Humanized anti-Tau (pS422) antibodies and methods of use

Abstract

The present invention provides humanized anti-Tau (pS422) antibodies and methods of use.

Description

[0001] This application is a divisional application of PCT application PCT / EP2016 / 064465, which was filed on June 22, 2016 and whose invention name is “Humanized anti-Tau (pS422) antibodies and methods of use”. The date on which the PCT application entered the Chinese national phase is December 22, 2017, and the application number is 201680036961.5. Field of the Invention

[0002] The present invention relates to a humanized anti-Tau (pS422) antibody that specifically binds to the phosphorylated Tau fragment of SEQ ID NO: 03, and use thereof for treating brain diseases. background

[0003] Human Tau (microtubule-associated protein Tau (neurofibrillary tangles, paired helical filaments-Tau (Pairedhelicalfilament-Tau), PHF-Tau)) is a microtubule-associated protein of neurons found primarily in axons, whose function is to promote tubulin polymerization and stabilize microtubules. Eight isoforms are found in the human brain (isoforms A, B, C, D, E, F, G, fetal-Tau), the longest isoform containing 441 amino acids (isoform F, Uniprot P10636-8). Tau and its properties are also described by Reynolds, CH et al., J. Neurochem. 69 (1997) 191-198.

[0004] Tau in its hyperphosphorylated form is the main component of paired helical filaments (PHFs), the structural units of neurofibrillary lesions in the brain of Alzheimer's disease (AD). Tau can be phosphorylated at its serine or threonine residues by several different kinases, including GSK3β, cdk5, MARK, and members of the MAP kinase family.

[0005] Tauopathies are characterized by abnormal hyperphosphorylation of Tau and according to Iqbal, K. et al. (Biochim. Biophysica Acta 1739 (2005) 198-210), tauopathies are:

[0006] Alzheimer disease, including the tangle-only form of the disease

[0007] Down syndrome, adult cases

[0008] Guam Parkinsonism dementia complex

[0009] Dementia pugilistica

[0010] Pick disease

[0011] Dementia with argyrophilic grains

[0012] Frontotemporal dementia

[0013] Cortico-basal degeneration

[0014] Pallido-ponto-nigral degeneration

[0015] Progressive supranuclear palsy

[0016] Gerstmann-Schwann disease with tangles -Scheinker disease with tangles).

[0017] To date, nearly 40 serine (S) / threonine (T) phosphorylation sites have been found in the brain of Alzheimer's disease (Hanger, DP et al., J. Biol. Chem. 282 (2007) 23645-23654). The development of Tau pathology in Alzheimer's disease is related to its phosphorylation state. However, most of the 40 phosphorylation sites are not related to disease pathology because they are also found in Tau extracted from healthy, fetal brain tissue. Only some phosphorylation sites are specific to the disease state, and it is speculated that they are responsible for the abnormal, aggregation and characteristic insolubility that define Tau in Alzheimer's brain PHFs (Morishima-Kawashima, M. et al., J. Biol. Chem. 270 (1995) 823-829). According to Pei, JJ et al., (J. Alzheimer's Disease 14 (2008) 385-392), the existing literature provides limited and unclear information on which of these sites are specific to AD brain. Pei used a series of phospho-specific antibodies to Tau and measured their levels in homogenates of the medial temporal cortex from 22 AD patients and 10 controls.

[0018] Bussiere, T. et al. (Acta Neuropathol. 97 (1999) 221-230) describe that phosphorylated serine 422 (pS422) on the Tau protein is a pathological epitope found in several diseases with neurofibrillary degeneration. Augustinack, JC et al. (Acta Neuropathol 103 (2002) 26-35) describe that pS422 is associated with the severity of neuronal pathology in Alzheimer's disease. Guillozet-Bongaarts, AJ Neurochem 97 (2006) 1005-1014 describe that phosphorylation of Tau at serine 422 is part of the maturation process of PHFs. Tau pS422 was also found to be associated with the development of pathology in various transgenic mouse models of Alzheimer's disease. Thus, Deters, N. et al., Biochem. Biophys. Res. Commun. 379 (2009) 400-405, mentioned that double transgenic Dom5 / pR5 mice showed a 7-fold increase in the number of hippocampal neurons containing a specific phosphorylated pathological S422 epitope. Goetz, J. et al. (Science 293 (2001) 1491-1495) reported that Tau phosphorylated at S422 appeared in the brain of Tau P301L transgenic mice injected with Aβ42 fibrils.

[0019] EP 2 009 104 relates to epitopes of Tau proteins present in the phosphorylation state of Tau proteins from Alzheimer's disease PHFs, and the use of said epitopes in the production of antibodies for specific detection of Alzheimer's Tau proteins. WO2002 / 062851 and US 7,446,180 relate to antibodies specific for abnormal truncated forms of Tau proteins and diagnostic and therapeutic aspects related to Alzheimer's disease and related tauopathies.

[0020] WO 1998 / 22120 relates to a method for treating a patient suffering from Alzheimer's disease, comprising the step of administering to the patient an antibody against phosphorylated Tau fragments of amino acids about 207 to about 222, amino acids about 224 to about 240, and amino acids about 390 to about 408. Animal studies using phosphorylated Tau fragments 379-408 [P-Ser396, 404] to vaccinate Tau transgenic mice are mentioned in Asuni, AA et al., J. Neuroscience 27 (2007) 9115-9129. US2008 / 0050383 relates to a method for treating and preventing Alzheimer's disease or other Tau diseases in a subject by administering Tau protein fragments.

[0021] Hasegawa, M., et al. (FEBS Lett. 384 (1996) 25-30) reported a monoclonal antibody (AP422) specific for phosphoserine 422 in the microtubule-associated protein tau.

[0022] In WO 2001 / 55725, antibodies specifically recognizing tau and antibodies specifically recognizing phospho-tau (181) are reported for use in the in vivo diagnosis of tauopathy and / or for use in a method for differentially diagnosing tauopathy relative to non-tauopathy in vivo.

[0023] In WO 2002 / 027017, antibodies prepared from polypeptide immunogens having phosphorylated serines are reported. WO 2002 / 062851 relates to antibodies specific for abnormal truncated forms of Tau protein and to diagnostic and therapeutic aspects related to Alzheimer's disease and related Tauopathies.

[0024] In WO 2004 / 016655, an antibody specific for central nervous system (CNS) Tau protein is reported, wherein the antibody specifically recognizes CNS Tau protein but does not recognize peripheral Tau protein, and wherein the antibody specifically recognizes an amino acid sequence of a connecting portion between an amino acid sequence encoded by exon 4 of a gene encoding Tau protein and an amino acid sequence encoded by exon 5 thereof as an epitope.

[0025] Monoclonal antibodies against Tau pS422 are described, for example, in EP 1 876 185. Polyclonal antibodies against Tau pS422 are commercially available (eg ProSci Inc. and Biosource International).

[0026] In WO 2006 / 055178, a method for inhibiting the phosphorylation of tau protein at Ser202 / Thr205 is reported, which comprises contacting a sample containing tau protein with an antibody or antigen-binding fragment that binds to an amyloid β-derived diffusible ligand, thereby inhibiting the phosphorylation of tau protein at Ser202 / Thr205.

[0027] Antibody preparations that specifically bind to tau phosphorylated at tyr394 and / or tyr310 are reported in WO 2007 / 019273. Animal studies in which phosphorylated Tau fragment 379-408 [P-Ser396, 404] was used to immunize Tau transgenic mice are mentioned in Asuni, AA et al., J. Neuroscience 27 (2007) 9115-9129.

[0028] EP 2 009 104 relates to an epitope of a Tau protein that appears in a phosphorylated state in the Tau protein from Alzheimer's disease PHFs, and to the use of the epitope for generating an antibody that specifically detects the Alzheimer's disease Tau protein.

[0029] US2008 / 0050383 relates to methods for treating and preventing Alzheimer's disease or other tauopathies in a subject by administering a tau protein fragment.

[0030] In WO 2010 / 037135, isolated, synthetic or recombinant polypeptides or peptides are reported, which comprise a first domain containing or consisting of a ligand or equivalent for a blood-brain barrier (BBB) ​​receptor and a second domain containing or consisting of an enzyme or composition that slows down the aggregation rate of protein aggregates, inhibits the formation of protein aggregates, or reverses, digests or dissolves protein aggregates. Antibodies capable of recognizing and binding tau protein in vitro and / or in vivo, particularly monoclonal antibodies or functional portions thereof, are reported in WO 2010 / 115843.

[0031] In WO 2011 / 026031, monoclonal antibodies or fragments thereof that specifically bind to tau oligomers and do not bind to soluble tau or tau fibrils are reported for use in treating protein diseases such as Alzheimer's disease, progressive supranuclear palsy and corticobasal degeneration. In WO 2011 / 053565, isolated antibodies that specifically bind to human tau protein phosphorylated at one or more of Ser (238) and Thr (245) are reported.

[0032] In WO 2012 / 045882, antibodies that specifically bind to phosphorylated epitopes on mammalian Tau proteins are reported, which are used to treat neurodegenerative disorders such as Tau protein pathology, and for treating or alleviating cognitive deficits. In WO 2012 / 049570, human monoclonal anti-tau antibodies or tau binding fragments thereof are reported. In WO 2012 / 106363, a method for preventing or treating Alzheimer's disease or other tau protein pathology in a subject is reported, which includes administering antibodies to people who need treatment for Alzheimer's disease or other tau protein pathology, the antibodies have specificity for abnormal forms of tau protein, the antibodies do not show binding and / or reactivity to normal tau protein, and are administered under conditions and in an amount effective to prevent or treat Alzheimer's disease or other tau protein pathology.

[0033] In WO 2012 / 149365, antibodies are reported which show reactivity to aggregated tau and substantially no reactivity to non-aggregated Tau, wherein the aggregated tau comprises at least two tau proteins cross-linked to each other at one or more cysteines directly or via a linker.

[0034] WO 2010 / 142423 reports a composition for treating Tau pathology such as Alzheimer's disease, which comprises an antibody that binds to Tau, a compound modified with serine that is phosphorylated at a specific position that specifically binds to a specific phosphorylated Tau and a fragment thereof, and a carrier.

[0035] In EP 1 876 185A, antibodies that recognize phosphorylated polypeptides are reported. In WO 2013 / 151762, humanized tau antibodies are reported. In WO 2014 / 016737, new chicken monoclonal antibodies against human phosphorylated tau and their uses are reported. In WO 2014 / 016737, new egg cloned antibodies against human phosphorylated tau and their uses are reported. In WO 2012 / 149365, antibodies selective for pathological tau dimers and pre-filamentous pathological tau oligomers and their uses in the treatment, diagnosis and monitoring of Tau protein diseases are reported.

[0036] Humanized anti-tau (pS422) antibodies and methods of use are reported in WO2015 / 091656. Bispecific anti-hapten / anti-blood-brain barrier receptor antibodies, complexes thereof and their use as blood-brain barrier shuttles are reported in WO2015 / 101586. SUMMARY OF THE INVENTION

[0037] The present invention provides anti-human Tau (pS422) antibodies, in particular humanized anti-human Tau (pS422) antibodies, and methods of using the same.

[0038] Humanized antibodies as reported herein are not obtainable by standard humanization methods. It is necessary to introduce non-standard mutations in the amino acid sequence to obtain humanized antibodies with binding properties and pharmacokinetic properties comparable to the parent rabbit antibody comprising a variable domain with an amino acid sequence of SEQ ID NO: 07 and SEQ ID NO: 11. This is particularly important because the antibodies as reported herein are intended to cross the human blood-brain barrier and act in the human brain. Therefore, in the current situation, the selection criteria for humanized antibodies that are usually applied are not strict enough for direct application.

[0039] One aspect as reported herein is a (humanized) antibody specifically binding to human Tau (pS422), wherein the antibody

[0040] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0041] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0042] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0043] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0044] v) specifically binds to human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0045] The antibodies as reported herein show selectivity with respect to human Tau phosphorylated at serine at position 422 over unphosphorylated wild-type human Tau and the Tau mutant S422A. The unphosphorylated wild-type human Tau and the Tau mutant S422A are not bound at all or are bound with lower affinity, respectively.

[0046] One aspect as reported herein is a (humanized) anti-human Tau (pS422) monoclonal antibody which is a humanized variant of the antibody Mab086 generated by grafting the HVRs of Mab086 onto human framework and constant regions, wherein selected human variable region framework residues are optionally substituted as follows:

[0047] Selected human variable region framework residues are replaced with the equivalent framework amino acid from the rabbit Mab 086 antibody when the amino acid (1) directly non-covalently binds the antigen, (2) is adjacent to an HVR region, (3) or interacts with an HVR region, or (4) participates in the VL-VH interface; or

[0048] The human framework amino acid at that position that is unusual in the human immunoglobulin is replaced by an amino acid at the equivalent position from a rabbit donor antibody or by an amino acid at the equivalent position from a more typical human immunoglobulin.

[0049] One aspect as reported herein is a (humanized) antibody that specifically binds to human Tau (pS422) comprising the amino acid sequence of SEQ ID NO: 02, wherein the antibody has inhibitory activity against Tau (pS422)-induced cytotoxicity, and wherein the antibody binds to a fragment of human Tau (pS422) protein, and wherein the fragment comprises amino acid residues 416-430 of SEQ ID NO: 02, and wherein the antibody comprises a human constant region, wherein the antibody is selected from any one of the following (1) to (2); (1)

[0051] (a) an antibody comprising a heavy chain having an amino acid sequence of SEQ ID NO: 08 as HVR-H1, an amino acid sequence of SEQ ID NO: 09 as HVR-H2, and an amino acid sequence of SEQ ID NO: 10 as HVR-H3, and a light chain having an amino acid sequence of SEQ ID NO: 70 as HVR-L1, an amino acid sequence of SEQ ID NO: 72 as HVR-L2, and an amino acid sequence of SEQ ID NO: 15 as HVR-L3;

[0052] or

[0053] (b) an antibody comprising a heavy chain having an amino acid sequence of SEQ ID NO: 08 as HVR-H1, an amino acid sequence of SEQ ID NO: 09 as HVR-H2, and an amino acid sequence of SEQ ID NO: 10 as HVR-H3, and a light chain having an amino acid sequence of SEQ ID NO: 12 as HVR-L1, an amino acid sequence of SEQ ID NO: 14 as HVR-L2, and an amino acid sequence of SEQ ID NO: 74 as HVR-L3;

[0054] or

[0055] (c) an antibody comprising a heavy chain having an amino acid sequence of SEQ ID NO: 08 as HVR-H1, an amino acid sequence of SEQ ID NO: 09 as HVR-H2, and an amino acid sequence of SEQ ID NO: 10 as HVR-H3, and a light chain having an amino acid sequence of SEQ ID NO: 71 as HVR-L1, an amino acid sequence of SEQ ID NO: 73 as HVR-L2, and an amino acid sequence of SEQ ID NO: 15 as HVR-L3;

[0056] or

[0057] (d) an antibody comprising a heavy chain having an amino acid sequence of SEQ ID NO: 77 as HVR-H1, an amino acid sequence of SEQ ID NO: 09 as HVR-H2, and an amino acid sequence of SEQ ID NO: 10 as HVR-H3, and a light chain having an amino acid sequence of SEQ ID NO: 12 as HVR-L1, an amino acid sequence of SEQ ID NO: 14 as HVR-L2, and an amino acid sequence of SEQ ID NO: 75 as HVR-L3;

[0058] (2) An antibody having one or more conservative amino acid substitutions in the antibody of (1), which has an activity equivalent to that of the antibody of (1).

[0059] One aspect as reported herein is a (humanized) antibody specifically binding to human Tau (pS422), wherein the antibody

[0060] a) comprising HVRs of SEQ ID NOs: 08, 18 and 10 in the heavy chain variable domain, or

[0061] b) comprising HVRs of SEQ ID NOs: 08, 09 and 10 in the heavy chain variable domain, or

[0062] c) HVR comprising SEQ ID NOs: 77, 09 and 10 in the heavy chain variable domain.

[0063] In a preferred embodiment the humanized antibodies as reported herein have a lysine (K) amino acid residue at position 32 (according to Kabat numbering) in the light chain variable domain.

[0064] In one embodiment, a humanized antibody

[0065] a) comprising HVRs of SEQ ID NOs: 71, 73 and 15 in the light chain variable domain, or

[0066] b) comprising HVRs of SEQ ID NOs: 70, 72 and 15 in the light chain variable domain, or

[0067] c) in the light chain variable domain comprises HVRs of SEQ ID NOs: 12, 14 and 79, or

[0068] d) HVRs comprising SEQ ID NOs: 71, 81 and 15 in the light chain variable domain.

[0069] In one embodiment, the humanized antibody as reported herein

[0070] a) comprising HVRs of SEQ ID NOs: 08, 18 and 10 in the heavy chain variable domain and HVRs of SEQ ID NOs: 13, 14 and 15 in the light chain variable domain, or

[0071] b) comprising HVRs of SEQ ID NOs: 08, 09 and 10 in the heavy chain variable domain and HVRs of SEQ ID NOs: 12, 05 and 15 in the light chain variable domain, or

[0072] c) comprising HVRs of SEQ ID NOs: 08, 09 and 10 in the heavy chain variable domain and HVRs of SEQ ID NOs: 13, 14 and 15 in the light chain variable domain, or

[0073] d) comprising HVRs of SEQ ID NOs: 08, 09 and 10 in the heavy chain variable domain and HVRs of SEQ ID NOs: 71, 73 and 15 in the light chain variable domain, or

[0074] e) comprising HVRs of SEQ ID NOs: 08, 09 and 10 in the heavy chain variable domain and HVRs of SEQ ID NOs: 70, 72 and 15 in the light chain variable domain, or

[0075] f) comprising HVRs of SEQ ID NOs: 77, 09 and 10 in the heavy chain variable domain and HVRs of SEQ ID NOs: 12, 14 and 79 in the light chain variable domain, or

[0076] g) comprising HVRs of SEQ ID NOs: 08, 09 and 10 in the heavy chain variable domain and HVRs of SEQ ID NOs: 12, 14 and 79 in the light chain variable domain, or

[0077] h) comprising HVRs of SEQ ID NOs: 77, 09 and 10 in the heavy chain variable domain and HVRs of SEQ ID NOs: 71, 81 and 15 in the light chain variable domain.

[0078] A preferred aspect is a humanized antibody that specifically binds to human Tau (pS422), wherein the antibody comprises HVRs from VH35H5 and VL31A1.

[0079] In one embodiment, such a humanized antibody that specifically binds to human Tau (pS422) comprises in the heavy chain variable domain HVRs of SEQ ID NOs: 08, 09 and 10 and in the light chain variable domain HVRs of SEQ ID NOs: 70, 72 and 15. SEQ ID NO: 70 corresponds to the sequence of HVR-L1 and has an amino acid residue lysine at position 32 according to Kabat.

[0080] In one embodiment, the humanized antibody that specifically binds to human Tau (pS422) comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO:65 and a light chain variable domain having the amino acid sequence of SEQ ID NO:66.

[0081] A preferred aspect is a humanized antibody that specifically binds to human Tau (pS422), wherein the antibody comprises HVRs from VH35H5 and VL49G1.

[0082] In a preferred embodiment, such a humanized antibody that specifically binds to human Tau (pS422) comprises in the heavy chain variable domain the HVRs of SEQ ID NOs: 08, 09 and 10 and in the light chain variable domain the HVRs of SEQ ID NOs: 71, 73 and 15. SEQ ID NO: 71 corresponds to the sequence of HVR-L1 and has an amino acid residue lysine at position 32 according to Kabat.

[0083] In one embodiment, the humanized antibody that specifically binds to human Tau (pS422) comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO:65 and a light chain variable domain having the amino acid sequence of SEQ ID NO:67.

[0084] A preferred aspect is a humanized antibody that specifically binds to human Tau (pS422), wherein the antibody comprises HVRs from VH35H5 and VL35F2.

[0085] In one embodiment, such a humanized antibody that specifically binds to human Tau (pS422) comprises in the heavy chain variable domain HVRs of SEQ ID NOs: 08, 09 and 10 and in the light chain variable domain HVRs of SEQ ID NOs: 12, 14 and 74. SEQ ID NO: 12 corresponds to the sequence of HVR-L1 and has an amino acid residue lysine at position 32 according to Kabat.

[0086] In one embodiment, the humanized antibody that specifically binds to human Tau (pS422) comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO:65 and a light chain variable domain having the amino acid sequence of SEQ ID NO:68.

[0087] A preferred aspect is a humanized antibody that specifically binds to human Tau (pS422), wherein the antibody comprises HVRs from VH76A6 and VL35G4.

[0088] In one embodiment, such a humanized antibody that specifically binds to human Tau (pS422) comprises in the heavy chain variable domain HVRs of SEQ ID NOs: 77, 09 and 10 and in the light chain variable domain HVRs of SEQ ID NOs: 12, 14 and 75. SEQ ID NO: 12 corresponds to the sequence of HVR-L1 and has an amino acid residue lysine at position 32 according to Kabat.

[0089] In one embodiment, the humanized antibody that specifically binds to human Tau (pS422) comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO:76 and a light chain variable domain having the amino acid sequence of SEQ ID NO:69.

[0090] In one embodiment, the humanized antibody as reported herein comprises

[0091] a) the heavy chain variable domain of SEQ ID NO: 20 and the light chain variable domain of SEQ ID NO: 17, or

[0092] b) the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 16, or

[0093] c) the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 17, or

[0094] d) the heavy chain variable domain of SEQ ID NO: 21 and the light chain variable domain of SEQ ID NO: 17, or

[0095] e) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 67, or

[0096] f) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 66, or

[0097] g) the heavy chain variable domain of SEQ ID NO: 76 and the light chain variable domain of SEQ ID NO: 78, or

[0098] h) the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 78, or

[0099] i) the heavy chain variable domain of SEQ ID NO: 76 and the light chain variable domain of SEQ ID NO: 80.

