Multiepitope vaccine for the treatment of Alzheimer's disease

Abstract

To provide a multiepitope vaccine for the treatment of Alzheimer's disease. [Solution] This disclosure provides peptide compositions and immunotherapy compositions comprising amyloid-beta (Aβ) peptide and tau peptide. This disclosure also provides methods for treating or preventing Alzheimer's disease or other diseases involving beta-amyloid deposition in subjects, including methods for removing deposits, inhibiting or reducing the aggregation of Aβ and / or tau, blocking uptake by neurons, removing amyloid, and inhibiting the transmission of tau seeds in subjects having or at risk of developing Alzheimer's disease or other diseases involving tau and / or amyloid-beta accumulation. The methods include administering a composition comprising amyloid-beta (Aβ) peptide and tau peptide to such patients.

Description

[Technical Field]

[0001] Related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 140,917 filed on 24 January 2021, U.S. Provisional Patent Application No. 63 / 062,903 filed on 7 August 2020, and U.S. Provisional Patent Application No. 63 / 027,150 filed on 19 May 2020, each of which is incorporated herein by reference in whole.

[0002] Sequence listing statement A computer-readable sequence listing is filed electronically with this application and is incorporated herein by reference in its entirety. The sequence listing was created on 19 May 2021 and is contained in a file named "20-050-WO_Sequence-Listing_ST25.txt" with a size of 183kb.

[0003] field This disclosure relates to the fields of immunology and pharmaceutical technology, particularly to the treatment of Alzheimer's disease and other diseases of protein misfolding. [Background technology]

[0004] background Alzheimer's disease (AD) is a progressive disease that leads to senile dementia. Broadly speaking, the disease is classified into two categories: late-onset, which occurs in old age (65 years or older), and early-onset, which occurs considerably earlier, i.e., between the ages of 35 and 60. In both types of the disease, the pathology is the same, but the abnormalities tend to be more severe and widespread in cases that begin at an earlier age. The disease is characterized by at least two types of lesions in the brain: neurofibrillary tangles and senile plaques. Neurofibrillary tangles are intracellular deposits of microtubule-associated tau protein consisting of two fibers that are intertwined in pairs. Senile plaques (i.e., amyloid plaques) are areas of irregular neural networks up to 150 μm in size, encompassing a central extracellular amyloid deposit, which can be visualized by microscopic analysis of sections of brain tissue. The accumulation of amyloid plaques in the central nervous system is associated with cerebral amyloid angiopathy (CAA) in Down syndrome and other cognitive disorders, as well as age-related macular degeneration in eye diseases.

[0005] The main component of amyloid plaques is a peptide called Aβ or β-amyloid peptide. The Aβ peptide is a 38-43 amino acid, 4 kDa internal fragment of a longer transmembrane glycoprotein called amyloid precursor protein (APP). As a result of proteolytic processing of APP by different secretory enzymes, Aβ is found primarily in both a short form of 40 amino acids and a long form ranging from 42 to 43 amino acids. The hydrophobic transmembrane domain portion of APP is found at the carboxyl terminus of Aβ and may be the main cause of Aβ's ability to aggregate into plaques, especially in the long form. The accumulation of amyloid plaques in the brain ultimately leads to neuronal cell death. Cognitive and physical symptoms associated with this type of neuronal deterioration characterize Alzheimer's disease.

[0006] Another protein reported to occur at increased levels in Alzheimer's patients compared to the general population is tau, a major component of neurofibrillary tangles, which, along with amyloid plaques, is a prominent feature of Alzheimer's disease. Tau tangles consist of abnormal fibrils with a measured diameter of 10 nm, occurring in pairs that spirally wind with a regular period of 80 nm. Tau within neurofibrillary tangles is abnormally phosphorylated (hyperphosphorylated) with phosphate groups bound to specific sites on the molecule. Significant involvement of neurofibrillary tangles is found in Alzheimer's disease in layer II neurons of the entorhinal cortex, the CA1 and uncinate gyrus regions of the hippocampus, the amygdala, and deeper layers of the neocortex (layers III, V, and surface VI). Tau pathology is known to correlate with cognitive decline.

[0007] Therefore, there is a need for new treatments and reagents for the prevention or treatment of Alzheimer's disease, in particular for treatments and reagents that can induce an immune response against Aβ and tau present in patients. [Overview of the project] [Means for solving the problem]

[0008] overview In some embodiments, the disclosure relates to a polypeptide comprising a first peptide containing 3 to 10 amino acids derived from residues 1 to 10 of SEQ ID NO: 01, linked to a second peptide containing 3 to 13 amino acids derived from residues 244 to 400 of SEQ ID NO: 02. For example, the second peptide may be derived from the microtubule-binding region (MTBR) of tau (residues 244 to 372 of SEQ ID NO: 02). The first peptide may be N-terminus of the second peptide, or C-terminus of the second peptide. In addition, the first peptide may contain one amino acid sequence from SEQ ID NOs: 3 to 38 or SEQ ID NOs: 1002 to 1057, and the second peptide may contain one amino acid sequence from SEQ ID NOs: 39 to 56, 83 to 86, or 146 to 996. For example, the first polypeptide may be DAEFRHD (SEQ ID NO: 06), DAEFR (SEQ ID NO: 08), or EFRHD (SEQ ID NO: 21), and the second polypeptide may be 5 to 13 amino acids, for example, QIVYKPV (SEQ ID NO: 39), EIVYKSV (SEQ ID NO: 42), EIVYKSP (SEQ ID NO: 43), EIVYKPV (SEQ ID NO: 44), NIKHVP (SEQ ID NO: 48), VKSKIGST (SEQ ID NO: 801), SKIGSTEN (SEQ ID NO: 817), TENLKHQP (SEQ ID NO: 695), ENLKHQPG (SEQ ID NO: 689), SKIGSTDNIKH (SEQ ID NO: 985), SKIGSKDNIKH (SEQ ID NO: 986), or SKIGSLDNIKH (SEQ ID NO: 988).

[0009] In other embodiments, the first peptide and the second peptide may be linked by a cleavable linker, which may be an amino acid sequence. If present, the cleavable peptide linker may be 1 to 10 amino acids long. In some embodiments, the linker comprises about 1 to 10 amino acids, about 1 to 9 amino acids, about 1 to 8 amino acids, about 1 to 7 amino acids, about 1 to 6 amino acids, about 1 to 5 amino acids, about 1 to 4 amino acids, about 1 to 3 amino acids, about 2 amino acids, or 1 amino acid. In some embodiments, the cleavable peptide linker is 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids. For example, the linker may be arginine-arginine (Arg-Arg), arginine-valine-arginine-arginine (Arg-Val-Arg-Arg (SEQ ID NO: 69)), valine-citrulline (Val-Cit), valine-arginine (Val-Arg), valine-lysine (Val-Lys), valine-alanine (Val-Ala), phenylalanine-lysine (Phe-Lys), glycine-alanine-glycine-alanine (Gly-Ala-Gly-Ala (SEQ ID NO: 80)), Ala-Gly-Ala-Gly (SEQ ID NO: 81) or Lys-Gly-Lys-Gly (SEQ ID NO: 82). In certain embodiments, the polypeptide may be DAEFRHDRRQIVYKPV (SEQ ID NO: 57), DAEFRHDRREIVYKSV (SEQ ID NO: 58), DAEFRHDRRVKSKIGSTGGC (SEQ ID NO: 997), DAEFRHDRRSKIGSTENGGC (SEQ ID NO: 998), DAEFRHDRRTENLKHQPGGC (SEQ ID NO: 999), DAEFRHDRRENLKHQPGGGC (SEQ ID NO: 1000), or DAEFRHDRRSKIGSKDNIKHGGC (SEQ ID NO: 1001).

[0010] In further embodiments, the polypeptide may contain a linker to the carrier at the C-terminus or N-terminus of the polypeptide. The linker, if present, may be 1 to 10 amino acids long. In some embodiments, the linker comprises about 1 to 10 amino acids, about 1 to 9 amino acids, about 1 to 8 amino acids, about 1 to 7 amino acids, about 1 to 6 amino acids, about 1 to 5 amino acids, about 1 to 4 amino acids, about 1 to 3 amino acids, about 2 amino acids, or 1 amino acid. In some embodiments, the linker is 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids. For example, the linker may contain amino acid sequences GG, GGG, AA, AAA, KK, KKK, SS, and SSS. In addition, if the linker to the carrier is at the C-terminus, it may contain a C-terminal cysteine ​​(C). Alternatively, if the linker to the carrier is at the N-terminus, it may contain an N-terminal cysteine ​​(C). For example, the polypeptide may contain the amino acid sequence DAEFRHDRRQIVYKPVXXC (SEQ ID NO: 70), where XX and C are present independently as needed, and if present, XX may be GG, AA, KK, SS, GAGA (SEQ ID NO: 80), AGAG (SEQ ID NO: 81), or KGKG (SEQ ID NO: 82). For example, the polypeptide may contain the amino acid sequence DAEFRHDRREIVYKSVXXC (SEQ ID NO: 79), where XX and C are present independently as needed, and if present, XX may be GG, AA, KK, SS, GAGA (SEQ ID NO: 80), AGAG (SEQ ID NO: 81), and KGKG (SEQ ID NO: 82).

[0011] In other embodiments, the Disclosure relates to an immunotherapy composition comprising the polypeptides of the Disclosure, wherein the polypeptides may be linked to a carrier. The carrier may include serum albumin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid (TT), diphtheria toxoid (DT), genetically modified cross-reactive substances (CRMs) of diphtheria toxin, CRM197, meningococcal outer membrane protein complex (OMPC) and H. influenzae protein D (HiD), rEPA (Pseudomonas aeruginosa exotoxin A), KLH (keyhole limpet hemocyanin), and flagellin.

[0012] Furthermore, embodiments of the present disclosure relate to pharmaceutical formulations comprising polypeptides or immunotherapy compositions of the present disclosure, comprising at least one adjuvant. The adjuvant may be aluminum hydroxide, aluminum phosphate, aluminum sulfate, 3-de-O-acylated monophosphoryl lipid A (MPL), QS-21, QS-18, QS-17, QS-7, TQL1055, Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), oil-in-water emulsions (such as squalene or peanut oil), CpG, polyglutamic acid, polylysine, AddaVax®, MF59®, and combinations thereof. In addition, the formulation may comprise liposomal formulations, diluents, or multiple antigen presentation systems (MAPs). The MAP may comprise one or more of the following: a Lys-based dendritic scaffold, a helper T cell epitope, an immunostimulatory lipophilic moiety, a cell-permeable peptide, a radical-induced polymerization, self-assembling nanoparticles as an antigen presentation platform, and gold nanoparticles.

[0013] Furthermore, embodiments of the present disclosure relate to an immunotherapy composition comprising a first peptide sequence comprising 3 to 10 amino acid residues derived from the first 10 N-terminal residues of SEQ ID NO: 01, and a second peptide sequence comprising 3 to 13 amino acids derived from residues 244 to 400 of SEQ ID NO: 02. The first peptide may comprise one amino acid sequence from SEQ ID NOs: 3 to 38 or SEQ ID NOs: 1002 to 1057, and the second peptide may comprise one amino acid sequence from SEQ ID NOs: 39 to 56, SEQ ID NOs: 83 to 86, or SEQ ID NOs: 146 to 996. Each of the first and second peptides may contain a linker to a carrier at the C-terminal or N-terminal portion of the polypeptide. If present, the linker may comprise an amino acid sequence selected from GG, GGG, AA, AAA, KK, KKK, SS, SSS, GAGA (SEQ ID NO: 80), AGAG (SEQ ID NO: 81), and KGKG (SEQ ID NO: 82), and may comprise a C-terminal cysteine ​​(C). In some embodiments, if the C-terminal residue in the immunogen is any of IVYKPV (SEQ ID NO: 194), VYKPV (SEQ ID NO: 195), YKPV (SEQ ID NO: 196), KPV, or PV, the linker is an amino acid linker that does not have an N-terminal glycine (e.g., GG, GAGA (SEQ ID NO: 80)). The carrier may include serum albumin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid (TT), diphtheria toxoid (DT), genetically modified cross-reactive substances (CRMs) of diphtheria toxin, CRM197, meningococcal outer membrane protein complex (OMPC) and H. influenzae protein D (HiD), rEPA (Pseudomonas aeruginosa exotoxin A), KLH (keyhole limpet hemocyanin), and flagellin.

[0014] In addition, the immunotherapy composition may include at least one pharmaceutically acceptable diluent and / or a multiple antigen presentation system (MAP). The MAP may include one or more of the following: a Lys-type dendritic scaffold, a helper T cell epitope, an immunostimulatory lipophilic moiety, a cell-permeable peptide, a radical-induced polymerization, self-assembling nanoparticles as an antigen presentation platform, and gold nanoparticles.

[0015] The immunotherapy composition may include an immunotherapy composition and at least one adjuvant, for example, the adjuvant may be an immunotherapy composition and a pharmaceutical composition comprising aluminum hydroxide, aluminum phosphate, aluminum sulfate, 3-de-O-acylated monophosphoryl lipid A (MPL), QS-21, QS-18, QS-17, QS-7, TQL1055, Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), oil-in-water emulsion (such as squalene or peanut oil), CpG, polyglutamic acid, polylysine, AddaVax®, MF59®, and combinations thereof.

[0016] Embodiments of this disclosure also cover nucleic acid sequences encoding polypeptides and immunotherapy compositions of this disclosure. Nucleic acids may be included in nucleic acid immunotherapy compositions comprising nucleic acids and at least one adjuvant.

[0017] Furthermore, embodiments of the present disclosure relate to methods for treating or preventing Alzheimer's disease in a subject, and methods for inhibiting or reducing the aggregation of at least one of Aβ and tau in a subject who has or is at risk of developing Alzheimer's disease. The methods include administering an immunotherapy composition, nucleic acid immunotherapy composition, or pharmaceutical formulation of the present disclosure to a subject.

[0018] The method of the present disclosure may include repeating the administration at least two, at least three, at least four, at least five, or at least six times, and may include repeating the administration at intervals of about 21 to about 28 days.

[0019] Furthermore, the methods of the present disclosure are directed to inducing an immune response in an animal. The methods include administering to the animal a polypeptide, immunotherapeutic composition, pharmaceutical formulation or nucleic acid immunotherapeutic composition of the present disclosure in a regimen effective to generate an immune response that includes an antibody that specifically binds to Aβ, tau, or both Aβ and tau. The immune response may include an antibody that specifically binds to the N-terminal region of Aβ and / or the microtubule region of tau.

[0020] In other embodiments, the present disclosure is directed to an immune kit that includes an immunotherapeutic composition of the present disclosure and may include an adjuvant, wherein the immunotherapeutic composition may be in a first container and the adjuvant may be in a second container.

[0021] Furthermore, the present disclosure is directed to a kit that includes a nucleic acid immunotherapeutic composition of the present disclosure and may include an adjuvant. The nucleic acid may be in a first container and the adjuvant may be in a second container. In certain embodiments, for example, the following items are provided. (Item 1) A polypeptide comprising a first peptide comprising 3 to 10 amino acids derived from residues 1 to 10 of SEQ ID NO: 01 linked to a second peptide comprising 3 to 13 amino acids derived from residues 244 to 400 of SEQ ID NO: 02. (Item 2) The polypeptide according to Item 1, wherein the second peptide is derived from the microtubule binding region (MTBR) of tau (residues 244 to 372 of SEQ ID NO: 02). (Item 3) The polypeptide according to Item 1, wherein the first peptide is N-terminal to the second peptide. (Item 4) The polypeptide according to Item 1, wherein the first peptide is C-terminal to the second peptide. (Item 5) (a) The first peptide is

Chemical formula

[0022] [Figure 1]Figure 1 shows the results of an experiment comparing the geometric mean titers of guinea pig serum against the immunogen DAEFRHDRRQIVYKPV (SEQ ID NO: 57) in a tau MTBR epitope containing monomeric Aβ amino acids 1-28 (DAEFRHDSGYEVHHQKLFFAEDVGSNKG; SEQ ID NO: 67), a soluble agglutinated species of Aβ (Aβ42), full-length tau, and N-terminal biotin-containing GGGSVQIVYKPVDLS (SEQ ID NO: 68).

