Protein-based therapies and diagnostics for tau protein-mediated pathologies in alzheimer's disease

Abstract

The present invention provides unique therapeutic and diagnostic antibodies and fragments, portions, derivatives and variants thereof and methods of making the same that bind to regions of tau protein that contribute to initiation and propagation of pathological tau-tau interactions. The invention also relates to methods of using those antibodies for diagnosis, prevention and treatment of Alzheimer's disease and related tauopathies. The invention also provides a method for prophylactic and therapeutic treatment of Alzheimer's disease and other neurodegenerative tauopathies. The method entails injection of antibodies and / or peptide vaccines that elicit an immune response against pathological tau and tau deposits in the brain of a patient. Suitable vaccines represent tau peptides carrying one or more tau therapeutic epitopes provided herein.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201810662577.7, filed on September 14, 2012, entitled "Protein-based therapy and diagnosis of TAU protein-mediated lesions in Alzheimer's disease".

[0002] This invention claims priority to U.S. Provisional Application No. 61 / 536,339, filed September 19, 2011, and U.S. Provisional Application No. 61 / 653,115, filed May 30, 2012, the contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list that has been submitted via EFS in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on September 13, 2012, is named SEQUENCE_LISTING.txt and is 155,400 bytes in size. Technical Field

[0005] The present invention is characterized by protein-based (e.g., antibody, peptide) methods and means for interfering with the production and clearance of certain forms of tau protein, said forms of tau protein being involved in the promotion and / or progression of pathological tau protein-tau protein aggregates in Alzheimer's disease; and methods for generating anti-tau protein antibodies suitable for the diagnosis and treatment of Alzheimer's disease. The invention further relates to methods and means for diagnosing Alzheimer's disease, including methods for grading and assessing treatment progress. background

[0006] Alzheimer's disease (AD) is a progressive neurodegenerative disorder that damages higher brain structures, such as those involved in memory and cognition. The disease leads to cognitive impairment and decline in memory, learning, language, and the ability to perform intentional and purposeful activities. AD is also accompanied by behavioral, emotional, interpersonal, and social regression. These cognitive and behavioral deficits make life difficult (Burns et al., 2002). Patients with advanced AD often cannot speak, understand language, or manage their own basic personal care, eventually requiring full-time care and monitoring, and often becoming dependent on family members and nursing homes. AD is a leading cause of dementia in the elderly, and its incidence is predicted to increase as the proportion of older people in the population grows. The total number of people with AD is projected to increase at least threefold between 2000 and 2050, making AD a global public health problem (Sloane et al., 2002). Clinical management of AD remains primarily supportive. That is, the goal of treating patients is to prevent, control, or mitigate complications and side effects from AD, and to improve their comfort and quality of life. There remains an unmet need for treatments that directly target the disease process and have disease-modifying effects.

[0007] The histological features of Alzheimer's disease (AD) include extraneuronal plaques and intracellular and extracellular neurofibrillary tangles in the brain. Plaques are primarily composed of β-amyloid (Aβ), while tangles contain pathological forms of tau protein, such as pathological tau protein conformational isoforms and their aggregates. The relationship between plaques and tangles and the disease process remains unclear, but studies have shown a link between amyloid and tau protein pathogenesis (Hardy et al., 1998; Oddo et al., 2004; Rapoport et al., 2002; Roberson et al., 2007; Shipton et al., 2011). The major role of Aβ in AD lesions was initially proposed in the hypothesis known as the "Aβ cascade," in which tau protein phosphorylation and tangle formation follow Aβ deposition, followed by neuronal death (Hardy and Allsop, 1991; Hardy and Selkoe, 2002; see Walsh and Selkoe, 2004 for a review; also see Seabrook et al., 2007). Therefore, initial treatments for AD primarily focused on targeting Aβ. However, documentation demonstrates a lack of association between the extent of Aβ lesions in the brain of AD patients and clinical disease progression (Braak and Braak, 1991). Furthermore, asymptomatic individuals have shown widespread, often diffuse, amyloid deposition at autopsy (Braak and Braak, 1991), and neuronal loss and amyloid deposition occur in different brain regions, at least in early AD (Carter and Lippa, 2001). Therefore, targeting Aβ alone is insufficient to alter the disease course in any or all patients. However, the most advanced disease-targeting therapies undergoing clinical trials in AD patients remain those aimed at the production and clearance of Aβ. These therapies include passive immunotherapies such as bapinuzumab, solanuzumab, and ponizumab; and the small-molecule γ-secretase inhibitor semacet (see Citron et al., 2010 for a review).

[0008] A well-established role of tau protein in Alzheimer's disease has been demonstrated in numerous studies. For example, Braak showed that the closest association between tau protein tangles and amyloid plaques and Alzheimer's disease degeneration is the presence of tau protein tangles rather than amyloid plaques (Braak and Braak, 1991). In another study, Aβ neurotoxicity in cultured neurons appeared to depend on tau protein (Rapoport et al., 2002). Recently, reducing endogenous tau protein prevented behavioral defects in transgenic mice expressing human amyloid precursor protein without altering their high Aβ levels (Roberson et al., 2007). Tau protein reduction also protected both transgenic and non-transgenic mice from excitotoxicity. Similarly, Santacruz et al. demonstrated that reducing tau protein levels restored memory function in a tau protein-related disease model (Santacruz et al., 2005). Therefore, therapies aimed at reducing tau protein represent an effective strategy for treating Alzheimer's disease and other tau protein-related conditions.

[0009] Tau proteins belong to the intrinsically disordered protein family, characterized by the absence of a rigid three-dimensional structure in their physiological context (Zilka et al., 2008). However, tau protein truncation and hyperphosphorylation can lead to pathological transformations from their intrinsically disordered state to a variety of soluble and insoluble misordered structures, including paired helical filaments (PHFs) and other aggregates (Wischik et al., 1988a; Wischik et al., 1988b; Novak et al., 1993; Skhrabana et al., 2006; Zilka et al., 2008; Kovacech et al., 2010). These structural changes result in the acquisition of toxic functions, loss of the physiological functions of the native protein, or both (Zilka et al., 2008; Kovacech et al., 2010).

[0010] The physiological function of tau protein is to mediate the assembly of tubulin monomers into microtubules that form the microtubule network of neurons (Buee et al., 2000). Tau protein binds to microtubules through repeating domains located in the C-terminal portion of the protein. These repeating domains (R1-R4) are distinct from each other but contain 31-32 highly conserved amino acids (Taniguchi et al., 2005b). In the human brain, there are six unique tau protein isotypes that differ from each other due to the presence or absence of certain amino acids in the N-terminal portion of the tau protein and the presence of three (R1, R3, and R4) or four (R1-R4) repeating domains at the C-terminus. See also Figure 1It shows six human subtypes (2N4R, 1N4R, 2N3R, 0N4R, 1N3R, and 0N3R). The most potent region of tau protein for inducing microtubule polymerization has been proposed to be the 274-KVQIINKK-281 region covering R1-R2 (SEQ ID NO: 113). Ibid. Furthermore, the pathological and physiological functions of tau protein appear to be influenced by specific structural conformations and inherent disordered structures adopted by the full-length protein subtypes and their fragments. For example, Kontsekova et al. described a conformational region within certain truncated tau protein molecules that is significantly related to the function of those truncated tau protein molecules in microtubule assembly (covering residues 297-IKHVPGGGSVQIVYKPVDLSKVTSKCGSL-325 (SEQ ID NO: 114)) (WO2004 / 007547).

[0011] Besides their physiological functions, tau protein repeat sequences are believed to be involved in the formation of pathological tau protein aggregates and other structures. Therefore, there is a need for tau protein-targeted therapies and diagnostic methods that can differentiate between physiological and pathological repeat sequence-mediated activities. For example, the pronase-resistant core of pathological paired helical filaments (PHF) consists of microtubule-binding regions of 3-repeated and 4-repeated tau protein isoforms (Jakes et al., 1991; Wischik et al., 1988a; Wischik et al., 1988b). Furthermore, Novak et al. showed that the protease-resistant core of PHF with a length of 93-95 amino acids is limited to three tandem repeat sequences (Novak et al., 1993). Von Bergen et al. identified a minimal tau peptide / interacting motif (306-VQIVYK-311; SEQ ID NO: 115) and a second site on the tau protein (275-VQIINK-280) (SEQ ID NO: 116), which form a β-sheet and have been described as potentially responsible for initiating the formation of pathological tau protein aggregates (PHF) (Von Bergen et al., 2000; EP 1214598; WO 2001 / 18546). For a functional map of the tau protein, see [link to tau protein functional map]. Figure 2 Therefore, the current strategy aims to produce anti-aggregation drugs that do not disrupt the intracellular role of tau protein in microtubule stabilization.

[0012] Furthermore, although tau protein is considered an intracellular cytoplasmic protein under physiological conditions, intracellular tau protein can be released into the extracellular space and promote neurodegeneration (Gómez-Ramos et al., 2006). Indeed, neuronal loss has been associated with the local anatomical distribution of neurofibrillary tangles (composed of tau protein) in the AD brain (West et al., 1994; Gomez-Isla et al., 1996, 1997). In addition, the levels of total tau protein and phosphorylated tau protein are increased in the cerebrospinal fluid (CSF) of AD patients (Hampel et al., 2010), and extracellular tau protein has been described as a “ghost tangle” in the brain that indicates the release of intracellular tau protein into the extracellular space (Frost and Diamond, 2009). Furthermore, extracellular tau protein aggregates can enter cells and stimulate intracellular tau protein fibrosis, thereby further seeding tau protein monomers for the production of pathological tau protein aggregates (Frost et al., 2009). The aforementioned studies have highlighted that extracellular insoluble tau proteins can act as transmissible agents to spread tau protein lesions in the brain in a prion-like manner (Frost et al., 2009; Frost and Diamond, 2009). Clearing extracellular tau protein tangles can alleviate tau protein-related extracellular and intracellular lesions. See, for example, Asuni et al., 2007. Therefore, there is a need for therapies that can reduce extracellular tau proteins by inhibiting their formation, promoting their clearance, or both, as well as therapies that reduce intracellular disease tau proteins.

[0013] In summary, although tau protein appears to play a pathological role in the clinical manifestations of AD, the development of drugs targeting tau protein has lagged behind, partly due to its importance in physiological microtubule dynamics and its complex biology (Dickey and Petrucelli, 2006). However, increasing understanding of the molecular mechanisms underlying pathological tau protein conversion has opened up the possibility of specifically targeting pathological tau protein modifications for therapeutic purposes. Therefore, numerous therapeutic approaches have emerged that directly or indirectly target the tau protein cascade (see, for example, Dickey and Petrucelli, 2006; Schneider and Mandelkow, 2008; Zilka et al., 2008 for review articles), including compounds that prevent or reverse tau protein aggregation (Wischik et al., 1996; Necula et al., 2005; Pickhardt et al., 2005; Taniguchi et al., 2005a; Larbig et al., 2007), and small molecule drugs that inhibit tau protein kinase or activate tau protein phosphatase (Iqbal and Grundke-lq). Iqbal, 2004; Noble et al., 2005; Iqbal and Grundke-lqbal, 2007), microtubule stabilizing agents (Zhang et al., 2005), agents that facilitate the proteolytic degradation of misfolded tau proteins (Dickey et al., 2005; Dickey et al., 2006; Dickey and Petrucelli, 2006), and immunosuppressive agents (Zilka et al., 2008), as well as immunotherapy strategies including active and passive immunity (Schneider and Mandelkow et al., 2008; Zilka et al., 2008; Tabira, T. Immunization Therapy for Alzheimer disease: A Comprehensive Review of Active Immunization Strategies. Tohoku J. Exp. Med., 220:95-106 (2010)).

[0014] More generally, since 2007, novel monoclonal antibodies (mAbs) have entered clinical trials at a rate of more than 40 per year. At the end of 2010, at least 25 mAbs and 5 Fc fusion proteins were in Phase 2 / 3 or Phase 3 clinical trials in the United States (Reichert, 2011). This trend suggests that passive immunotherapy is an evolving approach to treating human diseases including Alzheimer's disease (AD). See, for example, Citron et al., 2010. Indeed, despite the challenges of overcoming the blood-brain barrier (BBB) ​​in AD treatment, a growing number of preclinical and clinical studies report antibody-mediated therapies that can clear AD aggregates from the brain, proposing multiple mechanisms of action, such as (i) antibody uptake into the brain through altered BBB permeability or BBB leakage in AD; (ii) antibodies acting as a “peripheral sink” for soluble plaque-forming amyloid material; (iii) antibody-secreting cells entering the brain from the periphery for local antibody delivery; and (iv) intracellular and transcellular transport of IgG. For reviews, see, for example, Citron et al., 2010 and Asuni et al., 2007. Therefore, therapeutic antibodies targeting disease forms of tau protein represent a promising approach for treating and / or diagnosing AD and other tau protein lesions (WO 2004 / 007547, US 2008 / 0050383).

[0015] One approach to immunotherapy targeting tau protein lesions is based on the insight that anti-tau antibodies can prevent tau protein aggregation, clear tau protein aggregates, or both. Although studies have described antibodies that bind to tau protein sequences, and some of those antibodies have been reported to interfere with tau protein aggregation and clearance (Asuni et al., 2007), no monoclonal anti-tau antibodies have been reported to be undergoing in vivo preclinical or clinical trials for AD. In fact, one mAb was predicted to have three binding sites within the microtubule-binding domain of murine tau protein (i.e., at R3, R4, and possibly R1), but it does not block microtubule binding (Dingus et al., 1991). Dingus did not describe the effect of this antibody on tau protein aggregation, and therefore there is no reason to believe that Dingus would block tau protein aggregation. In other reports, mAbs that differentiate tau protein subtypes have been generated, but without suggesting any effect on tau protein aggregation (DeSilva et al., 2003; Ueno et al., 2007). Taniguchi et al. demonstrated that certain anti-tau protein mAbs targeting R1 or R2 inhibit tau protein aggregation into PHF in vitro while promoting tau protein-induced tubulin assembly (Taniguchi et al., 2005b). Taniguchi's RTA-1 and RTA-2 antibodies specifically bind to R1 and R2, respectively. Neither antibody binds to more than one tau protein repeat sequence, and neither antibody has been reported to have been tested for in vivo effects on tau protein aggregation or clearance. Although at least three anti-amyloid antibodies have been identified in clinical trials of passive immunotherapy for AD (i.e., antibody administration to patients), no clinical trial reports of passive tau protein immunotherapy for AD are available.

[0016] In several APP transgenic mouse studies of Alzheimer's disease (AD), an active immunization approach (i.e., a method in which the patient's own body generates immunity against the target) has been found to effectively clear Aβ deposits and reverse neuropathological lesions (see, for example, Schenk et al., 1999; Janus et al., 2000; Morgan et al., 2000; Sigurdsson et al., 2001). More recently, in mouse models of tau tangles, active immunization with phosphorylated tau epitopes (Tau protein 379-408 [P-Ser 396, 404]) has reduced the amount of tau protein aggregated in the brain and slowed the progression of behavioral phenotypes (Asuni et al., 2007; Boutajangout et al., 2010; US2008 / 0050383; US / 2010 / 00316564). The treated animals produced anti-tau protein antibodies that were detected in the brain and co-localized with antibodies recognizing pathological tau proteins (Asuni et al., 2007). This immunotherapy approach was substantially more effective in the early stages (5 months) of functional impairment in animals than in the later stages (8 months), suggesting that clearing pathological tau proteins in the early stages could have a therapeutic benefit (Asuni et al., 2007; Zilka et al., 2008). In fact, it has been learned that not all tau proteins are susceptible or even suitable for destruction and clearance. Some researchers have shown that disrupting tau protein aggregates increases the abundance of toxic intermediates, while others have shown that detectable tau protein aggregates are not necessarily toxic and can even be protective (Lee et al., 2005). Therefore, although immunotherapies targeting tau proteins have shown preclinical promise, there remains a need for therapeutic agents that specifically target and eliminate early abnormal forms of tau proteins that would yield enhanced and sustained benefits. However, it is also necessary to identify those tau protein species that are suitable targets for immunotherapy.

[0017] Therefore, another consideration in developing mAbs targeting tau protein is to identify and characterize various structural forms of tau protein (physiological, early-stage, and late-stage) and the targeted stages of tau protein lesions. Oddo et al. observed that in a transgenic mouse model of AD, mature tau protein aggregates remained intact despite Aβ immunotherapy clearing Aβ plaques and early tau protein lesions (Oddo et al., 2004). Similarly, although neurofibrillary tangles continued to accumulate, in the P301L tau protein model of tau protein lesions, a heritable (non-immunotherapy-related) decrease in tau protein expression improved memory (Santacruz et al., 2005).

[0018] Despite its prevalence, Alzheimer's disease (AD) remains one of the greatest unmet medical needs in neurology (Citron, 2010). The most common medical approach focuses on providing symptomatic treatments that are ineffective even after years of therapy. Novel treatments and strategies for AD need to go beyond symptom-based therapies to prevent cognitive decline and combat the underlying pathological processes of the disease. Specifically, there is a need to develop molecules, alone or in combination with other AD-targeting drugs, that interfere with at least some of the earliest stages of the disease. These molecules will provide new and advantageous options for early diagnosis (which itself can improve treatment outcomes), prevention, and treatment of AD. Invention Overview

[0019] In one embodiment, the present invention provides an isolated antibody, wherein the antibody binds to one or more tau epitopes and is capable of exhibiting two or more of the following properties:

[0020] a) It showed a higher affinity for pathological tau proteins than for physiological tau proteins;

[0021] b) Inhibit tau protein-tau protein aggregation; and

[0022] c) Mediates the uptake and degradation of pathological tau protein by microglia;

[0023] Furthermore, each tau epitope contains an aggregation-promoting region of the tau protein.

[0024] In one embodiment, the isolated antibody is such that one or more epitopes are each independently selected from epitopes at the following locations:

[0025] i. relative to tau 441 Positions 267-273 or residue KHQPGGG (SEQ ID NO:98);

[0026] ii. relative to tau 441 Positions 298-304 or residue KHVPGGG (SEQ ID NO:99)

[0027] iii. Relative to tau 441 Positions 329-335 or residues HHKPGGG (SEQ ID NO:100); and

[0028] iv. relative to tau 441 Positions 361-367 or residue THVPGGG (SEQ ID NO:101).

[0029] In some embodiments, the isolated antibody having the properties described in the preceding paragraphs is capable of binding to one or more forms of pathological tau protein selected from: disordered tau protein, erroneously disordered tau protein, sarkosyl-insoluble tau protein, neurofibrillary tangles, neurofibrillary networks, and neuritis plaques in brain biopsies from human Alzheimer's disease patients, brain samples from animal models of Alzheimer's disease, or both. In some embodiments, the isolated antibody is such that at least one of the epitopes it recognizes is a conformational epitope.

[0030] In one embodiment, the present invention provides an isolated antibody, wherein the antibody binds to one or more tau epitopes and is capable of exhibiting two or more of the following properties:

[0031] a) It showed a higher affinity for pathological tau proteins than for physiological tau proteins;

[0032] b) Inhibit tau protein-tau protein aggregation; and

[0033] c) Mediates the uptake and degradation of pathological tau protein by microglia;

[0034] Furthermore, each tau epitope contains an aggregation-promoting region of the tau protein.

[0035] In one embodiment, the isolated antibody is such that one or more epitopes are each independently selected from epitopes at the following locations:

[0036] i. relative to tau 441 Positions 268-273 or residue HQPGGG (SEQ ID NO:223);

[0037] ii. relative to tau 441 Positions 299-304 or residue HVPGGG (SEQ ID NO:154)

[0038] iii. Relative to tau 441 Positions 330-335 or residues HKPGGG (SEQ ID NO:224); and

[0039] iv. relative to tau 441 Positions 362-367 or residue HVPGGG (SEQ ID NO:154).

[0040] In one embodiment, the isolated antibody is such that one or more epitopes it binds to are each independently selected from epitopes at the following locations:

[0041] i. relative to tau441 Positions 268-273 or residue HQPGGG (SEQ ID NO:223);

[0042] ii. relative to tau 441 Positions 299-304 or residue HVPGGG (SEQ ID NO:154)

[0043] iii. Relative to tau 441 Positions 330-335 or residues HKPGGG (SEQ ID NO:224); and

[0044] iv. relative to tau 441 Positions 362-367 or residue HVPGGG (SEQ ID NO:154);

[0045] And the antibody comprises:

[0046] a) Antibody light chain variable region, wherein the antibody light chain variable region comprises:

[0047] i. QSLLNSRTRKNY (SEQ ID NO:117) or SEQ ID NO:247 for CDR1;

[0048] ii. WAS (SEQ ID NO:118) or SEQ ID NO:253 for CDR2; and

[0049] iii. KQSFYLRT (SEQ ID NO: 119) or any of SEQ ID NO: 255, 257, 258, 259 and 260 for CDR3; and

[0050] b) Antibody heavy chain variable region, wherein the antibody heavy chain variable region comprises:

[0051] iv. GYIFTDYVIS (SEQ ID NO:120), SEQ ID NO:261 or SEQ ID NO:262 for CDR1;

[0052] v. IFPRSGST (SEQ ID NO: 121), SEQ ID NO: 264, or SEQ ID NO: 265 for CDR2; and

[0053] vi. ARDYYGTSFAMDY (SEQ ID NO:122), SEQ ID NO:266, SEQ ID NO:267 or SEQ ID NO:269 for CDR3.

[0054] The present invention also provides an isolated antibody that binds to one or more epitopes on tau protein in a conformationally specific manner, wherein:

[0055] a) Each of the one or more epitopes is independently selected from epitopes in the following locations:

[0056] i. relative to tau 441 Positions 267-273 or residue KHQPGGG (SEQ ID NO:98);

[0057] ii. relative to tau 441 Positions 298-304 or residue KHVPGGG (SEQ ID NO:99)

[0058] iii. Relative to tau 441 Positions 329-335 or residues HHKPGGG (SEQ ID NO:100); and

[0059] iv. relative to tau 441 Positions 361-367 or residue THVPGGG (SEQ ID NO: 101);

[0060] b) The zero, one, two, or three of the stated tabletops are linear tabletops; and

[0061] c) The zero, two, three, or four epitopes are conformational epitopes.

[0062] The present invention also provides an isolated antibody that binds to one or more epitopes on tau protein in a conformationally specific manner, wherein:

[0063] a) Each of the one or more epitopes is independently selected from the following locations:

[0064] i. relative to tau 441 Positions 268-273 or residue HQPGGG (SEQ ID NO:223);

[0065] ii. relative to tau 441 Positions 299-304 or residue HVPGGG (SEQ ID NO:154)

[0066] iii. Relative to tau 441 Positions 330-335 or residues HKPGGG (SEQ ID NO:224); and

[0067] iv. relative to tau 441 Positions 362-367 or residue HVPGGG (SEQ ID NO:154).

[0068] b) The zero, one, two, or three of the stated tabletops are linear tabletops; and

[0069] c) The zero, two, three, or four epitopes are conformational epitopes.

[0070] In one embodiment, the antibody is DC8E8, which is an antibody produced from a hybridoma deposited at the American Type Culture Collection (ATC) under patent accession number PTA-11994.

[0071] In some embodiments, the isolated antibody binds to one or more epitopes on the tau protein that are identical to those bound by DC8E8. In one embodiment, the isolated antibody competes with the monoclonal antibody DC8E8 for binding to the tau protein.

[0072] The present invention also provides an isolated antibody comprising one or more complementarity-determining region (CDR) sequences selected from the following in its epitope-binding domain:

[0073] i.QSLLNSRTRKNY(SEQ ID NO:117)

[0074] ii.WAS (SEQ ID NO:118)

[0075] iii. KQSFYLRT (SEQ ID NO:119)

[0076] iv. GYIFTDYVIS (SEQ ID NO:120)

[0077] v.IFPRSGST(SEQ ID NO:121); and

[0078] vi.ARDYYGTSFAMDY (SEQ ID NO: 122).

[0079] This invention also specifies that any antibody described in any embodiment described in the preceding paragraphs may be such that the isolated antibody comprises:

[0080] a) Antibody light chain variable region, wherein the antibody light chain variable region comprises:

[0081] i. QSLLNSRTRKNY (SEQ ID NO:117) for CDR1;

[0082] ii. WAS (SEQ ID NO: 118) for CDR2; and

[0083] iii. KQSFYLRT (SEQ ID NO:119) for CDR3; and

[0084] b) Antibody heavy chain variable region, wherein the antibody heavy chain variable region comprises:

[0085] iv. GYIFTDYVIS (SEQ ID NO:120) for CDR1

[0086] v. IFPRSGST (SEQ ID NO: 121) for CDR2; and

[0087] vi. ARDYYGTSFAMDY (SEQ ID NO:122) for CDR3.

[0088] This invention also specifies that any antibody described in the previous embodiments may be such that the isolated antibody comprises:

[0089] a) One or more light chain CDR sequences from monoclonal antibody DC8E8, or one or more sequences having at least 80%, 90%, or 95% identity with one of these light chain CDRs after optimal alignment; and

[0090] b) One or more heavy chain CDR sequences from monoclonal antibody DC8E8 or one or more sequences that have at least 80%, 90% or 95% identity with one of these heavy chain CDRs after optimal alignment;

[0091] And among them:

[0092] i. The light chain CDR comprises a sequence selected from QSLLNSRTRKNY (SEQ ID NO: 117), WAS (SEQ ID NO: 118), and KQSFYLRT (SEQ ID NO: 119); and

[0093] ii. The heavy chain CDR comprises a sequence selected from GYIFTDYVIS (SEQ ID NO:120), IFPRSGST (SEQ ID NO:121), and ARDYYGTSFAMDY (SEQ ID NO:122).

[0094] This invention also specifies that any antibody described in the prior embodiments may be composed of or comprise of: Fab, Fab', F(ab')2, Fac, Fv fragments, any other antigen-binding fragments; or an antigen-binding antibody portion thereof; having one or more of the following immunobinding characteristics:

[0095] 1. The antibody binds to one or more tau epitopes in a conformation-specific manner, wherein:

[0096] a) Each of the one or more tau epitopes is independently selected from epitopes in the following locations:

[0097] i. relative to tau 441 Positions 267-273 or residue KHQPGGG (SEQ ID NO:98);

[0098] ii. relative to tau 441 Positions 298-304 or residue KHVPGGG (SEQ ID NO:99)

[0099] iii. Relative to tau 441 Positions 329-335 or residues HHKPGGG (SEQ ID NO:100); and

[0100] iv. relative to tau 441 Positions 361-367 or residue THVPGGG (SEQ ID NO: 101);

[0101] b) The zero, one, two, or three of the said epitopes are linear epitopes;

[0102] c) One, two, three, or four of the epitopes are conformational epitopes;

[0103] 2. The antibody binds to two or more tau epitopes and exhibits a higher affinity for pathological tau proteins than for physiological tau proteins, wherein the two tau epitopes are selected from epitopes at the following locations:

[0104] v. Positions 267-273 relative to tau441 or residue KHQPGGG (SEQ ID NO:98);

[0105] vi. Positions 298-304 relative to tau441 or residue KHVPGGG (SEQ ID NO:99)

[0106] vii. Positions 329-335 relative to tau441 or residue HHKPGGG (SEQ ID NO:100); and

[0107] viii. Positions 361-367 relative to tau441 or residue THVPGGG (SEQ ID NO:101).

[0108] This invention also specifies that any antibody described in the prior embodiments may be composed of or comprise of: Fab, Fab', F(ab')2, Fac, Fv fragments, any other antigen-binding fragments; or an antigen-binding antibody portion thereof; having one or more of the following immunobinding characteristics:

[0109] 1. The antibody binds to one or more tau epitopes in a conformation-specific manner, wherein:

[0110] a) Each of the one or more tau epitopes is independently selected from epitopes in the following locations:

[0111] i. relative to tau 441 Positions 268-273 or residue HQPGGG (SEQ ID NO:223);

[0112] ii. relative to tau 441 Positions 299-304 or residue HVPGGG (SEQ ID NO:154)

[0113] iii. Relative to tau 441 Positions 330-335 or residues HKPGGG (SEQ ID NO:224); and

[0114] iv. relative to tau 441 Positions 362-367 or residue HVPGGG (SEQ ID NO:154).

[0115] b) The zero, one, two, or three of the said epitopes are linear epitopes;

[0116] c) One, two, three, or four of the epitopes are conformational epitopes;

[0117] 2. The antibody binds to two or more tau epitopes and exhibits a higher affinity for pathological tau proteins than for physiological tau proteins, wherein the two tau epitopes are selected from epitopes at the following locations:

[0118] i. relative to tau 441 Positions 268-273 or residue HQPGGG (SEQ ID NO:223);

[0119] ii. relative to tau 441 Positions 299-304 or residue HVPGGG (SEQ ID NO:154)

[0120] iii. Relative to tau 441 Positions 330-335 or residues HKPGGG (SEQ ID NO:224); and

[0121] iv. relative to tau 441 Positions 362-367 or residue HVPGGG (SEQ ID NO:154).

[0122] This invention also relates to any isolated antibody that competes with any isolated antibody for binding to the tau protein as described in the previous embodiments. In one embodiment, the isolated antibody competes with the tau protein for binding when tested against the isolated DC8E8 assay.

[0123] In some embodiments, the antibody comprises a light chain including SEQ ID NO.:141. In some embodiments, the antibody comprises a light chain including SEQ ID NO.:138. In some embodiments, the antibody comprises a light chain including SEQ ID NO.:141 and a light chain including SEQ ID NO.:138.

[0124] This invention specifies that the antibodies provided by this invention can be selected from:

[0125] a) Monoclonal antibodies;

[0126] b) Polyclonal antibodies;

[0127] c) Recombinant antibodies;

[0128] d) Chimeric antibodies;

[0129] e) Humanized antibodies;

[0130] f) Human antibodies; and

[0131] The antigen-binding fragment or antigen-binding portion of any of (g)(a) to (f).

[0132] Any isolated antibody provided by this invention can be produced in mammals. In some embodiments, the isolated antibody is produced by a recombinant animal or by a recombinant host cell.

[0133] This invention specifies that any isolated anti-tau protein antibody provided herein can be detectably labeled with one or more labeling agents. In some embodiments, at least one labeling agent is selected from enzymes, radioisotopes, fluorophores, nuclear magnetic resonance markers, and heavy metals.

[0134] In some implementations, the antibody comprises at least one drug (combination drug) linked to the antibody molecule.

[0135] The present invention also provides isolated nucleic acids encoding at least one CDR or at least a binding domain or variable region of an immunoglobulin chain encoding any of the anti-tau protein antibodies described in the previous embodiments. Isolated vectors comprising any of those nucleic acids are also provided. In some embodiments, the present invention provides an isolated host cell comprising one or more of these isolated nucleic acids and a vector.

[0136] In some embodiments, the present invention provides an isolated cell line expressing any of the anti-tau protein antibodies described in the previous embodiments. In one embodiment, the isolated cell line is a hybridoma. In one embodiment, the isolated cell line is a hybridoma from which the monoclonal antibody DC8E8 is produced, and said cell line was deposited on July 13, 2011, at the American Center for Type Culture Collection (Manassas, VA, USA) under ATCC Patent Accession No. PTA-11994.

[0137] This invention provides the use of any anti-tau protein antibodies, nucleic acids, and cells provided herein as pharmaceuticals or for the manufacture of agents for the diagnosis, prevention, or treatment of Alzheimer's disease or related tau protein lesions.

[0138] In some embodiments, the antibody is included in a pharmaceutical composition, which further includes a pharmaceutically acceptable carrier and / or diluent. In one embodiment, the pharmaceutical composition comprises a combination of an antibody and a pharmaceutically acceptable carrier and / or diluent, wherein the combination comprises at least two different antibodies, and wherein each antibody is independently selected from the antibodies described in previous embodiments. In one embodiment, at least one antibody is DC8E8 or the human form of DC8E8 or a humanized form of DC8E8.

[0139] In some embodiments, the antibody is included in the composition, which further includes a diluent and / or a carrier. The composition may be a pharmaceutical composition, a diagnostic composition, or any other composition. In some embodiments, the composition may further include at least one compound or reagent selected from: detectable markers, keyhole limpet hemocyanin, tetanus toxoid or toxoids derived from other pathogenic bacteria, serum albumin, bovine serum albumin, immunoglobulin molecules or fragments thereof, thyroglobulin, ovoglobulin, universal T cell epitopes, cytokines, chemokines, interleukin-1-α (IL-1α), IL-1β, IL-2, IL-10, interferon-γ (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein 1α (MIP1α), MIP1β, and RANTES (regulated upon activation and expressed and secreted by normal T cells).

[0140] The present invention also provides an article of manufacture (e.g., a reagent kit) for pharmaceutical or diagnostic use, said article comprising packaging materials and a container comprising a solution of any one or more anti-tau protein antibodies provided herein in lyophilized form. In some embodiments, the container is part of a device or system for delivering antibodies to a subject.

[0141] In some embodiments, the present invention provides a medical device comprising an anti-tau protein antibody as provided herein (see above), wherein the device is adapted to contact or administer the antibody via at least one mode selected from the following: parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intra-abdominal, intracystic, intracartilaginous, intracavitary, intracavitary, intracavitary, intracavitary, intracerebellar, intravenous, intrasheath, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intrabladder, intralesional, intravenous, intradose, transvaginal, transrectal, transbuccal, sublingual, intranasal, and transcutaneous.

[0142] In one embodiment, the present invention relates to a method for treating a subject with Alzheimer's disease or related tau protein lesions or preventing their progression, the method comprising administering to the subject an effective amount of at least one anti-tau protein antibody provided herein. In some embodiments, the method is capable of reducing motor impairment, improving motor function, reducing cognitive impairment, improving cognitive function, or a combination thereof.

