Multi-epitope vaccines for the treatment of Alzheimer's disease

Abstract

The present disclosure provides peptide compositions and immunotherapy compositions containing amyloid-beta (Aβ) peptides and alpha-synuclein peptides. The present disclosure also provides methods for treating or preventing Alzheimer's disease or other diseases involving beta-amyloid deposits in a subject, including methods for clearing deposits, inhibiting or reducing Aβ and / or alpha-synuclein aggregation, blocking neuronal uptake, clearing amyloid, and inhibiting the propagation of alpha-synuclein seeds in a subject with or at risk of developing Alzheimer's disease or other diseases involving alpha-synuclein and / or amyloid-beta accumulation. The methods include administering a composition containing amyloid-beta (Aβ) peptides and alpha-synuclein peptides to such a patient.

Description

[Technical Field]

[0001] Related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 080,619, filed on 18 September 2020, which is incorporated herein by reference in its entirety.

[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 3 August 2021 and is contained in an ASCII text file named "20-1084-WO_Sequence-Listing_ST25.txt" with a size of 31kb.

[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. Senile plaques (i.e., amyloid plaques) are areas of irregular neural networks up to 150 μm in size, encompassing extracellular amyloid deposition in the center, which are visualized by microscopic analysis of sections of brain tissue. The accumulation of amyloid plaques in the central nervous system is also associated with cerebral amyloid angiopathy (CAA) in Down syndrome and other cognitive disorders, as well as age-related macular degeneration in the eye disease.

[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] Alpha-synuclein, a protein found in neurons and other cells, is a key component of the pathology that characterizes several neurodegenerative disorders collectively known as synucleinopathy, including Parkinson's disease, Lewy body dementia, and multiple system atrophy. While the normal physiological function of alpha-synuclein is only partially understood, evidence suggests that the protein's soluble form can interact with other proteins and certain intracellular membranes. In synucleinopathy, alpha-synuclein protein appears to aggregate abnormally within cells, contributing to the disease pathology. There is growing evidence that certain aggregate forms of alpha-synuclein can be transmitted between neurons, leading to the propagation of pathology that causes neuronal dysfunction and neuronal loss. Misfolding and aggregation of alpha-synuclein (SNCA) often occur in association with β-amyloid deposition in some neurodegenerative diseases, and alpha-synuclein aggregates and Aβ aggregates coexist in several neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease.

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

[0008] Abstract 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 10 amino acids derived from residues 81 to 140 of SEQ ID NO: 02. For example, the second peptide may be derived from the C-terminus of alpha-synuclein (residues 111 to 140 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 121 to 176, and the second peptide may contain one amino acid sequence from SEQ ID NOs. 39 to 109. 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 10 amino acids, for example, PDNEAYE (SEQ ID NO: 55) or DPDNEAYE (SEQ ID NO: 48).

[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: 113)), 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: 114)), Ala-Gly-Ala-Gly (SEQ ID NO: 115) or Lys-Gly-Lys-Gly (SEQ ID NO: 116). In certain embodiments, the polypeptide may be DAEFRHDRRPDNEAYEGGC (SEQ ID NO: 110) or DAEFRHDRRDPDNEAYEGGC (SEQ ID NO: 111).

[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 DAEFRHDRRX1PDNEAYEXXC (SEQ ID NO: 112), where X1 is optional and, if present, is D, and XX and C are independently optional and, if present, XX may be GG, AA, KK, SS, GAGA (SEQ ID NO: 114), AGAG (SEQ ID NO: 115), or KGKG (SEQ ID NO: 116).

[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 8 amino acids derived from residues 81 to 140 of SEQ ID NO: 02. The first peptide may comprise one amino acid sequence from SEQ ID NOs: 3 to 38 or 121 to 176, and the second peptide may comprise one amino acid sequence from SEQ ID NOs: 39 to 109. Each of the first and second peptides may contain a linker to a carrier at the C-terminal portion of the polypeptide. If present, the linker may comprise an amino acid sequence selected from GG, AA, KK, SS, GAGA (SEQ ID NO: 114), AGAG (SEQ ID NO: 115), and KGKG (SEQ ID NO: 116), and may comprise a C-terminal cysteine ​​(C). The carrier may contain serum albumin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid (TT), diphtheria toxoid (DT), genetically modified cross-reactive substances of diphtheria toxin (CRM), 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 alpha-synuclein 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 method of the present disclosure is directed to inducing an immune response in an animal. The method includes 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, alpha-synuclein, or both Aβ and alpha-synuclein. The immune response may include an antibody that specifically binds to the N-terminal region of Aβ and / or the C-terminal region of alpha-synuclein.

[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. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] [Figure 1] FIG. 1 shows the results of an experiment comparing the titers of guinea pig sera against the immunogens DAEFRHDRRPDNEAYEGGC (SEQ ID NO: 110) and DAEFRHDRRDPDNEAYEGGC (SEQ ID NO: 111). All immunogens included a GG C-terminal linker and cysteine for coupling to maleimide-activated CRM197 carrier. QS21 was utilized as an adjuvant in an AddaVax squalene-based water-in-oil nanoemulsion.

[0023] [Figure 2]Figure 2 shows the results of an experiment measuring the titers of mouse sera against the immunogens DAEFRHDRRPDNEAYEGGC (SEQ ID NO: 110) and DAEFRHDRRDPDNEAYEGGC (SEQ ID NO: 111). The peptides were coupled to maleimide-activated CRM197 carrier via the N-terminal cysteine. QS21 in PBS was used as an adjuvant.

