Multi-epitope vaccines for treating Alzheimer's disease

Abstract

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 tau and alpha-synuclein aggregation, blocking neuronal uptake, clearing amyloid, and inhibiting tau seed propagation and alpha-synuclein synucleinopathy in a subject having or at risk of developing Alzheimer's disease or other diseases involving tau and amyloid-beta and alpha-synuclein accumulation. The methods involve administering to such a patient a composition comprising amyloid-beta (Aβ) peptides, tau peptides, and alpha-synuclein peptides.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 062,919, filed August 7, 2021, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Statement A computer-readable form of the Sequence Listing is being filed with this application by electronic submission and is incorporated herein by reference in its entirety. The Sequence Listing is contained in an ASCII text file created on August 6, 2021, having the file name "20-1085-WO_Sequence-Listing_ST25.txt" and is 191 kb in size.

[0003] Field The present disclosure relates to the fields of immunology and medicine, 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 results in senile dementia. Broadly speaking, the disease is divided into two categories: late-onset, which occurs in old age (over 65 years of age), and early-onset, which occurs well before senility, i.e., between 35 and 60 years of age. While the pathology is the same in both types of disease, the abnormalities tend to be more severe and widespread in cases beginning at an earlier age. The disease is characterized by at least two types of lesions in the brain: neurofibrillary tangles and senile plaques. Neurofibrillary tangles are intracellular deposits of microtubule-associated tau protein, consisting of two filaments wound around each other in pairs. Senile plaques (i.e., amyloid plaques) are irregular, neuropil-like regions up to 150 μm in size, with a central extracellular amyloid deposit visible by microscopic analysis of brain tissue sections. The accumulation of amyloid plaques within the central nervous system is also associated with cerebral amyloid angiopathy (CAA) in Down's syndrome and other cognitive disorders, as well as the eye disease age-related macular degeneration.

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

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

[0007] Alpha-synuclein is a protein found in neurons and other cells and is a key component of the pathology that characterizes several neurodegenerative disorders, collectively referred to as synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. While our understanding of the normal physiological function of alpha-synuclein is limited, evidence suggests that soluble forms of this protein may interact with other proteins and certain intracellular membranes. In synucleinopathies, alpha-synuclein proteins abnormally aggregate within cells, which is thought to contribute to disease pathology. Increasing evidence suggests that certain aggregated forms of alpha-synuclein can be transmitted between neurons, resulting in the propagation of pathology that causes neuronal dysfunction and loss. Alpha-synuclein (SNCA) misfolding and aggregation can often accompany β-amyloid deposition in some neurodegenerative diseases, and alpha-synuclein and tau aggregates coexist in several neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease.

[0008] Thus, there is a need for new therapies and reagents for the prevention or treatment of Alzheimer's disease, particularly those that can elicit an immune response against Aβ, tau, and alpha-synuclein present in the patient. Summary of the Invention [Means for solving the problem]

[0009] overview In some embodiments, the disclosure is directed to a polypeptide comprising a first peptide comprising 3-10 amino acids from residues 1-10 or 12-25 of SEQ ID NO: 01, a second peptide comprising 3-13 amino acids from residues 244-400 of SEQ ID NO: 02, and a third peptide comprising 3-10 amino acids from residues 81-140 of SEQ ID NO: 58. In certain embodiments, the polypeptide further comprises a fourth peptide selected from one of (a) a peptide comprising 3-10 amino acids from residues 1-10 or 12-25 of SEQ ID NO: 01, (b) a peptide comprising 3-13 amino acids from residues 244-400 of SEQ ID NO: 02, and (c) a peptide comprising 3-10 amino acids from residues 81-140 of SEQ ID NO: 58. In some embodiments, the first peptide, second peptide, third peptide, and fourth peptide are arranged in any order in the polypeptide. For example, the second peptide may be derived from the microtubule-binding region (MTBR) of tau (residues 244-372 of SEQ ID NO: 02). Additionally, the first peptide may comprise one of the amino acid sequences of SEQ ID NOs: 3-38 or 1002-1057, the second peptide may comprise one of the amino acid sequences of SEQ ID NOs: 39-57 or 142-1000, the third peptide may comprise one of the amino acid sequences of SEQ ID NOs: 59-129, and the fourth peptide, if present, is one of any one of the amino acid sequences of SEQ ID NOs: 3-38, 1002-1057, 39-57, 142-1000, and 59-129. For example, the first polypeptide may be DAEFRHD (SEQ ID NO: 06) or EFRHDSG (SEQ ID NO: 19), the second polypeptide may be 5 to 10 amino acids, e.g., QIVYKPV (SEQ ID NO: 39) or NIKHVPG (SEQ ID NO: 57), the third polypeptide may be PDNEAYE (SEQ ID NO: 75) or DPDNEAY (SEQ ID NO: 69), and the fourth polypeptide, if present, may be NIKHVP (SEQ ID NO: 48) or QIVYKPV (SEQ ID NO: 39).

