ccgacgagca gcttaagtcc ggcaccgcct ccgtcgtgtg cctgctgaac 420

aacttctacc cccgcgaggc caaggtgcag tggaaggtgg acaacgccct gcagtccggc 480

aactcccagg aatccgtcac cgagcaggac tccaaggaca gcacctactc cctgtcctcc 540

accctgaccc tgtccaaggc cgactacgag aagcacaagg tgtacgcctg cgaagtgacc 600

caccagggcc tgtccagccc cgtgaccaag tccttcaacc ggggcgagtg c 651

<210> 35

<211> 651

<212> DNA

<213> Artificial sequence

<220>

<223> Humanization

<400> 35

gatatccaaa tgacccagtc cccatcctcc ctgtccgctt ctgtcggtga ccgcgtgacc 60

attacttgtc aagccagcca gtcggtgtcc tcatacctcg cctggtatca gcagaagccg 120

ggaaaggcgc ccaaactttt gatctacgcc gcctcgtacc tggcatccgg cgtgccgtca 180

agattcagcg ggtcgggctc cggaactgac ttcaccctga ccatcagcag cctgcagcct 240

gaggactttg ccacttacta ctgccagcag gcctacaccc ggaccgatat tgacaacacc 300

ttcggcgggg gaactaaggt cgaaatcaag cgtacggtag cggccccatc tgtcttcatc 360

ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 420

aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 480

aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 540

accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 600

catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg t 651

<210> 36

<211> 1341

<212> DNA

<213> Artificial sequence

<220>

<223> Humanization

<400> 36

gagctccaac tgcaagaatc cggccctggt ctggtcaagc cctccgaaac cctgagcctc 60

acctgtactg tgtccggggat cgacttgagc acctggagga tgaactggat tcgccagcca 120

ccgggaaagg gactggagtg gatcggcatt atcggtactg ggggacggac ctactacgcc 180

aattgggcaa agggcagagt gacgatttca aaggacacct cgaagaacca ggtgtccctg 240

aaactgtcct ccgtgactgc ggctgacacc gccgtgtact attgcgcccg gctggggagcc 300

aacaaccagg gctacccgct ggatctttgg ggacagggaa ccctcgtgac tgtctcgagt 360

gcctccacca agggcccctc cgtgttccct ctggcccctt gctcccggtc cacctccgag 420

tctaccgccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gacagtgtcc 480

tggaactctg gcgccctgac ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 540

ggcctgtact ccctgtcctc cgtcgtgacc gtgccctcct ccagcctggg caccaagacc 600

tacacctgta acgtggacca caagccctcc aacaccaagg tggacaagcg ggtggaatct 660

aagtacggcc ctccctgccc cccctgccct gcccctgaat ttctgggcgg accttccgtg 720

ttcctgttcc ccccaaagcc caaggacacc ctgatgatct cccggacccc cgaagtgacc 780

tgcgtggtgg tggacgtgtc ccaggaagat cccgaggtcc agttcaattg gtacgtggac 840

ggcgtggaag tgcacaatgc caagaccaag cccagagagg aacagttcaa ctccacctac 900

cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agagtacaag 960

tgcaaggtgt ccaacaaggg cctgccctcc agcatcgaaa agaccatctc caaggccaag 1020

ggccagcccc gcgagcccca ggtgtacacc ctgcccccta gccaggaaga gatgaccaag 1080

aaccaggtgt ccctgacctg tctggtcaag ggcttctacc cctccgacat tgccgtggaa 1140

tgggagtcca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 1200

gacggctccttcttcctgta ctctcggctg accgtggaca agtcccggtg gcaggaaggc 1260

aacgtcttct cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1320

ctgtccctga gcctgggcaa g 1341

<210> 37

<211> 1341

<212> DNA

<213> Artificial sequence

<220>

<223> Humanization

<400> 37

gagctccaac tgcaagaatc cggccctggt ctggtcaagc cctccgaaac cctgagcctc 60

acctgtactg tgtccggggat cgacttgagc acctggagga tgaactggat tcgccagcca 120

ccgggaaagg gactggagtg gatcggcatt atcggtactg ggggacggac ctactacgcc 180

aattgggcaa agggcagagt gacgatttca aaggacacct cgaagaacca ggtgtccctg 240

aaactgtcct ccgtgactgc ggctgacacc gccgtgtact attgcgcccg gctggggagcc 300