[0024] [Figure 3] Figure 3 shows the results of an experiment measuring the titers of guinea pig sera against the immunogens DAEFRHDRRPDNEAYEGGC (SEQ ID NO: 110) and DAEFRHDRRDPDNEAYEGGC (SEQ ID NO: 111). The peptides were coupled to maleimide-activated CRM197 carrier via the N-terminal cysteine. QS21 in PBS was used as an adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Description The present disclosure provides peptide compositions and immunotherapy compositions comprising amyloid-beta (Aβ) peptides and alpha-synuclein peptides. The present disclosure also provides methods of treating or preventing Alzheimer's disease or other diseases associated with beta-amyloid deposition in a subject, and in a subject having or at risk of developing Alzheimer's disease or other diseases involving alpha-synuclein and / or amyloid-beta accumulation, methods of inhibiting or reducing aggregation of Aβ and / or alpha-synuclein, blocking binding and / or uptake of Aβ and / or alpha-synuclein by neurons, preventing formation and removing deposits thereof, inhibiting transmission of alpha-synuclein species between cells, and inhibiting propagation of pathology between brain regions. The methods include administering to such patients a composition comprising amyloid-beta (Aβ) peptides and alpha-synuclein peptides.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] The terms “prevention,” “prevent,” or “preventing,” as used herein, mean bringing a subject into contact with (e.g., administering) the peptide or immunotherapy composition of the Disclosure before the onset of a disease, with or without pre-existing Aβ and / or alpha-synuclein 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 in contact with the peptide or immunotherapy composition, but not completely suppressing the onset of the disease. In some cases, prevention may occur within a limited time period following administration of the peptide or immunotherapy composition of the Disclosure. In other cases, prevention may occur during the duration of a treatment regimen that includes administration of the peptide or immunotherapy composition of the Disclosure.

[0035] The terms “reduce,” “reduce,” or “to reduce,” as used herein, mean reducing the amount of Aβ and / or alpha-synuclein present in or to the tissue of the subject, or inhibiting its increase, which includes reducing the amount of accumulated, aggregated, or deposited Aβ and / or alpha-synuclein present in or to the tissue of the 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 alpha-synuclein present in or to the tissue of the subject, or inhibiting its increase (e.g., reducing the rate of increase), means the amount of accumulated, aggregated, or deposited Aβ and / or alpha-synuclein 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 alpha-synuclein present in the subject, or an inhibition of its increase (e.g., reducing the rate of increase), refers to the amount of accumulated, aggregated, or deposited Aβ and / or alpha-synuclein 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 alpha-synuclein present in the subject, or an inhibition of its increase (e.g., reducing the rate of increase), refers to the amount of accumulated, aggregated, or deposited Aβ and / or alpha-synuclein present in the brain of the subject. In some embodiments, the reduced Aβ and / or alpha-synuclein are pathological forms of Aβ (e.g., extracellular plaque deposition of β-amyloid peptide (Aβ), neurite amyloid plaques) and / or alpha-synuclein (e.g., fibular alpha-synuclein-containing, oligomeric or fibrillary alpha-synuclein aggregates, and protofibril intermediates of alpha-synuclein oligomers). In yet other embodiments, pathological indicators of neurodegenerative diseases and / or synucleinopathy are reduced.

[0036] 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).

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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 alpha-synuclein peptides in a recipient. Such a response may be an active response induced by the administration of an immunogen (e.g., Aβ and / or alpha-synuclein peptides). Cellular immune responses are 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.

[0044] Amyloid beta (Aβ or A-beta)

[0045] 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 at 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 of Aβ, or residues 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 of Aβ. 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.

[0046] 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.

[0047] Alpha-synuclein

[0048] Alpha-synuclein is a highly conserved protein abundant in neurons, particularly in presynaptic terminals. Agglutinated alpha-synuclein proteins form brain lesions characteristic of neurodegenerative synucleinopathy. Furthermore, misfolding and aggregation can often occur in association with β-amyloid deposition in some neurodegenerative diseases, and alpha-synuclein coexists in several neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease.

[0049] Immunogen Aβ / alpha-synuclein polypeptide

[0050] 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 or 12, or more, consecutive amino acids derived from the Aβ and / or alpha-synuclein peptide region.

[0051] In some embodiments of this disclosure, the Aβ / alpha-synuclein immunogen may comprise an Aβ peptide comprising 3 to 10 amino acids derived from residues 1 to 10 of the N-terminal sequence of Aβ (SEQ ID NO: 01), linked to an alpha-synuclein peptide comprising 3 to 10 amino acids derived from residues 81 to 140 of alpha-synuclein (SEQ ID NO: 02). For example, the alpha-synuclein peptide may comprise 3 to 10 amino acids derived from the C-terminal region of alpha-synuclein (residues 111 to 140 of SEQ ID NO: 02). In some embodiments, the peptide is unphosphorylated. In some embodiments, the peptide is phosphorylated at serine (S), threonine (T), and / or tyrosine (Y) phosphorylation sites.

[0052] 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.

[0053] In certain embodiments, the Aβ peptide is DAEFRHD (SEQ ID NO: 06), DAEFR (SEQ ID NO: 08), or EFRHD (SEQ ID NO: 21).

[0054] The alpha-synuclein peptide may correspond to a peptide containing 3 to 10 amino acids derived from residues 81 to 140 of SEQ ID NO: 02. In some embodiments, the alpha-synuclein is not phosphorylated. In some embodiments, the alpha-synuclein is phosphorylated. In some compositions, the alpha-synuclein peptide is as follows: [ka] [ka] [ka] Selected from.