[0143] In some embodiments, the present invention relates to a method for improving at least one symptom associated with Alzheimer's disease or related tau protein lesions in a subject, the method comprising administering to the subject an effective amount of at least one anti-tau protein antibody provided herein.

[0144] In another embodiment, the present invention provides a method for diagnosing or screening subjects for the presence of Alzheimer's disease or related tau protein lesions, or for determining the risk of subjects exhibiting Alzheimer's disease or related tau protein lesions, the method comprising:

[0145] a) Contacting the subject or the subject's cells, tissues, organs, fluids, or any other sample with an effective amount of at least one anti-tau protein antibody as provided herein; and

[0146] b) Determine the presence of a complex comprising pathological tau protein and the antibody, wherein the presence of the complex is a diagnostic criterion for Alzheimer's disease or related tau protein lesions associated with the presence of pathological tau protein.

[0147] In one related embodiment, the present invention provides a method for monitoring a subject for the presence, progression, regression, or stabilization of Alzheimer's disease or related tau protein lesions, or for determining the stage of Alzheimer's disease or related tau protein lesions, the method comprising:

[0148] a) Contacting the subject or the subject's cells, tissues, organs, fluids, or any other sample with an effective amount of at least one anti-tau protein antibody provided herein; and

[0149] b) Determine the presence and / or characteristics of a complex comprising pathological tau protein and the antibody, wherein the presence of the complex is a diagnostic criterion for Alzheimer's disease or related tau protein lesions associated with the presence of pathological tau protein.

[0150] In some implementations, the antibody is administered intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally, intravenously, intrathecally, or as an aerosol.

[0151] In some embodiments of methods for treating Alzheimer's disease or related tau protein lesions in a subject or preventing their progression, and methods for improving at least one symptom associated with Alzheimer's disease or related tau protein lesions in a subject, the effective dose of each antibody is at least 1 mg per kg of the subject's body weight. In some embodiments, the effective dose of each antibody is at least 10 mg per kg of the subject's body weight. In some embodiments, at least one antibody is administered in multiple doses over a period of at least 6 months. In some embodiments, the antibody is administered peripherally to a human subject to exert its beneficial effect. In some embodiments, when administered peripherally to a human subject, the antibody binds to soluble tau protein, creatine acyl-insoluble tau protein, or both. In some embodiments, upon peripheral administration to a human subject, the antibody binds to tau protein, wherein the tau protein is in one or more pathological forms selected from: misordered tau protein in brain biopsies of human Alzheimer's patients, brain samples from animal models of Alzheimer's disease, misordered tau protein, sarcosyl-insoluble tau protein, neurofibrillary tangles, neurofibrillary networks, and neuritis plaques. In some embodiments, upon peripheral administration to a human subject, the antibody exerts one or more effector-mediated beneficial effects on the subject. In some embodiments, the antibody is delivered to the periphery by injecting / implanting antibody-expressing cells into the subject's brain. In some embodiments, the antibody-expressing cells are hybridoma cells. In some embodiments, the hybridoma cells are hybridomas expressing DC8E8.

[0152] In some relevant embodiments, the present invention provides an isolated peptide, wherein:

[0153] a) The isolated peptide is a fragment of tau protein with a length of at least 6 amino acid residues, at least 7 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 12 amino acid residues, or at least 30 amino acid residues; and

[0154] b) The isolated peptide contains a tau protein therapeutic epitope.

[0155] In some related implementations, therapeutic epitopes include therapeutic epitopes selected from those sites:

[0156] i. relative to tau 441 Positions 267-273 or residue KHQPGGG (SEQ ID NO:98);

[0157] ii. relative to tau 441 Positions 298-304 or residue KHVPGGG (SEQ ID NO:99)

[0158] iii. Relative to tau 441 Positions 329-335 or residues HHKPGGG (SEQ ID NO:100); and

[0159] iv. relative to tau 441 Positions 361-367 or residue THVPGGG (SEQ ID NO:101).

[0160] In some relevant embodiments, the present invention provides an isolated peptide, wherein:

[0161] a) The isolated peptide is a fragment of tau protein with a length of at least 6 amino acid residues, at least 7 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 12 amino acid residues, or at least 30 amino acid residues; and

[0162] b) The isolated peptide contains a tau protein therapeutic epitope.

[0163] In some related implementations, therapeutic epitopes include therapeutic epitopes selected from those sites:

[0164] i. relative to tau 441 Positions 268-273 or residue HQPGGG (SEQ ID NO:223);

[0165] ii. relative to tau 441 Positions 299-304 or residue HVPGGG (SEQ ID NO:154)

[0166] iii. Relative to tau 441 Positions 330-335 or residues HKPGGG (SEQ ID NO:224); and

[0167] iv. relative to tau 441Positions 362-367 or residue HVPGGG (SEQ ID NO:154).

[0168] In some relevant implementation schemes, the therapeutic epitope is selected from:

[0169] i. relative to tau 441 Positions 268-273 or residue HQPGGG (SEQ ID NO:223);

[0170] ii. relative to tau 441 Positions 299-304 or residue HVPGGG (SEQ ID NO:154)

[0171] iii. Relative to tau 441 Positions 330-335 or residues HKPGGG (SEQ ID NO:224); and

[0172] iv. relative to tau 441 Positions 362-367 or residue HVPGGG (SEQ ID NO:154).

[0173] In other embodiments, the isolated peptides are sequences selected from the following: SEQ ID NO:1-4, SEQ ID NO:9-101 and SEQ ID NO:108-112, NIKAVPGGGS (SEQ ID NO:200), NIKHVPGGGS (SEQ ID NO:201), IKHVPGGGS (SEQ ID NO:202), KHVPGGGSV (SEQ ID NO:203), HVPGGGSVQ (SEQ ID NO:204), VPGGGSVQ (SEQ ID NO:205), GWSIHSPGGGSC (SEQ ID NO:250), SVFQHLPGGGSC (SEQ ID NO:251), ANIKHVPGGGS (SEQ ID NO:144), DAIKHVPGGGS (SEQ ID NO:146), DNAKHVPGGGS (SEQ ID NO:149), DNIAHVPGGGS (SEQ ID NO:151), DNIKAVPGGGS (SEQ ID NO:149), and DNIKAVPGGGS (SEQ ID NO:149). NO:159), DNIKHAPGGGS (SEQ ID NO:161), and DNIKHVPGGGS (SEQ ID NO:171).

[0174] In other embodiments, the isolated peptide is selected from the following sequences: SEQ ID NO:270 (TENLKHQPGGGK); SEQ ID NO:271 (KHQPGGG), SEQ ID NO:272 (HQPGGG); SEQ ID NO:275 (ENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS), SEQ ID NO:276 (KHVPGGG), SEQ ID NO:277 (HVPGGG), SEQ ID NO:280 (DNIKHVPGGGSVQIVYKPV), SEQ ID NO:281 (HHKPGGG), SEQ ID NO:282 (HKPGGG) and SEQ ID NO:283 (THVPGGG).

[0175] In other embodiments, the isolated peptide is selected from the following sequences: SEQ ID NO:270 (TENLKHQPGGGK); SEQ ID NO:271 (KHQPGGG), SEQ ID NO:272 (HQPGGG); SEQ ID NO:275 (ENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS), SEQ ID NO:276 (KHVPGGG), SEQ ID NO:277 (HVPGGG), SEQ ID NO:280 (DNIKHVPGGGSVQIVYKPV), SEQ ID NO:281 (HHKPGGG), SEQ ID NO:282 (HKPGGG), and SEQ ID NO:283 (THVPGGG); and the therapeutic epitope is selected from:

[0176] i. relative to tau 441 Positions 268-273 or residue HQPGGG (SEQ ID NO:223);

[0177] ii. relative to tau 441 Positions 299-304 or residue HVPGGG (SEQ ID NO:154)

[0178] iii. Relative to tau 441 Positions 330-335 or residues HKPGGG (SEQ ID NO:224); and

[0179] iv. relative to tau 441 Positions 362-367 or residue HVPGGG (SEQ ID NO:154).

[0180] In other embodiments, the isolated peptide is selected from the sequences: SEQ ID NO:272 (HQPGGG) and SEQ ID NO:277 (HVPGGG).

[0181] In some embodiments, the isolated peptide is active in at least one assay selected from the following assays measuring the peptide's ability:

[0182] a) Competes with tau protein to bind to the monoclonal antibody DC8E8;

[0183] b) The ability to reduce the level of creatine acyl-insoluble tau protein in the body;

[0184] c) The body's ability to promote the clearance of tau protein from the brain;

[0185] d) The ability to reduce the level of at least one biochemical marker of AD in vivo;

[0186] e) The ability to reduce neurofibrillary tangles (NFT) load in vivo;

[0187] f) The ability to increase at least one neurobehavioral parameter in the body;

[0188] g) The ability to improve the AD process in subjects;

[0189] h) The ability to reduce the level of tau protein in the brain, cerebrospinal fluid, or both; and / or

[0190] i) The ability to act as an immunogen in the preparation of antibodies that can compete with monoclonal DC8E8 for binding to tau protein.

[0191] This invention also relates to compounds comprising any isolated peptides and portions provided herein. In some embodiments, the portion is located at the N-terminus, C-terminus, or attached to an internal amino acid of the peptide, and said portion is selected from one or more of the following: cysteine ​​residues, phosphate groups, keyhole hemocyanin, tetanus toxoid or toxoids derived from other pathogenic bacteria, serum albumin, bovine serum albumin, immunoglobulin molecules or fragments thereof, thyroglobulin, ovoglobulin, universal T cell epitopes, cytokines, chemokines, interleukin-1-α (IL-1α), IL-1β, IL-2, IL-10, interferon-γ (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein 1α (MIP1α), MIP1β, and RANTES (regulated upon activation and expressed and secreted by normal T cells).

[0192] Pharmaceutical compositions comprising one or more isolated peptides and / or compounds provided by the present invention, as well as pharmaceutically acceptable carriers and / or diluents and / or adjuvants, are also provided. In some embodiments, the pharmaceutical compositions are suitable for providing peptides or compounds in doses between 1 ng and 10 mg. In some embodiments, the pharmaceutical compositions are suitable for providing peptides or compounds in doses greater than 10 micrograms.

[0193] This invention also relates to an article of manufacture (e.g., a reagent kit) for pharmaceutical or diagnostic use, said article comprising packaging materials and a container comprising a solution of peptides and / or compounds in lyophilized form provided by this invention. In some embodiments, the container is part of a device or system for delivering peptides or compounds to a subject.

[0194] Medical devices comprising peptides, compounds, and / or peptide / compound compositions as provided by the present invention are also provided, wherein the devices are adapted to contact or administer the antibodies by at least one mode selected from the following: parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraperitoneal, intracystic, intracartilaginous, intracavitary, intracavitary, intracavitary, intracerebellar, intravenous, intrasheath, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intraperitoneal, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intrabladder, intralesional, intravenous, intradose, transvaginal, transrectal, transbuccal, sublingual, intranasal, and transcutaneous.

[0195] In relevant embodiments, the present invention provides a method for treating or preventing the progression of Alzheimer's disease or related tau protein lesions in a subject, the method comprising administering to the subject an effective amount of at least one peptide and / or at least one compound, such as those provided by the present invention. In some embodiments, the method is capable of reducing motor impairment, improving motor function, reducing cognitive impairment, improving cognitive function, or a combination thereof.

[0196] In a related embodiment, the present invention provides a method for improving at least one symptom associated with Alzheimer's disease or related tau protein lesions in a subject, the method comprising administering to the subject an effective amount of at least one peptide and / or at least one compound, such as those provided by the present invention.

[0197] Among these methods for treating, preventing, or improving at least one symptom associated with Alzheimer's disease or related tau protein lesions in a subject, there is a method for improving at least one symptom associated with Alzheimer's disease or related tau protein lesions in a subject, the method comprising administering to a human patient a peptide and / or compound, such as those provided by the present invention, and / or an adjuvant that enhances the immune response, the method achieving an immune response comprising antibodies against pathological tau protein, thereby treating at least one AD-related symptom in the human patient, preventing its progression, or improving said at least one symptom.

[0198] The present invention also provides a method for generating an antibody capable of competitively binding to tau protein with DC8E8, the method comprising immunizing a subject with at least one peptide and / or at least one compound as provided by the present invention. In some embodiments, at least one peptide is selected from any of the following: SEQ ID NO:1-4, SEQ ID NO:9-101 and SEQ ID NO:108-112, NIKHVPGGGS (SEQ ID NO:201), IKHVPGGGS (SEQ ID NO:202), KHVPGGGSV (SEQ ID NO:203), HVPGGGSVQ (SEQ ID NO:204), VPGGGSVQ (SEQ ID NO:205), GWSIHSPGGGSC (SEQ ID NO:250), SVFQHLPGGGSC (SEQ ID NO:251), ANIKHVPGGGS (SEQ ID NO:144), DAIKHVPGGGS (SEQ ID NO:146), DNAKHVPGGGS (SEQ ID NO:149), DNIAHVPGGGS (SEQ ID NO:151), DNIKAVPGGGS (SEQ ID NO:159), DNIKHAPGGGS (SEQ ID NO:159), SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:159, SEQ ID NO:151, SEQ ID NO:159, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:204, SEQ ID NO:205 ... SEQ ID NO:161) and DNIKHVPGGGS (SEQ ID NO:171). In one embodiment, the peptide is selected from SEQ ID NO:1-4. In another embodiment, the peptide is SEQ ID NO:108. In one embodiment, the peptide is GWSIHSPGGGSC (SEQ ID NO:250). In some embodiments, the peptide is SVFQHLPGGGSC (SEQ ID NO:251). In some embodiments, the peptide is selected from SEQ ID NO:270 (TENLKHQPGGGK); SEQ ID NO:271 (KHQPGGG), SEQ ID NO:272 (HQPGGG); SEQ ID NO:275 (ENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS), SEQ ID NO:276 (KHVPGGG), SEQ ID NO:277 (HVPGGG), SEQ ID NO:280 (DNIKHVPGGGSVQIVYKPV), SEQ ID NO:281 (HHKPGGG), SEQ ID NO:282 (HKPGGG), and SEQ ID NO:283 (THVPGGG). In other embodiments, the peptide is selected from SEQ ID NO:272 (HQPGGG) and SEQ ID NO:277 (HVPGGG).

[0199] A method for isolating DC8E8 or an antibody capable of competitively binding to tau protein with DC8E8 is also provided, the method comprising contacting DC8E8 or the antibody with peptides and / or compounds such as those provided by the present invention.

[0200] In relevant embodiments, the present invention provides a method for diagnosing or screening subjects for the presence of Alzheimer's disease or related tau protein lesions, or for determining the risk of subjects exhibiting Alzheimer's disease or related tau protein lesions, the method comprising:

[0201] a) Contacting the subject or the subject's cells, tissues, organs, fluids, or any other sample with an effective amount of at least one antibody, such as that provided by the present invention; and

[0202] b) Determine the presence of a complex comprising pathological tau protein and the antibody, wherein the presence of the complex is a diagnostic criterion for Alzheimer's disease or related tau protein lesions associated with the presence of pathological tau protein.

[0203] In some embodiments, the present invention provides a method for monitoring a subject for the presence, progression, regression, or stabilization of Alzheimer's disease or related tau protein lesions, or for determining the stage of Alzheimer's disease or related tau protein lesions in the subject, the method comprising:

[0204] a) Contacting the subject, or the subject's cells, tissues, organs, fluids, or any other sample, with an effective amount of at least one antibody as provided by at least one embodiment of the present invention (e.g., administration); and

[0205] b) Determine the presence and / or characteristics of a complex comprising pathological tau protein and the antibody, wherein the presence of the complex is a diagnostic criterion for Alzheimer's disease or related tau protein lesions associated with the presence of pathological tau protein.

[0206] In some embodiments of methods for monitoring subjects for the presence, progression, regression, or stabilization of Alzheimer's disease or related tau protein lesions, or for identifying stages of Alzheimer's disease or related tau protein lesions in a subject, the antibody, peptide, and / or compound are administered intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally, intraventricularly, intrathecally, or as an aerosol. In some embodiments, the effective amount of each peptide and / or compound is at least 1 μg, at least 10 μg, or at least 100 μg per dose. In some embodiments, the effective amount of each peptide and / or compound is at least 10 μg per dose in the presence of an adjuvant and at least 100 μg per dose in the absence of an adjuvant. In some embodiments, at least one peptide or compound is administered in multiple doses over a period of at least 6 months.

[0207] According to one relevant embodiment, the present invention provides a method for treating or preventing the progression of Alzheimer's disease or related tau protein lesions in a subject, the method comprising administering to the subject an effective amount of at least one antibody and / or at least one peptide and / or at least one compound provided by the present invention, in combination with at least one combination agent selected from the following: acetylcholinesterase inhibitors, N-methyl-D-aspartate (NMDA) receptor antagonists, transition metal chelators, growth factors, hormones, nonsteroidal anti-inflammatory drugs (NSAIDs), antioxidants, lipid-lowering agents, selective phosphodiesterase inhibitors, tau protein aggregation inhibitors, protein kinase inhibitors, heat shock protein inhibitors, anti-amyloid passive and active immunizing agents, anti-amyloid aggregation inhibitors, and secretase inhibitors. In some embodiments, the method can reduce motor injury, improve motor function, reduce cognitive impairment, improve cognitive function, or achieve a combination of these effects.

[0208] In one related embodiment, the present invention provides a method for improving at least one symptom associated with Alzheimer's disease or related tau protein lesions in a subject, the method comprising administering to the subject an effective amount of at least one antibody, at least one peptide, and / or at least one compound provided by the present invention, in combination with at least one combination agent selected from the following: acetylcholinesterase inhibitor, NMDA receptor antagonist, transition metal chelator, growth factor, hormone, nonsteroidal anti-inflammatory drug (NSAID), antioxidant, lipid-lowering agent, selective phosphodiesterase inhibitor, tau protein aggregation inhibitor, protein kinase inhibitor, heat shock protein inhibitor, anti-amyloid passive and active immunizing agent, anti-amyloid aggregation inhibitor, and secretase inhibitor.

[0209] In some embodiments of a method for treating, preventing, or improving at least one symptom associated with Alzheimer's disease or related tau protein lesions in a subject, the method comprises administering to a human patient an effective amount of at least one antibody, at least one peptide, and / or at least one compound and / or an adjuvant that enhances the immune response, in combination with at least one combination agent selected from the following: acetylcholinesterase inhibitors, NMDA receptor antagonists, transition metal chelators, growth factors, hormones, nonsteroidal anti-inflammatory drugs (NSAIDs), antioxidants, lipid-lowering agents, selective phosphodiesterase inhibitors, tau protein aggregation inhibitors, protein kinase inhibitors, heat shock protein inhibitors, anti-amyloid passive and active immunizing agents, anti-amyloid aggregation inhibitors, and secretase inhibitors; wherein the method achieves an immune response comprising an antibody against pathological tau protein, thereby treating at least one AD-related symptom in the human patient, preventing its progression, or improving said at least one symptom.

[0210] In some embodiments of methods for treating, preventing, or improving at least one symptom associated with Alzheimer's disease or related tau protein lesions in a subject, a combination of agents is administered before, simultaneously with, or after the administration of antibodies, peptides, and / or compounds such as those provided by the present invention.

[0211] In one related embodiment, the present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and / or diluent; and

[0212] a) such as the antibodies provided by the present invention; and / or

[0213] b) such peptides provided by the present invention; and / or

[0214] c) Compounds provided by the present invention;

[0215] And at least one combination of agents selected from: acetylcholinesterase inhibitors, NMDA receptor antagonists, transition metal chelators, growth factors, hormones, nonsteroidal anti-inflammatory drugs (NSAIDs), antioxidants, lipid-lowering agents, selective phosphodiesterase inhibitors, tau protein aggregation inhibitors, protein kinase inhibitors, heat shock protein inhibitors, anti-amyloid passive and active immunizing agents, anti-amyloid aggregation inhibitors, and secretase inhibitors. In some embodiments, the antibody is DC8E8. In some embodiments, the antibody contains at least one CDR from DC8E8. In some embodiments, the antibody contains at least one variable chain (light or heavy chain) from DC8E8. In some embodiments, humanized or human-form DC8E8 may be used. In some embodiments, at least one peptide is selected from any of the following: SEQ ID NO:1-4, SEQ ID NO:9-101 and SEQ ID NO:108-112, NIKHVPGGGS (SEQ ID NO:201), IKHVPGGGS (SEQ ID NO:202), KHVPGGGSV (SEQ ID NO:203), HVPGGGSVQ (SEQ ID NO:204), VPGGGSVQ (SEQ ID NO:205), GWSIHSPGGGSC (SEQ ID NO:250), SVFQHLPGGGSC (SEQ ID NO:251), ANIKHVPGGGS (SEQ ID NO:144), DAIKHVPGGGS (SEQ ID NO:146), DNAKHVPGGGS (SEQ ID NO:149), DNIAHVPGGGS (SEQ ID NO:151), DNIKAVPGGGS (SEQ ID NO:159), DNIKHAPGGGS (SEQ ID NO:159). SEQ ID NO:161) and DNIKHVPGGGS (SEQ ID NO:171). In one embodiment, the peptide is selected from SEQ ID NO:1-4. In another embodiment, the peptide is SEQ ID NO:108. In one embodiment, the peptide is GWSIHSPGGGSC (SEQ ID NO:250). In some embodiments, the peptide is SVFQHLPGGGSC (SEQ ID NO:251).In some embodiments, the peptide is selected from SEQ ID NO:270 (TENLKHQPGGGK); SEQ ID NO:271 (KHQPGGG), SEQ ID NO:272 (HQPGGG); SEQ ID NO:275 (ENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS), SEQ ID NO:276 (KHVPGGG), SEQ ID NO:277 (HVPGGG), SEQ ID NO:280 (DNIKHVPGGGSVQIVYKPV), SEQ ID NO:281 (HHKPGGG), SEQ ID NO:282 (HKPGGG), and SEQ ID NO:283 (THVPGGG). In other embodiments, the peptide is selected from SEQ ID NO:272 (HQPGGG) and SEQ ID NO:277 (HVPGGG).

[0216] Other objectives and advantages of the embodiments will be set forth in part in the description which follows, and in part will be apparent from the description or may be recognized by practicing the embodiments. The objectives and advantages of the embodiments will be realized and obtained by means of the elements and combinations particularly pointed out in the appended claims.

[0217] It should be understood that both the general description above and the detailed description below are exemplary and illustrative only and do not limit the implementation as requested.

[0218] The accompanying drawings, which are incorporated in and form part of this specification, illustrate several embodiments and, together with the description, serve to explain the principles of those embodiments. The disclosures discussed herein are provided solely because of their content prior to the filing date of this application. They are all incorporated herein by reference in their entirety for all purposes. Nothing herein should be construed as an admission that the invention is not entitled to any prior disclosure due to prior invention. Furthermore, the provided disclosure date may differ from the actual disclosure date, which may require independent verification. Brief description of the attached diagram

[0219] Figure 1 Schematic diagram of the six subtypes of human tau protein.

[0220] Figure 2 Schematic functional diagram of human tau protein (2N4R). Figure 2 “VQIINK” and “VQIVYK” are disclosed as SEQ ID NO 116 and 115, respectively.

[0221] Figure 3The figure shows the nucleotide and amino acid sequences of the variable region of DC8E8 and their alignment with the most closely related mouse germline sequence. The figure shows (A) nucleotides (SEQ ID NO: 165) and amino acids (SEQ ID NO 141 (for the variable light chain) and 117-119 (for each of its CDRs, according to IMGT), in order of appearance; (B) the sequence of the variable light (VL) chain region of DC8E8 (aligned with SEQ ID NO 166 and 168, in order of appearance); and (C) the V gene of the variable light chain of DC8E8 and its alignment with the most closely related mouse germline sequence IGKV8-21. * The comparison of 01 (the comparisons are disclosed in the order of appearance of SEQ ID NO 166 and 167 respectively; followed by the VL J gene of DC8E8 (SEQ ID NO: 168) and the most closely related mouse J gene IGKJ1) * (01) Alignment with (SEQ ID NO: 169). The figure shows the nucleotide (SEQ ID NO: 170) (in D) and amino acid sequences of the variable heavy chain of DC8E8 and its three CRDs (SEQ ID NO 171 and 120-122 in order of appearance). The following alignment of DC8E8 is shown in (F): First, the variable heavy (VH) chain V gene (SEQ ID NO 172) of DC8E8 is most closely aligned with the mouse germline sequence IGHV1-81. * 01 (SEQ ID NO 172); secondly, the variable heavy (VH) chain D gene of DC8E8 (SEQ ID NO 174) is most closely related to the mouse germline sequence IGHD2-14. * 01 (SEQ ID NO 175); and finally, the variable heavy (VH) chain J gene of DC8E8 (SEQ ID NO 176) is most closely related to the mouse germline sequence IGHJ4. * 01 (SEQ ID NO 177). Also shown are the sequences of the DC8E8κ light chain constant region (SEQ ID NO:178) (G) and the heavy chain constant region (SEQ ID NO:179) (H). The complementarity-determining regions (CDRs) are underlined in protein sequences (B) and (E) and identified according to the IMGT numbering system.

[0222] Figure 4 Alignment of the DC8E8 variable light (VL) chain sequence (SEQ ID NO 166 (V gene) and 168 (J gene) respectively) with the most closely related human germline VL genes (SEQ ID NO 180-181 respectively in order of appearance).

[0223] Figure 5Alignment of the DC8E8 variable heavy (VH) chain sequence (SEQ ID NO 172, 174 and 176 for genes V, D and J, respectively) with the most closely related human germline VH genes (SEQ ID NO 182-183 and 185 in order of appearance).

[0224] Figure 6: Epitope localization of DC8E8 by tau protein deletion mutants using ELISA. (A) Schematic diagram of tau proteins used for epitope localization of DC8E8, and (B) their amino acid sequences (SEQ ID NO 186-197, 102, 104 and 198-199 in order of appearance). (C) ELISA readings. DC8E8 recognizes the following tau proteins: Δ358-441, Δ421-441, Δ134-168, Δ1-220, Δ1-126, 2N4R, 2N3R, Δ(1-296; 392-441), and Δ(1-150; 392-441) / 4R. DC8E8 does not recognize the following tau proteins: Δ222-427, Δ306-400, Δ228-441, Δ300-312, Δ257-400, Δ137-441, and Δ283-441.

[0225] Figure 7: (A) and (B) are schematic diagrams of the synthetic peptides used for epitope localization (SEQ ID NO 206, 207, 208, 2, 210, 211, 212, 3, 214, 215, 4, 217, 26, 219, 36, 221, 222, 109, and 88, respectively, in order of appearance) and their sequences. (C) Epitope localization of DC8E8 by ELISA using synthetic peptides. (D) Schematic diagram of epitopes that DC8E8 can bind to within the tau protein. DC8E8 can bind to any one of four individual binding regions, each of which is an individual epitope named epitope 1 to 4. The four epitopes are located within the 1st (epitope 1), 2nd (epitope 2), 3rd (epitope 3), and 4th (epitope 4) repeating domains of the tau protein, respectively. As shown, each of the four DC8E8 epitopes is contained within one of the following amino acid sequences: 267-KHQPGGG-273 (SEQ ID NO:98) (within the first repeat domain of the tau protein), 298-KHVPGGG-304 (SEQ ID NO:99) (within the second repeat domain of the tau protein), 329-HHKPGGG-335 (SEQ ID NO:100) (within the third repeat domain of the tau protein), and 361-THVPGGG-367 (SEQ ID NO:101) (within the fourth repeat domain of the tau protein).

[0226] Figure 8: (A) Alignment of the human tau protein amino acid sequence (SEQ ID NO: 225) with tau protein sequences from other species (SEQ ID NOs 226-245 in order of appearance). The full-length human tau protein was used for alignment; only amino acids 265-368 from the aligned human tau protein are shown. Regions containing the human tau protein and the four individual DC8E8 epitopes on the aligned sequence are boxed and shown in bold. (B) A competitive ELISA showing six tau peptides (SEQ ID NO 201-205 and 200 in order of appearance) competing with tauΔ(1-150; 392-441) / 4R (SEQ ID NO: 199) for binding to antibody DC8E8, which recognizes at least one tau epitope involved in tau protein-tau protein aggregation. (C) A competitive ELISA showing seven tau peptides (SEQ ID NO 144, 146, 149, 151, 159, 161 and 171) competing with tauΔ(1-150; 392-441) / 4R for binding to antibody DC8E8, which recognizes at least one tau epitope involved in tau protein-tau protein aggregation.

[0227] Figure 9: (A) Surface plasmon resonance (SPR) used to characterize the combination of DC8E8 with tauΔ(1-150;392-441) / 4R and 2N4R. (B) Surface plasmon resonance (SPR) used to characterize the combination of DC8E8 with tauΔ(1-150;392-441) / 3R and 2N3R.

[0228] Figure 10: (A) Association and dissociation rates of DC8E8 binding to tauΔ(1-150; 392-441) / 4R and tau protein 2N4R, as determined by SPR. (B) Association and dissociation rates of DC8E8 binding to tauΔ(1-150; 392-441) / 3R and tau protein 2N3R, as determined by SPR. Concentration indications used in the measurements are shown in the figure. The dashed lines are interpolated from the measurement data used for kinetic parameter calculations using the computer program BIA Evaluation Software 4.1 (Biacore AB).

[0229] Figure 11: The monoclonal antibody DC8E8 can differentiate between preclinical AD, early-stage clinical AD, and fully developed late-stage AD. DC8E8 shows staining of pathological tau protein in the early stages (tau protein monomers, dimers) of Black's stage I in preclinical AD in humans. (A). The antibody recognizes the following stages: pathological tau protein oligomers (arrows) and pathological tau protein polymers (tangles) (arrows). (B). In fully developed Alzheimer's disease (late-stage - Black's stage VI), DC8E8 primarily recognizes pathological tau protein polymers in the form of neurofibrillary tangles (arrows), neuritis plaques (inside circles), and neuritis lines (inside pentagons). (C). Scale bar: 100 μm. The monoclonal antibody DC8E8 recognizes tangled structures at all stages of Alzheimer's disease development (D). DC8E8 recognizes tangled structures in the early developmental stages—monomers, dimers, and early oligomeric stages (D1), and the pre-oligomeric tangling stage (D2)—as well as pathological tau protein polymers in the late developmental stages—intracellular (D3) and extracellular neurofibrillary tangles (D4). Arrows indicate small oligomeric tau protein aggregates (D1) within pyramidal hippocampal neurons. Scale bar: 10 μm

[0230] Figure 12: (A) Monoclonal antibody DC8E8 recognizes neurofibrillary degeneration in transgenic rat SHR72. DC8E8 recognizes tau protein neurodegeneration at the oligomeric stage (arrow) and tangled stage (arrow). Furthermore, the antibody reacts with misfolded tau protein located within axonal fibers (inside the rectangle). (B) In the brains of age-matched control rats, the antibody does not show intraneuronal staining. Scale bar: 20 μm. As in human Alzheimer's disease, DC8E8 also recognizes tangled structures at all developmental stages in the brains of transgenic rats (SHR72). DC8E8 recognizes tangled structures in early developmental stages—monomers, dimers, and early oligomeric stages (C), and late oligomeric pre-tangled stages (D), as well as pathological tau protein polymers in late developmental stages—intracellular (E) and extracellular neurofibrillary tangles (nucleus loss) (F). The arrow in (C) indicates small oligomeric tau protein aggregates inside neurons (A). Scale bar: 10 μm

[0231] Figure 13 (A) Staining of neurofibrillary tangles in the cortex of SHR24 transgenic rats expressing tauΔ(1-150; 392-441) / 3R using DC8E8. (B) Recognition of neurofibrillary tangles in the brainstem of SHR72 transgenic rats expressing tauΔ(1-150; 392-441) / 4R using DC8E8. Tissue sections were contrast-stained with methyl green. Arrows – neurofibrillary tangles. Scale bar: 50 μm

[0232] Figure 14: Monoclonal antibody DC8E8 recognizes both soluble tau protein (A) and insoluble tau protein (B) in brain samples isolated from transgenic rat model SHR24 (homogeneous cortex) and Alzheimer's disease patients (including atypical cortical tissues of the hippocampus, entorhinal cortex, and temporal lobe cortex). Arrows - human truncated tau protein, nodule - rat endogenous tau protein. For the soluble tau protein fraction, 15 μg of protein was loaded per lane. For the insoluble tau protein fraction, the clumps were dissolved in 1 / 50 of a volume of 1x sodium dodecyl sulfate (SDS) sample loading buffer, and loaded to the same volume as in the case of the soluble fraction. Monoclonal antibody DC8E8 recognizes both soluble tau protein (A) and insoluble tau protein (B) in brain samples isolated from Alzheimer's disease patients (including atypical cortical tissues of the hippocampus, entorhinal cortex, and temporal lobe cortex) (C) and from transgenic rat model SHR72 (brainstem) (D). Arrows – physiological human tau protein (A) and rat endogenous tau protein (B); arrowhead – human truncated tau protein (tauΔ(1-150; 392-441) / 4R)(D) expressed transgenically in neurons of SHR72 rats. For the soluble tau protein fraction, 15 μg of total protein was loaded per lane. For the insoluble tau protein fraction, the clumps were dissolved in 1 / 50 of a volume of 1x sodium dodecyl sulfate (SDS) sample loading buffer, and loaded to the same volume as in the case of the soluble fraction.

[0233] Figure 15: DC8E8 inhibits pathological tau protein-tau protein interactions in a fluorescence-based tau protein fibrosis assay. TauΔ(1-150; 392-441) / 4R( Figure 15A ) or tauΔ(1-296; 392-441) / 4R( Figure 15B Heparin-induced conformational changes and fibrosis, as measured by thioflavin T fluorescence; test the ability of mAbs DC8E8, Rab50 and DC11 to prevent pathological conformational changes.