[0023] [Figure 2A] Figure 2A shows the results of an experiment comparing the titers of guinea pig serum against the single-peptide immunogens and dual-peptide immunogens of this disclosure for all forms of Aβ and full-length tau protein (DAEFRHD is SEQ ID NO: 6, QIVYKPV is SEQ ID NO: 39, and DAEFRHDRRQIVYKPV is SEQ ID NO: 57).

[0024] [Figure 2B] Figure 2B shows the results of experiments comparing the titers of guinea pig serum against the single-peptide immunogens and dual-peptide immunogens of this disclosure for all forms of Aβ and full-length tau protein (DAEFRHD is SEQ ID NO: 6, QIVYKPV is SEQ ID NO: 39, EIVYKSP is SEQ ID NO: 43, and DAEFRHDRRQIVYKPV is SEQ ID NO: 57).

[0025] [Figure 3A] Figure 3A shows the staining of Aβ and tau lesions in fresh-frozen human AD brain tissue using serum (1:300 dilution) derived from guinea pigs vaccinated with immunogen 9 (DAEFRHDRRQIVYKPVGGC, SEQ ID NO: 59).

[0026] [Figure 3B] Figure 3B shows the staining of Aβ and tau lesions in fresh-frozen human AD brain tissue using serum (1:300 dilution) derived from guinea pigs vaccinated with immunogen 9 (DAEFRHDRRQIVYKPVGGC, SEQ ID NO: 59).

[0027] [Figure 3C] Figure 3C shows the staining of Aβ and tau lesions in fresh-frozen human AD brain tissue using serum (1:1500 dilution) derived from guinea pigs vaccinated with immunogen 9 (DAEFRHDRRQIVYKPVGGC, SEQ ID NO: 59).

[0028] [Figure 3D] Figure 3D shows the lack of staining for Aβ and tau lesions in fresh-frozen human AD brain tissue using serum (1:300 dilution) derived from unvaccinated guinea pigs.

[0029] [Figure 3E] Figure 3E shows the lack of staining for Aβ and tau lesions in fresh-frozen human AD brain tissue using serum (1:300 dilution) derived from unvaccinated guinea pigs.

[0030] [Figure 3F] Figure 3F shows the lack of staining for Aβ and tau lesions in fresh-frozen human AD brain tissue using serum from unvaccinated guinea pigs (1:1500 dilution).

[0031] [Figure 4A] Figure 4A shows that guinea pig serum derived from animals vaccinated with the dual immunogenic peptide DAEFRHDRRQIVYKPVGGC (SEQ ID NO: 59, Immunogen 9) dose-dependently inhibited the binding of A-beta soluble aggregates to primary neurons.

[0032] [Figure 4B] Figure 4B shows examples of Ab staining of primary neurons in the presence or absence of guinea pig serum derived from animals vaccinated with the dual immunogenic peptide DAEFRHDRRQIVYKPVGGC (SEQ ID NO: 59, Immunogen 9).

[0033] [Figure 5A] Figure 5A shows that mice vaccinated with various linker-containing bipeptide antigens produce similar titers for Aβ and tau.

[0034] [Figure 5B] Figure 5B shows that mice vaccinated with a bipeptide antigen containing the tau sequence EIVYKSP (SEQ ID NO: 43) produce titers for Aβ and tau.

[0035] [Figure 6A] Figure 6A shows the staining of Aβ and tau lesions in fresh-frozen human AD brain tissue using serum (1:1000 dilution) derived from mice vaccinated with immunogen 19.

[0036] [Figure 6B] Figure 6B shows the staining of Aβ and tau lesions in fresh-frozen human AD brain tissue using serum (1:1000 dilution) derived from mice vaccinated with immunogen 19.

[0037] [Figure 6C] Figure 6C shows the lack of staining for Aβ and tau lesions in fresh-frozen human AD brain tissue using serum from unvaccinated mice (1:1000 dilution).

[0038] [Figure 6D] Figure 6D shows the staining of tau lesions in fresh-frozen human AD brain tissue using the anti-tau antibody m6H3 (0.1 μg / ml).

[0039] [Figure 6E] Figure 6E shows comparative staining of tau lesions in fresh-frozen human AD brain tissue using serum (1:1500 dilution) derived from guinea pigs vaccinated with immunogen 9 (DAEFRHDRRQIVYKPVGGC, SEQ ID NO: 59).

[0040] [Figure 7]Figures 7A and 7B show the titers of dual Aβ-tau immunogens under two injection paradigms. The titers for Aβ1-28, full-length tau, and carrier CRM protein under two injection schedules across the experiment (Figure 7A: showing weeks 0, 4, 12, and 24; Figure 7B: showing weeks 0, 8, and 24) are expressed as mean titer ± SEM.

[0041] [Figure 8A] Figure 8A shows the individual animal titers for Aβ and tau at 2 weeks after each injection for the injection schedule in Figure 7A, i.e., at weeks 2, 6, 14, and 26.

[0042] [Figure 8B] Figure 8B shows the individual animal titers for Aβ and tau at 2 weeks after each injection, i.e., at weeks 2, 14, and 26, for the injection schedule in Figure 7B.

[0043] [Figure 9A] Figure 9A shows the staining of Aβ and tau lesions in fresh-frozen human AD brain tissue using serum (1:300 dilution) from 26-week-old cynomolgus monkey #1001 vaccinated with immunogen 15 (DAEFRHDRRQIVYKPVGGC, SEQ ID NO: 59) in a four-injection vaccination schedule.

[0044] [Figure 9B] Figure 9B shows a magnified rectangular view of the staining of Aβ lesions in Figure 9A.

[0045] [Figure 9C] Figure 9C shows a magnified rectangular view of the tau lesion staining in Figure 9A.

[0046] [Figure 10A]Figure 10A shows the staining of Aβ lesions in fresh-frozen human AD brain tissue using serum (1:300 dilution) from cynomolgus monkey #1003 vaccinated with immunogen 15 (DAEFRHDRRQIVYKPVGGC, SEQ ID NO: 59).

[0047] [Figure 10B] Figure 10B shows slight staining of tau lesions in fresh-frozen human AD brain tissue using serum from cynomolgus monkey #1003 (1:300 dilution).

[0048] [Figure 11A] Figure 11A shows the staining of Aβ lesions in fresh-frozen human AD brain tissue using serum (1:300 dilution) from cynomolgus monkey #1501 vaccinated with immunogen 15 (DAEFRHDRRQIVYKPVGGC, SEQ ID NO: 59).

[0049] [Figure 11B] Figure 11B shows very slight staining of tau lesions in fresh-frozen human AD brain tissue using serum from cynomolgus monkey #1501 (1:300 dilution).

[0050] [Figure 12A] Figure 12A shows the staining of Aβ lesions in fresh-frozen human AD brain tissue using serum (1:300 dilution) from cynomolgus monkey #2501 vaccinated with immunogen 15 (DAEFRHDRRQIVYKPVGGC, SEQ ID NO: 59).

[0051] [Figure 12B] Figure 12B shows the lack of staining for tau lesions in fresh-frozen human AD brain tissue using serum from cynomolgus monkey #2501 (1:300 dilution).

[0052] [Figure 13]Figure 13 shows the titers of animals for Aβ and tau at 9 weeks after injection at 0 days, 4 weeks, and 8 weeks for the dual A-beta-tau constructs DAEFRHDRRVKSKIGSTGGC (SEQ ID NO: 997), DAEFRHDRRSKIGSTENGGC (SEQ ID NO: 998), DAEFRHDRRTENLKHQPGGC (SEQ ID NO: 999), DAEFRHDRRENLKHQPGGGC (SEQ ID NO: 1000), and DAEFRHDRRSKIGSKDNIKHGGC (SEQ ID NO: 1001).

[0053] [Figure 14] Figure 14 shows the blocking of tau binding to heparin by serum derived from the Aβ-tau constructs DAEFRHDRRVKSKIGSTGGC (SEQ ID NO: 997), DAEFRHDRRSKIGSTENGGC (SEQ ID NO: 998), DAEFRHDRRTENLKHQPGGC (SEQ ID NO: 999), DAEFRHDRRENLKHQPGGGC (SEQ ID NO: 1000), and DAEFRHDRRSKIGSKDNIKHGGC (SEQ ID NO: 1001).

[0054] [Figure 15A] Figure 15A shows tau competition by serum derived from the DAEFRHDRRVKSKIGSTGGC construct (SEQ ID NO: 997).

[0055] [Figure 15B] Figure 15B shows tau competition by serum derived from the DAEFRHDRRSKIGSTENGGC construct (SEQ ID NO: 998).

[0056] [Figure 15C] Figure 15C shows tau competition by serum derived from the DAEFRHDRRTENLKHQPGGC construct (SEQ ID NO: 999).

[0057] [Figure 15D] Figure 15D shows tau competition by serum derived from the DAEFRHDRRENLKHQPGGGC construct (SEQ ID NO: 1000).

[0058] [Figure 15E] Figure 15E shows tau competition by serum derived from the DAEFRHDRRSKIGSKDNIKHGGC construct (SEQ ID NO: 1001).

[0059] [Figure 15F] Figure 15F shows the competition for tau by serum from a control source.

[0060] [Figure 15G] Figure 15G shows tau competition by serum derived from the DAEFRHDRRSKIGSKDNIKHGGC construct (SEQ ID NO: 1001).

[0061] [Figure 16A] Figure 16A shows A beta / tau dual-vaccinated mouse serum 2.3_031521 (300x). An example of staining with DAEFRHDRRVKSKIGSTGGC (SEQ ID NO: 997).

[0062] [Figure 16B] Figure 16B shows A beta / tau dual-vaccinated mouse serum 3.1_031521 (300x). Example of staining with DAEFRHDRRSKIGSTENGGC (SEQ ID NO: 998).

[0063] [Figure 16C] Figure 16C shows A beta / tau dual-vaccinated mouse serum 8.4_031521 (300x). An example of staining with DAEFRHDRRTENLKHQPGGC (SEQ ID NO: 999).

[0064] [Figure 16D] Figure 16D shows A beta / tau dual-vaccinated mouse serum 9.2_031521 (300x). An example of staining with DAEFRHDRRENLKHQPGGGC (SEQ ID NO: 1000) is shown.

[0065] [Figure 16E]Figure 16E shows A beta / tau dual-vaccinated mouse serum 11.1_031521 (300x). Example of staining with DAEFRHDRRSKIGSKDNIKHGGC (SEQ ID NO: 1001). [Modes for carrying out the invention]

[0066] explanation This disclosure provides peptide compositions and immunotherapy compositions comprising amyloid-beta (Aβ) peptide and tau peptide. This disclosure also provides methods for treating or preventing Alzheimer's disease or other diseases involving beta-amyloid deposition in subjects, including methods for removing and preventing the formation of deposits, inhibiting or reducing the accumulation of Aβ and / or tau, blocking the binding and / or uptake of Aβ and / or tau by neurons, inhibiting the transmission of tau species between cells, and inhibiting the transmission of pathology between brain regions in subjects who have or are at risk of developing Alzheimer's disease or other diseases involving tau and / or amyloid-beta accumulation. This method includes administering a composition comprising amyloid-beta (Aβ) peptide and tau peptide to such patients.

[0067] Some terms are defined below. As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless the context explicitly indicates otherwise. For example, the term "a compound" or "at least one compound" may refer to multiple compounds, including mixtures thereof.

[0068] Unless otherwise evident from the context, the term “approximately” encompasses very small variations, such as values ​​within the standard limits of measurement error (e.g., SEM) of the stated value. For example, as used herein, the term “approximately” may, when referring to measurable values, such as parameters, quantities, or time periods, encompass variations of a given value and from the given value of + / - 10% or less, + / - 5% or less, or + / - 1% or less (or less or less). A specification of a range of values ​​includes all integers within or defining the range, and all subranges defined by the integers within the range. As used herein, statistical significance means p ≤ 0.05.

[0069] A composition or method that "comprising" or "including" one or more of the listed elements may also include other elements that are not specifically listed. For example, a composition that "comprises" or "includes" a polypeptide sequence may contain the sequence alone or in combination with other sequences or components.

[0070] If an individual has at least one known risk factor (e.g., age, genetic, biochemical, family history, and situational exposure) and is placed in a situation where individuals with that risk factor are at a statistically significantly higher risk of developing the disease than individuals without the risk factor, then the individual is at increased risk of developing the disease.

[0071] The term “patient” includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment, and includes treatment-naive subjects. Where used herein, the terms “subject” or “patient” refer to any single subject for which treatment is desired, including other mammalian subjects such as humans, cattle, dogs, guinea pigs, and rabbits. Any subject involved in a clinical research trial that does not show any clinical signs of disease, or a subject involved in an epidemiological study, or a subject used as a control, is also intended to be included as a subject.

[0072] The term "disease" refers to any abnormal condition that impairs physiological function. This term is used broadly to encompass any disorder, illness, abnormality, pathology, disease, condition, or syndrome that impairs physiological function, regardless of the nature of its etiology.

[0073] The term "symptoms" refers to subjective evidence of a disease, such as changes in gait, as perceived by the individual. "Signs" refers to objective evidence of a disease as observed by a physician.

[0074] As used herein, the terms “to treat” and “treatment” mean reducing or relieving one or more symptoms or effects associated with a disease, preventing, inhibiting or delaying the onset of one or more symptoms or effects of a disease, reducing the severity or frequency of one or more symptoms or effects of a disease, and / or increasing or moving toward a desired outcome as described herein.

[0075] The terms “prevention,” “prevent,” or “prevent” as used herein mean, when used herein, contacting (e.g., administering) the peptide(s) or immunotherapy composition of the Disclosed to a subject before the onset of a disease, with or without pre-existing Aβ and / or tau pathology (primary and secondary prevention), thereby delaying the onset of clinical symptoms and / or mitigating the symptoms of the disease after its onset compared to a case where the subject has not been contacted with the peptide(s) or immunotherapy composition, and not constituting complete suppression of the onset of the disease. In some cases, prevention may occur within a limited time period following administration of the peptide(s) or immunotherapy composition of the Disclosed. In other cases, prevention may occur during the duration of a treatment regimen that includes administration of the peptide(s) or immunotherapy composition of the Disclosed.

[0076] The terms “reduce,” “reduce,” or “to reduce,” as used herein, mean reducing the amount of Aβ and / or tau present in or to the tissue of a subject, or inhibiting the increase of Aβ and / or tau present in or to the tissue of a subject, which includes reducing the amount of accumulated, aggregated, or deposited Aβ and / or tau present in or to the tissue of a subject, or inhibiting its increase (e.g., reducing the rate of increase). In certain embodiments, reducing the amount of accumulated, aggregated, or deposited Aβ and / or tau present in or to the tissue of a subject, or inhibiting its increase (e.g., reducing the rate of increase), means the amount of accumulated, aggregated, or deposited Aβ and / or tau present in the central nervous system (CNS) of the subject. In certain embodiments, a reduction in the amount of accumulated, aggregated, or deposited Aβ and / or tau present in the subject, or a suppression of its increase (e.g., a reduction in the rate of increase), refers to the amount of accumulated, aggregated, or deposited Aβ and / or tau present in the periphery of the subject (e.g., the peripheral circulatory system). In certain embodiments, a reduction in the amount of accumulated, aggregated, or deposited Aβ and / or tau present in the subject, or a suppression of its increase (e.g., a reduction in the rate of increase), refers to the amount of accumulated, aggregated, or deposited Aβ and / or tau present in the brain of the subject. In some embodiments, the Aβ and / or tau reduced are pathological forms of Aβ (e.g., extracellular plaque deposition of β-amyloid peptide (Aβ), neurite amyloid plaques) and / or tau (e.g., tau neurofibrillary tangles, dystrophic neurites). In yet other embodiments, pathological indicators of neurodegenerative disease are reduced.