[0010] In other embodiments, two or more of the first peptide, second peptide, and third peptide, and, if present, the fourth peptide, may be linked by a cleavable linker, which may be an amino acid sequence. The cleavable peptide linker, if present, may be 1 to 10 amino acids in length. 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: 138)), 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: 139), Ala-Gly-Ala-Gly (SEQ ID NO: 140), or Lys-Gly-Lys-Gly (SEQ ID NO: 141). In certain embodiments, the polypeptide is [ka] may be.

[0011] In certain embodiments, the polypeptide further comprises a blocked amine at the N-terminus.

[0012] In further embodiments, the polypeptide may include a linker to a carrier at the C-terminal portion of the polypeptide or the N-terminal portion of the polypeptide. The linker, if present, may be 1 to 10 amino acids in length. 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 include the amino acid sequence GG, GGG, AA, AAA, KK, KKK, SS, SSS, GAGA (SEQ ID NO: 139), AGAG (SEQ ID NO: 140), or KGKG (SEQ ID NO: 141). Additionally, the linker to a carrier, if present at the C-terminus, may include a C-terminal cysteine ​​(C). For example, the polypeptide may comprise the amino acid sequence DAEFRHDRRPDNEAYERRQIVYKPVKKC (SEQ ID NO: 130), where KK and C are independently optional, and where KK, if present, may be replaced with GG, AA, SS, GAGA (SEQ ID NO: 139), AGAG (SEQ ID NO: 140), or KGKG (SEQ ID NO: 141). Alternatively, the linker to the carrier, if present at the N-terminus, may comprise an N-terminal cysteine ​​(C). For example, the sequence may be represented as a CXX polypeptide, where XX and C are independently optional, and where XX, if present, may be GG, AA, KK, SS, GAGA (SEQ ID NO: 139), AGAG (SEQ ID NO: 140), or KGKG (SEQ ID NO: 141).

[0013] In other embodiments, the present disclosure is directed to immunotherapy compositions comprising a polypeptide of the present disclosure, wherein the polypeptide is optionally linked to a carrier, which may include serum albumin, an immunoglobulin molecule, thyroglobulin, ovalbumin, tetanus toxoid (TT), diphtheria toxoid (DT), 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.

[0014] Still further, embodiments of the present disclosure are directed to pharmaceutical formulations comprising a polypeptide or immunotherapy composition of the present disclosure and 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 include a liposome formulation, a diluent, or a multiple antigen presentation system (MAP). The MAP may comprise 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.

[0015] Still further, embodiments of the present disclosure are directed to immunotherapeutic compositions comprising a first peptide sequence comprising 3-10 amino acid residues from the first 10 or 12-25 N-terminal residues of SEQ ID NO: 01, a second peptide sequence comprising 3-13 amino acids from residues 244-400 of SEQ ID NO: 02, and a third sequence comprising 3-10 amino acids from residues 81-140 of SEQ ID NO: 58. The first peptide may comprise the amino acid sequence of one of SEQ ID NOs: 3-38 or 1002-1057, the second peptide may comprise the amino acid sequence of one of SEQ ID NOs: 39-57 or 142-1000, the third peptide sequence may comprise the amino acid sequence of one of SEQ ID NOs: 59-129, and the fourth peptide sequence, if present, is any one of the amino acid sequences of SEQ ID NOs: 3-38, 1002-1057, 39-57, 142-1000, and 59-129. Each of the first peptide, the second peptide, the third peptide, and the fourth peptide may include a linker to the carrier at the C-terminal portion of the polypeptide or the N-terminal portion of the polypeptide. If present, the linker may include an amino acid sequence selected from GG, GGG, AA, AAA, KK, KKK, SS, SSS, GAGA (SEQ ID NO: 139), AGAG (SEQ ID NO: 140), and KGKG (SEQ ID NO: 141), and may include a C-terminal cysteine ​​(C). In some embodiments, when the C-terminal residue in the immunogen is IVYKPV (SEQ ID NO: 194), VYKPV (SEQ ID NO: 195), YKPV (SEQ ID NO: 196), KPV, or PV, the linker is an amino acid linker that does not have an N-terminal glycine (e.g., GG, GAGA (SEQ ID NO: 139)). Carriers may include serum albumin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid (TT), diphtheria toxoid (DT), 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.

[0016] In addition, the immunotherapeutic composition may include at least one pharmaceutically acceptable diluent and / or a multiple antigen presentation system (MAP), which may include 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.

[0017] The immunotherapeutic composition may be included in a pharmaceutical composition comprising the immunotherapeutic composition and at least one adjuvant, for example, the adjuvant is 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.