[0055] In some embodiments, the Aβ and alpha-synuclein peptides are linked to form a dual Aβ / alpha-synuclein polypeptide. The Aβ and alpha-synuclein 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 alpha-synuclein peptide can be arranged in any order on the dual Aβ / alpha-synuclein polypeptide. For example, the Aβ peptide may be located at the N-terminal portion of the dual polypeptide, and the alpha-synuclein peptide may be located at the C-terminal portion of the dual polypeptide. Alternatively, the alpha-synuclein peptide may be located at the N-terminal portion of the dual polypeptide, and the Aβ peptide may be located at the C-terminal portion of the alpha-synuclein peptide on the dual polypeptide side. References to the first or second peptide herein are not intended to suggest the order of the Aβ or alpha-synuclein peptide in the immunogen polypeptide.

[0056] In addition, the C-terminal portion of the Aβ peptide, alpha-synuclein peptide, or dual Aβ-alpha-synuclein polypeptide may include a linker for conjugating the peptide or polypeptide to a carrier. Examples of linkers for coupling the peptide or dual polypeptide to the carrier may include GG, GGG, KK, KKK, AA, AAA, SS, SSS, GAGA (SEQ ID NO: 114), AGAG (SEQ ID NO: 115), KGKG (SEQ ID NO: 116), 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, the linker includes one of the amino acid sequences AA, AAA, KK, KKK, SS, SSS, AGAG (SEQ ID NO: 115), GG, GGG, GAGA (SEQ ID NO: 114), and KGKG (SEQ ID NO: 116). In some embodiments, any of the Aβ peptide, alpha-synuclein peptide, and dual Aβ / alpha-synuclein polypeptide may contain a C-terminal cysteine, e.g., AEFRHDSGC (SEQ ID NO: 117) and DAEFRHDC (SEQ ID NO: 118), without a spacer. In some embodiments, any of the Aβ peptide, alpha-synuclein peptide, and dual Aβ / alpha-synuclein polypeptide may contain an N-terminal cysteine, without a spacer.

[0057] When Aβ and alpha-synuclein polypeptides are linked to form a dual Aβ / alpha-synuclein polypeptide, the linker may be a cleavable linker. As used herein, the term “cleavable linker” refers to any linker between antigen peptides that, by cleavage (e.g., by endopeptidase, protease, low pH, or any other means that may occur in or around the antigen-presenting cell), facilitates the separation of the Aβ peptide and the alpha-synuclein peptide from each other, 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: 113), 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; GAGA (SEQ ID NO: 114)), alanine-glycine-alanine-glycine (Ala-Gly-Ala-Gly; AGAG (SEQ ID NO: 115)), and lysine-glycine-lysine-glycine (Lys-Gly-Lys-Gly; KGKG (SEQ ID NO: 116)). In some compositions, the cleavable linker is arginine-arginine (Arg-Arg).

[0058] In some embodiments of the present disclosure, the dual Aβ / alpha-synuclein polypeptide comprises an amino acid sequence selected from DAEFRHDRRPDNEAYEGGC (SEQ ID NO: 110), DAEFRHDRRDPDNEAYEGGC (SEQ ID NO: 111), or DAEFRHDRRX1PDNEAYEXXC (SEQ ID NO: 112), where X1 is optionally present and, if present, D, and XX and C are independently optionally present and, if present, XX may be GG, AA, KK, SS, GAGA (SEQ ID NO: 114), AGAG (SEQ ID NO: 115), or KGKG (SEQ ID NO: 116).

[0059] In some embodiments, the dual Aβ / alpha-synuclein 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 an alpha-synuclein peptide, and if [the first peptide] is an alpha-synuclein peptide, then [the second peptide] is an Aβ peptide, and [linker 1], [linker 2] and [Cys] are present as needed, and [linker 1] and [linker 2] are the same or different linkers).

[0060] In a particular embodiment, the dual Aβ / alpha-synuclein 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 an alpha-synuclein peptide, and if [the first peptide] is an alpha-synuclein peptide, then [the second peptide] is an Aβ peptide, and [linker 1], [linker 2] and [Cys] are present as needed, and [linker 1] and [linker 2] are the same or different linkers).

[0061] Examples of Aβ peptides include either sequence numbers 3-38 or 121-176.

[0062] An example of an alpha-synuclein peptide is one of the sequence numbers 39-109.

[0063] [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: 113), 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: 114), alanine-glycine-alanine-glycine (Ala-Gly-Ala-Gly; SEQ ID NO: 115), or lysine-glycine-lysine-glycine (Lys-Gly-Lys-Gly; SEQ ID NO: 116).

[0064] 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: 177) or (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 178), 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: 114), AGAG (SEQ ID NO: 115), and KGKG (SEQ ID NO: 116).

[0065] [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.

[0066] Examples of the [first peptide]-[linker 1]-[second peptide]-[linker 2]-[Cys] dual Aβ / alpha-synuclein polypeptides of this disclosure include: [Table 1-1]

[0067] polypeptide immunogen

[0068] Aβ peptides, alpha-synuclein peptides, and dual Aβ / alpha-synuclein polypeptides are immunogens according to this disclosure. In some embodiments, the peptides and dual Aβ / alpha-synuclein polypeptides can be ligated to a suitable carrier to help induce an immune response. Thus, one or more peptides and dual Aβ / alpha-synuclein polypeptides of this disclosure can be ligated to a carrier. For example, each of the Aβ peptide, alpha-synuclein peptide, and Aβ / alpha-synuclein polypeptide may be linked to a carrier with or without a spacer amino acid (e.g., Gly-Gly, Ala-Ala, Lys-Lys, Ser-Ser, Gly-Ala-Gly-Ala (SEQ ID NO: 114), Ala-Gly-Ala-Gly (SEQ ID NO: 115), or Lys-Gly-Lys-Gly (SEQ ID NO: 116)), and optionally, in certain embodiments, the dual Aβ-alpha-synuclein polypeptide can be linked to a suitable carrier using its C-terminal cysteine ​​to provide a linker between the peptide and the carrier or between the dual Aβ / alpha-synuclein polypeptide and the carrier. In certain embodiments, the dual Aβ-alpha-synuclein polypeptide can be linked to a suitable carrier using its N-terminal cysteine ​​to provide a linker between the peptide and the carrier.

