Tau peptide immunogen construct

Abstract

The present invention relates to individual peptide immunogen constructs that target a portion of the tau protein for the treatment and / or prevention of tauopathy. The present invention also relates to compositions containing said peptide immunogen constructs, methods of manufacturing and using said peptide immunogen constructs, and antibodies generated by said peptide immunogen constructs. The disclosed peptide immunogen constructs contain about 15 or more amino acids. The peptide immunogen constructs contain B cell epitopes from a portion of the longest isoform of the human tau protein (GenBank: AGF19246.1) having the amino acid sequence of SEQ ID NO: 167 shown in Table 3.

Description

Technical Field

[0001] The present disclosure relates to peptide immunogen constructs based on parts of tau protein and formulations thereof for the prevention and treatment of tauopathy.

Background Art

[0002] Tau protein is abundant in nerve cells of the central nervous system and plays a role in stabilizing microtubules. Neurological conditions and dementias such as Alzheimer's disease are associated with tau protein that has become defective and unable to properly stabilize microtubules. Further neurodegenerative diseases called tauopathies are characterized by abundant fibrillar tau. These diseases include neurofibrillary tangle-type dementia, chronic traumatic encephalopathy (CTE), argyrophilic grain disease, progressive supranuclear palsy, corticobasal degeneration, globular glial tauopathy, and Pick's disease.

[0003] There is a need to develop tau-targeted therapies for use in treating patients suffering from diseases such as Alzheimer's disease and tauopathy.

Summary of the Invention

[0004] The present invention relates to individual peptide immunogen constructs that target parts of tau protein for the treatment and / or prevention of tauopathy. The present invention also relates to compositions containing the peptide immunogen constructs, methods of making and using the peptide immunogen constructs, and antibodies generated by the peptide immunogen constructs.

[0005] The disclosed peptide immunogen construct contains about 15 or more amino acids. The peptide immunogen construct contains a B cell epitope from a part of the longest isoform of the human tau protein (GenBank: AGF19246.1) having the amino acid sequence of SEQ ID NO: 167 shown in Table 3. The B cell epitope can bind to a heterologous T helper cell (Th) epitope derived from a pathogen protein via any heterologous spacer. The disclosed peptide immunogen construct stimulates the production of highly specific antibodies against tau. The disclosed peptide immunogen construct can be used as an immunotherapy for patients suffering from tauopathy.

[0006] The B cell epitope portion of the peptide immunogen construct has the amino acid sequence of the full-length tau protein (SEQ ID NO: 167). In some embodiments, the B cell epitope has the sequence of any one of SEQ ID NOs: 1-8, 36, 39, 40, 48, 50, 51, 52, 57, 58, 59, 62, 87, 88, 129, 49, 35, 99, 135, and 137, or has a sequence containing any one of these, as shown in any one of Table 1 or Tables 4-7.

[0007] The peptide immunogen constructs of the present disclosure may include heterologous Th epitope amino acid sequences (e.g., SEQ ID NOs: 138-166) derived from pathogenic proteins, as shown in Table 2. In some embodiments, the heterologous Th epitope consists of SEQ ID NO: 151 or includes SEQ ID NO: 151. In some embodiments, the heterologous Th epitope consists of SEQ ID NO: 149 or 152, or includes SEQ ID NO: 149 or 152. In certain embodiments, the heterologous Th epitopes are derived from natural pathogens such as diphtheria toxin (SEQ ID NO: 142), Plasmodium falciparum (SEQ ID NO: 143), and cholera toxin (SEQ ID NO: 145). In other embodiments, the heterologous Th epitopes are ideal artificial Th epitopes derived from measles virus fusion protein (MVF1-5) or hepatitis B surface antigen (HBsAg1-3) in the form of a single sequence or a combined sequence (e.g., SEQ ID NO: 149, 148, or 150).

[0008] In some embodiments, the peptide immunogen construct contains a B cell epitope from tau that is linked to a heterologous T helper cell (Th) epitope via any heterologous spacer. In certain embodiments, the peptide immunogen construct has or includes a B cell antigen site having an amino acid sequence from tau (e.g., any one of SEQ ID NOs: 1-8, 36, 39, 40, 48, 50, 51, 52, 57, 58, 59, 62, 87, 88, 129, 49, 35, 99, 135, and 137) linked to a heterologous Th epitope derived from a pathogenic protein (e.g., SEQ ID NOs: 138-166, e.g., 151, 152, or 149) via any heterologous spacer. In some embodiments, any heterologous spacer is a molecule or chemical structure capable of binding two amino acids and / or peptides together. In certain embodiments, the spacer is a natural amino acid, an unnatural amino acid, or a combination thereof. In a specific embodiment, the peptide immunogen construct has the amino acid sequence of one of SEQ ID NOs: 9-16 shown in Table 1 (e.g., 10 or 15) (or a construct in which the positions of the tau peptide and the Th peptide are reversed).

[0009] The present invention also relates to a composition comprising a tau peptide immunogen construct. In some embodiments, the disclosed composition comprises a plurality of tau peptide immunogen constructs to cover a plurality of B epitopes from tau. In certain embodiments, the composition comprises a mixture of tau peptide immunogen constructs (e.g., any combination of SEQ ID NOs: 9-16) comprising a plurality of heterologous Th epitopes derived from a pathogenic protein to cover a wide genetic background of a patient. A composition containing a mixture of peptide immunogen constructs can provide a higher percentage of response rate upon immunization for the treatment of tauopathy as compared to a composition containing only a single Th peptide immunogen construct.

[0010] The present disclosure also relates to a pharmaceutical composition for the treatment and / or prevention of tauopathy. In some embodiments, the pharmaceutical composition is disclosed in the form of a stabilized immunostimulatory complex formed via electrostatic association by mixing a CpG oligomer (e.g., CpG1, CpG2, or CpG3) with a composition comprising a peptide immunogen complex and contains the peptide immunogen construct. Such a stabilized immunostimulatory complex can further enhance the immunogenicity of the peptide immunogen construct. In some embodiments, the pharmaceutical composition contains an adjuvant such as an aluminum salt such as ALHYDROGEL, aluminum phosphate (ADJUPHOS), or an oil-in-water emulsion containing MONTANIDE ISA51 or 720.

[0011] The present invention also relates to antibodies against the disclosed tau peptide immunogen constructs. In particular, the peptide immunogen constructs of the present disclosure, when administered to a subject, can stimulate the production of highly specific antibodies that cross-react with tau amino acid sequences (e.g., any one of SEQ ID NOs: 1-8, 36, 39, 40, 48, 50, 51, 52, 57, 58, 59, 62, 87, 88, 129, 49, 35, 99, 135, and 137). The highly specific antibodies produced by the peptide immunogen constructs cross-react with recombinant tau-containing proteins. The disclosed antibodies bind to tau with high specificity, with little or no orientation towards heterologous Th epitopes used for immunogenic enhancement. This is in marked contrast to conventional proteins or other biological carriers used for such peptide antigen enhancement.

[0012] The present disclosure also includes methods for treating and / or preventing tauopathy using the disclosed peptide immunogen constructs and / or antibodies against the peptide immunogen constructs. In some embodiments, a method for treating and / or preventing tauopathy includes administering to a host a composition containing the disclosed peptide immunogen construct. In certain embodiments, the composition utilized in the method contains the disclosed peptide immunogen construct in the form of a stable immunostimulatory complex with a negatively charged oligonucleotide, such as a CpG oligomer, via electrostatic association, and the complex is optionally further supplemented with an inorganic salt or oil as an adjuvant for administration to a patient suffering from tauopathy. The disclosed methods also include dosing regimens, dosage forms, and routes for administering the peptide immunogen construct to a host having a risk of or suffering from tauopathy. BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0025] The present invention relates to individual peptide immunogen constructs that target a portion of the tau protein for the treatment and / or prevention of Alzheimer's disease and tauopathy. The present invention also relates to compositions containing the peptide immunogen constructs, methods for manufacturing and using the peptide immunogen constructs, and antibodies generated by the peptide immunogen constructs.

[0026] The disclosed peptide immunogen constructs contain about 15 or more amino acids. The peptide immunogen constructs contain B cell epitopes from a portion of the longest isoform of the human tau protein (GenBank: AGF19246.1) having the amino acid sequence shown in Table 3. The B cell epitopes can be linked via any heterologous spacer to a heterologous T helper cell (Th) epitope derived from a pathogen protein. The disclosed peptide immunogen constructs stimulate the production of highly specific antibodies against tau. The disclosed peptide immunogen constructs can be used as immunotherapy for patients suffering from Alzheimer's disease and / or tauopathy.

[0027] The B-cell epitope portion of the peptide immunogen construct has the amino acid sequence of the full-length tau protein (Table 3). In some embodiments, the B-cell epitope has or comprises any one of the target sequences (SEQ ID NOs: 1-8) listed in Table 1. In some embodiments, the B-cell epitope has or comprises a sequence of one of Tables 4-7 (e.g., any one of SEQ ID NOs: 17-137, e.g., p5543a, p5546a, p5547a, p5555a, p5557a, p5558a, p5559a, p5564a, p5565a, p5566a, p5569a, p5695a, p5696, or p5535a, which are SEQ ID NOs: 36, 39, 40, 48, 50, 51, 52, 57, 58, 59, 62, 87, 88, and 129, respectively, or p5556a, p5456a, p4579a, p5541a, or p4062a, which are SEQ ID NOs: 49, 35, 99, 135, and 137, respectively).