[0018] Embodiments of the present disclosure are also directed to nucleic acid sequences encoding the polypeptides and immunotherapeutic compositions of the present disclosure. The nucleic acids may be included in nucleic acid immunotherapeutic compositions comprising the nucleic acid and at least one adjuvant.

[0019] Still further, embodiments of the present disclosure are directed to methods for treating or effecting prevention of Alzheimer's disease in a subject, and for inhibiting or reducing aggregation of at least one of Aβ, tau, and alpha-synuclein in a subject having or at risk of developing Alzheimer's disease, comprising administering to the subject an immunotherapeutic composition, nucleic acid immunotherapeutic composition, or pharmaceutical formulation of the present disclosure.

[0020] The methods of the present disclosure may include repeating the administration at least two times, at least three times, at least four times, at least five times, or at least six times, and may include repeating the administration at intervals of about 21 to about 28 days.

[0021] Still further, methods of the present disclosure are directed to inducing an immune response in an animal. The methods include administering to the animal a polypeptide, immunotherapeutic composition, pharmaceutical formulation, or nucleic acid immunotherapeutic composition of the present disclosure in a regimen effective to generate an immune response comprising antibodies that specifically bind to Aβ, and / or tau and / or alpha-synuclein. The immune response may comprise antibodies that specifically bind to the N-terminal region of Aβ and / or the microtubule region of tau and / or the C-terminal region of alpha-synuclein.

[0022] In other embodiments, the present disclosure is directed to an immunization kit comprising the immunotherapeutic composition of the present disclosure and optionally an adjuvant, wherein the immunotherapeutic composition can be in a first container and the adjuvant can be in a second container.

[0023] Still further, the present disclosure is directed to a kit comprising the nucleic acid immunotherapy composition of the present disclosure, and optionally an adjuvant. The nucleic acid can be in a first container and the adjuvant can be in a second container. [Brief explanation of the drawings]

[0024] [Figure 1A] Figure 1A shows that serum from guinea pigs vaccinated with a tripeptide immunogen containing Aβ peptide, tau peptide, and alpha-synuclein peptide antigens produces titers against Aβ, tau, and alpha-synuclein. Three guinea pigs were injected with immunogen 25 (DAEFRHDRRQIVYKPVRRPDNEAYEKKC; SEQ ID NO: 131) on day 0, week 3, and week 7, and serum was collected one week after each injection (i.e., week 1, week 4, and week 8).

[0025] [Figure 1B] Figure 1B shows that serum from guinea pigs vaccinated with a tripeptide immunogen containing Aβ peptide, tau peptide, and alpha-synuclein peptide antigens produces titers against Aβ, tau, and alpha-synuclein. Three guinea pigs were injected with immunogen 27 (DAEFRHDRRNIKHVPGRRPDNEAYEKKC; SEQ ID NO: 133) on day 0, week 3, and week 7, and serum was collected one week after each injection (i.e., week 1, week 4, and week 8).

[0026] [Figure 1C] Figure 1C shows that serum from guinea pigs vaccinated with a tripeptide immunogen containing Aβ peptide, tau peptide, and alpha-synuclein peptide antigens produces titers against Aβ, tau, and alpha-synuclein. Three guinea pigs were injected with immunogen 26 (DAEFRHDRRPDNEAYERRNIKHVPGKKC; SEQ ID NO: 132) on day 0, week 3, and week 7, and serum was collected one week after each injection (i.e., week 1, week 4, and week 8).

[0027] [Figure 1D] Figure ID shows that serum from guinea pigs vaccinated with a tripeptide immunogen containing Aβ peptide, tau peptide, and alpha-synuclein peptide antigens produces titers against Aβ, tau, and alpha-synuclein. Three guinea pigs were injected with immunogen 24 (DAEFRHDRRPDNEAYERRQIVYKPVKKC; SEQ ID NO: 130) on day 0, week 3, and week 7, and serum was collected one week after each injection (i.e., week 1, week 4, and week 8).

[0028] [Figure 2A]Figure 2A shows that serum from guinea pigs vaccinated with a tripeptide immunogen containing Aβ peptide, tau peptide, and alpha-synuclein peptide antigens produces titers against Aβ, tau, and alpha-synuclein. Three guinea pigs were injected with immunogen 25 (DAEFRHDRRQIVYKPVRRPDNEAYEKKC; SEQ ID NO: 131) on day 0, week 3, week 7, and week 11, and serum was collected one week after each injection (i.e., week 1, week 4, week 8, and week 12).

[0029] [Figure 2B] Figure 2B shows that serum from guinea pigs vaccinated with a tripeptide immunogen containing Aβ peptide, tau peptide, and alpha-synuclein peptide antigens produces titers against Aβ, tau, and alpha-synuclein. Three guinea pigs were injected with immunogen 24 (DAEFRHDRRPDNEAYERRQIVYKPVKKC; SEQ ID NO: 130) on day 0, week 3, week 7, and week 11, and serum was collected one week after each injection (i.e., week 1, week 4, week 8, and week 12).