[0069] 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 coupling agents form thioethers rather than disulfide couplings. 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 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. Virus-like particles (VLPs), also called pseudovirions or virus-derived particles, represent a subunit structure composed of multiple copies of a viral capsid and / or envelope protein that can self-assemble in vivo into a spherically symmetric VLP (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).

[0070] 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 / alpha-synuclein polypeptides. For example, the dual Aβ / alpha-synuclein polypeptide of the immunogen may contain polypeptides having the Aβ antigen and alpha-synuclein in different orders, or may exist with or without an intrapeptide linker and / or a linker to the carrier.

[0071] 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.

[0072] nucleic acid

[0073] This disclosure further provides nucleic acids encoding either the amyloid-beta (Aβ) peptide or the alpha-synuclein 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 alpha-synuclein 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 alpha-synuclein peptide is a sequence 3 to 8 amino acid residues long, derived from residues 81 to 140 of SEQ ID NO: 02. Thus, a nucleic acid encoding any of SEQ ID NOs: 3 to 38 may be combined with a nucleic acid encoding any of SEQ ID NOs: 39 to 109 to provide components of the immunogens and pharmaceutical compositions of this disclosure. Similarly, one or more nucleic acids encoding either the A-beta and alpha-synuclein sequences may contain codons for RR-N-terminal or -RR-C-terminal dipeptides. In certain embodiments, the Aβ and alpha-synuclein 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 mitigating Alzheimer's disease. In other embodiments, the nucleic acid immunotherapy compositions disclosed herein provide compositions for reducing the pathogenic forms of Aβ and alpha-synuclein in and / or in the tissue of the subject. In some embodiments, the Aβ and / or alpha-synuclein reduced by the immunotherapy composition are pathological forms of Aβ (e.g., extracellular plaque deposition of β-amyloid peptide (Aβ), neurite amyloid plaques) and / or alpha-synuclein (e.g., frame-shaped neurofibrillary tangles of alpha-synuclein oligomers).In yet another embodiment, pathological indicators of neurodegenerative diseases and / or synucleinopathy are reduced by the nucleic acid immunotherapy composition. In yet another embodiment, the nucleic acid immunotherapy compositions disclosed herein provide compositions for reducing brain Aβ and brain alpha-synuclein.

[0074] 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 alpha-synuclein 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 alpha-synuclein 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.

[0075] 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, for example, Beissert et al., Molecular Therapy January 2020 28(1):119-128). In certain embodiments, the RNA vaccine may encode the Aβ peptide and alpha-synuclein peptide disclosed herein and may express the Aβ and alpha-synuclein peptides, 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 modification of the RNA that is 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'-cap may be modified to have the ability to stabilize the RNA when bound to it. In certain embodiments, further modifications may be extension or cleavage of naturally occurring poly(A) tails, or alteration of the 5'- or 3'-untranslated region (UTR). In some embodiments, the RNA, e.g., or mRNA vaccine, is formulated in an effective amount to produce an antigen-specific immune response in the subject. For example, the RNA vaccine formulation is administered to the subject to stimulate the subject's humoral and / or cellular immune systems against Aβ and alpha-synuclein 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 alpha-synuclein antigens.

[0076] 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 ndEd., Garland Publishing, Inc., New York, NY (1989);Watson, JD et al., Recombinant DNA, 2 nd 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.

[0077] 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).

[0078] 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.

[0079] Pharmaceutical composition

[0080] 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.

[0081] 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).

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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)).

[0090] 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).

[0091] 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.

[0092] Suitable subjects for treatment

[0093] The presence of Aβ plaques and / or neurofibrillary tangles has been found in several conditions, including Alzheimer's disease, Down syndrome, mild cognitive impairment, cerebral amyloid angiopathy, 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, amyotrophic lateral sclerosis / parkinsonist dementia complex of Guam, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), Parkinson's disease, progressive supranuclear palsy (PSP), atrophic age-related macular degeneration (AMD), and inclusion body myositis.

[0094] 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 alpha-synuclein, 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 alpha-synuclein (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 having mutations in Aβ and / or alpha-synuclein associated with neurological disorders. The methods are particularly suitable for the treatment or prevention of Alzheimer's disease.

[0095] Suitable candidates 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 those in the aging population, asymptomatic subjects with Aβ and / or alpha-synuclein pathology and known genetic risk for the disease. Such individuals include those with relatives experiencing 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 alpha-synuclein, 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 an increased risk of Alzheimer's disease (AD). Other markers of risk for Alzheimer's disease 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 aforementioned risk factors. In addition, several diagnostic tests are available to identify individuals with AD. These include measuring alpha-synuclein and αβ42 levels in CSF or blood. Elevated alpha-synuclein and decreased αβ42 levels indicate the presence of AD. Mutations in some genes, such as Ala30Pro or Ala53Thr, or 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.

[0096] 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.

[0097] Treatment and Usage

[0098] This disclosure provides a method for inhibiting or reducing the aggregation of Aβ or alpha-synuclein in subjects having or at risk of developing a neurodegenerative disease (e.g., Alzheimer's disease). The method comprises administering the compositions disclosed herein to the subject. 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.

[0099] 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 synucleinopathy, and / or inhibiting or delaying its 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 already suffering from 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 levels of Aβ plaques and / or synucleinopathy associated with toxicity and / or behavioral defects.

[0100] 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).

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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 vascular system 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.