[0100] In a preferred embodiment, the humanized antibody comprises the heavy chain variable domain of SEQ ID NO:65 and the light chain variable domain of SEQ ID NO:66 or 67 or 68.

[0101] In a preferred embodiment, the humanized antibody comprises the heavy chain variable domain of SEQ ID NO:76 and the light chain variable domain of SEQ ID NO:69.

[0102] In one embodiment, the humanized antibody comprises the heavy chain variable domain of SEQ ID NO:76 or 19 and the light chain variable domain of SEQ ID NO:78.

[0103] In one embodiment, the antibody is used to treat Alzheimer's disease.

[0104] In one embodiment, the antibody is silent for effector function. In one embodiment, the antibody does not have effector function. In one embodiment, the antibody is of human IgG1 subclass and has mutations L234A, L235A and P329G in both heavy chains (numbered according to the EU index of Kabat).

[0105] In one embodiment, the antibody

[0106] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0107] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0108] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0109] iv) specifically binds to aggregates of human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02).

[0110] In one embodiment, the antibody has the following EC 50 value

[0111] a) 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0112] b) 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0113] c) 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0114] d) 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0115] In one embodiment, the antibody specifically binds human Tau (pS422) (SEQ ID NO: 02) and does not bind human Tau (SEQ ID NO: 01).

[0116] In one embodiment, the antibody is a monoclonal antibody.

[0117] In one embodiment, the antibody is an antibody fragment that binds human Tau (pS422) and:

[0118] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0119] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0120] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0121] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0122] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0123] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0124] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0125] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0126] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0127] In one embodiment, the antibody is

[0128] a) a full length antibody of human IgG1 subclass, or

[0129] b) a full length antibody of human IgG4 subclass, or

[0130] c) a full length antibody of human IgG1 subclass having the mutations L234A, L235A and P329G,

[0131] d) a full-length antibody of human IgG4 subclass having the mutations S228P, L235E and P329G,

[0132] e) a full length antibody of human IgG1 subclass having the mutations L234A, L235A and P329G in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain, or

[0133] f) a full length antibody of human IgG4 subclass having in both heavy chains the mutations S228P and P329G and in one heavy chain the mutations T366W and S354C and in the respective other heavy chain the mutations T366S, L368A, Y407V and Y349C.

[0134] One aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that

[0135] a) the antibody comprises two antibody heavy chains, each comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0136] i) the variable domain comprises HVRs of SEQ ID NO: 08, SEQ ID NO: 18 and SEQ ID NO: 10,

[0137] ii) the constant region is a human IgG1 constant region, in which the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0138] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0139] b) the antibody comprises two antibody light chains, each comprising a light chain variable domain and a light chain constant domain, wherein

[0140] i) the variable domain comprises HVRs of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15,

[0141] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0142] and

[0143] c) Antibodies

[0144] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0145] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0146] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0147] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0148] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0149] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0150] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0151] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0152] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0153] One aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that

[0154] a) the antibody comprises two antibody heavy chains, each comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0155] i) the variable domain comprises HVRs of SEQ ID NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10,

[0156] ii) the constant region is a human IgG1 constant region, in which the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0157] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0158] b) the antibody comprises two antibody light chains, each comprising a light chain variable domain and a light chain constant domain, wherein

[0159] i) the variable domain comprises HVRs of SEQ ID NO: 12, SEQ ID NO: 05 and SEQ ID NO: 15,

[0160] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0161] and

[0162] c) Antibodies

[0163] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0164] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0165] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0166] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0167] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0168] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0169] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0170] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0171] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0172] One aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that

[0173] a) the antibody comprises two antibody heavy chains, each comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0174] i) the variable domain comprises HVRs of SEQ ID NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10,

[0175] ii) the constant region is a human IgG1 constant region, in which the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0176] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0177] b) the antibody comprises two antibody light chains, each comprising a light chain variable domain and a light chain constant domain, wherein

[0178] i) the variable domain comprises HVRs of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15,

[0179] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0180] and

[0181] c) Antibodies

[0182] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0183] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0184] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0185] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0186] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0187] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0188] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0189] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0190] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0191] One preferred aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that

[0192] a) the antibody comprises two antibody heavy chains, each comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0193] i) the variable domain comprises HVRs of SEQ ID NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10,

[0194] ii) the constant region is a human IgG1 constant region, in which the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0195] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0196] b) the antibody comprises two antibody light chains, each comprising a light chain variable domain and a light chain constant domain, wherein

[0197] i) the variable domain comprises HVRs of SEQ ID NO: 71, SEQ ID NO: 73 and SEQ ID NO: 15,

[0198] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0199] and

[0200] c) Antibodies

[0201] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0202] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0203] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0204] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0205] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0206] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0207] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0208] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0209] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0210] One preferred aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that

[0211] a) the antibody comprises two antibody heavy chains, each comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0212] i) the variable domain comprises HVRs of SEQ ID NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10,

[0213] ii) the constant region is a human IgG1 constant region, in which the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0214] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0215] b) the antibody comprises two antibody light chains, each comprising a light chain variable domain and a light chain constant domain, wherein

[0216] i) the variable domain comprises HVRs of SEQ ID NO: 70, SEQ ID NO: 72 and SEQ ID NO: 15,

[0217] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0218] and

[0219] c) Antibodies

[0220] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0221] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0222] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0223] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0224] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0225] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0226] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0227] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0228] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0229] One aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that

[0230] a) the antibody comprises two antibody heavy chains, each comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0231] i) the variable domain comprises HVRs of SEQ ID NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10,

[0232] ii) the constant region is a human IgG1 constant region, in which the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0233] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0234] b) the antibody comprises two antibody light chains, each comprising a light chain variable domain and a light chain constant domain, wherein

[0235] i) the variable domain comprises HVRs of SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 79,

[0236] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0237] and

[0238] c) Antibodies

[0239] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0240] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0241] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0242] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0243] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0244] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0245] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0246] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0247] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0248] One preferred aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that

[0249] a) the antibody comprises two antibody heavy chains, each comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0250] i) the variable domain comprises HVRs of SEQ ID NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10,

[0251] ii) the constant region is a human IgG1 constant region, in which the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0252] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0253] b) the antibody comprises two antibody light chains, each comprising a light chain variable domain and a light chain constant domain, wherein

[0254] i) the variable domain comprises HVRs of SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 74,

[0255] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0256] and

[0257] c) Antibodies

[0258] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0259] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0260] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0261] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0262] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0263] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0264] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0265] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0266] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0267] One preferred aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that:

[0268] a) the antibody comprises two antibody heavy chains, each comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0269] i) the variable domain comprises HVRs of SEQ ID NO: 77, SEQ ID NO: 09 and SEQ ID NO: 10,

[0270] ii) the constant region is a human IgG1 constant region, in which the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0271] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0272] b) the antibody comprises two antibody light chains, each comprising a light chain variable domain and a light chain constant domain, wherein

[0273] i) the variable domain comprises HVRs of SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 75,

[0274] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0275] and

[0276] c) Antibodies

[0277] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0278] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0279] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0280] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0281] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0282] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0283] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0284] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0285] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0286] One aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that:

[0287] a) The antibody comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0288] i) the variable domain has the amino acid sequence of SEQ ID NO: 20,

[0289] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0290] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0291] b) the antibody comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein

[0292] i) the variable domain has the amino acid sequence of SEQ ID NO: 17,

[0293] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0294] and

[0295] c) Antibodies

[0296] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0297] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0298] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0299] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0300] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0301] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0302] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0303] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0304] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0305] One aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that:

[0306] a) The antibody comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0307] i) the variable domain has the amino acid sequence of SEQ ID NO: 19,

[0308] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0309] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0310] b) the antibody comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein

[0311] i) the variable domain has the amino acid sequence of SEQ ID NO: 16,

[0312] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0313] and

[0314] c) Antibodies

[0315] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0316] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0317] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0318] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0319] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0320] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0321] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0322] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0323] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0324] One aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that:

[0325] a) The antibody comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0326] i) the variable domain has the amino acid sequence of SEQ ID NO: 19,

[0327] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0328] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0329] b) the antibody comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein

[0330] i) the variable domain has the amino acid sequence of SEQ ID NO: 17,

[0331] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0332] and

[0333] c) Antibodies

[0334] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0335] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0336] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0337] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0338] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0339] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0340] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0341] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0342] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0343] One aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that:

[0344] a) The antibody comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0345] i) the variable domain has the amino acid sequence of SEQ ID NO: 21,

[0346] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0347] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0348] b) the antibody comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein

[0349] i) the variable domain has the amino acid sequence of SEQ ID NO: 17,

[0350] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0351] and

[0352] c) Antibodies

[0353] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0354] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0355] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0356] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0357] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0358] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0359] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0360] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0361] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0362] One preferred aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that:

[0363] a) The antibody comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0364] i) the variable domain has the amino acid sequence of SEQ ID NO: 65,

[0365] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0366] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0367] b) the antibody comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein

[0368] i) the variable domain has the amino acid sequence of SEQ ID NO: 67,

[0369] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0370] and

[0371] c) Antibodies

[0372] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0373] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0374] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0375] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0376] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0377] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0378] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0379] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0380] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0381] One preferred aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that:

[0382] a) The antibody comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0383] i) the variable domain has the amino acid sequence of SEQ ID NO: 65,

[0384] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0385] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0386] b) the antibody comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein

[0387] i) the variable domain has the amino acid sequence of SEQ ID NO: 66,

[0388] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0389] and

[0390] c) Antibodies

[0391] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0392] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0393] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0394] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0395] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0396] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0397] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0398] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0399] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0400] One preferred aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that:

[0401] a) The antibody comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0402] i) the variable domain has the amino acid sequence of SEQ ID NO: 65,

[0403] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0404] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0405] b) the antibody comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein

[0406] i) the variable domain has the amino acid sequence of SEQ ID NO: 68,

[0407] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0408] and

[0409] c) Antibodies

[0410] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0411] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0412] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0413] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0414] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0415] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0416] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0417] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0418] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0419] One aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that:

[0420] a) The antibody comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0421] i) the variable domain has the amino acid sequence of SEQ ID NO: 19,

[0422] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0423] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0424] b) the antibody comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein

[0425] i) the variable domain has the amino acid sequence of SEQ ID NO: 78,

[0426] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0427] and

[0428] c) Antibodies

[0429] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0430] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0431] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0432] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0433] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0434] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0435] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0436] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0437] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0438] One preferred aspect as reported herein is a (humanized) anti-human Tau (pS422) antibody characterized in that:

[0439] a) The antibody comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein

[0440] i) the variable domain has the amino acid sequence of SEQ ID NO: 76,

[0441] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue or glycine-lysine dipeptide may or may not be present, and

[0442] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0443] b) the antibody comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein

[0444] i) the variable domain has the amino acid sequence of SEQ ID NO: 69,

[0445] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0446] and

[0447] c) Antibodies

[0448] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0449] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0450] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0451] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0452] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0453] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0454] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0455] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0456] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0457] In a preferred embodiment of all aspects the anti-human Tau (pS422) antibody is characterized in that the antibody has a lysine (K) amino acid residue at position 32 of the light chain variable domain (according to Kabat numbering).

[0458] In a preferred embodiment of all aspects the anti-human Tau (pS422) antibody is characterized in that the antibody has valine residues at positions 4, 24 and 78 in the heavy chain variable domain.

[0459] In a preferred embodiment of all aspects the anti-human Tau (pS422) antibody is characterized in that the antibody has an arginine residue at position 71 in the heavy chain variable domain.

[0460] One aspect as reported herein is an isolated nucleic acid encoding a (humanized) antibody as reported herein. The nucleic acid comprises a nucleic acid encoding an antibody heavy chain and a nucleic acid encoding an antibody light chain. The nucleic acid comprises at least one expression cassette. Preferably, the nucleic acid comprises two expression cassettes.

[0461] One aspect as reported herein is a host cell comprising a nucleic acid as reported herein.

[0462] One aspect as reported herein is a method for producing a (humanized) antibody comprising the step of culturing a host cell as reported herein such that the antibody is produced.

[0463] In one embodiment, the method further comprises the step of recovering the antibody from the cells or culture medium.

[0464] One aspect as reported herein is a pharmaceutical formulation comprising a (humanized) antibody as reported herein and a pharmaceutically acceptable carrier.

[0465] In one embodiment, the pharmaceutical formulation further comprises an additional therapeutic agent.

[0466] In one embodiment, the additional therapeutic agent is an anti-amyloid therapeutic agent. In one embodiment, the anti-amyloid therapeutic agent is an anti-human alpha-synuclein antibody or an anti-Abeta antibody.

[0467] One aspect as reported herein is a (humanized) antibody as reported herein for use as a medicament.

[0468] One aspect as reported herein is a (humanized) antibody as reported herein for use in the treatment of Alzheimer's disease.

[0469] One aspect as reported herein is a (humanized) antibody as reported herein for use in the treatment of prodromal Alzheimer's disease.

[0470] One aspect as reported herein is a (humanized) antibody as reported herein for use in the treatment of mild Alzheimer's disease.

[0471] One aspect as reported herein is a (humanized) antibody as reported herein for use in alleviating Tau(pS422)-induced neurodegeneration.

[0472] One aspect as reported herein is a (humanized) antibody as reported herein for use in maintaining cognition and function.

[0473] One aspect as reported herein is a (humanized) antibody as reported herein for use in slowing the rate of cognitive and functional decline.

[0474] One aspect as reported herein is a (humanized) antibody as reported herein for use in slowing down the rate of neurofibrillary tangle accumulation.

[0475] In one embodiment of the aforementioned aspects, the use is to reduce neurofibrillary tangle burden by clearing Tau (pS422).

[0476] In one embodiment of the aforementioned aspects, the use is established by preventing neurofibrillary tangles.

[0477] In one embodiment of the aforementioned aspects, the use is by removing / clearing neurofibrillary tangles.

[0478] In one embodiment, the preventing and / or removing is by promoting intracellular clearance of Tau aggregates.

[0479] In one embodiment of the aforementioned aspects, the use is by inhibiting the spread of neurofibrillary tangles. In one embodiment, the inhibition is by preventing the interneuronal transfer of pathological Tau forms / seeds.

[0480] Other aspects of the invention are methods of treatment comprising administering a (humanized) antibody as reported herein for treating Alzheimer's disease, for treating prodromal Alzheimer's disease, for treating mild Alzheimer's disease, for reducing Tau (pS422)-induced neurodegeneration, for maintaining cognition and function, for slowing the rate of cognitive and functional decline, and / or for slowing the rate of neurofibrillary tangles accumulation.

[0481] One aspect as reported herein is the use of a (humanized) antibody as reported herein for the preparation of a medicament.

[0482] In one embodiment, the medicament is for treating Alzheimer's disease.

[0483] In one embodiment, the medicament is for treating prodromal Alzheimer's disease.

[0484] In one embodiment, the medicament is for treating mild Alzheimer's disease.

[0485] In one embodiment, the medicament is for reducing Tau (pS422) induced neurodegeneration.

[0486] In one embodiment, the medicament is for maintaining cognition and function.

[0487] In one embodiment, the medicament is for slowing the rate of cognitive and functional decline.

[0488] One aspect as reported herein is a method for treating an individual suffering from Alzheimer's disease, the method comprising administering to the individual an effective amount of a (humanized) anti-human Tau (pS422) antibody as reported herein.

[0489] One aspect as reported herein is a method for reducing Tau (pS422)-induced neurodegeneration in an individual, comprising administering to the individual an effective amount of a (humanized) anti-human Tau (pS422) antibody as reported herein to reduce Tau (pS422)-induced neurodegeneration.

[0490] One aspect as reported herein is a method of maintaining cognition and function in an individual comprising administering to the individual an effective amount of a (humanized) anti-human Tau (pS422) antibody as reported herein to maintain cognition and function.

[0491] One aspect as reported herein is a method for slowing down the rate of cognitive and functional decline in an individual, comprising administering to the individual an effective amount of a (humanized) anti-human Tau (pS422) antibody as reported herein to slow down the rate of cognitive and functional decline.

[0492] One aspect as reported herein is the use of a (humanized) anti-human Tau (pS422) antibody as reported herein for alleviating Tau (pS422) induced neurodegeneration.

[0493] One aspect as reported herein is the use of a (humanized) anti-human Tau (pS422) antibody as reported herein in maintaining cognition and function.

[0494] One aspect as reported herein is the use of a (humanized) anti-human Tau (pS422) antibody as reported herein for slowing the rate of cognitive and functional decline.

[0495] The antibodies as reported herein may be used for the treatment of Alzheimer's disease.

[0496] Using the (humanized) antibodies as reported herein an inhibition / alleviation of the progression of Alzheimer's disease and neuropathology can be effected.

[0497] The (humanized) antibodies as reported herein may be used to protect an animal from developing Alzheimer's disease or even to stop the progression of Alzheimer's disease in an animal. In one embodiment, the animal is a human.

[0498] In one embodiment, the (humanized) antibody as reported herein i) binds to Tau (pS422) on brain sections of Tau (pS422) transgenic mice and Alzheimer's patients; and / or labels Tau (pS422) in Tau (pS422) transgenic cells.

[0499] The (humanized) antibodies as reported herein may be used for the treatment of Alzheimer's disease.

[0500] One aspect as reported herein is a (humanized) antibody that specifically binds to the amino acid sequence of SEQ ID NO: 03 in human Tau (pS422).

[0501] The (humanized) antibodies as reported herein specifically bind / recognize both early and late disease-associated forms of human Tau (pS422).

[0502] One aspect as reported herein is the use of a (humanized) antibody as reported herein for preventing the spread of human Tau (pS422)-associated Alzheimer's disease.

[0503] One aspect as reported herein is the use of a (humanized) antibody as reported herein for reducing lysosomal membrane breakdown.

[0504] One aspect as reported herein is the use of a (humanized) antibody as reported herein for stabilizing lysosomal membranes against human Tau (pS422)-induced destabilization and / or degradation.

[0505] One aspect as reported herein is the use of a (humanized) antibody as reported herein for preventing the progression of Alzheimer's disease.

[0506] The (humanized) antibodies as reported herein function via antibody-mediated inhibition of human Tau (pS422) seeding and cell-to-cell spread.