[0234] Figure 16 The inhibitory potential of DC8E8 in preventing the formation of tau protein dimers, trimers, and oligomers from truncated tau protein tauΔ(1-296; 392-441) / 4R by immunoblotting was analyzed using HRP-conjugated mAb DC25.

[0235] Figure 17: Uptake and degradation of TauΔ(1-150; 392-441) / 4R by microglia BV2 cells. TauΔ(1-150; 392-441) / 4R was added to mouse BV2 cells alone (1 μM) or in combination with the monoclonal antibody DC8E8 (1 μM tauΔ(1-150; 392-441) / 4R + 1 μM DC8E8). After incubation for various durations (2, 4, 6, and 12 hours), BV2 cells were washed with acid, cellular proteins were extracted, and the level of internalized tau protein was analyzed by Western blotting using the full-tau protein antibody DC25. TauΔ(1-150; 392-441) / 4R was immunolabeled in cell lysates (intracellular tau protein) (A) and in cell culture medium (extracellular tau protein) (B). DC8E8 antibody was observed using an anti-mouse HRP-conjugated antibody. Each lane is loaded with 20 μg of protein.

[0236] Figure 18 This indicates the stability (shelf life) of DC8E8 at 37°C as tested by ELISA. After several months of storage (1, 2, 3, and 4 months), the antibody recognizes tauΔ(1-150; 392-441) / 4R. The bars represent the serial dilutions of the antibody as indicated. Measurements should be performed in triplicate.

[0237] Figure 19 DC8E8 recognizes and targets misfolded (disease-causing) tau protein in brain tissue of human Alzheimer's disease patients. (A) Western blot analysis using a full-length tau protein DC25 antibody:

[0238] 1) Biochemical extraction of pathological tau protein from human Alzheimer's disease brain tissue (Greenberg and Davies, 1989);

[0239] 2) The mimic antibody (Rab50) does not recognize tau protein;

[0240] 3) DC8E8 recognizes and targets misfolded (affected) tau protein in the brain tissue of human Alzheimer's disease patients; and

[0241] (B) Ponceau S staining: 2) and 3) are used to control the amount of antibody (Rab50 and DC8E8) in the experiment.

[0242] Figure 20 DC8E8 recognizes and targets misfolded (disease-causing) tau protein in the brain tissue of an AD-prone SHR72 rat model. (A) Western blot analysis using a full-length tau protein DC25 antibody:

[0243] 4) Biochemical extraction of pathological tau protein from human Alzheimer's disease brain tissue (Greenberg and Davies, 1989);

[0244] 5) The mimic antibody (Rab50) does not recognize tau protein;

[0245] 6) DC8E8 recognizes and targets misfolded (affected) tau protein in the brain tissue of human Alzheimer's disease patients; and

[0246] (B) Ponceau S staining: 2) and 3) are used to control the amount of antibody (Rab50 and DC8E8) in the experiment.

[0247] Figure 21: In vivo, DC8E8 targets the pathological form of tau protein in the brain of transgenic rats (SHR72) and transports the pathological tau protein from the brain to the peripheral blood. (A) The concentrations of DC8E8 antibody in the serum of DC8E8-treated animals reached 466, 200, and 273 μg / ml, respectively. (B) The DC8E8-tau protein complex was observed to be transported from the brain to the peripheral blood. The pathological tau protein reached a mean serum concentration of 350 pg / ml. Active transport of tau protein by DC8E8 eliminates the pathological tau protein from the brain. On the other hand, tau protein was not detected in the serum of animals treated with a rabies virus mimic antibody (Rab50) (Macikova et al., 1992). The concentration of tau protein in the serum of treated animals was determined by Innotest hTAU ELISA (Innogenetics, Belgium). The figure shows the mean along with the standard mean error (SEM). Eight bars of rat AC each indicate different consecutive serum dilutions (from left to right, from 100-fold to 12,800-fold).

[0248] Figure 22: Removal of pathological tau protein from the brain of transgenic rats (SHR72) by DC8E8 monoclonal antibody. (A) Intracerebral administration of DC8E8 (left side) removes (arrow) pathological tau protein from neurons compared to simulated animals (right side). (B) Quantification of the amount of pathological tau protein in neurons of simulated and DC8E8-treated animals shows a fundamental reduction in the amount of pathological tau protein in animals treated with DC8E8 (p<0.0001).

[0249] Figure 23: Recognition of pathological error disordered tauΔ(1-150; 392-441) / 4R by the recombinant scFv fragment (scDC8E8v) of the monoclonal antibody DC8E8 expressed in bacteria. (A) Coomassie brilliant blue staining of crude lysate separated by 10% SDS-PAGE from control BL21 bacteria and bacteria with scDC8E8v expression plasmid: Lane 1, crude lysate of control BL21 bacteria; Lane 2, crude lysate of BL21 bacteria expressing scDC8E8v; and Lane M, protein molecular weight marker (Page Ruller pre-stained protein ladder #SM0672, Fermentas). (B) Nitrocellulose membranes containing tau protein stained with Ponceau S S: Lane 1, tauΔ(1-150; 392-441) / 4R, 500 ng; Lane 2, tauΔ(1-150; 392-441) / 4R, 250 ng; Lane 3, tauΔ(1-150; 392-441) / 4R, 125 ng; Lane 4, tauΔ228-441, 50 ng; and Lane M, protein molecular weight marker. (C) Western blot / nitrocellulose membranes containing tau protein, loaded as in B), detected with lysates from bacteria expressing scDC8E8v. (D) Western blot / nitrocellulose membranes containing tau protein, loaded as in B), developed with lysates from negative control bacteria.

[0250] Figure 24: The recombinant scFv fragment of monoclonal antibody DC8E8 (scDC8E8v) exhibits tau protein binding properties similar to those of the DC8E8 antibody in selectively recognizing tauΔ(1-150; 392-441) / 4R. (A) Kinetic affinity of scDC8E8v for ADtauΔ(1-150; 392-441) / 4R determined by SPR. (B) Kinetic affinity of scDC8E8v for tau protein 2N4R determined by SPR. (C) Rate constant (k) of scDC8E8v binding. 缔合 and k 解离 ) and association equilibrium constant.

[0251] Figure 25: Identification of residues in the scDC8E8v binding site that affect the misidentification of disordered tau protein by scDC8E8v / DC8E8. (A) Coomassie brilliant blue staining of polyacrylamide gels after protein isolation from crude lysate of BL21 bacteria containing scDC8E8v expression plasmid (wt) and its mutant forms. Each lane number corresponds to the corresponding clone number (e.g., lane 2 corresponds to 2-VL-R33A). Expressed single-chain proteins are indicated by an asterisk. Control bacterial cultures do not express single-chain proteins. (B) Nitrocellulose membranes containing tau protein stained with Ponceau S: Lane 1, tauΔ(1-150; 392-441) / 4R, 500 ng; Lane 2, tauΔ(1-150; 392-441) / 4R, 250 ng; Lane 3, tauΔ(1-150; 392-441) / 4R, 125 ng. (C) A protein blot of a nitrocellulose membrane containing tau protein, loaded as in (B), is detected by lysate from bacteria expressing scDC8E8v (wt gel) or one of its mutant forms (blot 1-VL-N31 A to 22-VH-G102A).

[0252] Figure 26: (A) Schematic diagram of tau protein 2N4R (SEQ ID NO: 102) with four DC8E8 epitopes of SEQ ID NO 98-101 shown in the shaded box within the magnified region (SEQ ID NO. 246) between residues 261 and 373. (1) Schematic diagram of an overlapping tau protein-derived peptide immunogen for use as an active vaccine or for purification of DC8E8 antibodies, etc., the immunogen comprising at least one of four regions of the tau protein recognized by the DC8E8 antibody; (2) General possibilities for other modified and designed peptides and compounds with optional portions. (B) Overview of immunoblotting analysis of insoluble tau protein prepared from the brainstem of transgenic rats (SHR72, expressing tauΔ(1-150; 392-441) / 4R) treated with self-use peptides SEQ ID NO 1-8 and 108. Immunoblot analysis was performed using various mAbs to determine the reduction of insoluble tau protein with the following AD-related epitopes: mAb DC25 (tau protein 347-353), mAb DC217 (tau protein pThr217), mAb DC209 (tau protein pThr231), mAb AT8 (tau protein pSer202 / pThr205), and mAb AT270 (tau protein pThr181). (C) Densitometric immunoblot analysis of insoluble tau protein prepared from the brainstem of rats treated with tau protein 251-PDLKNVKSKIGSTENLKHQPGGGKVQIINK-280 (SEQ ID NO:1) with autologous and adjuvant combination and control rats treated with autologous adjuvant alone. Mean values ​​are presented together with standard mean error.

[0253] Figure 27 Neurobehavioral assessment of transgenic rats (SHR72) simulating Alzheimer's disease (AD) treated with tau protein 251-PDLKNVKSKIGSTENLKHQPGGGKVQIINK-280 (SEQ ID NO:1). Transgenic rats were used for behavioral tests 10 days after the fifth immunogen dose. Figures represent mean ± SEM. All statistics were obtained using the nonparametric Mann-Whitney U test. (A) Bar walking test (3.5 bars). (B) Number of hindlimb slides (3.5 bars). (C) Neurological scales.

[0254] Figure 28 Vaccination of transgenic SHR72 rats with tau peptide SEQ ID NO:1 resulted in a 49% reduction in neurofibrillary tangles (NFT) loading. Antibody AT8 was used to assess NFTs in the brain tissue of transgenic SHR72 rats.

[0255] Figure 29Quantitative immunoblotting analysis of insoluble tau protein prepared from the brainstem of self-administered tau protein 256-VKSKIGSTENLKHQPGGGKVQIINKKLDLS-285 (SEQ ID NO:2) in transgenic rats (SHR72) treated with adjuvant or control rats treated with adjuvant alone. Mean values ​​are presented together with standard mean error.

[0256] Figure 30 Neurobehavioral assessment of transgenic rats (SHR72) treated with tau protein 256-VKSKIGSTENLKHQPGGGKVQIINKKLDLS-285 (SEQ ID NO:2). All statistical data were obtained using the nonparametric Mann-Whitney U test. (A) Bar walking test (3.5 bars). (B) Number of hind limb slides (3.5 bars). (C) Neurological scales.

[0257] Figure 31 Vaccination of transgenic SHR72 rats with tau peptide SEQ ID NO:2 resulted in a 60% reduction in neurofibrillary tangles (NFT) loading. Antibody AT8 was used to assess NFTs in the brain tissue of transgenic SHR72 rats.

[0258] Figure 32 Quantitative immunoblotting analysis of insoluble tau protein prepared from the brainstem of transgenic rats (SHR72) with phosphorylated Ser262 tau protein 256-VKSKIGSTENLKHQPGGGKVQIINKKLDLS-285 (SEQ ID NO:2) treated with adjuvant or control rats treated with adjuvant alone. Mean values ​​are presented together with standard mean error.

[0259] Figure 33 Vaccination of transgenic SHR72 rats with tau peptide SEQ ID NO:2 / phosphate group resulted in a 77% reduction in neurofibrillary tangles (NFT) loading. Antibody AT8 was used to assess NFTs in the brain tissue of transgenic SHR72 rats.

[0260] Figure 34 Quantitative immunoblotting analysis of insoluble tau protein prepared from the brainstem of transgenic rats (SHR72) treated with tau protein 259-KIGSTENLKHQPGGGKVQIINKKLDLSNVQ-288 (SEQ ID NO:3) along with adjuvant or control rats treated with adjuvant alone. Mean values ​​are presented together with standard mean error.

[0261] Figure 35Neurobehavioral assessment of transgenic rats (SHR72) treated with tau protein 259-KIGSTENLKHQPGGGKVQIINKKLDLSNVQ-288 (SEQ ID NO:3). All statistical data were obtained using the nonparametric Mann-Whitney U test. (A) Bar walking test (3.5 bars). (B) Number of hind limb slides (3.5 bars). (C) Neurological scales.

[0262] Figure 36 Vaccination of transgenic SHR72 rats with tau peptide SEQ ID NO:3 resulted in a 58% reduction in neurofibrillary tangles (NFT) loading. Antibody AT8 was used to assess NFTs in the brain tissue of transgenic SHR72 rats.

[0263] Figure 37 Quantitative immunoblotting analysis of insoluble tau protein prepared from the brainstem of transgenic rats (SHR72) treated with tau protein 275-VQIINKKLDL SNVQSKCGSKDNIKHVPGGG-304 (SEQ ID NO:4) or control rats treated with adjuvant alone. Mean values ​​are presented together with standard mean error.

[0264] Figure 38 Vaccination of transgenic rats with tau peptide SEQ ID NO:4 (SHR72) showed a moderate increase in neurobehavioral parameters. (A) Bar walking test (3.5 cm bar). (B) Number of hind limb slides (3.5 cm bar). (C) Neurological scales. Data are presented as mean with standard mean error.

[0265] Figure 39 Vaccination of transgenic SHR72 rats with tau peptide SEQ ID NO:4 resulted in a 66% reduction in neurofibrillary tangles (NFT) loading. Antibody AT8 was used to assess NFTs in the brain tissue of transgenic SHR72 rats.

[0266] Figure 40 Immunoblot analysis of insoluble tau protein prepared from the brainstem of transgenic rats (SHR72) immunized with adjuvant and containing phosphorylated threonine (SEQ ID NO:5) at position 217, or control rats treated with adjuvant alone, using tau protein 201-GSPGTPGSRSRTPSLPTPPT REPKKVAVVR-230. Mean values ​​are presented together with standard mean error.

[0267] Figure 41Neurobehavioral assessment of transgenic rats (SHR72) treated with tau protein 201-GSPGTPGSRSRTPSLPTPPT REPKKVAVVR-230 / carrying phosphorylated threonine (SEQ ID NO:5) at position 217. All statistics were obtained using the nonparametric Mann-Whitney U test. (A) Bar walking test (3.5 bars). (B) Number of hind limb slides (3.5 bars). (C) Neurological scales.

[0268] Figure 42 Vaccination of transgenic SHR72 rats with tau peptide SEQ ID NO:5 showed no effect on neurofibrillary tangles (NFT) loading. Antibody AT8 was used to assess NFTs in the brain tissue of transgenic SHR72 rats.

[0269] Figure 43 Immunoblot analysis of insoluble tau protein prepared from the brainstem of self-administered tau protein 379-RENAKAKTDHGAEIVYKSPW SGDTSPRHL-408 (SEQ ID NO:6) containing phosphorylated serine residues at positions 396 and 404, and from adjuvant-immunized transgenic rats (SHR72) or control rats treated with adjuvant alone. Mean values ​​are presented together with standard mean error.

[0270] Figure 44 Neurobehavioral assessment of transgenic rats (SHR72) treated with tau protein phosphorylated at Ser396 / Ser404 (SEQ ID NO:6). All statistics were obtained using the nonparametric Mann-Whitney U test. (A) Bar walking test (3.5 bars). (B) Number of hind limb slides (3.5 bars). (C) Neurological scales.

[0271] Figure 45 Vaccination of transgenic SHR72 rats with tau peptide SEQ ID NO:6 did not reduce neurofibrillary tangles (NFT) loading. Antibody AT8 was used to assess NFTs in the brain tissue of transgenic SHR72 rats.

[0272] Figure 46 Immunoblot analysis of insoluble tau protein prepared from the brainstem of rats (SHR72) immunized with adjuvant, containing a phosphorylated serine residue at position 202 and a phosphorylated threonine residue at position 205, using tau protein 181-TPPSSGEPPKSGDRSGYSSPGSPGTPGSRS-210 (SEQ ID NO:7) with phosphorylated serine residue at position 202 and phosphorylated threonine residue at position 205, or control rats treated with adjuvant alone. Mean values ​​are presented together with standard mean error.

[0273] Figure 47Neurobehavioral assessment of SHR72 rats treated with tau protein 181-TPPSSGEPPKSGDRSGYSSPGSPGTPGSRS-210 (SEQ ID NO:7) containing phosphorylated serine residue at position 202 and phosphorylated threonine residue at position 205. All statistical data were obtained using the nonparametric Mann-Whitney U test. (A) Bar walking test (3.5 bars). (B) Number of hindlimb slides (3.5 bars). (C) Neurological scales.

[0274] Figure 48 Vaccination of transgenic SHR72 rats with tau peptide SEQ ID NO:7 showed no effect on neurofibrillary tangles (NFT) loading. Antibody AT8 was used to assess NFTs in the brain tissue of transgenic SHR72 rats.

[0275] Figure 49 Immunoblot analysis of insoluble tau protein prepared from the brainstem of rats (SHR72) immunized with self-administered tau protein 300-VPGGGSVQIVYKPVDLSK-317 (SEQ ID NO:8) along with adjuvant or control rats treated with adjuvant alone. Mean values ​​are presented together with standard mean error.

[0276] Figure 50 Neurobehavioral assessment of transgenic AD rats (SHR72) treated with tau protein 300-VPGGGSVQIVYKPVDLSK-317 (SEQ ID NO:8). All statistical data were obtained using the nonparametric Mann-Whitney U test. (A) Bar walking test (3.5 bars). (B) Number of hind limb slides (3.5 bars). (C) Neurological scales.

[0277] Figure 51 Vaccination of transgenic SHR72 rats with tau peptide SEQ ID NO:8 did not reduce neurofibrillary tangles (NFT) loading. Antibody AT8 was used to assess NFTs in the brain tissue of transgenic SHR72 rats.

[0278] Figure 52 Vaccination of transgenic SHR72 rats with tau peptide (SEQ ID NO:108) significantly reduced the amount of insoluble pathological tau protein (p<0.001). Pathological insoluble tau protein was extracted from the brains of SHR72 transgenic rats immunized with tau peptide and analyzed by Western blotting. Mean values ​​and standard mean error are presented together.

[0279] Figure 53Vaccination of transgenic rats with tau peptide (SEQ ID NO:108) significantly improved neurobehavioral parameters (p<0.05). (A) Bar walking test (3.5cm bar). (B) Number of hind limb slides (3.5cm bar). (C) Neurological scales. Data are presented as mean with standard mean error.

[0280] Figure 54 Vaccination of transgenic SHR72 rats with tau peptide (SEQ ID NO:108) reduced neurofibrillary tangles (NFT) load by 60%. Antibody AT8 was used to evaluate NFTs in the brain tissue of transgenic SHR72 rats.

[0281] Figure 55 ELISA of antiserum produced from transgenic rats (SHR72) immunized with peptide tau protein 275-VQIINKKLDLSNVQSKCGSKDNIKHVPGGG-304 (SEQ ID NO:4) showed differences in the binding of antiserum to human pathological tauΔ(1-150; 392-441) / 4R and human pathological tau protein 2N4R.

[0282] Figure 56 Vaccination of transgenic SHR72 rats with tau peptide SEQ ID NO:108 induced the formation of antibodies that preferentially bind to pathological tau protein. Geometric mean antibody titers measured by ELISA showed that the antibodies induced by tau peptide SEQ ID NO:108 vaccination exhibited the highest binding activity against the immunogen (SEQ ID NO:108 peptide) and against pathological tauΔ(1-150; 392-441) / 4R. Physiological tau protein (tau2N4R), used as a control, was recognized more weakly.

[0283] Figure 57 Vaccination of transgenic SHR72 rats with tau peptide SEQ ID NO:108 preferentially induced the formation of IgG antibody isotypes specific to pathological tau protein. The isotype distribution of antibodies induced by tau peptide SEQ ID NO:108 is shown. The binding activity of serum from individual rats to pathological tauΔ(1-150; 392-441) / 4R was analyzed by ELISA after a 1:800 dilution.

[0284] Figure 58SPR affinity assay of antiserum produced by SHR72 rats immunized with peptide tau protein 275-VQIINKKLDLSNVQSKCGSKDNIKHVPGGG-304 (SEQ ID NO:4) for binding to human tau Δ(1-150; 392-441) / 4R and human tau protein 2N4R.

[0285] Figure 59 Immunohistochemical staining of brains from human Alzheimer's disease patients using rat antibodies produced by immunizing transgenic rats (SHR72) with tau protein 275-VQIINKKLDL SNVQSKCGSKDNIKHVPGGG-304 (SEQ ID NO:4). (A) Antiserum identifies neurofibrillary lesions in the hippocampus of Alzheimer's disease brains. (B) Neurofibrillary tangles are shown at higher magnification. Scale bar: 100 μm (A), 10 μm (B).

[0286] Figure 60 Vaccination of transgenic SHR72 rats with tau peptide SEQ ID NO:108 induced antibodies that recognize pathological tau protein in sections of brain tissue from human Alzheimer's disease patients. Representative immunostaining of sera from rats 3 (A), 5 (B), 6 (C), 7 (D), and 8 (E) showed that all tested rat serum antibodies recognized neurofibrillary tangles in the anterior α layer of the entorhinal cortex of Alzheimer's patients. Confluent sera from rats immunized with adjuvant only served as a negative control (F). Serial brain tissue sections from the entorhinal cortex were used. Scale bar: 50 μm.

[0287] Figure 61 Vaccination of transgenic SHR72 rats with tau peptide SEQ ID NO:108 induced specific antibodies recognizing pathological tau proteins in the brains of human Alzheimer's disease patients and transgenic SHR72 rats. Pathological tau proteins were extracted from human and rat brain tissue and analyzed by immunoblotting using confluent serum from transgenic SHR72 rats immunized with peptide SEQ ID NO:108. Serum antibodies recognized monomeric (lanes 1, 2, and 3) and oligomeric (lanes 2 and 3) pathological tau proteins, including the AD-characteristic A68 pathological tau protein.

[0288] Figure 62 Immunization of mice with tau peptide SEQ ID NO:109 induced antibody binding activity against pathological tauΔ(1-150; 392-441) / 4R significantly higher than that against physiological tau protein 2N4R (p = 0.0115). The figure represents a statistical evaluation of ELISA results from individual sera diluted 1:800. Mean values ​​are shown together with standard mean error.

[0289] Figure 63 The antibody induced by immunization of mice with tau peptide SEQ ID NO:110 showed statistically significantly higher binding activity against pathological tauΔ(1-150; 392-441) / 4R than against physiological tau protein 2N4R (p = 0.0029). The figure represents a statistical evaluation of ELISA results from individual sera diluted at 1:800. Mean values ​​are shown together with standard mean error.

[0290] Figure 64 The antibody induced by immunization of mice with tau peptide SEQ ID NO:111 showed statistically significantly higher binding activity against pathological tauΔ(1-150; 392-441) / 4R than against physiological tau protein 2N4R (p = 0.0007). The figure represents a statistical evaluation of ELISA results from individual sera diluted at 1:800. Mean values ​​are shown together with standard mean error.

[0291] Figure 65 The antibody induced by immunization of mice with tau peptide SEQ ID NO:112 showed statistically significantly higher binding activity against pathological tauΔ(1-150; 392-441) / 4R than against physiological tau protein 2N4R (p<0.001). The figure represents the statistical evaluation of ELISA results from individual sera diluted at 1:800. Mean values ​​are shown together with standard mean error.

[0292] Figure 66 The therapeutic epitopes GWSIHSPGGGSC (SEQ ID NO:250) and SVFQHLPGGGSC (SEQ ID NO:251) were designed to competitively bind to the pathological tauΔ(1-150; 392-441)4R antibody DC8E8.

[0293] Figure 67 The therapeutic epitopes GWSIHSPGGGSC (SEQ ID NO:250) and SVFQHLPGGGSC (SEQ ID NO:251) were designed to induce statistically significant antibodies that distinguish pathological tauΔ(1-150; 392-441)4R from physiological tau protein 2N4R, as determined by ELISA. Antibodies specific for tau protein: pathological tauΔ(1-150; 392-441)4R and physiological tau protein 2N4R were detected by ELISA in serum (at a 1:3200 dilution) from mice immunized with either peptide 250 or 251.

[0294] Figure 68The designed therapeutic epitopes GWSIHSPGGGSC (SEQ ID NO:250) and SVFQHLPGGGSC (SEQ ID NO:251) were used to induce the production of IgG1 isotype antibodies in the most robust immune systems.

[0295] Figure 69: Quantitative SPR (surface plasmon resonance) measurement results show that the therapeutic epitope 1 (GWSIHSPGGGSC, SEQ ID NO:250) was designed for treatment. Figure 69A ) and designed therapeutic epitope 2 (SVFQHLPGGGSC, SEQ ID NO:251) Figure 69B The antibodies induced by the 2N4R tau protein were statistically significantly differentiating between pathological tauΔ(1-150; 392-441) / 4R and physiological 2N4R tau protein (p<0.001 and p<0.01, respectively).

[0296] Figure 70: Immunohistochemical staining of human AD-affected brain tissue with serum generated targeting therapeutic epitopes 1 (GWSIHSPGGGSC, SEQ ID NO:250) and 2 (SVFQHLPGGGSC, SEQ ID NO:251). (A) Antiserum targeting therapeutic epitope 1 recognizes neurofibrillary lesions in the brain of AD patients. (C) High magnification of neurofibrillary tangles and neural fibrillary networks (arrows). (B) Antiserum targeting therapeutic epitope 2 recognizes neurofibrillary lesions in the brain of AD patients. (D) High magnification of stained neurofibrillary tangles and neural fibrillary networks (arrows). Antiserum targeting therapeutic epitopes 1 and 2 does not recognize normal tau protein in the control human brain (E, F). Scale bar: 50 μm (A, B, E, F), 20 μm (C, D). (G) Serum generated from the therapeutic epitope 2 (SVFQHLPGGGSC, SEQ ID NO:251) recognizes neurofibrillary lesions in transgenic rat SHR72. (H) In the brains of age-matched control rats, the antibody does not show intraneuronal staining. Serum recognizes the pre-oligotangled stage (I) and the intracellular stage (J). Scale bar: 20 μm (A, B), 10 μm (C, D).

[0297] Figure 71 Antibodies induced by therapeutic epitope 1 (GWSIHSPGGGSC, SEQ ID NO:250) and therapeutic epitope 2 (SVFQHLPGGGSC, SEQ ID NO:251) recognize soluble and creatine acyl insoluble pathological tau proteins isolated from human Alzheimer's disease brain tissue.

[0298] Figure 72Antibodies induced by therapeutic epitope 1 (GWSIHSPGGGSC, SEQ ID NO:250) and therapeutic epitope 2 (SVFQHLPGGGSC, SEQ ID NO:251) recognize soluble (lanes 1, 3, 5) and insoluble (lanes 2, 4, 6) pathological tau proteins isolated from the brain of an Alzheimer's disease rat model (SHR72).

[0299] Figure 73 Immunotherapy with therapeutic epitope 2 (SVFQHLPGGGSC, SEQ ID NO:251) significantly improved neurobehavioral parameters (neurological scales) in SHR72 rats. (A) Bar walking test. (B) Number of hind limb slides (p<0.05). (C) Neurological scales. Compared with transgenic control rats receiving adjuvant alone, rats treated with therapeutic epitope 2 (SEQ ID NO:251) showed: a) a 27% reduction in escape latency in the bar walking test, b) a 44% reduction in the number of hind limb slides (p<0.05), and c) a 26% reduction in neurological scale scores. All statistics were obtained using the nonparametric Mann-Whitney U test.

[0300] Figure 74 Immunotherapy with a designed therapeutic epitope 2 (SVFQHLPGGGSC, SEQ ID NO:251) showed a significant reduction in pathological tau protein levels in the brains of immunized Alzheimer's transgenic SHR72 rats. Compared to control transgenic rats receiving adjuvant alone, immunotherapy significantly (p<0.05) reduced the amount of pathologically insoluble tau protein in the immunized animals. Reduced levels of pathologically insoluble tau protein were observed at all analyzed tau epitopes (P<0.05).

[0301] Figure 75: (A) Schematic diagram of the synthetic peptides used for further evaluation of the minimum epitope (therapeutic core unit) and immunogenicity assay of DC8E8 and (B) their amino acid sequences.

[0302] Figure 76The DC8E8 minimal epitope (therapeutic core unit) was determined using a competitive ELISA with synthetic peptides. Ten tau peptides (SEQ ID NO: 270, 271, 272, 275, 276, 277, 280, 281, 282, and 283) containing at least six amino acids of the DC8E8 recognition sequence were able to compete with pathological tauΔ(1-150; 392-441) / 4R for binding to antibody DC8E8. Tau peptides (SEQ ID NO: 273, 274, 278, and 279) containing only five amino acids of the DC8E8 recognition sequence did not compete with tauΔ(1-150; 392-441) / 4R (SEQ ID NO: 199) for binding to antibody DC8E8.

[0303] Figure 77: Induction of tau protein-specific antibodies in C57BL mice after immunization with tau peptides. (A) The 12-mer, 7-mer, and 6-mer peptides (SEQ ID NO: 270, 271, and 272, respectively) were immunogenic. The immunized antibodies showed statistically significantly higher binding activity against pathological tauΔ(1-150; 392-441) / 4R than against physiological tau protein 2N4R (p<0.0079; p<0.0052; p<0.0079, respectively). The 5-mer peptides (SEQ ID NO: 273 and 274) were not immunogenic. (B) The 42-mer, 19-mer, 7-mer, and 6-mer peptides (SEQ ID NO: 275, 280, 276, and 277, respectively) were immunogenic. Antibodies induced by these peptides were statistically significant (p<0.0079, p<0.0159, p<0.0079, and p<0.0379, respectively) in distinguishing pathological tauΔ(1-150; 392-441) / 4R from physiological tau protein 2N4R. The 5-meric peptides SEQ ID NO:278 and 279 were not immunogenic. (C) The 7-meric peptides (SEQ ID NO:281 and 283) were immunogenic. Antisera against these peptides were statistically significant (p<0.0379 and p<0.0286, respectively) in distinguishing pathological tauΔ(1-150; 392-441) / 4R from physiological tau protein 2N4R. The levels of antibodies against both pathological and physiological tau proteins induced by the 6-meric peptide SEQ ID NO:282 were extremely low. The figure represents a statistical evaluation of the ELISA results of individual sera diluted at 1:800. The mean is shown together with the standard mean error.

[0304] Figure 78Geometric mean antibody titers of tau protein-specific antibodies after immunization of C57BL mice with tau peptides. Vaccination of C57BL mice with tau peptides SEQ ID NO:270, 271, 272, 275, 276, 277, 280, 281, and 283 induced the formation of tau protein-specific antibodies. Geometric mean antibody titers measured by ELISA showed that antibodies induced by vaccination with tau peptides SEQ ID NO:270, 271, 272, 275, 276, 277, 280, 281, and 283 exhibited higher binding activity against pathological tauΔ(1-150; 392-441) / 4R than against physiological tau protein (tau2N4R). Lower titers of tau protein-specific antibodies were detected after immunization of mice with tau peptides SEQ ID NO:273, 274, 278, 279, and 282.

[0305] Figure 79A and 79B This study shows the isotype distribution of antibodies induced by tau peptide. Immunization of C57 / BL mice with tau peptide carrying the minimal DC8E8 epitope preferentially induced the formation of IgG1 and IgG2b antibody isotypes specific to pathological tau protein. The binding activity of confluent serum from individual mice at a 1:800 dilution to pathological tauΔ(1-150; 392-441) / 4R was analyzed by ELISA.

[0306] Figure 80 Quantitative assessment of the binding affinity of antibodies induced in C75BL mice immunized with tau peptides for tauΔ(1-150; 392-441) / 4R and 2N4R. Surface plasmon resonance (SPR) measurements showed statistically significant antibody binding to tau peptides SEQ ID NO: 270, 271, 272, 275, 276, 277, 280, 281, and 283. ** ...p<0.001 and * ...p<0.01) to differentiate pathological tauΔ(1-150; 392-441) / 4R from physiological 2N4R tau protein. KA-Association equilibrium binding constant.

[0307] Figure 81In Western blot analysis, antibodies induced in mice immunized with tau peptides recognized pathological forms of tau protein. Vaccination of C57BL mice with tau peptides SEQ ID NO:270, 271, 272, 275, 276, 277, 280, 281, and 283 induced specific antibodies recognizing pathological tau protein isolated from human Alzheimer's disease brain tissue and from the brainstem of transgenic rat SHR72. Antiserum following immunization of mice with peptides SEQ ID NO:273, 274, 278, 279, and 282 did not recognize pathological forms of tau protein.

[0308] Figure 82A -C: Neurofibrillary tangles recognized by tau peptide-induced antibodies in human AD brain tissue. Antibodies recognizing neurofibrillary lesions in the hippocampus of the Alzheimer's disease brain were induced by vaccination of C57BL mice with tau peptide SEQ ID NO:270, 271, 272, 275, 276, 277, 280, 281, and 283. Serum from mice immunized with adjuvant only was used as a negative control. Brain tissue sections from the CA1 hippocampus were used. Scale bar: 100 μm.

[0309] Figure 83 An overview of immunohistochemical staining (and corresponding relative intensities) of brain tissue from human AD patients using serum antibodies generated from C57BL mice immunized with tau peptide SEQ ID NO:270,271,272,273,274,275,276,277,278,279,280,281,282 and 283. Invention Details

[0310] The term "antibody" refers to an immunoglobulin that is genetically engineered, naturally occurring, or wholly or partially synthesized or recombinant. All derivatives, portions, and fragments thereof that maintain antigen-binding properties and at least one tau protein-related characteristic property according to the invention are also included in this term. The term also covers any protein having a binding domain homologous or substantially homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources or be wholly or partially synthesized or recombinant. Antibodies may be monoclonal or polyclonal. Antibodies may be members of any immunoglobulin class, including any human class: IgG, IgM, IgA, IgD, and IgE. In some embodiments of the invention, derivatives of the IgG class are preferred.

[0311] The terms “isolated antibody” and “isolated peptide” refer to proteins or peptides derived from cDNA, recombinant RNA or any other synthetic source or a combination thereof; and also to proteins and peptides that, depending on their source or derived source, (1) do not associate with proteins found in nature, (2) do not contain other proteins from the same source, such as mouse proteins, (3) are expressed by cells from a different species, or (4) are not found in nature.