[0106] 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 is sufficient as needed. For chronic disorders, the vaccines / immunotherapies disclosed herein can be administered at regular intervals, e.g., weekly, bi-weekly, monthly, every 3 months, every 6 months, for at least 1, 5 or 10 years, or over the patient's lifetime.

[0107] 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.

[0108] 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.

[0109] 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).

[0110] The immunotherapeutic compositions disclosed herein may also be used in combination with other treatments for diseases associated with the accumulation of Aβ or alpha-synuclein, 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. 2010 / 0202968 and U.S. Patent No. 8,906,367, and / or anti-alpha-synuclein antibodies, for example, antibodies that specifically bind to any of the alpha-synuclein epitopes disclosed herein, ABBV-8E12, goslanemab, zagotenemab, RG-6100, BIIB076, or WO2014 / 165271, U.S. Patent No. 10,501,531, WO2017 / 191560, U.S. Patent No. 2019 / 0330314, WO2017 / 191561, U.S. Patent No. 2019 / 033 Any of the antibodies disclosed in No. 0316, WO2017 / 191559, and WO2018 / 204546; and / or anti-alpha-synuclein antibodies, for example, antibodies that specifically bind to any of the alpha-synuclein epitopes disclosed herein, or antibodies and / or other alpha-synuclein-binding compounds, for example, PRX002 / RO7046015, PRX002 / RG7935 (pracinezumab), NPT200-11 / UCB0599, NPT088, BIIB054 (simpanemab), ABBV-0805, MEDI-1341, NPT088, and Lu AF82422. In some combination therapy methods, the patient receives passive immunotherapy before the active immunotherapy method disclosed herein. Alternatively, the patient may receive passive and active immunotherapy during the same period of treatment. Or, 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.

[0111] The compositions of this disclosure may be used in the manufacture of pharmaceuticals for the treatment regimens described herein.

[0112] Treatment regimen

[0113] 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.

[0114] 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months later. Another regimen involves administration every two months for life. Alternatively, booster doses may be irregular, as indicated by monitoring of the immune response.

[0115] 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.

[0116] kit

[0117] 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.

[0118] 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.

[0119] use

[0120] 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.

[0121] 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.

[0122] All U.S. and international patent applications identified herein are incorporated herein by reference in their entirety. [Examples]

[0123] (Example 1) animal immunity

[0124] Female Swiss Webster mice were subcutaneously injected with 100 μl of test immunogen at two sites (200 μl total) on days 0, 14, 42, and 70. The test immunogen was prepared by mixing 25 μg of test immunogen with 25 μg of QS21 adjuvant in 200 μl of phosphate-buffered saline (PBS). Blood was collected from mice on days 21, 49, and 77 by wounding the tail and collecting 50 μl of blood, which was then treated with serum. The immunogens tested included DAEFRHDRRPDNEAYEGGC (SEQ ID NO: 110) and DAEFRHDRRDPDNEAYEGGC (SEQ ID NO: 111). The immunogen contained an Aβ peptide, an alpha-synuclein peptide, a C-terminal linker, and a C-terminal cysteine ​​(i.e., -Gly-Gly-Cys-), and was coupled to CRM-197 via a maleimide bond through the C-terminal cysteine.

[0125] 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 tested immunogens included DAEFRHDRRPDNEAYEGGC (SEQ ID NO: 110) and DAEFRHDRRDPDNEAYEGGC (SEQ ID NO: 111). The immunogens contained Aβ peptide, alpha-synuclein peptide, a C-terminal linker, and a C-terminal cysteine ​​(i.e., -Gly-Gly-Cys-), which was coupled to CRM-197 via a maleimide bond through the C-terminal cysteine.

[0126] 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.

[0127] The immunogen concentration was 0.5 mg / ml. Before administering each test immunogen, the injection site was visualized by applying a small amount of solution approximately 3 cm from each hind limb. 2 The 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 QS-21 in 100 μl Addavax). 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.

[0128] (Example 2) Measurement of antibody titer

[0129] For guinea pigs, whole blood samples were collected via the jugular vein at weeks 1, 4, 8, and 12, at a rate of 250–350 μl per sample. For mice, whole blood samples were collected via tail incision at weeks 1, 3, 7, and 11, at a rate of 50 μl per sample. At the end of the final blood collection week, the maximum volume of whole blood was collected via cardiac puncture and placed in a coagulation activator tube. 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 transferred individually to clean frozen vials. The serum supernatant was stored frozen at -80°C (±12°C).

[0130] Aβ titer (mouse)

[0131] Plates were coated with 2 μg / ml Aβ1-28 monomers in PBS at a concentration of 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 mouse serum was added to column 1 at a 100-fold dilution, while the rest of the row contained the 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-mouse IgG HRP was prepared in PBS Tween containing 0.1% BSA, then added to the washed wells. This was incubated for 1 hour and washed. OPD substrates were prepared using Thermo-Fisher OPD tablets, at a rate of 1 tablet per 10 mL. Thermo-Fisher substrate buffer was added at a 10-fold dilution, 100 μl 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 Molecular Devices Spectromax. Titer was defined as the dilution factor that gives 50% of the maximum OD, and extrapolation was performed when values ​​fell between dilution factors.

[0132] Alpha-synuclein titer (mouse)

[0133] Plates were coated with 2 μg / ml recombinant human alpha-synuclein in PBS at a concentration of 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 mouse serum was added to column 1 at a 100-fold dilution, while the rest of the row contained the 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-mouse IgG HRP was prepared in PBS Tween containing 0.1% BSA, then added to the washed wells. This was incubated for 1 hour and washed. OPD substrates were prepared using Thermo-Fisher OPD tablets, at a rate of 1 tablet per 10 mL. Thermo-Fisher substrate buffer was added at a 10-fold dilution, 100 μl 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 Molecular Devices Spectromax. Titer was defined as the dilution factor that gives 50% of the maximum OD, and extrapolation was performed when values ​​fell between dilution factors.