[0507] The (humanized) antibodies as reported herein protect lysosomes from fibrillar damage by binding to human Tau (pS422). Sequence Listing <110> F. Hoffmann-La Roche AG <120> Humanized anti-Tau (pS422) antibodies and methods of use <130> P32939 <150> EP15173511.5 <151> 2015-06-24 <160> 87 <170> PatentIn Version 3.5 <210> 1 <211> 441 <212> PRT <213> people <400> 1 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 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> 2 <211> 441 <212> PRT <213> Human <220> <221> MISC_FEATURE <222> (422)..(422) <223> X = Phosphoserine <400> 2 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 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 Xaa 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> 3 <211> 15 <212> PRT <213> Human <220> <221> MISC_FEATURE <222> (7)..(7) <223> X = Phosphoserine <400> 3 Ser Ile Asp Met Val Asp Xaa Pro Gln Leu Ala Thr Leu Ala Asp 1 5 10 15 <210> 4 <211> 12 <212> PRT <213> European rabbit <400> 4 Gln Ser Ser Gln Ser Val Arg Thr Asn Lys Leu Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> European rabbit <400> 5 Ser Ala Ser Thr Leu Asp Phe 1 5 <210> 6 <211> 13 <212> PRT <213> European rabbit <400> 6 Leu Gly Tyr Phe Asp Cys Ser Ile Ala Asp Cys Val Ala 1 5 10 <210> 7 <211> 112 <212> PRT <213> European rabbit <400> 7 Ala Gln Val Leu Thr Gln Thr Thr Ser Pro Val Ser Ala Ala Val Gly 1 5 10 15 Ser Thr Val Thr Ile Ser Cys Gln Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Ala Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val Gln 65 70 75 80 Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Cys Ser 85 90 95 Ile Ala Asp Cys Val Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 8 <211> 5 <212> PRT <213> European rabbit <400> 8 Ser Asn Ala Ile Asn 1 5 <210> 9 <211> 16 <212> PRT <213> European rabbit <400> 9 Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 <210> 10 <211> 3 <212> PRT <213> European rabbit (Oryctolagus cuniculus) <400> 10 Ser Asn Ile 1 <210> 11 <211> 108 <212> PRT <213> Rabbit <400> 11 Gln Ser Val 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 Phe Ser Leu Ser Ser Asn Ala 20 25 30 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Ala Ser Thr Thr Val Asp Leu Lys Met Thr 65 70 75 80 Ser Pro Thr Ala Glu Asp Thr Gly Thr Tyr Phe Cys Gly Lys Ser Asn 85 90 95 Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Leu 100 105 <210> 12 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 12 Arg Ser Ser Gln Ser Val Arg Thr Asn Lys Leu Ala 1 5 10 <210> 13 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 13 Arg Ser Ser Gln Ser Val Arg Thr Asn Arg Leu Ala 1 5 10 <210> 14 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 14 Ser Ala Ser Thr Leu Asp Tyr 1 5 <210> 15 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 15 Leu Gly Tyr Phe Asp Ser Ser Ala Asp Ile Val Ala 1 5 10 <210> 16 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 16 Ala Gln Val 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95 Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 17 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 17 Ala Gln Val 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Tyr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95 Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 18 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 18 Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 19 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 19 Glu Val Gln Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser Asn 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly 85 90 95 Lys Ser Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 20 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 20 Ala Gln Val 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95 Ile Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 21 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 21 Ala 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95 Ile Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> twenty two <211> 7 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> twenty two Ser Ala Ser Thr Leu Gln Ser 1 5 <210> twenty three <211> 7 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> twenty three Ser Ala Ser Thr Leu Glu Ser 1 5 <210> twenty four <211> 13 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> twenty four Leu Gly Tyr Phe Asp Ser Ser Ile Ala Asp Ser Val Ala 1 5 10 <210> 25 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 25 Leu Gly Tyr Phe Asp Ser Ser Ile Ala Asp Arg Val Ala 1 5 10 <210> 26 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 26 Leu Gly Tyr Phe Asp Pro Ser Ile Ala Asp Pro Val Ala 1 5 10 <210> 27 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 27 Leu Gly Tyr Phe Asp Ser Ser Ile Ala Asp Ile Val Ala 1 5 10 <210> 28 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 28 Leu Gly Tyr Phe Asp Pro Ser Ala Asp Pro Ile Ala 1 5 10 <210> 29 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 29 Leu Gly Tyr Phe Asp Pro Ser Ala Asp Pro Val Ala 1 5 10 <210> 30 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 30 Arg Ala Ser Gln Gly Val Arg Thr Asn Lys Leu Ala 1 5 10 <210> 31 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 31 Arg Ala Ser Gln Ser Val Arg Thr Asn Lys Leu Ala 1 5 10 <210> 32 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 32 Asp Ile Gln Met Thr Gln Ser Thr Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Cys Ser 85 90 95 Ile Ala Asp Cys Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 33 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 33 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Cys Ser 85 90 95 Ile Ala Asp Cys Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 34 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 34 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 Gly Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Cys Ser 85 90 95 Ile Ala Asp Cys Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 35 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 35 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 Gly Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95 Ile Ala Asp Ser Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 36 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 36 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 Gly Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95 Ile Ala Asp Arg Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 37 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 37 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Cys Ser 85 90 95 Ile Ala Asp Cys Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 38 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 38 Ala Gln Val 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Cys Ser 85 90 95 Ile Ala Asp Cys Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 39 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 39 Ala Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Pro Ser 85 90 95 Ala Asp Pro Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 40 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 40 Ala 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95 Ile Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 41 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 41 Ala Ile Gln Met Thr Gln Ser Thr Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95 Ile Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 42 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 42 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Pro Ser 85 90 95 Ile Ala Asp Pro Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 43 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 43 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Pro Ser 85 90 95 Ala Asp Pro Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 44 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Pro Ser 85 90 95 Ala Asp Pro Ile Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 45 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 45 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Pro Ser 85 90 95 Ala Asp Pro Ile Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 46 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 46 Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Asn Ala 20 25 30 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45 Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly Lys 85 90 95 Ser Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 47 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 47 Gln Ser Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Asn Ala 20 25 30 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45 Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly Lys 85 90 95 Ser Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 48 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 48 Gln Ser Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser Asn Ala 20 25 30 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45 Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly Lys 85 90 95 Ser Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 49 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 49 Gln Ser Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser Asn Ala 20 25 30 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45 Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Ser Thr Thr Leu Tyr Leu Gln Met Asn 65 70 75 80 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly Lys Ser Asn 85 90 95 Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 100 105 <210> 50 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 50 Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Asn Ala 20 25 30 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45 Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly Lys 85 90 95 Ser Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 51 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 51 Gln Ser Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Asn Ala 20 25 30 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45 Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly Lys 85 90 95 Ser Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 52 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 52 Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Asn Ala 20 25 30 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45 Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly Lys 85 90 95 Ser Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 53 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 53 Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Asn Ala 20 25 30 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45 Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly Lys 85 90 95 Ser Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 54 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 54 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Asn 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly 85 90 95 Lys Ser Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 55 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 55 Glu Val Gln Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser Asn 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly 85 90 95 Lys Ser Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 56 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 56 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Asn 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly 85 90 95 Lys Ser Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 57 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 57 Glu Val Gln Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser Asn 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly 85 90 95 Lys Ser Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 58 <211> 330 <212> PRT <213> Human <400> 58 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 59 <211> 107 <212> PRT <213> Human <400> 59 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 60 <211> 106 <212> PRT <213> Human <400> 60 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 61 <211> 37 <212> DNA <213> Artificial sequence <220> <223> rbHCfinal.up <400> 61 aagcttgcca ccatggagac tgggctgcgc tggcttc 37 <210> 62 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> rbHCfinal.do <400> 62 ccattggtga gggtgcccga g 21 <210> 63 <211> 34 <212> DNA <213> Artificial sequence <220> <223> rbLCfinal.up <400> 63 aagcttgcca ccatggacay gagggccccc actc 34 <210> 64 <211> 26 <212> DNA <213> Artificial sequence <220> <223> rbLCfinal.do <400> 64 cagagtrctg ctgaggttgt aggtac 26 <210> 65 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 65 Glu Val Gln Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Ser Leu Ser Ser Asn 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly 85 90 95 Lys Ser Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 66 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Humanized sequence <400> 66 Ala Gln Val 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 Ser Ser Gln Ser Val Arg Asn Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Asp Leu Asp Tyr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95 Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 67 <211> 111 <212> PRT <213> Synthetic Sequence <220> <223> Humanized Sequence <400> 67 Ala Gln Val 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 Ser Ser Gln Ser Val Arg Ser Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Asn Leu Asp Tyr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95 Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 68 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 68 Ala Gln Val 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Pro 85 90 95 Leu Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 69 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Humanized Sequence <400> 69 Ala Gln Val 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Tyr Phe Asp Pro His 85 90 95 Gln Asp Val Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 70 <211> 12 <212> PRT <213> Artificial sequence <220> <223> HVR-L1 <400> 70 Arg Ser Ser Gln Ser Val Arg Asn Asn Lys Leu Ala 1 5 10 <210> 71 <211> 12 <212> PRT <213> Artificial sequence <220> <223> HVR-L1 <400> 71 Arg Ser Ser Gln Ser Val Arg Ser Asn Lys Leu Ala 1 5 10 <210> 72 <211> 7 <212> PRT <213> Artificial sequence <220> <223> HVR-L2 <400> 72 Ser Ala Ser Asp Leu Asp Tyr 1 5 <210> 73 <211> 7 <212> PRT <213> Artificial sequence <220> <223> HVR-L2 <400> 73 Ser Ala Ser Asn Leu Asp Tyr 1 5 <210> 74 <211> 12 <212> PRT <213> Artificial sequence <220> <223> HVR-L3 <400> 74 Leu Gly Tyr Phe Asp Ser Pro Leu Asp Ile Val Ala 1 5 10 <210> 75 <211> 12 <212> PRT <213> Artificial sequence <220> <223> HVR-L3 <400> 75 Ala Gly Tyr Phe Asp Pro His Gln Asp Val Val Ala 1 5 10 <210> 76 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable domain <400> 76 Glu Val Gln Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Asn Ser 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly 85 90 95 Lys Ser Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 77 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HVR-H1 <400> 77 Asn Ser Ala Ile Asn 1 5 <210> 78 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL35G4 <400> 78 Ala Gln Val 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Tyr Phe Glu Ala Pro 85 90 95 Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 79 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> HVR-L3 <400> 79 Ala Gly Tyr Phe Glu Ala Pro Ala Asp Ile Val Ala 1 5 10 <210> 80 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL145B12 <400> 80 Ala Gln Val 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 Ser Ser Gln Ser Val Arg Ser Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ala Ala Ser Asn Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95 Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 81 <211> 7 <212> PRT <213> Artificial sequence <220> <223> HVR-L2 <400> 81 Alas, Alas, Serenity, Leu, Asp 1 5 <210> 82 <211> 124 <212> PRT <213> Artificial sequence <220> <223> 299-009 VH humanized variant_DANG <400> 82 Gln Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45 Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys 85 90 95 Arg Tyr Gly Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Asn Gly Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 83 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> 299 - 009 VH Humanized Variant_DASG <400> 83 Gln Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45 Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys 85 90 95 Arg Tyr Gly Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Ser Gly Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 84 <211> 124 <212> PRT <213> Synthetic Sequence <220> <223> 299-009 VH Humanized Variant_DAQG <400> 84 Gln Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45 Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys 85 90 95 Arg Tyr Gly Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Gln Gly Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 85 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> 299 - 009 VL Humanized Variant_NYA <400> 85 Ala Ile Gln Leu 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 Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr 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 Asn Tyr Ala Ser Ser Asn 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 86 <211> 111 <212> PRT <213> artificial sequence <220> <223> VL4G1 <400> 86 Ala Gln Val 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 Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Pro 85 90 95 Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 87 <211> 12 <212> PRT <213> Artificial sequence <220> <223> VL4G1-HVR-L3 <400> 87 Leu Gly Tyr Phe Asp Ser Pro Ala Asp Ile Val Ala 1 5 10 BRIEF DESCRIPTION OF THE DRAWINGS

[0508] Figure 1 : Pharmacokinetic study results in cynomolgus monkeys; solid diamonds: VH35H5 / VL31A1; hollow squares: VH35H5 / VL35F2; solid squares: VH76A6 / VL35G4; solid triangles: VH32 / VL22; solid circles: VH00 / VL00; X-axis: time after administration [h]; y-axis: serum concentration, dose-norm. [(μg / mL) / (mg / kg)].

[0509] Figure 2 : Zoom to Figure 1 A time range of 0-200 hours.

[0510] Figure 3: Biochemical binding of different combinations of humanized VH and VL to (A) phosphorylated tau peptide, (B) phosphorylated full-length human tau, (C) non-phosphorylated tau peptide, (D) non-phosphorylated full-length human tau; (1) = VH00 / VL00, (2) = VH32 / VL21, (3) = VH20 / VL22, (4) = VH32 / VL22, (5) = VH33 / VL22; coating concentration: phosphorylated tau peptide: 50 ng / ml, all other targets: 1 μg / ml; (comparable results were obtained if phosphorylated tau peptide was coated at 1 μg / ml (data not shown)).

[0511] Figure 4 : Biochemical binding of different combinations of humanized VH and VL to (A) = full-length human tau S422A mutant, (B) = aggregated human Tau (pS422); (1) = VH00 / VL00, (2) = VH32 / VL21, (3) = VH20 / VL22, (4) = VH32 / VL22, (5) = VH33 / VL22; coating concentration: phosphorylated tau peptide: 50 ng / ml, all other targets: 1 μg / ml; (comparable results were obtained if phosphorylated tau peptide was coated at 1 μg / ml (data not shown)).

[0512] Figure 5 : Western blot showing the selectivity of selected humanized VH / VL combinations; (1) = VH00 / VL00, (2) = VH32 / VL21, (3) = VH20 / VL22, (4) = VH32 / VL22, (5) = VH33 / VL22.

[0513] Figure 6 : Binding to hyperphosphorylated tau in brain extracts of Alzheimer's disease patients; (1) = VH00 / VL00, (2) = VH32 / VL21, (3) = VH32 / VL22.

[0514] Figure 7 : Quantification of Tau (pS442) levels in mouse brain extracts; maximum-minimum values, median and mean (+); significance calculated using Student's t-test, *p<0.05, **p<0.01, ***p<0.001.

[0515] A: x-axis: Tau (pS422) [ng / mg brain]; y-axis: 1: baseline, 2: vehicle, 3: anti-Tau (pS422) antibody (VH35H5 / VL31A1) @1.7 mg / kg, 4: anti-Tau (pS422) antibody (VH35H5 / VL31A1) @5 mg / kg, 5: anti-Tau (pS422) antibody (VH35H5 / VL31A1) @15 mg / kg.

[0516] B: x-axis: Tau (pS422) [ng / mg brain]; y-axis: 1: baseline, 2: vehicle, 3: anti-Tau (pS422) antibody (VH35H5 / VL49G1) @1.7 mg / kg, 4: anti-Tau (pS422) antibody (VH35H5 / VL49G1) @5 mg / kg, 5: anti-Tau (pS422) antibody (VH35H5 / VL49G1) @15 mg / kg.

[0517] Figure 8 : Quantification of total Tau in mouse brain extracts; maximum-minimum, median and mean (+).

[0518] A: x-axis: total Tau [ng / mg brain]; y-axis: 1: baseline, 2: vehicle, 3: anti-Tau (pS422) antibody (VH35H5 / VL49G1) @1.7 mg / kg, 4: anti-Tau (pS422) antibody (VH35H5 / VL49G1) @5 mg / kg, 5: anti-Tau (pS422) antibody (VH35H5 / VL49G1) @15 mg / kg.

[0519] B: x-axis: total Tau [ng / mg brain]; y-axis: 1: baseline, 2: vehicle, 3: anti-Tau (pS422) antibody (VH35H5 / VL31A1) @ 1.7 mg / kg, 4: anti-Tau (pS422) antibody (VH35H5 / VL31A1) @ 5 mg / kg, 5: anti-Tau (pS422) antibody (VH35H5 / VL31A1) @ 15 mg / kg.

[0520] Fig. 9 : Quantitative IHC analysis of Tau (pS422) pathology in TauPS2APP mice treated with anti-Tau (pS422) antibody (VH35H5 / VL31A1); maximum-minimum values, median and mean (+), significance calculated using Student's t-test, **p<0.01, ***p<0.001.

[0521] A: cortex; x-axis: Tau (pS422) area occupancy [%]; y-axis: 1: baseline, 2: vehicle, 3: anti-Tau (pS422) antibody (VH35H5 / VL31A1) @1.7 mg / kg, 4: anti-Tau (pS422) antibody (VH35H5 / VL31A1) @5 mg / kg, 5: anti-Tau (pS422) antibody (VH35H5 / VL31A1) @15 mg / kg.

[0522] B: Hippocampus; x-axis: area occupancy [%]; y-axis: 1: baseline, 2: vehicle, 3: anti-Tau (pS422) antibody (VH35H5 / VL31A1) @1.7 mg / kg, 4: anti-Tau (pS422) antibody (VH35H5 / VL31A1) @5 mg / kg, 5: anti-Tau (pS422) antibody (VH35H5 / VL31A1) @15 mg / kg.

[0523] Sequence Description

[0524] SEQ ID NO: 01 Human Tau protein isoform F (441 residues)

[0525] SEQ ID NO: 02 Human Tau protein isoform F phosphorylated at the serine residue at position 422 (residue 441)

[0526] SEQ ID NO:03 Fragment of human Tau protein with phosphorylated serine at position 7 (corresponding to position 422 of SEQ ID NO:01) (residues 416 to 430 of SEQ ID NO:01): Ser-Ile-Asp-Met-Val-Asp-Ser(PO3H2)-Pro-Gln-Leu-Ala-Thr-Leu-Ala-Asp SEQ ID NO:04 Rabbit antibody 086CDRL1-QSSQSVRTNKLA

[0527] SEQ ID NO:05 Rabbit antibody 086CDRL2-SASTLDF

[0528] SEQ ID NO: 06 Rabbit antibody 086CDRL3-LGYFDCSIADCVA

[0529] SEQ ID NO: 07 Rabbit antibody 086VL00

[0530] SEQ ID NO: 08 Rabbit antibody 086CDRH1-SNAIN

[0531] SEQ ID NO: 09 Rabbit antibody 086CDRH2-YIAVSGNTYYASWAKG

[0532] SEQ ID NO: 10 Rabbit antibody 086CDRH3-SNI

[0533] SEQ ID NO: 11 rabbit antibody 086VH00

[0534] SEQ ID NO: 12 Humanized CDRL1 variant 1-RSSQSVRTNKLA

[0535] SEQ ID NO: 13 Humanized CDRL1 variant 2-RSSQSVRTNRLA

[0536] SEQ ID NO: 14 Humanized CDRL2 variant 1-SASTLDY

[0537] SEQ ID NO: 15 Humanized CDRL3 variant 1-LGYFDSSADIVA

[0538] SEQ ID NO: 16 Humanized VL variant 1-VL21

[0539] SEQ ID NO: 17 Humanized VL variant 2-VL22

[0540] SEQ ID NO: 18 Humanized CDRH2-YIAVSGNTYYADSVKG

[0541] SEQ ID NO: 19 Humanized VH variant 1-VH32

[0542] SEQ ID NO:20 Humanized VH variant 2-VH20

[0543] SEQ ID NO:21 Humanized VH variant 3-VH33

[0544] SEQ ID NO:22 Humanized CDRL2 variant 2-SASTLQS

[0545] SEQ ID NO:23 Humanized CDRL2 variant 3-SASTLES

[0546] SEQ ID NO:24 Humanized CDRL3 variant 2-LGYFDSSIADSVA

[0547] SEQ ID NO:25 Humanized CDRL3 variant 3-LGYFDSSIADRVA

[0548] SEQ ID NO:26 Humanized CDRL3 variant 4-LGYFDPSIADPVA

[0549] SEQ ID NO:27 Humanized CDRL3 variant 5-LGYFDSSIADIVA

[0550] SEQ ID NO:28 Humanized CDRL3 variant 6-LGYFDPSADPIA

[0551] SEQ ID NO:29 Humanized CDRL3 variant 7-LGYFDPSADPVA

[0552] SEQ ID NO:30 Humanized CDRL1 variant 3 -RASQGVRTNKLA SEQ ID NO:31 Humanized CDRL1 variant 4 -RASQSVRTNKLA SEQ ID NO:32 Humanized VL variant 4 -VL01

[0553] SEQ ID NO:33 Humanized VL variant 5-VL09

[0554] SEQ ID NO:34 Humanized VL variant 6-VL12

[0555] SEQ ID NO:35 Humanized VL variant 7-VL15

[0556] SEQ ID NO:36 Humanized VL variant 8-VL16

[0557] SEQ ID NO:37 Humanized VL variant 9-VL17

[0558] SEQ ID NO:38 Humanized VL variants 10-VL19

[0559] SEQ ID NO:39 Humanized VL variants 11-VL28

[0560] SEQ ID NO:40 Humanized VL variant 12-VL33

[0561] SEQ ID NO:41 Humanized VL variant 13-VL35

[0562] SEQ ID NO:42 Humanized VL variant 14-VL39

[0563] SEQ ID NO:43 Humanized VL variant 15-VL40

[0564] SEQ ID NO:44 Humanized VL variant 16-VL41

[0565] SEQ ID NO:45 Humanized VL variant 17-VL42

[0566] SEQ ID NO:46 Humanized VH variant 4-VH01

[0567] SEQ ID NO:47 Humanized VH variant 5-VH02

[0568] SEQ ID NO:48 Humanized VH variant 6-VH03

[0569] SEQ ID NO:49 Humanized VH variant 7-VH04

[0570] SEQ ID NO:50 Humanized VH variant 8-VH14

[0571] SEQ ID NO:51 Humanized VH variant 9-VH15

[0572] SEQ ID NO:52 Humanized VH variants 10-VH18

[0573] SEQ ID NO:53 Humanized VH variants 11-VH19

[0574] SEQ ID NO:54 Humanized VH variant 12-VH22

[0575] SEQ ID NO:55 Humanized VH variant 13-VH23

[0576] SEQ ID NO:56 Humanized VH variants 14-VH24

[0577] SEQ ID NO:57 Humanized VH variant 15-VH31

[0578] SEQ ID NO:65 Humanized VH variant 16-VH35H5

[0579] SEQ ID NO:66 Humanized VL variant 18-VL31A1

[0580] SEQ ID NO:67 Humanized VL variant 19-VL49G1

[0581] SEQ ID NO:68 Humanized VL variant 20-VL35F2

[0582] SEQ ID NO:69 Humanized VL variant 21-VL53A2

[0583] SEQ ID NO:70 Humanized CDRL1 variant 5

[0584] SEQ ID NO:71 Humanized CDRL1 variant 6

[0585] SEQ ID NO:72 Humanized CDRL2 variant 4

[0586] SEQ ID NO:73 Humanized CDRL2 variant 5

[0587] SEQ ID NO:74 Humanized CDRL3 variant 8

[0588] SEQ ID NO:75 Humanized CDRL3 variant 9

[0589] SEQ ID NO:76 Humanized VH variant 17-VH76A6

[0590] SEQ ID NO: 77 Humanized CDRH1 variant 1

[0591] SEQ ID NO:78 Humanized VL variant 22-VL35G4

[0592] SEQ ID NO:79 Humanized CDRL3 variant 10

[0593] SEQ ID NO:80 Humanized VL variant 23-VL145B12 SEQ ID NO:81 Humanized CDRL2 variant 6

[0594] SEQ ID NO:82 Anti-transferrin receptor antibody heavy chain 1

[0595] SEQ ID NO:83 Anti-transferrin receptor antibody heavy chain 2

[0596] SEQ ID NO:84 Anti-transferrin receptor antibody heavy chain 3

[0597] SEQ ID NO:85 Anti-transferrin receptor antibody light chain

[0598] SEQ ID NO:86 Humanized VL variant 24-VL4G1

[0599] SEQ ID NO:87 Humanized CDRL3 variant 11 sequence correspondence table

[0600]

[0601]

[0602] DETAILED DESCRIPTION OF THE INVENTION

[0603] The present invention relates to a binding peptide or protein or a functional part thereof, in particular to an antibody, in particular a monoclonal antibody or a functional part thereof, in particular a binding peptide or antibody, which recognizes and specifically binds to a phospho-epitope on a mammalian, in particular human Tau protein or a fragment thereof, in particular a conformational isomer of the pathological protein tau, but in one embodiment, does not bind to a corresponding non-phosphorylated epitope and / or an irrelevant epitope, wherein the binding peptide or antibody binds to an epitope on a mammalian, in particular human Tau protein as shown in SEQ ID NO: 02, which consists of Tau amino acid residues 416-430, which contains phosphorylated Ser (pS422) at position 422.

[0604] I. Definitions

[0605] For the purposes of this article, an "acceptor human framework" is a framework that comprises an amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus sequence framework as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0606] "Affinity" refers to the sum of the strengths of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). It can generally be expressed by the dissociation constant (K d ) represents the affinity of molecule X for its partner Y. Affinity can be measured by common methods known in the art, including those described herein. Specific schematic and exemplary embodiments for measuring binding affinity are described below.

[0607] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) wherein such alterations result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alterations.

[0608] The terms "anti-human Tau (pS422) antibody" and "antibody that specifically binds to human Tau (pS422)" refer to an antibody that is capable of binding to human Tau (pS422) with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent in targeting human Tau (pS422). In one embodiment, the extent of binding of the anti-human Tau (pS422) antibody to an unrelated, non-human Tau (pS422) protein is less than about 10% of the binding of the antibody to human Tau (pS422), as measured, for example, by radioimmunoassay (RIA).