aacaacgccg gctacccgct ggatctttgg ggacagggaa ccctcgtgac tgtctcgagt 360

gcctccacca agggcccctc cgtgttccct ctggcccctt gctcccggtc cacctccgag 420

tctaccgccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gacagtgtcc 480

tggaactctg gcgccctgac ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 540

ggcctgtact ccctgtcctc cgtcgtgacc gtgccctcct ccagcctggg caccaagacc 600

tacacctgta acgtggacca caagccctcc aacaccaagg tggacaagcg ggtggaatct 660

aagtacggcc ctccctgccc cccctgccct gcccctgaat ttctgggcgg accttccgtg 720

ttcctgttcc ccccaaagcc caaggacacc ctgatgatct cccggacccc cgaagtgacc 780

tgcgtggtgg tggacgtgtc ccaggaagat cccgaggtcc agttcaattg gtacgtggac 840

ggcgtggaag tgcacaatgc caagaccaag cccagagagg aacagttcaa ctccacctac 900

cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agagtacaag 960

tgcaaggtgt ccaacaaggg cctgccctcc agcatcgaaa agaccatctc caaggccaag 1020

ggccagcccc gcgagcccca ggtgtacacc ctgcccccta gccaggaaga gatgaccaag 1080

aaccaggtgt ccctgacctg tctggtcaag ggcttctacc cctccgacat tgccgtggaa 1140

tgggagtcca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 1200

gacggctccttcttcctgta ctctcggctg accgtggaca agtcccggtg gcaggaaggc 1260

aacgtcttct cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1320

ctgtccctga gcctgggcaa g 1341

<210> 38

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> Humanization

<400> 38

gagctccaac tgcaagaatc cggccctggt ctggtcaagc cctccgaaac cctgagcctc 60

acctgtactg tgtccggggat cgacttgagc acctggagga tgaactggat tcgccagcca 120

ccgggaaagg gactggagtg gatcggcatt atcggtactg ggggacggac ctactacgcc 180

aattgggcaa agggcagagt gacgatttca aaggacacct cgaagaacca ggtgtccctg 240

aaactgtcct ccgtgactgc ggctgacacc gccgtgtact attgcgcccg gctggggagcc 300

aacaaccagg gctacccgct ggatctttgg ggacagggaa ccctcgtgac tgtctcgagt 360

gcctccacca agggcccctc cgtgttcccg ctcgctccat catcgaagtc taccagcgga 420

ggcactgcgg ctctcggttg cctcgtgaag gactacttcc cggagccggt gaccgtgtcg 480

tggaacagcg gagccctgac cagcggggtg cacacctttc cggccgtctt gcagtcaagc 540

ggcctttact ccctgtcatc agtggtgact gtcccgtcca gctcattggg aacccaaacc 600

tacatctgca atgtgaatca caaacctagc aacaccaagg ttgacaagaa agtcgagccc 660

aaatcgtgtg acaagactca cacttgtccg ccgtgcccgg cacccgaact gctgggaggt 720

cccagcgtct ttctgttccc tccaaagccg aaagacacgc tgatgatctc ccgcaccccg 780

gaggtcactt gcgtggtcgt ggacgtgtca catgaggacc cagaggtgaa gttcaattgg 840

tacgtggatg gcgtcgaagt ccacaatgcc aaaactaagc ccagagaaga acagtacaat 900

tcgacctacc gcgtcgtgtc cgtgctcacg gtgttgcatc aggattggct gaacgggaag 960

gaatacaagt gcaaagtgtc caacaaggcg ctgccggcac cgatcgagaa aactatctcc 1020

aaagcgaagg gacagcctag ggaacctcaa gtctacacgc tgccaccatc acgggatgaa 1080

ctgactaaga atcaagtctc actgacttgt ctggtgaagg ggttttaccc tagcgacatt 1140

gccgtggagt gggaatccaa cggccagcca gagaacaact acaagactac ccctccagtg 1200

ctcgactcgg atggatcgtt cttcctttac tcgaagctca ccgtggataa gtcccggtgg 1260

cagcagggaa acgtgttctc ctgctcggtg atgcatgaag ccctccataa ccactatacc 1320

caaaagtcgc tgtccctgtc gccgggaaag 1350

<210> 39

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> Humanization

<400> 39

gagctccaac tgcaagaatc cggccctggt ctggtcaagc cctccgaaac cctgagcctc 60

acctgtactg tgtccggggat cgacttgagc acctggagga tgaactggat tcgccagcca 120

ccgggaaagg gactggagtg gatcggcatt atcggtactg ggggacggac ctactacgcc 180

aattgggcaa agggcagagt gacgatttca aaggacacct cgaagaacca ggtgtccctg 240