[0028] As shown in Table 2, the peptide immunogen construct of the present disclosure may include a heterologous Th epitope amino acid sequence (e.g., SEQ ID NOs: 138-166) derived from a pathogenic protein. In some embodiments, the Th epitope has or comprises the sequence of SEQ ID NO: 151, 152, or 149. In certain embodiments, the heterologous Th epitope is derived from natural pathogens such as diphtheria toxin (SEQ ID NO: 142), Plasmodium falciparum (SEQ ID NO: 143), cholera toxin (SEQ ID NO: 145). In other embodiments, the heterologous Th epitope is an ideal artificial Th epitope derived from measles virus fusion protein (MVF1-5) or hepatitis B surface antigen (HBsAg1-3) in the form of a single sequence or a combined sequence (e.g., SEQ ID NO: 149, 148 or 150).

[0029] In some embodiments, the peptide immunogen construct contains a B cell epitope from tau that is linked to a heterologous T helper cell (Th) epitope via any heterologous spacer. In certain embodiments, the peptide immunogen construct contains a B cell antigen site having or comprising an amino acid sequence from tau (see, e.g., Table 1 (one of SEQ ID NOs: 1-8) and Tables 4-7 (e.g., one of SEQ ID NOs: 36, 39, 40, 48, 50, 51, 52, 57, 58, 59, 62, 87, 88, 129, 49, 35, 99, 135, and 137)) that is linked to a heterologous Th epitope derived from a pathogenic protein (e.g., SEQ ID NOs: 138-166, e.g., 151, 152, or 149) via any heterologous spacer. In some embodiments, any heterologous spacer is a molecule or chemical structure that can link two amino acids and / or peptides together. In certain embodiments, the spacer is a natural amino acid, a non-natural amino acid, or a combination thereof. In a specific embodiment, the peptide immunogen construct has the amino acid sequence shown in Table 1 (e.g., any one of the sequences of SEQ ID NOs: 9-16 (e.g., 10 or 15), or a construct in which the positions of the tau peptide and the Th peptide are reversed).

[0030] The present invention also relates to a composition comprising a tau peptide immunogen construct. In some embodiments, the disclosed composition contains a plurality of tau peptide immunogen constructs to cover a plurality of B epitopes from tau. In certain embodiments, the composition contains a mixture of tau peptide immunogen constructs that contain a plurality of heterologous Th epitopes derived from a pathogenic protein to cover a broad genetic background of a patient. A composition containing a mixture of peptide immunogen constructs can result in a higher percentage of response rates upon immunization for the treatment of Alzheimer's disease and / or tauopathy as compared to a composition containing only a single peptide immunogen construct.

[0031] The present disclosure also relates to pharmaceutical compositions for the treatment and / or prevention of Alzheimer's disease and / or tauopathy. In some embodiments, the pharmaceutical composition is disclosed in the form of a stabilized immunostimulatory complex formed via electrostatic association by mixing a CpG oligomer with a composition comprising a peptide immunogen complex and contains a peptide immunogen construct. Such a stabilized immunostimulatory complex can further enhance the immunogenicity of the peptide immunogen construct. In some embodiments, the pharmaceutical composition contains an adjuvant such as an aluminum salt such as ALHYDROGEL, aluminum phosphate (ADJUPHOS), or a water-in-oil emulsion containing MONTANIDE ISA51 or 720.

[0032] The present invention also relates to antibodies against the disclosed tau peptide immunogen construct. In particular, the peptide immunogen constructs of the present disclosure can stimulate the production of highly specific antibodies that cross-react with the tau amino acid sequence when administered to a subject. The highly specific antibodies produced by the peptide immunogen construct cross-react with recombinant tau-containing proteins. The disclosed antibodies bind to tau with high specificity with little or no orientation towards the heterologous Th epitopes used for immunogenic enhancement. This is in marked contrast to conventional proteins or other biological carriers used for such peptide antigenicity enhancement.

[0033] The present disclosure also includes methods for treating and / or preventing Alzheimer's disease and / or tauopathy using the disclosed peptide immunogen constructs and / or antibodies against the peptide immunogen constructs. In some embodiments, the method for treating and / or preventing Alzheimer's disease and / or tauopathy includes administering to a host a composition containing the disclosed peptide immunogen construct. In certain embodiments, the composition utilized in the method contains the disclosed peptide immunogen construct in the form of a stable immunostimulatory complex with a negatively charged oligonucleotide, such as a CpG oligomer, via electrostatic association, and the complex is optionally further supplemented with an inorganic salt or an oil as an adjuvant for administration to a patient suffering from Alzheimer's disease and / or tauopathy. The disclosed methods also include dosing regimens, dosage forms, and routes for administering the peptide immunogen construct to a host having a risk of or suffering from Alzheimer's disease and / or tauopathy.

[0034] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references or portions of references cited in this application are hereby expressly incorporated by reference in their entirety for all purposes.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Thus, "including A or B" means including A or B, or A and B. Further, it should be understood that all amino acid sizes, and all molecular weight or molecular mass values given for polypeptides are approximate and are provided for illustrative purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed methods, the appropriate methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0036] Tau peptide immunogen construct The present disclosure provides a peptide immunogen construct containing a B cell epitope, comprising an amino acid sequence from tau covalently linked directly or via any heterologous spacer to a heterologous T helper cell (Th) epitope.

[0037] As used herein, the terms "tau peptide immunogen construct" or "peptide immunogen construct" refer to a peptide comprising (a) a B cell epitope having about 8 or more amino acid residues (e.g., about 8 - 40, 9 - 30, 10 - 25, 8 - 20, 9 - 18 or 10 - 15, or about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids) from the full-length sequence of the longest tau isoform (SEQ ID NO: 167), (b) a heterologous Th epitope, and (c) any heterologous spacer.

[0038] In certain embodiments, the tau peptide immunogen construct has the formula: (Th) m -(A) n -(tau fragment)-X or (tau fragment)-(A) n -(Th) m -X (wherein Th is a heterologous T helper epitope, A is a heterologous spacer, (tau fragment) is a B cell epitope having from about 8 to about 40 amino acid residues from SEQ ID NO: 167 (e.g., about 8 - 40, 9 - 30, 10 - 25, 8 - 20, 9 - 18 or 10 - 15, or about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids), X is the α-COOH or α-CONH2 of an amino acid, m is from 1 to about 4, n is from 0 to about 10).

[0039] The various components of the disclosed tau peptide immunogen constructs are described below.

[0040] a. tau fragment The disclosed peptide immunogen construct contains about 8 or more amino acids. The peptide immunogen construct contains a B cell epitope from a part of the longest isoform of the human tau protein (GenBank: AGF19246.1) having the amino acid sequence of SEQ ID NO: 167 shown in Table 3. The B cell epitope can bind to a heterologous T helper cell (Th) epitope derived from a pathogen protein via any heterologous spacer. The disclosed peptide immunogen construct stimulates the production of highly specific antibodies against tau. The disclosed peptide immunogen construct can be used as an immunotherapy for patients suffering from Alzheimer's disease and / or tauopathy.

[0041] The B cell epitope portion of the peptide immunogen construct has the amino acid sequence of the full-length tau protein (SEQ ID NO: 167). In some embodiments, the B cell epitope has or comprises the sequence shown in Table 1 (one of SEQ ID NOs: 1-8) or Tables 4-7 (e.g., one of SEQ ID NOs: 36, 39, 40, 48, 50, 51, 52, 57, 58, 59, 62, 87, 88, 129, 49, 35, 99, 135, and 137).

[0042] In some embodiments, the tau peptide of the peptide immunogen construct comprises or consists of any one of the peptides in Table 1 and Tables 4-7.

[0043] In some embodiments, the tau peptide of the peptide immunogen construct comprises, or consists of, any one of the peptides in Table 1 and Tables 4-7, and the peptide comprises 1, 2, 3, 4, or 5 additional amino acids of the tau protein of SEQ ID NO: 167 at the N-terminus of the peptide of Table 1 or Tables 4-7. In some embodiments, the tau peptide of the peptide immunogen construct comprises, or consists of, any one of the peptides in Table 1 and Tables 4-7, and the peptide comprises 1, 2, 3, 4, or 5 additional amino acids of the tau protein of SEQ ID NO: 167 at the C-terminus of the peptide of Table 1 or Tables 4-7. In some embodiments, the tau peptide of the peptide immunogen construct comprises, or consists of, any one of the peptides in Table 1 and Tables 4-7, and the peptide comprises 1, 2, 3, 4, or 5 additional amino acids of the tau protein of SEQ ID NO: 167 at the N-terminus and / or C-terminus of the peptide of Table 1 or Tables 4-7. The additional amino acids in the latter peptides are to be understood as amino acids of the tau protein of SEQ ID NO: 167 that are contiguous with the peptide sequences of Table 1 and Tables 4-7.

[0044] In some embodiments, the tau peptide of the peptide immunogen construct comprises, or consists of, about 8 to about 40 amino acid residues of SEQ ID NO: 167 (e.g., about 8-40, 9-30, 10-25, 8-20, 9-18 or 10-15, or about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids). In some embodiments, the tau peptide of the peptide immunogen construct is any one of the peptides in Table 1 and Tables 4-7, e.g., any one of SEQ ID NOs: 1-8, 36, 39, 40, 48, 50, 51, 52, 57, 58, 59, 62, 87, 88, 129, 49, 35, 99, 135, and 137, or comprises them, and optionally, the peptide has one or more of the above additional amino acids.