[0077] The term “epitope” or “antigen determinant” refers to a site on an antigen to which B cells and / or T cells respond, or to a site on an antigen to which an antibody binds. Epitopes can be formed both from continuous amino acids and from discontinuous amino acids juxtaposed by tertiary folding of proteins. Epitopes formed from continuous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 amino acids in their unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0078] An “immunogenic agent,” “immunogen,” or “antigen” may, if used in conjunction with an adjuvant as needed, induce an immune response to itself or a modified / processed version thereof when administered to an animal. The terms “immunogenic agent,” “immunogen,” or “antigen” refer to a compound or composition comprising a peptide, polypeptide, or protein that, when administered in an appropriate amount (“immunologically effective dose”), is “antigenic” or “immunogenic,” i.e., can induce, trigger, increase or boost a cellular and / or humoral immune response and can be recognized by the products of that response (T cells, antibodies). An immunogen may be a peptide, or a combination of two or more identical or different peptides, which contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 amino acids in a linear or spatial conformation.

[0079] Immunogens may be effective when administered alone, in combination with another substance (which may be administered once or at intervals), or ligated to or fused with it. Immunogenic agents or immunogens may comprise antigenic peptides or polypeptides ligated to carriers as described herein.

[0080] Nucleic acids, such as DNA or RNA, that encode an antigenic peptide or polypeptide are referred to as "DNA [or RNA] immunogens" because the encoded peptide or polypeptide is expressed in vivo after administration of the DNA or RNA. The peptide or polypeptide may be recombinantly expressed from a vaccine vector, which may be naked DNA or RNA containing a peptide or polypeptide coding sequence operably linked to a promoter, for example, an expression vector or cassette as described herein.

[0081] The term "adjuvant" refers to a compound that, when administered in combination with an antigen, enhances the immune response to that antigen, but, when administered alone, does not produce an immune response to that antigen. Adjuvants can enhance the immune response through several mechanisms, including lymphocyte recruitment, B cell and / or T cell stimulation, and macrophage stimulation. Adjuvants may be natural compounds, modified or derivative versions of natural compounds, or synthetic compounds.

[0082] The terms “peptide” and “polypeptide” are used interchangeably herein and refer to chains of two or more consecutive amino acids. Where a distinction is made, the context will clarify the meaning. For example, when two or more peptides described herein are linked together to form a dimer or polymeric peptide, the term polypeptide may be used to indicate a “poly” or “two or more” peptide.

[0083] The term "pharmaceutically acceptable" means that a carrier, diluent, excipient, adjuvant, or adjuvant is compatible with the other components of a pharmaceutical formulation and is not substantially harmful to its recipient.

[0084] The terms “immunotherapy” or “immune response” refer to the development of a beneficial humoral (antibody-mediated) and / or cellular response (mediated by antigen-specific T cells or their secretions) to Aβ and / or tau peptides in a recipient. Such a response may be an active response induced by the administration of an immunogen (e.g., Aβ and / or tau peptides). A cellular immune response may be triggered by the presentation of polypeptide epitopes associated with class I or class II MHC molecules, leading to antigen-specific CD4 + Helper T cells and / or CD8 + It activates cytotoxic T cells. The response may also include activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, or other components of innate immunity. The presence of a cell-mediated immune response is confirmed by proliferation assays (CD4 + This can be determined by T cell (T cell) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T cells from immunized syngenes and measuring the protective or therapeutic effect in a second subject.

[0085] Amyloid beta (Aβ)

[0086] Aβ (also referred herein as beta-amyloid peptide or A-beta) peptides are approximately 4 kDa internal fragments of APP consisting of 38–43 amino acids (Aβ39, Aβ40, Aβ41, Aβ42, and Aβ43). Aβ40 consists, for example, of residues 672–711 of APP, and Aβ42 consists of residues 673–713 of APP. As a result of proteolytic processing of APP by different secretory enzymes in vivo or in situ, Aβ is found in both a "short form" of 40 amino acids in length and a "long form" in the range of 42–43 amino acids in length. The epitope or antigenic determinant, as described herein, is located within the N-terminus of the Aβ peptide and includes residues within amino acids 1-10 and 12-25 of Aβ, for example, residues 1-3, 1-4, 1-5, 1-6, 1-7 or 3-7, 2-4, 2-5, 2-6, 2-7 or 2-8 of Aβ, residues 3-5, 3-6, 3-7, 3-8 or 3-9 of Aβ, or residues 4-7, 4-8, 4-9 or 4-10 of Aβ, or residues 12-24, 12-23, 12-22, 13-25, 13-24, 13-23, 13-22, 14-25, 14-24, 14-23, 14-22, 15-25, 15-24, 15-23 or 15-22. For example, they originate from residues 12-17, 12-18, 12-19, 12-20, 12-21, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 14-17, 14-18, 14-19, 14-20, 14-21, 14-22, 14-23, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, or 15-24 of Aβ42. Examples of additional epitopes or antigenic determinants include residues 16-18, 16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 16-25, 17-19, 17-20, 17-21, 17-22, 17-23, 17-24, or 17-25 of Aβ42. Other examples of epitopes or antigenic determinants include residues 18-20, 18-21, 18-22, 18-23, 18-24, 18-25, 19-21, 19-22, 19-23, 19-24, 19-25, 20-22, 20-23, 20-24, 20-25, 21-23, 21-24, or 21-25 of Aβ42.

[0087] Aβ (A-beta) is the main component of the characteristic plaques of Alzheimer's disease. Aβ is produced by the processing of the larger protein APP by two enzymes called beta-secretase and gamma-secretase. Known mutations in APP associated with Alzheimer's disease occur either near the beta-secretase or gamma-secretase site, or within Aβ itself. The hydrophobic transmembrane domain portion of APP is found at the carboxyl terminus of Aβ and may be the main cause of Aβ's ability to aggregate into plaques, especially in its long form. The accumulation of amyloid plaques in the brain ultimately leads to neuronal cell death. The physical symptoms associated with this type of neurodegeneration characterize Alzheimer's disease.

[0088] Tau

[0089] Tau is a protein with a molecular weight of approximately 50,000 that is normally present in nerve axons and other structures, contributing to microtubule stability. Tau protein (or τ protein) is a group of six highly soluble protein isoforms produced by alternative splicing from the MAPT (microtubule-associated protein tau) gene. They primarily play a role in maintaining microtubule stability in axons and are abundant in neurons of the central nervous system (CNS). They are less common elsewhere, but are also expressed at very low levels in astrocytes and oligodendrocytes of the CNS. Neuropathologies and dementias such as Alzheimer's disease and Parkinson's disease are associated with tau protein, which forms highly phosphorylated, insoluble aggregates called neurofibrillary tangles. Pathogenic tau species can cause toxic effects through direct binding to and / or intracellular accumulation and / or initiation of misfolding processes (seeding), and can be transmitted from one cell to another via intercellular communication. Toxicity can also be caused by neurofibrillary tangles (NFTs), which lead to cell death and cognitive decline. Other tauopathies include, for example, progressive supranuclear palsy, corticobasal syndrome, certain types of frontotemporal dementia, and chronic traumatic encephalopathy.

[0090] Immunogen Aβ / tau polypeptide

[0091] Drugs used for active immunization can induce an immune response in a patient and function as immunotherapy. Drugs used for active immunization may be, for example, the same type of immunogen used to produce monoclonal antibodies in experimental animals and may contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, or more, consecutive amino acids derived from the Aβ and / or tau peptide region. In each of the embodiments of the peptide described herein, the peptide may contain, be, or essentially consist of the enumerated sequence.

[0092] In some embodiments of this disclosure, the Aβ / tau immunogen may comprise an Aβ peptide comprising 3 to 10 amino acids derived from residues 1 to 10 or 12 to 25 of the N-terminal sequence of Aβ (SEQ ID NO: 01), linked to a tau peptide comprising 3 to 10 amino acids derived from residues 244 to 400 of the long form of tau (SEQ ID NO: 02). For example, the tau peptide may comprise 3 to 13 amino acids derived from the microtubule-binding region of tau (residues 344 to 372 of SEQ ID NO: 02).

[0093] In some embodiments of this disclosure, the Aβ peptide may comprise 3 to 10 amino acids derived from residues 1 to 10 or 12 to 25 of DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 01). For example, the Aβ peptide may be: [ka] [ka] [ka] [ka] Selected from.

[0094] In certain embodiments, the Aβ peptide is DAEFRHD (SEQ ID NO: 06), DAEFR (SEQ ID NO: 08), or EFRHD (SEQ ID NO: 21).

[0095] The tau peptide may correspond to a peptide containing 3 to 13 amino acids derived from residues 244 to 400 of SEQ ID NO: 02. In some embodiments, the fragment is not phosphorylated. In some embodiments, the fragment is phosphorylated. In some embodiments, the tau peptide contains an amino acid sequence represented by the consensus motif (Q / E)IVYK(S / P) (SEQ ID NO: 996). In some embodiments, the tau peptide contains an amino acid sequence represented by the consensus motif KXXSXXNX(K / H)H (SEQ ID NO: 995), where X is any amino acid. In some embodiments, the tau peptide is selected from SEQ ID NOs: 146 to 996. In some embodiments, the tau peptide is: [ka] [ka] Selected from. In each of these embodiments, the peptide may contain, be composed of, or be essentially composed of the listed sequences.

[0096] In some embodiments, Aβ and tau peptides are linked to form a dual Aβ / tau polypeptide. The Aβ and tau peptides may be linked by an intrapeptide linker. For example, the polypeptide linker is located between the C-terminus of the first peptide and the N-terminus of the second peptide. With or without an intrapeptide linker, the Aβ peptide and tau peptide can be positioned in any order on the dual Aβ / tau polypeptide. For example, the Aβ peptide may be positioned at the N-terminal portion of the dual polypeptide, and the tau peptide at the C-terminal portion. Alternatively, the tau peptide may be positioned at the N-terminal portion of the dual polypeptide, and the Aβ peptide at the C-terminal portion of the tau peptide on the dual polypeptide side. References to the first or second peptides herein are not intended to suggest an order of Aβ or tau peptides in the immunogen polypeptide.

[0097] In addition, the C-terminal portion of the Aβ peptide, tau peptide, or dual Aβ-tau polypeptide may include a linker for conjugating the peptide or polypeptide to a carrier. The linker for coupling the peptide or dual polypeptide to the carrier may include, for example, GG, GGG, KK, KKK, AA, AAA, SS, SSS, GAGA (SEQ ID NO: 80), AGAG (SEQ ID NO: 81), KGKG (SEQ ID NO: 82), etc., between the peptide or dual polypeptide and the carrier, and may further include a C-terminal or N-terminal cysteine ​​to provide a short peptide linker (e.g., GGC-, KKC-, AAC-, or SSC-). In some embodiments, if the C-terminal residue in the immunogen is one of IVYKPV (SEQ ID NO: 194), VYKPV (SEQ ID NO: 195), YKPV (SEQ ID NO: 196), KPV, or PV, the linker is an amino acid linker that does not have an N-terminal glycine (e.g., GG, GAGA (SEQ ID NO: 80)). In some embodiments, the linker includes one of the amino acid sequences AA, AAA, KK, KKK, SS, SSS, AGAG (SEQ ID NO: 81), GG, GGG, GAGA (SEQ ID NO: 80), and KGKG (SEQ ID NO: 82). In some embodiments, any of the Aβ peptide, tau peptide, and dual Aβ / tau polypeptide may contain a C-terminal cysteine ​​without a spacer. In some embodiments, any of the Aβ peptide, tau peptide, and dual Aβ / tau polypeptide may contain an N-terminal cysteine ​​without a spacer.

[0098] When Aβ and tau polypeptides are linked to form a dual Aβ / tau polypeptide, the linker may be a cleavable linker. As used herein, the term “cleavable linker” refers to any linker between antigen peptides that facilitates the separation of the Aβ peptide and the tau peptide from each other by cleavage (e.g., by endopeptidase, protease, low pH, or any other means that may occur in or around the antigen-presenting cell), or otherwise makes them more sensitive to separation from each other, thereby being processed by the antigen-presenting cell more than equivalent peptides lacking such a cleavable linker. In some compositions, the cleavable linker is a protease-sensitive dipeptide or oligopeptide cleavable linker. In certain embodiments, the cleavable linker is sensitive to cleavage by proteases of the trypsin family of proteases. In some compositions, the cleavable linker contains an amino acid sequence selected from the group consisting of arginine-arginine (Arg-Arg), arginine-valine-arginine-arginine (Arg-Val-Arg-Arg; SEQ ID NO: 69), valine-citrulline (Val-Cit), valine-arginine (Val-Arg), valine-lysine (Val-Lys), valine-alanine (Val-Ala), phenylalanine-lysine (Phe-Lys), GAGA (SEQ ID NO: 80), AGAG (SEQ ID NO: 81), and KGKG (SEQ ID NO: 82). In some compositions, the cleavable linker is arginine-arginine (Arg-Arg).

[0099] In some embodiments of the present disclosure, the dual Aβ / tau polypeptide comprises, consists of, or is essentially derived from, an amino acid sequence selected from DAEFRHDRRQIVYKPV (SEQ ID NO: 57), or DAEFRHDRREIVYKSV (SEQ ID NO: 58), or DAEFRHDRRQIVYKPVXXC (SEQ ID NO: 70) (wherein XX and the C-terminal cysteine ​​are each independently and as necessary), or DAEFRHDRREIVYKSVXXC (SEQ ID NO: 79) (wherein XX and C are each independently and as necessary, and where present, XX may be GG, AA, KK, SS, GAGA (SEQ ID NO: 80), AGAG (SEQ ID NO: 81), and KGKG (SEQ ID NO: 82).

[0100] In some embodiments, the dual Aβ / tau polypeptide is as follows: [Peptide 1]-[Linker 1]-[Peptide 2]-[Linker 2]-[Cys] (Here, if [the first peptide] is an Aβ peptide, then [the second peptide] is a tau peptide; if [the first peptide] is a tau peptide, then [the second peptide] is an Aβ peptide; [linker 1], [linker 2], and [Cys] are present as needed, and [linker 1] and [linker 2] are the same or different linkers).

[0101] In a particular embodiment, the dual Aβ / tau polypeptide is as follows: [Cys]-[Linker 2]-[First Peptide]-[Linker 1]-[Second Peptide] (Here, if [the first peptide] is an Aβ peptide, then [the second peptide] is a tau peptide; if [the first peptide] is a tau peptide, then [the second peptide] is an Aβ peptide; [linker 1], [linker 2], and [Cys] are present as needed, and [linker 1] and [linker 2] are the same or different linkers).

[0102] Examples of Aβ peptides include either SEQ ID NOs: 3-38 or 1002-1057.

[0103] Examples of tau peptides include any one of the following: SEQ ID NOs: 39-56, 83-86, or 146-996.

[0104] [Linker 1] may be present as needed and, if present, may be a cleavable linker. If present, the cleavable linker may be 1 to 10 amino acids long. In some embodiments, the linker comprises about 1 to 10 amino acids, about 1 to 9 amino acids, about 1 to 8 amino acids, about 1 to 7 amino acids, about 1 to 6 amino acids, about 1 to 5 amino acids, about 1 to 4 amino acids, about 1 to 3 amino acids, about 2 amino acids, or 1 amino acid. In some embodiments, the cleavable linker is 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids. In some embodiments, the linker may be a cleavable linker having an amino acid sequence selected from the group consisting of arginine-arginine (Arg-Arg), arginine-valine-arginine-arginine (Arg-Val-Arg-Arg; SEQ ID NO: 69), valine-citrulline (Val-Cit), valine-arginine (Val-Arg), valine-lysine (Val-Lys), valine-alanine (Val-Ala), phenylalanine-lysine (Phe-Lys), glycine-alanine-glycine-alanine (Gly-Ala-Gly-Ala; SEQ ID NO: 80), alanine-glycine-alanine-glycine (Gly-Ala-Gly-Ala; SEQ ID NO: 81), and lysine-glycine-lysine-glycine (Lys-Gly-Lys-Gly; SEQ ID NO: 82).