[0312] Furthermore, the antibodies according to the invention also include "conserved sequence modifications," i.e., modifications to the nucleotide and amino acid sequences, that do not affect or alter the aforementioned characteristics of the antibodies according to the invention. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved amino acid substitutions include substitutions of amino acid residues by replacing them with amino acid residues having similar side chains. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the predicted non-essential amino acid residues in anti-tau protein antibodies can be replaced, for example, by another amino acid residue from the same side chain family.

[0313] "Antibody fragment" and "antibody moiety" encompass a portion of a full-length antibody, typically at least its antigen-binding portion / domain or variable region. Examples of antibody fragments include double-chain antibodies (diabody), single-chain antibody molecules, immunotoxins, and multispecific antibodies formed from antibody fragments. Furthermore, antibody fragments include single-chain polypeptides that possess the characteristics of a VH chain binding to pathological tau protein (i.e., capable of assembling together with a VL chain) or a VL chain binding to pathological tau protein (i.e., capable of assembling together with a VH chain) to form a functional antigen-binding bag and thereby provide the property of binding to pathological tau protein. The term also includes fragments that do not provide effector function (e.g., ADCC / CDC) on their own, but provide this function after combination with an appropriate antibody constant domain.

[0314] The term "chimeric antibody" refers to a monoclonal antibody typically prepared using recombinant DNA technology, comprising a variable region (i.e., binding region) from one source or species and at least a portion of a constant region from a different source or species. Chimeric antibodies comprising a murine variable region and a human constant region are particularly preferred. The murine / human chimeric antibody is the product of an expressed immunoglobulin gene containing a DNA segment encoding a murine immunoglobulin variable region and a DNA segment encoding a human immunoglobulin constant region. Other forms of "chimeric antibodies" covered by this invention are those whose class or subclass has been modified or altered from that of the original antibody. Such "chimeric" antibodies are also referred to as "class-changing antibodies." Methods for generating chimeric antibodies involve conventional recombinant DNA and gene transfection techniques known in the art. See, for example, Morrison, SL, et al., Proc. Natl. Acad Sci. USA 81(1984) 6851-6855; U.S. Patent Nos. 5,202,238 and 5,204,244.

[0315] The term "humanized antibody" refers to an antibody in which the frame region (FR) and / or complementarity-determining region (CDR) have been modified to contain a CDR of an immunoglobulin having a different specificity than that of the parental immunoglobulin. In one embodiment, a murine CDR is grafted into the frame region of a human antibody to prepare a "humanized antibody". See, for example, Riechmann, L. et al., Nature 332 (1988) 323-327; and Neuberger, MS et al., Nature 314 (1985) 268-270. Particularly preferred are CDRs that correspond to sequences representing epitopes that recognize antigens and are described herein as "therapeutic epitopes" on the tau protein.

[0316] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The constant region of an antibody may be, for example, a constant region of the human IgG1 type. This region may be atypical and described, for example, by Johnson, G. and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218 and the databases mentioned therein, and is preferred for certain embodiments, provided that the induced ADCC and, for example, CDC properties according to the invention are preserved.

[0317] As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from host cells, like NSO or CHO cells, or from animals transfected with human immunoglobulin genes (e.g., mice, such as XENOMOUSE, a genetically modified mouse that produces antibodies with amino acid sequences of human antibodies (e.g., human frame (FR) and human constant region amino acid sequences)), or antibodies expressed using a recombinant expression vector transfected into host cells. The recombinant human antibody has variable and constant regions derived from human germline immunoglobulin sequences in a rearranged form. The recombinant human antibody according to the invention has been highly mutated in somatic cells. Therefore, the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, although derived from and related to human germline VH and VL sequences, cannot be naturally present in vivo within the human antibody germline lineage.

[0318] The term "effective function" includes, but is not limited to, C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptor; BCR).

[0319] The term "epitope" is used here to refer to a binding site recognized by a binding protein or antibody. An epitope can be any molecule or set thereof, including but not limited to amino acids, amino acid side chains, sugars, and lipids, and can have a specific three-dimensional structure or conformation. Therefore, an epitope can comprise any portion of a tau peptide / protein molecule, including primary, secondary, tertiary, or quaternary structures, as those terms are generally known in the art. A "linear epitope" consists of a continuous sequence of amino acid residues. A linear epitope is an epitope present on physiological tau proteins (e.g., present in tau protein 2N / 4R). A "conformational epitope" is an epitope to which an antibody or binding protein binds in a conformation-specific manner. In the case of protein-based epitopes, binding can depend on the secondary, tertiary, or quaternary structure of the protein carrying the epitope. In other words, the antibody binds in a structure-specific, tertiary-structure-specific, or quaternary-structure-specific manner. A conformational epitope is an epitope present in pathological tau proteins (e.g., present in tauΔ(1-150; 392-441) / 4R).

[0320] The term "therapeutic epitope" refers to regions within the tau protein identified in this paper that promote tau-tau aggregation when in certain conformations (recognized by the DC8E8 antibody). Antibodies (and other binding proteins) binding to one or more of these regions inhibit early and late stages of tau aggregation, including the conversion of tau monomers to dimers and to higher aggregate forms; that is, antibody inhibition of the conversion from physiological tau protein to pathological tau protein. These regions within the tau protein may be involved in promoting tau fibrillation into paired helical filaments (PHFs) by promoting the formation of β-sheets in adjacent regions of the tau protein. The therapeutic epitopes are contained within 267-KHQPGGG-273 (within the first repeat domain of the tau protein), 298-KHVPGGG-304 (within the second repeat domain of the tau protein), 329-HHKPGGG-335 (within the third repeat domain of the tau protein), and 361-THVPGGG-367 (within the fourth repeat domain of the tau protein). In some embodiments, the therapeutic epitopes are each contained within 268-HQPGGG-273 (within the first repeat domain of the tau protein), 299-HVPGGG-304 (within the second repeat domain of the tau protein), 330-HKPGGG-335 (within the third repeat domain of the tau protein), and 362-HVPGGG-367, respectively.

[0321] The term "showing a higher affinity for pathological tau proteins than for physiological tau proteins" means that the antibody interacts more strongly with at least one form of pathological tau protein than with at least one form of physiological tau protein. This interaction can be measured, for example, by ELISA or surface plasmon resonance (SPR) as described in the following examples.

[0322] The terms "specific binding" and "specific to" are interchangeable and mean that an antibody or its antigen-binding fragment (or other binding protein) forms a complex with an antigen or epitope that is relatively stable under physiological conditions. Specific binding is characterized by a dissociation constant of approximately 1 × 10⁻⁶. -6 M or smaller, for example less than about 100 nM, and primarily, for example, less than 10 nM. Methods for determining whether two molecules specifically bind are known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. Typically, the antibody or antigen-binding fragment thereof provided by the present invention has a dissociation constant of at least about 1 × 10⁻⁶ for binding the antigen or epitope. -6 M or smaller, but molecules whose dissociation constants for binding with other molecules are not the dissociation constants stated therein.

[0323] "Preferred binding" refers to the binding affinity for pathological tau proteins being higher than that for physiological tau proteins. For example, the affinity for tauΔ(1-150; 392-441) / 4R is higher than that for 2N4R.

[0324] "Universal T-cell epitopes" are selected from influenza hemagglutinin: HA307-319 (PKYVKQNTLKLAT) (SEQ ID NO:123); PADRE (AKXVAAWTLKAAA) (SEQ ID NO:124); malaria CS: T3 epitope (EKKIAKMEKASSVFNV) (SEQ ID NO:125); hepatitis B surface antigen: HBsA919_28 (FFLLTRILTI) (SEQ ID NO:126); heat shock protein 65: hsp65153_171 (DQSIGDLIAEAMDKVGNEG) (SEQ ID NO:127); BCG vaccine (QVHFQPLPPAWKL) (SEQ ID NO:128); tetanus toxoid: TI830-844 (QYIKANSKFIGITEL) (SEQ ID NO:123). Sequences of tetanus toxoid: TI947-967(FNNFTVSFWLRVPKVSASHLE) (SEQ ID NO:130); and HIV gp120T1(KQIINMWQEVGKAMYA) (SEQ ID NO:131).

[0325] The term "intrinsically disordered tau protein" refers to a normal / physiological form of tau protein that lacks any defined 3D structure. It is present in the healthy brain (Kovacech et al., 2010).

[0326] "Misconduct-disordered tau protein" refers to a form of tau protein that differs from the conformation of normal / physiologically inherently disordered tau protein and lacks a robust / defined 3D structure. Misconduct-disordered truncated tau proteins can induce neurofibrillary degeneration in vivo. They are not present in the healthy brain (Kovacech et al., 2010). "Misordered tau protein" refers to a structurally pathological form of tau protein that assembles into polymers of the neurofibrillary fibrillary fungus (PHF) that form neurofibrillary free radicals (NFTs). Misordered tau proteins are not present in the healthy brain (Kovacech et al., 2010).

[0327] “SHR24” refers to a transgenic rat strain expressing type IIB tau protein (151-391 / R3). These transgenic rats exhibit progressive, age-dependent neurofibrillary degeneration in the cortical brain regions. Neurofibrillary tangles (NFTs) in SHR24 rats meet several key histological criteria for identifying neurofibrillary degeneration in human Alzheimer's disease, including argyrophilia, Congo red birefringence, and thioflavin S reactivity. These criteria can be used to analyze neurofibrillary degeneration in subjects receiving any embodiment of this invention. Neurofibrillary tangles are also identified using antibodies used to detect pathological tau proteins in the human brain (including DC11, which recognizes abnormal tau protein conformations) and antibodies specific to hyperphosphorylated forms of tau protein. Furthermore, neurofibrillary degeneration is characterized by the widespread formation of sarcosyl-insoluble tau protein complexes composed of rat endogenous and truncated tau protein species (Filipcik et al., 2010).

[0328] “SHR72” refers to a transgenic rat strain that expresses human truncated tauΔ(1-150; 392-441) / 4R in several brain regions and spinal cord, according to international patent application PCT WO 2004 / 007547. The origin of this rat strain was described by Zilka et al., 2006, and the pathology of tau protein was described by Koson et al., 2008.

[0329] "Type IA Tau protein" refers to a tau protein that is doubly truncated at both the N-terminus and C-terminus, specifically the truncated tau43 containing at least the first 236 N-terminal amino acids and at least the last 45 C-terminal amino acids. This molecule is detectable in brain tissue affected by Alzheimer's disease but not in normal healthy brain tissue (WO2004 / 007547A2).

[0330] "Type IB Tau protein" refers to a tau protein that is doubly truncated at both the N-terminus and C-terminus, specifically the truncated tau44 containing at least the first 236 N-terminal amino acids and at least the last 45 C-terminal amino acids. This molecule is detectable in brain tissue affected by Alzheimer's disease but not in normal healthy brain tissue (WO2004 / 007547A2).

[0331] "Type IIA Tau protein" refers to a tau protein that is doubly truncated at both the N-terminus and C-terminus, specifically the truncated tau43 containing at least the first 68 N-terminal amino acids and at least the last 40 C-terminal amino acids. This molecule is detectable in brain tissue affected by Alzheimer's disease but not in normal healthy brain tissue (WO2004 / 007547A2).

[0332] "Type IIB Tau protein" refers to a tau protein that is doubly truncated at both the N-terminus and C-terminus, specifically the truncated tau44 containing at least the first 68 N-terminal amino acids and at least the last 20 C-terminal amino acids. This molecule is detectable in brain tissue affected by Alzheimer's disease but not in normal healthy brain tissue (WO2004 / 007547A2).

[0333] As used herein, the term "treatment," etc., refers to achieving a desired pharmacological and / or physiological effect. This effect may be preventative or therapeutic in terms of complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or adverse effects attributable to said disease. As used herein, "treatment" also covers any treatment of AD or related tau protein lesions in mammals, particularly humans, and includes: (a) preventing the disease from occurring in subjects who are susceptible to the disease or at risk of acquiring the disease but have not yet been diagnosed with said disease; (b) suppressing the disease, i.e., halting its development; and (c) alleviating the disease, i.e., causing disease remission. Preferred embodiments of "treatment" are further discussed below. In some embodiments, "treatment" refers to the administration of a therapeutic agent to a patient suspected of having or already having AD or another tau protein lesion. It may also refer to the reduction, elimination, or at least partial inhibition of the disease and / or one or more symptoms associated with the disease and / or its complications, and to the application of any beneficial effect on said one or more symptoms.

[0334] "Prevention" is applied to patients who are susceptible to a specific disease or otherwise at risk for a specific disease. Everyone in the general population is at risk for Alzheimer's disease (AD). Some individuals have an increased genetic risk of AD. Prevention can eliminate or reduce the risk or delay the onset of the disease. Delay in onset or progression can be measured based on standard disease progression time in similar populations or individuals.

[0335] "Tau protein lesions" refer to diseases associated with the formation of pathological tau proteins.

[0336] "Physiological tau protein" refers to any one of the six subtypes of tau protein in normal individuals, namely:

[0337] 2N4R (SEQ ID NO:102)

[0338] 1N4R (SEQ ID NO:103)

[0339] 2N3R (SEQ ID NO:104)

[0340] 0N4R (SEQ ID NO:105)

[0341] 1N3R (SEQ ID NO:106)

[0342] 0N3R (SEQ ID NO:107)

[0343] This definition excludes tau proteins that carry any phosphorylation associated with Alzheimer's disease and other tau protein disorders.

[0344] "Pathological tau protein" includes pathological tau protein conformational isoforms and structures and encompasses all of the following: type IA, IB, IIA, and IIB tau proteins, out-of-order tau proteins, error-disordered tau proteins (monomers, dimers, trimers, oligomers), error-disordered soluble tau proteins, sarcosyl-insoluble tau proteins, extracellular tau protein deposits, tau protein aggregates, paired helical fibers, neurofibrillary lesions (including neurofibrillary lesions, tangles, threads, fibers, axonal globules), highly phosphorylated truncated tau proteins and full-length tau proteins, or any other form of tau protein associated with AD or another tau protein lesion.

[0345] "Link" refers to a portion being attached to a peptide, antibody, or compound. This portion may be coupled, compounded, covalently or non-covalently linked. The portion may be chemically cross-linked with, expressed, or synthesized in the form of a peptide or antibody fusion.

[0346] "Part" refers to any compound, organic matter, peptide, protein, nucleic acid, carrier, or adjuvant that can be attached to a peptide, antibody, or binding protein, but is not the peptide, antibody, or binding protein itself for which protection is sought.

[0347] "Immunogenicity" refers to certain properties that can trigger an immune response. Immune responses can be mediated by antibodies, cells, or both.

[0348] "Adjuvant" refers to a substance that can increase, amplify, or regulate the immune response to an accompanying peptide.

[0349] "Other treatments" refers to additional treatments that are acceptable to the subject patient.

[0350] "Clearance" refers to reducing the level or detecting pathological tau protein and / or pathological tau protein structures. Clearance does not necessarily mean the complete disappearance of pathological tau protein; that is, pathological tau protein may be partially eliminated.

[0351] The term “promote” encompasses inducing, enhancing, or increasing.

[0352] "Brain tissue" refers to any neuronal tissue, such as that from the brain, brainstem, and spinal cord.

[0353] The terms "specific binding" and "high affinity" refer to antibody binding to the predetermined antigen, i.e., the tau epitope defined above. Typically, the dissociation constant (KD) for antibody binding is 10. -6M or smaller, and the KD of the antibody binding the predetermined antigen is at least twice as small as the KD of its binding to non-specific antigens other than the predetermined antigen (e.g., BSA, casein, or any other designated polypeptide). The terms “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably herein with the term “antibody that specifically binds to an antigen.” As used herein, “highly specific” binding means that the antibody has a relative KD of [missing value] against misordered tau protein. D Compared to the binding of antibodies to other ligands or normal full-length tau protein, K D At least 4 times smaller.

[0354] The term "prokaryote" is intended to include all bacteria that can be transformed or transfected with DNA or RNA molecules that can be used to express the antibodies or one or more corresponding immunoglobulin chains of the present invention. Prokaryotic hosts may include Gram-negative and Gram-positive bacteria, such as *Escherichia coli*, *Salmonella typhimurium*, *Serratia marcescens*, and *Bacillus subtilis*. The term "eukaryote" is intended to include yeast, higher plants, insects, and, for example, mammalian cells, primarily, for example, HEK 293, NSO, and CHO cells.

[0355] The term "chemical derivative" describes a molecule that contains an additional chemical part that is not normally part of a base molecule. This part may improve the solubility, half-life, absorption, etc., of the base molecule. Alternatively, the part may reduce unwanted side effects or decrease the toxicity of the base molecule.

[0356] The terms “nucleic acid,” “nucleic acid sequence,” “polynucleotide,” “oligonucleotide,” “polynucleotide sequence,” and “nucleotide sequence” are used interchangeably in this specification and refer to an exact nucleotide sequence, whether modified or unmodified, that defines a fragment or region of a nucleic acid, which may or may not contain non-natural nucleotides, and is double-stranded DNA, single-stranded DNA, or a transcript of said DNA.

[0357] As used herein, the terms “isolated polynucleotide” or “isolated nucleic acid” will mean a polynucleotide of a genome, cDNA or synthetic source or a combination thereof, which, depending on its origin (1) is not associated with all or part of the polynucleotide found in nature, (2) is operatively linked to a polynucleotide to which it is not linked in nature, or (3) is not present in nature as part of a larger sequence.

[0358] Antibodies for diagnostics, passive immunization, drug delivery, and AD therapy

[0359] This article describes novel isolated antibodies specific to one or more tau epitopes displayed by pathological forms of tau protein. These epitopes are located in the regions of tau protein first designated as having an effect in the pathological tau protein aggregate, namely within: 267-KHQPGGG-273 (SEQ ID NO:98) (i.e., epitope 1 is located within 267-KHQPGGG-273, which belongs to the first repeat domain of tau protein), 298-KHVPGGG-304 (SEQ ID NO:99) (epitaxe 2, within the second repeat domain of tau protein), 329-HHKPGGG-335 (SEQ ID NO:100) (epitaxe 3, within the third repeat domain of tau protein), and 361-THVPGGG-367 (SEQ ID NO:101) (epitaxe 4, within the fourth repeat domain of tau protein). These antibodies can recognize misordered and error-disordered tau proteins in the human AD brain and in transgenic rat models of AD and related tau protein lesions, wherein the rat models express human error-disordered truncated tauΔ(1-150; 392-441) / 3R or tauΔ(1-150; 392-441) / 4R. The isolated antibodies can also interfere with one or more of the various tau protein-mediated activities that promote AD lesions, including: (i) conversion from misordered or physiological tau proteins to error-disordered tau proteins; (ii) formation of “pathological tau protein” monomers, dimers, trimers, and other tau protein polymers; (iii) formation of insoluble tau protein aggregates; and (iv) promotion of extracellular tau protein clearance.

[0360] The invention disclosed herein is based in part on the discovery that an antibody specifically binding to one of four previously unidentified functional domains of tau protein can inhibit the formation of pathological tau protein aggregates and detect various pathological forms of tau protein (some of which are the earliest forms formed in the disease, such as pathological monomers) selected from 267-KHQPGGG-273 (SEQ ID NO: 98) (within the first repeat domain of tau protein), 298-KHVPGGG-304 (SEQ ID NO: 99) (within the second repeat domain of tau protein), 329-HHKPGGG-335 (SEQ ID NO: 100) (within the third repeat domain of tau protein) and 361-THVPGGG-367 (SEQ ID NO: 101) (within the fourth repeat domain of tau protein). Hybridomas generated by human error-disordered tau protein II (151-391 / 4R), also referred to herein as tauΔ(1-150; 392-441) / 4R, were screened using both immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). The resulting group included a mouse monoclonal antibody (mAb) DC8E8 with an IgG1 subclass. Epitope localization of DC8E8 revealed that it binds to four previously unidentified epitopes on human tau protein. Furthermore, further functional analysis of DC8E8 revealed that each epitope represents a different functional region within the tau protein. These regions, now described as novel targets for AD diagnosis and therapy and thus referred to as “therapeutic epitopes,” are contained within 267-KHQPGGG-273 (SEQ ID NO:98) (within the first repeat domain of the tau protein), 298-KHVPGGG-304 (SEQ ID NO:99) (within the second repeat domain of the tau protein), 329-HHKPGGG-335 (SEQ ID NO:100) (within the third repeat domain of the tau protein), and 361-THVPGGG-367 (SEQ ID NO:101) (within the fourth repeat domain of the tau protein). In some implementations, one or more of the therapeutic epitopes are included within 268-HQPGGG-273 (SEQ ID NO:223) (within the first repeat domain of the tau protein), 299-HVPGGG-304 (SEQ ID NO:154) (within the second repeat domain of the tau protein), 330-HKPGGG-335 (SEQ ID NO:224) (within the third repeat domain of the tau protein), and 362-HVPGGG-367 (SEQ ID NO:154) (within the fourth repeat domain of the tau protein).In some embodiments, at least one therapeutic epitope is contained within 299-HVPGGG-304 (SEQ ID NO: 154) (within the second repeat domain of the tau protein). In some embodiments, one or more therapeutic epitopes are 299-HVPGGG-304 (SEQ ID NO: 154).

[0361] In fact, DC8E8 can distinguish between pathological and normal tau proteins, indicating that at least one of these four epitopes is a conformational epitope. In other words, DC8E8 reveals that at least one of the regions covered by each of the four therapeutic epitopes exhibits a conformation in pathological tau proteins that differs from its shape in inherently disordered tau proteins (normal tau proteins). DC8E8 can sense or detect changes because its affinity for binding pathological tau proteins is higher than its affinity for binding physiological tau proteins. Furthermore, DC8E8 binding to tau proteins can inhibit tau protein-tau protein interactions that lead to the formation of pathological tau protein aggregates, as measured by DC8E8's ability to inhibit the formation of insoluble tau protein aggregates in vitro. For example, DC8E8 binding to normal tau proteins can prevent one or more of the aforementioned conformational / shape changes in the regions covering the therapeutic epitopes.

[0362] Furthermore, DC8E8 binding to one or more of these regions or therapeutic epitopes of normal tau protein inhibits certain other conformational changes required for the production of pathological tau protein elsewhere in the molecule. Without being bound by any particular mechanism, one or more of these epitopes / regions within the intended tau protein, recognized by DC8E8, act as promoters of tau-tau protein aggregation within the tau protein. For example, the structure / shape / conformation of one or more of these epitopes affects the structure of neighboring regions such that fixing their shape within the tau protein molecule by binding to them with DC8E8 interferes with the ability or tendency of neighboring regions (e.g., 274-281) to form β-sheets, which are required for tau-tau protein aggregation. Therefore, the binding of intended DC8E8 to one of these four regions within normal tau protein can prevent one of the earliest pathological changes identified in tau protein to date: changes required to promote or that promote or allow β-sheet formation within tau protein. Furthermore, it is also intended that DC8E8 binding to one of these four regions within the disordered / pathological tau protein (i.e., after one or more of the four regions have become pathological conformations) can still inhibit pathological tau protein-tau protein aggregation, at least because it still inhibits β-sheet formation, blocks tau protein-tau protein physical interactions, or both.

[0363] Therefore, DC8E8 is used as a tool to identify novel targets or functional regions within tau protein, specifying that four particular DC8E8 binding sites on tau protein play a role in Alzheimer's disease. This is based on the recognition that one or more of these tau protein sites are involved in the formation of pathological tau protein monomers and polymers, at least because the binding of DC8E8 to one or more of them can inhibit those processes. Furthermore, antibodies (e.g., DC8E8) binding to one or more of these therapeutic epitopes can promote the clearance of pathological tau protein from the extracellular environment, at least because they can mediate the uptake and degradation of pathological tau protein by microglia in vitro; reduce extracellular and intracellular tau protein in the brain in vivo; or both. In other words, these antibodies can help reduce the brain damage caused by the pathological form of tau protein.

[0364] Therefore, this article describes antibodies that specifically bind to one or more therapeutic epitopes on the tau protein, wherein each therapeutic epitope is located within amino acid residues 267-KHQPGGG-273 (SEQ ID NO:98) (epitope 1, within the first repeat domain of the tau protein), 298-KHVPGGG-304 (SEQ ID NO:99) (epitope 2, within the second repeat domain of the tau protein), 329-HHKPGGG-335 (SEQ ID NO:100) (epitope 3, within the third repeat domain of the tau protein), and 361-THVPGGG-367 (SEQ ID NO:101) (epitope 4, within the fourth repeat domain of the tau protein). In some embodiments, therapeutic epitopes 1 to 4 are contained within 268-HQPGGG-273 (SEQ ID NO: 223) (within the first repeat domain of the tau protein), 299-HVPGGG-304 (SEQ ID NO: 154) (within the second repeat domain of the tau protein), 330-HKPGGG-335 (SEQ ID NO: 224) (within the third repeat domain of the tau protein), and 362-HVPGGG-367 (SEQ ID NO: 154) (within the fourth repeat domain of the tau protein). The antibody may be monoclonal or polyclonal. It also includes antigen-binding antibody moieties, antibody fragments, antibody variants, engineered proteins, and polymer backbones. These include any protein or peptide molecule comprising at least a portion of an immunoglobulin molecule, said at least a portion such as, but not limited to, at least one heavy or light chain complementarity-determining region (CDR) or its ligand-binding moiety, heavy or light chain variable region, heavy or light chain constant region, frame region, or any portion thereof.

[0365] As a non-limiting example, suitable antibodies, antibody portions, fragments, or variants provided by the present invention can bind to at least one of the therapeutic epitopes. The term "antibody" also includes antibody digestion fragments, designated antibody portions, and variants thereof, portions of antibodies that mimic the structure and / or function of antibody mimics or designated fragments or portions thereof, including single-chain antibodies and fragments thereof. Functional fragments include antigen-binding fragments that bind to one or more therapeutic epitopes. For example, antibody fragments capable of binding therapeutic epitopes include, but are not limited to, Fab (e.g., obtained by papain digestion), Fab' (e.g., obtained by pepsin digestion and partial reduction), and F(ab')2 (e.g., obtained by pepsin digestion), facb (e.g., obtained by plasmin digestion), pFc' (e.g., obtained by pepsin or plasmin digestion), Fd (e.g., obtained by pepsin digestion, partial reduction, and reaggregation), Fv, or scFv (e.g., obtained by molecular biology techniques) fragments provided by the present invention. See also William E. Paul (ed.), Fundamental Immunology, 6th ed., Lippincott Williams & Wilkins, NY, NY (2008), the entire bibliography of which is incorporated herein by reference. Certain fragments may be generated by enzymatic cleavage, synthesis, or recombination techniques as commonly known in the art or as provided herein. Antibodies may also be generated in various truncated forms using antibody genes with one or more stop codons introduced upstream of the natural termination site. For example, a combinatorial gene encoding the F(ab')2 heavy chain portion may be engineered to include DNA sequences encoding the CH1 domain and / or hinge region of the heavy chain. The various portions of an antibody may be chemically joined together using conventional techniques or prepared as adjacent proteins using conventional genetic engineering techniques.

[0366] The basic structural unit of an antibody is known to be a tetramer. Each tetramer consists primarily of two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region with approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as κ and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgA, and IgE, respectively. Within both light and heavy chains, the variable and constant regions are linked by a "J" region with approximately 12 or more amino acids, with the heavy chain also including a "D" region with approximately 10 additional amino acids. See generally Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)) (incorporated hereinstantly for all purposes). The variable regions of each light / heavy chain pair form antibody binding sites. Thus, a complete antibody has two binding sites. The two binding sites are identical except in bifunctional or bispecific antibodies. All chains exhibit the same general structure of relatively conserved frame regions (FRs) joined by three hypervariable regions (also known as complementarity-determining regions or CDRs). The CDRs from the two chains of each pair are aligned through the frame regions, enabling binding of specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chains contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acids for each domain are specified according to the definitions in the IMGT. Alternative definitions are also known to those skilled in the art. See, for example, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia and Lesk J. Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).

[0367] In some embodiments, the subject antibody comprises a light chain having an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity with any of SEQ ID NO: 141, 143, 152, and 153. In some embodiments, the subject antibody comprises a light chain comprising an amino acid sequence that differs from any of SEQ ID NO: 141, 143, 152, and 153 by only 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. Those skilled in the art can determine which amino acids in the variable region of the light chain can be altered. For example, by comparing the amino acid sequences of the variable regions of the light chains of antibodies having the same specificity, those skilled in the art can determine which amino acids can be altered without changing the specificity. For a comparison of the CDR amino acid sequences of the exemplary DC8E8 antibody light chain, see the examples. Furthermore, an antigen binding assay can be used to determine whether the specificity has been altered. In some embodiments, the subject antibody comprises a light chain including the amino acid sequence described in any of SEQ ID NO: 141, 143, 152 and 153.

[0368] In some embodiments, the subject antibody comprises a heavy chain having an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity with any of SEQ ID NO: 138, 140, 147, and 148. In some embodiments, the subject antibody comprises a heavy chain comprising an amino acid sequence that differs from any of SEQ ID NO: 138, 140, 147, and 148 by only 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. Those skilled in the art can determine which amino acids in the variable region of the heavy chain can be altered. For example, by comparing the amino acid sequences of the variable regions of the heavy chains of antibodies having the same specificity, those skilled in the art can determine which amino acids can be altered without changing the specificity. For a comparison of the CDR amino acid sequences of the exemplary DC8E8 antibody heavy chain, see, for example… Figure 3 E and 25B. Additionally, antigen binding assays can be used to determine whether specificity has changed. In some embodiments, the subject antibody comprises a heavy chain including the amino acid sequence described in any of SEQ ID NO: 138, 140, 147, and 148.

[0369] In some embodiments, the subject antibody comprises a light chain comprising the amino acid sequence described in SEQ ID NO:141 and a heavy chain comprising the amino acid sequence described in SEQ ID NO:138. In some embodiments, the subject antibody comprises a light chain comprising the amino acid sequence described in SEQ ID NO:141 and a heavy chain comprising the amino acid sequence described in SEQ ID NO:140. In some embodiments, the subject antibody comprises a light chain comprising the amino acid sequence described in SEQ ID NO:141 and a heavy chain comprising the amino acid sequence described in SEQ ID NO:147. In some embodiments, the subject antibody comprises a light chain comprising the amino acid sequence described in SEQ ID NO:141 and a heavy chain comprising the amino acid sequence described in SEQ ID NO:148. In some embodiments, the subject antibody comprises a light chain comprising the amino acid sequence described in SEQ ID NO:143 and a heavy chain comprising the amino acid sequence described in SEQ ID NO:138. In some embodiments, the subject antibody comprises a light chain comprising the amino acid sequence described in SEQ ID NO:143 and a heavy chain comprising the amino acid sequence described in SEQ ID NO:140. In some embodiments, the subject antibody comprises a light chain comprising the amino acid sequence described in SEQ ID NO:143 and a heavy chain comprising the amino acid sequence described in SEQ ID NO:147. In some embodiments, the subject antibody comprises a light chain comprising the amino acid sequence described in SEQ ID NO:143 and a heavy chain comprising the amino acid sequence described in SEQ ID NO:148. In some embodiments, the subject antibody comprises a light chain comprising the amino acid sequence described in SEQ ID NO:152 and a heavy chain comprising the amino acid sequence described in SEQ ID NO:138. In some embodiments, the subject antibody comprises a light chain comprising the amino acid sequence described in SEQ ID NO:152 and a heavy chain comprising the amino acid sequence described in SEQ ID NO:140. In some embodiments, the subject antibody comprises a light chain comprising the amino acid sequence described in SEQ ID NO:152 and a heavy chain comprising the amino acid sequence described in SEQ ID NO:147. In some embodiments, the subject antibody comprises a light chain comprising the amino acid sequence described in SEQ ID NO:152 and a heavy chain comprising the amino acid sequence described in SEQ ID NO:148. In some embodiments, the subject antibody comprises a light chain including the amino acid sequence described in SEQ ID NO:153 and a heavy chain including the amino acid sequence described in SEQ ID NO:138.In some embodiments, the subject antibody comprises a light chain including the amino acid sequence described in SEQ ID NO:153 and a heavy chain including the amino acid sequence described in SEQ ID NO:140. In some embodiments, the subject antibody comprises a light chain including the amino acid sequence described in SEQ ID NO:153 and a heavy chain including the amino acid sequence described in SEQ ID NO:147. In some embodiments, the subject antibody comprises a light chain including the amino acid sequence described in SEQ ID NO:153 and a heavy chain including the amino acid sequence described in SEQ ID NO:148.

[0370] In some embodiments, the subject antibody comprises a light chain variable region including at least one, at least two, or three CDRs selected from SEQ ID NO. 117-119. In some embodiments, the subject antibody comprises a heavy chain variable region including at least one, at least two, or three CDRs selected from SEQ ID NO. 120-122. Embodiments in which any of these six CDRs are modified as described in Example 14 are also provided. In some embodiments, at least one modified CDR in the light chain is selected from any of SEQ ID NO: 247 for CDR1, SEQ ID NO: 253 for CDR2, and SEQ ID NO: 255, 257, 258, 259, and 260 for CDR3. In some implementations, at least one modified CDR in the heavy chain is selected from SEQ ID NO:261 or SEQ ID NO:262 for CDR1, SEQ ID NO:264 or SEQ ID NO:265 for CDR2, and SEQ ID NO:266, SEQ ID NO:267 or SEQ ID NO:269 for CDR3.

[0371] Bispecific or bifunctional antibodies are artificial hybrid antibodies with two different pairs of heavy / light chains and two different binding sites. Bispecific antibodies can be generated by various methods, including hybridoma fusion or linking Fab' fragments. See, for example, Songsivilai and Lachmann Clin. Exp. Immunol. 79:315-321 (1990), Kostelny et al. J. Immunol. 148:1547-1553 (1992). The generation of bispecific antibodies can be a relatively labor-intensive process compared to the generation of conventional antibodies, and the yield and purity of bispecific antibodies are typically lower. Bispecific antibodies do not exist in the form of fragments with a single binding site (e.g., Fab, Fab', and Fv).