[0609] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0610] "Antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0611] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. In one embodiment, an antibody that binds to the same epitope as the reference antibody blocks the binding of the reference antibody to its antigen by 50% or more. In one embodiment, an antibody that binds to the same epitope as the reference antibody blocks the binding of the reference antibody to its antigen by 80% or more. In one embodiment, an antibody that binds to the same epitope as the reference antibody blocks the binding of the reference antibody to its antigen by 90% or more. In one embodiment, an antibody that binds to the same epitope as the reference antibody blocks the binding of the reference antibody to its antigen by 95% or more. In a preferred embodiment, an antibody that binds to the same epitope as the reference antibody has a binding interaction with the same residue on the antigen as the reference antibody.

[0612] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0613] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0614] "Effector functions" refer to those biological activities attributed to the Fc region of an antibody that vary with the antibody class. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0615] An "effective amount" of an active agent (eg, a pharmaceutical agent) refers to an amount effective to achieve the desired therapeutic or prophylactic result, at dosages and for periods of time to achieve the result.

[0616] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl end of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise specified herein, the numbering of the amino acid residues in the Fc region or constant region is according to the EU numbering system as described in Kabat, EA et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242, also referred to as the EU index.

[0617] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain is typically composed of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR sequences and FR sequences typically appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0618] The terms "full-length antibody", "intact antibody" and "complete antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a natural antibody structure or having a heavy chain containing an Fc region as defined herein. The term "full-length antibody" refers to a multimeric polypeptide consisting of two antibody light chain polypeptides and two antibody heavy chain polypeptides connected by a disulfide bond, wherein in the two antibody heavy chain polypeptides, a C-terminal lysine residue (K) may or may not be present. The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be exactly the same as parental cells in terms of nucleic acid content, but may contain mutations. Mutant progeny are included herein, and the mutant progeny have the same function or biological activity for screening or selection as the initially transformed cells.

[0619] "People's consensus framework" is such a framework, which represents the most frequently occurring amino acid residues in the options of human immunoglobulin VL or VH framework sequences. Typically, the options of human immunoglobulin VL or VH framework sequences are from a subgroup of variable domain sequences. Typically, the sequence subgroup is a subgroup as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Bethesda MD (1991), NIH Publication 91-3242, Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI as described in Kabat et al. above. In one embodiment, for VH, the subgroup is subgroup III as described in Kabat et al. above.

[0620] "Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will substantially comprise all of at least one and generally two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of non-human antibodies and all or substantially all of the FR regions correspond to those of human antibodies. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0621] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops"), and / or contain antigen contact residues ("antigen contacts"). Typically, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

[0622] The HVRs in this article include

[0623] (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and Lesk, AM, J. Mol. Biol. 196 (1987) 901-917);

[0624] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242);

[0625] (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and

[0626] (d) a combination of (a), (b) and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0627] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0628] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules.

[0629] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0630] An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

[0631] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained within cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0632] "Isolated nucleic acid encoding anti-human TAU (pS422) antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors and such nucleic acid molecules present at one or more locations in a host cell.

[0633] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, each antibody constituting the population is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or occurring during the production of monoclonal antibody preparations), such variants are generally present in a minute amount. Contrary to the polyclonal antibody preparations generally including different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by obtaining from a substantially homogeneous antibody population, and should not be interpreted as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0634] "Native antibodies" refer to naturally occurring immunoglobulin molecules with indefinite structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons consisting of two identical light chains and two identical heavy chains bound by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody can be divided into one of two types, called κ (κ) and λ (λ), based on the amino acid sequence of its constant domain.

[0635] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or precautions concerning the use of such therapeutic products.

[0636] Relative to a reference polypeptide sequence, "percent amino acid sequence identity (%)" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence after aligning the sequences and introducing spaces as needed to achieve maximum sequence homology percentage and not considering any conservative substitutions as part of sequence identity. Alignment for determining amino acid sequence identity percentages can be achieved in a variety of ways within the capabilities of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithm required for achieving maximum alignment within the full-length sequence being compared. However, for the purposes of this article, amino acid sequence identity % values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is authorized by Genentech, Inc., and the source code has been submitted with user documentation to the U.S. Copyright Office, Washington, D.C. 20559, where it is registered with U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California or can be assembled from source code. The ALIGN-2 program should be assembled for use on a UNIX operating system (including Digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0637] In case ALIGN-2 is used to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which may alternatively be described as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

[0638] Multiply 100 by the fraction X / Y

[0639] wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and wherein Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0640] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0641] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0642] As used herein, the term "human Tau (pS422)" refers to natural human Tau (pS422) (UniProt P37840). The term includes "full length", unprocessed human Tau (pS422) and any form of human Tau (pS422) obtained by processing in a cell. The term also includes variants of naturally occurring human Tau (pS422), for example, mutants, splice variants or allelic variants. The amino acid sequence of human Tau (pS422) is shown in SEQ ID NO: 02.

[0643] As used herein, the noun "treatment" (and its grammatical variants such as the verb "treat" or the participle "treat") refers to a clinical intervention intended to alter the natural course of the individual being treated, and can be used for prevention or implemented during the course of clinical pathology. The desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay disease development or to slow the progression of the disease.

[0644] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in the binding of an antibody to an antigen. The variable domains of natural antibody heavy and light chains (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt, TJ et al., Kuby Immunology, 6th edition, WH Freeman and Co., NY (2007) p. 91). A single VH domain or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using VH or VL domains from antibodies that bind to the antigen to screen libraries of complementary VL domains or VH domains, respectively. See, for example, Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0645] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0646] II. Compositions and Methods

[0647] A. Exemplary Humanized Anti-Human Tau (pS422) Antibodies

[0648] Humanized antibodies as reported herein cannot be obtained by standard humanization methods. It is necessary to introduce non-standard mutations in the amino acid sequence to obtain humanized antibodies with binding properties and pharmacokinetic properties comparable to the parent rabbit antibody. This is particularly important because antibodies as reported herein are intended to cross the human blood-brain barrier and are effective in the human brain. Therefore, the stringency of the commonly used standards for selecting humanized antibodies is not enough to be directly used in this case.

[0649] It has been found that in order to obtain suitable and developable humanized antibodies, the two cysteines that form disulfide bonds in CDRL3 (light chain CDR3) must be replaced by serine and isoleucine, respectively. In addition to ensuring the correct orientation of the same CDRL3, the isoleucine residue present in the middle of rabbit CDRL3 was deleted, resulting in a humanized CDRL3 with one less amino acid residue than the parent rabbit CDRL3.

[0650] It was also found that in order to maintain the in vivo pharmacokinetic properties, the amino acid residue at position 32 in the light chain should be lysine (according to Kabat numbering). Removal of the disulfide bond in light chain HVR-L3 did not affect the in vivo kinetic behavior.

[0651] It has further been found that it is advantageous to maintain three valine amino acid residues at heavy chain positions 4, 24 and 78 (according to Kabat numbering). Without being limited by this theory, it is hypothesized that these residues are required to ensure correct presentation of the antigen binding loop of the heavy chain variable region. In addition, the presence of an arginine residue at position 71 (according to Kabat numbering) of the heavy chain variable domain is advantageous.

[0652] HVR-L3 contains two aspartate residues, which may be hot spots for deamidation, especially during long-term storage. In the light chain variable domain variant VL35G4, one of these two aspartate residues has been altered.

[0653] All numbering as used herein is based on the Kabat variable domain numbering scheme.

[0654] In the table below, the properties of the different humanized variants of the rabbit light chain variable domain in combination with the humanized heavy chain variable domains VH14 and VH20, respectively, are shown. The binding partner is human Tau (pS422).

[0655]

[0656]

[0657] Reference values ​​VH00 and VL00 (rabbit antibody):

[0658] 25℃: kd=2.6E-04; t / 2=44min.

[0659] 37°C: ka=3.7E+04, kd=5.25E-03, KD=1.4E-08, t / 2=22min.

[0660] In the table below, the properties of different humanized variants of the rabbit light chain variable domain in combination with the humanized light chain variable domains VL17 and VL19, respectively, are shown.

[0661]

[0662]

[0663] Reference values ​​VH00 and VL00 (rabbit antibody):

[0664] 25℃: kd=2.6E-04; t / 2=44min.

[0665] 37°C: ka=3.7E+04, kd=5.25E-03, KD=1.4E-08, t / 2=22min.

[0666] In the table below the kinetic constants for different VH / VL combinations (determined according to Example 8) are shown.

[0667]

[0668] Biochemical binding of different combinations of humanized VH and VL was shown in Figure 3 and 4 middle.

[0669] The binding specificity (EC50 values ​​in [ng / ml]) of the different VH / VL combinations is shown in the table below.

[0670]

[0671] from Figure 5 The Western blot shown in Figure 4 shows the sensitivity of selected humanized VH / VL combinations to the human tau mutant S422A. All humanized variants bind selectively to human tau phosphorylated at S422. There is a low level of cross-reactivity to non-S422 phosphorylated epitopes of the parental rabbit antibody, but at this point, the humanized variants shown have weaker cross-reactivity than the parental rabbit antibody.

[0672] exist Figure 6 In , binding to PHF-tau in brain extracts of Alzheimer's patients is shown for the parental rabbit antibody and for selected humanized anti-human Tau (pS422) antibodies.

[0673] Based on the above, the combination of VH32 / VL22 was selected as the humanized antibody.

[0674] The following mean clearances were found in cynomolgus monkeys with the parental VH / VL combination and the humanized VH / VL combination (normal clearance for human IgG is 0.18-0.36 ml / hr / kg).

[0675]

[0676] nd = not determined

[0677] It can be seen that the VH32 / VL22 combination had an increased clearance rate, which was dose-dependent.

[0678] In the table below, kinetic constants for additional VH / VL combinations generated to account for the increased dose-dependent clearance of VH32 / VL22 with the Tau (pS422) fragment (determined according to Example 10) are shown.

[0679]

[0680] In the table below the kinetic constants of VH / VL combinations as Fab fragments with full-length Tau (pS422) and Tau (pS422) fragments (determined according to Example 11 and Example 12) are shown.

[0681]

[0682] In the table below the binding specificity (selectivity for phosphorylated versus non-phosphorylated Tau) of the different VH / VL combinations determined by ELISA is shown.

[0683]

[0684] The following mean clearances were found in cynomolgus monkeys with the different VH / VL combinations (normal clearance for human IgG is 0.18-0.36 ml / hr / kg; nd = not determined).

[0685]

[0686] The following average clearance rates in mice were found with different VH / VL combinations (normal clearance for human IgG is 0.18-0.36 ml / hr / kg).

[0687]

[0688] In one embodiment of all aspects as reported herein, the humanized anti-human Tau (pS422) antibody has a mean clearance of less than 0.6 ml / hr / kg after intravenous administration at a dose of up to 25 mg / kg. In one embodiment, at a dose of up to 25 mg / kg, the mean clearance is 0.3 ml / hr / kg or less.

[0689] In a preferred aspect, the present invention provides a (humanized) anti-human Tau (pS422) antibody comprising at least one, or two, or three, or four, or five, or six HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 71; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 73; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

[0690] In a preferred aspect, the present invention provides a (humanized) anti-human Tau (pS422) antibody comprising at least one, or two, or three, or four, or five, or six HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 72; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

[0691] In a preferred aspect, the present invention provides a (humanized) anti-human Tau (pS422) antibody comprising at least one, or two, or three, or four, or five, or six HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

[0692] In a preferred aspect, the present invention provides a (humanized) anti-human Tau (pS422) antibody comprising at least one, or two, or three, or four, or five, or six HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75.

[0693] In one aspect, the present invention provides a (humanized) anti-human Tau (pS422) antibody comprising at least one, or two, or three, or four, or five, or six HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 77; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 79.

[0694] In one aspect, the invention provides a (humanized) antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 09; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10.

[0695] In one aspect, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:08; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:09; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10.

[0696] In another embodiment, the antibody further comprises at least one, at least two, or all three VLHVR sequences selected from:

[0697] i) (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 71; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 73; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15; or

[0698] ii) (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 72; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15; or

[0699] iii) (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 79.

[0700] In yet another embodiment, the antibody comprises

[0701] i) (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 71; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 73; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15; or

[0702] ii) (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 72; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15; or

[0703] iii) (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 79.

[0704] In one aspect, the invention provides a (humanized) antibody comprising

[0705] i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 71; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 73; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15, or

[0706] ii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 72; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15, or

[0707] iii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 79.

[0708] In another embodiment, VH or VL contains a substitution (eg, a conservative substitution), insertion, or deletion relative to the reference sequence, but an anti-human Tau (pS422) antibody comprising the sequence retains the ability to bind to human Tau (pS422).

[0709] In a further aspect of the invention, the anti-human Tau (pS422) antibody according to any of the above embodiments is a monoclonal antibody, including chimeric, humanized or human antibodies. In one embodiment, the anti-human Tau (pS422) antibody is an antibody fragment, e.g., Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, e.g., a complete IgG1 or IgG 4 antibody or other antibody types or isotypes as defined herein.

[0710] The (humanized) antibodies as reported herein reduce Tau (pS422) levels in the brain of transgenic TauPS2APP mice.

[0711] In a further aspect, the (humanized) anti-human Tau (pS422) antibody according to any of the above embodiments may incorporate any of the features as described in the following sections 1-7, alone or in combination:

[0712] 1. Antibody affinity

[0713] In certain embodiments, the antibodies provided herein have a concentration of ≤100 nM, ≤50 nM, or between 1 nM and 100 nM (e.g., 10 -7 M or less, for example, from 10 -7 M to 10 -9 M) dissociation constant (KD).

[0714] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed with a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured by titrating the antibody in the presence of an unlabeled antigen titration series using a minimal concentration of ( 125 I)-labeled antigen equilibrated Fab, and then the bound antigen was captured by anti-Fab antibody-coated plates for measurement (see, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). To establish the assay conditions, Multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and subsequently blocked with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 I]-antigen is mixed with a serial dilution of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12 in Presta, LG et al., Cancer Res. 57 (1997) 4593-4599). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution is then removed and the plate is washed with 0.1% polysorbate 20 (TWEEN- ) Wash eight times. When the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20TM ; Packard), and place the plate on TOPCOUNT TM The concentrations of each Fab that gave less than or equal to 20% of maximal binding were selected for competition binding assays.

[0715] According to another embodiment, Kd is Surface plasmon resonance measurements. For example, using -2000 or -3000 (BIAcore, Inc., Piscataway, NJ) assays were performed at 25°C using a CM5 chip with antigen immobilized at ~10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5 μg / ml (~0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to obtain approximately 10 response units (RU) of coupled protein. After injection of the antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were added to a 4% PBS solution with 0.05% polysorbate 20 (TWEEN- ) surfactant-free PBS (PBST) was injected at a flow rate of approximately 25 μl / min at 25°C. Using a simple one-to-one Langmuir binding model ([ The association rate (kon) and dissociation rate (koff) were calculated by simultaneously fitting the association and dissociation sensor curves (SPR software version 3.2). The equilibrium dissociation constant (Kd) was calculated as the ratio koff / kon (see, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). If the on-rate determined by the surface plasmon resonance above exceeds 10 6 M -1 s -1 , the binding rate can be determined by using the fluorescence quenching technique in a spectrometer such as a stopped-flow spectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO with a stirred vessel. TMThe increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) at 25°C of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 was measured in the presence of increasing antigen concentrations measured in a spectrophotometer (ThermoSpectronic).

[0716] 2. Antibody fragments

[0717] In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv and scFv fragments and other fragments described below. For a review of certain antibody fragments, see Hudson, PJ et al., Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see, for example, Plueckthun, A., cited in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), (Springer-Verlag, New York (1994), pp. 269-315; see also WO 93 / 16185; US 5,571,894 and US 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see US 5,869,046.

[0718] Diabodies are antibody fragments with two antigen binding sites, which can be bivalent or bispecific. See, for example, EP 0 404 097; WO 1993 / 01161; Hudson, PJ et al., Nat. Med. 9 (2003) 129-134; and Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448. Triabodies and tetrabodies are also described in Hudson, PJ et al., Nat. Med. 9 (2003) 129-134).

[0719] Single domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US 6,248,516).

[0720] Antibody fragments can be produced by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

[0721] 3. Humanized Antibodies

[0722] In general, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies include one or more variable domains, and their HVR (e.g., CDR) (or portions thereof) are derived from non-human antibodies and FR (or portions thereof) are derived from human antibody sequences. Humanized antibodies will optionally also include at least a portion or the full length of a human constant region. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0723] Humanized antibodies and methods for making them are reviewed, for example, in Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, and for example in Riechmann, I. et al., Nature 332 (1988) 323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033; US 5,821,337, US 7,527,791, US 6,982,321 and US 7,087,409; Kashmiri, SV et al., Methods 36 (2005) 25-34 (describing specificity determining region (SDR) transplantation); Padlan, EA, Mol. Immunol. 28 (1991) 489-498 (describing "surface remodeling"); Dall'Acqua, WF et al., Methods 36 (2005) 43-60 (describing "FR shuffling"); and Osbourn, J. et al., Methods 36 (2005) 61-68; and further described in Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing the "guided selection" approach for FR shuffling).

[0724] The human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (e.g., see Sims, MJ et al., J. Immunol. 151 (1993) 2296-2308); framework regions derived from the consensus sequence of the light chain variable region or the heavy chain variable region of a particular subgroup of human antibodies (e.g., see Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, LG et al., J. Immunol. 151 (1993) 2623-2632); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regions derived from screening FR libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 ​​(1997) 10678-10684 and Rosok, MJ et al., J. Biol. Chem. 271 (1996) 22611-22618).

[0725] 4. Multispecific Antibodies

[0726] In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity to at least two different sites. In certain embodiments, one of these binding specificities is directed to human Tau (pS422), and the other specificity is directed to any other antigen. In certain embodiments, bispecific antibodies can bind to 2 different epitopes of human Tau (pS422). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0727] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983) 537-540, WO 93 / 08829 and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) and “knob-in-knob” engineering (see, for example, US 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic steering effects to produce antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (e.g., see US 4,676,980 and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553); using "diabody" technology to prepare bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (sFv) dimers (see, e.g., Gruber, M. et al., J. Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies as described, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

[0728] Also included herein are engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies" (see, e.g., US 2006 / 0025576).

[0729] The antibodies or fragments herein also include "Dual Acting Fab" or "DAF", which comprise antigen binding sites that bind to human Tau (pS422) and another different antigen (see, for example, US 2008 / 0069820).

[0730] The antibodies or fragments herein also include the multispecific antibodies described in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792 and WO 2010 / 145793.

[0731] 5. Antibody variants

[0732] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody may be prepared by introducing suitable modifications to the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions of residues from within the amino acid sequence of the antibody and / or insertion of residues into the amino acid sequence and / or replacement of residues in the amino acid sequence. Any combination of deletions, insertions, and replacements may be produced to obtain the final construct, as long as the final construct possesses desired characteristics, such as antigen binding.

[0733] a) Substitution variants, insertion variants and deletion variants

[0734] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. The target sites for substitution mutagenesis include HVR and FR. Conservative substitutions are shown in the following table under the heading of "preferred substitutions". More substantial changes are provided in the following table under the heading of "exemplary substitutions", and reference to the amino acid side chain categories are further described below. Amino acid substitutions can be introduced into the target antibody and the product can be screened for desired activity, for example, retained / improved antigen binding, reduced immunogenicity or improved ADCC or CDC.

[0735] surface

[0736]

[0737]

[0738] Amino acids can be grouped according to common side chain properties:

[0739] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0740] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn; Gln;

[0741] (3) Acidic: Asp, Glu;

[0742] (4) Basic: His, Lys, Arg;

[0743] (5) Residues that affect chain direction: Gly, Pro;

[0744] (6) Aromatic: Trp, Tyr, Phe.

[0745] Non-conservative substitutions will involve exchanging a member of one of these classes for a member of another class.

[0746] A substitution variant type involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody). Typically, the resulting variant selected for further study will have modifications (e.g., improvements) relative to the parent antibody in certain biological properties (e.g., increased affinity, reduced immunogenicity) and / or will have certain biological properties that are substantially retained by the parent antibody. An exemplary substitution variant is an affinity matured antibody, which can be conveniently produced, for example, using affinity maturation techniques based on phage display such as those described herein. In short, one or more HVR residues are mutated and the variant antibody is displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0747] Changes (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such changes can be made in HVR "hot spots" (i.e., residues encoded by codons that undergo mutations at high frequency during the somatic maturation process (see, e.g., Chowdhury, PS, Methods Mol. Biol. 207 (2008) 179-196) and / or residues that contact antigens, and the resulting variant VH or VL is tested for binding affinity. This has been described, for example, in Hoogenboom, HR et al., cited in Methods in Molecular Biology 178 (2002) 1-37 describes the realization of affinity maturation by constructing a secondary library and reselecting therefrom. In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling or oligonucleotide directed mutagenesis). A secondary library is then produced. The library is then screened to identify any antibody variant with the desired affinity. Another method of introducing diversity involves an HVR-guided scheme, in which several HVR residues (e.g., 4-6 residues at a time) are randomly grouped. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often targeted in particular.

[0748] In certain embodiments, substitutions, insertions or deletions may occur in one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigens. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may, for example, be outside the antigen contact residues in HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two or three amino acid substitutions.

[0749] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described in Cunningham, BC and Wells, JA, Science 244 (1989) 1081-1085. In this method, a residue or target residue group (e.g., charged residues such as arg, asp, his, lys and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is determined to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they contain desired properties.

[0750] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions ranging in length from 1 residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusion of the N-terminus or C-terminus of an antibody to an enzyme (e.g., an enzyme for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

[0751] b) Glycosylation variants

[0752] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibodies are glycosylated. Adding or deleting glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0753] In the case where the antibody comprises an Fc region, the sugar connected thereto can be changed. Natural antibodies produced by mammalian cells generally contain branched biantennary oligosaccharides, which are usually connected to Asn297 of the CH2 domain of the Fc region by means of N-linkage (e.g., see Wright, A. and Morrison, SL, TIBTECH 15 (1997) 26-32). Oligosaccharides can include a variety of sugars, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose connected to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to produce antibody variants with improved certain properties.