[0105] Linker 2 is a linker that is present as needed and, if present, couples the polypeptide to the carrier. The linker, if present, can be 1 to 10 amino acids long. In some embodiments, the linker comprises about 1 to 10 amino acids, about 1 to 9 amino acids, about 1 to 8 amino acids, about 1 to 7 amino acids, about 1 to 6 amino acids, about 1 to 5 amino acids, about 1 to 4 amino acids, about 1 to 3 amino acids, about 2 amino acids, or 1 amino acid. In some embodiments, the linker is 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids. In some embodiments, the amino acid composition of the linker can mimic the composition of linkers found in natural multi-domain proteins, where certain amino acids occupy a large proportion, a small proportion, or an equal proportion in the natural linker compared to their abundance in the whole protein. For example, threonine (Thr), serine (Ser), proline (Pro), glycine (Gly), aspartic acid (Asp), lysine (Lys), glutamine (Gln), asparagine (Asn), arginine (Arg), phenylalanine (Phe), glutamic acid (Glu), and alanine (Ala) account for large proportions in natural linkers. In contrast, isoleucine (Ile), tyrosine (Tyr), tryptophan (Trp), and cysteine ​​(Cys) account for smaller proportions. Generally, the amino acids that account for large proportions are polar uncharged or charged residues, which make up about 50% of the naturally encoded amino acids, with Pro, Thr, and Gln being the most preferred amino acids for natural linkers. In some embodiments, the amino acid composition of the linker can mimic the composition of linkers commonly found in recombinant proteins, which can generally be classified as flexible or rigid linkers. For example, flexible linkers found in recombinant proteins are generally composed of small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, and their small size provides flexibility, enabling the mobility to connect functional domains.For example, the incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing the interaction between the linker and the immunogen. In some embodiments, the linker includes a stretch of Gly and Ser residues ("GS" linker). Examples of widely used flexible linkers are (Gly-Gly-Ser)n, (Gly-Gly-Gly-Ser)n (SEQ ID NO: 1062), or (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 1063), where n = 1 to 3. By adjusting the copy number "n", the linker can be optimized to achieve sufficient isolation of the functional immunogen domain, for example, to maximize the immunogenic response. Many other flexible linkers have been designed for recombinant fusion proteins that may be used herein. In some embodiments, the linker may be rich in small or polar amino acids such as Gly and Ser, but may also contain further amino acids such as Thr and Ala to maintain flexibility, while polar amino acids such as Lys and Glu improve solubility. See, for example, Chen, X. et al., "Fusion Protein Linkers: Property, Design and Functionality" Adv Drug Deliv Rev., 15; 65(10): 1357-1369 (203). In certain embodiments, if present, the linker may be an amino acid sequence selected from the group consisting of GG, GGG, KK, KKK, AA, AAA, SS, SSS, GAGA (SEQ ID NO: 80), AGAG (SEQ ID NO: 81), and KGKG (SEQ ID NO: 82).

[0106] [Cys] may be present as needed and can help conjugate the polypeptide to a carrier. If present, Cys can be located at the C-terminal or N-terminal portion of the polypeptide.

[0107] Examples of the [first peptide]-[linker 1]-[second peptide]-[linker 2]-[Cys] dual Aβ / tau polypeptides of this disclosure include: [Table 1]

[0108] polypeptide immunogen

[0109] Aβ peptides, tau peptides, and dual Aβ / tau polypeptides are immunogens according to this disclosure. In some embodiments, the peptides and dual Aβ-tau polypeptides can be ligated to a suitable carrier to help induce an immune response. Thus, one or more peptides and dual Aβ-tau polypeptides of this disclosure can be ligated to a carrier. For example, each of the Aβ peptide, tau peptide, and Aβ-tau polypeptide may be ligated to a carrier with or without a spacer amino acid (e.g., Gly-Gly, Gly-Gly-Gly, Ala-Ala, Ala-Ala-Ala, Lys-Lys, Lys-Lys-Lys, Ser-Ser, Ser-Ser-Ser, Gly-Ala-Gly-Ala (SEQ ID NO: 80), Ala-Gly-Ala-Gly (SEQ ID NO: 81), or Lys-Gly-Lys-Gly (SEQ ID NO: 82)). In certain embodiments, the dual Aβ-tau polypeptide can be linked to a suitable linker using a C-terminal cysteine ​​to provide a linker between the peptide(s) and the carrier or between the dual Aβ / tau polypeptide and the carrier. In certain embodiments, the dual Aβ-tau polypeptide can be linked to a suitable linker using an N-terminal cysteine ​​to provide a linker between the peptide(s) and the carrier. In some embodiments, if the C-terminal residue in the immunogen is one of IVYKPV (SEQ ID NO: 194), VYKPV (SEQ ID NO: 195), YKPV (SEQ ID NO: 196), KPV, or PV, the linker is an amino acid linker that does not have an N-terminal glycine (e.g., GG, GAGA (SEQ ID NO: 80)).

[0110] Suitable carriers include, but are not limited to, serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or toxoids or attenuated toxin derivatives derived from other pathogenic bacteria, such as diphtheria (e.g., CRM197), E. coli, cholera, or H. pylori. T cell epitopes are also suitable carrier molecules. Some conjugates can be formed by linking the peptide immunogen of the present invention to immunostimulatory polymer molecules (e.g., tripalmitoyl-S-glycerol cysteine ​​(Pam3Cys), mannan (mannose polymer), or glucan (β1-2 polymer)), cytokines (e.g., IL-1, IL-1 alpha and β peptides, IL-2, γ-INF, IL-10, GM-CSF) and chemokines (e.g., MIP1-α and β, and RANTES). Further carriers include virus-like particles. In some compositions, immunogenic peptides can also be linked to a carrier by chemical crosslinking. Techniques for linking immunogens to a carrier include the formation of disulfide linkages using N-succinimidyl 3-(2-pyridylthio)propionate (SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptide lacks a sulfhydryl group, this can be provided by the addition of a cysteine ​​residue). These reagents create disulfide linkages between themselves and a peptide cysteine ​​residue on a certain protein, and amide linkages through epsilon-amino or other free amino groups in amino acids on lysine. In some embodiments, chemical crosslinking may include the use of N-hydroxysuccinimide (NHS) ester and SBAP (succinimidyl 3-(bromoacetamide)propionate), a short (6.2 angstroms) crosslinking agent for amine-to-sulfhydryl conjugation via a bromoacetyl reactive group.Various such disulfide / amide forming agents are described by Jansen et al., "Immunotoxins: Hybrid Molecules Combining High Specificity and Potent Cytotoxicity" Immunological Reviews 62:185-216 (February 1982). Other bifunctional crosslinking agents form thioethers rather than disulfide linkages. Many of these thioether forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid and 2-iodoacetic acid, and 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid. The carboxyl groups can be activated by mixing them with succinimide or sodium 1-hydroxyl-2-nitro-4-sulfonate salts. Virus-like particles (VLPs), also called pseudovirions or virus-derived particles, represent subunit structures formed from multiple copies of viral capsid and / or envelope proteins that can self-assemble in vivo into spherically symmetric VLPs (Powilleit, et al., (2007) PLoS ONE 2(5):e415). Alternatively, peptide immunogens can be linked to at least one artificial T cell epitope capable of binding to a large portion of MHC class II molecules, e.g., a pan-DR epitope ("PADRE"). Pan-DR-binding peptides (PADRE) are described in US5,736,142, WO95 / 07707 and Alexander, et al, Immunity, 1:751-761 (1994).

[0111] Active immunogens can exist in a multimeric form in which multiple copies of the immunogen (peptide of polypeptide) are present on a carrier as a single covalent molecule. In some embodiments, the carrier comprises various forms of dual Aβ / tau polypeptides. For example, the dual Aβ / tau polypeptide of the immunogen may contain polypeptides having the Aβ antigen and the tau antigen in different orders, or may exist with or without an intrapeptide linker and / or a linker to the carrier.

[0112] In some compositions, immunogenic peptides can also be expressed as fusion proteins with a carrier. In certain compositions, immunogenic peptides can be linked to a carrier at the amino terminus, carboxyl terminus, or internally. In some compositions, the carrier is CRM197. In some compositions, the carrier is diphtheria toxoid.

[0113] nucleic acid

[0114] This disclosure further provides nucleic acids encoding either the amyloid-beta (Aβ) peptide or the tau peptide disclosed herein. The nucleic acid immunotherapy compositions disclosed herein comprise, consist of, or are essentially composed of, a first nucleic acid sequence encoding the amyloid-beta (Aβ) peptide and a second nucleic acid sequence encoding the tau peptide. For example, the Aβ peptide is a sequence 3 to 10 amino acid residues long, derived from the first 10 N-terminal residues of SEQ ID NO: 01, and the tau peptide is a sequence 3 to 13 amino acid residues long, derived from residues 244 to 400 of SEQ ID NO: 02. Thus, nucleic acids encoding either SEQ ID NOs: 3 to 38 or SEQ ID NOs: 1002 to 1057 may be combined with nucleic acids encoding either SEQ ID NOs: 39 to 56, 83 to 86, or 146 to 996 to provide components of the immunogens and pharmaceutical compositions of this disclosure. Similarly, one or more nucleic acids encoding either the A-beta and tau sequences may contain codons for RR-N-terminal or -RR C-terminal dipeptides. In certain embodiments, the Aβ and tau peptide sequences may be encoded by the same nucleic acid sequence or by separate nucleic acid sequences. In some embodiments, the nucleic acid sequence may also encode a linker to a carrier described herein and / or a C-terminal cysteine. In addition, if a single nucleic acid sequence encodes both peptides, the sequence may also encode an intrapeptide linker described herein. The nucleic acid compositions (pharmaceutical compositions) described herein can be used in methods for treating or preventing and / or resolving Alzheimer's disease. In other embodiments, the nucleic acid immunotherapy compositions disclosed herein provide compositions for reducing the pathogenic forms of Aβ and / or tau in a subject and / or in the tissue of the subject. In some embodiments, the Aβ and / or tau reduced by the immunotherapy composition are the pathological forms (may be plural) of Aβ (e.g., extracellular plaque deposition of β-amyloid peptide (Aβ), neurite amyloid plaques) and / or tau (e.g., flamed neurofibrillary tangles of tau, neurofibrillary tangles of tau).In further embodiments, pathological indicators of neurodegenerative diseases are reduced by nucleic acid immunotherapy compositions. In another embodiment, the nucleic acid immunotherapy compositions disclosed herein provide compositions for reducing brain Aβ and brain tau.

[0115] Nucleic acids, such as DNA, that encode immunogens and are used as vaccines may be called "DNA immunogens" or "DNA vaccines" because the encoded polypeptide is expressed in vivo after the DNA is administered. DNA vaccines are intended to induce antibodies against the target protein they encode in a target by incorporating the DNA encoding the target protein into a vector (plasmid or virus), administering the vector to a target, and then, upon administration, stimulating the target's immune system to express the target protein. DNA vaccines remain in the target's body for a long period after administration, slowly continuing to produce the encoded protein. In this way, an excessive immune response can be avoided. DNA vaccines can also be modified using genetic engineering techniques. If necessary, such nucleic acids may further encode signal peptides and be expressed together with the signal peptides linked to the peptides. The coding sequence of a nucleic acid can be operably linked to regulatory sequences to ensure the expression of the coding sequence, such as promoters, enhancers, ribosome binding sites, and transcription termination signals. Nucleic acids encoding Aβ and tau may occur in isolated forms or be cloned into one or more vectors. Nucleic acids can be synthesized, for example, by solid-state synthesis or PCR of overlapping oligonucleotides. Nucleic acids encoding Aβ and tau peptides and polypeptides, with and without linkers or cleavable linkers, and with and without protein-based carriers, can be linked together as a single continuous nucleic acid, for example, within an expression vector.

[0116] DNA is more stable than RNA, but it carries several potential safety risks, such as the induction of anti-DNA antibodies; therefore, in some embodiments, the nucleic acid may be RNA. RNA nucleic acids that encode immunogens and are used as vaccines may be called “RNA immunogens,” “RNA vaccines,” or “mRNA vaccines” because the encoded polypeptide is expressed in vivo after administration of the RNA. Ribonucleic acid (RNA) vaccines can safely induce target cellular mechanisms to produce one or more polypeptides of interest. In some embodiments, RNA vaccines may be non-replicating mRNA (messenger RNA) or self-amplifying RNA derived from viruses. mRNA vaccines encode the antigen of interest and contain 5' and 3' untranslated regions (UTRs), while self-amplifying RNA encodes not only the antigen but also the viral replication mechanism that enables intracellular RNA amplification and abundant protein expression. In vitro transcribed mRNA may be produced from a linear DNA template using T7, T3, or Sp6 phage RNA polymerase. The resulting product may contain 5'- and 3'-UTR sequences, a 5' cap, and an open reading frame adjacent to the poly(A) tail encoding the peptide of interest disclosed herein. In some embodiments, the RNA vaccine may contain trans-amplified RNA (see, e.g., Beissert et al., Molecular Therapy January 2020 28(1):119-128). In certain embodiments, the RNA vaccine may encode the Aβ peptide and tau peptide disclosed herein and may express the Aβ and tau peptide, particularly when transferred to cells such as immature antigen-presenting cells. The RNA may also contain sequences encoding other polypeptide sequences, such as immunostimulatory elements. In some embodiments, the RNA of the RNA vaccine may be modified RNA. The term “modified” in the context of RNA may include any modifications of the RNA that are not naturally present in the RNA. For example, modified RNA may refer to RNA having a 5'-cap, however, the RNA may include further modifications.The 5'-caps can be modified to have the ability to stabilize RNA when bound to them. In certain embodiments, further modifications may include elongation or cleavage of naturally occurring poly(A) tails, or alteration of the 5'- or 3'-untranslated region (UTR). In some embodiments, RNA, e.g., or mRNA vaccines, are formulated in an effective amount to produce an antigen-specific immune response in a subject. For example, an RNA vaccine formulation is administered to a subject to stimulate the subject's humoral and / or cellular immune systems against Aβ and tau antigens, and therefore may further contain one or more adjuvants, diluents, carriers, and / or excipients, and is applied to the subject via any preferred route to induce a protective and / or therapeutic immune response against Aβ and tau antigens.

[0117] All of the basic texts disclosing general methods of molecular biology, which are incorporated herein by reference, are Sambrook, J et al., Molecular Cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989;Ausubel, FM et al. Current Protocols in Molecular Biology, Vol. 2, Wiley-Interscience, New York (current edition);Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990);Glover, DM, ed, DNA Cloning: A Practical Approach, vol. I & II, IRL Press, 1985;Albers, B. et al., Molecular Biology of the Cell, 2 nd Ed., Garland Publishing, Inc., New York, NY (1989);Watson, JD et al., Recombinant DNA, 2nd Ed., Scientific American Books, New York, 1992; and Old, RW et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2. nd Ed., University of California Press, Berkeley, Calif. (1981) is one example.

[0118] For example, techniques for manipulating nucleic acids, such as inducing mutations in sequences, subcloning, probe labeling, sequencing, and hybridization, are well described in scientific literature and patent documents. See, for example, Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Tijssen, ed. Elsevier, NY (1993).

[0119] Nucleic acids, vectors, capsids, polypeptides, etc., can be analyzed and quantified by any of several common methods well known to those skilled in the art. These include, for example, NMR, spectrophotography, X-ray imaging, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and ultradiffusion chromatography; various immunological methods, such as fluid or gel precipitation reactions, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Southern analysis, Northern analysis, dot blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and analytical biochemistry methods such as affinity chromatography.

[0120] Pharmaceutical composition

[0121] Each of the peptides and immunogens described herein may be present in a pharmaceutical composition administered with a pharmaceutically acceptable adjuvant and a pharmaceutically acceptable excipient. The adjuvant increases the titer and / or binding affinity of the induced antibody compared to the situation when the peptide is used alone. Various adjuvants can be used in combination with the immunogens of this disclosure to induce an immune response. Some adjuvants enhance the endogenous response to the immunogen without causing conformational changes in the immunogen that affect the qualitative form of the response. The adjuvant may be a natural compound, a modified or derivative of a natural compound, or a synthetic compound.

[0122] Some adjuvants include aluminum salts, such as aluminum hydroxide and aluminum phosphate, and 3-de-O-acylated monophosphoryl lipid A (MPL®) (see GB2220211 (RIBI ImmunoChem Research Inc., Hamilton, Montana, now part of Corixa)). As used herein, MPL refers to both the natural and synthetic versions of MPL. Examples of synthetic versions include PHAD®, 3D-PHAD®, and 3D(6A)-PHAD® (Avanti Polar Lipids, Alabaster, Alabama).