[0372] This invention does not relate to antibodies in their natural form, i.e., those not derived from their natural environment but obtained through purification from natural sources, or through genetic recombination or chemical synthesis, and therefore they may carry non-natural amino acids. Thus, as used herein, the 20 common amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd edition, eds. ESGolub and DRGren, Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Stereoisomers of the 20 common amino acids (e.g., D-amino acids Nle, Nva, Cha, Orn, Hle, Chg, Hch, or Har), non-natural amino acids (e.g., α-, α-disubstituted amino acids, N-alkyl amino acids), lactate, and other unconventional amino acids may also be components suitable for the polypeptides of this invention. Examples of unconventional amino acids include (i.e., not limited to) 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, according to standard usage and convention, the left-hand direction is the direction of the amino terminus and the right-hand direction is the direction of the carboxyl terminus.

[0373] Similarly, this disclosure does not relate to nucleotide sequences in their natural chromosomal environment, i.e., in their natural state. The sequences of the present invention have been isolated and purified, i.e., they have been sampled directly or, for example, indirectly by copying, wherein their environment has been at least partially modified. Isolated nucleic acids, obtained, for example, by means of host cells using recombinant genetics or by chemical synthesis, are also provided.

[0374] In this disclosure, the "percentage of identity" between two nucleic acid or amino acid sequences means the percentage of identical nucleotide or amino acid residues between the two sequences to be compared, obtained after optimal alignment, wherein this percentage is statistically significant only and the differences between the two sequences are randomly distributed along their length. Traditionally, the comparison of two nucleic acid or amino acid sequences is performed by comparing the sequences after optimal alignment, wherein the comparison can be performed by segmentation or by using an "alignment window". Besides manual comparison, the optimal alignment of sequences for comparison can be achieved using the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2: 482], the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol. 48: 443], the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85: 2444], or by using computer software that employs these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, WI), or by using comparison software BLAST NR or BLAST P).

[0375] The percentage of identity between two nucleic acid or amino acid sequences is determined by comparing two optimal alignment sequences, where the nucleic acid or amino acid sequence to be compared may have additions or deletions compared to a reference sequence to achieve optimal alignment between the two sequences. The percentage of identity is calculated by determining the number of identical amino acid or nucleotide residues between the two sequences, for example, between two complete sequences, dividing the total number of positions in the alignment window by the number of identical positions, and multiplying the result by 100 to obtain the percentage of identity between the two sequences.

[0376] For example, the BLAST program “BLAST 2sequences” (Tatusova et al., “Blast 2sequences-a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol., 1999, Lett. 174: 247-250), available at http: / / www.ncbi.nlm.nih.gov / gorf / bl2.html, can be used with default parameters (specifically for the parameters “open vacancy penalty”: 5 and “extended vacancy penalty”: 2; the selected matrix is, for example, the “BLOSUM 62” matrix recommended by the program); the percentage of identity between the two sequences to be compared is calculated directly by the program.

[0377] For amino acid sequences exhibiting at least 80%, such as 85%, 90%, 95%, and 98%, identity with a reference amino acid sequence, preferred examples include those containing the reference sequence, certain modifications (particularly deletion, addition, or substitution of at least one amino acid), truncated portions, or extended portions. In cases of substitution of one or more continuous or discontinuous amino acids, substitution by an "equivalent" amino acid substitution is preferred. Here, the expression "equivalent amino acid" is intended to indicate any amino acid that may substitute a structural amino acid without altering the biological activity of the corresponding antibody and those specific examples defined below.

[0378] Equivalent amino acids can be determined based on their structural homology with the amino acid they substituted or based on the results of comparative tests of biological activity among the various antibodies that may be generated. As a non-limiting example, the table below outlines possible substitutions that can be performed without significantly altering the biological activity of the corresponding modified antibody; reverse substitution is naturally possible under the same conditions.

[0379] Original residues replace Ala(A) Val, Gly, Pro Arg(R) Lys, His Asn(N) Gln Asp(D) Glu Cys(C) Ser Gln(Q) Asn Glu(E) Asp Gly(G) Ala His(H) Arg Ile(I) Leu Leu(L) Ile, Val, Met Lys(K) Arg Met(M) Leu Phe(F) Tyr Pro(P) Ala Ser(S) Thr, Cys Thr(T) Ser Trp(W) Tyr Tyr(Y) Phe,Trp Val(V) LeU, Ala

[0380] This invention provides an antibody derived from a mouse hybridoma cell line deposited on July 13, 2011, at the American Center for Type Culture Collection (ATCC, 10801 University Blvd, Manassas, VA, USA) with ATCC patent accession number PTA-11994 (granted on July 29, 2011), as described in Examples 1-2. Other suitable antibodies may be derived from cell lines, hybrid cell lines, immortalized cells, or clonal populations of immortalized cells as known in the art. See, for example, Ausubel et al. (eds.), Current Protocols in Molecular Biology, (John Wiley & Sons, Inc., New York, NY (1987–2001)); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., (Cold Spring Harbor, NY (1989)) and Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed.), Volumes 1–3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 2000 (collectively referred to as "Sambrook"); Harlow and Lane, Antibodies, A Laboratory Manual, (Cold Spring Harbor, NY (1989)); Colligan et al. (eds.), Current Protocols in Immunology, (John Wiley & Sons, Inc., NY (1994–2001)); Colligan et al., Current Protocols in Protein Science, (John Wiley & Sons, Inc., New York, NY (1994–2001)); Wiley & Sons, NY, NY, (1997–2001), each incorporated herein by reference in its entirety.

[0381] In a method for generating antibodies provided by the present invention, a hybridoma is generated by fusing a suitable immortalized cell line (e.g., a myeloma cell line) with one of a variety of antibody-producing cells. Suitable immortalized cell lines include, but are not limited to, Sp2 / 0, Sp2 / 0-AG14, P3 / NS1 / Ag4-1, NSO, P3X63Ag8.653, MCP-11, S-194, heterologous myeloma, their fusion products or any cells or fusion cells derived therefrom, or any other suitable cell lines as known in the art and / or commercially available for this purpose (e.g., ATCC). Suitable antibody-producing cells include, but are not limited to, isolated or cloned spleen cells, peripheral blood cells, lymphocytes, tonsillar cells or other immune cells, or cells containing B cells, or any other cells expressing a constant or variable heavy or light chain, or frame or CDR sequence, in the form of endogenous or heterologous nucleic acids, wherein the nucleic acids are recombinant or endogenous viruses, bacteria, algae, prokaryotes, amphibians, insects, reptiles, fish, mammals, rodents, equines, sheep, goats, sheep, primates, eukaryotes, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-stranded, double-stranded or triple-stranded, hybrids, etc., or any combination thereof. See, for example, Ausubel (above) and Colligan, Immunology (above), Chapter 2, the references of which are incorporated herein by reference in their entirety.

[0382] Other methods for generating antibodies according to the various embodiments described above include, but are not limited to, methods for selecting recombinant antibodies from peptide or protein libraries, said libraries including those commercially available from Cambridge antibody Technologies, Cambridgeshire, UK; MorphoSys, Martinsreid / Planegg, Del.; Biovation, Aberdeen, Scotland, UK; Biolnvent, Lund, Sweden; Dyax Corp., Enzon, Affymax / Biosite; Xoma, Berkeley, Calif.; Ixsys; Applied Molecular Biology. Those methods, such as those used in Evolution; methods that rely on immunizing transgenic animals capable of producing multiple sets of selectable human antibodies (typically, these mice contain at least one transgene containing DNA from at least one functionally rearranged or functionally rearranged human immunoglobulin locus; the endogenous immunoglobulin locus in the mice may be destroyed or deleted to eliminate the animal’s ability to produce antibodies encoded by the endogenous gene); selection methods, including ribosome display, single-cell antibody production techniques (e.g., lymphocyte antibody selection methods (“SLAM”)) and B cell selection; attenuated immunization using cyclophosphamide treatment; and any other conventional methods in the art, including but not limited to those described in U.S. Publication Application No. 2005 / 0142609, which are incorporated herein by reference in their entirety).

[0383] In some implementations, the antibody is an optimal full-length chimeric or humanized antibody produced by any or a combination of known techniques, such as those listed and illustrated below: Chapters 3, 4, and 5 of *Business Insights, Preclinical Development of Monoclonal Antibodies and Related Biologicals—Emerging Technologies and New Therapeutic Candidates* by James Shirvill (2010), the entire contents of which are incorporated herein by reference. The techniques mentioned include: CDR transplantation, such as UBC's SLAM technology, PDL's SMART technology, Arana Therapeutics plc's superhumanization, framework assembly, techniques for preparing composite human antibodies, BioAtla LLC's ATLAB platform, humanization, mutation lineage guidance (MLG) strategies, deimmunization strategies, humanization strategies, humanization techniques (e.g., XOMA's HE technology), FcX, and Biolex Therapeutics. Pittsboro Inc. (NC, US) LEX system, Potelligent methods (e.g., BioWa), Complegent technology, BestMAb, ImmunoBody, EB66, Synageva expression platform, Xencor Inc. XmAb, glycoengineered antibodies (e.g., Seattle Genetics Inc. (Bothell, WA, US)), "Wox" (tryptophan oxidation) antibodies (e.g., InNexus Biotechnology Inc. (Vancouver, BC, Canada)), etc.In some implementations, the antibody is a fully human monoclonal antibody and can be generated by one or a combination of technology platforms, such as those listed and illustrated below: Chapter 4 of "Business Insights, Preclinical Development of Monoclonal Antibodie and Related Biologicals - Emerging technologies and new therapeutic candidates" by James Shirvill (2010), and said technology platforms include, but are not limited to: phage display (e.g., PDL, Dyax Corp; Cambridge, MA, US); molecular-based antibody screening (MBAS) (e.g., Affitech A / S as described in EP0547201 and US 6,730,483); cell-based antibody selection (CBAS) platforms; human combinatorial antibody libraries (HuCAL; e.g., MorphoSys AG); MAbstract platforms (e.g., Crucell). NV), including those using the PER.C6 cell line; Adimab platform; XenoMouse; UltiMAb platform; SEBVI platform; Veloclmmune platform, open monoclonal antibody platform, Xenerex platform; cloned human response platform (e.g., IQ Therapeutics) and "instant immune antibodies"; Viventia platform (e.g., Fusogenics, UnLock, ImmunoMine); "natural human antibody" platform (e.g., OncoMab, Patrys, Acceptys); MablgX (e.g., Kenta Biotech); reverse translation medicine platform (e.g., Neuimmune Therapeutics); I-STAR (e.g., Theraclone Sciences); CellSpot (e.g., Trellis Bioscience); iBioLaunch (e.g., iBio Inc.), etc.

[0384] In some implementations, the antibody is modified by linking it to a non-antibody reagent using one or more technology platforms and methods described in Chapter 5 of "Business Insights, Preclinical Development of Monoclonal Antibodie and Related Biologicals - Emerging technologies and new therapeutic candidates" by James Shirvill (2010). These technology platforms and methods include: antibody-drug conjugates (e.g., ADC, Seattle Genetics); targeted antibody payloads (TAP; Immunogen Inc); proteolytically activated antibodies (Probody) (e.g., CytomX Therapeutics); antibody masking (e.g., BioTransformations); targeted photodynamic therapy (e.g., PhotoBiotics); AlbudAb (e.g., GSK); hyFc (e.g., Genexine); ligand traps (e.g., BioLogix); CovX-Body (e.g., CovX); dynamic crosslinking (e.g., InNexus Biotechnology); LEC technology (e.g., Pivotal Biosciences, Morphotek), etc.

[0385] In some embodiments, the antibody or its encoding cDNA may be further modified. Therefore, in another embodiment, the present invention provides a method for generating antibodies according to various embodiments, wherein the method includes any step of generating a chimeric antibody, a humanized antibody, or an analogue of any of those antibodies. In some embodiments, the generation of chimeric antibodies is as described in International Application WO89 / 09622. Methods for generating humanized antibodies are described, for example, in U.S. Patent No. 6,548,640 or Canadian Patent No. 1340879 (CDR Transplantation).

[0386] Furthermore, the antibody or its encoding cDNA may be further modified. Therefore, in another embodiment, the present invention provides a method comprising any step of generating a single-chain antibody, a Fab fragment, a bispecific antibody, a fusion antibody, a labeled antibody, or an analogue of any of those antibodies. As discussed above, in addition to complete antibodies, the antibodies of the present invention may also exist in various forms; including, for example, Fv, Fab, and F(ab)2, as well as in single-chain form. See, for example, International Application WO88 / 09344. Furthermore, double-chain antibodies and V-like domain-binding molecules are well known to those skilled in the art; see, for example, U.S. Patent No. 7,166,697.

[0387] In some embodiments, the antibody (e.g., DC8E8) is modified or serves as the basis for preparing a binding molecule having one or more antigen-binding properties as described for the DC8E8 antibody. These binding proteins can be prepared by one or more techniques, such as those listed and illustrated below: Chapter 6 of *Business Insights, Preclinical Development of Monoclonal Antibodies and Related Biologicals—Emerging Technologies and New Therapeutic Candidates* by James Shirvill (2010), including: Fab, TetraMAB (e.g., Galileo Oncologics); scFv; Imuna (e.g., ESBA Tech AG); [scFv]2, a binding molecule comprising any two of the four therapeutic epitopes of DC8E8; BiTE (Affitech, Micromet AG); affinity antibodies (e.g., Avipep Pty); TandAb (e.g., Affimed Therapeutics); flexible antibodies (e.g., Affirmed); V-NAR (e.g., AdAlta); nanobodies (Ablynx NV); domain antibodies (e.g., Diversys). Ltd.GSK (US Patent Nos. 6,248,516 and EP0368684); hybrids (e.g., Elusys Therapeutics Inc.); monoclonal antibodies (e.g., GenMab A / S); domain-exchange antibodies (e.g., Calmune Corporation, Science. June 27, 2003; 300(5628):2065-71); small modular immunopharmaceuticals (SMIPs) and SCORPION molecules (e.g., Trubion Pharmaceuticals); dual variable domain immunoglobulin DVD-Ig (Abbott Laboratories), etc.

[0388] Conventional techniques known in the art can be used to further modify the antibodies of the present invention or their corresponding immunoglobulin chains, such as by using amino acid deletions, insertions, substitutions, additions, and / or recombinations and / or any other modifications known in the art, alone or in combination. See, for example, the examples provided below. Methods for introducing said modifications into a DNA sequence latent beneath the amino acid sequence of the immunoglobulin chain are known to those skilled in the art. See, for example, Sambrook (above) and Ausubel (above). Modification of the antibodies of the present invention includes chemical and / or enzymatic derivatization at one or more constitutive amino acids, including side-chain modifications, backbone modifications, and N-terminal and C-terminal modifications, including acetylation, hydroxylation, methylation, amidation, and the attachment or removal of carbohydrate or lipid moieties, cofactors, etc. Similarly, the present invention covers chimeric proteins that produce said antibodies or fragments thereof containing a heterologous molecule (such as an immunostimulatory ligand) fused at the amino terminus to the carboxyl terminus. For corresponding technical details, see, for example, International Application WO00 / 30680, which is incorporated herein by reference in its entirety.

[0389] In one embodiment, the present invention relates to a method for generating an antibody or a binding fragment thereof or an immunoglobulin chain, the method comprising:

[0390] (a) Culturing cells as described above; and

[0391] (b) Isolate the antibody or its binding fragment or immunoglobulin chain from the culture.

[0392] In some embodiments, separation includes contacting an antibody-containing sample with a peptide provided by the present invention to which the antibody binds.

[0393] The transformed host can be grown in a fermenter and cultured according to techniques known in the art to achieve optimal cell growth. Once expressed, the complete antibody of the present invention, its dimer, individual light and heavy chains, or other immunoglobulin forms can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column chromatography, column chromatography, gel electrophoresis, etc.; see Scopes, “Protein Purification”, Springer Verlag, NY (1982). The antibody or its corresponding immunoglobulin chain can then be separated from the growth medium, cell lysate, or cell membrane fraction. The separation and purification of, for example, the recombinant expressed antibody or immunoglobulin chain provided by the present invention can be carried out by any conventional means, such as preparative chromatographic separation and immunoassay, as those involving monoclonal or polyclonal antibodies using constant regions of the antibody of the present invention.

[0394] Generally pure immunoglobulins with at least about 90% to 95% homogeneity are preferred, and homogeneity of 98% to 99% or higher is most preferred for pharmaceutical use. Once partially purified or purified to the desired homogeneity, the antibody can then be used for therapeutic (including in vitro) purposes or for developing and conducting assays.

[0395] This invention also provides antibodies conjugated to other parts for purposes such as drug targeting and imaging applications. The conjugation can be performed chemically at the linker site after antibody expression, or the conjugation product can be engineered into the antibody of this invention at the DNA level. The DNA is then expressed in a suitable host system, and the expressed protein is collected and reverted if necessary.

[0396] This invention also relates to a method for generating cells capable of expressing the antibodies of the present invention or their corresponding immunoglobulin chains, said method comprising genetically engineering the cells with the polynucleotides or vectors of the present invention. Cells obtained by the method of the present invention can, for example, be used to test the interaction between the antibodies of the present invention and their antigens.

[0397] This invention also provides antibody-producing cell lines and recombinant cells as sources of antibodies provided by this invention. This invention further relates to diagnostic assays and kits comprising antibodies or equivalent binding molecules provided by this invention, and to treatment methods based thereon.

[0398] This invention also provides a method for generating antibodies that can compete with DC8E8 and also inhibit pathological tau protein-tau protein interactions. These antibodies can be screened by their ability to sufficiently compete with DC8E8 for binding to tau proteins and to bind one, two, three, or all four of the “therapeutic epitopes” identified herein.

[0399] This invention also relates to polynucleotides encoding one or more antibody-based pharmaceutical agents provided by this invention. In some cases, the nucleotides encode, for example, at least a binding domain or variable region of the immunoglobulin chain of the aforementioned antibody. Typically, the variable region encoded by the polynucleotide comprises at least one complementarity-determining region (CDR) of the VH and / or VL of the variable region of the antibody. Those skilled in the art will understand that each variable domain (heavy chain VH and light chain VL) of an antibody comprises three hypervariable regions flanked by four relatively conserved frame regions or “FRs,” sometimes referred to as complementarity-determining regions or “CDRs,” and referring to the amino acid residues of the antibody responsible for antigen binding. According to the Kabat numbering system, the hypervariable region or CDR of the human IgG subtype of the antibody contains amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and amino acid residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health. As described in Health, Bethesda, Md. (1991); and / or residues from the hypervariable ring, namely residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain, as described by Chothia et al., J. Mol. Biol. 196 (1987), 901-917. In the IMGT unique numbering system, conserved amino acids always have the same position, such as cysteine ​​23 (CYS 1), tryptophan 41 (conserved TRP), hydrophobic amino acid 89, cysteine ​​104 (CYS 2), phenylalanine or tryptophan 118 (J-PHE or J-TRP). See, for example, Lefranc M.-P., Immunology Today 18, 509 (1997); Lefranc M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P., Pommié, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L, Thouvenin-Contet, V., and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003).The unique IMGT numbering provides normalized limits for the frame regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104, and FR4-IMGT: 118 to 128) and complementary determinant regions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65, and CDR3-IMGT: 105 to 117. The unique IMGT numbering is used in 2D graphical representations designated as IMGT Colliers de Perles. See, for example, Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002); Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007). It is also used to represent 3D structures. See, for example, IMGT / 3Dstructure-DB Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004). Framework or FR residues are those variable domain residues that, in addition to the hypervariable region, support it.

[0400] Therefore, the present invention also relates to an isolated nucleic acid characterized by being selected from the following nucleic acids (including any degenerate genetic code):

[0401] a) Nucleic acid, DNA, or RNA encoding the antibody of this invention;

[0402] b) Nucleic acids that are complementary to the nucleic acids defined in a);

[0403] c) A nucleic acid having at least 18 nucleotides capable of hybridizing under highly stringent conditions with at least one CDR selected from SEQ ID NO. 117-122 and SEQ ID NO. 247, 253, 255, 257-259, 122, 261, 262, 264, 265-267 and 269; and

[0404] d) A sequence having at least 18 nucleotides that is capable of at least 80%, for example 85%, 90%, 95% and 98%, identity with the light chain having nucleic acid sequence SEQ ID No. 165 and / or the heavy chain having nucleic acid sequence SEQ ID No. 170 under highly stringent conditions, or after optimal alignment with sequence SEQ ID No. 165 and / or SEQ ID 170, for example, a nucleic acid that hybridizes with at least one CDR from SEQ ID No. 165 and / or SEQ ID 170 according to the IMGT number.

[0405] Nucleic acid sequences exhibiting at least 80%, such as 85%, 90%, 95%, and 98%, of identity with the preferred sequence after optimal alignment imply certain modifications to the reference nucleic acid sequence, such as specific deletions, truncations, extensions, chimeric fusions, and / or substitutions, particularly punctual modifications. In some embodiments, these sequences are sequences encoding the same amino acid sequence as the reference sequence, which is related to the degeneracy of the genetic code or may be complementary sequences that specifically hybridize to the reference sequence under highly stringent conditions (particularly those defined below).

[0406] Hybridization under highly stringent conditions means selecting conditions related to temperature and ionic strength in a manner that allows hybridization between two complementary DNA fragments to be maintained. For illustrative purposes only, the highly stringent conditions for the hybridization step, for the purpose of defining the aforementioned polynucleotide fragments, are advantageously as follows.

[0407] DNA-DNA or DNA-RNA hybridization is performed in two steps: (1) pre-hybridization for 3 hours at 42°C in phosphate buffer (20 mM, pH 7.5) containing 5X SSC (1X SSC corresponds to a solution of 0.15 M NaCl + 0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10X Denhardt's solution, 5% dextran sulfate, and 1% salmon sperm DNA; (2) primary hybridization for 20 hours at a temperature depending on the probe length (i.e., 42°C for probes >100 nucleotides), followed by two 20-minute washes at 20°C in 2X SSC + 2% SDS and one 20-minute wash at 20°C in 0.1X SSC + 0.1% SDS. For probes >100 nucleotides, a final 30-minute wash at 60°C in 0.1X SSC + 0.1% SDS is performed. The highly stringent hybridization conditions described above for determining the size of polynucleotides can be adapted by those skilled in the art for longer or shorter oligonucleotides according to the procedures described in Sambrook et al. (Molecular cloning: a laboratory manual, ColdSpring Harbor Laboratory; 3rd edition, 2001).

[0408] The affinity or cohesion of an antibody to an antigen can be determined experimentally using any suitable method; see, for example, Pope ME, Soste MV, Eyford BA, Anderson NL, Pearson TW. (2009) J Immunol Methods. 341(1-2):86-96 and the methods described herein. The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH). Therefore, the affinity and other antigen binding parameters (e.g., K+) are standardized using standardized solutions of the antibody and antigen and standardized buffers. D Measurements were performed using IC50.

[0409] This invention also specifies that the variable domains of antibodies having the aforementioned variable domains can be used to construct other peptides or antibodies having desired specificity and biological function. Therefore, this invention also covers peptides and antibodies comprising at least one CDR containing the aforementioned variable domains and advantageously having binding properties substantially the same or similar to those of the antibodies described in the appended examples. Those skilled in the art will understand that antibodies can be constructed using the variable domains or CDRs described herein, according to methods known in the art, such as those described in European patent applications EP 0 451 216 A1 and EP 0 549 581 A1. Furthermore, those skilled in the art will understand that binding affinity can be enhanced by amino acid substitutions within the CDR or within a hypervariable ring partially overlapping with a CDR as defined by Kabat (Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917). Therefore, this invention also relates to antibodies in which one or more of the mentioned CDRs comprise one or more, for example, no more than two amino acid substitutions. In some embodiments, the antibodies of this invention contain, for example, at least one CDR within one or two of their immunoglobulin chains. Figure 3 Two or all three CDRs of the variable region described in B and 3E. In some embodiments, the antibody of the present invention contains, as in one or two of its immunoglobulin chains, the following... Figure 25B The two or all three CDRs mentioned in the document.

[0410] The polynucleotide or nucleic acid encoding the aforementioned antibody may be, for example, DNA, cDNA, RNA, or a synthetically produced DNA or RNA or a recombinant chimeric nucleic acid molecule containing any of those polynucleotides (alone or in combination). In some embodiments, the polynucleotide is part of a vector. The vector may contain other genes, such as marker genes that allow selection of the vector in a suitable host cell and under suitable conditions.

[0411] In some embodiments, the polynucleotide is operatively linked to one or more expression control sequences, thereby allowing expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide involves transcription into translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, such as mammalian cells, are known to those skilled in the art. These typically include regulatory sequences ensuring transcription initiation and optionally polyA signals ensuring transcription termination and transcript stabilization. Other regulatory elements may include transcriptional enhancers as well as translational enhancers and / or naturally associated or heterologous promoter regions.

[0412] In this regard, those skilled in the art will understand that polynucleotides encoding at least a variable domain of the light chain and / or heavy chain may encode a variable domain of two or only one immunoglobulin chain. Similarly, the polynucleotide may be controlled by the same promoter or may be separately controlled for expression. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, the PL, lac, trp, or tac promoters in *E. coli*, and examples of regulatory elements that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoters in yeast or the CMV promoter, SV40 promoter, RSV promoter, CMV enhancer, SV40 enhancer, or globulin intron in mammalian and other animal cells.

[0413] In addition to the elements responsible for initiating transcription, the regulatory elements may also include transcription termination signals downstream of the polynucleotide, such as SV40 polyA sites or tk polyA sites. Furthermore, depending on the expression system used, a leader sequence capable of guiding the polypeptide into a cellular compartment or causing it to be secreted into a culture medium may be added to the coding sequence of the polynucleotide and is known in the art. The leader sequence is assembled in appropriate phase with translation, initiation, and termination sequences, and optionally with a leader sequence capable of guiding the translation of a protein or a portion thereof to be secreted into the periplasmic space or extracellular culture medium. In some embodiments, the heterologous sequence may encode a fusion protein comprising a C-terminal or N-terminal identification peptide that imparts desired characteristics, such as stabilizing the expressed recombinant product or simplifying purification. In this case, suitable expression vectors are known in the art and include, without limitation, the Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), and pSPORT1 (GIBCO BRL).

[0414] In some implementations, the expression control sequence may be a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells, but a prokaryotic host control sequence may also be used. Once the vector has been incorporated into a suitable host, i.e., the host is maintained under conditions suitable for high-level expression of the nucleotide sequence, and, if necessary, the immunoglobulin light chain, heavy chain, light chain / heavy chain dimer, or intact antibody, binding fragment, or other immunoglobulin forms can be subsequently collected and purified. See, for example, Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY, (1979).

[0415] Furthermore, the present invention provides polynucleotides containing variable domains of immunoglobulin chains encoding antibodies of the present invention, conventionally used in genetic engineering; and optionally, vectors, particularly plasmids, granules, viruses, and bacteriophages, containing variable domains of other immunoglobulin chains encoding antibodies of the present invention. In some embodiments, the vectors are expression vectors and / or gene transfer or targeting vectors. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpesvirus, or bovine papillomavirus can be used to deliver the polynucleotides or vectors of the present invention to a target cell population. Any method known to those skilled in the art can be used to construct recombinant viral vectors. See, for example, the techniques described in Sambrook (above) and Ausubel (above). Alternatively, the polynucleotides and vectors provided by the present invention can be reconstructed in liposomes for delivery to target cells. Vectors containing polynucleotides provided by the present invention (e.g., variable domains of heavy and / or light chains encoding immunoglobulin chains and expression control sequences) can be transferred into host cells by known methods that vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts.

[0416] Furthermore, this invention relates to host cells transformed with the polynucleotides or vectors provided by this invention. The host cells can be prokaryotic or eukaryotic cells. The polynucleotides or vectors present in the host cells can be integrated into the host cell's genome or can be maintained extrachromosomally. The host cells can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plant, animal, or human cells. Preferred fungal cells are those of the genus *Saccharomyces*, particularly those of the species *Saccharomyces cerevisiae*. Depending on the host used in the recombinant production process, the antibody or immunoglobulin chain encoded by the polynucleotides of this invention can be glycosylated or can be non-glycosylated. Some antibodies or corresponding immunoglobulin chains provided by this invention may also include initial methionine amino acid residues. The polynucleotides of this invention can be used to transform or transfect hosts using any technique generally known to those skilled in the art. Furthermore, methods for preparing fusion-operatedly linked genes and expressing them in, for example, mammalian cells and bacteria are well known in the art. See, for example, Sambrook. The genetic constructs and methods described herein can be used to express antibodies or their corresponding immunoglobulin chains provided by the present invention in eukaryotic or prokaryotic hosts. Generally, expression vectors containing promoter sequences that facilitate efficient transcription of the inserted polynucleotides are used in conjunction with a host. Expression vectors typically contain an origin of replication, a promoter, and a terminator, as well as specific genes that provide phenotypic selection for transformed cells. Suitable source cells for immunoglobulin expression and secretion, along with host cells, are available from numerous sources, such as the American Collection of Type Cultures (“Catalogue of Cell Lines and Hybridomas”, 5th edition (1985) Manassas, VA, USA, and other available editions, which are incorporated herein by reference). Furthermore, transgenic animals (e.g., mammals) containing cells of the present invention can be used for large-scale production of the antibodies of the present invention.

[0417] Furthermore, this invention covers small peptides, including those containing the binding molecules described above, such as those containing the CDR3 region of the variable region of any of the mentioned antibodies, particularly the CDR3 of the heavy chain, as it has often been observed that for some antibodies, the heavy chain CDR3 (HCDR3) is a region with greater variability and primarily involved in antigen-antibody interactions. The peptides can be synthesized or generated by recombinant means to produce binding agents suitable according to the invention. Such methods are known to those skilled in the art. Peptides can be synthesized, for example, using commercially available automated peptide synthesizers. Peptides can also be generated using recombinant techniques by incorporating the DNA expressing the peptide into an expression vector and transforming cells with said expression vector to produce the peptide.

[0418] The aforementioned fusion protein may further include a protease-cleavable linker or cleavage site, which may be referred to as a spacer portion. These spacer portions may be insoluble or soluble (Diener et al., Science 231 (1986), 148) and may be selected to allow the drug to be released from the antibody at the target site. Examples of therapeutic agents that can be conjugated to the antibodies of the present invention for immunotherapy are drugs, radioisotopes, lectins, and toxins. Drugs that can be conjugated to the antibodies and antigens of the present invention include compounds classically referred to as drugs, such as mitomycin C, daunorubicin, and vinblastine. When using the antibodies or antigens of the present invention conjugated with radioisotopes for, for example, immunotherapy, certain isotopes may be preferred over others, depending on factors such as leukocyte distribution and isotype stability and emission. Depending on the autoimmune response, some emitters may be superior to others. Generally, α- and β-particle-emitting radioisotopes are preferred in immunotherapy. In certain preferred cases, the radioactive isotope is a short-range, high-energy alpha emitter, such as... 212 Bi. Examples of radioisotopes that can be incorporated into the antibodies or antigens of the present invention for therapeutic purposes are: 125 I, 131 I, 90 Y、 67 Cu、 212 Bi、 212 At、 211 Pb, 47 Sc、 109 Pd and 188 Re. In some cases, radioactive marking is 64 Cu. Other therapeutic agents that can be coupled to the antibodies or antigens of the present invention, as well as in vitro and in vivo therapeutic regimens, are known or can be determined by those skilled in the art. Where appropriate, those skilled in the art may use the polynucleotides of the present invention encoding any of the above-described antibodies, antigens, or corresponding carriers (and as sources thereof) rather than their own proteinaceous substances.

[0419] This invention also relates to the preparation of compositions, such as those provided herein, of binding molecules or antibodies for the in vivo inhibition of the formation of disordered and / or misordered tau proteins in subjects or for the additional reduction of the levels of disordered and / or misordered tau proteins in subjects; or for the use in the in vitro extraction of pathological tau protein compounds or precursors thereof from autologous fluids. These methods can be used to improve cognition or to slow or reverse disease-related cognitive decline. Antibodies or binding molecules or their chemical derivatives provided by this invention can be administered directly to blood or CSF and chelated in subsequent steps by affinity capture from the blood or CSF, whereby misordered and disordered tau proteins are chelated together with the aforementioned binding molecules. Therefore, this invention also relates to a method for treating or preventing the onset or progression of Alzheimer's disease or related tau protein disorders in a subject, the method comprising removing blood or CSF from the subject's body, testing the blood and CSF, and returning the blood and CSF to the subject, respectively, thereby obtaining the treatment or prevention.

[0420] Molecules and particles containing antibodies, peptides, or binding molecules / proteins of the present invention also have diagnostic utility. The present invention provides antibodies that identify and distinguish different forms of tau protein present at different stages of Alzheimer's disease. These antibodies are capable of detecting tau protein (and its various conformational changes) both in vitro and in vivo. The antibodies can distinguish physiological tau protein from pathological tau protein in a variety of assays, including biochemical, immunoprecipitation, ELISA, Western blotting, and immunohistochemical assays (e.g., fresh, fixed, frozen, paraffin-embedded), and in vivo imaging using, for example, radiolabeled DC8E8 (including fragments of DC8E8, such as single-chain DC8E8) that would distinguish physiological from pathological tau protein (see Examples). They can do so in solid and fluid (e.g., blood, plasma, CSF, tissue homogenate), animal (e.g., rodent, human) samples, and biopsies. Some of these assays are described in the following examples. Other conventional methods for detecting proteins are known to those skilled in the art and are therefore conventionally adaptable to the antibodies, peptides, and tau protein binding molecules provided by the present invention. The antibodies of this invention can be labeled (e.g., fluorescent, radioactive, enzyme-based, NMR, heavy metal-labeled) and used for in vivo or in vitro detection of specific targets, including in vitro immunochemical assays (see, for example, the examples below). Furthermore, in vivo, they can be used in a manner similar to nuclear medicine imaging techniques to detect tissues, cells, or other materials with disordered tau proteins and their deposits. Targeting intracellular and extracellular disordered tau proteins and neurofibrillary lesions with diagnostic imaging probes detectable by MRI or PET will provide a more definitive biomarker for pre-death diagnosis of AD and a means for monitoring the efficacy of therapies targeting tau proteins. Therefore, this invention provides the use of the antibodies described herein in the preparation of compositions for tau protein detection and / or targeting diagnostic agents to pathological tau proteins and neurofibrillary lesions of the brain to achieve the diagnosis of AD, and the use of the antibodies described herein in methods for tau protein detection and / or targeting diagnostic agents to pathological tau proteins and neurofibrillary lesions of the brain to achieve the diagnosis of AD. These compositions and methods can be used as part of treatment regimens for AD and related tau protein lesions.