[0030] [Figure 2C] Figure 2C shows that serum from guinea pigs vaccinated with a tripeptide immunogen containing Aβ peptide, tau peptide, and alpha-synuclein peptide antigens produces titers against Aβ, tau, and alpha-synuclein. Three guinea pigs were injected with immunogen 27 (DAEFRHDRRNIKHVPGRRPDNEAYEKKC; SEQ ID NO: 133) on day 0, week 3, week 7, and week 11, and serum was collected one week after each injection (i.e., week 1, week 4, week 8, and week 12).

[0031] [Figure 2D]Figure 2D shows that serum from guinea pigs vaccinated with a tripeptide immunogen containing Aβ peptide, tau peptide, and alpha-synuclein peptide antigens produces titers against Aβ, tau, and alpha-synuclein. Three guinea pigs were injected with immunogen 26 (DAEFRHDRRPDNEAYERRNIKHVPGKKC; SEQ ID NO: 132) on day 0, week 3, week 7, and week 11, and serum was collected one week after each injection (i.e., week 1, week 4, week 8, and week 12).

[0032] [Figure 3] Figure 3 shows that sera from mice vaccinated with one of four tripeptide immunogens, each containing an Aβ peptide, a tau peptide, and an alpha-synuclein peptide antigen, produce titers against Aβ, tau, and alpha-synuclein. Four Swiss Webster mice per immunogen were injected on days 0 and 10, and sera were collected on day 16. Tri2 is DAEFRHDRRPDNEAYERRENLKHQPGGGC (SEQ ID NO: 1058), Tri1 is DAEFRHDRRPDNEAYERRENLKHQPGRRDPDNEAYEGGC (SEQ ID NO: 1059), Tri4 is DAEFRHDRRPDNEAYERRENLKHQPGGGC (SEQ ID NO: 1060), and Tri3 is DAEFRHDRRRENLKHQPGRRPDNEAYEGGC (SEQ ID NO: 1061) (see also Table 4). DETAILED DESCRIPTION OF THE INVENTION

[0033] explanation The present disclosure provides peptide compositions and immunotherapeutic compositions containing amyloid-beta (Aβ) peptides, tau 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 and preventing deposit formation, inhibiting or reducing Aβ and / or tau and / or alpha-synuclein aggregation, blocking the binding and / or uptake of Aβ and / or tau and / or alpha-synuclein by neurons, inhibiting the transmission of tau species between cells, and inhibiting the propagation of pathology between brain regions in subjects with or at risk of developing Alzheimer's disease or other diseases involving tau and / or amyloid-beta accumulation. The methods include administering a composition containing amyloid-beta (Aβ) peptides, tau peptides, and alpha-synuclein peptides to such patients.

[0034] Certain terms are defined below. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include multiple compounds, including mixtures thereof.

[0035] Unless otherwise clear from the context, the term "about" encompasses slight variations, such as values ​​within the standard limits of error (e.g., SEM) of the measurement of the stated value. For example, when the term "about" is used herein to refer to a measurable value, such as a parameter, amount, or time period, it can encompass a variation of + / -10% or less, + / -5% or less, or + / -1% or less (or less or less) of the stated value and from the stated value. The designation of a range of values ​​includes all integers within or defining the range, and all subranges defined by integers within the range. As used herein, statistical significance means p≦0.05.

[0036] A composition or method that "comprising" or "including" one or more recited elements may include other elements not specifically recited. For example, a composition that "comprises" or "includes" a polypeptide sequence may contain the sequence alone or in combination with other sequences or components.

[0037] An individual is at increased risk of a disease if the subject has at least one known risk factor (e.g., age, genetic, biochemical, family history, and situational exposure) that places the individual with that risk factor at a statistically significantly higher risk of developing the disease than an individual without the risk factor.

[0038] The term "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment, including treatment-naive subjects. As used herein, the term "subject" or "patient" refers to any single subject for whom treatment is desired, including other mammalian subjects, such as humans, cows, dogs, guinea pigs, rabbits, etc. Any subject involved in a clinical research trial, or subject involved in an epidemiological study, or subject used as a control, who does not show any clinical signs of disease is also intended to be included as a subject.

[0039] The term "disease" refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, disorder, pathology, condition, or syndrome in which physiological function is impaired, regardless of the nature of the etiology.

[0040] The term "symptom" refers to subjective evidence of disease, such as a change in gait, perceived by a subject. "Sign" refers to objective evidence of disease observed by a physician.

[0041] As used herein, the terms "treat" and "treatment" refer to reducing or ameliorating 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 trending toward a desired outcome as described herein.