[0045] b. Heterologous T helper cell epitope (Th epitope) The present disclosure provides a peptide immunogen construct containing a B cell epitope from tau covalently linked directly or via any heterologous spacer to a heterologous T helper (Th) epitope.

[0046] The heterologous Th epitope in the tau peptide immunogen construct enhances the immunogenicity of the tau fragment, which through rational design promotes the production of specific high-titer antibodies against the optimized target B cell epitope (i.e., the tau fragment).

[0047] As used herein, the term "heterologous" refers to an amino acid sequence that is not part of the wild-type sequence of tau or is not homologous to the wild-type sequence. Thus, a heterologous Th epitope is a Th epitope derived from an amino acid sequence that is not naturally found in tau (i.e., the Th epitope is not self-derived with respect to tau). Since the Th epitope is heterologous with respect to tau, when the heterologous Th epitope is covalently linked to a tau fragment, the native amino acid sequence of tau is not extended in either the N-terminal or C-terminal direction.

[0048] The heterologous Th epitope of the present disclosure can be any Th epitope that does not have an amino acid sequence naturally found in tau. The Th epitope can have an amino acid sequence derived from any species (e.g., human, pig, cow, dog, rat, mouse, guinea pig, etc.). The Th epitope can also have a promiscuous binding motif for MHC class II molecules of multiple species. In certain embodiments, the Th epitope contains multiple promiscuous MHC class II binding motifs that allow for maximal activation of helper T cells that initiate and regulate the immune response. The Th epitope is preferably immunologically silent by itself, i.e., since there are few, if any, antibodies directed against the Th epitope generated by the tau peptide immunogen construct, a very focused immune response directed against the target B cell epitope of the tau fragment is enabled.

[0049] Epitopes of the present disclosure include, but are not limited to, amino acid sequences derived from foreign pathogens as exemplified in Table 2 (SEQ ID NOs: 138 to 166, such as 151, 152 or 149). Furthermore, Th epitopes include idealized artificial Th epitopes and combinations of idealized artificial Th epitopes (e.g., SEQ ID NOs: 139 and 146 to 152). Heterologous Th epitope peptides presented as combinatorial sequences (e.g., SEQ ID NOs: 147 to 150) contain a mixture of amino acid residues shown at specific positions within the peptide framework based on the variable residues of homologs to that specific peptide. An aggregate of combinatorial peptides can be synthesized in one process by adding a mixture of designated protected amino acids instead of one specific amino acid at the designated positions during the synthesis process. Such an aggregate of combinatorial heterologous Th epitope peptides can cover a wide range of Th epitopes for animals with diverse genetic backgrounds. Representative combinatorial sequences of heterologous Th epitope peptides include SEQ ID NOs: 147 to 150 shown in Table 2. The epitope peptides of the present invention provide broad reactivity and immunogenicity to genetically diverse populations of animals and patients.

[0050] Tau peptide immunogen constructs containing Th epitopes can be generated simultaneously in a single solid-phase peptide synthesis tandem with tau fragments. Th epitopes also include immunological analogs of Th epitopes. Immunological Th analogs include immune-enhancing analogs, cross-reactive analogs, and segments of any one of these Th epitopes sufficient to enhance or stimulate an immune response to tau fragments.

[0051] Functional immunological analogs of the Th epitope peptides are also effective and are included as part of the present invention. Functional immunological Th analogs can include conservative substitutions, additions, deletions, and insertions of 1 to about 5 amino acid residues in the Th epitope that do not substantially modify the Th-stimulating function of the Th epitope. Conservative substitutions, additions, and insertions can be achieved using natural or non-natural amino acids, as described above for the tau fragment. Table 2 identifies another variation of functional analogs to the Th epitope peptide. In particular, the Th of MvF1 and MvF2 of SEQ ID NOs: 139 and 146 are functional analogs of MvF4 and MvF5 of SEQ ID NOs: 149 and 151, which differ in that in the amino acid frame, two amino acids are either deleted (SEQ ID NOs: 139 and 146) or included (SEQ ID NOs: 149 and 151) at the N-terminus and C-terminus, respectively. The differences between these two sets of similar sequences will not affect the function of the Th epitope contained within these sequences. Thus, functional immunological Th analogs include several versions of Th epitopes derived from measles virus fusion protein MvF1-4 Th (SEQ ID NOs: 139, 146, 147, 149, and 151), as well as from hepatitis B surface protein HBsAg1-3 Th (SEQ ID NOs: 148, 150, and 152).

[0052] The Th epitopes in the tau peptide immunogen construct can be covalently attached to either the N-terminus or the C-terminus of the tau peptide. In some embodiments, the Th epitope is covalently attached to the N-terminus of the tau peptide. In other embodiments, the Th epitope is covalently attached to the C-terminus of the tau peptide. In certain embodiments, multiple Th epitopes are covalently attached to the tau fragment. When multiple Th epitopes are attached to the tau fragment, each Th epitope can have the same amino acid sequence or a different amino acid sequence. Further, when multiple Th epitopes are attached to the tau fragment, the Th epitopes can be arranged in any order. For example, the Th epitopes can be attached sequentially to the N-terminus of the tau fragment, or sequentially to the C-terminus of the tau fragment, or one Th epitope can be covalently attached to the N-terminus of the tau fragment while another Th epitope is covalently attached to the C-terminus of the tau fragment. There is no limitation on the arrangement of the Th epitopes with respect to the tau fragment. It should be understood that for the immunogen constructs shown in Table 1, the present disclosure includes corresponding immunogen constructs in which the positions of the tau peptide and the Th epitope are inverted from the orientations shown in Table 1.

[0053] In some embodiments, the Th epitope is directly covalently attached to the tau fragment. In other embodiments, the Th epitope is covalently attached to the tau fragment via a heterologous spacer, which is described in more detail below.

[0054] c. Heterologous spacer The disclosed tau constructs optionally contain a heterologous spacer that covalently attaches a B cell epitope derived from tau to a heterologous helper T cell (Th) epitope.

[0055] As described above, the term "heterologous" refers to an amino acid sequence that is not part of the wild-type sequence of tau or is not homologous. Thus, the native amino acid sequence of tau is not extended in either the N-terminal or C-terminal direction when a heterologous spacer is covalently attached to a B-cell epitope derived from tau, as the spacer is heterologous to the tau sequence.

[0056] A spacer is any molecule or chemical structure that can join two amino acids and / or peptides together. The spacer can vary widely in length or polarity depending on the application. The attachment of the spacer can be through an amide bond or a carboxyl - bond, although other functional groups are also possible. The spacer can contain a compound, a natural amino acid, or a non-natural amino acid.

[0057] The spacer can provide structural features to the tau peptide immunogen construct. Structurally, the spacer physically separates the Th epitope from the B cell epitope of the tau fragment. By physically separating with the spacer, any artificial secondary structure caused by the binding of the Th epitope to the B cell epitope can be disrupted. Furthermore, by physically separating the epitopes with the spacer, interference between the Th cell response and / or B cell response can be eliminated. Additionally, the spacer can be designed such that the secondary structure of the peptide immunogen construct is generated or modified. For example, the spacer can be designed to act as a flexible hinge to facilitate the separation of the Th epitope and the B cell epitope. The flexible hinge spacer can also enable more efficient interaction between the presented peptide immunogen and the appropriate Th cells and B cells, enhancing the immune response to the Th epitope and the B cell epitope. Examples of sequences encoding flexible hinges are often found in the immunoglobulin heavy chain hinge region, which is rich in proline. One particularly useful flexible hinge that can be used as a spacer is provided by the sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 168), wherein Xaa is any amino acid, preferably aspartic acid.

[0058] The spacer can also provide functional features to the tau peptide immunogen construct. For example, the spacer can be designed to change the overall charge of the tau peptide immunogen construct, which can affect the solubility of the peptide immunogen construct. Furthermore, the change in the overall charge of the tau peptide immunogen construct can affect the ability of the peptide immunogen construct to associate with other compounds and reagents. As will be discussed in more detail below, the tau peptide immunogen construct can be formed into a stable immunostimulatory complex with a highly charged oligonucleotide such as a CpG oligomer via electrostatic association. The overall charge of the tau peptide immunogen construct is important for the formation of these stable immunostimulatory complexes.

[0059] Examples of compounds that can be used as spacers include, but are not limited to, (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric acid (AVA), 6-aminocaproic acid (Ahx), 8-amino-3,6-dioxaoctanoic acid (AEEA, mini-PEG1), 12-amino-4,7,10-trioxadodecanoic acid (mini-PEG2), 15-amino-4,7,10,13-tetraoxapentadecanoic acid (mini-PEG3), trioxatridecane-succinic acid (Ttds), 12-aminododecanoic acid, Fmoc-5-amino-3-oxapentanoic acid (O1Pen), and the like.

[0060] Natural amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0061] Examples of non-natural amino acids include ε-N-lysine, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, γ-aminobutyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca, 6-aminohexanoic acid), hydroxyproline, mercaptopropionic acid (MPA), 3-nitro-tyrosine, pyroglutamic acid, and the like.