[0123] QS-21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja saponaria Molina tree found in South America (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995)). QS-21 products include Stimulon® (Antigenics, Inc., New York, NY; now Agenus, Inc., Lexington, MA) and QS-21 Vaccine Adjuvant (Desert King, San Diego, CA). QS-21 has been disclosed, characterized, and evaluated in US patents 5,057,540 and 8,034,348, which are incorporated herein by reference. In addition, QS-21 has been evaluated in numerous clinical studies at various dosages.NCT00960531 (clinicaltrials.gov / ct2 / show / study / NCT00960531), Huell et al., Curr Alzheimer Res. 2017 Jul; 14(7): 696-708 (evaluating 50mcg of QS-21 with various doses of vaccine ACC-001); Gilman et al., "Clinical effects of Abeta immunization (AN1792) in patients with AD in an interrupted trial" Neurology. 2005 May 10; 64(9):1553-62; Wald et al., "Safety and immunogenicity of long HSV-2 peptides complexed with rhHsc70 in HSV-2 seropositive persons" Vaccine 2011; 29(47):8520-8529; and Cunningham et al., "Efficacy of the See "Herpes Zoster Subunit Vaccine in Adults 70 Years of Age or Older." NEJM. 2016 Sep 15; 375(11):1019-32. QS-21 is used in FDA-approved vaccines, including SHINGRIX. SHINGRIX contains 50 mcg of QS-21. In certain embodiments, the amount of QS-21 ranges from approximately 10 μg to approximately 500 μg.

[0124] TQL1055 is an analog of QS-21 (Adjuvance Technologies, Lincoln, NE). Semi-synthetic TQL1055 is characterized as having higher purity, increased stability, reduced topical tolerance, and reduced systemic tolerance compared to QS-21. TQL1055 is disclosed, characterized, and evaluated in US20180327436A1, WO2018191598A1, WO2018200656A1, and WO2019079160A1, the disclosures of which are incorporated herein by reference. US20180327436A1 teaches that TQ1055 was more than 2.5 times superior to 20 μg of QS-21, but there was no improvement over 50 μg of TQ1055. However, unlike QS-21, there was no increase in either weight loss or RBC hemolysis with increasing doses of TQL1055. WO2018200656A1 teaches that the optimal amount of TQ1055 can reduce the amount of antigen and achieve excellent titer. In certain embodiments, the amount of TQL1055 is approximately 10 μg to approximately 500 μg.

[0125] Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil) combined with immunostimulants such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)), Pluronic® polymer, and mycobacteria killers, as needed. The Ribi adjuvant is an oil-in-water emulsion. Ribi contains saline solution containing Tween® 80 and emulsified metabolizable oil (squalene). Ribi also contains purified mycobacterial products and bacterial monophosphoryl lipid A, which act as immunostimulants. Other adjuvants may include CpG oligonucleotides (see WO98 / 40100), cytokines (e.g., IL-1, IL-1 alpha and β peptides, IL-2, γ-INF, IL-10, GM-CSF), chemokines (e.g., MIP1-α and β, as well as RANTES), saponins, RNA, and / or TLR agonists (e.g., TLR4 agonists such as MPL and synthetic MPL molecules), aminoalkylglucosaminide phosphates, and other TLR agonists. Adjuvants may be administered as components of a therapeutic composition with the active agent, or may be administered before, together with, or separately after the administration of the therapeutic agent.

[0126] In various embodiments of this disclosure, the adjuvant is QS-21 (Stimulon®). In some compositions, the adjuvant is MPL. In certain embodiments, the amount of MPL is about 10 μg to about 500 μg. In some compositions, the adjuvant is TQL1055. In certain embodiments, the amount of TQL1055 is about 10 μg to about 500 μg. In some compositions, the adjuvant is QS21. In certain embodiments, the amount of QS21 is about 10 μg to about 500 μg. In some compositions, the adjuvant is a combination of MPL and QS-21. In some compositions, the adjuvant is a combination of MPL and TQL1055. In some compositions, the adjuvant can be a liposomal formulation.

[0127] In addition, some embodiments of this disclosure may include multiple antigen-presenting systems (MAPs). Multiple antigen-presenting peptide vaccine systems have been developed to avoid the adverse effects associated with conventional vaccines (i.e., live-attenuated pathogens, toxic pathogens, or inactivated pathogens), carrier proteins, and cytotoxic adjuvants. Multiple antigen-presenting peptide vaccine systems have been developed using two main approaches: (1) the addition of functional components, e.g., T cell epitopes, cell-permeable peptides, and lipophilic moieties; and (2) a synthetic approach using size-defined nanomaterials, e.g., self-assembling peptides, non-peptide dendrimers, and gold nanoparticles, as antigen-presenting platforms. The use of multiple antigen-presenting peptide (MAP) systems can improve the sometimes insufficient immunogenicity of subunit peptide vaccines. In MAP systems, multiple copies of the antigen peptide are simultaneously bound to the α- and ε-amino groups of a non-immunogenic Lys-type dendritic scaffold, helping to confer stability from degradation, thus enhancing molecular recognition by immune cells and induction of a stronger immune response compared to small antigen peptides alone. In some compositions, the MAP comprises one or more of the following: a Lys-based dendritic scaffold, a helper T cell epitope, an immunostimulatory lipophilic moiety, a cell-permeable peptide, a radical-induced polymerization, self-assembling nanoparticles as an antigen presentation platform, and gold nanoparticles.

[0128] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions may be provided in unit dosage forms (i.e., doses for single administration). Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. Formulation depends on the chosen route of administration. For injection, the peptides of this disclosure may be formulated in aqueous solution, preferably in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, the peptide composition may be in a lyophilized form for use with a suitable vehicle, e.g., sterile pyrogen-free water.

[0129] Peptides (and, if necessary, carriers fused to the peptide(s)) can also be administered in the form of nucleic acids encoding the peptide(s) and expressed in situ in the subject. The nucleic acid segment encoding the immunogen is typically ligated to regulatory elements, such as promoters and enhancers that enable the expression of the DNA segment in the intended target cells of the subject. For expression in blood cells, or as desired for induction of an immune response, promoter and enhancer elements derived from light-chain or heavy-chain immunoglobulin genes, or the CMV major intermediate-early promoter and enhancer are preferred for directing expression. The ligated regulatory elements and coding sequences are often cloned into a vector.

[0130] DNA and RNA can be delivered in naked form (i.e., without colloidal or inclusion material). Alternatively, retroviruses (see, e.g., Boris-Lawrie and Teumin, Cur. Opin. Genet. Develop. 3(1):102-109 (1993)); adenovirus vectors (see, e.g., Bett et al, J. Virol. 67(10);5911-21 (1993)); adeno-associated virus vectors (see, e.g., Zhou et al., J. Exp. Med. 179(6):1867-75 (1994)); viral vectors derived from the pox family, including vaccinia virus and tripoxvirus; and viral vectors derived from the alphavirus genus, such as those derived from Sindbis virus and Semlik Forest virus (see, e.g., Dubensky et al., J. Virol. 70(1):508-519) Several viral vector systems can be used, including rhabdoviruses such as (see 1996), Venezuelan encephalitis virus (see US5,643,576), and vesicular stomatitis virus (see WO96 / 34625), as well as papillomavirus (WO94 / 12629; Ohe et al., Human Gene Therapy 6(3):325-333 (1995); and Xiao & Brandsma, Nucleic Acids. Res. 24(13):2620-2622 (1996)).

[0131] DNA and RNA encoding immunogens, or vectors containing them, can be packaged in liposomes, nanoparticles, or lipoprotein complexes. Other suitable polymers include, for example, protamine liposomes, polysaccharide particles, cationic nanoemulsions, cationic polymers, cationic polymer liposomes, cationic lipid nanoparticles, cationic lipids, cholesterol nanoparticles, cationic lipid-cholesterol, PEG nanoparticles, or dendrimer nanoparticles. Further suitable lipids and related analogs are described in US5,208,036, US5,264,618, US5,279,833, and US5,283,185, each of which is incorporated herein by reference in whole. The immunogen-encoding vectors and DNA can also be adsorbed or associated with microparticle carriers, such as polymethyl methacrylate polymers, as well as polylactides and poly(lactide-co-glycolides) (see, e.g., McGee et al., J. Micro Encap. Mar-Apr 1997; 14(2):197-210).

[0132] Pharmaceutically acceptable carrier compositions may include, but are not limited to, water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, and additives including surfactants that are acceptable as pharmaceutical additives.

[0133] Suitable subjects for treatment

[0134] The presence of Aβ plaques and / or neurofibrillary tangles is associated with Alzheimer's disease, Down syndrome, mild cognitive impairment, cerebral amyloid angiopathy, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, and globular glial tauopathy. It has been found in several diseases, including tauopathy, amyotrophic lateral sclerosis / parkinsonian dementia complex in Guam, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variants of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), spheroid glial tauopathy (GGT), Parkinson's disease, progressive supranuclear palsy (PSP), atrophic age-related macular degeneration (AMD), and inclusion body myositis.

[0135] The compositions and methods of this disclosure can be used in the treatment or prevention of any of these diseases. Due to the broad association between neurological disorders and Aβ and / or tau, the compositions and methods of this disclosure can be used in the treatment or prevention of any subject exhibiting elevated levels of Aβ and / or tau (e.g., in CSF) compared to the mean values ​​in individuals without neurological disorders. The compositions and methods of this disclosure can also be used in the treatment or prevention of neurological disorders in individuals with mutations in Aβ and / or tau associated with neurological disorders. The methods are particularly suitable for the treatment or prevention of Alzheimer's disease.

[0136] Suitable subjects for treatment include individuals at risk of the disease but without symptoms, and patients currently exhibiting symptoms, including treatment-naive subjects who have not been previously treated for the disease. Subjects at risk of the disease include subjects in the aging population, asymptomatic subjects with Aβ and / or tau pathology and known genetic risk for the disease. Such individuals include those with relatives who have experienced the disease, and those whose risk has been determined by analysis of genetic or biochemical markers. Genetic markers of risk include mutations in Aβ and / or tau, as well as mutations in other genes associated with neurological disorders. For example, heterozygous ApoE4 alleles, and even more so homozygous ApoE4 alleles, are associated with the risk of Alzheimer's disease (AD). Other markers of Alzheimer's disease risk include mutations in the APP gene, particularly at position 717, as well as mutations at positions 670 and 671, known as the Hardy and Swedish mutations, respectively, mutations in the presenilin gene PS1 and PS2, and a family history of AD, hypercholesterolemia, or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be identified by PET imaging due to characteristic dementia and the presence of the risk factors described above. In addition, several diagnostic tests are available to identify individuals with AD. These include measuring CSF or blood tau or phosphorylated tau levels, and Aβ42 levels. Elevated tau or phosphorylated tau levels and decreased Aβ42 levels indicate the presence of AD. Some mutations, such as Ala30Pro or Ala53Thr, or mutations in other Parkinson's disease-related genes (LRRK2 or PARK8), such as leucine-rich repeat kinase, are associated with Parkinson's disease. Individuals may also be diagnosed with any of the neurological disorders mentioned above according to the DSM IV TR criteria.

[0137] In asymptomatic subjects, treatment can be initiated at any age (e.g., 10, 20, 30, or older). However, it is usually not necessary to start treatment until the subject reaches 20, 30, 40, 50, 60, 70, 80, or 90 years of age. Treatment typically requires multiple doses over a period of time. Treatment can be monitored by assaying antibody levels over time. If the response declines, booster medication is prescribed. In patients with potential Down syndrome, treatment can be initiated prenatally or immediately after birth by administering the therapeutic agent to the mother.

[0138] Treatment and Usage Instructions

[0139] This disclosure provides a method for inhibiting or reducing the aggregation of A-beta and / or tau in subjects having or at risk of developing neurodegenerative diseases (e.g., Alzheimer's disease). The method comprises administering the compositions disclosed herein to subjects. The therapeutically effective dose is the dosage that, when administered over an effective period, achieves the desired immunological or clinical effect. The dosage regimen may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered at set intervals (e.g., weekly, monthly), or the dose may be proportionally reduced as indicated by the urgency of the treatment situation.

[0140] For prophylactic use, the compositions described herein may be administered to subjects who are susceptible to or otherwise at risk of the disease (e.g., Alzheimer's disease) in regimens (dosage, frequency, and route of administration) that are effective in reducing the risk of the disease, lowering the severity of the disease, or delaying the onset of at least one sign or symptom of the disease. In particular, the regimens are effective in inhibiting or delaying Aβ plaque formation, and / or inhibiting or delaying tau or phosphorylated tau and the counter-filaments formed therefrom in the brain, and / or inhibiting or delaying their toxic effects, and / or inhibiting or delaying the onset of behavioral defects. For therapeutic use, the compositions described herein may be administered to subjects suspected of having the disease (e.g., Alzheimer's disease) or patients who already have the disease in regimens (dosage, frequency, and route of administration) that are effective in improving at least one sign or symptom of the disease, or at least preventing its further worsening. In particular, the regimen is preferably effective in reducing or at least inhibiting further increases in the levels of Aβ plaques and / or tau, phosphorylated tau, or counter-filaments formed therefrom, associated toxicity, and / or behavioral defects.

[0141] A regimen is considered therapeutically or prophylactically effective if the treated individuals achieve a more favorable outcome than the mean outcome in a control population of equivalent subjects not treated by the method of the present invention, or if the better outcome is demonstrated at a level of p<0.05, 0.01, or even 0.001 in the treated subjects compared to the control subjects in a comparative clinical trial (e.g., a Phase II, Phase II / III, or Phase III trial).

[0142] The effective dose varies depending on many different factors, such as the means of administration, the target site, the patient's physiological state, whether the patient is an ApoE carrier, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic.

[0143] In some embodiments, the effective dose is a total dose of 25 μg to 1000 μg, or 50 μg to 1000 μg. In some embodiments, the effective dose is a total dose of 100 μg. In some embodiments, the effective dose is a dose of 25 μg administered to the subject in a total of two doses. In some embodiments, the effective dose is a dose of 100 μg administered to the subject in a total of two doses. In some embodiments, the effective dose is a dose of 400 μg administered to the subject in a total of two doses. In some embodiments, the effective dose is a dose of 500 μg administered to the subject in a total of two doses. In some embodiments, the RNA (e.g., mRNA) vaccine is administered to the subject by intradermal injection, intramuscular injection, or intranasal administration.

[0144] In some embodiments, the amount of drug for active immunotherapy varies from 1 to 1,000 micrograms (μg), or 0.1 to 500 μg, or 10 to 500 μg, or 50 to 250 μg per patient, and may be 1 to 100 μg or 1 to 10 μg per injection for human administration. The timing of injections can vary very greatly, from once daily to once weekly, once monthly, once a year, or once every 10 years. A typical regimen consists of immunization followed by booster injections at time intervals such as 6 weeks or 2 months. Another regimen consists of immunization followed by one or more booster injections 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months later. Another regimen necessarily involves injections every 2 months throughout life. Alternatively, booster injections may be irregular, as indicated by monitoring of the immune response. The frequency of administration may be once or multiple times, provided that side effects are within a clinically acceptable range.

[0145] In some embodiments, the compositions or methods disclosed herein involve administering a nucleic acid vaccine comprising one or more DNA or RNA polynucleotides having open reading frames encoding a first peptide and a second peptide, with the nucleic acid vaccine dosage administered to the subject ranging from 10 μg / kg to 400 μg / kg. In some embodiments, the RNA polynucleotide dosage per dose is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg. The dosages are 100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg, 80-300 μg, 100-300 μg, 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg, 300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or 300-400 μg. In some embodiments, the nucleic acid is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid is administered to the subject on day 0. In some embodiments, the second dose of nucleic acid is administered to the subject on day 7, day 14, or day 21.

[0146] The compositions described herein are preferably administered via the peripheral route (i.e., the route by which the administered composition passes through the blood-brain barrier and reaches the intended site in the brain, spinal cord or eye, resulting in a robust immune response and / or an induced antibody population). For peripheral diseases, the induced antibodies leave the vasculature to reach the intended peripheral organ. Routes of administration include oral, subcutaneous, intranasal, intradermal or intramuscular. Some routes for active immunization are subcutaneous and intramuscular. Intramuscular and subcutaneous administrations can be performed at a single site or multiple sites. Intramuscular injections are most typically performed in the muscles of the arm or leg. In some methods, the agent is directly injected into a specific tissue where deposits have accumulated.