[0134] Aβ titer (guinea pig)

[0135] Plates were coated with 2 μg / ml Aβ1-28 monomers in PBS at a concentration of 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 guinea pig serum was added to column 1 at a 100-fold dilution, while the rest of the row contained the 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 washed. OPD substrates were prepared using Thermo-Fisher OPD tablets at a rate of 1 tablet per 10 mL. Thermo-Fisher substrate buffer was added at a 10-fold dilution, 100 μl 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 Molecular Devices Spectromax. Titer was defined as the dilution factor that gives 50% of the maximum OD, and extrapolation was performed when values ​​fell between dilution factors.

[0136] Alpha-synuclein titer (guinea pig)

[0137] Plates were coated with 100 μl / well of recombinant human alpha-synuclein at 2 μg / ml 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 0.1% BSA in 200 μl of PBS Tween was added to row A. Negative guinea pig serum was added to column 1 at a 100-fold dilution, while the rest of the row contained the test serum at a 100-fold dilution. The rows were serially diluted 1:2 under the plate to dilutions 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, then added to the washed wells. This was incubated for 1 hour and washed. OPD substrates were prepared using Thermo-Fisher OPD tablets at a rate of 1 tablet per 10 mL. Thermo-Fisher substrate buffer was added at a 10-fold dilution, 100 μl 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 Molecular Devices Spectromax. Titer was defined as the dilution factor that gives 50% of the maximum OD, and extrapolation was performed when values ​​fell between dilution factors.

[0138] Table 1 shows the antibody titers observed in guinea pigs immunized as described above. Immunization was performed using QS21 in Addavax. The reported titers are from blood samples taken after the second injection. These results are shown in Figure 1.

[0139] [Table 1-2]

[0140] Table 2 shows the antibody titers observed in mice immunized as described above. Immunization was performed using QS21. The reported titers are from blood samples taken after the third injection. These results are shown in Figure 2.

[0141] [Table 2]

[0142] (Example 3) Staining of Alzheimer's brain tissue with serum derived from animals immunized with the vaccine disclosed herein.

[0143] Endogenous peroxidase is blocked by placing the tissue sections in a solution of glucose oxidase and beta-D-glucose in the presence of sodium azide. Once the tissue sections are prepared, staining is performed with identified serum from animals immunized with the vaccine disclosed herein, at two dilutions (1:300 and 1:1500), using appropriate secondary antibodies for the animal species and the DAKO DAB Detection Kit, according to the manufacturer's instructions. Staining is performed using an automated Leica Bond Stainer. The results indicate that serum from animals immunized with the vaccine disclosed herein contains antibodies specific to A-beta and / or alpha-synuclein in human brain tissue from Alzheimer's patients.

[0144] conclusion

[0145] We developed dual-immunogen Aβ-alpha-synuclein vaccine constructs and demonstrated that these constructs produced balanced titers against Aβ and alpha-synuclein in mice, guinea pigs, and cynomolgus monkeys. The antibodies were immunoreactive to both Aβ plaques and neurofibrillary alpha-synuclein 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 alpha-synuclein. These results support the development of a dual-immunogen vaccine, a single agent capable of targeting pathogenic forms of Aβ and alpha-synuclein. These results support the development of a dual Aβ-alpha-synuclein vaccine capable of targeting pathogenic Aβ and alpha-synuclein for the prevention and / or treatment of AD.

[0146] 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.

[0147] In each of the embodiments of the peptides described herein, the peptide may include, consist of, or essentially consist of the listed sequences. Accordingly, the following sequences, which may be part of a composition comprising the amyloid-beta (Aβ) peptide and alpha-synuclein peptide disclosed herein, are incorporated into this disclosure (see Table 3). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

Claims

1. i) a first peptide comprising 3 to 10 amino acids derived from residues 1 to 10 of SEQ ID NO: 01; and a second peptide comprising 3 to 10 amino acids from residues 111 to 132 of SEQ ID NO: 02, and an intrapeptide linker connecting the first peptide and the second peptide, and optionally ii) a linker and a carrier at either the C-terminal or N-terminal portion of the polypeptide A polypeptide comprising:

2. The polypeptide of claim 1 , wherein the first peptide is N-terminal to the second peptide.

3. The polypeptide of claim 1 , wherein the first peptide is C-terminal to the second peptide.

4. A polypeptide described in any of claims 1 to 3, wherein the 3 to 10 amino acids of the first polypeptide include the amino acid sequence EFR, and the 3 to 10 amino acids of the second polypeptide include the amino acid sequence DNE.

5. (a) the first peptide is 【Chemistry 7-1】 【Chemistry 7-2】 and comprising an amino acid sequence selected from the group consisting of: (b) the second peptide is 【Chemistry 8-1】 【Chemistry 8-2】 comprising an amino acid sequence selected from the group consisting of: A polypeptide according to any one of claims 1 to 4.

6. The polypeptide of any one of claims 1 to 5, wherein the first peptide and the second peptide are linked by a cleavable linker.

7. The polypeptide of claim 6 , wherein the cleavable linker comprises an amino acid sequence.

8. 8. The polypeptide of claim 7, wherein the amino acid sequence comprises arginine-arginine (Arg-Arg), arginine-valine-arginine-arginine (Arg-Val-Arg-Arg (SEQ ID NO: 113)), 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: 114), alanine-glycine-alanine-glycine (Ala-Gly-Ala-Gly; SEQ ID NO: 115), or lysine-glycine-lysine-glycine (Lys-Gly-Lys-Gly; SEQ ID NO: 116).