[0754] c) Fc region variants

[0755] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. The Fc region variant can include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0756] In certain embodiments, the present invention contemplates antibody variants that possess some but not all effector functions, which make the antibody variants favorable candidates for applications in which the in vivo half-life of the antibody is important, while certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be implemented to confirm that CDC and / or ADCC activity is reduced / consumed. For example, Fc receptor (FcR) binding assays can be implemented to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. The main cell mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Ravetch, JV and Kinet, JP, Table 3 on page 464 of Annu. Rev. Immunol. 9 (1991) 457-492 summarizes FcR expression on hematopoietic cells. Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in US 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502); US 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assays can be used (see, e.g., ACTI for flow cytometry). TM Nonradioactive cytotoxicity assays (Cell Technology, Inc. Mountain View, CA; and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, MS et al., Blood 101 (2003) 1045-1052; and Cragg, MS and MJ Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivo clearance / half-life can also be determined using methods known in the art (see, e.g., Petkova, SB et al., Int. Immunol. 18 (2006: 1759-1769).

[0757] Antibodies with reduced effector function include those with one or more Fc region residues 238, 265, 269, 270, 297, 327 and 329 replaced (US 6,737,056). Such Fc region mutants include those with substitutions at two or more positions of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc region mutants in which residues 265 and 297 are replaced with alanine (US 7,332,581).

[0758] Certain antibody variants with improved or impaired FcR binding are described. (See, e.g., US 6,737,056; WO 2004 / 056312, and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604)

[0759] In some embodiments, alterations are made in the Fc region that result in altered (ie, impaired) CIq binding and / or complement dependent cytotoxicity (CDC), e.g., as described in US 6,194,551, WO 99 / 51642, and Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184.

[0760] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US2005 / 0014934, which is responsible for the transfer of maternal IgG to the fetus (Guyer, RL et al., J. Immunol. 117 (1976) 587-593 and Kim, JK et al., J. Immunol. 24 (1994) 2429-2434). These antibodies comprise an Fc region with one or more substitutions, wherein the substitutions improve the binding of the Fc region to FcRn. Such Fc region variants include those having a substitution at one or more Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434 (e.g., Fc region residue 434 substitution) (U.S. Pat. No. 7,371,826).

[0761] See also Duncan, AR and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821 and WO 94 / 29351 for other examples of Fc region variants.

[0762] d) Cysteine ​​engineered antibody variants

[0763] In certain embodiments, it may be desirable to produce cysteine ​​engineered antibodies, e.g., "thio MAbs," wherein one or more residues of the antibody are replaced with cysteine ​​residues. In specific embodiments, the replaced residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive sulfhydryls are thus placed at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties to produce immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine ​​engineered antibodies may be produced, for example, as described in US 7,521,541.

[0764] e) Antibody derivatives

[0765] In certain embodiments, the antibodies provided herein can be further modified to contain additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly 1,3-dioxolane, poly 1,3,6-triazine ... In some embodiments, the invention relates to a polyol or a mixture of polyols. Polyols may be present in the form of polyols such as polyols, e.g., polyols ...

[0766] In another embodiment, a conjugate of an antibody and a non-protein moiety is provided, wherein the non-protein moiety can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can have any wavelength, and includes but is not limited to such wavelength, which does not harm ordinary cells, but heats the non-protein moiety to a temperature that kills cells adjacent to the antibody-non-protein moiety.

[0767] B. Recombinant Methods and Compositions

[0768] Recombinant methods and compositions can be used to produce antibodies, for example as described in US 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-human Tau (pS422) antibody described herein is provided. This nucleic acid can encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acids is provided. In such an embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid, the nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid, the nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid, the nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method for preparing an anti-human Tau (pS422) antibody is provided, wherein the method comprises culturing a host cell as provided above under conditions suitable for expression of the antibody, the host cell comprising a nucleic acid encoding the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0769] To recombinantly produce anti-human Tau (pS422) antibodies, nucleic acids encoding the antibodies (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that specifically bind to genes encoding antibody heavy and light chains).

[0770] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expressing antibody fragments and polypeptides in bacteria, for example, see US 5,648,237, US5,789,199 and US 5,840,523. (See also Charlton, KA, cited in Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes expression of antibody fragments in Escherichia coli). After expression, antibodies can be separated from bacterial cell paste in soluble fractions and antibodies can be further purified.

[0771] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning hosts or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0772] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0773] Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (describing PLANTIBODIES producing antibodies in transgenic plants). TM technology).

[0774] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to suspension culture can be useful. Other examples of mammalian host cell lines that can be used are monkey kidney CV1 line (COS-7) transformed by SV40; human embryonic kidney line (such as 293 or 293 cells described in, for example, Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse supporting cells (such as TM4 cells described in, for example, Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); such as in, for example, Mather, JP et al., Annals TRI cells as described in NY Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR -CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for producing antibodies, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0775] C. Assay

[0776] The physical / chemical properties and / or biological activities of the anti-human Tau (pS422) antibodies provided herein can be identified, screened or characterized by a variety of assays known in the art.

[0777] 1. Binding Assays and Other Assays

[0778] In one aspect, antibodies of the invention are tested for their antigen binding activity, etc., for example, by known methods such as ELISA, αLISA, Western blot, antibody or reverse phase arrays, etc.

[0779] In an exemplary ELISA or αLISA assay, Tau (pS422) in a solution (e.g., cell supernatant, cell or tissue lysate, body fluid, etc.) is bound by a capture antibody that specifically binds to a first epitope on Tau (pS422) or a certain conformation of Tau (pS422), and a detection antibody is coupled to a detection entity that specifically binds to a second epitope or conformation of Tau (pS422). The result readout is based on the detection entity (chemiluminescence, fluorescence, luminescence caused by energy transfer, etc.). In some cases, the same antibody can be used as a capture and detection antibody in the same assay to detect aggregated forms of Tau (pS422) (see, e.g., Tokuda, T. et al., Neurology 75 (2010) 1766-1772).

[0780] In the case of an antibody array, the antibodies are dotted on a glass or nitrocellulose chip. The slide is sealed and incubated with a solution containing Tau (pS422), washed to remove unbound antibodies and the bound antibodies are detected with fluorescently labeled corresponding secondary antibodies. The fluorescence signal is measured by a fluorescent slide scanner. Similarly, for a reverse phase array, recombinant Tau (pS422), cell supernatants, cell or tissue lysates, body fluids, etc. are dotted on a glass or nitrocellulose chip. The slide is sealed and the individual arrays are incubated with antibodies against specific epitopes on Tau (pS422). Unbound antibodies are washed away and the bound antibodies are detected using fluorescently labeled corresponding secondary antibodies. The fluorescence signal is measured by a fluorescent slide scanner (Dernick, G. et al., J. Lipid Res. 52 (2011) 2323-2331).

[0781] In the example of Western blotting, aggregated recombinant Tau (pS422) or Tau (pS422) derived from, for example, cell supernatants, cell or tissue lysates, body fluids, etc., is separated by molecular weight in SDS PAGE or native gel conditions and blotted onto nitrocellulose or PVDF membranes. After blocking, the membrane is incubated with an antibody specific to the amino acid sequence or conformation of Tau (pS422). The membrane is then washed to remove unbound antibodies. The bound antibody is detected with a corresponding secondary antibody coupled to the detection entity for chemiluminescence or fluorescence or other detection methods. Antibodies specific to the amino acid sequence of Tau (pS422) will bind to various aggregated forms and therefore various molecular weights of Tau (pS422), as long as the epitope is not obscured by aggregation. On the other hand, conformation-specific antibodies will detect only certain aggregated forms of Tau (pS422), showing bands only at specific molecular weights (see, e.g., Towbin, H. et al., Proc. Natl. Acad. Sci. USA 76 (1979) 4350-4353; Burnette, WN, Anal. Biochem. 112 (1981) 195-203).

[0782] On the other hand, competition assays can be used to identify antibodies that compete with the (humanized) antibodies as reported herein for binding to human Tau (pS422). In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) as reported herein. Detailed exemplary methods for mapping antibody binding epitopes are provided in Morris, GE (ed.), Epitipe Mapping Protocols, in: Methods in Molecular Biology, Vol. 66, Humana Press, Totowa, NJ (1996).

[0783] In an exemplary competition assay, immobilized human Tau (pS422) is incubated in a solution containing a first labeled antibody that binds to human Tau (pS422) and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to human Tau (pS422). As a control, immobilized human Tau (pS422) is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to human Tau (pS422), excess unbound antibody is removed, and the amount of label associated with immobilized human Tau (pS422) is measured. If the amount of label associated with immobilized human Tau (pS422) is significantly reduced in the test sample relative to the control sample, it indicates that the second antibody competes with the first antibody for binding to human Tau (pS422) (see, e.g., Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988)).

[0784] 2. Activity Assay

[0785] In one aspect, an assay for identifying an anti-human Tau (pS422) antibody having biological activity is provided. The biological activity may include, for example, protection from / reduction / inhibition of cytotoxicity caused by Tau (pS422), and / or protection from / reduction / inhibition of cell-to-cell transmission of oligomeric human Tau (pS422), and / or reduction of Tau (pS422)-induced caspase activity in LUHMES cells. Antibodies having this biological activity in vivo and / or in vitro are also provided.

[0786] In certain embodiments, the antibodies of the invention are tested for such biological activities.

[0787] Protective bioactivity can be evaluated by adding conditioned medium containing secreted Tau (pS422) that causes cell death of recipient neuronal cells. This toxicity can be reversed by adding protective antibodies as described herein. The toxicity of secreted Tau (pS422) has been established previously (Emmanouilidou, E. et al., J. Neurosci., 30 (2010) 6838-6851).

[0788] D. Methods and compositions for diagnosis and detection

[0789] In certain embodiments, any of the anti-human Tau (pS422) antibodies provided herein can be used to detect the presence of human Tau (pS422) in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as brain tissue.

[0790] In one embodiment, an anti-human Tau (pS422) antibody for use in a diagnostic or detection method is provided. In yet another aspect, a method for detecting the presence of human Tau (pS422) in a biological sample is provided. In certain embodiments, the method comprises: contacting a biological sample with an anti-human Tau (pS422) antibody as described herein under conditions that allow the anti-human Tau (pS422) antibody to bind to human Tau (pS422), and detecting whether a complex is formed between the anti-human Tau (pS422) antibody and human Tau (pS422). This method can be an in vitro or in vivo method. In one embodiment, the anti-human Tau (pS422) antibody is used to select a subject suitable for treatment with an anti-human Tau (pS422) antibody, for example, where human Tau (pS422) is a biomarker for selecting patients.

[0791] Exemplary disorders that can be diagnosed using the antibodies of the invention include neurodegeneration with brain iron accumulation type 1 (NBIA1), pure autonomic failure, Down's syndrome, complex of Guam, and various Lewy body disorders such as diffuse Lewy body disease (DLBD), Lewy body variant of Alzheimer's disease (LBVAD), certain forms of Gaucher's disease, and Parkinson's disease dementia (PDD).

[0792] In certain embodiments, a labeled anti-human Tau (pS422) antibody is provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and indirectly detected (e.g., by enzymatic reactions or molecular interactions) moieties such as enzymes or ligands. Exemplary labels include, but are not limited to, radioisotopes. 32 P. 14 C. 125 I. 3 H and 131I. Fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (US 4,737,456), luciferin, 2,3-dihydrophthalazine dione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, which are coupled to enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase or microperoxidase, biotin / avidin, spin labels, phage labels, stable free radicals, etc.

[0793] E. Pharmaceutical preparations

[0794] Pharmaceutical formulations of anti-human Tau (pS422) antibodies as described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. (ed.) (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citric acid and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (such as octadecylbenzyldimethylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzalkonium bromide; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides ; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as poly (vinyl pyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other sugars including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannose, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes) and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rhuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rhuPH20, are described in US2005 / 0260186 and US2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

[0795] Exemplary lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006 / 044908, the latter formulations comprising a histidine-acetate buffer.

[0796] The formulations herein may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in amounts effective for the intended purpose.

[0797] The active ingredient can be embedded in, for example, microcapsules (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules) prepared by coacervation techniques or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. (ed.) (1980).

[0798] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles (eg, films, or microcapsules).

[0799] Formulations to be used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0800] For example, where the indication to be treated is Alzheimer's disease or prodromal Alzheimer's disease, the pharmaceutical preparation may also contain one or more additional active ingredients, such as donepezil, memantine, rivastigmine, galantamine, rivastigmine, ergoloid mesylates, anti-Abeta antibiotics and anti-α-synuclein antibodies.

[0801] F. Methods of Treatment and Compositions

[0802] Any of the anti-human Tau (pS422) antibodies provided herein can be used in therapeutic methods.

[0803] In one aspect, an anti-human Tau (pS422) antibody for use as a medicament is provided. In other aspects, an anti-human Tau (pS422) antibody for the treatment of Alzheimer's disease is provided. In certain embodiments, an anti-human Tau (pS422) antibody for use in a method of treatment is provided. In certain embodiments, the present invention provides an anti-human Tau (pS422) antibody for use in a method of treating an individual with Alzheimer's disease, the method comprising administering an effective amount of an anti-human Tau (pS422) antibody to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, for example, a therapeutic agent as described below. In other embodiments, the present invention provides an anti-human Tau (pS422) antibody for inhibiting cytotoxicity caused by Tau (pS422) in human neurons and glial cells, or inhibiting cell-to-cell transmission of oligomeric human Tau (pS422) between neurons and glial cells, or reducing Tau (pS422)-induced caspase activity. In certain embodiments, the present invention provides an anti-human Tau (pS422) antibody for use in a method of inhibiting cytotoxicity caused by Tau (pS422) in human neurons and glial cells, or inhibiting cell-to-cell transmission of oligomeric human Tau (pS422) between neurons and glial cells, or reducing Tau (pS422)-induced caspase activity in an individual, the method comprising administering an effective amount of the anti-human Tau (pS422) antibody to the individual to inhibit cytotoxicity caused by Tau (pS422) in human neurons and glial cells, or inhibiting cell-to-cell transmission of oligomeric human Tau (pS422) between neurons and glial cells, or reducing Tau (pS422)-induced caspase activity. The "individual" according to any of the above embodiments is preferably a human.

[0804] In yet another aspect, the present invention provides the use of anti-human Tau (pS422) antibodies in the manufacture or formulation of a medicament. In one embodiment, the medicament is used to treat Alzheimer's disease. In yet another embodiment, the medicament is used in a method for treating Alzheimer's disease, the method comprising administering an effective amount of the medicament to an individual suffering from Alzheimer's disease. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. In yet another embodiment, the medicament is used to inhibit cytotoxicity caused by Tau (pS422) in human neurons and glial cells, or to inhibit cell-to-cell transmission of oligomeric human Tau (pS422) between neurons and glial cells, or to reduce Tau (pS422)-induced caspase activity. In a further embodiment, the drug is used in a method for inhibiting cytotoxicity caused by Tau (pS422) in human neurons and glial cells, or inhibiting cell-to-cell transmission of oligomeric human Tau (pS422) between neurons and glial cells, or reducing Tau (pS422)-induced caspase activity in an individual, the method comprising administering to the individual an effective amount of the drug to inhibit cytotoxicity caused by Tau (pS422) in human neurons and glial cells, or inhibiting cell-to-cell transmission of oligomeric human Tau (pS422) between neurons and glial cells, or reducing Tau (pS422)-induced caspase activity. The "individual" according to any of the above embodiments is preferably a human.

[0805] In other aspects, the present invention provides methods for treating Alzheimer's disease. In one embodiment, the method comprises administering an effective amount of an anti-human Tau (pS422) antibody to an individual suffering from the Alzheimer's disease. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent as described below. The "individual" according to any of the above embodiments can be a human.

[0806] In other aspects, the present invention provides a method for inhibiting cytotoxicity caused by Tau (pS422) in human neurons and glial cells, or inhibiting cell-to-cell transmission of oligomeric human Tau (pS422) between neurons and glial cells, or reducing Tau (pS422)-induced caspase in an individual. In one embodiment, the method comprises administering an effective amount of an anti-human Tau (pS422) antibody to the individual to inhibit cytotoxicity caused by Tau (pS422) in human neurons and glial cells, or inhibiting cell-to-cell transmission of oligomeric human Tau (pS422) between neurons and glial cells, or reducing Tau (pS422)-induced caspase activity. In one embodiment, the "individual" is a human.

[0807] In yet another aspect, the present invention provides a pharmaceutical preparation comprising any anti-human Tau (pS422) antibody provided herein, for example, for use in any of the aforementioned treatment methods. In one embodiment, the pharmaceutical preparation comprises any anti-human Tau (pS422) antibody provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical preparation comprises any anti-human Tau (pS422) antibody provided herein and at least one additional therapeutic agent (e.g., as described below).

[0808] The antibodies of the invention can be used alone or in combination with other drugs in therapy. For example, the antibodies of the invention can be co-administered with at least one additional therapeutic agent.

[0809] Such combination therapies as indicated above encompass both combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case administration of the antibodies of the invention may be performed before, simultaneously with, and / or after administration of the additional therapeutic agent or agents. In one embodiment, administration of the anti-human Tau (pS422) antibody and administration of the additional therapeutic agent are performed within about one month, or within about one, two, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

[0810] Antibodies of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal and (if local treatment is required) intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be performed by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusions.

[0811] The antibodies of the present invention will be formulated, dosed and administered in a manner consistent with good medical practice. Factors considered in this case include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the drug, the method of administration, the administration plan and other factors known to medical practitioners. The antibody is not necessarily formulated with one or more active agents currently used to prevent or treat the condition in question, but is optionally formulated with them. The effective amount of such other drugs depends on the amount of antibodies present in the formulation, the type of disease or therapy and other factors discussed above. These drugs are generally used in the same dosages as described herein and using the same administration routes as described herein, or in about 1% to 99% of the dosages described herein, or in any dosage and any route determined to be suitable empirically / clinically.

[0812] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prevention or treatment purposes, previous therapy, the patient's clinical history and response to the antibody, and the decision of the attending physician. The antibody is appropriately administered to the patient once or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.5 mg / kg-10 mg / kg) of antibody can be a starting candidate dose for administration to the patient, whether, for example, administered by one or more independent administrations or by continuous infusion. Depending on the factors mentioned above, a common daily dose may be from about 1 μg / kg to 100 mg / kg or more. For repeated administration over a period of several days or longer, depending on the disease, treatment will generally continue until the desired inhibitory effect on disease symptoms occurs. An exemplary dosage of the antibody will be in the range of about 0.05 mg / kg to about 10 mg / kg. Therefore, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every 3 weeks (e.g., so that the patient receives about 2 to about 20 or, for example, about 6 doses of the antibody). A higher initial administration dose may be administered, followed by one or more lower doses. However, other dosage regimens may be used. The progress of this therapy is easily monitored by conventional techniques and assays.

[0813] For example, where the indication being treated is Alzheimer's disease or prodromal Alzheimer's disease, the additional therapeutic agent may be selected from donepezil, memantine, rivastigmine, galantamine, rivastigmine, ergoloid mesylates, anti-Abeta antibacterial agents, and anti-α-synuclein antibodies.

[0814] It will be appreciated that any of the aforementioned preparations or treatment methods may be implemented using the immunoconjugates of the present invention instead of or in addition to the anti-human Tau (pS422) antibodies.

[0815] III. Manufactures

[0816] In another aspect of the invention, an article of manufacture is provided, the article of manufacture containing the above-described substances useful for treating, preventing and / or diagnosing a condition. The article of manufacture includes a container and or a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container holds a composition that is effective for treating, preventing and / or diagnosing a condition when it is itself or in combination with another composition and can have a sterile access port (for example, the container can be an intravenous bag or a vial with a stopper that can be penetrated by a hypodermic needle). At least one active substance in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat a selected disease. In addition, the article of manufacture may include (a) a first container containing a composition, wherein the composition comprises an antibody of the invention; and (b) a second container containing a composition, wherein the composition comprises other cytotoxic agents or therapeutic agents. In this embodiment of the invention, the article of manufacture may also include a package insert indicating that the composition can be used to treat a specific disease. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may also include other materials that are popular from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

[0817] It will be appreciated that any of the foregoing articles of manufacture may comprise an immunoconjugate of the invention instead of or in addition to an anti-human Tau (pS422) antibody.

[0818] IV. Specific Implementation Methods

[0819] 1. A humanized antibody that specifically binds to human Tau (pS422), wherein the antibody

[0820] a) comprising HVRs of SEQ ID NOs: 08, 09 and 10 in the heavy chain variable domain, or

[0821] b) HVR comprising SEQ ID NOs: 77, 09 and 10 in the heavy chain variable domain.

[0822] 2. The humanized antibody according to item 1, which further

[0823] a) comprising HVRs of SEQ ID NOs: 71, 73 and 15 in the light chain variable domain, or

[0824] b) comprising HVRs of SEQ ID NOs: 70, 72 and 15 in the light chain variable domain, or

[0825] c) in the light chain variable domain comprises HVRs of SEQ ID NOs: 12, 14 and 79, or

[0826] d) comprising HVRs of SEQ ID NOs: 71, 81 and 15 in the light chain variable domain, or

[0827] e) an HVR comprising SEQ ID NOs: 12, 14 and 74 in the light chain variable domain, or

[0828] f) HVR comprising SEQ ID NOs: 12, 14 and 75 in the light chain variable domain.