[0042] The terms "prevention," "prevent," or "preventing," as used herein, refer to contacting (e.g., administering) a peptide(s) or immunotherapeutic composition of the present disclosure to a subject prior to the onset of disease with or without pre-existing Aβ and / or tau pathology (primary and secondary prevention), thereby delaying the onset of clinical symptoms and / or reducing disease symptoms after disease onset compared to when the subject has not been contacted with the peptide or immunotherapeutic composition, but not completely inhibiting the onset of disease. In some cases, prevention may occur for a limited time after administration of a peptide or immunotherapeutic composition of the present disclosure. In other cases, prevention may occur during the duration of a treatment regimen that includes administering a peptide or immunotherapeutic composition of the present disclosure.

[0043] The terms "reduce," "reduce," or "reducing," as used herein, refer to decreasing the amount of Aβ and / or tau and / or alpha-synuclein present in a subject or a subject's tissue, or inhibiting an increase in the amount of Aβ and / or tau and / or alpha-synuclein present in a subject or a subject's tissue, including decreasing the amount of, or inhibiting an increase (e.g., decreasing the rate of increase) of, accumulated, aggregated, or deposited Aβ and / or tau and / or alpha-synuclein present in a subject or a subject's tissue. In certain embodiments, a decrease in the amount of, or inhibiting an increase (e.g., decreasing the rate of increase) of accumulated, aggregated, or deposited Aβ and / or tau and / or alpha-synuclein present in a subject refers to the amount of accumulated, aggregated, or deposited Aβ and / or tau and / or alpha-synuclein present in the subject's central nervous system (CNS). In certain embodiments, a reduction in the amount of accumulated, aggregated, or deposited Aβ and / or tau and / or alpha-synuclein present in a subject, or an inhibition of its increase (e.g., a reduction in the rate of increase) refers to the amount of accumulated, aggregated, or deposited Aβ and / or tau and / or alpha-synuclein present in the subject's periphery (e.g., peripheral circulatory system). In certain embodiments, a reduction in the amount of accumulated, aggregated, or deposited Aβ and / or tau and / or alpha-synuclein present in a subject, or an inhibition of its increase (e.g., a reduction in the rate of increase) refers to the amount of accumulated, aggregated, or deposited Aβ and / or tau and / or alpha-synuclein present in the subject's brain.In some embodiments, the Aβ and / or tau and / or alpha-synuclein that is reduced is pathological form(s) of Aβ (e.g., extracellular plaque deposits of β-amyloid peptide (Aβ), neuritic amyloid plaques), and / or tau (e.g., tau neurofibrillary tangles, dystrophic neurites), and / or alpha-synuclein (e.g., fibrillar alpha-synuclein inclusions, oligomeric or fibrillar alpha-synuclein aggregates, and protofibrillar intermediates of alpha-synuclein oligomers). In yet other embodiments, pathological indicators of neurodegenerative diseases and / or synucleinopathies are reduced.

[0044] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which B cells and / or T cells respond, or to which an antibody binds. Epitopes can be formed both from contiguous amino acids or from non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically contains 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 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).

[0045] An "immunogenic agent" or "immunogen" or "antigen," when administered to an animal, optionally in conjunction with an adjuvant, is capable of inducing an immune response against itself or a modified / engineered version of itself. The term "immunogenic agent" or "immunogen" or "antigen" refers to a compound or composition, including a peptide, polypeptide, or protein, that, when administered in an appropriate amount (an "immunologically effective amount"), is "antigenic" or "immunogenic," i.e., capable of inducing, eliciting, augmenting, or boosting a cellular and / or humoral immune response and being recognized by the products of that response (T cells, antibodies). An immunogen can be a peptide, or a combination of two or more of the same 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.

[0046] Immunogens can be effective when given alone or in combination with, linked to, or fused to another substance, which can be administered once or over several intervals. The immunogenic agent or immunogen can include an antigenic peptide or polypeptide linked to a carrier as described herein.

[0047] 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 can be recombinantly expressed from a vaccine vector, which can be naked DNA or RNA, such as an expression vector or cassette described herein, that includes a peptide or polypeptide coding sequence operably linked to a promoter.

[0048] The term "adjuvant" refers to a compound that, when administered in combination with an antigen, enhances the immune response to the antigen, but does not produce an immune response to the antigen when administered alone. Adjuvants can enhance the immune response through several mechanisms, including lymphocyte recruitment, stimulation of B cells and / or T cells, and stimulation of macrophages. Adjuvants can be natural compounds, modified versions or derivatives of natural compounds, or synthetic compounds.

[0049] The terms "peptide" and "polypeptide" are used interchangeably herein to refer to a chain of two or more consecutive amino acids. If and when a distinction is made, the context will clarify the meaning. For example, when two or more peptides described herein are joined to form a dimeric or multimeric peptide, polypeptide may be used to refer to "poly" or "two or more" peptides.

[0050] The term "pharmaceutically acceptable" means the carrier, diluent, excipient, adjuvant or auxiliary material must be compatible with the other ingredients of the pharmaceutical formulation and not substantially deleterious to the recipient thereof.