aaactgtcct ccgtgactgc ggctgacacc gccgtgtact attgcgcccg gctggggagcc 300

aacaacgccg gctacccgct ggatctttgg ggacagggaa ccctcgtgac tgtctcgagt 360

gcctccacca agggcccctc cgtgttcccg ctcgctccat catcgaagtc taccagcgga 420

ggcactgcgg ctctcggttg cctcgtgaag gactacttcc cggagccggt gaccgtgtcg 480

tggaacagcg gagccctgac cagcggggtg cacacctttc cggccgtctt gcagtcaagc 540

ggcctttact ccctgtcatc agtggtgact gtcccgtcca gctcattggg aacccaaacc 600

tacatctgca atgtgaatca caaacctagc aacaccaagg ttgacaagaa agtcgagccc 660

aaatcgtgtg acaagactca cacttgtccg ccgtgcccgg cacccgaact gctgggaggt 720

cccagcgtct ttctgttccc tccaaagccg aaagacacgc tgatgatctc ccgcaccccg 780

gaggtcactt gcgtggtcgt ggacgtgtca catgaggacc cagaggtgaa gttcaattgg 840

tacgtggatg gcgtcgaagt ccacaatgcc aaaactaagc ccagagaaga acagtacaat 900

tcgacctacc gcgtcgtgtc cgtgctcacg gtgttgcatc aggattggct gaacgggaag 960

gaatacaagt gcaaagtgtc caacaaggcg ctgccggcac cgatcgagaa aactatctcc 1020

aaagcgaagg gacagcctag ggaacctcaa gtctacacgc tgccaccatc acgggatgaa 1080

ctgactaaga atcaagtctc actgacttgt ctggtgaagg ggttttaccc tagcgacatt 1140

gccgtggagt gggaatccaa cggccagcca gagaacaact acaagactac ccctccagtg 1200

ctcgactcgg atggatcgtt cttcctttac tcgaagctca ccgtggataa gtcccggtgg 1260

cagcagggaa acgtgttctc ctgctcggtg atgcatgaag ccctccataa ccactatacc 1320

caaaagtcgc tgtccctgtc gccgggaaag 1350

<210> 40

<211> 132

<212> PRT

<213> Rabbit

<220>

<221> MISC_FEATURE

<222> (1)..(22)

<223> Signal sequence

<400> 40

Met Asp Met Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Pro Gly Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro Ala Ser

20 25 30

Val Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser

35 40 45

Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

50 55 60

Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Ala Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

85 90 95

Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Gly Tyr Thr Arg Thr Asp Ile Asp Asn Thr Phe Gly Gly Gly Thr Lys

115 120 125

Val Val Val Glu

130

<210> 41

<211> 136

<212> PRT

<213> Rabbit

<220>

<221> MISC_FEATURE

<222> (1)..(19)

<223> Signal sequence

<400> 41

Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro

20 25 30

Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser

35 40 45

Thr Trp Arg Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

50 55 60

Trp Ile Gly Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Ala Asn Trp

65 70 75 80

Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu

85 90 95

Lys Val Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala

100 105 110

Arg Leu Gly Ala Asn Asn Asn Gly Tyr Pro Leu Asp Leu Trp Gly Pro

115 120 125

Gly Thr Leu Val Thr Val Ser Ser

130 135

<210> 42

<211> 396

<212> DNA

<213> Rabbit

<220>

<221> misc_feature

<222> (1)..(66)

<223> Signal sequence

<400> 42

atggacatga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60

agatgtgatg ttgtgatgac ccagactcca gcctccgtgg aggcagctgt gggaggcaca 120

gtcaccatca agtgccaggc cagtcagagc gttagtagtt acttagcctg gtatcagcag 180

aaaccagggc agcctcccaa gctcctgatc tatgctgcat cctatctggc atccggggtc 240

ccatcacggt tcaaaggcag tggatctggg acagagttca ctctcaccat cagcgacctg 300

gagtgtgccg atgctgccac ttattactgt caacagggtt atactaggac tgatattgat 360

aatactttcg gcggagggac caaggtggtg gtcgaa 396

<210> 43

<211> 408

<212> DNA

<213> Rabbit

<220>

<221> misc_feature

<222> (1)..(57)