[0829] 3. The humanized antibody according to any one of items 1 to 2,

[0830] a) comprising HVRs of SEQ ID NOs: 08, 09 and 10 in the heavy chain variable domain and HVRs of SEQ ID NOs: 71, 73 and 15 in the light chain variable domain, or

[0831] b) HVRs comprising SEQ ID NOs: 08, 09 and 10 in the heavy chain variable domain and HVRs comprising SEQ ID NOs: 70, 72 and 15 in the light chain variable domain, or

[0832] c) HVRs comprising SEQ ID NOs: 77, 09 and 10 in the heavy chain variable domain and HVRs comprising SEQ ID NOs: 12, 14 and 79 in the light chain variable domain, or

[0833] d) comprising HVRs of SEQ ID NOs: 08, 09 and 10 in the heavy chain variable domain and HVRs of SEQ ID NOs: 12, 14 and 79 in the light chain variable domain, or

[0834] e) HVRs comprising SEQ ID NOs: 77, 09 and 10 in the heavy chain variable domain and HVRs comprising SEQ ID NOs: 71, 81 and 15 in the light chain variable domain, or

[0835] f) HVRs comprising SEQ ID NOs: 08, 09 and 10 in the heavy chain variable domain and HVRs comprising SEQ ID NOs: 12, 14 and 74 in the light chain variable domain, or

[0836] g) HVRs comprising SEQ ID NOs: 77, 09 and 10 in the heavy chain variable domain and HVRs comprising SEQ ID NOs: 12, 14 and 75 in the light chain variable domain.

[0837] 4. A humanized antibody according to any one of items 1 to 3, comprising

[0838] a) the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 67, or

[0839] b) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 66, or

[0840] c) the heavy chain variable domain of SEQ ID NO: 76 and the light chain variable domain of SEQ ID NO: 78, or

[0841] d) the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 78, or

[0842] e) the heavy chain variable domain of SEQ ID NO: 76 and the light chain variable domain of SEQ ID NO: 80, or

[0843] f) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 67, or

[0844] g) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 68, or

[0845] h) the heavy chain variable domain of SEQ ID NO: 76 and the light chain variable domain of SEQ ID NO: 69.

[0846] 5. Humanized bispecific antibodies, including

[0847] i) a first binding site selected from

[0848] a) the heavy chain variable domain of SEQ ID NO: 20 and the light chain variable domain of SEQ ID NO: 17, or

[0849] b) the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 16, or

[0850] c) the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 17, or

[0851] d) the heavy chain variable domain of SEQ ID NO: 21 and the light chain variable domain of SEQ ID NO: 17, or

[0852] e) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 67, or

[0853] f) the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 66, or

[0854] g) the heavy chain variable domain of SEQ ID NO: 76 and the light chain variable domain of SEQ ID NO: 78, or

[0855] h) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 78, or

[0856] i) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 66, or

[0857] j) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 68, or

[0858] k) the heavy chain variable domain of SEQ ID NO: 76 and the light chain variable domain of SEQ ID NO: 69, or

[0859] 1) a heavy chain variable domain of SEQ ID NO: 76 and a light chain variable domain of SEQ ID NO: 80,

[0860] and

[0861] ii) a second binding site selected from

[0862] a) the heavy chain variable domain of SEQ ID NO: 82 and the light chain variable domain of SEQ ID NO: 85, or

[0863] b) the heavy chain variable domain of SEQ ID NO: 83 and the light chain variable domain of SEQ ID NO: 85, or

[0864] c) the heavy chain variable domain of SEQ ID NO:84 and the light chain variable domain of SEQ ID NO:85.

[0865] 6. The humanized antibody according to any one of items 1 to 5, wherein the antibody is used to treat Alzheimer's disease.

[0866] 7. The humanized antibody according to any one of items 1 to 6, wherein the antibody is effector function-silenced.

[0867] 8. The humanized antibody according to any one of items 1 to 7, wherein the antibody has no effector function.

[0868] 9. The humanized antibody according to any one of items 1 to 8, wherein the antibody

[0869] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0870] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0871] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0872] iv) specifically binds to aggregates of human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02).

[0873] 10. A humanized antibody according to any one of items 2 to 9, wherein the antibody has an EC50 value of

[0874] a) 6 ng / mL or less for the human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0875] b) 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0876] c) 30 ng / mL or less aggregates for human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0877] d) 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0878] 11. The humanized antibody according to any one of items 1 to 10, wherein the antibody specifically binds to human Tau (pS422) (SEQ ID NO: 02) and does not bind to human Tau (SEQ ID NO: 01).

[0879] 12. The humanized antibody according to any one of items 1 to 11, wherein the antibody has valine residues at positions 4, 24 and 78 of the heavy chain variable domain.

[0880] 13. The humanized antibody according to any one of items 1 to 12, wherein the antibody has an arginine residue at position 71 of the heavy chain variable domain.

[0881] 14. The humanized antibody according to any one of items 1 to 13, wherein the antibody is a monoclonal antibody.

[0882] 15. The humanized antibody according to any one of items 1 to 14, wherein the antibody is an antibody fragment that binds to human Tau (pS422) and

[0883] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0884] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0885] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0886] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0887] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0888] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0889] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0890] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0891] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0892] 16. The humanized antibody according to any one of items 1 to 14, wherein the antibody is

[0893] a) a full length antibody of human IgG1 subclass, or

[0894] b) a full length antibody of human IgG4 subclass, or

[0895] c) a full length antibody of human IgG1 subclass having the mutations L234A, L235A and P329G,

[0896] d) a full length antibody of human IgG4 subclass having the mutations S228P, L235E and P329G,

[0897] e) a full length antibody of human IgG1 subclass having the mutations L234A, L235A and P329G in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the corresponding other heavy chain, or

[0898] f) a full length antibody of human IgG4 subclass having in both heavy chains the mutations S228P and P329G and in one heavy chain the mutations T366W and S354C and in the respective other heavy chain the mutations T366S, L368A, Y407V and Y349C.

[0899] 17. A humanized anti-human Tau (pS422) antibody, wherein

[0900] a) the antibody comprises two antibody heavy chains, each of which comprises a heavy chain variable domain and a heavy chain constant region, wherein

[0901] i) the variable domain comprises HVRs of SEQ ID NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10,

[0902] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue may or may not be present, and

[0903] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0904] b) the antibody comprises two antibody light chains, each of which comprises a light chain variable domain and a light chain constant domain, wherein

[0905] i) the variable domain comprises HVRs of SEQ ID NO: 71, SEQ ID NO: 73 and SEQ ID NO: 15,

[0906] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0907] and

[0908] c) the antibody

[0909] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0910] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0911] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0912] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0913] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0914] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0915] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0916] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0917] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0918] 18. A humanized anti-human Tau (pS422) antibody, wherein

[0919] a) the antibody comprises two antibody heavy chains, each of which comprises a heavy chain variable domain and a heavy chain constant region, wherein

[0920] i) the variable domain comprises HVRs of SEQ ID NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10,

[0921] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue may or may not be present, and

[0922] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0923] b) the antibody comprises two antibody light chains, each of which comprises a light chain variable domain and a light chain constant domain, wherein

[0924] i) the variable domain comprises HVRs of SEQ ID NO: 70, SEQ ID NO: 72 and SEQ ID NO: 15,

[0925] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0926] and

[0927] c) the antibody

[0928] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0929] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0930] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0931] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0932] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0933] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0934] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0935] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0936] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0937] 19. A humanized anti-human Tau (pS422) antibody, wherein

[0938] a) the antibody comprises two antibody heavy chains, each of which comprises a heavy chain variable domain and a heavy chain constant region, wherein

[0939] i) the variable domain comprises HVRs of SEQ ID NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10,

[0940] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue may or may not be present, and

[0941] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0942] b) the antibody comprises two antibody light chains, each of which comprises a light chain variable domain and a light chain constant domain, wherein

[0943] i) the variable domain comprises HVRs of SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 74,

[0944] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0945] and

[0946] c) the antibody

[0947] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0948] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0949] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0950] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0951] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0952] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0953] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0954] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0955] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0956] 20. A humanized anti-human Tau (pS422) antibody, wherein

[0957] a) the antibody comprises two antibody heavy chains, each of which comprises a heavy chain variable domain and a heavy chain constant region, wherein

[0958] i) the variable domain comprises HVRs of SEQ ID NO: 77, SEQ ID NO: 09 and SEQ ID NO: 10,

[0959] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue may or may not be present, and

[0960] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0961] b) the antibody comprises two antibody light chains, each of which comprises a light chain variable domain and a light chain constant domain, wherein

[0962] i) the variable domain comprises HVRs of SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 75,

[0963] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0964] and

[0965] c) the antibody

[0966] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0967] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0968] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0969] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0970] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0971] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0972] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0973] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0974] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0975] 21. A humanized anti-human Tau (pS422) antibody, wherein

[0976] a) the antibody comprises two antibody heavy chains, each of which comprises a heavy chain variable domain and a heavy chain constant region, wherein

[0977] i) the variable domain comprises the amino acid sequence of SEQ ID NO: 65,

[0978] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue may or may not be present, and

[0979] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0980] b) the antibody comprises two antibody light chains, each of which comprises a light chain variable domain and a light chain constant domain, wherein

[0981] i) the variable domain has the amino acid sequence of SEQ ID NO: 67,

[0982] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[0983] and

[0984] c) the antibody

[0985] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[0986] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[0987] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[0988] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[0989] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[0990] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[0991] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0992] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[0993] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[0994] 22. A humanized anti-human Tau (pS422) antibody, wherein

[0995] a) the antibody comprises two antibody heavy chains, each of which comprises a heavy chain variable domain and a heavy chain constant region, wherein

[0996] i) the variable domain has the amino acid sequence of SEQ ID NO: 65,

[0997] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue may or may not be present, and

[0998] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[0999] b) the antibody comprises two antibody light chains, each of which comprises a light chain variable domain and a light chain constant domain, wherein

[1000] i) the variable domain has the amino acid sequence of SEQ ID NO: 66,

[1001] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[1002] and

[1003] c) the antibody

[1004] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[1005] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[1006] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[1007] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[1008] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[1009] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[1010] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[1011] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[1012] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[1013] 23. A humanized anti-human Tau (pS422) antibody, wherein

[1014] a) the antibody comprises two antibody heavy chains, each of which comprises a heavy chain variable domain and a heavy chain constant region, wherein

[1015] i) the variable domain has the amino acid sequence of SEQ ID NO: 65,

[1016] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue may or may not be present, and

[1017] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[1018] b) the antibody comprises two antibody light chains, each of which comprises a light chain variable domain and a light chain constant domain, wherein

[1019] i) the variable domain has the amino acid sequence of SEQ ID NO: 68,

[1020] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[1021] and

[1022] c) the antibody

[1023] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[1024] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[1025] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[1026] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[1027] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[1028] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[1029] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[1030] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[1031] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[1032] 24. A humanized anti-human Tau (pS422) antibody, wherein

[1033] a) the antibody comprises two antibody heavy chains, each of which comprises a heavy chain variable domain and a heavy chain constant region, wherein

[1034] i) the variable domain has the amino acid sequence of SEQ ID NO: 76,

[1035] ii) the constant region is a human IgG1 constant region, wherein the C-terminal lysine residue may or may not be present, and

[1036] iii) the constant region comprises the amino acid changes L234A, L235A and P329G,

[1037] b) the antibody comprises two antibody light chains, each of which comprises a light chain variable domain and a light chain constant domain, wherein

[1038] i) the variable domain has the amino acid sequence of SEQ ID NO: 69,

[1039] ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region,

[1040] and

[1041] c) the antibody

[1042] i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or

[1043] ii) does not bind full-length human Tau (SEQ ID NO:01) at 1 μg / mL, and / or

[1044] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[1045] iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, and / or

[1046] v) specifically binds to full-length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or

[1047] vi) having an EC50 value of 6 ng / mL or less for a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[1048] vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[1049] viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[1050] ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[1051] 25. The humanized antibody according to any one of items 1 to 24, wherein the antibody has a lysine (K) amino acid residue at position 32 of the light chain variable domain (according to Kabat numbering).

[1052] 26. An isolated nucleic acid encoding the humanized antibody according to any one of items 1 to 25.

[1053] 27. A host cell comprising the nucleic acid according to item 26.

[1054] 28. A method for producing a humanized antibody according to any one of items 1 to 25, comprising the step of culturing a host cell as reported herein, thereby producing the humanized antibody.

[1055] 29. The method according to item 28, further comprising the step of recovering the humanized antibody from the cells or culture medium.

[1056] 30. A pharmaceutical preparation comprising the humanized antibody according to any one of items 1 to 25 and a pharmaceutically acceptable carrier.

[1057] 31. The pharmaceutical formulation according to item 30, further comprising an additional therapeutic agent.

[1058] 32. The pharmaceutical formulation of item 31, wherein the additional therapeutic agent is an anti-amyloid therapeutic agent.

[1059] 33. The pharmaceutical formulation according to item 32, wherein the anti-amyloid therapeutic agent is an anti-human α-synuclein antibody or an anti-Abeta antibody.

[1060] 34. A humanized antibody according to any one of items 1 to 25 for use as a medicament.

[1061] 35. The humanized antibody according to any one of items 1 to 25, for use in treating Alzheimer's disease.

[1062] 36. The humanized antibody according to any one of items 1 to 25, for use in the treatment of prodromal Alzheimer's disease.

[1063] 37. The humanized antibody according to any one of items 1 to 25, for use in treating mild Alzheimer's disease.

[1064] 38. The humanized antibody according to any one of items 1 to 25, for use in alleviating Tau (pS422)-induced neurodegeneration.

[1065] 39. The humanized antibody according to any one of items 1 to 25, for use in maintaining cognition and function.

[1066] 40. A humanized antibody according to any one of items 1 to 25 for use in slowing the rate of cognitive and functional decline.

[1067] 41. Use of the humanized antibody according to any one of items 1 to 25 for preparing a medicament.

[1068] 42. The use according to any one of items 34 and 41, wherein the medicament is used to treat Alzheimer's disease.

[1069] 43. The use according to any one of items 32 and 41 to 42, wherein the medicament is for treating prodromal Alzheimer's disease.

[1070] 44. The use according to any one of items 34 and 41 to 43, wherein the medicament is used to treat mild Alzheimer's disease.

[1071] 45. The use according to any one of items 34 and 41 to 44, wherein the medicament is used to reduce neurodegeneration caused by Tau (pS422).

[1072] 46. ​​The use according to any one of items 34 and 41 to 45, wherein the medicament is used to maintain cognition and function.

[1073] 47. The use according to any one of items 34 and 41 to 46, wherein the medicament is used to slow the rate of cognitive and functional decline.

[1074] 48. A method of treating an individual suffering from Alzheimer's disease, comprising administering to the individual an effective amount of the humanized anti-human Tau (pS422) antibody according to any one of items 1 to 25.

[1075] 49. A method for reducing neurodegeneration caused by Tau (pS422) in an individual, comprising administering to the individual an effective amount of the humanized anti-human Tau (pS422) antibody according to any one of items 1 to 25 to reduce neurodegeneration caused by Tau (pS422).

[1076] 50. A method of maintaining cognition and function in an individual, comprising administering to the individual an effective amount of the humanized anti-human Tau (pS422) antibody according to any one of items 1 to 25 to maintain cognition and function.

[1077] 51. A method for slowing the rate of cognitive and functional decline in an individual, comprising administering to the individual an effective amount of a humanized anti-human Tau (pS422) antibody according to any one of items 1 to 25 to slow the rate of cognitive and functional decline.

[1078] 52. Use of the humanized anti-human Tau (pS422) antibody according to any one of items 1 to 25 in reducing neurodegeneration caused by Tau (pS422).

[1079] 53. Use of the humanized anti-human Tau (pS422) antibody according to any one of items 1 to 25 in maintaining cognition and function.

[1080] 54. Use of the humanized anti-human Tau (pS422) antibody according to any one of items 1 to 25 in slowing the rate of cognitive and functional decline.

[1081] 55. The humanized antibody according to any one of items 1 to 25, wherein the antibody i) binds to Tau (pS422) on brain sections of Tau (pS422) transgenic mice and Alzheimer's disease patients; and / or labels Tau (pS422) in Tau (pS422) transgenic cells.

[1082] 56. The humanized antibody according to any one of items 1 to 25, wherein the antibody specifically binds / recognizes early and late disease-associated forms of human Tau (pS422).

[1083] 57. Use of the humanized antibody according to any one of items 1 to 25 for preventing the spread of human Tau (pS422)-related Alzheimer's disease.

[1084] 58. Use of the humanized antibody according to any one of items 1 to 25 for reducing lysosomal membrane disintegration.

[1085] 59. Use of the humanized antibody according to any one of items 1 to 25 for stabilizing lysosomal membranes against the destabilization and / or degradation caused by human Tau (pS422).

[1086] 60. Use of the humanized antibody according to any one of items 1 to 25 for preventing the progression of Alzheimer's disease.

[1087] The present invention also relates to the following embodiments:

[1088] 1. A humanized antibody that specifically binds to human Tau (pS422), wherein the antibody comprises:

[1089] In the heavy chain variable domain, the HVRs of SEQ ID NOs: 08, 09 and 10, and

[1090] In the light chain variable domain,

[1091] HVRs of SEQ ID NOs: 71, 73 and 15, or

[1092] HVRs of SEQ ID NOs: 70, 72 and 15, or

[1093] HVRs of SEQ ID NOs: 12, 14 and 74.

[1094] 2. The humanized antibody according to embodiment 1, comprising:

[1095] a) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 67, or

[1096] b) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 66, or

[1097] c) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 68.

[1098] 3. The humanized antibody according to any one of embodiments 1 to 2, wherein the antibody is effector function-silenced.

[1099] 4. The humanized antibody according to any one of embodiments 1 to 3, wherein the antibody

[1100] i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, and / or

[1101] ii) does not bind full-length human Tau (SEQ ID NO: 01) at 1 μg / mL, and / or

[1102] iii) specifically binds to full-length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 02), and / or

[1103] iv) specifically binds to human Tau aggregates phosphorylated at serine at position 422 (SEQ ID NO: 02).

[1104] 5. The humanized antibody according to any one of embodiments 1 to 4, wherein the antibody has an EC50 value of

[1105] a) 6 ng / mL or less for the human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03, and / or

[1106] b) 4.5 ng / mL or less for full-length human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[1107] c) 30 ng / mL or less for aggregates of human Tau (pS422) having the amino acid sequence of SEQ ID NO: 02, and / or

[1108] d) 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

[1109] 6. The humanized antibody of any one of embodiments 1 to 5, wherein the antibody specifically binds to human Tau (pS422) (SEQ ID NO: 02) and does not bind to human Tau (SEQ ID NO: 01).

[1110] 7. The humanized antibody according to any one of embodiments 1 to 6, wherein the antibody is

[1111] a) a full length antibody of human subclass IgG1, or

[1112] b) a full length antibody of human subclass IgG4, or

[1113] c) a full length antibody of human subclass IgG1 having the mutations L234A, L235A and P329G,

[1114] d) a full length antibody of human subclass IgG4 having the mutations S228P, L235E and P329G,

[1115] e) a human subclass IgG1 full length antibody having the mutations L234A, L235A and P329G in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the corresponding other heavy chain, or

[1116] f) a human subclass IgG4 full length antibody having the mutations S228P, L235E and P329G in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain.

[1117] 8. A bispecific antibody comprising

[1118] i) a first binding site selected from

[1119] a) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 66, or

[1120] b) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 67, or

[1121] c) a heavy chain variable domain of SEQ ID NO: 65 and a light chain variable domain of SEQ ID NO: 68,

[1122] and

[1123] ii) a second binding site selected from

[1124] a) the heavy chain variable domain of SEQ ID NO: 82 and the light chain variable domain of SEQ ID NO: 85, or

[1125] b) the heavy chain variable domain of SEQ ID NO: 83 and the light chain variable domain of SEQ ID NO: 85, or

[1126] c) the heavy chain variable domain of SEQ ID NO:84 and the light chain variable domain of SEQ ID NO:85.

[1127] 9. A pharmaceutical formulation comprising the antibody according to any one of embodiments 1 to 8 and optionally a pharmaceutically acceptable carrier.

[1128] 10. The antibody according to any one of embodiments 1 to 8 for use as a medicament.

[1129] 11. The antibody according to any one of embodiments 1 to 8 for use in treating Alzheimer's disease.

[1130] 12. The antibody according to any one of embodiments 1 to 8 for use in treating prodromal Alzheimer's disease.

[1131] 13. Use of the antibody according to any one of embodiments 1 to 8 in the preparation of a medicament.

[1132] 14. Use of the anti-human Tau (pS422) antibody according to any one of embodiments 1 to 8 in reducing Tau (pS422)-induced neurodegeneration.

[1133] 15. A method of treatment comprising administering an antibody according to any one of embodiments 1 to 8 for treating Alzheimer's disease, for treating prodromal Alzheimer's disease, for treating mild Alzheimer's disease, for reducing Tau (pS422)-induced neurodegeneration, for maintaining cognition and function, for slowing the rate of cognitive and functional decline, and / or for slowing the rate of neurofibrillary tangles accumulation.

[1134] V. Examples

[1135] The following are examples of methods and compositions of the present invention.It is understood that various other embodiments can be practiced, given the general description provided above.

[1136] Materials and methods

[1137] Recombinant DNA technology

[1138] DNA was manipulated using standard methods as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturers' instructions.

[1139] Gene and oligonucleotide synthesis

[1140] The desired gene fragments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthetic gene fragments were cloned into E. coli plasmids for propagation / amplification. The DNA sequence of the subcloned gene fragments was verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. Individual oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).

[1141] Reagents

[1142] If not stated otherwise, all commercial chemicals, antibodies or kits were used as provided according to the manufacturers' protocols.