[0147] The number of doses administered can be adjusted to provide a more robust immune response (e.g., a higher titer). For acute disorders or acute exacerbations of chronic disorders, often 1 to 10 doses are sufficient. Sometimes, for acute disorders or acute exacerbations of chronic disorders, a single bolus dose in divided form may be sufficient as needed. For chronic disorders, the vaccines / immunotherapies disclosed herein can be administered at regular intervals, e.g., weekly, biweekly, monthly, every 3 months, every 6 months, for at least 1, 5 or 10 years, or over the patient's lifetime.

[0148] The effective amount of DNA or RNA encoding the immunogen can be from about 1 nanogram to about 1 gram per kilogram of recipient body weight, or about 0.1 μg / kg to about 10 mg / kg, or about 1 μg / kg to about 1 mg / kg. A dosage form suitable for internal administration preferably contains from about 0.1 μg to 100 μg of the active ingredient per unit (for the latter dosage range). The active ingredient can vary from 0.5 to 95% by weight based on the total weight of the composition. Alternatively, the effective dose of antigen-loaded dendritic cells is about 10 4 ~10 8 cells. Those skilled in immunotherapy can adjust these doses without undue experimentation.

[0149] Nucleic acid compositions may be administered in a simple manner, for example, by injection via a simple and effective route. Routes may include, but are not limited to, intradermal "gene gun" delivery or intramuscular injection. Modified dendritic cells are administered subcutaneously, intravenously, or intramuscularly. Other possible routes include oral administration, intrathecal administration, inhalation, transdermal application, or rectal administration.

[0150] Depending on the route of administration, the composition may be coated with a material that protects the compound from the action of enzymes, acids, and other natural conditions that could inactivate the compound. Therefore, it may be necessary to coat the composition with a material that prevents inactivation, or to co-administer the composition with such a material. For example, in an enzyme inhibitor of a nuclease or protease (e.g., pancreatic trypsin inhibitors, diisopropyl fluorophosphate, and trazilol), or in a suitable carrier such as liposomes (including water-in-oil emulsions) and conventional liposomes (Strejan et al., J. Neuroimmunol 7(1):27-41, 1984).

[0151] The immunotherapeutic compositions disclosed herein may also be used in combination with other treatments for diseases associated with the accumulation of Aβ or tau, such as anti-Aβ antibodies, including antibodies that specifically bind to any of the Aβ epitopes disclosed herein. For example, aducanumab, or any of the antibodies disclosed in U.S. Patent Publication No. 20100202968 and U.S. Patent No. 8,906,367, and / or anti-tau antibodies, for example, antibodies that specifically bind to any of the tau epitopes disclosed herein, such as ABBV-8E12, goslanemab, zagotenemab, RG-6100, BIIB076, or any of the antibodies disclosed in WO2014 / 165271, US10,501,531, WO2017 / 191559, WO2017 / 191560, WO2017 / 191561, US20190330314, US20190330316 and WO2018 / 204546. In some combination therapy methods, the patient receives passive immunotherapy before the active immunotherapy method disclosed herein. In other methods, the patient receives passive and active immunotherapy during the same period of treatment. Alternatively, the patient may receive active immunotherapy before passive immunotherapy. The combination may also include small molecule therapies and non-immunogenic therapies, such as RAZADYNE® (galantamine), EXELON® (rivastigmine), and ARICEPT® (donepezil), as well as other compositions that improve the function of nerve cells in the brain.

[0152] The compositions of this disclosure may be used in the manufacture of pharmaceuticals for the treatment regimens described herein.

[0153] Treatment regimen

[0154] The desired outcomes of the treatment methods disclosed herein vary according to the disease and patient profile and are determinable to those skilled in the art. Desired outcomes include improvement in the patient's health status. Generally, desired outcomes include measurable indicators, such as reduction or elimination of pathogenic amyloid fibrils, reduced or inhibited amyloid aggregation and / or amyloid fibril deposition, and an increased immune response to pathogenic and / or aggregated amyloid fibrils. Remission of the specific symptoms of amyloid disease is also a desired outcome. Where used herein, relative terms such as “improve,” “increase,” or “reduce” refer to values ​​compared to a control, e.g., a measurement in the same individual before the commencement of the treatment described herein, or a measurement in a control individual or control group. A control individual is an individual suffering from the same amyloid disease as the treated individual, but who has not been treated with the disclosed immunotherapy / vaccine formulation, but is approximately the same age as the treated individual (to ensure that the disease stage is equivalent in the treated and control individuals). Alternatively, the control individual is a healthy individual of approximately the same age as the individual being treated. Changes or improvements in response to treatment are generally described by p-values ​​less than or equal to 0.1, less than 0.05, less than 0.01, less than 0.005, or less than 0.001, which are statistically significant and can be considered significant.

[0155] The effective dose of the compositions disclosed herein for a treatment of interest varies depending on many different factors, including the means of administration, the target site, the patient's physiological state, whether the patient is human or animal, any other drugs administered, and whether the treatment is prophylactic or therapeutic. Treatment doses can be dose-set to optimize safety and efficacy. The amount of immunogen may also depend on whether an adjuvant is also administered, with higher doses required in the absence of an adjuvant. The amount of immunogen for administration sometimes varies from 1 to 500 μg per patient, and more commonly from 5 to 500 μg per injection for human administration. Occasionally, higher doses of 1 to 2 mg per dose are used. Typically, about 10, 20, 50, or 100 μg are used for each human dose. The timing of administration can vary very greatly, from once daily to once a year or once every ten years. On any given day on which immunogen is administered, the dose is higher than 1 μg / patient, usually higher than 10 μg / patient if an adjuvant is also administered, and higher than 10 μg / patient in the absence of an adjuvant, and usually higher than 100 μg / patient. A typical regimen consists of immunization followed by booster doses at 6-week intervals. Another regimen consists of immunization followed by booster doses at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months later. Another regimen involves lifelong administration of medication every 2 months. Alternatively, booster doses may be irregular, as indicated by monitoring of the immune response.

[0156] When administered in combination with a second treatment for Alzheimer's disease, such as Razadyne® (galantamine), Exelon® (rivastigmine), and Aricept® (donepezil), the second treatment may be administered as required, in accordance with the product label or considering the treatment with the compositions of this disclosure.

[0157] kit

[0158] This disclosure further provides the compositions disclosed herein and related materials, such as kits (e.g., containers) including instructions for use (e.g., accompanying documents). The instructions for use may include, for example, instructions for the administration of the compositions and, optionally, one or more additional agents. Containers for peptide and / or nucleic acid compositions may be unit doses, bulk packages (e.g., multi-dose packages) or subunit doses.

[0159] The package insert refers to the instructions customarily included on the market packaging of a therapeutic product, containing information about indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic product. The kit may also include a second container containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. This may also include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0160] use

[0161] Each of the peptides, polypeptides, immunogens, and pharmaceutical compositions described herein may be used for the treatment of one or more of the diseases described herein. In addition, each of the peptides, polypeptides, immunogens, and pharmaceutical compositions described herein may be used for the treatment of one or more of the diseases described herein. Each of the peptides, polypeptides, immunogens, and pharmaceutical compositions described herein may be used in a method for treating one or more of the diseases described herein or for manufacturing a pharmaceutical for use in such treatment.

[0162] The following is provided for illustrative purposes only and is not intended to limit the scope of the invention as described in the broad terms above.

[0163] All U.S. and international patent applications identified herein are incorporated herein by reference in their entirety. [Examples]

[0164] (Example 1) Guinea pig immunity

[0165] Guinea pigs were intramuscularly injected with 50 μg of test immunogen and 25 μg of QS21 in 200 μl of Addavax on days 0, 21, 49, and 77. Blood was collected 7 days after immunization. The peptides tested included DAEFRHD (SEQ ID NO: 06), QIVYKPV (SEQ ID NO: 39), and DAEFRHDRRQIVYKPV (SEQ ID NO: 57). The specific immunogens were DAEFRHDC (SEQ ID NO: 71), QIVYKPVGGC (SEQ ID NO: 72), and DAEFRHDRRQIVYKPVGGC (SEQ ID NO: 59). The peptides were linked to CRM-197 via a maleimide bond through the C-terminal cysteine.

[0166] Female guinea pigs were at least 5 weeks old at the start of the study and weighed approximately 350-500g. Appropriate animal housing and research procedures for animal rearing and care were carried out in an accredited facility in accordance with the guidelines of the United States Department of Agriculture (USDA) and the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International.

[0167] The immunogen concentration was 0.5 mg / ml. Before administering each test immunogen, the injection site was visualized by applying approximately 3 cm of the sample to each hind limb. 2The area was shaved and wiped with ethanol. Each animal received a 200 μl (0.25 μg / μl) test immunogen dose, divided into two separate sites, 100 μl per injection (i.e., the animals received 50 μg immunogen in 100 μl PBS + 25 μg QS21 in 100 μl MF59). A 25G–27G needle was inserted intramuscularly into the hind limb to a depth of approximately 0.25–0.5 cm, and 100 μl was injected at each site. The injection sites were rotated between four separate sites per hind limb, with each dose spaced at least 2 cm apart.

[0168] (Example 2) Measurement of antibody titer

[0169] Whole blood samples of 250–350 μl each were collected via the jugular vein into coagulation activator tubes at weeks 1, 4, and 8. At the end of week 12, the maximum volume of whole blood was collected via cardiac puncture into coagulation activator tubes. All blood samples were allowed to coagulate at room temperature for more than 30 minutes, centrifuged at ambient temperature (approximately 20–25°C) at 3,000 RPM for 10–15 minutes, and the serum supernatant was individually transferred to clean frozen vials. The serum supernatant was stored frozen at -80°C (±12°C).

[0170] Guinea pig blood titer for soluble Aβ aggregates

[0171] A preparation of soluble aggregated Aβ at 2.5 μg / ml (the HFIP film of Aβ42 was resuspended, incubated overnight with shaking, and centrifuged to remove insoluble aggregates) was coated onto plates at 100 μl / well in PBS and incubated overnight at room temperature. The plates were blocked with 1% BSA in PBS for 1 hour. The plates were aspirated and 200 μl of 0.1% BSA in PBS Tween was added to rows A1 - 4. Negative GP serum was added to 1 at a 100-fold dilution, while 2 - 4 contained test serum at a 100-fold dilution. Rows B - H contained 100 μl of 0.1% BSA in PBS Tween. The rows were serially diluted 1:2 under the plate to a dilution of 100-fold to 12,800-fold. The wells were incubated for 2 hours at room temperature, then washed, and a 5,000-fold dilution of anti-guinea pig IgG HRP in PBS Tween containing 0.1% BSA was prepared and 100 μl was added to the washed wells. This was incubated for 1 hour and washed. Using Thermo-Fisher OPD tablets, the OPD substrate was prepared at 1 tablet per 10 ml. Thermo fisher substrate buffer was added at a 10-fold dilution and 100 μl was added to each well and incubated for 15 minutes. 50 μl of 2N H2SO4 was added to stop the reaction and the plates were read at 490 nM using a Molecular Devices Spectromax. The titer was defined as the dilution factor that gave 50% of the maximum OD, and was extrapolated if it fell between dilution factors.

[0172] Titer of guinea pig blood sampling for tau

[0173] 2 μg / ml of recombinant WT tau 4R2N was coated onto plates using 100 μl / well in PBS and incubated overnight at room temperature. The plates were blocked with 1% BSA in PBS for 1 hour. The plates were aspirated and 200 μl of 0.1% BSA in PBS Tween was added to row A. Negative GP serum was added at a 100-fold dilution to column 1, while the rest of the row contained 100-fold diluted test sera. The rows were serially diluted 1:2 under the plate to a dilution of 100-fold to 12,800-fold. The wells were incubated for 2 hours at room temperature, then washed, and a 5,000-fold dilution of anti-guinea pig IgG HRP in PBS Tween containing 0.1% BSA was prepared and 100 μl was added to the washed wells. This was incubated for 1 hour and washed. OPD substrate was prepared using Thermo-Fisher OPD tablets at 1 tablet per 10 ml. Thermo fisher substrate buffer was added at a 10-fold dilution and 100 μl was added to each well and incubated for 15 minutes. 50 μl of 2N H2SO4 was added to stop the reaction and the plates were read at 490 nM using a Molecular Devices spectromax. The titer was defined as the dilution factor that gave 50% of the maximum OD, and extrapolation was performed if it fell between dilution factors.

[0174] Titers of guinea pig blood collections for Aβ1-28 or Aβ1-15

[0175] Aβ1-15 and Aβ1-28 were used in different parts of this study. None of them formed aggregates. Plates were coated with 2 μg / ml Aβ monomer in PBS at 100 μl / well and incubated overnight at room temperature. The plates were blocked with 1% BSA in PBS for 1 hour. The plates were aspirated, and 0.1% BSA in 200 μl of PBS Tween was added to row A. Negative GP serum was added to column 1 at a 100-fold dilution, while the rest of the row contained test serum at a 100-fold dilution. The rows were serially diluted 1:2 under the plate to dilutions ranging from 100-fold to 12800-fold. The wells were incubated at room temperature for 2 hours, then washed, and 100 μl of a 5000-fold dilution of anti-guinea pig IgG HRP was prepared in PBS Tween containing 0.1% BSA and added to the washed wells. This was incubated for 1 hour and then washed. OPD substrates were prepared using Thermo-Fisher OPD tablets, with one tablet per 10 ml. Thermo-Fisher substrate buffer was added at a 10-fold dilution, 100 μl of which was added to each well, and incubated for 15 minutes. The reaction was stopped by adding 50 μl of 2N H2SO4, and the plate was read at 490 nM using the molecular device Spectromax. Titer was defined as the dilution factor that yielded 50% of the maximum OD, and extrapolation was performed when values ​​fell between dilution factors.

[0176] Tau titer measurement for peptides containing that epitope

[0177] Thermofisher neutavidin plates were rehydrated with 0.05% Tween in TBS and aspirated. Biotin-containing peptide GGGSVQIVYKPVDLS (SEQ ID NO: 68) was prepared at a 500-fold dilution in PBS Tween containing 0.1% BSA. 100 μl was added per well for 1 hour, followed by washing. 200 μl of 0.1% BSA in PBS Tween was added to row A of the plate. Negative GP serum was added at a 100-fold dilution to column 1, while the rest of the row contained test serum at a 100-fold dilution. The rows were serially diluted 1:2 below the plate to dilutions ranging from 100-fold to 12800-fold. The wells were incubated at room temperature for 2 hours, followed by washing. A 5000-fold dilution of anti-guinea pig IgG HRP was prepared in PBS Tween containing 0.1% BSA, and 100 μl was added to the washed wells. This was incubated for 1 hour and then washed. OPD substrates were prepared using Thermo-Fisher OPD tablets, with one tablet per 10 ml. Thermo-Fisher substrate buffer was added at a 10-fold dilution, 100 μl of which was added to each well, and incubated for 15 minutes. The reaction was stopped by adding 50 μl of 2N H2SO4, and the plate was read at 490 nM using the molecular device Spectromax. Titer was defined as the dilution factor that yielded 50% of the maximum OD, and extrapolation was performed when values ​​fell between dilution factors.

[0178] Figure 1 shows a comparison of the geometric mean titers of guinea pig serum against the immunogen DAEFRHDRRQIVYKPV (SEQ ID NO: 57), which was titrated using GGGSVQIVYKPVDLS (SEQ ID NO: 68), which contains monomeric Aβ1-28, soluble Aβ aggregates, full-length tau, and tau MTBR epitopes.

[0179] Figure 2 shows the results for single-peptide immunogens Aβ and tau peptides (DAEFRHD (SEQ ID NO: 06) and QIVYKPV (SEQ ID NO: 39)) and the bipeptide immunogen DAEFRHDRRQIVYKPV (SEQ ID NO: 57) in Aβ1-28 and full-length tau.

[0180] (Example 3) Staining of Alzheimer's brain tissue with serum derived from guinea pigs immunized with the vaccine disclosed herein.