[0051] The term "immunotherapy" or "immune response" refers to the development in a recipient of a beneficial humoral response (antibody-mediated) and / or cellular response (mediated by antigen-specific T cells or their secretory products) to Aβ and / or tau peptides. Such a response can be an active response induced by administration of an immunogen (e.g., Aβ and / or tau and / or alpha-synuclein peptides). A cellular immune response is elicited by presentation of polypeptide epitopes in association with class I or class II MHC molecules to induce antigen-specific CD4 + Helper T cells and / or CD8 +Activates cytotoxic T cells. The response may also include activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglial cells, eosinophils, or other components of innate immunity. The presence of a cell-mediated immune response can be determined by proliferation assays (CD4 + 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 an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

[0052] Amyloid beta (Aβ)

[0053] Aβ (also referred to herein as beta amyloid peptide or Abeta) peptides are 38-43 amino acid, approximately 4 kDa internal fragments of APP (Aβ39, Aβ40, Aβ41, Aβ42, and Aβ43). Aβ40, for example, consists 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 40 amino acid long "short form" and a "long form" ranging from 42-43 amino acids in length. Epitopes or antigenic determinants, as described herein, are located within the N-terminus of the Aβ peptide and include residues within amino acids 1-10 and 12-25 of Aβ, e.g., residues 1-3, 1-4, 1-5, 1-6, 1-7, or 3-7 of Aβ42. Additional examples of epitopes or antigenic determinants include 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 Aβ 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, 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. Additional examples of 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.

[0054] Aβ (Abeta) is the primary component of the plaques characteristic of Alzheimer's disease. Aβ is generated by processing of the larger protein APP by two enzymes, beta-secretase and gamma-secretase. Known mutations in APP associated with Alzheimer's disease occur near the beta-secretase or gamma-secretase sites or within Aβ. A portion of the hydrophobic transmembrane domain of APP, found at the carboxy terminus of Aβ, may be primarily responsible for Aβ's ability to aggregate into plaques, particularly in the long form. The accumulation of amyloid plaques in the brain ultimately leads to neuronal cell death. Physical symptoms associated with this type of neurodegeneration characterize Alzheimer's disease.

[0055] Tau

[0056] Tau is a protein with a molecular weight of approximately 50,000 normally present in nerve axons and contributes to the stability of microtubules. Tau proteins (or τ proteins) are a group of six highly soluble protein isoforms generated by alternative splicing from the gene MAPT (microtubule-associated protein tau). They primarily play a role in maintaining microtubule stability in axons and are abundant in neurons of the central nervous system (CNS). They are less common elsewhere, but are also expressed at very low levels in astrocytes and oligodendrocytes of the CNS. Neuropathologies and dementias, such as Alzheimer's disease and Parkinson's disease, are associated with tau protein becoming hyperphosphorylated, insoluble aggregates called neurofibrillary tangles. Pathogenic tau species cause toxic effects by directly binding to cells and / or accumulating inside cells and / or initiating a misfolding process (seeding), and can spread from one cell to another via cell-to-cell communication. Toxicity can also occur through neurofibrillary tangles (NFTs), which lead to cell death and cognitive decline. Other tauopathies include, for example, progressive supranuclear palsy, corticobasal syndrome, some frontotemporal dementias, and chronic traumatic encephalopathy.

[0057] Alpha-synuclein

[0058] Alpha-synuclein is a highly conserved protein abundant in neurons, particularly in presynaptic terminals. Aggregated alpha-synuclein proteins form brain lesions characteristic of neurodegenerative synucleinopathies. Furthermore, misfolding and aggregation can often accompany β-amyloid deposition in some neurodegenerative diseases, and alpha-synuclein and tau aggregates coexist in several neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease.

[0059] Immunogenic Aβ / tau / alpha-synuclein polypeptides

[0060] The agent used for active immunization can induce an immune response in patients and can function as an immunotherapy.The agent used for active immunization can be, for example, the same type of immunogen used to raise monoclonal antibodies in experimental animals, and can include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 or more consecutive amino acids from regions of Aβ and / or tau peptide and / or alpha-synuclein peptide.In each of the peptide embodiments described herein, the peptide can include, consist of, or essentially consist of the listed sequence.

[0061] In some embodiments of the present disclosure, an Aβ / tau / alpha-synuclein immunogen may comprise an Aβ peptide comprising 3-10 amino acids from residues 1-10 or 12-25 of the N-terminal sequence of Aβ (SEQ ID NO: 01) linked to a tau peptide comprising 3-13 amino acids from residues 244-400 of the long form of tau (SEQ ID NO: 02), and an alpha-synuclein peptide comprising 3-10 amino acids from residues 81-140 of SEQ ID NO: 58. For example, the tau peptide may comprise 3-13 amino acids from the microtubule-binding region of tau (residues 344-372 of SEQ ID NO: 02).