[0062] The spacer in the tau peptide immunogen construct can be covalently linked to either the N-terminus or the C-terminus of the tau epitope and the tau peptide. In some embodiments, the spacer is covalently linked to the C-terminus of the Th epitope and the N-terminus of the tau peptide. In other embodiments, the spacer is covalently linked to the C-terminus of the tau peptide and the N-terminus of the Th epitope. In certain embodiments, for example, when multiple Th epitopes are present in the peptide immunogen construct, multiple spacers can be used. When multiple spacers are used, each spacer may be the same as or different from each other. Further, when multiple Th epitopes are present in the peptide immunogen construct, the Th epitopes can be separated by spacers that may be the same as or different from the spacers used to separate the B cell epitope from the Th epitope. There is no limitation on the arrangement of the spacer with respect to the Th epitope or the tau fragment.

[0063] In certain embodiments, the heterologous spacer is a natural amino acid or a non-natural amino acid. In other embodiments, the spacer contains multiple natural or non-natural amino acids. In specific embodiments, the spacer is Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 169).

[0064] d. Specific embodiments of the tau peptide immunogen construct The tau peptide immunogen construct has the formula: (Th) m -(A) n -(tau fragment)-X or (tau fragment)-(A) n -(Th) m -X (wherein, Th is a heterologous T helper epitope, A is a heterologous spacer, (Tau fragment) is a B cell epitope having from about 8 to about 40 amino acid residues (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids) from the full-length tau protein of SEQ ID NO: 167, X is the α-COOH or α-CONH2 of an amino acid, m is from 1 to about 4, n is from 0 to about 10).

[0065] In certain embodiments, the heterologous Th epitope in the tau peptide immunogen construct has an amino acid sequence selected from any one of those described in Table 2. In certain embodiments, the tau peptide immunogen construct comprises a plurality of Th epitopes.

[0066] In certain embodiments, any heterologous spacer is selected from any one of Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 169), and combinations thereof. In certain embodiments, the heterologous spacer is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 169).

[0067] In certain embodiments, the tau fragment has from about 8 to about 40 (e.g., 10 - 30, 10 - 25, 12 - 20, or 12 - 15) amino acid residues from the full-length tau protein of SEQ ID NO: 167. In certain embodiments, the tau fragment has an amino acid sequence shown in Table 1 (one of SEQ ID NOs: 1 - 8) or one of Tables 4 - 7 (e.g., one of SEQ ID NOs: 36, 39, 40, 48, 50, 51, 52, 57, 58, 59, 62, 87, 88, 129, 49, 35, 99, 135, and 137).

[0068] In some embodiments, the tau peptide immunogen construct a. MAEPRQEFEVMEDHAGTYGLGDKKK-eK-ISITEIKGVIVHRIETILF-NH2; SEQ ID NO: 9 b. DHAGTYGLGDKKK-eK-ISITEIKGVIVHRIETILF-NH2; SEQ ID NO: 10 c. ISITEIKGVIVHRIETILF-eK-KKKNITHVPGGGNKK-NH2; SEQ ID NO: 11 d. ISITEIKGVIVHRIETILF-eK-KKKKDNIKHVPGGGSVQIVYK-NH2; SEQ ID NO: 12 e. ISITEIKGVIVHRIETILF-eK-KKKTKIATPRGAAPP-NH2; SEQ ID NO: 13 f. ISITEIKGVIVHRIETILF-eK-KKKVVRTPPKSPSSAKSRL-NH2; SEQ ID NO: 14 g. ISITEIKGVIVHRIETILF-eK-KKKIKHVPGGGSVQIVYK-NH2; and SEQ ID NO: 15 h. ISITEIKGVIVHRIETILF-eK-KKKVSGDTSPRHLSNVSST-NH2 SEQ ID NO: 16 is selected from the group consisting of.

[0069] In some embodiments, any one T cell epitope of SEQ ID NOs: 9-16 (e.g., 10 or 15) is replaced with any one T cell epitope of SEQ ID NOs: 138-166. In some embodiments, the positions of any one B cell epitope and T cell epitope of SEQ ID NOs: 9-16 (e.g., 10 or 15) are reversed within the molecule (i.e., if the B cell epitope is at the N-terminus of the original molecule, it is located at the C-terminus of the molecule in the reversed molecule, etc.).

[0070] a. Variants, homologs, and functional analogs Antibodies against preferred epitopes of tau protein and / or variants and analogs of the above immunogenic peptides that cross-react can also be used. Analogs, including allelic variants, species variants, and induced variants, often differ from the native peptide at one, two, or several positions by conservative substitutions. Analogs typically exhibit at least 80 or 90% sequence identity with the native peptide. Some analogs include non-natural amino acids or modifications of the N-terminal or C-terminal amino acids at one, two, or several positions.

[0071] Variants that are functional analogs can have conservative substitutions of amino acid positions, changes in overall charge, covalent attachment to another moiety, or amino acid addition, insertion, or deletion, and / or any combination of these.

[0072] A conservative substitution is when one amino acid residue is replaced by another amino acid residue with similar chemical properties. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0073] In certain embodiments, the functional analog has at least 50% identity to the original amino acid sequence. In another embodiment, the functional analog has at least 80% identity to the original amino acid sequence. In yet another embodiment, the functional analog has at least 85% identity to the original amino acid sequence. In yet another embodiment, the functional analog has at least 90% identity to the original amino acid sequence.

[0074] Variants also include mutations to phosphorylated residues. For example, variants can include different residues within a phosphorylated peptide. Variant immunogenic tau peptides can also include mimetic phosphorylated peptides. Mimetic phosphorylated peptides are generated by substituting one or more of the phosphorylated serine, threonine, and tyrosine residues of the tau peptide with acidic amino acid residues such as glutamic acid and aspartic acid.

[0075] Composition The present invention also provides a composition comprising the disclosed tau immunogen construct.

[0076] a. Peptide composition Compositions containing the disclosed tau peptide immunogen construct can be in liquid or solid form. Liquid compositions can include water, buffers, solvents, salts, and / or any other acceptable reagents that do not change the structural or functional properties of the tau peptide immunogen construct. Peptide compositions can include one or more of the disclosed tau peptide immunogen constructs.

[0077] b. Pharmaceutical composition The present invention also relates to a pharmaceutical composition comprising the disclosed tau peptide immunogen construct.

[0078] Pharmaceutical compositions can contain a carrier and / or other additives within a pharmaceutically acceptable delivery system. Thus, pharmaceutical compositions can contain a pharmaceutically effective amount of the tau peptide immunogen construct together with a pharmaceutically acceptable carrier, adjuvant, and / or other excipients, such as diluents, additives, stabilizers, preservatives, solubilizers, buffers, etc.

[0079] The pharmaceutical composition may contain one or more adjuvants that act to accelerate, prolong, or enhance the immune response against the tau peptide immunogen construct without having any specific antigenic effect per se. Adjuvants used in the pharmaceutical composition include oils, aluminum salts, virosomes, aluminum phosphate (e.g., ADJU-PHOS®), aluminum hydroxide (e.g., ALHYDROGEL®), liposyn, saponin, squalene, L121, Emulsigen®, monophosphoryl lipid A (MPL), QS21, ISA35, ISA206, ISA50V, ISA51, ISA720, and other adjuvants and emulsifiers.

[0080] In some embodiments, the pharmaceutical composition contains MONTANIDE™ ISA51 (an oil adjuvant composition consisting of vegetable oil and mannide oleate for generating water-in-oil emulsions), Tween® 80 (also known as polysorbate 80 or polyoxyethylene (20) sorbitan monooleate), CpG oligonucleotides, and / or any combination thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (i.e., w / o / w) emulsion containing Emulsigen or Emulsigen D as an adjuvant.

[0081] The pharmaceutical composition can be formulated as an immediate-release or sustained-release preparation. Further, the pharmaceutical composition can be formulated to induce systemic or local mucosal immunity through capture of the immunogen and co-administration with microparticles. Such delivery systems are readily determined by those skilled in the art.

[0082] The pharmaceutical composition can be prepared as an injectable, either as a liquid solution or a suspension. A liquid vehicle containing the tau peptide immunogen construct can also be prepared prior to injection. The pharmaceutical composition can be administered by any suitable mode of application, e.g., i.d., i.v., i.p., i.m., intranasal, oral, subcutaneous, etc., and with any suitable delivery device. In certain embodiments, the pharmaceutical composition is formulated for intravenous, subcutaneous, intradermal, or intramuscular administration. Pharmaceutical compositions suitable for other methods of administration, including oral and intranasal administration, can also be prepared.

[0083] The pharmaceutical composition can be formulated as an immediate release or sustained release formulation. Further, the pharmaceutical composition can be formulated to induce systemic or local mucosal immunity through capture of the immunogen and co - administration with microparticles. Such delivery systems can be readily determined by those skilled in the art.

[0084] The pharmaceutical composition can also be formulated in suitable dosage units. In some embodiments, the pharmaceutical composition contains from about 0.5 μg to about 1 mg of the tau peptide immunogen construct per kg body weight. The effective dose of the pharmaceutical composition varies depending on many different factors including the means of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Usually, the patient is human, but non - human mammals, including transgenic mammals, can also be treated. When administered in multiple doses, the pharmaceutical composition can be conveniently divided into appropriate amounts for each unit of administration. The dosage will vary according to the age, weight, and general health of the subject, as is well known in the art of therapy.

[0085] In some embodiments, the pharmaceutical composition contains a plurality of tau peptide immunogen constructs. The pharmaceutical composition contains a mixture of a plurality of tau peptide immunogen constructs and can synergistically enhance the immunogenicity of the constructs. The pharmaceutical composition containing a plurality of tau peptide immunogen constructs is more effective in a larger genetic population because it broadly covers MHC class II, and thus can improve the immune response against the tau peptide immunogen construct.