[1143] Example 1

[1144] Preparation and purification of rabbit antibodies

[1145] immunity

[1146] New Zealand White (NZW) rabbits from Charles River Laboratories International, Inc. were used for immunization. The phosphopeptide Tau (416-430) [pS422] coupled to keyhole limpet hemocyanin (KLH) was dissolved in K3PO4 buffer pH 7.0 at a concentration of 1 mg / ml and mixed with complete Freund's adjuvant (CFA) (1:1) until a stable emulsion was produced. Three rabbits received an intradermal (id) injection of 2 ml of the emulsion, followed by a second intramuscular (im) and a third subcutaneous (sc) injection, each with 1 ml at one-week intervals. A fourth im injection of 1 ml was given two weeks later, followed by two further sc injections of 1 ml at four-week intervals. Peripheral whole blood samples of 10 ml were collected from each animal 4-6 days after the third, fourth, fifth, and sixth injections and used for single cell sorting in FACS. An additional 0.5 ml of serum from each animal was collected at the same time and used to determine Tau(416-463)[pS422]-specific antibody responses.

[1147] Antibody response

[1148] Antibody responses to immunization were determined using ELISA by serial dilution of serum, with 30 ng / well of biotinylated Tau (416-430) [pS422] incubated overnight in 1x PBS at 4°C on streptavidin pre-coated 96-well microtiter plates (MC1347, Micro Coat Biotechnologie GmbH, Bernried, Germany). For detection, goat anti-rabbit IgG linked to horseradish peroxidase (The Jackson laboratory) was used at a dilution of 1:16000. Ready-to-use BM Blue POD substrate (precipitated tetramethylbenzidine (TMB)) from Roche Diagnostics GmbH was used for visualization. The reaction was stopped by 1N HCl and measured at 450 / 690 nm in a Tecan Infinite.

[1149] B-cell clones

[1150] Plate coating

[1151] Sterile streptavidin-coated 6-well plates (cell culture grade) were incubated with a mixture of 3 biotinylated control peptides (unphosphorylated Tau (416-430), MCAK_human (88-102) [95-pSer], and MAP2_human (1802-1816) [pSer-1802]) or with the biotinylated phosphopeptide Tau (416-430) [pS422] (each at a concentration of 0.5-1 μg / ml in PBS) at room temperature for 1 hour. The plates were washed three times with sterile PBS before use. Cell culture 6-well plates were coated with 2 μg / ml KLH (keyhole limpet hemocyanin) in carbonate buffer (0.1 M sodium bicarbonate, 34 mM disodium hydrogen carbonate, pH 9.55) overnight at 4°C. The plates were washed three times in sterile PBS before use.

[1152] Isolation of rabbit peripheral blood mononuclear cells (PBMC)

[1153] Whole blood containing EDTA was diluted two-fold with 1xPBS before density centrifugation on a lympholyte mammal (Cedarlane Laboratories), which was performed to isolate rabbit PBMCs. PBMCs were washed twice before staining with antibodies.

[1154] EL-4B5 medium

[1155] RPMI 1640 (Pan Biotech, Aidenbach, Germany) supplemented with 10% FCS (Hyclone, Logan, UT, USA), 2 mM glutamine, 1% penicillin / streptomycin solution (PAA, Pasching, Austria), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany) and 0.05 mM β-mercaptoethanol (Gibco, Paisley, Scotland).

[1156] Macrophage / monocyte clearance

[1157] Sterile 6-well plates (cell culture grade) were used to remove macrophages and monocytes by nonspecific adhesion. The wells were coated with KLH (keyhole limpet hemocyanin) or with streptavidin and control peptide. Each well was filled with a maximum of 4 ml of culture medium and up to 6 x 10 6 Peripheral blood mononuclear cells from immunized rabbits were collected and allowed to bind for 1 hour in an incubator at 37° C. 50% of the cells in the supernatant were used for the panning step; the remaining 50% of the cells were directly subjected to immunofluorescence staining and single cell sorting.

[1158] Panning B cells on peptides

[1159] Six-well tissue culture plates coated with streptavidin and biotinylated peptide Tau(416-430)[pS422] were seeded with up to 6x10 6 Cells / 4 ml medium were added and allowed to bind for 1 hour at 37°C in an incubator. Non-adherent cells were removed by carefully washing the wells 1-2 times with 1xPBS. The remaining adherent cells were detached by trypsin in an incubator at 37°C for 10 minutes and then washed twice in medium. Cells were kept on ice until immunofluorescence staining was performed.

[1160] Immunofluorescence staining and single-cell sorting

[1161] Anti-rabbit IgG FITC for single cell sorting comes from AbD Serotec (STAR121F, Düsseldorf, Germany). For surface staining, cells from the clearing and panning steps were rolled and incubated with anti-rabbit IgG FITC antibodies in PBS in the dark at 4 ° C for 30 minutes in a cold room. After centrifugation, the supernatant was removed by aspiration. PBMCs were centrifuged and washed for 2 cycles with ice-cold PBS. Finally, PBMCs were resuspended in ice-cold PBS and FACS analysis was performed immediately. Propidium iodide (BD Pharmingen, San Diego, CA, USA) at a concentration of 5 μg / ml was added before FACS analysis to distinguish dead cells from live cells. FACS was performed using Becton Dickinson FACSAria (BD Biosciences, USA) with FACSDiva software, and single, FITC-labeled live cells were deposited in 96-well plates.

[1162] B-cell culture

[1163] B cell cultures were prepared by methods similar to those described by Zubler, RH et al., J. Immunol. 134 (1985) 3662-3668. Briefly, single sorted B cells were cultured in 96-well plates with 210 μl / well EL-4B5 medium and Pansorbin cells (1:20000) (Calbiochem (Merck), Darmstadt, Germany), 5% rabbit thymocyte supernatant and γ-irradiated EL-4-B5 mouse thymoma cells (2×10 4 The cells were cultured at 37°C in an incubator with 5% CO2 for 7 days. For screening, the B cell culture supernatant was removed and the cells were immediately harvested for variable region gene cloning or frozen at -80°C in 100 μl RLT buffer (Qiagen, Hilden, Germany).

[1164] B-cell clone screening

[1165] B cell culture supernatants were screened for binding to biotinylated Tau (416-430) [pS422] by ELISA. Non-phosphorylated Tau (416-430), KLH (keyhole limpet hemocyanin) and the irrelevant phosphopeptide MCAK_human (88-102) [95-pSer] were used as control antigens. For the preparation of ELISA plates, streptavidin pre-coated microtiter plates were incubated with 50 ng / ml of biotinylated Tau (415-430) [pS422] for 1 hour at room temperature. Coating of KLH or control peptide was performed at 1 μg / mL. B cell supernatants were diluted 1:5 to 1:10 and incubated with antigen-coated microtiter plates for 60 minutes. After intensive washing, the binding of rabbit antibodies was detected with a sheep anti-rabbit IgG digoxigenin-conjugated detection antibody (Chemicon AQ301D). After incubation with TMB at room temperature, absorbance was measured at 370 nm-492 nm. B cell clones that produced signals above background with biotinylated Tau (416-430) [pS422] but not with KLH and MCAK_human (88-102) [95-pSer] were further considered and subjected to variable region gene cloning.

[1166] PCR amplification and sequencing of the V-domain

[1167] Total RNA used 8 / 96 RNA kit (Macherey & Nagel; 740709.4, 740698) was prepared according to the manufacturer's protocol. All steps were performed on an epMotion 5075 liquid handling system (Eppendorf). RNA was eluted with 60 μl of RNAse-free water. 6 μl of RNA was used to generate cDNA by reverse transcriptase reaction using Superscript III First-Strand Synthesis SuperMix (Invitrogen 18080-400) and oligo dT primers according to the manufacturer's instructions. 4 μl of cDNA was used to amplify immunoglobulin heavy and light chain variable regions (VH and VL) using AccuPrime Super Mix (Invitrogen 12344-040) in a final volume of 50 μl using primers rbHCfinal.up and rbHCfinal.do for heavy chain and primers rbLCfinal.up and rbLCfinal.do for light chain (see table below). PCR conditions were as follows: hot start at 94°C for 5 min; 35 cycles of 94°C, 20 s, 70°C, 20 s, and 68°C, 45 s; and final extension at 68°C for 7 min.

[1168] surface:

[1169]

[1170]

[1171] 8 μl of the 50 μl PCR solution was loaded onto 48E-Gel 2% (Invitrogen G8008-02). Positive PCR reactions were used Extract II kit (Macherey & Nagel; 740609250) according to the manufacturer's protocol and eluted in 50 μl elution buffer. 12 μl of purified PCR product was directly sequenced in both directions using rbHCfinal.up and rbHCfinal.do for the heavy chain and rbLCfinal.up and rbLCfinal.do for the light chain (see table above).

[1172] Recombinant expression of rabbit monoclonal antibodies and rabbit / mouse chimeric antibodies

[1173] For recombinant expression of rabbit monoclonal antibodies, PCR products encoding VH or VL were cloned into expression vectors as cDNA by overhang cloning method (Haun, RS et al., Bio Techniques 13 (1992) 515-518; Li, MZ et al., Nature Methods 4 (2007) 251-256). Linearized expression plasmids encoding rabbit κ or γ constant regions and VL or VH inserts were amplified by PCR using overlapping primers. The purified PCR product was incubated with T4 DNA polymerase, which produces single-stranded overhangs. The reaction was terminated by adding dCTP. Next, the plasmid and insert were combined and incubated with RecA, which induces site-specific recombination. The recombinant plasmids were transformed into E. coli. The next day, growing clones were selected and the correctly recombined plasmids were tested by plasmid preparation, restriction analysis and DNA sequencing. For antibody expression, the isolated HC and LC plasmids were transiently co-transfected into HEK293 cells, and the supernatant was harvested after 1 week. To clone and express rabbit-mouse chimeric antibodies, VH and VL regions were amplified by PCR and subcloned into expression vectors containing mouse constant kappa regions or mouse constant gamma 1 regions. Rabbit / mouse chimeric HC and LC plasmids were isolated, tested for correct insertion by restriction analysis and DNA sequencing, and transiently co-transfected into HEK293 cells. Supernatants were collected one week after transfection.

[1174] Antibody purification

[1175] In MabSelectSuRe TMRecombinantly expressed rabbit antibodies were purified from cell culture supernatant on a MabSelect SuRe column (GE Healthcare). Before sample loading, the column was equilibrated with 25 mmol / L Tris-HCl, 25 mmol / L NaCl, pH 7.4. Antibody elution was completed with 50 mmol / L acetate pH 3.14. The eluted sample was immediately loaded on a desalting column (Sephadex G25, GE Healthcare) and eluted in 20 mmol / L His-HCl, 140 mmol / L NaCl pH 6.0. This buffer was also used to store the purified antibodies. The general storage temperature during the purification process was 4°C, room temperature, and -80°C after aliquoting. Recombinantly expressed rabbit / mouse chimeric antibodies from cell culture supernatant were purified on MabSelect SuRe TM The antibody was purified on a column (GE Healthcare). Before sample loading, the column was balanced with 1x PBS, pH 7.4. Antibody elution was completed with 100mmol / L citrate pH 3.0. The eluted sample was immediately neutralized with 2mol / L Tris / HCl pH 9.0. The antibody was then loaded on a size exclusion column (Superdex 200, GE Healthcare) and eluted in 20mmol / L His-HCl, 140mmol / L NaCl pH 6.0. This buffer was also used to store the purified antibody. The general storage temperature during the purification process was 4°C, room temperature, and -80°C after aliquoting.

[1176] Example 2

[1177] Anti-Tau pS422 monoclonal rabbit antibody is highly selective for Tau phosphorylated at pS422 and binds to fibrillar aggregates of TaupS422

[1178] ELISA

[1179] Rabbit monoclonal antibodies were recombinantly expressed in HEK293 cells. Cell culture supernatants or purified rabbit antibodies were tested for binding to biotinylated Tau (416-430) [pS422], non-phosphorylated Tau (416-430), KLH and the irrelevant phosphopeptide MCAK_human (88-102) [95-pSer] by ELISA. To prepare ELISA plates, streptavidin pre-coated microtiter plates were incubated with 50 ng / ml of biotinylated Tau (415-430) [pS422] at room temperature for 1 hour. Coating with KLH or control peptide was performed at 1 μg / mL. Different concentrations of rabbit anti-Tau pS422 antibody (Abcam AB51071) or supernatant containing rabbit antibodies were incubated in antigen-labeled microtiter plates for 60 minutes. After intensive washing, the binding of the rabbit antibody was detected with a sheep anti-rabbit IgG digoxigenin-conjugated detection antibody (Chemicon AQ301D). After incubation with TMB at room temperature, the absorbance at 370nm-492nm was measured. Antibody binding was characterized by its EC50 value. Antibodies that bind to biotinylated Tau (416-430) [pS422] and non-phosphorylated Tau (416-430) peptides were characterized by their EC50 values. Cross-reactivity with KLH or MCAK phosphopeptides was estimated by single-point measurements at high concentrations (i.e., 1:5 dilution of cell culture supernatant). The results are shown in the table below. It was found that the EC50 value for binding to Tau phosphopeptide was more than 100 times lower than the EC50 value for binding to Tau peptide, indicating at least 100-fold selectivity for phosphorylated Tau fragments compared to non-phosphorylated Tau peptides. For all antibodies, binding to KLH and MCAK control phosphopeptides was at background levels, which was approximately 1 < 3% of the maximum measured with Tau phosphopeptide.

[1180] surface:

[1181]

[1182] Specificity for soluble and aggregated full-length Tau pS422 was also tested. Fibrillar aggregates of Tau pS422 (300 μg / ml) were coated on polystyrene-based Maxisorb microtiter plates (Nunc) at room temperature (RT) overnight. In a similar manner, soluble full-length Tau and Tau pS422 were coated on Maxisorb microtiter plates. Rabbit anti-Tau pS422 antibody control (Abcam AB51071) or purified rabbit antibody was added and incubated for 60 minutes at a concentration of up to 1000 ng / ml. After intensive washing, binding to the rabbit antibody was detected with a sheep anti-rabbit IgG digoxin-conjugated detection antibody (Chemicon AQ301D). After incubation with TMB at room temperature, the absorbance was measured at 370nm-492nm. Antibody binding was characterized by its EC50 value. The results are shown in the table below.

[1183] surface:

[1184]

[1185]

[1186] Rabbit monoclonal antibodies bind to Tau-pS422 protein with an EC50 value of less than 1 ng / ml. The EC50 values ​​of the detected fibrillar TaupS422 range from 0.4 ng / ml to 14 ng / ml. The signal binding to the non-phosphorylated full-length Tau protein is indistinguishable from the background level. Therefore, it is estimated that each antibody binds to Tau pS422 and fibrillar Tau pS422 with at least 100-fold selectivity compared to Tau.

[1187] BIAcore TM

[1188] By BIAcore TM The analysis further investigated and confirmed the binding of TaupS422 aggregates to fibrils. The measurements were performed using a BIAcore 3000 instrument at 37°C. The system and sample buffer was HBS-EP (10 mM HEPES, 50 mM NaCl, 3.4 mM EDTA, 0.005% polysorbate 20 (v / v)). BIAcore TM The CM5 sensor chip was pre-treated. 0.1% SDS, 50 mM NaOH, 10 mM HCl and 100 mM H3PO4 were injected sequentially for 30 seconds for flow cells FC1, FC2, FC3 and FC4. TMWizard v.4.1 was used according to the manufacturer's instructions for the amine coupling procedure. After EDC / NHS activation of the sensor surface, non-phosphoselective anti-Tau mAb <tau>M-4 / 53-IgG was immobilized on sensor cells FC2, FC3, and FC4. As a control, an antibody against CK-MM (creatine kinase isoform), which recognizes an irrelevant antigen, was captured in cell FC1. mAb <tau>M-4 / 53-IgG and antibodies against CK-MM were diluted in 10mM NaAc pH 5.0 at 30μg / ml and injected at 10μl / min for 7min contact time to immobilize 10.000RU of antibody capture system. The surface was deactivated by 1M ethanolamine. The sensor was adapted by 5 cycles of phosphorylated filamentous Tau protein (stock solution 0.3mg / ml, diluted 1:100 in HBS-EP) as the analyte in solution at 10μl / min for 2min. Regeneration was performed at 30μl / min for 3min with 10mM glycine pH 2.5. It is assumed that the analyte binding mAb 4 / 53 does not dissociate the pTau filaments, because no dissociation of the pTau filaments from mAb4 / 53 was observed. For all further measurement cycles, 0.3 mg / ml pTau filaments were diluted 1:100 in HBS-EP buffer and injected at 10 μl / min for 1 min in order to present pTau to the corresponding antibody analyte in a heterogeneous sandwich-mode. The antibody analyte was diluted to a concentration of 100 nM in HBS-EP buffer and injected into the system at 20 μl / min for 3 min. After 3 min dissociation, the sensor surface was regenerated by 2 injections of 10 mM glycine pH 2.5 at 100 μl / min for 1 min followed by washing with 100 μl / min HBS for 15 s. The association and dissociation phases of the interaction were monitored. Since the antibody analyte in solution is bivalent, the avidity-burdened antibody-pTau kinetics are characterized by a biphasic dissociation model consisting of a rapid affinity-based early dissociation step, followed by an avidity-stabilized but rate-limiting kinetic step in the subsequent complex dissociation. 10 seconds (early) and 50 seconds (late) after the end of the analyte injection, kd and t / 2 (diss) were quantified if possible. Kinetic measurements were evaluated using a dual reference procedure. First, the signal from the FC1 reference was subtracted to correct for buffer volume effects and nonspecific binding. Second, the 0 nM analyte injection was subtracted to correct for the dissociation of the primary antibody from the respective capture system. The kinetic rates were calculated using the Langmuir 1.1 dissociation fitting model, according to BIAcore TM Evaluation software v.4.1 was used for evaluation. The half-life of antigen / antibody complex stability (min) was calculated according to the formula ln(2) / 60*kd.

[1189] The results are summarized in the table below.

[1190] surface:

[1191]

[1192]

[1193] Example 3

[1194] Binding of anti-Tau pS422 monoclonal rabbit antibody to intracellular pTau in brain slices from patients with Alzheimer's disease

[1195] Cryosections of human brain tissue from AD patients were used to investigate the specific and sensitive immunohistochemical detection of pTau pathology in Alzheimer's disease brain tissue by monoclonal rabbit anti-Tau pS422 antibody by immunofluorescence staining experiments. Rabbit IgGs were detected by goat anti-rabbit Alexa Conjugated secondary antibody (Invitrogen / Molecular Probes A11034) detection. For clones Mab 005, Mab 019, Mab 020, Mab 085, Mab 086 and Mab 097, specific and sensitive staining of pTau deposition and filaments was evident. Intracellular pTau deposition, such as large neurofibrillary tangles and extended neutrophil filaments, was significant. For all clones studied, a minimum effective concentration ranging between 0.08 and 0.016 μg / ml was determined, which showed a high sensitivity to the binding of real human pTau deposition.

[1196] Example 4

[1197] Humanization of Rabbit Anti-Human Tau (pS422) Antibody

[1198] As used herein, "variable domain" (variable domain of light chain (VL), variable domain of heavy chain (VH)) refers to each of the light chain and heavy chain domain pairs that are directly involved in the binding of the antibody to the Tau (pS422) antigen. The variable light and heavy chain domains have the same general structure and each domain comprises four framework (FR) regions whose sequences are extensively conserved connected by three "hypervariable regions".

[1199] Computer simulation (in silico) analyzes the structure of the VH and VL domains of rabbit antibody Mab 086, and compares with the structural database (IMGT) of people VH and VL domains.Select a group of V domains with the most similar structure to be used for the CDR of rabbit antibody to be grafted onto the people VH and VL domains of selection.In addition, considering the similarity of primary sequence, the selection of people V domains is narrowed by comparing the primary sequence of the VH and VL domains of rabbit antibody with the people V domain repertoire.In some humanized variants, back mutations to rabbit parent residues are introduced in people's framework regions.Similarly, in the case of being suitable for possibly increasing the affinity to antigen, the sudden change of CDR is introduced in some variants to maintain the CDR tertiary structure, and remove undesirable features such as cysteine ​​residues or residues that can be modified after antibody purification.

[1200] Heavy and light chain vectors containing each of the humanized variants were co-transfected in a matrix into HEK293 suspension cells in microtiter plates to obtain cell cultures expressing full-size IgGs of all possible light / heavy chain combinations. After 5 days of culture at 37°C, the supernatant was harvested and purified by protein A affinity chromatography on a microtiter scale.

[1201] Example 5

[1202] Generation of recombinant expression vectors

[1203] a) Production of vectors for expressing immunoglobulin heavy chains using human IgG1 constant regions

[1204] The humanized heavy chain encoding a fusion gene comprising a human IgG1 constant region (CH1, hinge, CH2, CH3) and a humanized anti-human Tau (pS422) antibody VH domain derived from the rabbit antibody Mab086 was assembled by fusing DNA fragments encoding each anti-human Tau (pS422)-specific antibody VH domain to sequence elements encoding the human IgG1 constant region.

[1205] The human IgG1 constant region has the following amino acid sequence:

[1206]

[1207] The expression vector also contains an origin of replication from the vector pUC18, which allows the plasmid to replicate in E. coli, and a β-lactamase gene that confers ampicillin resistance in E. coli.

[1208] The transcription unit of the antibody heavy chain contains the following functional elements in the 5' to 3' direction:

[1209] - the immediate early enhancer and promoter from human cytomegalovirus (P-CMV), including intron A,

[1210] - human heavy chain immunoglobulin 5'-untranslated region (5'UTR),

[1211] - mouse immunoglobulin heavy chain signal sequence,

[1212] - a nucleic acid encoding a heavy chain variable (VH) domain,

[1213] - a nucleic acid encoding a human IgG1 constant region, and

[1214] - Bovine growth hormone polyadenylation sequence (BGH pA).

[1215] b) Production of vectors expressing immunoglobulin light chains using human Ig-κ constant regions

[1216] The humanized κ light chain encoding a fusion gene comprising a human Ig-κ constant region (CL-κ) and an anti-human Tau (pS422) antibody VL (κ) domain derived from the rabbit antibody Mab 086 was assembled by fusing DNA fragments encoding each anti-human Tau (pS422) antibody VL (κ) domain to sequence elements encoding the human Ig-κ constant region.