[0062] In some embodiments of the present disclosure, the Aβ peptide can include 3-10 amino acids from residues 1-10 or 12-25 of DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 01). For example, the Aβ peptide can include the following: [ka] [ka] [ka] [ka] is selected from.

[0063] In certain embodiments, the Aβ peptide is DAEFRHD (SEQ ID NO: 06), DAEFR (SEQ ID NO: 08), or EFRHD (SEQ ID NO: 21).

[0064] The tau peptide may correspond to a peptide comprising 3-13 amino acids from residues 244-400 of SEQ ID NO: 02. In some embodiments, the fragment is unphosphorylated. In some embodiments, the fragment is phosphorylated. In some embodiments, the tau peptide comprises an amino acid sequence represented by the consensus motif (Q / E)IVYK(S / P) (SEQ ID NO: 996). In some embodiments, the tau peptide comprises an amino acid sequence represented by the consensus motif KXXSXXNX(K / H)H (SEQ ID NO: 995), where X is any amino acid. In some embodiments, the tau peptide is selected from SEQ ID NOs: 146-1000. In some embodiments, the tau peptide is selected from the following: [ka] [ka] is selected from.

[0065] The alpha-synuclein peptide may correspond to a peptide comprising 3 to 10 amino acids from residues 81 to 140 of SEQ ID NO: 58. In some embodiments, the alpha-synuclein is unphosphorylated. In some embodiments, the alpha-synuclein is phosphorylated. In some compositions, the alpha-synuclein peptide is one of the following: [ka] [ka] [ka] is selected from. In each of these embodiments, the peptide may comprise, consist of, or consist essentially of the recited sequence.

[0066] In some embodiments, Aβ and / or tau and / or alpha-synuclein peptides are linked to form a multiple Aβ / tau / alpha-synuclein polypeptide. The Aβ, tau, and alpha-synuclein peptides may be linked by an intrapeptide linker. For example, the polypeptide linker is located between the C-terminus of a first peptide and the N-terminus of a second peptide. With or without an intrapeptide linker, the Aβ and / or tau and / or alpha-synuclein peptides may be arranged in any order in the multiple Aβ / tau / alpha-synuclein polypeptide. For example, the Aβ peptide may be arranged at the N-terminal end of the multiple polypeptides, and the alpha-synuclein peptide may be arranged at the C-terminal end of the multiple polypeptides. Alternatively, the tau peptide may be arranged at the N-terminal end of the multiple polypeptides, and the Aβ peptide may be arranged at the C-terminal end of the tau peptide. Reference herein to a first peptide or a second peptide or a third peptide or a fourth peptide is not intended to imply an order of Aβ and / or tau and / or alpha-synuclein peptides in the immunogenic polypeptide.

[0067] Additionally, the C-terminal portion of the Aβ peptide, tau peptide, alpha-synuclein, or multiple Aβ / tau / alpha-synuclein polypeptide can include a linker for conjugating the peptide or polypeptide to a carrier. Linkers for coupling the peptide or multiple polypeptides to a carrier can include, for example, GG, GGG, KK, KKK, AA, AAA, SS, SSS, GAGA (SEQ ID NO: 139), AGAG (SEQ ID NO: 140), KGKG (SEQ ID NO: 141), etc., between the peptide or dual polypeptide and the carrier, and can further include a C- or N-terminal cysteine ​​to provide a short peptide linker (e.g., GGC-, KKC-, AAC-, or SSC-). In some embodiments, when the C-terminal residue in the immunogen is any of IVYKPV (SEQ ID NO: 194), VYKPV (SEQ ID NO: 195), YKPV (SEQ ID NO: 196), KPV, or PV, the linker is an amino acid linker that does not have an N-terminal glycine (e.g., GG, GAGA (SEQ ID NO: 139), and KGKG (SEQ ID NO: 141). In some embodiments, the linker is an amino acid linker that does not have an N-terminal glycine (e.g., AA, AAA, KK, KKK, SS, SSS, AGAG (SEQ ID NO: 140), GG, GGG, GAGA (SEQ ID NO: 139), and KGKG (SEQ ID NO: 141). G (SEQ ID NO: 141). In some embodiments, any of the Aβ peptides, tau peptides, alpha-synuclein peptides, and multiple Aβ / tau / alpha-synuclein polypeptides may include a C-terminal cysteine ​​without a spacer. In some embodiments, any of the Aβ peptides, tau peptides, alpha-synuclein peptides, and multiple Aβ / tau / alpha-synuclein polypeptides may include an N-terminal cysteine ​​without a spacer.