[0421] This invention provides antibodies suitable for use in immunoassays, in which the antibodies can be utilized in a liquid phase or bound to a solid-phase carrier. Examples of immunoassays utilizing the antibodies of this invention are competitive and non-competitive immunoassays, either directly or indirectly. Examples of such immunoassays are radioimmunoassays (RIA), sandwich immunoassays (immunometric assays), flow cytometry, and Western blotting. The antibodies of this invention can be bound to one of many different carriers and used to isolate cells that are specifically bound to them. Examples of known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose, and magnetite. For the purposes of this invention, the carrier can be soluble or insoluble. Many different labeling and labeling methods exist that are known to those skilled in the art.

[0422] Examples of various types of labels that can be used in this invention include enzymes, radioisotopes and radionuclides, colloidal metals, fluorescent compounds, chemiluminescent compounds, biotinylated groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pairs, binding sites of secondary antibodies, metal-binding domains, epitope tags), and chemiluminescent / electrochemical / bioluminescent compounds. Enzymes include peroxidases (e.g., HRP), luciferases, alkaline phosphatases, α-D-galactosidase, glucose oxidase, glucosylamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose-6-phosphate dehydrogenase. Alternatively, the label may be biotin, digoxigenin, or 5-bromodeoxyuridine. Fluorescent labels can also be combined with antibodies and tau protein-binding proteins provided by the present invention, the labels including rhodamine, lanthanide phosphors, fluorescein and its derivatives, fluorescent dyes, rhodamine and its derivatives, green fluorescent protein (GFP), red fluorescent protein (RFP) and other fluorescent proteins, dansyl, and umbelliferone. In the conjugates, the antibodies / binding proteins of the present invention can be prepared by methods known to those skilled in the art. They can then be conjugated directly; via spacer groups or linking groups, such as polyaldehyde, glutaraldehyde, ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DPTA); or in the presence of other conjugates such as those conventionally known in the art, to enzymes or fluorescent labels. Conjugates carrying fluorescein labels can be prepared, for example, by reacting with isothiocyanates. In some cases, the label or labeling material can also be therapeutic.

[0423] Other conjugates may include chemiluminescent labels, such as luminol and dioxane; bioluminescent labels, such as luciferase and luciferin; or radioactive labels, such as iodine. 123 ,iodine 125 ,iodine 126 ,iodine133,131 ,bromine 77 ,technetium 99m ,indium 111 ,indium 113m ,gallium 67 ,gallium 68 ,ruthenium 95 ,ruthenium 97 ,ruthenium 103 ,ruthenium 105 ,mercury 107 ,mercury 203 ,rhenium 99m ,rhenium 101 ,rhenium 105 ,scandium 47 ,tellurium 121m ,tellurium 122m ,tellurium 125m ,thulium 165 ,thulium 167 ,thulium 168 ,fluorine 18 ,yttrium 199 and iodine 131 Existing methods for labeling antibodies with radioisotopes directly or via chelating agents such as EDTA or DTPA, as known to those skilled in the art, can be used in the same manner as for diagnosing radioisotopes. See, for example, the use of [I] via chloramine-T technology. 125 Na label [Hunter WM and Greenwood F.C. (1962) Nature 194:495]; technetium as described by Crockford et al. (US Patent 4,424,200) 99m Marking; and as described by Hnatowich (US Patent 4,479,930) via DTPA.

[0424] This invention also provides antibodies and other tau protein-binding molecules that can be used in methods for diagnosing diseases in individuals. These methods involve obtaining a bodily fluid sample from the individual, which may be a blood sample, lymph sample, or any other bodily fluid sample, and contacting the bodily fluid sample with the antibody of this invention under conditions that allow for the formation of an antibody-antigen complex. The presence and / or amount of the complex is then determined using methods known in the art; a level significantly higher than that formed in a control sample indicates the presence of disease in the tested individual. Therefore, this invention relates to an in vitro immunoassay comprising the antibody of this invention.

[0425] Furthermore, this invention relates to in vivo imaging techniques employing any tau protein-binding molecule of the invention. For example, positron emission tomography (PET), a medical imaging technique that produces three-dimensional images of body parts, is based on the detection of radiation from positron emissions. Typically, biomolecules are radiolabeled, for example, they incorporate radioactive tracer isotopes. After the labeled biomolecule is administered to the subject, usually by injection into the bloodstream, the radiolabeled biomolecule becomes enriched in the target tissue. The subject is then placed in an imaging scanner that detects positron emissions. In one embodiment, the labeled (e.g., tau protein-binding molecule) is administered to the subject... 64 Cu-labeled binding molecules (such as antibodies) are detected by placing the subject in an imaging scanner and detecting positron emission to the subject, thereby detecting the binding molecules and thus misordered or out-of-order tau proteins, whereby the detection of emission indicates a neurological disorder. Therefore, the present invention covers a method for PET imaging comprising administering the present invention to a subject. 64 The steps of binding molecules labeled with Cu or equivalents.

[0426] This invention also provides articles of manufacture such as pharmaceutical and diagnostic packaging or kits, said articles comprising one or more containers filled with one or more of the components described above (i.e., binding molecules, antibodies or their binding fragments, polynucleotides, carriers, or cells) as provided by this invention. Accompanying said containers may be a tabular report issued by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, reflecting approval for human administration by the agency manufacturing, using, or selling the product. Additionally or alternatively, the kit includes reagents and / or instructions for use in appropriate diagnostic assays. The compositions or kits of this invention are suitable for the diagnosis, prevention, and treatment of Alzheimer's disease and related tau protein lesions.

[0427] The bioactivity of the binding molecules, such as antibodies, provided by this invention indicates that they possess sufficient affinity to be candidates for achieving drug localization / delivery to cells or tissues. Targeting and binding to mislabeled, disordered tau protein deposits can be suitable for delivering therapeutic or diagnostic active agents and gene therapies / gene delivery. Therefore, this invention provides the use of the antibodies described herein in the preparation of compositions for detecting and / or targeting therapeutic or diagnostic agents to pathological tau proteins and neurofibrillary lesions of the brain, and the use of the antibodies described herein in methods for detecting and / or targeting therapeutic or diagnostic agents to pathological tau proteins and neurofibrillary lesions of the brain. These compositions and methods can be used as part of treatment regimens for AD and related tau protein lesions.

[0428] Therefore, the present invention relates to compositions comprising one or more of the compounds mentioned above, including binding molecules, antibodies, binding fragments; their chemical derivatives; polynucleotides, carriers, and cells. Certain compositions may further comprise one or more pharmaceutically acceptable carriers and one or more pharmaceutically acceptable diluents. Certain chemical derivatives comprise chemical portions that are not normally part of the underlying molecules or cells (e.g., antibodies, binding molecules, polynucleotides, carriers, and cells) but are linked to them by conventional methods. These portions may, for example, improve the solubility, half-life, visualization, detectability, and / or absorption of the underlying molecules or cells. Alternatively, the portions may reduce unwanted side effects or decrease the toxicity of the underlying molecules.

[0429] This invention also provides pharmaceutical compositions, depending on their intended use, comprising the antibodies provided herein in combination with other agents, such as those with interleukins or interferons. For example, for the treatment of Alzheimer's disease, additional agents may be selected from groups consisting of small organic molecules, anti-tau protein antibodies, anti-β-amyloid antibodies, and combinations thereof. Other agents include, but are not limited to, acetylcholinesterase inhibitors, NMDA receptor antagonists, transition metal chelators, growth factors, hormones, nonsteroidal anti-inflammatory drugs (NSAIDs), antioxidants, lipid-lowering agents, selective phosphodiesterase inhibitors, tau protein aggregation inhibitors, protein kinase inhibitors, heat shock protein inhibitors, anti-amyloid passive and active immunizing agents, anti-amyloid aggregation inhibitors, and secretase inhibitors. Therefore, in one embodiment, the present invention relates to the use of the binding molecules, antibodies, or binding fragments of the present invention, or binding molecules having substantially the same binding specificity as any of them, the polynucleotides, carriers, or cells of the present invention for the preparation of pharmaceutical or diagnostic compositions for treating Alzheimer's disease or related tau protein lesions or preventing their progression; improving symptoms associated with Alzheimer's disease or related tau protein lesions; diagnosing or screening subjects for the presence of Alzheimer's disease or related tau protein lesions to determine the risk of subjects exhibiting Alzheimer's disease or related tau protein lesions.

[0430] Peptides for diagnostics, active immunization, and AD therapy

[0431] This invention is based in part on the discovery that certain fragments of tau protein, when injected into a rat model of Alzheimer's disease (AD), are active in inducing an immune response against pathological tau proteins, and are expected to do so in humans. These immunogenic tau protein fragments, comprising one or more tau protein regions identified by DC8E8 as promoters or at least participants in the manifestation and progression of AD, are found to (i) promote the clearance of extracellular tau protein deposits in the AD brain (rat model); (ii) induce the production of protective antibodies against AD in animal models; and / or (iii) slow the progression of AD in recipient subjects, as measured by one or more biochemical and neurological assays performed in animal models. They may also directly and physically interfere with the ability of tau protein to form pathological tau protein-tau protein interactions along these regions.

[0432] This invention provides immunogens or immunogenic peptides derived from newly identified tau protein regions that are important for the formation of the PHF core and promote PHF assembly in vitro. Strategically targeting these regions (“therapeutic epitopes”) can lead to successful treatment of AD and related tau protein lesions. Therapeutic immunogens can be screened in animal models such as the transgenic rat model described below.

[0433] In one embodiment of the invention, the tau peptide comprises, for example, one of the following amino acid sequences, each containing one of four therapeutic epitopes: a) SEQ ID NO:98 tau protein 267-KHQPGGG-273, b) SEQ ID NO:99 tau protein 298-KHVPGGG-304, c) SEQ ID NO:100 tau protein 329-HHKPGGG-335, and d) SEQ ID NO:101 tau protein 361-THVPGGG-367 (numbered according to the longest human tau protein subtype tau protein 2N4R with a length of 441 residues, see SEQ ID NO:102). In another embodiment, the tau peptide comprises at least one therapeutic epitope, wherein the therapeutic epitope is selected from SEQ ID NO:223 tau protein 268-HQPGGG-273, SEQ ID NO:154 tau protein 299-HVPGGG-304, SEQ ID NO:224 tau protein 330-HKPGGG-335 and SEQ ID NO:154 tau protein 362-HVPGGG-367.

[0434] This invention provides immunogens of 30 amino acids in length, as shown in Table 1, for any of the SEQ ID NOs. Each immunogen included in Table 1 is an isolated fragment of tau protein containing a therapeutic epitope located within SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, and SEQ ID NO:101.

[0435] Table 1: Tau protein 30-meric peptides, each carrying a therapeutic epitope

[0436]

[0437]

[0438]

[0439]

[0440] In some embodiments, the immunogenic peptide is selected from SEQ ID NO:1 tau protein 251-PDLKNVKSKIGSTENLKHQPGGGKVQIINK-280; SEQ ID NO:2 tau protein 256-VKSKIGSTENLKHQPGGGKVQIINKKLDLS-285; SEQ ID NO:3 tau protein 259-KIGSTENLKHQPGGGKVQIINK KLDLSNVQ-288; and SEQ ID NO:4 tau protein 275-VQIINKKLDLSNVQSKCGSKDNIKHVPGGG-304.

[0441] The present invention also specifies that the shorter and longer immunogenic peptides containing one or more of the amino acid sequences SEQ ID NO:98267-KHQPGGG-273, SEQ ID NO:99298-KHVPGGG-304, SEQ ID NO:100329-HHKPGGG-335, or SEQ ID NO:101361-THVPGGG-367 used in the present invention may be derived from any of the six human tau protein subtypes. In one embodiment, the immunogenic peptide comprises at least one therapeutic epitope, wherein the therapeutic epitope is selected from SEQ ID NO:223 tau protein 268-HQPGGG-273, SEQ ID NO:154 tau protein 299-HVPGGG-304, SEQ ID NO:224 tau protein 330-HKPGGG-335, and SEQ ID NO:154 tau protein 362-HVPGGG-367. In one embodiment, the immunogenic peptide comprises a sequence selected from SEQ ID NO:109 Tau protein 314-DLSKVTSKCGSLGNIHHKPGGGQVEVKSE-342; SEQ ID NO:110 Tau protein 352-SKIGSLDNITHVPGGGNKKIETHKLTFREN-380; SEQ ID NO:111 Tau protein 325-LGNIHHKPGGGQ-336; SEQ ID NO:112 Tau protein 357-LDNITHVPGGGN-368; SEQ ID NO:108 Tau protein 294-305KDNIKHVPGGGS.In some embodiments, at least one immunogenic peptide is selected from any of the following: SEQ ID NO:1-4, SEQ ID NO:9-101 and SEQ ID NO:108-112, NIKAVPGGGS (SEQ ID NO:200), NIKHVPGGGS (SEQ ID NO:201), IKHVPGGGS (SEQ ID NO:202), KHVPGGGSV (SEQ ID NO:203), HVPGGGSVQ (SEQ ID NO:204), VPGGGSVQ (SEQ ID NO:205), GWSIHSPGGGSC (SEQ ID NO:250) and SVFQHLPGGGSC (SEQ ID NO:251), ANIKHVPGGGS (SEQ ID NO:144), DAIKHVPGGGS (SEQ ID NO:146), DNAKHVPGGGS (SEQ ID NO:149), DNIAHVPGGGS (SEQ ID NO:148), and DNIAHVPGGGS (SEQ ID NO:149). NO:151), DNIKAVPGGGS (SEQ ID NO:159), DNIKHAPGGGS (SEQ ID NO:161) and DNIKHVPGGGS (SEQ ID NO:171).

[0442] The amino acid sequence corresponding to the human tau protein subtype is given in SEQ ID NO:102-107.

[0443] SEQ ID NO:102(2N4R):

[0444]

[0445] SEQ ID NO:103(1N4R):

[0446]

[0447] SEQ ID NO:104(2N3R):

[0448]

[0449] SEQ ID NO:105(0N4R):

[0450]

[0451] SEQ ID NO:106(1N3R):

[0452]

[0453] SEQ ID NO:107(0N3R):

[0454]

[0455] Peptide-based vaccines are attractive for inducing an immune response in diseases for which no conventional vaccines are available (Brown, 1994; Ben Yedicia et al., 1997). However, in many cases, small peptides are undesirable immunogens because they act as haptens lacking the necessary Th cell epitopes and / or captured inefficiently by antigen-presenting cells (APCs). In one embodiment of the invention, the immunogenic epitope may be a protective epitope comprising tau peptides or a longer polypeptide analogue along with other amino acids.

[0456] Some of the agents described herein for inducing immune responses contain epitopes suitable for inducing an immune response against pathological tau proteins and tau protein deposits, but are too small to be immunogenic. In this case, the peptide immunogen can be linked to a suitable carrier to aid in initiating an immune response. In some embodiments, suitable carriers include serum albumin, keyhole hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or toxoids or attenuated toxin derivatives from other pathogenic bacteria such as diphtheria, Escherichia coli, cholera, or Helicobacter pylori (H. pylori). Other carriers for stimulating or enhancing immune responses include cytokines such as IL-1, IL-1α and β peptides, IL-2, γINF, IL-10, GM-CSF; and chemokines such as M1P1α and M1P1β, and RANTES. Immunogenic agents can also be linked to peptides that enhance transtissue transport, as described in O'Mahony, WO 97 / 17613 and WO 97 / 17614.

[0457] Immunogenic agents can be chemically crosslinked to a carrier. Techniques for linking immunogens to carriers include the use of 3-(2-pyridyl-thio)propionic acid N-succinimide ester (SPDP) and 4-(N-cis-butenylimide-methyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) to form disulfide bonds (which can be provided by adding cysteine ​​residues if the peptide lacks a thiohydrogen group). These agents form a disulfide bond between themselves and a cysteine ​​residue of a peptide on a protein and an amide bond through the ε-amino group on lysine or other free amino groups in other amino acids. Several of these disulfide / amide forming agents are described in Immun. Rev. 62, 185 (1982). Other bifunctional coupling agents form thioethers instead of disulfide bonds. Many of these thioether forming agents are commercially available and include reactive esters of 6-cis-butenyliminohexanoic acid, 2-bromoacetic acid and 2-iodoacetic acid, and 4-(N-cis-butenylimino-methyl)cyclohexane-1-carboxylic acid. The carboxyl groups can be activated by combining them with succinimide or sodium salt of 1-hydroxy-2-nitro-4-sulfonate.

[0458] Immunogenic peptides can also be expressed as fusion proteins along with a vector. The immunogenic peptide can be attached to the vector at the N-terminus, C-terminus, or any site within the peptide (internal). In some embodiments, multiple repeating sequences of the immunogenic peptide may be present in the fusion protein.

[0459] For example, immunogens comprising fusion proteins containing a tau peptide carrying a protective B-cell epitope linked to a non-natural Pan DR Th cellular epitope, the epitope inducing a B-cell response against the protective epitope. In another alternative, the invention provides immunogens that can be engineered as polymers (Jackson et al., 1997), multi-antigen peptide systems (MAPs) (Tam and Recent, 1996), immunostimulatory complexes (ISCOMs) (Barr, IG, and Mitchell, 1996), and possibly other branched amphipathic peptides (Wilkinson et al., 1998) or chimeric peptides generated through collinearization of epitopes (Marussig et al., 1997).

[0460] In some embodiments, the therapeutic peptide may be administered alone or in combination, with or without being bound to a pharmaceutically acceptable carrier (including KLH, tetanus toxoid, albumin-binding protein, bovine serum albumin, dendritic (MAP; Biol. Chem. 358:581)) and adjuvant substances, or combinations thereof, or mixtures thereof, as described in O'Hagan et al. (2003) (particularly endogenous immune-enhancing compounds and the formulation systems described therein) and Wilson-Welderer et al. (2009) (particularly those indicated in Tables 2 and 3 of the documents).

[0461] In some embodiments, the immunogenic agents of the present invention can be chemically cross-linked or conjugated to a suitable carrier to enhance the immune response against pathological tau proteins, including tau protein deposits. In some embodiments, pharmaceutically acceptable carriers for binding or conjugation are keyhole cyanin (KLH), tetanus toxoid, bovine serum albumin (BSA), immunoglobulin (Ig) molecules, thyroglobulin, or ovoglobulin. Other carriers for stimulating the immune response include cytokines (such as IL-1, IL-2, IL-10, IFNγ, GM-CSF) and chemokines (such as M1P1α and M1P1β).

[0462] Tau peptides or analogues can be synthesized via solid-phase peptide synthesis or recombinant expression, or obtained from natural sources. Automated peptide synthesizers are commercially available from numerous vendors, such as Applied Biosystems, EZBiolab, or Antagene. Recombinant expression systems may include bacteria (such as E. coli), yeast, insect cells, or mammalian cells. Procedures for manipulating DNA and preparing DNA constructs for recombinant expression are described by Sambrook et al. (1989), and methods for producing recombinant proteins are described in detail in Current Protocols in Protein Science (Chapter 5, “Production of Recombinant Proteins”, Units 5.1–5.24, DOI: 10.1002 / 0471140864, ​​also available online at onlinelibrary.wiley.com / book / 10.1002 / 0471140864 / toc).

[0463] The immunogenic agent of the present invention can be expressed by a virus or bacteria as a vector / carrier. The nucleic acid encoding the immunogenic peptide is incorporated into the genome or free organism of the virus or bacteria. Ultimately, the immunogenic peptide can be expressed as a secretory protein or a fusion protein with the outer surface protein of the virus, or it can be presented as a transmembrane protein of the bacteria. The viruses or bacteria used in the method are generally non-pathogenic or attenuated. Suitable viruses include adenovirus, HSV, Venezuelan equine encephalitis virus and other alpha viruses, vesicular stomatitis virus and other rhabdoviruses, vaccinia, and fowlpox. Suitable bacteria include Salmonella and Shigella. Alternatively, fusion of the immunogenic peptide with HBsAg of HBV is suitable.

[0464] Another aspect of the invention relates to therapeutic agents or immunogens that are analogs of various peptides (e.g., SEQ ID No: 1-4; 9-97) or fragments thereof, which may also be described in various embodiments.

[0465] This invention is also based on the discovery of novel peptides referred to herein as therapeutic epitopes. Although having a primary sequence different from that of tau protein and tau protein fragments, the invention is characterized in that the therapeutic epitopes can have a shape (e.g., inherent disordered structure, tertiary structure, conformation) that mimics one or more of the aforementioned tau protein "therapeutic epitopes." By mimicking one or more of these regions, these therapeutic epitopes can be adapted to generate antibodies against them, such as antibodies that compete with DC8E8. These peptides are capable of competitively binding to the disclosed DC8E8 antibodies against tau protein or tau protein fragments.

[0466] This also includes immunogenic, designed therapeutic epitopes that, when injected into rat models of AD, can induce an immune response to pathological tau protein and are intended to do so in humans. Furthermore, it is also disclosed that, in response to immunization with one or more designed therapeutic epitopes, mouse antibodies / antiserums capable of (i) recognizing one or more epitopes that are DC8E8 or mimicking DC8E8; (ii) distinguishing pathological tau protein from normal tau protein; and / or (iii) recognizing neurofibrillary lesions in the human AD brain and / or in transgenic rat models of AD.

[0467] The present invention also provides compositions for the prevention, treatment, and / or diagnosis of Alzheimer's disease, wherein the compositions comprise (i) a means for treating Alzheimer's disease in a subject by inhibiting tau protein-tau protein aggregation; and (2) a pharmaceutically acceptable carrier and / or diluent. The present invention also provides compositions for the prevention, treatment, and / or diagnosis of Alzheimer's disease, wherein the compositions comprise (i) a means for treating Alzheimer's disease in a subject by binding to one or more “therapeutic epitopes” in pathological tau proteins; and (2) a pharmaceutically acceptable carrier and / or diluent. The present invention also provides compositions for the prevention, treatment, and / or diagnosis of Alzheimer's disease, wherein the compositions comprise (i) a means for reducing tau protein-tau protein aggregation by binding to one or more “therapeutic epitopes” in pathological tau proteins; and (2) a pharmaceutically acceptable carrier and / or diluent.

[0468] preparation

[0469] The pharmaceutical preparations of the present invention can be administered in the form of a therapeutic agent (e.g., an antibody or peptide as described above) and one or more other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15th edition, Mack Publishing Company, Easton, Pa., 1980). These preparations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN), DNA conjugates, anhydrous adsorbent pastes, oil-in-water and water-in-oil emulsions, carbon wax (polyethylene glycol of various molecular weights) emulsions, semi-solid gels, and semi-solid mixtures containing carbon waxes. Any of the above mixtures may be suitable for treatments and therapies according to the present invention, provided that the active ingredient in the preparation is not inactivated by formulation and that the preparation is physiologically compatible with and tolerable to the route of administration. For additional information relating to formulations, excipients, and carriers known to pharmaceutical chemists, see also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” lnt. J. Pharm. 203(1-2):1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts.” J. Pharm Sci. 89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and citations therein.

[0470] A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been well described in a number of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999), HC Ansel et al., 7th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000), AHKibbe et al., 3rd edition, Amer. Pharmaceutical Assoc.

[0471] The chosen formulation depends on the intended administration method and therapeutic application. The formulation may also include pharmaceutically acceptable non-toxic carriers or diluents, defined as media commonly used to formulate pharmaceutical compositions for animal or human administration. Diluents are selected so as not to affect the bioactivity of the composition. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextran solution, and Hank's solution. Pharmaceutical compositions or formulations may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers. However, some agents suitable for animal administration, such as Complete Freund's adjuvant, are generally not included in compositions intended for human use.

[0472] Suitable carriers for pharmaceutical applications are known in the art and include phosphate-buffered saline solutions, water, emulsions (such as oil / water emulsions), various types of wetting agents, sterile solutions, etc. Compositions comprising these carriers can be formulated using known conventional methods. Further carriers are described below.

[0473] adjuvant

[0474] The therapeutic agents and immunogens of the present invention can be administered in combination with adjuvants, which are substances that do not induce an adaptive immune response themselves but amplify or modulate the response to accompanying antigens. Various adjuvants can be combined with therapeutic peptides and antibodies in the present invention to elicit an immune response. Preferably, the adjuvant amplifies the intrinsic response to the immunogen without causing conformational changes in the qualitative form of the immunogen that would affect the response.

[0475] In some embodiments, the adjuvant is an aluminum salt (alum), such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate (Hunter, 2002). The adjuvant may be used with or without other specific immunostimulatory agents, such as 3-de-O-acylated monophosphoryl lipid A (MPL) or 3-DMP, polymeric or monomeric amino acids, such as polyglutamic acid or polylysine. The adjuvant may be used with or without other specific immunostimulatory agents, such as muramyl peptides (N-acetylmurayl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normurayl-L-alanyl-D-isoglutamine (norMDP), N-acetylmurayl-L-alanyl-D-isoglutamine acyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucosamine-N-acetylmurayl-L-AI-D-isoglu-L-Ala-dipalmitoyloxypropylamide (DTP-DPP)theramide) TM Other bacterial cell wall components. Other adjuvants are oil-in-water emulsions and include (a) MF59 (belonging to Van Nest et al. WO 90 / 14837, which is incorporated herein by reference in its entirety), containing 5% squalene, 0.5% Tween 80 and 0.5% Spandex 85 (optionally containing various amounts of MTP-PE), used with a microfluidic system such as a 110Y type (Microfluidics, Newton). (a) a microfluidic system formulated into submicron particles, containing 10% squalene, 0.4% Tween 80, 5% Pronic block polymer L121, and thr-MDP, microfluidized into a submicron emulsion or vortexed to produce a larger particle size emulsion, and (b) a Ribi™ adjuvant system (RAS) (RibiInunoChem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components derived from the group consisting of monophospholipase A (MPL), trehalose dimethicone ester (TDM), and cell wall skeleton (CWS), such as MPL+CWS (Detox™). In some embodiments, the adjuvant is a saponin, such as Stimulon. TM (QS21, Aquila, Worcester, Mass.) or particles derived from them, such as ISCOM (immunostimulatory complex) and ISCANATRIX. Other adjuvants include complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA), cytokines such as interleukins (IL-1, IL-2, and IL-12), macrophage colony-stimulating factor (M-CSF), and tumor necrosis factor (TNF).

[0476] Alternatively, tau peptides and their immunogenic analogs can be coupled to adjuvants. For example, as described for hepatitis B antigen vaccination (Livingston, J. Immunol. 159, 1383-1392 (1997)), tau peptide "A" in lipopeptide form can be prepared by directly coupling palmitic acid or other lipids to the N-terminus of "A". However, such coupling should not substantially alter the conformation of tau peptide "A" to the extent that it affects the nature of the immune response to it.

[0477] Adjuvants may be administered as a single composition with the immunogen, or may be administered before, simultaneously with, or after the immunogen. The immunogen and adjuvant may be packaged and supplied in the same vial or packaged in separate vials and mixed prior to use. The immunogen and adjuvant are typically packaged with a label indicating the intended therapeutic application. If the immunogen and adjuvant are packaged separately, the package usually includes instructions for mixing prior to use. The choice of adjuvant and / or carrier depends on the stability of the immunogenic formulation containing the adjuvant, the route of administration, the timing of administration, the efficacy of the adjuvant on the inoculated species, and, in humans, a pharmaceutically acceptable adjuvant that has been or may be approved for human use by the relevant regulatory body. For example, complete Freund's adjuvant is not suitable for human use. However, alum, MPL or incomplete Freund's adjuvant, alone or in combination with any of alum, QS21 and MPL and all of their combinations (Chang et al., Advanced Drug Delivery Reviews 32:173-186 (1998), which is incorporated herein by reference in its entirety) is suitable for human administration.

[0478] Pharmaceutical compositions may also include large, slowly metabolizing macromolecules such as proteins, polysaccharides (e.g., polyglucosamine), polylactic acid, polyglycolic acid and copolymers (e.g., latex-functionalized agarose, agarose, cellulose, etc.), polymerized amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). Additionally, these carriers can act as immunostimulatory agents (i.e., adjuvants).

[0479] combination

[0480] This invention provides compositions and treatment methods combining the antibodies and peptides described herein with other therapeutic agents for Alzheimer's disease (AD) and related tau protein lesions. For example, current tau protein-related therapeutic strategies primarily focus on drugs that inhibit tau protein kinases or activate phosphatases (Iqbal and Grundke-Iqbal, 2004, 2005, 2007; Noble et al., 2005), drugs that stabilize microtubules (Zhang et al., 2005), drugs that promote the proteolytic degradation of misfolded tau proteins (Dickey et al., 2005; Dickey and Petrucelli, 2006; Dickey et al., 2006), compounds that prevent or reverse tau protein aggregation (Wischik et al., 1996; Pickhardt et al., 2005; Taniguchi et al., 2005; Necula et al., 2005; Larbig et al., 2007), or vaccine-mediated clearance of aggregated tau proteins (Asuni et al., 2007). Therefore, the present invention specifies that multiple targeting (e.g., targeting both tau protein and β-amyloid protein) can substantially increase treatment efficiency.

[0481] In cases of Alzheimer's disease and related tau protein lesions in which pathological soluble and insoluble tau proteins (tau protein deposits) are present in the brain, the agents of the present invention may also be administered together with other agents that enhance the ability of the agents of the present invention to cross the blood-brain barrier.

[0482] Application method

[0483] Agents used to induce an immune response (passive or active), to reduce tau protein levels, or for any of the preventive, therapeutic, or diagnostic (in vivo) methods described herein may be administered parenterally, locally, intradermally, intravenously, orally, subcutaneously, intraperitoneally, intranasally, or intramuscularly to achieve preventive and / or therapeutic treatment. The subcutaneous route is typical, but other routes can be equally effective. Another typical route is intramuscular injection. This type of injection is most commonly performed in the muscles of the arm or leg. Intravenous injection, as well as intraperitoneal, intra-arterial, intracranial, or intradermal injections, are also effective in generating an immune response. In some methods, the agent is injected directly into specific tissues where deposits have accumulated.

[0484] Aerosol formulations, such as nasal sprays, comprise purified aqueous or other solutions of active pharmaceutical ingredients, preservatives, and isotropic agents. The formulation is, for example, adjusted to a pH and isotropic state compatible with the nasal mucosa. Formulations for rectal or vaginal administration may be presented as suppositories with suitable carriers.

[0485] For parenteral administration, the therapeutic peptides of the present invention can be administered in the form of injectable solutions or suspensions, wherein the solutions or suspensions are solutions or suspensions of the substance in a physiologically acceptable diluent having a pharmaceutical carrier, which may be a sterile liquid (such as water, oil, physiological saline, glycerol, or ethanol). Additionally, excipients such as wetting agents or emulsifiers, surfactants, pH buffers, etc., may be present in the composition. Other components of the pharmaceutical composition are those of petroleum, animal, plant, or synthetic origin. Peanut oil, soybean oil, and mineral oil are all examples of suitable substances. Generally, diols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injectable solutions. The pharmaceutical agents of the present invention can also be administered in the form of reservoir injections or implantable formulations formulated in a manner that allows for sustained release of the active ingredient. An exemplary composition comprises a 5 mg / mL monoclonal antibody prepared in an aqueous buffer consisting of 50 mM histidine and 150 mM NaCl, adjusted to pH 6.0 with HCl.

[0486] Preparations intended for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol / water solutions, emulsions, or suspensions, including physiological saline and buffer media. Parenteral media include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or non-volatile oils. Intravenous media include fluids and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases.

[0487] Typically, the compositions are prepared as injectable solutions or suspensions; they may also be prepared as solid forms suitable for dissolving or suspending in a liquid medium prior to injection. As discussed above, formulations may also be emulsified or encapsulated in liposomes or microparticles (such as polylactide, polyglycolic acid lactide, or copolymers) to achieve adjuvant enhancement (see Langer, Science 249, 1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997)). The pharmaceutical preparations of the present invention can be administered in reservoir-type injections or implantable formulations that allow for sustained or pulsatile release of the active ingredient.

[0488] Other formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal dressings.

[0489] For suppositories, the binder and carrier include, for example, polyalkylene glycols or triglycerides; the suppositories may be formed from a mixture containing an active ingredient in the range of 0.5% to 10%, for example, 1% to 2%. Oral formulations include excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders and contain 10% to 95%, for example, 25% to 70% of the active ingredient.

[0490] Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administering the agent with cholera toxin or its detoxified derivatives or subunits or other similar bacterial toxins (see Glenn et al., Nature 391, 851 (1998)). Co-administration can be achieved by using components in the form of mixtures or as linker molecules obtained through chemical cross-linking or expression as fusion proteins.