<223> Signal sequence

<400> 43

atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60

tcgctggagg agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc 120

acagtctctg gaatcgacct cagtacttgg agaatgaact gggtccgcca ggctccaggg 180

aaggggctgg aatggatcgg aatcattggt actggtggta gaacatacta cgcgaactgg 240

gcaaaaggcc gattcaccat ctccaaaacc tcgaccacgg tggatctgaa ggtcaccagt 300

ccgacaaccg aggacacggc cacctatttc tgtgccagat tgggtgctaa taataatggt 360

tatcctttgg acttgtgggg cccggggacc ctggtcaccg tctcgagt 408

<210> 44

<211> 107

<212> PRT

<213> Homo sapiens

<400> 44

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 45

<211> 114

<212> PRT

<213> Homo sapiens

<400> 45

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser

20 25 30

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser

<210> 46

<211> 321

<212> DNA

<213> Homo sapiens

<400> 46

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 47

<211> 342

<212> DNA

<213> Homo sapiens

<400> 47

cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtagtagtt actactgggg ctggatccgc 120

cagcccccag ggaaggggct ggagtggatt gggagtatct attatagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagtcacc atatccgtag acacgtccaa gaaccagttc 240

tccctgaagc tgagctctgt gaccgccgca gacacggctg tgtattactg tgcgagatac 300

tttgactact ggggccaagg aaccctggtc accgtctcct ca 342

<210> 48

<211> 12

<212> PRT

<213> Rabbit

<400> 48

Leu Gly Ala Asn Asn Asn Gly Tyr Pro Leu Asp Leu

1 5 10

<210> 49

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 49

Leu Gly Ala Asn Asn Gln Gly Tyr Pro Leu Asp Leu

1 5 10

<210> 50

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 50

Leu Gly Ala Asn Asn Ala Gly Tyr Pro Leu Asp Leu

1 5 10

<210> 51

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 51

Leu Gly Ala Asn Asn Asp Gly Tyr Pro Leu Asp Leu

1 5 10

<210> 52

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 52

Leu Gly Ala Asn Asn Ser Gly Tyr Pro Leu Asp Leu

1 5 10

<210> 53

<211> 12

<212> PRT

<213> Rabbit

<400> 53

Gln Gln Gly Tyr Thr Arg Thr Asp Ile Asp Asn Thr

1 5 10

<210> 54

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Humanization

<400> 54

Gln Gln Ala Tyr Thr Arg Thr Asp Ile Asp Asn Thr

1 5 10

<210> 55

<211> 441

<212> PRT

<213> Homo sapiens

<400> 55

Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly

1 5 10 15

Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His

20 25 30

Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu

35 40 45

Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser

50 55 60

Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val

65 70 75 80

Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu

85 90 95

Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro

100 105 110

Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val

115 120 125

Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly

130 135 140

Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro

145 150 155 160

Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro

165 170 175

Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly

180 185 190

Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser

195 200 205

Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Pro Thr Arg Glu Pro Lys

210 215 220

Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys

225 230 235 240

Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val

245 250 255

Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly

260 265 270

Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln

275 280 285

Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly

290 295 300

Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser

305 310 315 320

Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln

325 330 335

Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser

340 345 350

Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn

355 360 365

Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala

370 375 380

Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser

385 390 395 400

Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser

405 410 415

Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val

420 425 430

Ser Ala Ser Leu Ala Lys Gln Gly Leu

435 440

<210> 56

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Modified

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Acetylation and nitrosylation

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Nitrosylation

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (5)..(6)

<223> Biotin conjugation position

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> Amidation

<400> 56

Tyr Ser Ser Pro Cys Ser Pro Gly Thr Pro

1 5 10

<210> 57

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Modified

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Acetylation

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> Biotin conjugation position

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> Amidation

<400> 57

Arg Thr Pro Pro Lys Ser Pro Ser Ser Cys

1 5 10

<210> 58

<211> 217

<212> PRT

<213> Mouse

<400> 58

Asp Val Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Gln Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Tyr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Asn Ile His Pro Val Glu Glu