[0086] In some embodiments, the pharmaceutical composition contains the tau peptide immunogen constructs shown in Table 1, as well as homologs, analogs, and / or combinations thereof. In some embodiments, the pharmaceutical composition contains a tau peptide immunogen construct containing a tau peptide from one of Tables 1 and 4-7.

[0087] The pharmaceutical composition containing the tau peptide immunogen construct can be used to induce an immune response and produce antibodies in a host upon administration.

[0088] c. Immunostimulatory complex The present disclosure also relates to a pharmaceutical composition containing a tau peptide immunogen construct in the form of an immunostimulatory complex with a CpG oligonucleotide. Such an immunostimulatory complex is specifically adapted to act as an adjuvant and as a peptide immunogen stabilizer. The immunostimulatory complex is in the form of particles, which can efficiently present the tau peptide immunogen to the cells of the immune system to generate an immune response. The immunostimulatory complex may be formulated as a suspension for parenteral administration. The immunostimulatory complex can also be formulated in the form of a w / o emulsion as a suspension combined with an inorganic salt or an in-situ gelling polymer to efficiently deliver the tau peptide immunogen to the cells of the host immune system after parenteral administration.

[0089] A stabilized immunostimulatory complex can be formed by complexing a tau peptide immunogen construct with an anionic molecule, oligonucleotide, polynucleotide, or a combination thereof via electrostatic association. The stabilized immunostimulatory complex may be incorporated into a pharmaceutical composition as an immunogen delivery system.

[0090] In certain embodiments, the tau peptide immunogen construct is designed to contain a cationic moiety that is positively charged at a pH in the range of 5.0 - 8.0. The net charge of the cationic moiety of the tau peptide immunogen construct or mixture of constructs is calculated by assigning a charge of +1 to each lysine (K), arginine (R), or histidine (H) in the sequence, a charge of -1 to each aspartic acid (D) or glutamic acid (E), and a charge of 0 to other amino acids. The charges are summed within the cationic moiety of the tau peptide immunogen construct and represented as the net average charge. Suitable peptide immunogens have a cationic moiety with a net average positive charge of +1. Preferably, the peptide immunogen has a net positive charge in a range greater than +2. In some embodiments, the cationic moiety of the tau peptide immunogen construct is a heterologous spacer. In certain embodiments, the cationic moiety of the tau peptide immunogen construct has a charge of +4 when the spacer sequence is (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 169).

[0091] As used herein, "anionic molecule" refers to any molecule that is negatively charged at a pH in the range of 5.0 - 8.0. In certain embodiments, the anionic molecule is an oligomer or polymer. The net negative charge on the oligomer or polymer is calculated by assigning a charge of -1 to each phosphodiester or phosphorothioate group in the oligomer. Suitable anionic oligonucleotides are single-stranded DNA molecules having 8 - 64 nucleotide bases, and the number of CpG motif repeats ranges from 1 - 10. Preferably, the CpG immunostimulatory single-stranded DNA molecule contains 18 - 48 nucleotide bases, and the number of CpG motif repeats ranges from 3 - 8.

[0092] More preferably, the anionic oligonucleotide has the formula: 5’X 1 CGX 2 3’, wherein C and G are not methylated, and X 1 is selected from the group consisting of A (adenine), G (guanine), and T (thymine), and X 2 is C (cytosine) or T (thymine). Alternatively, the anionic oligonucleotide has the formula: 5’(X 3 )2CG(X 4 )23’, wherein C and G are not methylated, and X 3 is selected from the group consisting of A, T, or G, and X 4 is C or T.

[0093] In some embodiments, the CpG oligonucleotide is as described in WO03 / 068169, the content of which is incorporated by reference. In some embodiments, the CpG comprises or consists of the sequence of CpG1 (tcgtcgtttt gtcgttttgt cgtttttgtcg tt; SEQ ID NO: 170), CpG2 (tcgtcgtttt gtcgttttgt cgtt; SEQ ID NO: 171), or CpG3 (tcgtcgtttt gtcgtttttgt cgtt; SEQ ID NO: 173). In some embodiments, the CpG oligonucleotide (e.g., CpG1, CpG2, or CpG3) is a phosphorothioate oligonucleotide.

[0094] The resulting immunostimulatory complex is typically in the form of particles having a size in the range of 1 to 50 microns and is a function of many factors including the relative charge stoichiometry and molecular weight of the interacting species. The micronized immunostimulatory complex has the advantage of providing in vivo adjuvantation and upregulation of specific immune responses. Furthermore, the stabilized immunostimulatory complex is suitable for preparing pharmaceutical compositions by various processes including water-in-oil emulsions, inorganic salt suspensions, and polymeric gels.

[0095] antibody The present invention also provides antibodies induced by tau peptide immunogen constructs.

[0096] The disclosed tau peptide immunogen constructs include tau fragments, heterologous Th epitopes, and optional heterologous spacers, and can induce an immune response and antibody production when administered to a host. By the design of the tau peptide immunogen construct, tolerance to self-tau can be disrupted, and the production of site-specific antibodies that recognize conformational rather than linear epitopes can be induced.

[0097] Antibodies produced by tau peptide immunogen constructs recognize and bind to tau in monomeric, dimeric, trimeric, and oligomeric forms.

[0098] Surprisingly, antibodies induced by tau peptide immunogen constructs can prevent the aggregation of tau (anti-aggregation activity) and can also dissociate pre-formed tau aggregates (disaggregation activity).

[0099] The immune response obtained from animals immunized with the tau peptide immunogen construct of the present invention demonstrated the ability of the construct to generate strong site-specific antibodies that react with tau in monomeric, dimeric, trimeric, and oligomeric forms.

[0100] Methods The present invention also relates to methods for manufacturing and using tau peptide immunogen constructs, compositions, and pharmaceutical compositions.

[0101] a. Method for manufacturing a tau peptide immunogen construct The tau peptide immunogen constructs of the present disclosure can be made by chemical synthesis methods well known to those skilled in the art (see, e.g., Fields et al., Chapter 3 in Synthetic Peptides: A User’s Guide, ed. Grant, W.H. Freeman & Co., New York, NY, 1992, p. 77). The tau peptide immunogen constructs can be synthesized, for example, using the automated Merrifield technique of solid-phase synthesis using α-NH2 protected by either t-Boc or F-moc chemistry with side-chain protected amino acids on an Applied Biosystems Peptide Synthesizer Model 430A or 431. Preparation of tau peptide immunogen constructs containing combinatorial library peptides for Th epitopes can be accomplished by providing a mixture of alternative amino acids for coupling at a given variable position.

[0102] After the desired tau peptide immunogen construct is fully assembled, the resin can be processed according to standard procedures to cleave the peptide from the resin and deblock the functional groups on the amino acid side chains. The free peptide can be purified by HPLC and characterized biochemically, for example, by amino acid analysis or sequencing. Methods for peptide purification and characterization are well known to those skilled in the art. The quality of the peptides produced by this chemical process can be controlled and defined, and as a result, the reproducibility, immunogenicity, and yield of the tau peptide immunogen constructs can be ensured.

[0103] The range of structural variability that allows for the retention of the intended immunological activity has been found to be much more flexible than the range of structural variability that allows for the retention of the specific drug activity by small molecule drugs, or the desired activity and the retention of unwanted toxicity observed in macromolecular drugs co-produced with biologically derived drugs. Thus, peptide analogs that are deliberately designed, or that are inevitably produced by errors in the synthesis process as a mixture of deletion sequence by-products having chromatographic and immunological properties similar to the intended peptide, are often as effective as a purified preparation of the desired peptide. If discrimination QC procedures are developed to monitor both the manufacturing process and the product evaluation process to ensure the reproducibility and effectiveness of the final product using these peptides, mixtures of designed and undesigned analogs are effective.

[0104] Tau peptide immunogen constructs can also be made using recombinant DNA techniques including nucleic acid molecules, vectors, and / or host cells. Thus, nucleic acid molecules encoding tau peptide immunogen constructs and immunologically functional analogs thereof are also encompassed in this disclosure as part of the present invention. Similarly, vectors such as expression vectors containing nucleic acid molecules, and host cells carrying the vectors are also encompassed in this disclosure as part of the present invention.

[0105] Various exemplary embodiments also include methods of producing tau peptide immunogen constructs and immunologically functional analogs thereof. For example, the method can include incubating a host cell containing an expression vector containing a nucleic acid molecule encoding a tau peptide immunogen construct and / or an immunologically functional analog thereof under conditions such that the peptide and / or analog is expressed. Longer synthetic peptide immunogens can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct the gene encoding the peptide of the present invention, the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence with codons optimized preferably for the organism in which the gene is to be expressed. Next, a synthetic gene is typically created by synthesizing oligonucleotides encoding the peptide and optionally any regulatory elements. The synthetic gene is inserted into a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

[0106] b. Method for producing immunostimulatory complex Various exemplary embodiments also include a method of generating an immunostimulatory complex comprising a tau peptide immunogen construct and a CpG oligodeoxynucleotide (ODN) molecule. The stabilized immunostimulatory complex (ISC) is derived from the cationic portion of the tau peptide immunogen construct and the polyanionic CpG ODN molecule. The self-assembly system is driven by electrostatic neutralization of charges. The degree of association is determined by the stoichiometry of the molar charge ratio of the cationic portion of the tau peptide immunogen construct to the anionic oligomer. The non-covalent electrostatic association of the tau peptide immunogen construct and CpG ODN is a completely reproducible process. The peptide / CpG ODN immunostimulatory complex aggregates facilitate presentation to "professional" antigen-presenting cells (APCs) of the immune system and thus further enhance the immunogenicity of the complex. These complexes are readily characterized for quality control during manufacture. The peptide / CpG ISC is well tolerated in vivo. This novel particulate system comprising CpG ODN and a peptide immunogen construct derived from a tau fragment is designed to utilize the generalized B cell mitogenicity associated with the use of CpG ODN and to further promote a balanced Th-1 / Th-2 type response.