[0491] Alternatively, percutaneous delivery can be achieved using skin patches or transfer devices (Paul et al., Eur. J. Immunol. 25, 3521-24 (1995); Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998)). Subcutaneous administration of the subject antibody, peptide, or compound is achieved using standard methods and devices, such as needles and syringes, subcutaneous injection port delivery systems, etc. Intramuscular administration is achieved using standard devices, such as needles and syringes, continuous delivery systems, etc. In some embodiments, the subject antibody, peptide, and / or compound is delivered via a continuous delivery system. The term “continuous delivery system” is used interchangeably herein with “controlled delivery system” and encompasses continuous (e.g., controlled) delivery medical devices (e.g., pumps) combined with catheters, injection devices, etc. (a wide variety of which is known in the art). Mechanical or motorized infusion pumps may also be adapted for use with the present invention. Examples of the apparatus include, for example, those described in U.S. Patent Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966, etc. Generally, the drug delivery method of the present invention can be achieved using any of a variety of refillable pump systems. The pump provides consistent, controlled release over time.

[0492] The medication can also be administered via the standard procedure currently used, which involves drilling a small hole in the skull to administer the drug. In a preferred aspect, intravenous or oral administration of the binding molecules, particularly the antibodies or antibody-based drugs of the present invention, is permitted to cross the blood-brain barrier.

[0493] In pharmaceutical dosage forms, the pharmaceutical agent (antibodies, peptides, and compounds provided by this invention) may be administered in its pharmaceutically acceptable salt form, or may be used alone or in appropriate combination with other pharmaceutically active compounds. The methods and excipients described herein are exemplary only and are in no way limiting. The subject antibody, peptide, or compound may be formulated into an injectable preparation by dissolving, suspending, or emulsifying it in an aqueous or non-aqueous solvent, such as vegetable oil or other similar oil, synthetic aliphatic acid glycerides, esters of higher fatty acids, or propylene glycol; and, if necessary, with conventional additives, such as solubilizers, isotonists, suspending agents, emulsifiers, stabilizers, and preservatives. Unit dosage forms for injection or intravenous administration may contain the active pharmaceutical agent in the form of a composition, said composition being a solution in sterile water, physiological saline, or another pharmaceutically acceptable carrier. As used herein, the terms "unit dosage form" or "dosage" refer to a physical individual unit suitable as a single dose for use in human and animal subjects, each unit containing a predetermined amount of the subject antibody and a pharmaceutically acceptable diluent, carrier, or mediator, said predetermined amount being calculated in an amount sufficient to produce the desired effect. The specifications of the unit dosage forms of the present invention depend on the specific compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0494] An immune response to pathological tau protein and tau protein deposits can also be induced by administering a nucleic acid encoding a therapeutic tau peptide. This nucleic acid can be DNA or RNA. The nucleic acid segment encoding the immunogen is linked to regulatory elements, such as promoters and enhancers, that allow the DNA segment to be expressed in predetermined target cells of the patient. Typically, promoter and enhancer elements from immunoglobulin genes (light or heavy chains) or CMV major-early promoters and enhancers are suitable for directing expression in blood cells, which are suitable targets for inducing an immune response. The linked regulatory elements and coding sequences are often cloned into a vector.

[0495] Many viral vector systems are available, including retroviral systems (see, for example, Lawrie et al., Cur. Opin. Genet. Develop. 3:102-109 (1993), which is incorporated herein by reference in its entirety); adenoviral vectors (Bett et al., J. Virol. 67:591 1 (1993), which is incorporated herein by reference in its entirety); adeno-associated virus vectors (Zhou et al., J. Exp. Med. 179:1867 (1994), which is incorporated herein by reference in its entirety); viral vectors from the pox family, including vaccinia virus and fowlpox virus; and viral vectors from the alphavirus genus, such as those derived from Sindbis and Semliki Forest Virus (Dubensky et al., J. Virol. Develop. 3:102-109 (1993)). 70:508-519 (1996), which is incorporated herein by reference in its entirety; Venezuelan equine encephalitis virus (see U.S. Patent No. 5,643,576, belonging to Johnston et al., which is incorporated herein by reference in its entirety) and rhabdoviruses, such as vesicular stomatitis virus (see WO 96 / 34625, belonging to Rose, which is incorporated herein by reference in its entirety) and papillomaviruses (Ohe et al., Human Gene Therapy 6:325-333 (1995); WO 94 / 12629, belonging to Woo et al.; and Xiao and Brandsma, Nucleic Acids. Res. 24:2630-2622 (1996), which is incorporated herein by reference in its entirety).

[0496] DNA encoding an immunogen, or a vector containing it, can be packaged into liposomes. Suitable lipids and related analogues are described in U.S. Patent Nos. 5,208,036, 5,264,618, 5,279,833, and 5,283,185. Vectors and DNA encoding immunogens can also be adsorbed onto or associated with particulate carriers, examples of which include polymethyl methacrylate polymers and polylactide and poly(lactide-co-glycolic acid), see, for example, McGee et al., J. Micro Encap. (1996).

[0497] Gene therapy vectors or naked DNA can be delivered in vivo by administration to individual patients, typically via systemic administration (e.g., intravenous, intraperitoneal, nasal, gastric, intradermal, intramuscular, subdermal, or intracranial infusion) or local application (see, for example, U.S. Patent No. 5,399,346). DNA can also be administered using a gene gun (see U.S. Patent No. 6,436,709). In this application, DNA encoding an immunogen is deposited on the surface of microscopic metal beads. The particles are accelerated using a shock wave or expanding helium gas and penetrate tissue to a depth of several cell layers (reviewed in Haynes et al., 1996). For example, Accel, manufactured by Agacetus, Inc. (Middleton, WI),... TM Gene delivery devices or the Helios gene gun manufactured by Bio-Rad Laboratories, Inc. (Hercules, CA) are suitable. For therapeutic purposes, DNA can also be delivered via electroporation (e.g., as described in Trollet et al., 2008 and its references). Alternatively, exposed DNA can be introduced into the bloodstream through the skin by spotting it onto the skin using chemical or mechanical stimulation (see WO 95 / 05853) or tattooing (e.g., as described by van den Berg et al., 2009).

[0498] In one variation, the DNA or vector encoding the immunogen can be delivered ex vivo to cells (such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirate, tissue biopsy) or pluripotent donor hematopoietic stem cells), and then, for example, after verifying immunogen expression and usually after selecting cells that already have the vector, the cells are re-implanted into the patient.

[0499] Another promising but potentially risky approach for human treatment has been to transfect dendritic cells (DCs, via direct DNA delivery or using viral strategies) to generate their own antigens (Xing et al., 2005), thereby providing a continuous supply of complete antigens presented via MHC I.

[0500] Subjects who can withstand treatment

[0501] Subjects eligible for treatment include individuals at risk of Alzheimer's disease or related tau protein lesions who are asymptomatic, as well as patients who have developed symptoms. In the case of Alzheimer's disease, virtually anyone is at risk of developing Alzheimer's disease if they live long enough. Therefore, the treatment or therapy of the present invention can be administered preventively to the general population even without any assessment of the subject patient's risk. The vaccine provided in this patent is particularly suitable for individuals with a known genetic risk of Alzheimer's disease. Such individuals include those with relatives who have the disease and those identified as at risk due to the presence of genetic or biochemical markers. Genetic markers of risk for early-onset familial Alzheimer's disease include mutations in the APP gene, the presenilin genes PS1 and PS2, and markers for late-onset Alzheimer's disease in the ApoE4 gene (recently reviewed by Bertram and Tanzi, 2008). Other risk factors include a family history of AD, hypercholesterolemia, or atherosclerosis. Individuals currently diagnosed with Alzheimer's disease can be identified based on characteristic dementia and the presence of the aforementioned risk factors. In addition, many diagnostic tests can be used to identify individuals with AD. These tests include measuring the levels of total tau protein, phosphate tau protein, and amyloid β (1-42) in the CSF. Elevated levels of tau protein and / or phosphate tau protein, and decreased levels of amyloid β (1-42) indicate the presence of AD. Individuals with Alzheimer's disease can also be diagnosed using the MMSE, ADRDA, or other criteria.

[0502] In asymptomatic patients, treatment can begin at any age (e.g., 10, 20, 30 years). However, treatment may not be necessary until the patient reaches 40, 50, 60, or 70 years of age. Treatment may require multiple doses administered over a period of time. Treatment can be monitored by measuring the activation of antibodies, or T-cell or B-cell responses to the therapeutic agent (e.g., tau peptide), over time. If the response declines, it indicates the need for a booster dose. In patients with underlying Down syndrome at high risk of AD or related tau protein lesions, treatment can be initiated prenatally or shortly after birth by administering the therapeutic agent to the mother.

[0503] In some implementations, DC8E8 (or its chimeric, humanized, human, or other derivatives / parts / fragments) is an antibody or passive vaccine intended for use in elderly immunosenile Alzheimer's disease (AD) patients exhibiting significantly reduced levels of the co-stimulatory molecule CD28 on T cells. Reduced levels of the co-stimulatory molecule CD28 indicate an impaired immune response (Saurwein-Teissl et al., 2002). Often, CD45RA is also involved. + CD8 + CD28 -T cell clone (immunophytic type: CD8) + CD28 - CD45RA + It produces large amounts of the pro-inflammatory cytokine IFN-γ and small amounts of IL-5. These clones accumulate during normal aging and induce an imbalance in the production of Th1 and Th2 cytokines. Therefore, the accumulating CD8... + CD28 - CD45RA + T cell clones, along with the declining population of natural B cells (Siegrist and Aspinall, 2009), are major contributors to the decline in immune function affecting about one-third of the older population (Weng et al., 2009; Saurwein-Teissl et al., 2002).

[0504] Therefore, passive immunotherapy (e.g., using DC8E8 (or its chimeric, humanized, human or other derivatives / parts / fragments)) provides a means of bypassing the immune system decline in a large number of AD patients and targeting the pathological tau protein that causes neurofibrillary degeneration.

[0505] In some implementations, a tau protein therapeutic epitope (or a peptide comprising a tau protein therapeutic epitope as described herein) is used as an active vaccine intended for use in elderly, immune-active Alzheimer's disease patients. The immune-active patient's immunophenotype is CD8. + CD28 + CD45RA + Therefore, CD8 + The level of the co-stimulatory molecule CD28 on T cells will be measured and used as a selection marker for patients who are actively vaccinated.

[0506] In addition, prior to treatment, antibodies against *Borrelia*, *Treponema*, *Chlamydia*, herpesvirus, and other brain pathogens will be tested in CSF and blood samples from patients to exclude individuals with chronic infectious and inflammatory CNS conditions that can mimic or worsen AD symptoms (Balin et al., 2008; Itzhaki and Wozniak, 2008; Miklossy, 2008; Andreasen, 2010). CNS infections often impair the function of the blood-brain barrier (BBB), especially *Chlamydia* infection of brain endothelial cells, which can lead to increased influx of monocytes into the brain parenchyma and thus affect local immune responses (Balin et al., 2008). Older subjects with higher levels of cytomegalovirus (CMV) IgG have also been shown to experience a faster rate of cognitive decline (Itzhaki and Wozniak, 2008). Therefore, to prevent adverse effects following immunization with a drug provided by the present invention (e.g., uncontrolled immune response to normal tau protein), Alzheimer's disease patients with CNS infection or those who test positive for antibodies against the aforementioned pathogens will be treated with highly selective vaccines.

[0507] Prior to treatment, antibodies against *Treponema pallidum*, *Treponema hygroscopicum*, *Chlamydia*, herpesviruses, and other brain pathogens can be tested in the patient's CSF and blood to exclude individuals with chronic infectious and inflammatory CNS conditions that may mimic or worsen AD symptoms (Balin et al., 2008; Itzhaki and Wozniak, 2008; Miklossy, 2008; Andreasen, 2010). To prevent potential adverse effects from various chronic infections, this group of patients will be treated with more selective antibodies or vaccines containing therapeutic epitopes. In some cases, active vaccines are designed epitopes that induce the production of highly selective antibodies targeting therapeutic epitopes on pathological tau proteins (see examples, for example). In some embodiments, the vaccine does not contain any amino acid sequences common to normal / physiological tau proteins.

[0508] Treatment plan

[0509] In preventative applications, a pharmaceutical composition or agent is administered to a patient susceptible to a specific disease or otherwise at risk of developing the disease in an amount sufficient to eliminate or reduce the risk of disease or delay the onset of the disease. In therapeutic applications, a composition or agent is administered to a patient suspected of or already suffering from the disease in an amount sufficient to cure or at least partially suppress the symptoms of the disease and its complications. An amount sufficient to achieve this effect is defined as a therapeutic or pharmaceutically effective dose. In both preventative and therapeutic regimens, the agent is typically administered at several doses until a sufficient immune response has been achieved. Typically, the immune response is monitored, and if the immune response begins to decline, the dose is repeated.

[0510] The effective dosage of the compositions of the present invention for treating the above-mentioned conditions varies depending on many factors, including the means of administration, target site, patient's physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is preventative or therapeutic. Typically, the patient is human. Treatment doses need to be titrated to optimize safety and efficacy. Therefore, treatment with antibodies or tau protein-binding proteins will generally require multiple doses administered over a period of time. For passive immunization with antibodies, the dose is in the range of about 0.0001 to 100 mg per kg of host body weight, and more typically 0.01 to 5 mg. In some applications, the amounts of antibodies or tau protein-binding proteins may be administered at doses of at least 0.1 mg, at least 0.5 mg, 1 mg, or any combination of doses between 0.1 mg and 10 mg per kg of body weight. In some methods, the antibodies or tau protein-binding proteins may be administered in multiple doses (equal or different) over a period of at least 1 month, at least 3 months, or at least 6 months. The total dose over any treatment period may be, for example, between 4 and 6, but other numbers may be used depending on the factors discussed above. Treatment may be monitored by any of the methods described below.

[0511] The amount of immunogen depends on whether an adjuvant is also administered, with higher doses required in the absence of an adjuvant. The amount of immunogen administered can vary from 1 μg to 500 μg per patient and is more commonly 5–500 μg per injection for human administration. Sometimes, higher doses of 1–2 mg are used per injection. Typically, each human injection uses approximately 10, 20, 50, or 100 μg. The timing of injections can vary significantly from once daily to once a year to once every ten years. On any given day when an immunogen dose is administered, if an adjuvant is also administered, the dose is greater than 1 μg / patient and usually greater than 10 μg / patient, and in the absence of an adjuvant, the dose is greater than 10 μg / patient and usually greater than 100 μg / patient. A typical regimen consists of immunization followed by booster injections at six weekly intervals. Another regimen consists of immunization followed by booster injections at 1, 2, and 12 months. Another regimen requires continuous injections every two months for life. Alternatively, booster injections may be administered intermittently, as indicated by monitoring of the immune response. In some implementations, the active vaccine will be formulated with a suitable carrier (KLH preferred) and aluminum hydroxide as an adjuvant. Preferably, 100 μg peptide / dose / patient (but 1 μg, 10 μg, 100 μg, and 1 mg will also be used in the preclinical phase and 10 μg, 100 μg, and 200 μg will be used in the Phase I toxicity study) will be administered at 5 doses every 4 weeks.

[0512] The dosage of nucleic acid encoding the immunogen ranges from approximately 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg of DNA per patient. The dosage of infectious viral vectors ranges from 10-10 μg per dose. 9 One or more viral changes. Treatment can be monitored by measuring the activation of antibodies, or T-cell or B-cell responses to the therapeutic agent over time. A decreased response may indicate the need for a booster dose.

[0513] Finally, the dosage regimen will be determined by the attending physician and based on clinical factors. As is known in the medical field, the dosage for any given patient depends on many factors, including the patient's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health condition, and any other medications administered concurrently. Typical doses may range, for example, from 0.001 to 1000 mg; however, doses below or above this exemplary range are contemplated, especially considering the factors mentioned above. Generally, regimens in the form of periodically administered pharmaceutical compositions should range from 1 mg to 10 mg units per day. If the regimen is a continuous infusion, then it should also be in the range of 1 mg to 10 mg per kilogram of body weight. Progress can be monitored through periodic assessments.

[0514] Furthermore, co-administration or sequential administration of other agents may be desirable. In some embodiments, the therapeutically effective dose or effective amount refers to the amount of active ingredient sufficient to improve symptoms or ailments. The therapeutic efficacy and toxicity of the compound can be determined in cell cultures or laboratory animals using standard pharmaceutical procedures, such as ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. In some embodiments, the therapeutic agent in the composition is present in an amount sufficient to restore normal behavior and / or cognitive properties in the case of Alzheimer's disease and related tau protein lesions.

[0515] This invention provides various measures that can be used to assess the effectiveness of treatment with any of the pharmaceutical agents provided by this invention. Examples include, but are not limited to, decreased levels of one or more pathological forms of tau protein (e.g., in the brain); increased clearance of pathological tau protein from the brain and / or CSF; improved cognitive abilities such as cognitive function (tested by, for example, the Clinical Dementia Rating Scale-CDR, the Alzheimer's Disease Assessment Scale-Cognitive Subscale-ADAS-Cog Mini Mental State Test-MMSE); improved motor function tests (e.g., grip strength test, time to stand and walk (TUG) test, TUG manual, talk-while-walking test, Unified Parkinson's Disease Rating Scale-UPDRS); improved performance in basic activities of daily living (ADL) tests (e.g., hygiene, dressing, self-preparation, eating, meal preparation, making phone calls, going out, finances and communication; dementia disability assessment tests); and decreased severity / rating of AD impairment in grip strength, motor and psychomotor ataxia (which are directly related to the animal models and assays described below in the examples), memory decline, aphasia, cognitive aphasia, disorientation in time and space, and depression.

[0516] For the purpose of assessing treatment effectiveness, the levels and distribution of tau protein (in the brain and in body fluids) can be determined by any of the methods described herein and / or by any other methods available for detecting tau protein. For example, the levels of tau protein can be measured in vivo (using positron emission tomography) using the novel imaging radiotracer 18F-THK523, which selectively binds to tau protein and tau lesions in vitro, ex vivo (tissue sections), and in vivo (transgenic mice) (Fodero-Tavoletti et al., 2011, Brain). Tau protein in cerebrospinal fluid and blood can be identified using ELISA kits that recognize total tau protein or phosphate-modified tau protein.

[0517] In fact, the neurobehavioral impairments in transgenic rats are similar to the motor impairments in Alzheimer's disease patients, which has implications for clinical trials and treatment protocols using any of the therapeutic agents provided in this article (including, but not limited to, agents used for active vaccination). In humans, the clinical manifestations of Alzheimer's disease are progressive memory impairment and cognitive decline, behavioral changes and psychological symptoms (disorders of mood, affect, appetite, wake-sleep cycles, agitation, and depression), and motor impairment (psychomotor ataxia, myoclonus, gait impairment, muscle weakness, extrapyramidal features such as bradykinesia, rigidity, and resting tremor) (Goldman et al., 1999; Boyle et al., 2009). Numerous studies have reported that motor signs are commonly observed in Alzheimer's disease (AD) and become more prominent as the disease progresses (Goldman et al., 1999; Wilson et al., 2003; Louis et al., 2004; Pettersson et al., 2005; Scarmeas et al., 2004; Scarmeas et al., 2005; Waite et al., 2005; Alfaro-Acha et al., 2006; Wang et al., 2006; Buchman et al., 2007a; Boyle et al., 2009). Notably, motor signs can precede cognitive impairment and predict cognitive and functional decline, hospitalization, and death in Alzheimer's disease (Morris et al., 1989; Soininen et al., 1992; Kraemer et al., 1994; Chui et al., 1994; Scarmeas et al., 2004; Scarmeas et al., 2005). It has been shown that muscle weakness precedes cognitive impairment (Buchman et al., 2007b; Boyle et al., 2009).

[0518] The manifestation of motor signs in AD has been associated with neurodegeneration and neuronal loss in the brainstem (Zarow et al., 2003; Burns et al., 2005; Grudzien et al., 2007; Simic et al., 2009; Wai et al., 2009; Braak and DelTredici, 2011). Furthermore, several studies have shown that neurofibrillary degeneration originates in the brainstem and precedes cortical neurodegeneration (Hertz, 1989; Simic et al., 2009; Braak and DelTredici, 2011).

[0519] These findings demonstrate that motor impairment represents a key marker in the pathogenesis of Alzheimer's disease (AD). Furthermore, functional impairment of some motor domains can precede dementia and predict cognitive decline. Active immunotherapy with the peptides described herein (including therapeutic epitopes) improved motor impairment in transgenic rats expressing pathological human tau protein. Therefore, direct targeting of brainstem lesions via active immunotherapy can prevent, slow, or delay motor and cognitive impairment in human AD patients. Consequently, testing of motor function can be included in a suite of tests that can be used to evaluate the clinical efficacy of the agents described herein (e.g., tau protein scavengers, active and passive vaccines).

[0520] Furthermore, those skilled in the art are aware of the well-established association between the levels and distribution of pathological tau proteins (e.g., NFTs in the cortex / hippocampus) and disease progression. The density of pathological tau proteins (NFT lesions) has been associated with cognitive deficits and the severity of Alzheimer's disease (Braak and Braak, 1991; Bierer et al., 1995; Berg et al., 1998; Duyckaerts et al., 1998; Giannakopoulos et al., 1998, 2003). Pathological tau proteins (e.g., NFTs, neurofibrillary networks) in the entorhinal cortex and hippocampus are negatively correlated with longitudinal memory changes (Reitz et al., 2009). Similarly, in the brainstem, pathological tau proteins (NFTs) are present very early in the dorsal raphe nuclei; subsequently, another raphe nucleus is affected. These lesions explain the serotonin activation defects seen in AD (Duyckaerts et al., 2009). Extrapyramidal symptoms have been associated with tau protein lesions in the substantia nigra (Liu et al., 1997). Therefore, therapeutic agents that can affect one or more of these AD distribution patterns may have a beneficial effect in AD. Example

[0521] Example 1: Preparation of recombinant human TAU protein

[0522] Human full-length tau protein (2N4R, 2N3R) and tau protein deletion mutants: recombinant tau protein produced by self-cloning T40 (Goedert, 1989) Figure 1 (6) The clones were subcloned into the expression plasmid pET-17b (Novagen) and expressed in bacteria. Each tau protein deletion mutant was verified by DNA sequencing. All tau protein deletion mutants and tau peptides were numbered according to the longest human tau protein isoform, 2N4R, which is 441 amino acids in length and is therefore also called tau. 441(D'Souza, 2005). Tau protein deletion mutants and peptides derived from subtype 2N3R are labeled with "3R" to indicate the deletion of the second microtubule-binding repeat sequence (amino acids 275-305 of 2N4R). The production of tau protein involves the following steps: a) expression of tau protein in bacteria; b) purification of tau protein by ion exchange chromatography; c) purification of tau protein by gel filtration; d) concentration and storage of the isolated tau protein; and e) immunoaffinity purification (an exception is made only for tauΔ(1-150; 392-441) / 4R used in microglial cell uptake experiments, see Example 10, Figure 17).

[0523] a) Bacterial expression of full-length human tau protein (2N4R or 2N3R) and recombinant tau protein deletion mutants: Human tau protein (or above) expression plasmids were transformed into *E. coli* producing strain BL21(DE3). Bacterial cells containing appropriate expression plasmids were cultured and induced as described in *Molecular Cloning: A Laboratory Manual* by Sambrook and Russell (2001). Single BL21(DE3) bacterial colonies transformed with pET-17b plasmids driving tau protein or fragment expression were grown at 300 rpm in 500 ml Luria broth at 37°C and induced by addition of isopropyl-β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM. After incubation at 37°C for 3 hours, bacteria were collected by centrifugation at 3,000 x g for 15 minutes at 4°C.

[0524] b) Basic and neutral tau proteins (full-length tau protein subtypes tauΔ358-441, tauΔ306-400, tauΔ421-441, tauΔ300-312, tauΔ134-168, tauΔ1-220, tauΔ1-126, tauΔ(1-150; 392-441) / 4R, tauΔ(1-150; 392-441) / 3R, and tauΔ(1-296; 392-441) / 4R) were purified by cation exchange chromatography, essentially as previously described (Krajciova et al., 2008). Following expression, the bacterial clumps were resuspended in 10 ml of dissolution buffer (50 mM 1,4-piperazine diethanesulfonic acid (PIPES) (pH 6.9), 50 mM sodium chloride (NaCl), 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 5% (v / v) glycerol), rapidly frozen in liquid nitrogen, and stored at -80°C until tau protein purification. For tau protein purification, the frozen bacterial suspension was rapidly thawed and placed on ice. The bacterial cell walls were disrupted by sonication on ice using a Sonopuls HD 2200 with a TT-13 tip (Bandelin, Germany) (set to 50% working cycles, 50 W power output, 6 cycles for 30 s, 30 s pause). The dissolved product was clarified by centrifugation (21,000 x g for 15 minutes at 4°C), and the supernatant was filtered through a 0.45 μm membrane filter. Large-scale purification of recombinant tau protein was performed at 6°C using a workstation (Amersham Biosciences, Sweden). The filtered dissolved product was loaded onto a 5 mL HiTrap SP HP column (GE Healthcare, Uppsala, Sweden) equilibrated with dissolution buffer at a flow rate of 3 mL / min and washed extensively with 60 mL of dissolution buffer until the baseline stabilized at 280 nm. The bound tau protein was eluted with a gradient (0-30% over 15 mL) of buffer B (replenished with 1 M NaCl). Individual 1 mL fractions were collected and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To remove nucleic acids co-purified with positively charged tau protein, the fractions containing tau protein were pooled and purified using a second cation exchange chromatography step on a 5 mL HiTrap SP HP column (GE Healthcare, Uppsala, Sweden) with a gentler gradient of buffer B (0-30% over 45 mL).

[0525] c) As previously described (Csokova et al. 2004), acidic tau proteins (tauΔ222-427, tauΔ228-441, tauΔ257-400, tauΔ137-441, tauΔ283-441) were purified by anion exchange chromatography. After expression, the bacterial clumps were resuspended in 10 ml of histidine dissolution buffer (20 mM histidine (pH 6.0), 50 mM NaCl, 1 mM EDTA, 5 mM DTT, 0.1 mM PMSF, and 5% (v / v) glycerol). The bacterial cell walls were disrupted by sonication on ice using a Sonopuls HD 2200 with a tip TT-13 (Bandelin, Germany) (set to 50% working cycles, 50 W power output, 6 cycles for 30 s, 30 s pause). The lysate was clarified by centrifugation (21,000 x g for 15 min at 4 °C). The bacterial lysate was precipitated with 1% streptomycin sulfate (Medexport, Russia), incubated on ice for 5 min, clarified by centrifugation (21,000 x g for 15 min at 4 °C), and filtered through a 0.45 μm membrane filter. The filtered streptomycin precipitate was loaded onto a 5 mL HiTrap QSepharose HP column (Amersham Biosciences, Sweden) at a flow rate of 3 mL / min and washed extensively with 30–50 mL of histidine lysis buffer until the A280 baseline stabilized. Tau protein was eluted using a two-step salt gradient in histidine lysis buffer (0.05–0.5 M NaCl in 40 mL, followed by 0.5–1 M NaCl in 20 mL).

[0526] d) In the final gel filtration purification step (the same for all tau proteins), the pooled tau protein fraction obtained by ion exchange chromatography is injected at 3 ml / min onto a gel filtration column (HiLoad 26 / 60 Superdex 200 preparative grade column, GE Healthcare). For basic / neutral or acidic tau proteins, the fraction is diluted in PIPES or histidine dissolution buffer supplemented with 100 mM NaCl, respectively. The pooled eluted tau protein is then eluted.

[0527] e) For the tau protein concentration after gel filtration purification, the pooled fractions were diluted with 1.5 volumes of 2.5% glycerol and reloaded onto a HiTrap SP HP column (for basic and neutral tau proteins) or a HiTrap Q HP column (for acidic tau proteins). The recombinant tau protein was then concentrated by elution from the column using a 1M NaCl step gradient. Finally, the buffer was exchanged using a 5 mL HiTrap desalting column (GE Healthcare) to argon-saturated phosphate-buffered saline (PBS, 8.09 mM disodium phosphate (Na₂HPO₄), 1.47 mM potassium dihydrogen phosphate (KH₂PO₄), 136.89 mM NaCl, 2.7 mM potassium chloride (KCl)). Protein quantification of the purified sample was performed using a quinolinic acid (BCA) quantification kit (Pierce, USA) with bovine serum albumin (BSA) as a standard. The Tau protein was divided into working aliquots, rapidly frozen in liquid nitrogen, and stored at -70°C.

[0528] f) To remove potential bacterial contaminants, recombinant tau protein was purified using the following modified method to measure tau protein uptake by microglia (Example 10, Figure 17). Following the first cation exchange chromatography step, the fraction containing tau protein was pooled and 1 / 20 volume of ice-cold 5% polyethyleneimine was added with stirring. Stirring was continued on ice for another 30 minutes. The sample was centrifuged at 20,000 x g for 15 minutes at 4°C. The supernatant was collected and injected at 3 ml / min into a HiLoad 26 / 60 Superdex 200 preparative-grade column (GE Healthcare) in PIPES dissolution buffer supplemented with 100 mM NaCl but lacking DTT or any other reducing agent. Following gel filtration, fractions containing tau protein were pooled and loaded onto an immunoaffinity column (at a flow rate of 0.5 ml / min) containing DC25 antibody (epitapht 347-353 of 2N4R tau protein, Axon Neuroscience, Vienna, Austria) immobilized on CNBr-activated agarose. The column was pre-equilibrated in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20 (TBS-Tween). After binding, the column was washed with 5 column volumes of TBS-Tween, and the bound tau protein was eluted with 0.1 M glycine (pH 2.7). The collected fractions were neutralized and pooled by adding 1 / 30 volume of 1 M Tris-HCl (pH 8.8). Finally, the buffer was exchanged for PBS (saturated with argon) using a 5 ml HiTrap desalting column (GE Healthcare). Protein quantification of purified samples was performed using a quinolinic acid (BCA) quantitative kit (Pierce, USA) with BSA as the standard. Proteins were aliquoted into working aliquots, rapidly frozen in liquid nitrogen, and stored at -70°C.

[0529] The purified DC25 antibody (Axon Neuroscience, Vienna, Austria) for use in the DC25 affinity column (above) was prepared as follows. Serum-free DC25 hybridoma culture supernatant was adjusted to pH 7.5 by adding 0.2 volumes of PBS, pre-clarified by centrifugation at 20,000 x g for 10 min at 4 °C, and filtered through a 0.2 μm filter. The pre-clarified DC25 hybridoma culture supernatant was loaded onto a PBS-equilibrated HiTrap Protein G HP column (5 ml, GE Healthcare) at 1 ml / min. After loading, the column was washed with 4 column volumes of PBS, and the bound antibody was eluted with 100 mM glycine (pH 2.7). The eluted fractions were neutralized with 1 M Tris-HCl (pH 9), pooled, and the buffer was exchanged for PBS using a HiTrap desalting column (5 ml, GE Healthcare). The purified DC25 antibody was stored in aliquots at -70°C.

[0530] Example 2: Preparation of hybridoma cell lines generating monoclonal antibodies against human TAUΔ(1-150:392-441) / 4R, screening of monoclonal antibodies by ELISA, and initial characterization of monoclonal antibody DC8E8.

[0531] Six-week-old Balb / c mice were subcutaneously sensitized with a complete Freund's adjuvant (SIGMA) containing 50 μg of recombinant tauΔ(1-150; 392-441) / 4R (prepared as described in Example 1), and were synergized five times at five-week intervals with an incomplete Freund's adjuvant containing 50 μg of the same antigen. Three days prior to fusion, mice were intravenously injected with PBS containing 50 μg of the same antigen. Spleen cells from immunized mice were fused with NS / 0 myeloma cells according to the method of Kontsekova et al. (1988). Spleen cells (10... 8 ) and 2x10 7 A mixture of 5:1 NS / 0 myeloma cells was confluent and fused for 1 minute in 1 ml of serum-free Dulbecco's modified Eagle's medium (DMEN) supplemented with 10% dimethyl sulfoxide. The fused cells were then seeded at 2.5 x 10⁶ cells per well in a 96-well plate. 5Spleen cells were then resuspended at a density of 1000 cells / ml in DMEM containing 20% ​​horse serum, L-glutamine (2 mM), hypoxanthine (0.1 mM), aminopterin (0.04 mM), thymidine (0.016 mM), and gentamicin (40 U / ml). Cells were incubated at 37°C for 10 days, and hybridomas producing specific monoclonal antibodies against tauΔ(1-150; 392-441) / 4R were selected by enzyme-linked immunosorbent assay (ELISA).

[0532] An ELISA was used to detect monoclonal antibodies against tauΔ(1-150; 392-441) / 4R (a disordered form of tau protein) in hybridoma culture supernatant. Microtiter plates were spread overnight at 37°C with PBS containing tauΔ(1-150; 392-441) / 4R (5 μg / ml, 50 μl / well). After blocking with 1% skim milk powder to reduce nonspecific binding, the plates were washed with PBS-0.05% Tween 20 and incubated at 37°C for 1 hour with 50 μl / well of hybridoma culture supernatant. The bound monoclonal antibodies were detected using sheep anti-mouse immunoglobulin (Ig) conjugated with horseradish peroxidase (HRP, DAKO). The reaction was developed using o-phenylenediamine solution as a peroxidase substrate and stopped with 50 μl of 2M H₂SO₄. Absorbance at 492 nm was measured using a Multiscan MCC / 340 ELISA reader (Labsystems). A reading at least twice the absorbance of the negative control (PBS) was considered positive. Positive hybridoma cultures were further tested by immunohistochemistry (according to the method of Zilka et al., 2003) and subcloning in soft agar according to the procedure described by Kontsekova et al. (1991).

[0533] The monoclonal antibody DC8E8 (produced from a mouse hybridoma cell line deposited at the American Center for Type Culture Collection on July 13, 2011, with ATCC patent accession number PTA-11994) was identified in the thus generated and selected positive hybridoma cultures. DC8E8 was further characterized as described below. The antibody isotype was determined to be mouse IgG1 by ELISA using a mouse Ig isotype assay kit (ISO-2, SIGMA).