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Arg Thr Asp

85 90 95

Ile Asp Asn Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr

100 105 110

Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu

115 120 125

Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro

130 135 140

Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn

145 150 155 160

Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr

165 170 175

Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His

180 185 190

Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile

195 200 205

Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 59

<211> 442

<212> PRT

<213> Mouse

<400> 59

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Trp

20 25 30

Arg Met Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Gly Thr Gly Gly Arg Thr Tyr Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Phe Ser Ile Ser Lys Asp Ser Thr Gln Val Phe Leu Lys Met

65 70 75 80

Asn Ser Leu Gln Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Leu

85 90 95

Gly Ala Asn Asn Asn Gly Tyr Pro Leu Asp Leu Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Ser Val Thr Val Pro Ser Ser Thr

180 185 190

Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro

210 215 220

Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys

245 250 255

Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp

260 265 270

Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser

305 310 315 320

Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

325 330 335

Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln

340 345 350

Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe

355 360 365

Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu

370 375 380

Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe

385 390 395 400

Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn

405 410 415

Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr

420 425 430

Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210> 60

<211> 651

<212> DNA

<213> Mouse

<400> 60

gatgtcgtga tgactcagtc acccgcaagc ctggctgtgt cgctgggaca gcgggctact 60

atctcgtgcc aagcgtccca gtctgtctcc tcgtacttgg cctggtatca gcagaagcca 120

ggacagccgc cgaaactcct catctacgcc gcctcatacc ttgcgtccgg agtgccttcg 180

agattcaaag gaagcggaag cggcactgag tttaccctga acatccaccc ggtggaagaa 240

gaggacgcag ccacgtacta ctgtcaacaa gggtacaccc gcaccgatat tgacaatacc 300

ttcggtggcg ggactaagct ggaaatcaag cgtacggatg ctgcaccaac tgtatccatc 360

ttcccaccat ccagtgagca gttaacatct ggaggtgcct cagtcgtgtg cttcttgaac 420

aacttctacc ccaaagacat caatgtcaag tggaagattg atggcagtga acgacaaaat 480

ggcgtcctga acagttggac tgatcaggac agcaaagaca gcacctacag catgagcagc 540

accctcacgt tgaccaagga cgagtatgaa cgacataaca gctatacctg tgaggccact 600

cacaagacat caacttcacc cattgtcaag agcttcaaca ggaatgagtg t 651

<210> 61

<211> 1326

<212> DNA

<213> Mouse

<400> 61

caagtgcagc tgaaagaatc aggaccggga ctggtggcac caagccagtc cctgtcgatc 60

acctgtactg tgtccggaat cgacctctcc acctggcgca tgaattgggt ccggcagcct 120

cccggaaagg gcctcgaatg gattggcatc atcggtactg ggggcagaac ttactacgcc 180

aattgggcga aggggcgctt ctcaatctcg aaagactcga cccaagtgtt cttgaagatg 240

aacagccttc agaccgagga taccgctacc tacttttgcg cgaggctggg agccaacaac 300

aacggttacc cgctcgatct gtggggagcc ggaacgactg tgactgtctc gagtgccaaa 360

acgacacccc catctgtcta tccactggcc cctggatctg ctgcccaaac taactccatg 420

gtgaccctgg gatgcctggt caagggctat ttccctgagc cagtgacagt gacctggaac 480

tctggatccc tgtccagcgg tgtgcacacc ttcccagctg tcctgcagtc tgacctctac 540

actctgagca gctcagtgac tgtcccctcc agcacctggc ccagcgagac cgtcacctgc 600

aacgttgccc acccggccag cagcaccaag gtggacaaga aaattgtgcc cagggattgt 660

ggttgtaagc cttgcatatg tacagtccca gaagtatcat ctgtcttcat cttcccccca 720

aagcccaagg atgtgctcac cattactctg actcctaagg tcacgtgtgt tgtggtagac 780

atcagcaagg atgatcccga ggtccagttc agctggtttg tagatgatgt ggaggtgcac 840

acagctcaga cgcaaccccg ggaggagcag ttcaacagca ctttccgctc agtcagtgaa 900

cttcccatca tgcaccagga ctggctcaat ggcaaggagt tcaaatgcag ggtcaacagt 960

gcagctttcc ctgcccccat cgagaaaacc atctccaaaa ccaaaggcag accgaaggct 1020

ccacaggtgt acaccattcc acctcccaag gagcagatgg ccaaggataa agtcagtctg 1080

acctgcatga taacagactt cttccctgaa gacattactg tggagtggca gtggaatggg 1140

cagccagcgg agaactacaa gaacactcag cccatcatgg acacagatgg ctcttacttc 1200