[0181] Autopsy blocks of fresh-frozen human brain tissue (approximately 0.5 g) were embedded in an optimal cutting temperature compound (OCT compound) and dissected using a cryostat to produce 10 μm sections. The sections were placed in a solution of glucose oxidase and beta-D-glucose in the presence of sodium azide to block endogenous peroxidase. Once the tissue sections were prepared, staining was performed with identified guinea pig serum derived from guinea pigs immunized with the vaccine disclosed herein, at two dilutions (1:300 and 1:1500), using rabbit anti-guinea pig secondary antibody and the DAKO DAB Detection Kit, according to the manufacturer's instructions. Staining was performed using an automated Leica Bond Stainer. The results indicate that the guinea pig serum immunized with the vaccine disclosed herein contains antibodies specific to Aβ and tau in human brain tissue from Alzheimer's patients (see Figures 3A–3F).

[0182] (Example 4) Serum derived from vaccinated animals blocks the binding of soluble Aβ aggregates to neurons.

[0183] Primary E18 rat hippocampal neurons were cultured as previously described (Zago, et al. "Neutralization of Soluble, Synaptotoxic Amyloid β Species by Antibodies Is Epitope Specific," J Neurosci. 2012 Feb 22; 32(8): 2696-2702). To block soluble Aβ aggregates from binding to neurites, soluble Aβ aggregates were pre-incubated on culture DIV14-21 with or without guinea pig vaccine serum. Guinea pig serum was isolated from animals vaccinated with the dual immunogenic peptide DAEFRHDRRQIVYKPVGGC (SEQ ID NO: 59, Immunogen 9). Fresh, unlabeled, biotinylated, or (9:1) soluble Aβ was prepared 1 day prior and incubated overnight at 4°C. Using NeuroBasal-Phenol Red-Free (NB-NPR) medium, each diluted serum sample (1:1000, 1:300, and 1:100) and soluble Aβ solution were prepared at twice the final concentration in half the final treatment volume. These were then combined with soluble Aβ at twice the final concentration in half the final volume and 2-fold diluted guinea pig vaccine serum in half the final volume to create a total final treatment volume at 1x the final concentration. This was thoroughly mixed and pre-incubated at 37°C for 30 minutes. E18 neurons were washed with 150 μL / well of NB-NPR before additional binding treatment. Guinea pig serum derived from vaccinated animals / Aβ-treated neurons was added to the E18 neurons at 60 μL / well and incubated at 37°C for 30 minutes under normal incubator conditions (5% CO2; 9% O2). The cells were washed twice with 150 μL / well of NB-NPR and fixed with 4% paraformaldehyde in 1× DPBS for 20 minutes. The cells were permeabilized with 0.1 TX-100 for 5 minutes and blocked with 10% normal goat serum (NGS) at room temperature (RT) for 1 hour. The cells were incubated overnight at 4°C in 100 μL / well of 1× DPBS containing 1% BSA and 1% NGS with MAP2 and NeuN primary antibodies. The following day, the cells were washed twice with 150 μL / well of 1× DPBS for 5 minutes each time.Secondary antibody was added to 100 μL / well of 1× DPBS + 1% BSA + 1% NGS at room temperature for 1 hour. To quantify soluble aggregated Aβ neurite binding, high-content imaging (HCI) analysis was performed using an Operetta HCI CLS instrument (Perkin Elmer; modified Neuroite Outgrowth algorithm: 40× H2O objective; 40 fields per well; n=3 per condition; data shown as mean (±) SD). Dendritic neurites were tracked using MAP2 and NeuN (Abcam) neuron markers, and the number of cells per optical field was counted. Spots of Aβ-soluble aggregates on neurites were detected using streptavidin 488 or polyclonal Aβ antibody (Thermo; Millipore). Data were reported as Aβ-soluble aggregate spot / neuron (or as integrated intensity).

[0184] Approximately 80–150 neurons were observed per well under each tested condition. The results demonstrate that guinea pig serum derived from animals vaccinated with the dual immunogenic peptide DAEFRHDRRQIVYKPVGGC (SEQ ID NO: 59, Immunogen 9) dose-dependently inhibited the binding of A-beta to neurons (see Figure 4).

[0185] (Example 5) Mice vaccinated with a bipeptide antigen produce titers against Aβ and tau.

[0186] On days 0, 14, and 28, Swiss-Webster female mice were injected with 25 μg of a bipeptide immunogen (Table 2) and 25 μg of QS21 (Desert King) in a total of 200 μl of PBS per injection. Each mouse received 200 μl subcutaneously. Blood was collected from the mice on days 21 and 35. [Table 2]

[0187] The immunogens containing the tau peptide EIVYKSP (SEQ ID NO: 43, see Figure 5B) showed overall greater variability in tau titer than the immunogens having the tau MTBR sequence QIVYKPV (SEQ ID NO: 39) (except for immunogen 23). Therefore, the basic microtubule-binding region (MTBR) peptide containing DAEFRHDRRQIVYKPV (SEQ ID NO: 57) was also tested (Figure 5A). The titers of three immunogens with different linkers compared to the GGC linker (no linker, AA, and KK) are shown in Figure 5.

[0188] The titers observed from the second blood collection are listed in Tables 3 and 4.

Table 3

Table 4

[0189] The titers observed in guinea pigs immunized with immunogen 9, DAEFRHDRRQIVYKPVGGC (SEQ ID NO: 59) are listed in Table 5 below.

Table 5

[0190] (Example 6) Serum from vaccinated animals stains Aβ plaques and tau lesions in human brain tissue.

[0191] Fresh frozen human brain tissue from necropped Alzheimer's disease donors or non-disease controls was embedded in OCT and 10 μm frozen sections were generated by cryostat cutting. Endogenous peroxidase was blocked by incubating the tissue sections in a solution of glucose oxidase and beta-D-glucose in the presence of sodium azide. Staining was performed with serum from vaccinated mice. Mice were either vaccinated with the following biantigen peptides, DAEFRHDRRQIVYKPVGGC (SEQ ID NO: 59, immunogen 9); DAEFRHDRRQIVYKPVC (SEQ ID NO: 60, immunogen 10); DAEFRHDRRQIVYKPVAAC (SEQ ID NO: 61, immunogen 18); and DAEFRHDRRQIVYKPVKKC (SEQ ID NO: 62, immunogen 19), or were control mice. Staining was performed at a 1:1000 dilution in an automated Leica Bond Rx Stainer (Leica Biosystems). Antibody binding was detected using the Bond Polymer Refine Detection Kit (DS9800, Leica Biosystems). This was based on anti-mouse polymer detection, DAB visualization, and hematoxylin counterstaining. After coverslips, stained tissue slides were digitally imaged using a Hamamatsu NanoZoomer 2.0HT slide scanner (Hamamatsu Corporation) with NDP.Scan 2.5.85 software. The digitized images were visually examined and analyzed using NDP.view 2.7.43.0 software.

[0192] The results demonstrate that Aβ plaques and tau neurofibrillary tangles were identified based on their typical histopathological features. Such lesions were not present in tissue incubated with control mouse serum. Furthermore, non-disease tissue did not exhibit such lesion staining after incubation with serum derived from vaccinated mice. Figures 6A–6E and Table 6 summarize the results of Aβ and tau staining using mouse serum from animals vaccinated with a dual antigen peptide (human brain tissue from AD patients was stained using serum diluted 1:1000). [Table 6-1] [Table 6-2]

[0193] (Example 7) Determination of immunization and titer in cynomolgus monkeys

[0194] The study described in this example was designed to evaluate the progression of Aβ and tau titers in cynomolgus monkeys using the dual antigen peptide DAEFRHDRRQIVYKPVGGC (SEQ ID NO: 59). Two injection schedules and titer persistence were also evaluated.

[0195] immunity

[0196] Several bimodal Aβ-tau linear immunogens with dendritic cell cleavage sites were screened in mice for balanced titers against Aβ and tau proteins (see Example 5 above). Subsets of immunogens were further evaluated in guinea pigs (see Examples 1-5 above) and cynomolgus monkeys (this example). Two groups of four monkeys each were immunized intramuscularly with 50 μg of immunogen (SEQ ID NO: 59, DAEFRHDRRQIVYKPVGGC) ligated to CRM197 carrier protein and 50 μg of adjuvant QS21. Group 1 was injected at weeks 0, 4, 12, and 24, and blood was collected every two weeks until week 38. Group 2 was injected at weeks 0, 8, and 24, and blood was collected every two weeks until week 38. Serum titer levels were determined for Aβ and full-length tau. Fresh-frozen human AD or control brain sections were stained with serum from immunized and control animals. The activity of guinea pig immunoserum was also evaluated for the binding of soluble Aβ oligomers in primary rat hippocampal neurons.

[0197] Potency protocol

[0198] Measurement of antibody titers against Aβ1-28 in cynomolgus monkeys

[0199] The titer of cynomolgus monkey blood was measured by enzyme-linked immunosorbent assay (ELISA). Plates were coated overnight with 2 μg / mL of Aβ1-28 (SEQ ID NO: 67) in phosphate-buffered saline (PBS), followed by blocking with 1% bovine serum albumin (BSA) in PBS for 1 hour. Pre-collected cynomolgus monkey blood was used as a negative control, and known positive antisera from previous mouse studies were used as positive controls at the same dilution ratio as the test serum. Blood was diluted starting at 1:100 in PBS / 0.1% BSA / 0.1% Tween 20 (0 / 1% Tween 20) (PBS / BSA / T) and then serially diluted 1:2 under the plate. Plates were washed with TBS / Tween 20, and goat anti-monkey immunoglobulin G (IgG) (heavy chain + light chain) horseradish peroxidase (HRP) (IgG[H+L]HRP; Invitrogen) was added and incubated at room temperature for 1 hour. Plates were washed with TBS / Tween 20, and antibody binding was detected with o-phenylenediamine dihydrochloride (OPD) substrate (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Plates were read at 490 nM using Molecular Devices Spectromax. Titer was defined as a dilution factor giving 50% of the maximum OD or 4 times the background (as defined in the figure or table), and extrapolated if it fell between dilution factors.

[0200] Measurement of antibody titers against tau in cynomolgus monkeys

[0201] The titer of cynomolgus monkey blood was measured by enzyme-linked immunosorbent assay (ELISA) against full-length recombinant tau (Proteos, Kalamazoo, MI; SEQ ID NO: 02). Plates were coated overnight with 2 μg / mL tau in phosphate-buffered saline (PBS) and then blocked for 1 hour with 1% bovine serum albumin (BSA) in PBS. Guinea pig serum collected prior to the study was used as a negative control, and known positive antisera from previous mouse studies were used as positive controls at the same dilution ratios as the test serum. Blood was diluted starting at 1:100 in PBS / 0.1% BSA / 0.1% Tween 20 (PBS / BSA / T) and then serially diluted 1:2 under the plate. Plates were washed with TBS / Tween 20, and goat anti-monkey immunoglobulin G (IgG) (heavy chain + light chain) horseradish peroxidase (HRP) (IgG[H+L]HRP; Invitrogen) was added and incubated at room temperature for 1 hour. Plates were washed with TBS / Tween 20, and antibody binding was detected with o-phenylenediamine dihydrochloride (OPD) substrate (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Plates were read at 490 nM using Molecular Devices Spectromax. Titer was defined as a dilution factor giving 50% of the maximum OD or 4 times the background (as defined in the figure or table), and extrapolated if it fell between dilution factors.

[0202] Titer of cynomolgus monkey blood samples on the carrier protein CRM197

[0203] The titer of cynomolgus monkey blood was measured by enzyme-linked immunosorbent assay (ELISA) against the CRM197 (FinaBio, Maryland) carrier protein. Plates were coated overnight with 2 μg / mL tau in phosphate-buffered saline (PBS), followed by blocking with 1% bovine serum albumin (BSA) in PBS for 1 hour. Guinea pig serum collected prior to the study was used as a negative control, and known positive antisera from previous mouse studies was used as a positive control at the same dilution ratio as the test serum. Blood was diluted starting at 1:100 in PBS / 0.1% BSA / 0.1% Tween 20 (PBS / BSA / T) and serially diluted 1:2 under the plate. Plates were washed with TBS / Tween 20, and goat anti-monkey immunoglobulin G (IgG) (heavy chain + light chain) and horseradish peroxidase (HRP) (IgG[H+L]HRP; Invitrogen) were added and incubated at room temperature for 1 hour. Plates were washed with TBS / Tween 20, and antibody binding was detected using o-phenylenediamine dihydrochloride (OPD) substrate (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Plates were read at 490 nM using Molecular Devices Spectromax. Titer was defined as a dilution factor giving 50% of the maximum OD or 4 times the background (as defined in the figure or table), and extrapolated if it fell between dilution factors.

[0204] result

[0205] The titer levels were similar for Aβ and tau. Monkeys in Group 1 using the vaccination schedule had a larger area under the curve in titer than monkeys in Group 2, but under these conditions, there was no significant improvement in maximum titer (Figures 7A and 7B). Overall titer levels were lower than in the guinea pig study described above. This change may be due to the change in adjuvant from QS21 in ADDVAX in guinea pigs to QS21 in phosphate-buffered saline (PBS) in cynomolgus monkeys to align with the approved vaccine. The persistence of titer was moderate for both the four-injection and three-injection vaccination schedules (Figures 8A and 8B). These results demonstrate that these immunogens can produce a balanced immune response against Aβ and tau in primates. Titer levels and persistence may be improved by optimizing the administration schedule and / or formulation.

[0206] (Example 8) Immunohistochemistry in brain sections of AD and control

[0207] We evaluated the ability of serum derived from immunized monkeys to bind to pathogenic Aβ plaques and tau tangles in human brain tissue derived from subjects with Alzheimer's disease (AD). Binding to both Aβ plaques and tau tangles is expected to reduce plaque load and tau transmission, thereby potentially reducing the signs and symptoms of AD.

[0208] Autopsy blocks of fresh-frozen human brain tissue were embedded in an optimal cutting temperature compound (OCT compound) and dissected using a cryostat to produce 10-micrometer sections. The sections were placed in a solution of glucose oxidase and beta-D-glucose in the presence of sodium azide to block endogenous peroxidase. Once the tissue sections were prepared, staining with identified cynomolgus monkey immunoserum was performed at two dilutions (300-fold in 5% goat serum containing 0.25% Triton®, at room temperature for 1 hour). Binding was detected at room temperature for 1 hour with purified, unlabeled mouse anti-monkey IgG secondary antibody (Mybiosource, 3 mg / mL) and at room temperature for 1 hour with goat anti-mouse IgG secondary antibody (Jackson, 200-fold), and at room temperature for 1 hour with avidin-biotin complex (ABC; Vector PK-4000) and DAKO DAB Detection Kit according to the manufacturer's instructions. The staining process was performed using an automated Leica Bond Stainer.

[0209] Table 7 summarizes the staining findings in the cynomolgus monkey study. All animals except 1002 exhibited positive staining for Aβ. Only animal 1001 showed strong tau staining (Figures 9A-9C), while 1003 (Figures 10A-10B) and 1501 (Figures 11A-11B) showed only slight staining. Animal 2501, for example, did not show significant tau staining (Figures 12A-12B). There did not appear to be a correlation between tau staining and tau titer, as evidenced by animal 1001, which showed strong tau staining despite lacking a strong tau titer. [Table 7]

[0210] Under in vivo conditions, immunoserum binds avidly to Aβ plaques and tau tangles in human AD brain sections at concentrations expected to be reached in the CNS. At 26 weeks, the monkey with the strongest titer stained Aβ, while one monkey with low to moderate tau titer stained both Aβ and tau.

[0211] These results demonstrate that serum antibody binding in cynomolgus monkey antibody responses is in equilibrium and that the antibodies bind to pathogenic Aβ plaques and tau tangles.

[0212] (Example 9) Serum-mediated blocking of soluble Aβ aggregates from binding to neurons and T cell activity.