[0107] The CpG ODN in the disclosed pharmaceutical composition binds 100% to the immunogen in a process mediated by electrostatic neutralization of opposite charges, resulting in the formation of micron-sized particles. This particulate form allows the dose of CpG to be significantly reduced compared to the conventional use of CpG adjuvants, reduces the likelihood of harmful innate immune responses, and promotes an alternative immunogen processing pathway involving antigen-presenting cells (APCs). Thus, such formulations are conceptually novel and offer potential advantages by promoting the stimulation of immune responses by alternative mechanisms.

[0108] c. Method for manufacturing a pharmaceutical composition Various exemplary embodiments also include pharmaceutical compositions containing a tau peptide immunogen construct. In certain embodiments, the pharmaceutical composition uses an oil-in-water emulsion and a suspension containing mineral salts.

[0109] For a pharmaceutical composition to be used by many people and for the prevention of tau aggregation to also be part of the purpose of administration, safety becomes another important factor to be considered. In clinical trials, oil-in-water emulsions are used in humans in many formulations, but for safety reasons, alum remains the main adjuvant used in formulations. Therefore, alum or its mineral salt, aluminum phosphate (ADJUPHOS), is frequently used as an adjuvant in the preparation of clinical applications.

[0110] Other adjuvants and immunostimulants include 3-de-O-acylated monophosphoryl lipid A (MPL) or 3-DMP, polymers or monomeric amino acids such as polyglutamic acid or polylysine. Such adjuvants can be used with or without other specific immunostimulants such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1’-2’ dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide (trademark)), or other bacterial cell wall components. Oil-in-water emulsions include MF59 (see WO90 / 14837 of Van Nest et al., which is incorporated herein by reference in its entirety), containing 5% squalene, 0.5% Tween® 80, and 0.5% Span® 85 (optionally containing varying amounts of MTP-PE), formulated into submicron particles using a microfluidizer; SAF containing 10% squalene, 0.4% Tween® 80, 5% of the pluronic®-block polymer L121, and thr-MDP, which is microfluidized into a submicron emulsion or vortexed into an emulsion of larger particle size; and Ribi® adjuvant system (RAS) (Ribi ImmunoChem, Hamilton, Mont.) containing one or more bacterial cell wall components selected from the group consisting of 2% squalene, 0.2% Tween® 80, and monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox®).Other adjuvants include complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), and cytokines such as interleukin (IL-1, IL-2, and IL-12), granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF).

[0111] The choice of adjuvant is determined by the stability of the immunogenic formulation containing the adjuvant, the route of administration, the dosing schedule, and the effectiveness of the adjuvant for the species being vaccinated. In the case of humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for administration to humans by the appropriate regulatory agency. For example, alum, MPL, or incomplete Freund's adjuvant (Chang et al., Advanced Drug Delivery Reviews 32:173-186 (1998), which is incorporated herein by reference in its entirety) are suitable for administration to humans either alone or, optionally, in any combination thereof.

[0112] The composition may include a pharmaceutically acceptable non-toxic carrier or diluent, defined as a vehicle commonly used to formulate pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution, and Hank's solution. Additionally, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0113] The pharmaceutical composition may also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid and copolymers (e.g., latex-functionalized sepharose, agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). Additionally, these carriers can function as immunostimulants (adjuvants).

[0114] The pharmaceutical composition of the present invention can further include a suitable delivery vehicle. Suitable delivery vehicles include, but are not limited to, viruses, bacteria, biodegradable microspheres, microparticles, nanoparticles, liposomes, collagen minipellets, and coacervates.

[0115] d. Method of using the pharmaceutical composition The present disclosure also includes a method of using a pharmaceutical composition containing a tau peptide immunogen construct.

[0116] In certain embodiments, a pharmaceutical composition containing a tau peptide immunogen construct can be used for the following purposes. (a) Inhibiting tau aggregation in a host; (b) Inducing disaggregation of pre-formed tau aggregates in a host; (c) Reducing neurodegeneration caused by exogenous tau aggregates in a host; (d) Reducing neurodegeneration in tau overexpressing cells; (e) Decreasing the tau concentration in the serum of a host; (f) Decreasing the level of oligomeric tau in the brain of a host; (g) Reducing neuropathology and restoring motor activity in a host, etc.

[0117] The above methods include administering to a subject in need thereof a pharmaceutical composition comprising a pharmacologically effective amount of a tau peptide immunogen construct.

[0118] As used herein, "tauopathy" encompasses any neurodegenerative disease associated with the pathological aggregation of the microtubule protein tau in the brain. Thus, in addition to both familial and sporadic Alzheimer's disease, other tauopathies that can be treated using the methods of the present invention include frontotemporal dementia, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, neurofibrillary change dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis / Parkinsonism / dementia complex, pantothenate kinase-associated neurodegeneration, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary changes, postencephalitic Parkinsonism, and chronic traumatic encephalopathy, but is not limited thereto. The method can further be used for the prevention and treatment of Lewy body disease.

[0119] Another aspect of the disclosure relates to a method for promoting the removal of tau aggregates from the brain of a subject. The method comprises administering to the subject one or more immunogenic tau peptides of any one of Table 1 or Tables 4-7, or one or more antibodies that recognize an epitope of such a peptide, under conditions effective to promote the removal of tau aggregates from the brain of the subject.

[0120] Removal of tau aggregates includes removal of neurofibrillary changes and / or pathological tau precursors relative to neurofibrillary changes. Neurofibrillary changes are associated in many cases with neurodegenerative diseases such as, for example, sporadic and familial Alzheimer's disease, amyotrophic lateral sclerosis, argyrophilic grain dementia, Pick's disease, chronic traumatic encephalopathy, diffuse neurofibrillary tangles with calcification, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, hereditary frontotemporal dementia, Parkinsonism linked to chromosome 17 (FTDP-17), inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, Pick's disease, prion protein cerebral amyloid angiopathy, sporadic corticobasal degeneration, progressive supranuclear palsy, subacute sclerosing panencephalitis, myotonic dystrophy, motor neuron diseases with neurofibrillary changes, neurofibrillary change dementias, and progressive subcortical gliosis.

[0121] Another aspect of the disclosure relates to a method of retarding the progression of tau pathology-related behavioral phenotypes in a subject. The method includes administering to the subject one or more immunogenic tau peptides of Table 1 or any one or more of Tables 4-7, or one or more antibodies that recognize an immunogenic tau epitope of such a peptide, under conditions effective to retard the subject's tau pathology-related behavioral phenotype.

[0122] As used herein, tau pathology-related behavioral phenotypes include, but are not limited to, cognitive impairment, early personality change and disinhibition, emotional blunting, apathy, mutism, apraxia, perseveration, stereotyped movements / behaviors, oral tendencies, confusion, inability to plan or organize sequential tasks, self-centered / rude, antisocial traits, lack of empathy, echolalia, paraphasic speech with frequent errors but relatively preserved comprehension, decreased comprehension and anomia, slowly progressive gait instability, retropulsion, freezing, frequent falls, non-levodopa-responsive axial rigidity, supranuclear gaze palsy, square wave jerks, slow vertical saccadic movements, pseudobulbar palsy, limb apraxia, dystonia, cortical sensory loss, and tremors.

[0123] According to the method of the present disclosure, in one embodiment, an immunogenic tau peptide or a combination of immunogenic tau peptides is administered to a subject in need thereof. Suitable immunogenic tau peptide fragments of the tau protein include one or more antigenic epitopes that mimic the pathological form of the tau protein. Exemplary immunogenic tau epitopes are phosphorylated at one or more amino acids that are phosphorylated in the pathological form of tau but not in the normal or non-pathological form of tau.

[0124] In some embodiments, administration of the immunogenic tau peptide induces an active immune response in the subject against the immunogenic tau peptide and the pathological form of tau, thereby promoting the removal of associated tau aggregates, slowing the progression of tau pathology-related behavior, and treating the underlying tauopathy. According to this aspect of the invention, the immune response involves the generation of beneficial humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secreted products) responses against the immunogenic tau peptide.

[0125] The presence of a humoral immune response can be determined and monitored by testing a biological sample (e.g., blood, plasma, serum, urine, saliva, feces, CSF, or lymph) from the subject for the presence of antibodies against the immunogenic tau peptide. Methods for detecting antibodies in biological samples are well known in the art and include, for example, ELISA, dot blot, SDS-PAGE gel, or ELISPOT. The presence of a cell-mediated immune response can be determined by proliferation assays (CD4+ T cells) or CTL (cytotoxic T lymphocyte) assays that are readily known in the art.