gtctacagca agctcaatgt gcagaagagc aactgggagg caggaaatac tttcacctgc 1260

tctgtgttac atgagggcct gcacaaccac catactgaga agagcctctc ccactctcct 1320

ggtaaa 1326

<210> 62

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Modified

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Acetylation

<220>

<221> MISC_FEATURE

<222> (1)..(2)

<223> Biotin conjugation position

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> Amidation

<400> 62

Cys Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro

1 5 10

<210> 63

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Modified

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Acetylation

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> Biotin conjugation position

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> Amidation

<400> 63

Arg Thr Pro Ser Leu Pro Thr Pro Cys

1 5

<210> 64

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Modified

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Acetylation

<220>

<221> MISC_FEATURE

<222> (1)..(2)

<223> Biotin conjugation position

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> Phosphorylation

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> Amidation

<400> 64

Cys Ser Pro Val Val Ser Gly Asp Thr Ser

1 5 10

<210> 65

<211> 10

<212> PRT

<213> Artificial sequence

<400> 65

Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro

1 5 10

Claims (13)

1. An isolated Tau-binding antibody or a Tau-binding antibody fragment thereof, wherein the Tau-binding antibody or the Tau-binding antibody fragment comprises... The light chain variable region includes CDR1 selected from SEQ ID No: 1, CDR2 selected from SEQ ID No: 2, and CDR3 selected from SEQ ID No: 53; and The heavy chain variable region includes CDR1 selected from SEQ ID No: 4, CDR2 selected from SEQ ID No: 5, and CDR3 selected from SEQ ID No: 49. 2. An isolated Tau-binding antibody or a Tau-binding antibody fragment thereof, wherein the Tau-binding antibody or the Tau-binding antibody fragment comprises... The light chain variable region shown in SEQ ID No: 11, and The heavy chain variable region shown in SEQ ID No: 14. 3. The Tau-binding antibody of claim 2 or an antibody fragment thereof binding to Tau, wherein the antibody or the antibody fragment thereof binding to Tau comprises the light chain of SEQ ID No: 17 and the heavy chain of SEQ ID No: 20. 4. The Tau-binding antibody or the antibody fragment binding Tau as claimed in any one of claims 1, 2 or 3, wherein the Tau-binding antibody or the antibody fragment binding Tau is a monoclonal humanized antibody. 5. The Tau-binding antibody or the antibody fragment thereof of any one of claims 1, 2 or 3, wherein the Tau-binding antibody or the antibody fragment thereof binds to a soluble form of human Tau, a pairing helical filament (PHF) of human Tau, or both a soluble form of human Tau and a pairing helical filament (PHF) of human Tau. 6. One or more isolated nucleic acid molecules encoding the Tau-binding antibody or a fragment of an antibody binding Tau as claimed in any one of claims 1, 2 or 3. 7. A cloning or expression vector comprising one or more nucleic acid molecules of claim 6. 8. A host cell comprising one or more nucleic acid molecules of claim 6 or one or more cloning or expression vectors of claim 7. 9. A method for manufacturing a Tau-binding antibody or an antibody fragment binding Tau according to any one of claims 1, 2, or 3, the method comprising at least the following steps: a) Culturing the host cell of claim 8, and b) Isolate the Tau-binding antibody or the antibody fragment that binds Tau. 10. Use of the isolated Tau-binding antibody or a Tau-binding antibody fragment thereof, according to any one of claims 1, 2 or 3, in the preparation of a medicament for treating tau proteinosis. 11. The use of claim 10, wherein the tau protein disease is Alzheimer's disease or progressive supranuclear palsy. 12. Use of the isolated Tau-binding antibody or a Tau-binding antibody fragment thereof, according to any one of claims 1, 2 or 3, in the preparation of a medicament for the diagnosis of tau proteinosis. 13. The use of claim 12, wherein the tau protein disease is Alzheimer's disease or progressive supranuclear palsy.