[0213] Primary hippocampal neurons isolated from E18 rats were cultured as described above. Fresh, unlabeled, biotinylated, or (9:1) soluble Aβ was prepared one day prior to the assay and incubated overnight at 4°C. Neurons were rinsed with 150 μL / well of NB-NPR before adding Aβ / serum treatment. IgG cuts from cynomolgus monkey blood samples from vaccinated animals were added to E18 neurons at 60 μL / well and incubated at 37°C for 30 minutes under normal incubator conditions (5% CO2; 9% O2). Cells were washed twice with 150 μL / well of NB-NPR and fixed with 4% paraformaldehyde in 1× DPBS for 20 minutes. Cells were permeabilized with 0.1 TX-100 for 5 minutes and blocked with 10% normal goat serum (NGS; Thermofisher) at room temperature (RT) for 1 hour. Cells were incubated overnight at 4°C in 100 μL / well of 1× DPBS containing 1% BSA and 1% NGS with MAP2 and NeuN primary antibodies.

[0214] The following day, cells were washed twice with 150 μL / well of 1× DPBS for 5 minutes each time. Secondary antibody in 100 μL / well of 1× DPBS + 1% BSA + 1% NGS was added at room temperature for 1 hour. Spots of Aβ-soluble agglutinates were detected using streptavidin 488 or polyclonal Aβ antibody (Thermofisher; Millipore). To quantify the neurite binding of soluble agglutinated Aβ, high-content imaging (HCI) analysis was performed using an Operetta HCI CLS instrument (Perkin Elmer; modified Neuroite Outgrowth algorithm: 40× H2O objective; 40 fields per well; n=3 per condition; data shown as mean (±) SD). Dendritic neurites were tracked using MAP2 and NeuN (Abcam) neuron markers, and the number of cell bodies per optical field was counted. Nerve process data were reported as Aβ-soluble aggregate spots / neuron (or as integrated intensity). Approximately 80–150 neurons were observed per well for each condition tested (Zago, et al., 2012).

[0215] T cell reactivity was also tested. Immunoserum inhibited the binding of soluble Aβ aggregates to hippocampal neurons. Immunogens did not induce a cytotoxic T cell response to Aβ or tau.

[0216] (Example 10) Animals vaccinated with a bipeptide antigen produce titers against Aβ and tau.

[0217] The study described in this example was designed to evaluate dual Aβ and tau antigen peptides in mice. The dual Aβ-tau construct in this example effectively demonstrated high titers for both antigens, blocking of tau binding to heparin, and staining / binding to Aβ and tau peptides in brain tissue derived from human Alzheimer's disease patients. Furthermore, the titer for tau of the engineered tau immunogen (dual #11) was comparable to, or in some cases superior to, other tau immunogens, despite containing a non-natural tau sequence. This demonstrates the usefulness of the engineered immunogen in vaccine construction. [Table 8] [Table 9]

[0218] Conjugation: The peptide was prepared by Biopeptide (San Diego, CA). CRM-bromoacetate was supplied by Fina Biosolutions (Rockville, MD). The peptide was coupled with CRM according to the following protocol.

[0219] 1M Tris HCl (pH 8.0), MilliQ DI water, 50mM borate, 100mM NaCl, and 5mM EDTA (pH 8.5) were aseptically filtered and degassed. 1 mg of each peptide was dissolved in 0.2 ml of degassed water, and 0.1 ml of degassed Tris HCl was added. Subsequently, 0.2 ml of stock CRM-bromoacetate (total 1 mg) was added, and finally 0.5 ml of borate buffer was added. This was incubated on a nutrient regulator at 4°C for 24 hours and mixed. The sample was desalted in PBS, and 5 μl was electrophoresed on a 10% Tris gel to confirm conjugation.

[0220] Animal injection: Four female Swiss Webster mice were used in each group. The immunogen preparation consisted of 25 μg of immunogen, 25 μg of QS21, and 150 μl of 0.02% Tween 80 / PBS per injection. Each mouse received 200 μl subcutaneously. Mice were injected on day 0, from day 0 to week 4, and from day 0 to week 8. Blood was collected for titer measurement from day 0 to week 5, and the animals were sacrificed from day 0 to week 9 for final blood collection (see Figure 13).

[0221] Titer assay: Mouse serum titers were measured by enzyme-linked immunosorbent assay (ELISA). Plates were coated overnight with 2 μg / mL of A-beta 1-28 (Anaspec, San Jose, CA) or recombinant tau (in phosphate-buffered saline (PBS), Proteos, Kalamazoo, MI), followed by blocking with 1% bovine serum albumin (BSA) in PBS for 1 hour. Normal mouse serum was used as a negative control, and known positive antisera from previous mouse studies were used as positive controls at the same dilution ratios as the test serum. Blood was diluted starting at 1:100 in PBS / 0.1% BSA / 0.1% Tween 20 (PBS / BSA / T) and then serially diluted 1:2 under the plate. Plates were washed with TBS / Tween 20, and goat anti-mouse immunoglobulin G (IgG) (heavy chain + light chain) horseradish peroxidase (HRP) (Thermo Fisher) 1:5000 was added and incubated at room temperature for 1 hour. Plates were washed with TBS / Tween 20, and antibody binding was detected with o-phenylenediamine dihydrochloride (OPD) substrate (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Plates were read at 490 nM using Molecular Devices Spectromax. Titer was defined as a dilution factor giving 4 times the background (as defined in the figure or table), and extrapolated if it fell between dilution factors.

[0222] MTBR join

[0223] Certain antibodies that bind to MTBRs can bind to two or more MTBR regions through homology between different MTBR regions. Using MTBR1-4 peptides purchased from Anaspec (San Jose, CA), we measured the titer of antiserum against four MTBR regions. [ka]

[0224] Mouse serum titers are measured by enzyme-linked immunosorbent assay (ELISA). Plates are coated overnight with various MTBR peptides at 2 μg / mL in phosphate-buffered saline (PBS), followed by blocking with 1% bovine serum albumin (BSA) in PBS for 1 hour. Normal mouse serum is used as a negative control. Starting with 1:100 blood, plates are washed with TBS / Tween 20 in PBS / 0.1% BSA / 0.1% Tween 20 (PBS / BSA / T), and goat anti-mouse immunoglobulin G (IgG) (heavy chain + light chain) horseradish peroxidase (HRP) (Thermo Fisher) 1:5000 is added and incubated at room temperature for 1 hour. Plates are washed with TBS / Tween 20, and antibody binding is detected using o-phenylenediamine dihydrochloride (OPD) substrate (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. The plate is read at 490 nM using Molecular Devices Spectromax. Titer is defined as a dilution factor that gives 4 times the background (as defined in the figure or table), and extrapolation is performed if the value falls between dilution factors.

[0225] Blocking the binding of tau to heparin

[0226] As a possible surrogate marker of serum's ability to block tau uptake into cells, we developed an ELISA to measure the blocking of tau binding to heparin plates. Recombinant tau was biotinylated in-house. Heparin-coated plates (Bioworld, Dublin, OH) were blocked with 2% BSA / PBS for 1 hour. In a separate deep-well polypropylene 96-well plate (ThermoFisher), serum was diluted 25- to 3200-fold with 60 μl total 2% BSA / PBS. To this 60 μl, 200 ng / ml of biotinylated tau in 2% BSA / PBS was added to the serum so that the final serum concentration was 50- to 6400-fold, resulting in a tau concentration of 100 ng / ml. The serum-tau mixture was incubated for 2 hours, then 100 μl / well was transferred to the blocked heparin plates and incubated for 1 hour. The plates were washed with 0.1% Tween 20 / TBS, and a 1:5000 mixture of goat anti-mouse immunoglobulin G (IgG) (heavy chain + light chain) and horseradish peroxidase (HRP) (ThermoFisher) was added and incubated at room temperature for 1 hour. The plates were washed with TBS / Tween 20, 100 μl of ThermoFisher TMB was added and incubated for 8 minutes, stopped with H2SO4 and read at 450°C.

[0227] IHC binding in serum

[0228] Autopsy blocks of fresh-frozen human brain tissue were embedded in an optimal cutting temperature compound (OCT compound) and dissected using a cryostat to produce 10 μm sections. The sections were placed in a solution of glucose oxidase and beta-D-glucose in the presence of sodium azide to block endogenous peroxidase. Once the tissue sections were prepared, staining with identified mouse immunoserum was performed in 5% goat serum containing 0.25% Triton at a 1:500 dilution at room temperature for 1 hour. To image plaque and tangle binding, biotin-SP-conjugated goat anti-mouse IgG, obtained from Jackson (Lot #115-065-166), was incubated with the sections at a 200-fold dilution. The DAKO DAB Detection Kit was used according to the manufacturer's instructions. Staining was performed using an automated Leica Bond Stainer. The results indicate that serum derived from guinea pigs immunized with the vaccines disclosed herein contains antibodies specific to A-beta and tau in human brain tissue from Alzheimer's patients.

[0229] Serum-mediated blocking of A-beta binding to neurons

[0230] E18 primary rat hippocampal neurons are cultured as previously described (Zago, et al. "Neutralization of Soluble, Synaptotoxic Amyloid β Species by Antibodies Is Epitope Specific," J Neurosci. 2012 Feb 22; 32(8): 2696-2702). To block soluble Aβ aggregates from binding to neurites, soluble Aβ aggregates are pre-incubated on culture medium DIV14-21 with or without vaccine serum. Fresh, unlabeled, biotinylated, or (9:1) soluble Aβ is prepared one day prior and incubated overnight at 4°C. Diluted serum samples and soluble Aβ solutions are prepared in NeuroBasal-Phenol Red-Free (NB-NPR) medium at twice the final concentration in half the final processing volume. This is combined with soluble Aβ at a 2x concentration in half the final volume and 2x diluted vaccine serum in half the final volume to create a total treatment volume of 1x the final concentration, which is thoroughly mixed and pre-incubated at 37°C for 30 minutes. E18 neurons are washed with 150 μL / well of NB-NPR before adding the binding treatment. Serum derived from vaccinated animals / Aβ-treated cells is added to the E18 neurons at 60 μL / well and incubated at 37°C for 30 minutes under normal incubator conditions (5% CO2; 9% O2). Cells are washed twice with 150 μL / well of NB-NPR and fixed with 4% paraformaldehyde in 1× DPBS for 20 minutes. Cells are permeabilized with 0.1 TX-100 for 5 minutes and blocked with 10% normal goat serum (NGS) at room temperature (RT) for 1 hour. Incubate the cells overnight at 4°C in 100 μL / well of 1× DPBS containing 1% BSA and 1% NGS with MAP2 and NeuN primary antibodies. The following day, wash the cells twice with 150 μL / well of 1× DPBS for 5 minutes each time. Add the secondary antibody in 100 μL / well of 1× DPBS + 1% BSA + 1% NGS at room temperature for 1 hour.To quantify the neurite binding of soluble aggregated Aβ, high-content imaging (HCI) analysis was performed using an Operetta HCI CLS instrument (Perkin Elmer; modified Neuroite Outgrowth algorithm: 40 × H2O objective; 40 fields per well; n=3 per condition; data shown as mean (±) SD). Dendritic neurites were tracked using MAP2 and NeuN (Abcam) neuron markers, and the number of cells per optical field was counted. Spots of Aβ-soluble aggregates on neurites were detected using streptavidin 488 or polyclonal Aβ antibody (Thermo; Millipore). Data were reported as Aβ-soluble aggregate spots / neuron (or as integrated intensity). Approximately 80–150 neurons were observed per well for each condition tested.

[0231] result

[0232] Data on the titers and staining of A-beta and tau are summarized in Tables 10 and 11. [Table 10] [Table 11]

[0233] potency

[0234] As can be seen in Figure 13 and Tables 10-13, most animals in all groups produced high and balanced A-beta and tau titers, although some animals had less variable titer responses. [Table 12] [Table 13-1] [Table 13-2]

[0235] Blocking the binding of tau to heparin

[0236] All constructs demonstrated the ability to block tau binding to heparin, with constructs DAEFRHDRRVKSKIGSTGGC (SEQ ID NO: 997) and DAEFRHDRRENLKHQPGGGC (SEQ ID NO: 1000) showing the strongest blocking. See Figure 15, Table 10, and Table 11.

[0237] IHC about Alzheimer's brain

[0238] Most animals exhibited pathogenic A-beta and tau staining. See Figure 16.

[0239] (Example 11) immunogen

[0240] Immunogens were selected for the evaluation of vaccine peptide constructs. The constructs include Aβ immunogen and tau immunogen. Some immunogens contain tau peptides with 3-10 amino acids derived from tau. Other immunogens contain engineered tau immunogen.

[0241] Manipulated tau immunogen

[0242] We designed and selected certain immunogenic peptides such that (i) they produce antibodies that bind within the microtubule-binding repeats (MTBRs) of human tau protein, (ii) they are unlikely to generate undesirable T cell-mediated autoimmune responses, and (iii) they are unlikely to produce antibodies that would cross-react with other human proteins.

[0243] First, to identify the amino acid residues crucial for generating antibodies that bind to the MTBR, we performed sequence analysis and 3D modeling of the tau MTBR. Using the results of these analyses, we designed synthetic tau immunogenic peptides containing conserved residues and swapped scattered residues. The resulting manipulated synthetic peptides are listed in Table 14. [Table 14]

[0244] Next, to assess the potential for undesirable T cell-mediated autoimmune responses, the engineered peptides were subjected to in silico analysis, and MHC II binding was predicted using the IEMB (Immune Epitope Database) of the National Institute of Allergy and Infectious Diseases / La Jolla Immunology Institute. MHC class II binding is considered a good indicator of sequences containing T cell epitopes. An allele panel was used to predict MHC II binding. Engineered peptides that predicted a maximum inhibitory concentration (IC50) above a specified cutoff were considered to have a low likelihood of MHC II binding and were selected for further analysis.

[0245] Finally, engineered peptides with low predicted MHC II binding were evaluated to predict whether the anti-tau MTBR antibodies produced by these peptides might exhibit undesirable cross-reactivity with other human proteins. The sequences of the engineered peptides were subjected to bioinformatics analysis against a non-redundant human proteome database to determine homology with human proteins. Engineered peptide sequences with low homology to secreted or cell surface proteins were selected as top candidates for use as antigens. The top candidate engineered tau immunogenic peptides are listed in Table 15. [Table 15]

[0246] Dual Aβ-tau constructs containing the engineered tau immunogen (e.g., dual #11) showed results comparable to, or in some cases better than, other tau immunogens, despite containing non-natural tau sequences. Figure 13 shows high titers for both A-beta and tau antigens containing the engineered tau sequences. Figure 14 shows blocking of tau binding to heparin. Figure 15G shows the binding of the obtained tau antibody to the MTBR region peptide. Figure 16E shows staining / binding to Aβ and tau peptides in brain tissue derived from human Alzheimer's patients. This demonstrates the usefulness of the engineered immunogen in vaccine construction.

[0247] conclusion

[0248] We developed dual immunogen Aβ-tau vaccine constructs and demonstrated that these constructs produced balanced titers against Aβ and tau in mice, guinea pigs, and cynomolgus monkeys. The antibodies were immunoreactive to both Aβ plaques and tau neurofibrillary tau tangles in human AD brain sections and blocked the binding of soluble Aβ aggregates (oligomers) to neurons without inducing a T-cell response to Aβ or tau. These results support the development of dual-immunogen vaccines, which are single agents capable of targeting pathogenic forms of Aβ and tau. These results support the development of dual Aβ-tau vaccines capable of targeting pathogenic Aβ and tau for the prevention and / or treatment of AD.

[0249] Although various specific embodiments of the present invention have been described herein, it should be understood that the present invention is not limited to these exact embodiments, and that various changes or modifications can be applied herein by those skilled in the art without departing from the scope and spirit of the invention.

[0250] In each of the embodiments of the peptides described herein, the peptide may include, consist of, or essentially consist of the cited sequence. Accordingly, the following sequences, which may be part of a composition comprising the amyloid-beta (Aβ) peptide and tau peptide disclosed herein, are incorporated into this disclosure (see Table 16). [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 16-5] [Table 16-6] [Table 16-7] [Table 16-8] [Table 16-9] [Table 16-10] [Table 16-11] [Table 16-12] Table 16-13 Table 16-14 Table 16-15 Table 16-16 Table 16-17 Table 16-18 Table 16-19 Table 16-20 Table 16-21 Table 16-22

Claims

[Claim 1] The invention described in the specification.

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