9. The polypeptide of any one of claims 1 to 8, further comprising a linker to a carrier at either the C-terminal portion of the polypeptide or the N-terminal portion of the polypeptide.

10. 10. The polypeptide of claim 9, wherein the linker comprises an amino acid sequence selected from the group consisting of GG, GGG, AA, AAA, KK, KKK, SS, SSS, GAGA (SEQ ID NO: 114), AGAG (SEQ ID NO: 115), and KGKG (SEQ ID NO: 116).

11. The polypeptide of any one of claims 1 to 10, wherein the polypeptide, or the linker to the carrier, if present, further comprises a C-terminal cysteine (C).

12. The polypeptide of any one of claims 1 to 11, wherein the first peptide is DAEFRHD (SEQ ID NO: 06), DAEFR (SEQ ID NO: 08) or EFRHD (SEQ ID NO: 21).

13. A polypeptide described in any of claims 1 to 12, wherein the 3 to 10 amino acids of the first polypeptide comprise the amino acid sequence EFRHD (sequence number 21), and the 3 to 10 amino acids of the second polypeptide comprise the amino acid sequence DNEAY (sequence number 62).

14. The polypeptide of any one of claims 1 to 13, wherein the second peptide comprises 5 to 10 amino acids.

15. The polypeptide of any one of claims 1 to 13, wherein the second peptide comprises the amino acid sequence PDNEAYE (SEQ ID NO: 55).

16. A polypeptide described in any of claims 1 to 13, wherein the second peptide comprises the amino acid sequence DPDNEAYE (sequence number 48).

17. 2. The polypeptide of claim 1, comprising the amino acid sequence DAEFRHDRRPDNEAYEGGC (SEQ ID NO: 110).

18. The polypeptide of claim 1, comprising the amino acid sequence DAEFRHDRRDPDNEAYEGGC (sequence number 111).

19. DAEFRHDRRRX １ PDNEAYEXXC (SEQ ID NO: 112), wherein X １ is optionally present and, when present, is D; XX and C are independently optionally present, and, when present, XX can be GG, AA, KK, SS, GAGA (SEQ ID NO: 114), AGAG (SEQ ID NO: 115), or KGKG (SEQ ID NO: 116).

20. An immunotherapeutic composition comprising a polypeptide according to any one of claims 1 to 19, wherein said polypeptide is linked to a carrier.

21. 21. The immunotherapy composition of claim 20, wherein the carrier comprises serum albumin, an immunoglobulin molecule, thyroglobulin, ovalbumin, tetanus toxoid (TT), diphtheria toxoid (DT), a genetically modified cross-reacting material of diphtheria toxin (CRM), CRM197, meningococcal outer membrane protein complex (OMPC) and H. influenzae protein D (HiD), rEPA (Pseudomonas aeruginosa exotoxin A), KLH (keyhole limpet hemocyanin), and flagellin.

22. The immunotherapy composition described in claim 21, wherein the carrier is CRM197.

23. The immunotherapy composition of claim 22, wherein the carrier is diphtheria toxoid.

24. A pharmaceutical formulation comprising (a) a polypeptide according to any one of claims 1 to 19 or an immunotherapeutic composition according to any one of claims 20 to 23, and (b) at least one adjuvant.

25. 25. The pharmaceutical formulation of claim 24, wherein the adjuvant is selected from the group consisting of aluminum hydroxide, aluminum phosphate, aluminum sulfate, 3 De-O-acylated monophosphoryl lipid A (MPL), QS-21, TQL1055, QS-18, QS-17, QS-7, 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.

26. The pharmaceutical formulation of claim 25, wherein the adjuvant is QS-21 or TQL1055.

27. The pharmaceutical formulation of claim 25, wherein the adjuvant is MPL.

28. The pharmaceutical formulation of claim 25, wherein the adjuvant is a combination of MPL and QS-21, or a combination of MPL and TQL1055.

29. The pharmaceutical formulation of any one of claims 24 to 28, wherein the adjuvant comprises a liposomal formulation.

30. The pharmaceutical formulation according to any one of claims 24 to 29, wherein the composition comprises at least one pharmaceutically acceptable diluent.

31. The pharmaceutical preparation according to any one of claims 24 to 30, comprising a multiple antigen-presenting system (MAP).

32. 32. The pharmaceutical formulation of claim 31, wherein the MAP comprises one or more of a Lys-based dendritic scaffold, a helper T cell epitope, an immunostimulatory lipophilic moiety, a cell-penetrating peptide, a radical-induced polymerization, a self-assembling nanoparticle as an antigen-presenting platform, and a gold nanoparticle.

33. 1. An immunotherapeutic composition comprising: a first peptide sequence comprising 3 to 10 amino acid residues from the first 10 N-terminal residues of SEQ ID NO: 01; and a second peptide sequence comprising 3 to 10 amino acids from residues 111-132 of SEQ ID NO: 02, wherein the 3 to 10 amino acids of the first polypeptide comprise the amino acid sequence EFRHD (SEQ ID NO: 21) and the 3 to 10 amino acids of the second polypeptide comprise the amino acid sequence DNEAY (SEQ ID NO: 62).

34. (a) the first peptide sequence is 【Chemistry 9】 and comprising an amino acid sequence selected from the group consisting of: (b) the second peptide sequence is 【Chemistry 10-1】 【Chemistry 10-2】 and comprising an amino acid sequence selected from the group consisting of: Each of the first peptide sequence and the second peptide sequence may optionally include a C-terminal cysteine. The immunotherapeutic composition of claim 33.

35. 35. The immunotherapy composition of any one of claims 33-34, wherein at least one of the first peptide and the second peptide further comprises a linker to a carrier at either the C-terminal portion of the polypeptide or the N-terminal portion of the polypeptide.