[0126] The isolated immunogenic tau peptide of the present invention is any one of the amino acid sequences in Tables 1 and 4-7 below, includes, or comprises the same. In preferred embodiments, the peptides in Tables 4-7 have at least one value greater than 1.

[0127] Specific embodiments (1) The tau peptide immunogen construct has the formula: (Th) m -(A) n -(tau fragment)-X or (tau fragment)-(A) n -(Th) m -X (wherein, Th is a heterologous T helper epitope, A is a heterologous spacer, (tau fragment) is a B cell epitope having about 8 to about 40 amino acid residues (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids) from the full-length tau protein of SEQ ID NO: 167, X is the α-COOH or α-CONH2 of an amino acid, m is from 1 to about 4, n is from 0 to about 10).

[0128] (2) The tau fragment is selected from the group consisting of SEQ ID NOs: 1 to 8, 36, 39, 40, 48, 50, 51, 52, 57, 58, 59, 62, 87, 88, 129, 49, 35, 99, 135, and 137, the tau peptide immunogen construct according to (1).

[0129] (3) The Th epitope is selected from the group consisting of SEQ ID NOs: 138 to 166, for example, 151, 152, or 149, the tau peptide immunogen construct according to any one of (1) or (2).

[0130] (4) The peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 9 to 16 (e.g., 10 or 15) (or a construct in which the positions of the tau peptide and the Th epitope are reversed), the tau peptide immunogen construct according to any one of (1) to (3).

[0131] (5) A tau peptide immunogen construct, An about 8- to about 40-amino acid residue (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids) B cell epitope from the full-length tau protein of SEQ ID NO: 167, A T helper epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 138-166, And any heterologous spacer selected from the group consisting of amino acids, Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 169), Wherein the B cell epitope is covalently linked to the T helper epitope directly or via the any heterologous spacer, the tau peptide immunogen construct.

[0132] (6) The B cell epitope is selected from the group consisting of SEQ ID NOs: 1-8, 36, 39, 40, 48, 50, 51, 52, 57, 58, 59, 62, 87, 88, 129, 49, 35, 99, 135, and 137, the tau peptide immunogen construct according to (5).

[0133] (7) The T helper epitope is selected from the group consisting of SEQ ID NOs: 138-166, the tau peptide immunogen construct according to (5) or (6).

[0134] (8) The any heterologous spacer is (α,ε-N)Lys, or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 169), the tau peptide immunogen construct according to any one of (5)-(7).

[0135] (9) The T helper epitope is covalently linked to the amino terminus of the B cell epitope, the tau peptide immunogen construct according to any one of (5)-(8).

[0136] (10) The tau peptide immunogenic construct according to any one of (5) to (8), wherein the T helper epitope is covalently bound to the amino terminus of the B cell epitope via any of the heterologous spacers.

[0137] (11) A composition comprising the tau peptide immunogenic construct according to any one of (1) to (10).

[0138] (12) A pharmaceutical composition, a. The peptide immunogenic construct according to any one of (1) to (10), and b. A pharmaceutically acceptable delivery vehicle and / or adjuvant, the pharmaceutical composition comprising the same.

[0139] (13) a. The tau peptide immunogenic construct is selected from the group consisting of SEQ ID NOs: 9 to 16, or a tau peptide immunogenic construct in which the positions of the B cell epitope and the T cell epitope are reversed as compared to one of SEQ ID NOs: 9 to 16 (e.g., 10 or 15), b. The tau peptide immunogenic construct is mixed with a CpG oligodeoxynucleotide (ODN), for example, CpG1, CpG2, or CpG3, to form a stable immunostimulatory complex, the pharmaceutical composition according to (12).

[0140] (14) An isolated antibody or an epitope-binding fragment thereof that specifically binds to the B cell epitope of the tau peptide immunogenic construct according to any one of (1) to (10).

[0141] (15) The isolated antibody or an epitope-binding fragment thereof according to (14) bound to the tau peptide immunogenic construct.

[0142] (16) A composition comprising the isolated antibody or an epitope-binding fragment thereof according to (14) or (15).

[0143] (17) A tau peptide immunogen construct, a. MAEPRQEFEVMEDHAGTYGLGDKKK-eK-ISITEIKGVIVHRIETILF-NH2; SEQ ID NO: 9 b. DHAGTYGLGDKKK-eK-ISITEIKGVIVHRIETILF-NH2; SEQ ID NO: 10 c. ISITEIKGVIVHRIETILF-eK-KKKNITHVPGGGNKK-NH2; SEQ ID NO: 11 d. ISITEIKGVIVHRIETILF-eK-KKKKDNIKHVPGGGSVQIVYK-NH2; SEQ ID NO: 12 e. ISITEIKGVIVHRIETILF-eK-KKKTKIATPRGAAPP-NH2; SEQ ID NO: 13 f. ISITEIKGVIVHRIETILF-eK-KKKVVRTPPKSPSSAKSRL-NH2; SEQ ID NO: 14 g. ISITEIKGVIVHRIETILF-eK-KKKIKHVPGGGSVQIVYK-NH2; and SEQ ID NO: 15 h. ISITEIKGVIVHRIETILF-eK-KKKVSGDTSPRHLSNVSST-NH2 SEQ ID NO: 16 The tau peptide immunogen construct selected from the group consisting of.

[0144] (18) A composition comprising the tau peptide immunogen construct according to (17).

[0145] (19) A pharmaceutical composition, a. The tau peptide immunogen construct according to (17), and b. A pharmaceutically acceptable delivery vehicle and / or adjuvant, the pharmaceutical composition comprising.

[0146] (20) The pharmaceutical composition according to (19), wherein the tau peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN), for example, CpG1, CpG2, or CpG3, to form a stable immunostimulatory complex.

[0147] (21) The pharmaceutical composition according to (19) or (20), wherein the composition comprises an aluminum adjuvant (aluminum phosphate or aluminum hydroxide) or another adjuvant described herein.

[0148] (22) A method for preventing, inhibiting, reducing the severity, delaying, or treating tauopathy in a subject, the method comprising administering to the subject a tau peptide immunogen construct or a composition of any one of (1)-(21).

[0149] (23) The tauopathy according to (22), which is selected from the group consisting of Alzheimer's disease, Lewy body disease, frontotemporal dementia, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, neurofibrillary change type dementia, diffuse neurofibrillary tangles with calcification, argentophilic grain dementia, amyotrophic lateral sclerosis / Parkinsonism / dementia complex, punchdrunk, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary changes, postencephalitic Parkinsonism, and chronic traumatic encephalopathy.

Example

[0150] Example 1 Accumulation of misfolded tau in the brain correlates with the clinical decline of Alzheimer's disease (AD), but appears decades earlier than cognitive symptoms (Congdon and Sigurdsson, Nat. Rev. Neurol, 2018). Targeting pathological tau before the clinical onset of AD may help prevent the disease and / or its progression. We have developed a vaccine-based immunotherapy for the prevention and treatment of Alzheimer's disease. The results of the preclinical characterization of the tau vaccine are shown in Table A below. The peptides described in the figure can be cross-referenced based on column 1 of Table 1.

Table A

[0151] The vaccine-based platform is shown in Figure 1 and includes a B cell epitope, a spacer or linker, and a Th peptide carrier. B cells recognize the B cell epitope and generate antibodies against peptides that mimic a biological target (e.g., tau). The spacer chemically couples the B cell epitope to the Th peptide, thereby optimizing the presentation of the B cell epitope to the immune system. The Th peptide carrier activates T helper cells, warns B cells to initiate the production of antibodies against the B cell epitope, while avoiding inflammation and off-target activity. The primary structure of tau annotated with functional domains and targeting peptides is shown at the bottom of Figure 1. The peptides shown in this figure and other figures described in this specification are as follows.

[0152] The construct was formulated with 100 ug / mL Adju-Phos CpG1 + 300 ug / 0.25 mL peptide. Guinea pigs were given five intramuscular injections at three-week intervals and terminal bleeds were taken at week 15. Antibodies against T helper peptide, adjuvant components, and tau were quantified by ELISA in serial dilutions. Furthermore, antibody binding was characterized against recombinant and brain-derived tau preparations by Western blot, dot blot, and Biolayer Interference (BLI). Evaluation of in vitro function was performed by tau FRET aggregation and pHrodo uptake assays in HEK293 and B103 cells.

[0153] The results of the characterization of antibody binding are shown in FIGS. 2A and 2B. The binding ability of vaccine-derived antibodies against three forms of recombinant tau (monomer, oligomer, and PFF) and Sarkosyl extract (Br) from postmortem brain tissue was evaluated by dot blot (FIG. 2A). Diverse binding profiles were observed against the various forms of tau. The results of BLI showed that the antibody binds from the construct with a Kd in the nM range to tau forms and that the three constructs have a slower off-rate than bepranemab (FIG. 2B). Representative binding curves of Bep and construct A to PFF are presented.

[0154] The results of the aggregation assay using the tau biosensor line are shown in FIGS. 3A and 3B. The tau biosensor line (Frost et al., J. Biol. Chem., 2009) was used to evaluate the functional inhibition of vaccine-derived antibodies compared to anti-tau mAb, semorinemab, and bepranemab. Assay conditions included the addition of Lipofectamine (FIG. 3A) and no Lipofectamine (FIG. 3B). Antibodies that inhibit aggregation were produced only under conditions without Lipofectamine.