[1217] The human Ig-κ constant region has the following amino acid sequence:

[1218]

[1219] The expression vector also contains an origin of replication from the vector pUC18, which allows the plasmid to replicate in E. coli, and a β-lactamase gene that confers ampicillin resistance in E. coli.

[1220] The transcription unit of the antibody kappa light chain contains the following functional elements in the 5' to 3' direction:

[1221] - the immediate early enhancer and promoter from human cytomegalovirus (P-CMV), including intron A,

[1222] - human heavy chain immunoglobulin 5'-untranslated region (5'UTR),

[1223] - mouse immunoglobulin heavy chain signal sequence,

[1224] - a nucleic acid encoding a light chain variable (VL) domain,

[1225] - a nucleic acid encoding a human Ig-κ constant region, and

[1226] - Bovine growth hormone polyadenylation sequence (BGH pA).

[1227] c) Production of vectors for expressing immunoglobulin light chains using human Ig-λ constant regions

[1228] The humanized λ light chain encoding a fusion gene comprising a human Ig-λ constant region (CL-λ) and an anti-human Tau (pS422) antibody VL (λ) domain derived from the rabbit antibody Mab 086 was assembled by fusing DNA fragments encoding each anti-human Tau (pS422) antibody VL (λ) domain to sequence elements encoding the human Ig-λ constant region.

[1229] The human Ig-λ constant region has the following amino acid sequence:

[1230]

[1231] The expression vector also contains an origin of replication from the vector pUC18, which allows the plasmid to replicate in E. coli, and a β-lactamase gene that confers ampicillin resistance in E. coli.

[1232] The transcription unit of the antibody lambda light chain contains the following functional elements in the 5' to 3' direction:

[1233] - the immediate early enhancer and promoter from human cytomegalovirus (P-CMV), including intron A,

[1234] - human heavy chain immunoglobulin 5'-untranslated region (5'UTR),

[1235] - mouse immunoglobulin heavy chain signal sequence,

[1236] - a nucleic acid encoding a variable light chain (VL) domain,

[1237] - a nucleic acid encoding a human Ig-λ constant region, and

[1238] - Bovine growth hormone polyadenylation sequence (BGH pA).

[1239] d) Generation of vectors for expression of immunoglobulin kappa light chains using human Ig-kappa constant regions

[1240] Human Ig-κ light chain encoding a fusion gene comprising a human Ig-κ constant region (CL-κ) and an anti-human Tau (S422) antibody VL (κ) domain derived from the rabbit antibody Mab 086, assembled by fusing DNA fragments encoding the respective anti-human Tau (pS422)-antibody VL (κ) domains to sequence elements encoding the human Ig-κ constant region. The construct is in a genomic structure, i.e., introns are present in the signal peptide and between the VL (κ) and CL-κ domains.

[1241] The expression vector also contains an origin of replication from the vector pUC18, which allows the plasmid to replicate in E. coli, and a β-lactamase gene that confers ampicillin resistance in E. coli.

[1242] The transcription unit of the antibody kappa light chain contains the following functional elements in the 5' to 3' direction:

[1243] - Immediate early enhancer and promoter from human cytomegalovirus (P-CMV)

[1244] - human heavy chain immunoglobulin 5'-untranslated region (5'UTR),

[1245] - mouse immunoglobulin heavy chain signal sequence,

[1246] - a nucleic acid encoding a light chain variable (VL) domain,

[1247] - human IgG kappa constant region, and

[1248] - Bovine growth hormone polyadenylation sequence (BGH pA).

[1249] e) Production of vector for expressing immunoglobulin lambda light chain using human Ig-λ constant region

[1250] Human Ig-λ light chain encoding a fusion gene comprising a human Ig-λ constant region (CL-λ) and an anti-human Tau (S422) antibody VL (λ) domain derived from the rabbit antibody Mab 086, assembled by fusing DNA fragments encoding the respective anti-human Tau (pS422)-antibody VL (λ) domains to sequence elements encoding the human Ig-λ constant region. The construct is in a genomic structure, i.e., introns are present in the signal peptide and between the VL (λ) and CL-λ domains.

[1251] The expression vector also contains an origin of replication from the vector pUC18, which allows the plasmid to replicate in E. coli, and a β-lactamase gene that confers ampicillin resistance in E. coli.

[1252] The transcription unit of the antibody lambda light chain contains the following functional elements in the 5' to 3' direction:

[1253] - the immediate early enhancer and promoter from human cytomegalovirus (P-CMV),

[1254] - human heavy chain immunoglobulin 5'-untranslated region (5'UTR),

[1255] - mouse immunoglobulin heavy chain signal sequence,

[1256] - a nucleic acid encoding a light chain variable (VL) domain,

[1257] - human IgG lambda constant region, and

[1258] - Bovine growth hormone polyadenylation sequence (BGH pA).

[1259] Example 6

[1260] Recombinant production of anti-human Tau (pS422) antibodies

[1261] Antibodies were produced in transiently transfected HEK293 cells (human embryonic kidney cell line 293-derived) cultured in F17 medium (Invitrogen Corp.). For transfection of each vector as described in Example 5, "293-Free" transfection reagent (Novagen) was used. Antibodies were expressed from individual expression plasmids. Transfection was performed as indicated in the manufacturer's instructions. Three to seven days after transfection, cell culture supernatants containing recombinant antibodies were harvested. Supernatants were stored at reduced temperatures (e.g., -80°C) until purification.

[1262] General information on the recombinant expression of human immunoglobulins in eg HEK293 cells is given in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

[1263] Example 7

[1264] Purification of recombinant anti-human Tau (pS422) antibody

[1265] The culture supernatant containing the antibody was filtered and purified by two chromatography steps.

[1266] Antibodies were captured by affinity chromatography using HiTrap MabSelectSuRe (GE Healthcare) equilibrated with PBS (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl), pH 7.4. Unbound proteins were removed by washing with equilibration buffer, and antibodies were recovered with 25 mM citrate buffer, pH 3.1, which was adjusted to pH 6.0 with 1 M Tris-base, pH 9.0 immediately after elution.

[1267] Using Superdex 200 TM Size exclusion chromatography on ELISA (GE Healthcare) was used as a second purification step. Size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. The antibody-containing solution was concentrated using an Ultrafree-CL centrifugal filter device equipped with a Biomax-SK membrane (Millipore, Billerica, MA, USA) and stored at -80°C.

[1268] Example 8

[1269] Kinetic screening

[1270] Kinetic screening was performed on a BIAcore 4000 instrument equipped with a BIAcore CM5 sensor according to Schraeml et al. (Schraeml, M. and M. Biehl, Methods Mol. Biol. 901 (2012) 171-181). The BIAcore 4000 instrument was under the control of software version V1.1. The BIAcore CM5 series S chip was installed into the instrument and liquid dynamics treatment and pretreatment were performed according to the manufacturer's instructions. The instrument buffer was HBS-EP buffer (10mM HEPES (pH 7.4), 150mM NaCl, 1mM EDTA, 0.05% (w / v) P20). The antibody capture system was prepared on the sensor surface. Polyclonal goat anti-human antibodies (Jackson Lab.) with human IgG-Fc specificity were fixed to points 1, 2, 4 and 5 in flow channels 1, 2, 3 and 4 of the instrument at 10,000 RU at 30 μg / ml in 10 mM sodium acetate buffer (pH 5) using NHS / EDC chemistry. Antibodies were captured at points 1 and 5 in each flow channel. Points 2 and 4 were used as reference points. The sensor was deactivated with a 1 M ethanolamine solution. Humanized antibody derivatives were applied at concentrations of 44 nM to 70 nM in an instrument buffer supplemented with 1 mg / ml CMD (carboxymethyl dextran). Antibodies were injected at a flow rate of 30 μl / min for 2 min. The capture level (CL) of antibodies present on the surface was measured in relative response units (RU). The analyte, phosphorylated human tau protein, non-phosphorylated human tau protein and phosphorylated human tau mutant protein T422S in the solution were injected at 300nM at a flow rate of 30μl / min for 3min. The dissociation was monitored for 5min. The capture system was regenerated by injecting 10mM glycine buffer pH 1.7 into all flow channels at 30μL / min for 1min. Two reporting points (signals recorded shortly before the end of the analyte injection (expressed as the late binding period (BL)) and signals recorded shortly before the end of the dissociation time (stable late period (SL))) were used to characterize the kinetic screening performance. In addition, the dissociation rate constant kd (1 / s) was calculated according to the Langmuir model and the antibody / antigen complex half-life was calculated in minutes according to the formula ln(2) / (60*kd). The molar ratio (MR) is calculated according to the formula MR = (binding late (RU)) / (capture level (RU)) * (MW (antibody) / (MW (antigen)). In the case of a sensor configuration with an appropriate amount of antibody ligand capture level, each antibody should be able to functionally bind at least one analyte in solution, which is represented by a molar ratio of MR = 1.0. The molar ratio is then also an indication of the valency pattern of analyte binding. For an antibody that binds two analytes, the maximum valency may be MR = 2, with a valency of one for each Fab.

[1271] In another embodiment, the kinetic rates are determined at 25° C. and 37° C. using the same experimental setup but using a variety of concentration series of each analyte in solution at 0 nM (buffer), 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM and 300 nM. Kinetic data are calculated from the concentration-dependent binding behavior using the BIAcore evaluation software according to the manufacturer's instructions and the Langmuir 1.1 model with RMAX global.

[1272] Example 9

[1273] ELISA

[1274] Non-biotinylated peptides / proteins / aggregates in PBS were added to uncoated Maxisorb plates, and biotinylated peptides / proteins / aggregates in PBS were added to streptavidin-coated Maxisorb plates and incubated overnight. The supernatant was discarded and the wells were washed three times with 90 μl of washing buffer (1x PBS / 0.1% Tween 20). The remaining reactive spots were blocked with blocking buffer (1xPBS / 2% BSA (bovine serum albumin fraction V, fatty acid-free, Roche, Cat. No.: 10735078001) / 0.05% Tween 20) by incubation for 1 h. The supernatant was discarded and the wells were washed three times with 90 μl of washing buffer. Sample and control antibodies were prepared at 12 dilutions (1:2) in ELISA buffer (1xPBS / 0.5% BSA (bovine serum albumin fraction V, fatty acid-free, Roche, Cat. No.: 10735078001) / 0.05% Tween 20) with a starting concentration of 500 ng / mL. The incubation time was 60 minutes at room temperature on a shaker. The supernatant was discarded and the wells were washed three times with 90 μl of wash buffer. The secondary antibody solution was prepared in ELISA buffer. A total of 25 μl of the antibody mixture was transferred to all wells of the assay plate and the plate was then incubated on a shaker at room temperature for 60 minutes. The supernatant was discarded and the wells were washed three times with 90 μl of wash buffer. 25 μl of ABTS solution was added to all wells. The absorbance was read at 405nm-492nm.

[1275] Example 10

[1276] Kinetic measurements of pTau peptide binding to anti-pTau antibodies (capture assay)

[1277] The binding of pTau fragments to anti-pTau antibodies was studied by surface plasmon resonance using a BIACORE T200 instrument (GE Healthcare). At pH 5.0, approximately 10,000 resonance units (RU) of the capture system (10 μg / ml goat anti-human Fc antibody; Jackson Immuno Research; Order code: JIR109-005-098) were coupled to a CM5 chip (GE Healthcare BR-1005-30) using an amine coupling kit provided by GE Healthcare. As a running buffer, HBS-N pH 7.4 (10 mM HEPES, 150 mM NaCl, pH 7.4, GE Healthcare) was used. To measure the samples, the running and dilution buffer was PBS-T at pH 7.4 (10 mM phosphate buffered saline containing 0.05% Tween 20). The sample block was set to 12 ° C. The flow cell was set to 25 ° C and perfused twice with the flow buffer.

[1278] Anti-pTau antibodies were captured by injecting a 10 μg / ml solution for 300 seconds at a flow rate of 10 μl / min. Binding was measured by injecting pTau fragments at various concentrations in solution (starting from 300 nM at a dilution of 1:3) for 180 seconds at a flow rate of 30 μl / min. The dissociation phase was monitored for up to 300 seconds and triggered by switching from the sample solution to the running buffer. The surface was regenerated by washing with a 0.85% phosphoric acid solution for 60 seconds, followed by a 5 mM sodium hydroxide solution for 60 seconds at a flow rate of 10 μl / min. Bulk refractive index differences were corrected by subtracting the response obtained from the goat anti-human Fc antibody surface. Blank injections were also subtracted (= double reference). To calculate KD and other kinetic parameters, the Langmuir 1:1 model was used.

[1279] Embodiment 11

[1280] Kinetic measurements of anti-pTau antibody Fab fragments binding to pTau fragments (direct assay)

[1281] The binding of anti-pTau antibody Fab fragment samples to pTau fragments was studied by surface plasmon resonance using a BIACORE T200 instrument (GE Healthcare). Approximately 20 resonance units (RU) of biotinylated pTau fragments (0.2 μg / ml; pTau (416-430) [Bi-XUUU-416; pSer-422] were coupled to S-series SA chips (GE Healthcare BR-1005-31). The running and dilution buffer used for immobilization was HBS-N pH 7.4 (10 mM HEPES, 150 mM NaCl, pH 7.4, GE Healthcare). For the following kinetic characterization, the running and dilution buffer was PBS-T at pH 7.4 (10 mM phosphate buffered saline, including 0.05% Tween 20). The sample block was set to 12°C. The flow cell was set to 25°C and perfused twice with flow buffer.

[1282] Binding was measured by injecting samples of anti-pTau antibody Fab fragments at different concentrations in solution (starting from 100 nM in a 1:3 dilution) for 180 seconds at a flow rate of 30 μl / min. The dissociation phase was monitored for up to 600 seconds and triggered by switching from the sample solution to the running buffer. The surface was regenerated by washing with a single injection of 10 mM glycine solution pH 1.7 for 60 seconds with a flow rate of 10 μl / min. Bulk refractive index differences were corrected by subtracting the response obtained from a blank surface. The blank injection was also subtracted (= double reference). For the calculation of KD and other kinetic parameters, the Langmuir 1:1 model was used.

[1283] Example 12

[1284] Kinetic measurements of anti-pTau antibody Fab fragments binding to full-length pTau (direct assay)

[1285] The binding of anti-pTau antibody Fab fragment samples to full-length pTau protein was studied by using the surface plasmon resonance of a BIACORE T200 instrument (GE Healthcare). Approximately 200 resonance units (RU) of biotinylated pTau protein (2 μg / ml) were coupled to an S series SA chip (GE Healthcare BR-1005-31). The running and dilution buffer for fixation was HBS-N pH 7.4 (10 mM HEPES, 150 mM NaCl, pH 7.4, GE Healthcare). For the following kinetic characterization, the running and dilution buffer was PBS-T (10 mM phosphate buffered saline, including 0.05% Tween 20) at pH 7.4. The sample block was set to 12 ° C. The flow cell was set to 25 ° C and perfused twice with running buffer.

[1286] Binding was measured by injecting samples of anti-pTau antibody Fab fragments at different concentrations in solution (starting from 300 nM at a dilution of 1:3) for 180 seconds at a flow rate of 30 μl / min. The dissociation phase was monitored for up to 600 seconds. And triggered by switchin...

Claims

1. Humanized bispecific antibody, including i) a first binding site having a) the heavy chain variable domain of SEQ ID NO: 20 and the light chain variable domain of SEQ ID NO: 17, or b) the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 16, or c) the heavy chain variable domain of SEQ ID NO: 21 and the light chain variable domain of SEQ ID NO: 17, or d) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 67, or e) the heavy chain variable domain of SEQ ID NO: 65 and the light chain variable domain of SEQ ID NO: 66, or f) the heavy chain variable domain of SEQ ID NO: 76 and the light chain variable domain of SEQ ID NO: 78, or g) the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 78, or h) a heavy chain variable domain of SEQ ID NO: 76 and a light chain variable domain of SEQ ID NO: 80; and ii) a second binding site selected from a) the heavy chain variable domain of SEQ ID NO: 82 and the light chain variable domain of SEQ ID NO: 85, or b) the heavy chain variable domain of SEQ ID NO: 83 and the light chain variable domain of SEQ ID NO: 85, or c) the heavy chain variable domain of SEQ ID NO:84 and the light chain variable domain of SEQ ID NO:

85.

2. The humanized antibody according to claim 1, wherein the antibody is effector function silent.

3. The humanized antibody according to claim 1, wherein the antibody i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or ii) does not bind to human Tau having an amino acid sequence of SEQ ID NO: 01 at 1 μg / mL, and / or iii) specifically binds to human Tau phosphorylated at serine at position 422 having an amino acid sequence of SEQ ID NO: 02, and / or iv) specifically binds to aggregates of human Tau phosphorylated at serine at position 422 having the amino acid sequence of SEQ ID NO:

02.

4. The humanized antibody according to claim 1, wherein the antibody has an EC50 value of a) 6 ng / mL or less for a polypeptide having the amino acid sequence of SEQ ID NO: 03, and / or b) 4.5 ng / mL or less for human Tau phosphorylated at serine at position 422 having the amino acid sequence of SEQ ID NO: 02, and / or c) 30 ng / mL or less for aggregates of human Tau phosphorylated at serine at position 422 having the amino acid sequence of SEQ ID NO: 02, and / or d) 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

5. The humanized antibody according to claim 1, wherein the antibody specifically binds to human Tau phosphorylated at serine at position 422 having the amino acid sequence of SEQ ID NO: 02 and does not bind to human Tau having the amino acid sequence of SEQ ID NO:

01.

6. The humanized antibody according to claim 3, wherein the antibody specifically binds to human Tau phosphorylated at serine at position 422 having the amino acid sequence of SEQ ID NO: 02 and does not bind to human Tau having the amino acid sequence of SEQ ID NO:

01.

7. The humanized antibody according to claim 4, wherein the antibody specifically binds to human Tau phosphorylated at serine at position 422 having the amino acid sequence of SEQ ID NO: 02 and does not bind to human Tau having the amino acid sequence of SEQ ID NO:

01.

8. The humanized antibody according to any one of claims 1 to 7, wherein the antibody is a monoclonal antibody.

9. The humanized antibody of claim 3, wherein the antibody is an antibody fragment that binds to human Tau phosphorylated at serine at position 422 and i) specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 03, and / or ii) does not bind to human Tau having an amino acid sequence of SEQ ID NO: 01 at 1 μg / mL, and / or iii) specifically binds to human Tau phosphorylated at serine at position 422 having an amino acid sequence of SEQ ID NO: 02, and / or iv) specifically binds to aggregates of human Tau phosphorylated at serine at position 422 having an amino acid sequence of SEQ ID NO: 02, and / or v) specifically binds to full-length human Tau having an amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and / or vi) having an EC50 value of 6 ng / mL or less for a human Tau fragment phosphorylated at serine at position 422 having an amino acid sequence of SEQ ID NO: 03, and / or vii) has an EC50 value of 4.5 ng / mL or less for full-length human Tau phosphorylated at serine at position 422 having the amino acid sequence of SEQ ID NO: 02, and / or viii) having an EC50 value of 30 ng / mL or less for aggregates of human Tau phosphorylated at serine at position 422 having the amino acid sequence of SEQ ID NO: 02, and / or ix) having an EC50 value of 125 ng / mL or less for human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A.

10. The humanized antibody according to claim 1, wherein the antibody is a) a full-length antibody of human IgG1 subclass, or b) a full length antibody of human IgG4 subclass, or c) a full length antibody of human IgG1 subclass having the mutations L234A, L235A and P329G, d) a full length antibody of the human IgG4 subclass having the mutations S228P, L235E and P329G, e) a full length antibody of the human IgG1 subclass having the mutations L234A, L235A and P329G in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the corresponding other heavy chain, or f) a full length antibody of human IgG4 subclass having in both heavy chains the mutations S228P and P329G and in one heavy chain the mutations T366W and S354C and in the respective other heavy chain the mutations T366S, L368A, Y407V and Y349C.

11. The humanized antibody according to claim 8, wherein the antibody is a) a full-length antibody of human IgG1 subclass, or b) a full length antibody of human IgG4 subclass, or c) a full length antibody of the human IgG1 subclass having the mutations L234A, L235A and P329G, d) a full length antibody of the human IgG4 subclass having the mutations S228P, L235E and P329G, e) a full length antibody of the human IgG1 subclass having the mutations L234A, L235A and P329G in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain, or f) a full length antibody of human IgG4 subclass having in both heavy chains the mutations S228P and P329G and in one heavy chain the mutations T366W and S354C and in the respective other heavy chain the mutations T366S, L368A, Y407V and Y349C.

12. An isolated nucleic acid encoding the humanized antibody according to any one of claims 1 to 11.

13. A host cell comprising the nucleic acid according to claim 12.

14. A method for producing a humanized antibody according to any one of claims 1 to 11, comprising the step of culturing the host cell according to claim 13 to produce the humanized antibody.

15. The method according to claim 14, further comprising the step of recovering the humanized antibody from the cells or culture medium.

16. A pharmaceutical preparation comprising the humanized antibody according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

17. The pharmaceutical formulation of claim 16, further comprising an additional therapeutic agent.

18. The pharmaceutical formulation of claim 17, wherein the additional therapeutic agent is an anti-amyloid therapeutic agent.

19. The pharmaceutical formulation of claim 18, wherein the anti-amyloid therapeutic agent is an anti-human α-synuclein antibody or an anti-Abeta antibody.

20. Use of the humanized antibody according to any one of claims 1 to 11 for preparing a medicament, wherein the medicament is used for treating Alzheimer's disease.

21. Use of a humanized antibody according to any one of claims 1 to 11 for the preparation of a medicament, wherein the medicament is used for the treatment of prodromal Alzheimer's disease.

22. Use of the humanized antibody according to any one of claims 1 to 11 for preparing a medicament, wherein the medicament is used to treat mild Alzheimer's disease.

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