[0534] Example 3: Sequencing of DC8E8 and its humanized variable region obtained through CDR transplantation

[0535] a) Determine the nucleotide and amino acid sequences of the light and heavy chain variable regions of DC8E8. Figure 3The variable region nucleotide sequence of DC8E8 was determined by DNA sequencing of cDNA synthesized from total RNA extracted from the mouse hybridoma cell line PTA-11994 (ATCC). Figure 3 (A and 3D), the cell line expresses the DC8E8 monoclonal antibody. Total RNA was extracted using reagents (Invitrogen, USA). First-strand cDNA was synthesized using the "High-Capacity cDNA Reverse Transcription" kit according to the manufacturer's protocol (Applied Biosystems, USA). The reagent composition of the 2x reverse transcription master mixture was as follows (per 20 μL reaction): 2 μL 10x RT buffer; 0.8 μL 25x dNTP mixture (100 mM); 2 μL 10x RT random primers (50 μM); 1 μL MultiScribe... TM Reverse transcriptase (50 U / μL); 4.2 μL nuclease-free H2O. For reverse transcription, mix 10 μL of 2x reverse transcription master mixture with the RNA sample (2 μg / 10 μL) and synthesize cDNA under the following conditions: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min, and finally cool to 4 °C. (The last sentence appears to be incomplete and possibly refers to a different process.) High-fidelity DNA polymerases (Finnzymes, Finland) were used to perform polymerase chain reaction (PCR) to amplify genes encoding variable regions of the light and heavy chains. Forward primers (8E8L-positive 5'-ACATTGTGATGTCACAGTCTCCATCCTCC-3' (SEQ ID NO:132) and 8E8H-positive 5'-CTCCTCCAATTGCAGCAGTCTGG-3' (SEQ ID NO:133) were designed based on the N-terminal protein sequences of the DC8E8 light chain (DIVMSQSPSS) (SEQ ID NO:134) and the heavy chain (QVQLQQSGPE) (SEQ ID NO:135). The N-terminal protein sequences were determined using Edman degradation (light chain) and MALDI in-source attenuation (heavy chain). Using this information, proteins most similar to the light and heavy chains were identified in Genebank based on their corresponding nucleotide sequences. Next, the most probable nucleotide sequences of the mouse V genes (light and heavy) were identified in the IMGT / LIGM-DB database (www.imgt.org). These genes were used to design forward primers (corrected using the N-terminal protein sequence of DC8E8). The reverse primers for the light and heavy chains (κ-antisense 5'-GGAATTCGTTGAAGCTCTTGACAATGGGTG-3' (SEQ ID NO:136) and G1-antisense 5'-GGAATTCACATATGCAAGGCTTACAACCAC-3 (SEQ ID NO:137)) are derived from the constant regions of the κ and IgG1 chains, respectively.

[0536] The DNA sequences obtained from sequencing the PCR product and the variable regions of the light and heavy chains of DC8E8 are shown in [the diagram / image / etc.]. Figure 3 In A and 3D. DC8E8 is most closely associated with the mouse germline light chain IGKV8-21. * 01 and heavy chain IGHV1-81 * The comparison of 0 and 1 is shown in Figure 3 In C and 3F. The complementarity-determining regions (CDRs) are underlined in the DC8E8 light and heavy chain protein sequences (respectively). Figure 3 B and 3E). CDR and framework region (FR) are identified according to the immunogenetic (IMGT) numbering system (see, for example, Lefranc MP. The IMGT unique numbering for immunoglobulins, T-cell receptors, and Ig-like domains. The Immunologist 7, 132-136, 1999 (1999)).

[0537] b) Humanization of DC8E8. To identify candidate immunoglobulins suitable for generating humanized DC8E8 by transplantation of the complementarity-determining region (CDR) of mouse DC8E8, a set of selected human immunoglobulin genes extracted from IMGT / LIGM-DB flat file version 201112-6 (www.imgt.org) were analyzed using ClustalX2 to perform paired alignment of DC8E8 nucleotide sequences to identify human germline genes with the highest sequence identity to DC8E8. IgKv4-1*01 was identified as the closest human germline gene to the DC8E8 light chain. Figure 4 ), and IgHVI-69*10 was identified as the closest germline gene of the DC8E8 heavy chain ( Figure 5 The following methods (Method 1 and Method 2) are designed and can be used to prepare one or more humanized forms of DC8E8 antibodies. After expression in a suitable antibody expression system (e.g., a mammalian expression vector for antibody expression in vitro (e.g., HEK293 cells) or in vivo (transgenic animals)), the activity (e.g., biochemical and therapeutic activity) of the resulting humanized recombinant antibody can be tested according to any method used to characterize the activity of DC8E8.

[0538] Method 1: CDR transplantation and, if necessary, mutations in the frame region (FR) (CDRs are indicated in bold and underlined, FR mutations are indicated in bold):

[0539] Heavy chain variable regions (SEQ ID NO 138-140 in order of appearance):

[0540]

[0541] Light chain variable regions (SEQ ID NO 141-143 in order of appearance):

[0542]

[0543] Method 2: Obtain mice with the highest sequence identity to DC8E8 ( Figure 3 ) and people ( Figure 4 and 5 Reproductive immunoglobulins were sequenced and compared with the DC8E8 protein sequence. The CDR region was identified following the IMGT numbering system. Based on the study by MacCallum et al., J. Mol. Biol. 1996, the most likely antigen-contact residues within the DC8E8 binding site were identified.

[0544] The following combination criteria were used to identify various amino acid mutation candidates in the humanized form of DC8E8:

[0545] i. They exist in CDRs and may come into contact with antigens.

[0546] ii. They exist in the cursor area.

[0547] iii. Whether they mutate in the mouse germline.

[0548] Two levels of identification for mutation candidates based on the above criteria:

[0549] (in bold):

[0550] -Different residues (non-similar amino acids) between DC8E8 and the closest mouse germline.

[0551] - Residues in the CDR that are in contact with the antigen. In the following DC8E8 sequence, the CDR is indicated in lowercase bold italics.

[0552] (use Bold and underlined express):

[0553] - Residues (non-similar amino acids) that are identical between DC8E8 and the closest mouse germline, but different in the closest human germline and located in the vernier region.

[0554] -Different residues (similar / conserved amino acids) between DC8E8 and the closest mouse germline.

[0555] Two humanized sequences in each strand were identified as having mutations predicted to affect the activity of DC8E8:

[0556] SEQ ID No. 147, 152: Only X-type residues will mutate.

[0557] SEQ ID No. 148, 153: Type residues and Both types of residues will mutate.

[0558] Heavy chain variable regions (SEQ ID NO 138, 145, 139 in order of appearance):

[0559]

[0560] Light chain variable regions (SEQ ID NO 141, 150, 142 in order of appearance):

[0561]

[0562] Example 4: Localization of the DC8E8 epitope using recombinant TAU ​​protein deletion mutant and TAU protein-derived peptide.

[0563] Human tau protein 2N4R deletion mutants and tau protein-derived peptides (Antagene, Inc. (Sunnyvale, CA) and EZBiolab, (USA)) were used for epitope localization of DC8E8 using ELISA (Figures 6, 7, and 8). Recombinant human tau protein isotypes (2N4R; 2N3R) and tau protein deletion mutants were prepared as described in Example 1. Figure 6A , 6B Peptides with a purity higher than 85% were synthesized by EZBiolabs (USA). Figure 7A , 7B ).

[0564] Spread microtiter plates overnight at 37°C with recombinant tau protein or tau peptide (5 μg / ml in PBS, 50 μl / well). After blocking with 1% skim milk powder to reduce nonspecific binding, wash the plates with PBS-0.05% Tween 20 and incubate at 37°C for 1 hour with 50 μl / well of DC8E8 hybridoma culture supernatant. Detection of bound monoclonal antibodies was performed using HRP-conjugated sheep anti-mouse Ig (DAKO). The reaction was developed using o-phenylenediamine solution as a peroxidase substrate and terminated with 50 μl of 2M H2SO4. Absorbance was measured at 492 nm using a Multiscan MCC / 340 ELISA reader (Labsystems). A reading at least twice the absorbance of the negative control (PBS) was considered positive.

[0565] DC8E8 recognizes the following human tau proteins: Δ358-441, Δ421-441, Δ134-168, Δ1-220, Δ1-126, Δ(1-296; 392-441) / 4R, and Δ(1-150; 392-441) / 4R, but fails to recognize tau proteins with deletions of Δ222-427, Δ306-400, Δ228-441, Δ300-312, Δ257-400, Δ137-441, and Δ283-441. Figure 6B , 6C DC8E8 recognizes physiological tau protein subtypes 2N4R and 2N3R to a lesser extent than it recognizes pathological / misordered tau Δ(1-296; 392-441) / 4R, tau Δ(1-150; 392-441) / 4R, and tau protein 2N4R tau protein deletion mutants (Δ358-441, Δ421-441, Δ134-168, Δ1-220, Δ1-126). Figure 6C More detailed epitope localization using tau peptides revealed that DC8E8 does not recognize tau peptides 240-270, 270-300, and 301-330. Figure 7A ,7B In summary, these findings indicate that DC8E8 possesses four binding sites or epitopes on human tau protein, each located within a microtubule-binding repeat domain of the tau protein, and each epitope is located within one of the following tau protein sequences: 267-KHQPGGG-273 (SEQ ID NO: 98) (the first repeat domain of tau protein), 298-KHVPGGG-304 (SEQ ID NO: 99) (the second repeat domain of tau protein), 329-HHKPGGG-335 (SEQ ID NO: 100) (the third repeat domain of tau protein), and 361-THVPGGG-367 (SEQ ID NO: 101) (the fourth repeat domain of tau protein). Figure 7D Furthermore, because DC8E8 binds to truncated forms of tau protein more readily than to full-length triplet and quadruplet tau proteins, these results also indicate that DC8E8 binds to disease-related forms of tau protein more readily than to physiological tau proteins (tau39(2N3R) and tau40(2N4R)). Moreover, since tau proteins are thought to conformate from physiological tau protein (intrinsically disordered) to disease tau protein (erroneous disorder and misordered sequence, Kovacech et al., 2010), these results suggest that one or more binding sites of DC8E8 (DC8E8 epitopes) have a different conformation in physiological tau protein than in disease tau protein, and that DC8E8 can detect that conformational change.

[0566] Because these tau protein repeat domains are conserved across species ( Figure 8A Therefore, DC8E8 can react with tau proteins from various species such as rats, mice, cows, chimpanzees, frogs, and others. Tau proteins from various animal species were compared using the software ClustalW2 (available, for example, at www.ebi.ac.uk / Tools / msa / clustalw2 / ). Human tau protein is represented by the longest tau protein isoform expressed in human brain neurons (2N4R, 441 amino acids). Tau proteins from other species were selected from public databases. Sequences within each of the four epitopes recognized by the DC8E8 antibody were framed.

[0567] Further point mutations and deletions were performed on certain tau protein-derived peptides (octamers, nonamers, and decanters) to further identify the DC8E8 epitope, as assessed by the ability of each peptide to competitively bind DC8E8 with tauΔ(1-150; 392-441 / 4R). Peptides with a purity higher than 85% were synthesized by EZBiolabs (USA). Competitive ELISA was performed according to the following standard protocol. ELISA plates (IWAKI high-binding plates, #3801-096, Bertoni GmbH, Austria) were plated overnight at 4°C with 100 μl / well of PBS containing 5 μg / ml of recombinant purified tauΔ(1-150; 392-441 / 4R). IWAKI high-binding plates were washed four times with PBS / Tween 20 (0.05% v / v) and blocked with PBS / Tween 20 for 2 hours at 25°C. Each peptide was dissolved separately in PBS at a final concentration of 5 mM. Serial dilutions (2-fold) of the peptides in PBS / Tween 20 were prepared in a polypropylene plate (Greiner, #651201) with conical bottoms (concentration ranges of 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, and 2.5 μM). 100 μl of each dilution was added to each well. The purified DC8E8 monoclonal antibody (purified as described below in Example 5) was diluted to 2 μg / mL in PBS / Tween 20, and 100 μl of this diluted antibody was mixed with each of the serial dilutions of the peptides to produce a 200 μl mixture containing 100 ng of antibody in each 100 μl of the corresponding test peptide at concentrations of 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM. The antibody / peptide mixture was incubated for 1 hour at 25°C on a rotating platform set to 250 rpm. 100 μL (100 μL) of antibody / peptide mixture was transferred from a polypropylene plate to an IWAKI high-binding plate coated with tauΔ (1-150; 392-441 / 4R) and blocked with PBS / Tween 20, and incubated at 25°C on a rotating platform set to 250 rpm for 1 hour. The plate was washed four times with PBS / Tween 20. The sample (in the plate) was then incubated with 100 μL of polyclonal goat anti-mouse immunoglobulin / HRP (Dako, #P0447) diluted 1:4,000 in PBS / Tween 20 on a rotating platform (set to 250 rpm) at 25°C for 1 hour. The plate was washed four times with PBS / Tween. Next, the sample / plate was incubated for 10 minutes at 25°C in the dark with 100 μl of o-PDA (o-phenylenediamine, SIGMA, P1526) in a 1.5 mg / 2 ml solution of 0.1 M sodium acetate (pH 6.0) (Roth, #6779) supplemented with 1.5 μl / 2 ml of 30% H2O2 (SIGMA, H-0904).The reaction was terminated by adding 100 μl of 2M H2SO4 (Merck, 1.00731.1000). The extent of the reaction was tracked by reading the absorbance of the sample / plate at 490 nm (e.g., using a Victor Multilabel Counter (Wallac)).

[0568] Figure 8B The results of a competitive ELISA using the following six peptides are shown: NIKAVPGGGS (SEQ ID NO:200), NIKHVPGGGS (SEQ ID NO:201), IKHVPGGGS (SEQ ID NO:202), KHVPGGGSV (SEQ ID NO:203), HVPGGGSVQ (SEQ ID NO:204), and VPGGGSVQ (SEQ ID NO:205). The peptide KHVPGGGSV (SEQ ID NO:203), which covers the therapeutic epitope of tau protein 2, and... H VPGGGSVQ (SEQ ID NO:204) competes with at least one original therapeutic epitope present on tauΔ (1-150; 392-441 / 4R). Removal of the underlined histidine from the epitope of SEQ ID NO:204 results in loss of competitive activity (see peptide VPGGGSVQ, SEQ ID NO:205). A point mutation changing histidine to alanine (at position 299 of the corresponding tau protein in "epitope 2") results in loss of competitive activity (peptide NIK). A VPGGGS (SEQ ID NO: 200). Peptides containing 2 or 3 amino acids before "histidine 299" (towards the N-terminus) also compete with the original epitope (peptides IKHVPGGGS (SEQ ID NO: 202) and NIKHVPGGGS (SEQ ID NO: 201), respectively). These results indicate that the minimal epitope of DC8E8 belonging to the second tau protein repeat sequence (epitope 2) is within the hexamer sequence, namely HVPGGG (SEQ ID NO: 154).

[0569] The aforementioned localization experiments demonstrated the presence of the amino acid sequence PGGG at one or more epitopes of the DC8E8 antibody. Furthermore, this amino acid sequence is present at all four epitopes on the tau protein that are bound by DC8E8 (see SEQ ID NO: 98, 99, 100, 101). To determine the residues in the N-terminal region of the DC8E8 epitope, an alanine scan was performed on the tau peptide 295-DNIKHVPGGGS-305, which contains the DC8E8 epitope belonging to the second repeat domain of the tau protein (within 298-KHVPGGG-304, SEQ ID NO: 99).

[0570] The binding affinity of mutant peptides to DC8E8 was assessed by competing with tauΔ (1-150; 392-441 / 4R) for each peptide. Seven peptides with a purity higher than 85% were synthesized by EZBiolabs (USA): ANIKHVPGGGS (SEQ ID NO: 144), DAIKHVPGGGS (SEQ ID NO: 146), DNAKHVPGGGS (SEQ ID NO: 149), DNIAHVPGGGS (SEQ ID NO: 151), DNIKAVPGGGS (SEQ ID NO: 159), DNIKHAPGGGS (SEQ ID NO: 161), and the peptide DNIKHVPGGGS (SEQ ID NO: 171) with the original sequence. A competitive ELISA was performed according to the following standard protocol. ELISA plates (IWAKI high-binding plates, #3801-096, Bertoni GmbH, Austria) were plated overnight at 4°C with 100 μl / well of PBS containing 5 μg / ml recombinant purified tauΔ (1-150; 392-441 / 4R). The IWAKI high-binding plates were washed four times with PBS / Tween 20 (0.05% v / v) and blocked with PBS / Tween 20 for 2 hours at 25°C. Each peptide was dissolved separately in PBS at a final concentration of 5 mM. Serial dilutions (2-fold) of the peptides in PBS / Tween 20 were prepared in polypropylene plates with conical bottoms (Greiner, #651201) at 100 μl / well. 100 μl of each dilution was added to each well. The purified DC8E8 monoclonal antibody (purified as described below in Example 5) was diluted to a concentration of 2 μg / ml in PBS / Tween 20, and 100 μl of this diluted antibody was mixed with each of the sequential dilutions of the peptide to produce a 200 μl mixture, wherein each 100 μl of the corresponding test peptide contained 100 ng of antibody at concentrations of 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM. The antibody / peptide mixture was incubated at 25°C on a rotating platform set to 250 rpm for 1 hour. 100 μl (100 μl) of the antibody / peptide mixture was transferred from a polypropylene plate to an IWAKI high-binding plate coated with tauΔ (1-150; 392-441 / 4R) and blocked by PBS / Tween 20, and incubated at 25°C on a rotating platform set to 250 rpm for 1 hour. Wash the plate four times with PBS / Tween 20.The sample (in the plate) was incubated for 1 hour at 25°C on a rotating platform (set to 250 rpm) with 100 μl of polyclonal goat anti-mouse immunoglobulin / HRP (Dako, #P0447) diluted 1:4,000 in PBS / Tween 20. The plate was washed four times with PBS / Tween. Then, the sample / plate was incubated for 10 minutes at 25°C in the dark with 100 μl of o-PDA (o-phenylenediamine, SIGMA, P1526) in a 1.5 mg / 2 ml solution of 0.1 M sodium acetate (pH 6.0) (Roth, #6779) supplemented with 1.5 μl / 2 ml of 30% H2O2 (SIGMA, H-0904). The reaction was terminated by adding 100 μl of 2 M H2SO4 (Merck, 1.00731.1000). The degree of reaction is tracked by reading the absorbance of the sample / plate at 490 nm (e.g., using a Victor Multilabel Counter (Wallac)).

[0571] Figure 8C The results of a competitive ELISA using the following seven peptides are shown: ANIKHVPGGGS (SEQ ID NO: 144), DAIKHVPGGGS (SEQ ID NO: 146), DNAKHVPGGGS (SEQ ID NO: 149), DNIAHVPGGGS (SEQ ID NO: 151), DNIKAVPGGGS (SEQ ID NO: 159), DNIKHAPGGGS (SEQ ID NO: 161), and DNIKHVPGGGS (SEQ ID NO: 171). A point mutation changing histidine to alanine (at position 299 of the corresponding tau protein in epitope 2) resulted in complete loss of activity of the peptide DNIKHVPGGGS in competitive binding to DC8E8 against tauΔ (1-150; 392-441 / 4R). A VPGGGS (SEQ ID NO: 159). Mutations that change amino acids D, N, I, K, and V to alanine do not eliminate the competitive activity of the corresponding mutant peptides (peptides ANIKHVPGGGS (SEQ ID NO: 144), DAIKHVPGGGS (SEQ ID NO: 146), DNAKHVPGGGS (SEQ ID NO: 149), DNIAHVPGGGS (SEQ ID NO: 151), and DNIKHAPGGGS (SEQ ID NO: 161)). These results indicate that the minimal epitope of DC8E8 belonging to the second tau protein repeat sequence (epitope 2) is located in the hexamer sequence, namely HVPGGG (SEQ ID NO: 154), and that DC8E8 binds to HXPGGG (SEQ ID NO: 164).

[0572] Example 5: DC8E8 identifies disordered TAUΔ(1-150; 151-391) / 4R, as evaluated by surface plasmon resonance.

[0573] Surface plasmon resonance (SPR) can be used to detect protein binding and determine the thermodynamic parameters of protein complexes (e.g., antibody-antigen complexes) by directly monitoring binding events in real time. This technique is routinely used to characterize both diagnostic and therapeutic antibodies (see, for example, Karlsson and Larsson, Affinity Measurement Using SurfacePlasmon Resonance, Methods in Molecular Biology, Vol. 248: Antibody Engineering: Methods and Protocols, edited by BKCLo). Humana Press Inc., Totowa, NJ, (2008)).

[0574] For SPR experiments, the DC8E8 monoclonal antibody (mAb) was purified from serum-free hybridoma supernatant on a protein G affinity column as follows. The hybridoma supernatant was adjusted to pH 7.5, pre-clarified by centrifugation, filtered through a 0.45 μm membrane filter, and loaded onto a 5 mL protein G agarose column. DC8E8 mAb was eluted from the column with 0.1 M glycine hydrochloride (pH 2.7). The eluted fraction was immediately neutralized with 1 M Tris-HCl (pH 9.0). The fraction obtained from PBS dialyzing was concentrated by ultrafiltration and stored at -70°C. The absorbance at 280 nm was measured, and the result was calculated using the formula c(mg / mL) = A. 280nm / 1.43 determines the antibody concentration.

[0575] A BIACORE 3000 instrument (Biacore AB, Uppsala) with a CM5 sensor chip was used for SPR assays. Amine coupling reagents (EDC, NHS, ethanolamine (pH 8.5)), P20 detergent, and 10 mM sodium acetate (pH 5.0) were obtained from Biacore AB. These experiments were performed at 25°C in PBS (pH 7.4) with 0.005% P20 as the operating buffer (PBS-P). Typically, 5,000 RU (reaction units) of polyclonal anti-mouse antibody (Z0420; DakoCytomation, Glostrup, Denmark) were simultaneously coupled in two flow cells at pH 5.0 via a primary amine, with one cell used as a reference measurement.

[0576] In each analytical cycle, purified DC8E8 was captured in the analytical flow cell to achieve an immobilization level of 230-250 RU. For K A Measurement and for the kinetic rate constant (k 缔合 and k 解离 The determination of tau protein (the tau protein targeted for testing DC8E8 affinity) was performed by injecting a two-fold serial dilution of tau protein (the tau protein targeted for testing DC8E8 affinity) or PBS-P as a control onto the sensor chip at a flow rate of 50 μl / min. Following Myszka, 1999, the kinetic binding data were subjected to dual reference and fitted against a two-phase reaction model using BIA evaluation software 4.1 (Biacore AB). The overall kinetic rate constant was estimated, the local maximum reaction was fitted, and the volumetric reaction was set to zero.

[0577] To quantify the affinity of DC8E8 for each tested tau protein, the association equilibrium binding constants (K0) of DC8E8 to the tetrad tau protein isoform 2N4R, the tripled tau protein isoform 2N3R, and the error-disordered tauΔ(1-150; 392-441) / 4R and error-disordered tauΔ(1-150; 392-441) / 3R were determined. A All tau proteins used in SPR were prepared according to Example 1. DC8E8 showed the highest affinity for the tetrad tauΔ(1-150; 392-441) / 4R, followed by the full-length tetrad tau protein isoform 2N4R, then the triplet tauΔ(1-150; 392-441) / 3R, and finally the triplet full-length tau protein isoform 2N3R. Figure 9A (B). Confirm these results: (1) DC8E8 is specific for error-disordered forms of tau protein, and (2) DC8E8 is more selective for error-disordered tau protein (i.e., diseased or pathological tau protein) than for full-length tau protein (i.e., normal or physiological tau protein).

[0578] Real-time monitoring of binding events using SPR enabled the measurement of association (k-association) and dissociation (k-dissociation) kinetics between DC8E8 and several tau proteins. The binding kinetics of DC8E8 revealed conformational changes in the disordered tauΔ(1-150; 392-441) / 4R and tauΔ(1-150; 392-441) / 3R compared to the physiological 2N4R tau protein, indicated by the greater accessibility of the DC8E8 epitope in the disordered tau protein. This was reflected in the faster binding and higher k-association of the disordered tau proteins compared to their full-length counterparts. Furthermore, the additional DC8E8 binding site on the tetrad tau protein species resulted in a 10-fold slower dissociation of the 4R tau protein species from the DC8E8 complex and a correspondingly 10-fold decrease in k-dissociation. Figure 10A B; the dashed line is calculated and interpolated using the self-measured data from the computer program BIAEvaluation v4.1.

[0579] Example 6: DC8E8 identifies all neurofibrillary degeneration stages in the human Alzheimer's disease brain.

[0580] Human brain tissue was obtained from a Dutch brain bank (on paraffin blocks). The blocks were cut using a microtome. Paraffin sections (8 μm) of the hippocampal-entorhinal cortex from Alzheimer's disease brains (Brack's stage VI) and non-dementia controls (Brack's stages I and III) were treated for 1 minute at room temperature (25°C) with 99% cold (+4°C) formic acid. The tissue sections were incubated overnight in a blocking solution (50 nM Tris-HCl containing 5% BSA and 0.3% Triton X-100) and then incubated with purified primary antibody DC8E8 (7.8 mg / ml; prepared as described in Example 5) diluted 1:2,000 in the blocking solution. Subsequently, the sections were incubated at room temperature for 1 hour with biotinylated secondary antibody (Vectastain Elite ABC kit, Vector Laboratories) followed by incubation with avidin-biotin peroxidase complex for 60 minutes (Vectastain Elite ABC kit, Vector Laboratories), both at room temperature (25°C). The immunoreaction was observed using a peroxidase substrate kit (Vector VIP, Vector laboratories, Ca, USA) and contrasted with methyl green (Vector Laboratories). The sections were examined using an Olympus BX71 microscope.

[0581] The monoclonal antibody DC8E8 can differentiate between preclinical AD, early-stage clinical AD, and fully developed late-stage AD. Immunohistochemical studies show that DC8E8 detects pathological tau protein in the early stages (tau protein monomers and dimers) of Black's stage I in preclinical AD in humans. Figure 11A The brain contains only a limited number of neurofibrillary tangles (NFTs) in the entorhinal cortex and none in the hippocampus (Black's stage I). In clinically early-stage Alzheimer's disease (Black's stage III) where a few NFTs are found in the hippocampus, DC8E8 mAb recognizes two phases: pathological tau protein oligomers (arrows) and pathological tau protein polymers (tangles). Figure 11B In the fully developed Alzheimer's brain with extensive neurofibrillary degeneration, DC8E8 primarily recognizes pathological tau protein polymers in the form of neurofibrillary tangles, neuritis plaques, and neuritis lines. Figure 11C Therefore, mAb DC8E8 recognizes all stages of neurofibrillary progression in human Alzheimer's disease brain tissue, including monomeric, dimeric, early oligomeric stages (Fig. 11D1) and late oligomeric pre-tangle stages (Fig. 11D2), as well as late-stage development of pathological tau protein polymers—intracellular (Fig. 11D3) and extracellular neurofibrillary tangles (Fig. 11D4). Thus, this reactivity of mAb DC8E8 is suitable for both diagnostic and therapeutic applications of this antibody.

[0582] Example 7: As seen in human Alzheimer's disease, DC8E8 recognizes all neurofibrillary degeneration stages in the brain of transgenic rat SHR72.

[0583] The SHR24 transgenic rat strain expresses the protein tauΔ(1-150; 392-441) / 3R as described in international patent application PCT WO 2004 / 007547. The generation and characterization of th...

Claims

1. A pharmaceutically acceptable salt of an immunogenic peptide, wherein the immunogenic peptide consists of KDNIKHVPGGGS (SEQ ID NO: 108) or CKDNIKHVPGGGS (SEQ ID NO: 218).

2. A pharmaceutically acceptable salt of an immunogenic peptide linked to a carrier, wherein the immunogenic peptide consists of KDNIKHVPGGGS (SEQ ID NO: 108) or CKDNIKHVPGGGS (SEQ ID NO: 218).

3. The pharmaceutically acceptable salt of an immunogenic peptide linked to a carrier of claim 2, wherein the carrier is serum albumin; keyhole limpet hemocyanin (KLH); an immunoglobulin molecule or fragment thereof; thyroglobulin; ovalbumin; ovoglobulin; tetanus toxoid protein; a toxoid from diphtheria, E. coli, cholera, or Helicobacter pylori; a universal T-cell epitope; or a cytokine.

4. The pharmaceutically acceptable salt of an immunogenic peptide linked to a carrier of claim 2 or claim 3, wherein the carrier is bovine serum albumin, IL-1, IL-2, IFNy, IL-10, GM-CSF, MIP1a, MIP1b, or RANTES.

5. The pharmaceutically acceptable salt of an immunogenic peptide linked to a carrier of claim 4, wherein IL-1 is IL-1a or IL-1b.

6. The pharmaceutically acceptable salt of an immunogenic peptide linked to a carrier of any one of claims 2-5, wherein the carrier is linked to an N-terminal or internal amino acid of the immunogenic peptide.

7. The pharmaceutically acceptable salt of an immunogenic peptide linked to a carrier of claim 6, wherein the carrier is linked to an N-terminal amino acid of the immunogenic peptide.

8. The pharmaceutically acceptable salt of an immunogenic peptide linked to a carrier of any one of claims 2-7, wherein the immunogenic peptide is linked to the carrier as a fusion protein.

9. The pharmaceutically acceptable salt of an immunogenic peptide linked to a carrier of any one of claims 2-7, wherein the carrier is linked to an N-terminal amino acid of the immunogenic peptide by a cross-linking agent.

10. The pharmaceutically acceptable salt of an immunogenic peptide linked to a carrier of claim 9, wherein the cross-linking agent comprises N-[y-maleimidobutyryloxy]succinimide ester (GMBS).

11. The pharmaceutically acceptable salt of an immunogenic peptide linked to a carrier of any one of claims 2-10, wherein the carrier is KLH.

12. The pharmaceutically acceptable salt of an immunogenic peptide linked to a carrier of any one of claims 2-10, wherein the carrier is tetanus toxoid protein.

13. The pharmaceutically acceptable salt of an immunogenic peptide linked to a carrier of claim 2, wherein the immunogenic peptide consists of SEQ ID NO: 218, the carrier is KLH, and the carrier is linked to an N-terminal amino acid of the pharmaceutically acceptable salt of the immunogenic peptide by a cross-linking agent comprising GMBS.

14. An immunogenic composition comprising a pharmaceutically acceptable salt of the immunogenic peptide of any one of claims 1-13 or the pharmaceutically acceptable salt of the immunogenic peptide linked to a carrier and a pharmaceutically acceptable excipient, diluent, and / or adjuvant.

15. The immunogenic composition of claim 14, wherein the adjuvant comprises an aluminum salt.

16. The immunogenic composition of claim 15, wherein the adjuvant comprises aluminum hydroxide.

17. Use of the immunogenic composition of any one of claims 14-16 in the manufacture of a medicament for ameliorating at least one symptom associated with Alzheimer's disease in a subject in need thereof.

18. The use of claim 17, wherein the at least one symptom comprises one or more of: progressive memory impairment, cognitive decline, language decline, behavioral changes, psychological symptoms, impaired motor function, impaired performance of instrumental activities of daily living, and disturbance in orientation in time and space.

19. The use of claim 18, wherein (a) the psychological symptoms comprise one or more of disturbances in mood, emotional disturbances, disturbances in appetite, disturbances in wake sleep cycle, confusion, agitation, and depression; and / or (b) the impaired motor function comprises one or more of apraxia, myoclonus, impaired gait, decreased muscle strength, and extrapyramidal features.

20. The use of claim 19, wherein the extrapyramidal features comprise one or more of bradykinesia, rigidity, and resting tremor.

21. Use of the immunogenic composition of any one of claims 14-16 in the manufacture of a medicament for treating or preventing Alzheimer's disease in a subject in need thereof.

22. Use of the immunogenic composition of any one of claims 14-16 in combination with at least one additional agent in the manufacture of a medicament or combination medicament for ameliorating at least one symptom associated with Alzheimer's disease in a subject in need thereof.

23. Use of the immunogenic composition of any one of claims 14-16 in combination with at least one additional agent in the manufacture of a medicament for treating or preventing Alzheimer's disease in a subject in need thereof.

24. The use of claim 22 or claim 23, wherein the at least one additional agent comprises one or more of: an acetylcholinesterase inhibitor, an NMDA receptor antagonist, a transition metal chelator, a growth factor, a hormone, a non-steroidal anti-inflammatory drug (NSAID), an antioxidant, a lipid lowering agent, a selective phosphodiesterase inhibitor, a tau protein aggregation inhibitor, a protein kinase inhibitor, a heat shock protein inhibitor, an anti-amyloid passive agent, an anti-amyloid active immunization agent, an anti-amyloid aggregation inhibitor, and a secretase inhibitor.

25. The use of any one of claims 22-24, wherein the at least one additional agent is to be administered prior to the immunogenic composition.

26. The use of any one of claims 22-24, wherein the at least one additional agent is to be administered concurrently with the immunogenic composition.

27. The use of any one of claims 22-24, wherein the at least one additional agent is to be administered after the immunogenic composition.

28. The use of any one of claims 17-27, wherein the medicament is to be administered by at least one mode selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, cerebrocisternal, intracerebroventricular, intrathecal, intra-colon, intra- cervical, intragastric, intrahepatic, myocardial, intraosseous, intra- pelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesicular, intravesicular, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

29. The use of claim 28, wherein the medicament is to be administered subcutaneously.

30. The use of any one of claims 17-29, wherein the medicament comprises at least 10 μg of a pharmaceutically acceptable salt of the immunogenic peptide.

31. The use of any one of claims 17-30, wherein the medicament is administered in multiple doses.

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