[0155] The results of the tau uptake assay are shown in Figures 4A and 4B. B103 cells were exposed to the pHrodo-labeled tau preparation for 6 hours. When taken up, pHrodo-tau emits fluorescence, which could be quantified using an IncuCyte live cell imaging device. Constructs A, B, C, and F inhibited the uptake of monomeric tau in a dose response (Figure 4A). All constructs decreased PFF uptake in a dose-dependent manner (Figure 4B). The potencies of the constructs varied, but all showed more potent inhibition of PFF than monomeric tau uptake.

[0156] Antibodies from tau candidates show diverse binding profiles against various forms of tau. No significant immunogenicity was observed against Th1 peptide or CpG1. In functional assays, it has been shown that binding antibodies prevent aggregation by inhibiting uptake.

[0157] Example 2 Vaccination experiments were conducted in WT mice and P301L mice. Figure 5A shows that P301L mice vaccinated with p5555kb generate strong titers against monomeric and PFF tau. Figure 5B shows that vaccination with p5555kb reduces tau accumulation in P301L. Figure 5C shows that lysates from the brains of P301L mice vaccinated with p5555kb show evidence of a decrease in aggregation induction in the tau biosensor line. These results indicate that both WT mice and P301L mice show a positive response to vaccination with p5555kb.

[0158] Experimental Example 3 The immunogenicities of p5555kb and p5187kb were evaluated in rats. The results are shown in Figure 6. ELISA analysis showed that the titers were comparable to those in guinea pig experiments. Thus, these constructs have immunogenicity in various species.

[0159] In further experiments, isotype analysis of antibodies generated in rats was performed. Ten male Sprague Dawley rats weighing 200 - 250 g per group were used in two groups (20 rats). The vaccine was administered at 0, 3, 6, 9, and 12 weeks (intramuscularly, 0.25 mL / dose). The blood sampling schedule was before administration at 0, 3, 6, 9, and 12 weeks, and the final blood sampling was at 15 weeks. Blood samples were separated into serum and placed on dry ice before subsequent thawing and analysis. Group 1: p5555kb, 10 animals, 30 μg / 0.25 mL / dose / IM (Adjuphos, CpG1), Group 2: p5187, 10 animals, 30 μg / 0.25 mL / dose / IM (Adjuphos, CpG1). The results are shown in Figures 7 - 10, (i) the total IgG titer of Group 1 (p5555kb) was significantly higher at time points after day 42, (ii) IgG1 was the dominant isotype in all groups, followed by IgG2b, IgG2c, and IgG2a, suggesting a TH-2 response, (iii) the antibody titers of Groups 1 and 2 reached a plateau (104 - 5) after the time point of day 42 (second boost).

[0160] In additional experiments, Western blot analysis was performed to evaluate the binding of antibodies to brain tissues (normal hippocampus, normal temporal lobe, lesioned hippocampus, and lesioned temporal lobe). This data is consistent with the data in guinea pigs, and p5187 and p5555 rat IgG bind well to human brain lysates.

[0161] In the tau aggregation assay, rat TgG from p5555 and p5187 was shown to inhibit tau aggregation at levels equivalent to those of the monoclonal antibodies SemorinemAb and BeprenemAb (Figure 11).

[0162] In further experiments, the immunogenicity of p5555kb and p5187kb was evaluated in mice. The results are shown in Figure 12, indicating that strong titers against the monomeric forms of PFF and tau were obtained with p5555 and p5187.

Table 1

Table 2-1

Table 2-2

Table 3

Table 4

Table 5

Table 6

Table 7

Claims

**Claim 1** A tau peptide immunogen construct having the formula: (Th) ｍ -(A) ｎ -(tau fragment)-X or (Tau fragment) - (A) ｎ - (Th) ｍ - X wherein Th is a heterologous T helper epitope, A is a heterologous spacer, (tau fragment) is a B cell epitope having from about 10 to about 40 amino acid residues from the full-length tau protein of SEQ ID NO: 167, X is the α-COOH or α-CONH of an amino acid ２ and m is 1 to about 4, n is 0 to about 10), said tau peptide immunogen construct. **Claim 2** The tau peptide immunogen construct according to claim 1, wherein the tau fragment is selected from the group consisting of SEQ ID NOs: 1-8, 36, 39, 40, 48, 50, 51, 52, 57, 58, 59, 62, 87, 88, 129, 49, 35, 99, 135, and 137. **Claim 3** The tau peptide immunogen construct according to any one of claims 1 or 2, wherein the Th epitope is selected from the group consisting of SEQ ID NOs: 138-166, such as 151, 152, or 149. **Claim 4** The tau peptide immunogen construct according to claim 1, wherein the peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 9-16 (such as 10 or 15), or is a construct in which the positions of the tau peptide and the Th peptide are reversed. **Claim 5** A tau peptide immunogen construct, a B cell epitope having from about 8 to about 40 amino acid residues (such as about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids) from the full-length tau protein of SEQ ID NO: 167, a T helper epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 138-166, and any heterologous spacer selected from the group consisting of the amino acids, Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 169), wherein the B cell epitope is covalently attached to the T helper epitope directly or via said any heterologous spacer, said tau peptide immunogen construct. **Claim 6** The tau peptide immunogen construct according to claim 5, wherein the B cell epitope is selected from the group consisting of SEQ ID NOs: 1 to 8, 36, 39, 40, 48, 50, 51, 52, 57, 58, 59, 62, 87, 88, 129, 49, 35, 99, 135, and 137.

7. The tau peptide immunogen construct according to claim 5 or 6, wherein the T helper epitope is selected from the group consisting of SEQ ID NOs: 138 to 166.

8. The tau peptide immunogen construct according to claim 5 or 6, wherein the optional heterologous spacer is (α,ε-N)Lys or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 169).

9. The tau peptide immunogen construct according to claim 5 or 6, wherein the T helper epitope is covalently linked to the amino terminus of the B cell epitope.

10. The tau peptide immunogen construct according to claim 5 or 6, wherein the T helper epitope is covalently linked to the amino terminus of the B cell epitope via the optional heterologous spacer.

11. A composition comprising the tau peptide immunogen construct according to any one of claims 1, 2, 4, 5, or 6.

12. A pharmaceutical composition, a. the tau peptide immunogen construct according to any one of claims 1, 2, 4, 5, or 6, and b. a pharmaceutically acceptable delivery vehicle and / or adjuvant, the pharmaceutical composition comprising the same.

13. a. The tau peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 9 to 16 (e.g., 10 or 15), or is a tau peptide immunogen construct in which the positions of the B cell epitope and the T cell epitope are reversed as compared to one of SEQ ID NOs: 9 to 16 (e.g., 10 or 15), b. The tau peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN), e.g., CpG1, CpG2, or CpG3, to form a stable immunostimulatory complex, the pharmaceutical composition according to claim 12.

14. An isolated antibody or an epitope-binding fragment thereof that specifically binds to the B cell epitope of the tau peptide immunogen construct according to any one of claims 1, 2, 4, 5, or 6.

15. The isolated antibody or epitope-binding fragment thereof according to claim 14, which binds to the tau peptide immunogen construct.

16. A composition comprising the isolated antibody or epitope-binding fragment thereof according to claim 14.

17. A tau peptide immunogen construct, a. MAEPRQEFEVMEDHAGTYGLGDKKK-eK-ISITEIKGVIVHRIETILF-NH2; SEQ ID NO: 9 b. DHAGTYGLGDKKK-eK-ISITEIKGVIVHRIETILF-NH2; SEQ ID NO: 10 c. ISITEIKGVIVHRIETILF-eK-KKKNITHVPGGGNK-K-NH2; SEQ ID NO: 11 d. ISITEIKGVIVHRIETILF-eK-KKKKDNIKHVPGGGSVQIVYK-NH2; SEQ ID NO: 12 e. ISITEIKGVIVHRIETILF-eK-KKKTKATPRAAP-NH2; SEQ ID NO: 13 f. ISITEIKGVIVHRIETILF-eK-KKKVVRTPPKSPSSAKSRL-NH2; SEQ ID NO: 14 g. ISITEIKGVIVHRIETILF-eK-KKKIKHVPGGGSVQIVYK-NH2; and SEQ ID NO: 15 h. ISITEIKGVIVHRIETILF-eK-KKKVSGDTSPRHLSSNVSSST-NH2 SEQ ID NO: 16 The tau peptide immunogen construct selected from the group consisting of.

18. A composition comprising the tau peptide immunogen construct according to claim 17.

19. A pharmaceutical composition, a. The tau peptide immunogen construct according to claim 17, and b. A pharmaceutically acceptable delivery vehicle and / or adjuvant, the pharmaceutical composition comprising.

20. The pharmaceutical composition according to claim 19, wherein the tau peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) (for example, CpG1, CpG2, or CpG3) to form a stable immunostimulatory complex.

21. The pharmaceutical composition according to claim 19 or 20, wherein the composition comprises an aluminum-based adjuvant (aluminum phosphate or aluminum hydroxide) or another adjuvant described herein.

22. A method for preventing, inhibiting, reducing the severity, delaying, or treating tauopathy in a subject, the method comprising administering to the subject a tau peptide immunogen construct, or a composition of any one of the preceding claims.

23. The tauopathy is selected from the group consisting of Alzheimer's disease, Lewy body disease, frontotemporal dementia, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, neurofibrillary change dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis / Parkinsonism / dementia complex, punch-drunk, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, The method according to claim 22, selected from the group consisting of prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary changes, post-encephalitic Parkinsonism, and chronic traumatic encephalopathy.

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