36. 36. The immunotherapy composition of claim 35, wherein the linker comprises an amino acid sequence selected from the group consisting of GG, GGG, AA, AAA, KK, KKK, SS, SSS, GAGA (SEQ ID NO: 114), AGAG (SEQ ID NO: 115), and KGKG (SEQ ID NO: 116).

37. 37. The immunotherapy composition of claim 36, wherein the linker to the carrier can optionally include a C-terminal cysteine (C).

38. 38. The immunotherapy composition of any one of claims 36-37, wherein the carrier comprises serum albumin, an immunoglobulin molecule, thyroglobulin, ovalbumin, tetanus toxoid (TT), diphtheria toxoid (DT), a genetically modified cross-reacting material of diphtheria toxin (CRM), CRM197, meningococcal outer membrane protein complex (OMPC) and H. influenzae protein D (HiD), rEPA (Pseudomonas aeruginosa exotoxin A), KLH (keyhole limpet hemocyanin), and flagellin.

39. The immunotherapy composition described in claim 38, wherein the carrier is CRM197.

40. The immunotherapy composition of claim 38, wherein the carrier is diphtheria toxoid.

41. The immunotherapeutic composition of any one of claims 33 to 40, further comprising at least one pharmaceutically acceptable diluent.

42. The immunotherapeutic composition of any one of claims 33 to 41, further comprising a multiple antigen-presenting system (MAP).

43. 43. The immunotherapy composition of claim 42, wherein the MAP comprises one or more of a Lys-based dendritic scaffold, a helper T cell epitope, an immunostimulatory lipophilic moiety, a cell-penetrating peptide, radical-induced polymerization, a self-assembling nanoparticle as an antigen-presenting platform, and a gold nanoparticle.

44. A pharmaceutical composition comprising an immunotherapeutic composition according to any one of claims 33 to 43 and at least one adjuvant.

45. 45. The pharmaceutical composition of claim 44, wherein the adjuvant is selected from the group consisting of aluminum hydroxide, aluminum phosphate, aluminum sulfate, 3 De-O-acylated monophosphoryl lipid A (MPL), QS-21, TQL1055, QS-18, QS-17, QS-7, 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.

46. The pharmaceutical composition described in claim 45, wherein the adjuvant is QS-21 or TQL1055.

47. The pharmaceutical composition of claim 45, wherein the adjuvant is MPL.

48. The pharmaceutical composition described in claim 45, wherein the adjuvant is a combination of MPL and QS-21, or a combination of MPL and TQL1055.

49. A nucleic acid comprising a nucleic acid sequence encoding a polypeptide according to any one of claims 1 to 19 of an immunotherapeutic composition according to claims 33 to 37.

50. A nucleic acid immunotherapy composition comprising the nucleic acid of claim 49 and at least one adjuvant.

51. A composition comprising an immunotherapeutic composition described in any one of claims 20 to 23 and 33 to 43 or a pharmaceutical formulation described in any one of claims 24 to 32 and 44 to 48, characterized in that the composition is administered to a subject.

52. 49. An immunotherapeutic composition according to any one of claims 20 to 23 and 33 to 43 or a pharmaceutical preparation according to any one of claims 24 to 32 and 44 to 48 for inhibiting or reducing aggregation of at least one of Aβ and alpha-synuclein in a subject having or at risk of developing Alzheimer's disease, characterized in that the immunotherapeutic composition or pharmaceutical preparation is administered to said subject.

53. A nucleic acid immunotherapy composition as described in claim 50 for treating or preventing Alzheimer's disease in a subject, characterized in that the nucleic acid immunotherapy composition is administered to the subject.

54. A nucleic acid immunotherapy composition as described in claim 50 for inhibiting or reducing aggregation of at least one of Aβ and alpha-synuclein in a subject having or at risk of developing Alzheimer's disease, characterized in that the nucleic acid immunotherapy composition is administered to the subject.

55. The composition described in claim 51, the immunotherapy composition or pharmaceutical preparation described in claim 52, or the nucleic acid immunotherapy composition described in claim 53 or 54, characterized in that the administration is repeated at least twice, at least three times, at least four times, at least five times, or at least six times.

56. The composition, immunotherapy composition, pharmaceutical formulation, or nucleic acid immunotherapy composition described in claim 55, characterized in that the administration is repeated at intervals of about 21 to about 28 days.

57. 51. A composition comprising the polypeptide of claims 1-19, the immunotherapy composition of claims 20-23 and 33-43, the pharmaceutical formulation of claims 24-32 and 44-48, or the nucleic acid immunotherapy composition of claim 50, for use in a regimen effective to generate an immune response comprising an antibody that specifically binds to Aβ, alpha-synuclein, or both Aβ and alpha-synuclein.

58. 58. The composition, immunotherapy composition, pharmaceutical formulation, or nucleic acid immunotherapy composition of claim 57, wherein the immune response comprises an antibody that specifically binds to Aβ and an antibody that specifically binds to alpha-synuclein.

59. 59. The composition, immunotherapy composition, pharmaceutical formulation, or nucleic acid immunotherapy composition of any of claims 57-58, wherein said generating said immune response comprises an antibody that specifically binds to the N-terminal region of Aβ and / or the C-terminal region of alpha-synuclein.

60. An immunization kit comprising the immunotherapeutic composition of any one of claims 20 to 23 and 33 to 43.

61. The kit described in claim 60, further comprising an adjuvant.

62. The kit of claim 61, wherein the immunotherapy composition is in a first container and the adjuvant is in a second container.

63. 51. A kit comprising the nucleic acid immunotherapy composition of claim 50.

64. The kit described in claim 63, further comprising an adjuvant.

65. The kit described in claim 64, wherein the nucleic acid is in a first container and the adjuvant is in a second container.

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