[0068] When Aβ, tau, and / or alpha-synuclein polypeptides are linked to form multiple Aβ / tau / alpha-synuclein polypeptides, the linker may be a cleavable linker. As used herein, the term "cleavable linker" refers to any linker between antigen peptides that facilitates or otherwise makes the Aβ, tau, and / or alpha-synuclein peptides more susceptible to separation from each other by cleavage (e.g., by endopeptidases, proteases, low pH, or any other means that may occur in or around antigen-presenting cells), thereby making them more susceptible to separation by antigen-presenting cells than comparable 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 susceptible to cleavage by a protease from the trypsin family of proteases. In some compositions, the cleavable linker comprises 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: 138), 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: 139)), Ala-Gly-Ala-Gly; AGAG (SEQ ID NO: 140), and Lys-Gly-Lys-Gly; KGKG (SEQ ID NO: 141). In some compositions, the cleavable linker is arginine-arginine (Arg-Arg).

[0069] In some embodiments of the present disclosure, the multiple Aβ / tau / alpha-synuclein polypeptide comprises, consists of, or consists essentially of an amino acid sequence selected from SEQ ID NOs: 130-137 and SEQ ID NOs: 1058-1063.

[0070] In some embodiments, the multiple Aβ / tau / alpha-synuclein polypeptide is: Formula I: [P1]-[CL1]-[P2]-[CL2]-[P3]-[L1]-[Cys], Formula II: [P1]-[CL1]-[P2]-[CL2]-[P3]-[CL3]-[P4]-[L1]-[Cys], wherein each of P1, P2, P3 and P4 can be independently selected from Aβ peptides, tau peptides and alpha-synuclein peptides, so long as each of Aβ peptides, tau peptides and alpha-synuclein peptides is selected. In embodiments, P1 is an Aβ peptide, e.g., when the first peptide [P1] is an Aβ peptide, the second peptide [P2] is a tau peptide, and the third peptide [P3] is an alpha-synuclein peptide; or when [P1] is an Aβ peptide, [P2] is a tau Aβ peptide, and the fourth peptide [P4] is a tau peptide; or when [P1] is an Aβ peptide, [P2] is an alpha-synuclein peptide and [P3] is a tau peptide; or when [P1] is an Aβ peptide, [P2] is an alpha-synuclein peptide, [P3] is a tau peptide, and [P4] is a tau peptide; each of [CL1], [CL2], and [CL3] is a cleavable linker; [L1] is a linker; and [CL1], [CL2], [CL3], [L1], and [Cys] are optionally present.

[0071] Examples of Aβ peptides include any one of SEQ ID NOs: 3-38 or 1002-1057.

[0072] Examples of tau peptides include any one of SEQ ID NOs: 39-57 or 142-1000.

[0073] Examples of alpha-synuclein peptides include any one of SEQ ID NOs: 59-129.

[0074] [CL1], [CL2], and [CL3] are optionally present and, if present, may be cleavable linkers. If present, the cleavable linker may be 1 to 10 amino acids in length. 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: 138), 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: 139), alanine-glycine-alanine-glycine (Ala-Gly-Ala-Gly; SEQ ID NO: 140), and lysine-glycine-lysine-glycine (Lys-Gly-Lys-Gly; SEQ ID NO: 141).

[0075] [L1] is optionally present and, if present, is a linker that couples the polypeptide to the carrier. The linker, if present, can be 1 to 10 amino acids in length. 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, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the amino acid composition of the linker can mimic the composition of linkers found in naturally occurring multidomain proteins, with certain amino acids being over-represented, under-represented, or equivalent in the naturally occurring linker relative 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) are predominant in natural linkers. In contrast, isoleucine (Ile), tyrosine (Tyr), tryptophan (Trp), and cysteine ​​(Cys) are predominant. Generally, predominant amino acids are polar uncharged or charged residues, which account for approximately 50% of 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 non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, whose small size provides flexibility and allows 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 interactions between the linker and the immunogen. In some embodiments, the linker comprises a stretch of Gly and Ser residues ("GS" linker). Examples of widely used flexible linkers are (Gly-Gly-Ser), (Gly-Gly-Gly-Ser) (SEQ ID NO: 1064), or (Gly-Gly-Gly-Gly-Ser) (SEQ ID NO: 1065), where n = 1 to 3. By adjusting the copy number "n," the linker can be optimized to achieve sufficient separation of functional immunogen domains, for example, to maximize the immunogenic response. Many other flexible linkers have been designed for recombinant fusion proteins that can 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 additional amino acids such as Thr and Ala to maintain flexibility, and polar amino acids such as Lys and Glu to improve solubility. See, e.g., Chen, X. et al., "Fusion Protein Linkers: Property, Design and Functionality" Adv Drug Deliv Rev., 15; 65(10): 1357-1369 (203). In certain embodiments, the linker, if present, may be an amino acid sequence selected from the group consisting of GG, GGG, KK, KKK, AA, AAA, SS, SSS, GAGA (SEQ ID NO: 139), AGAG (SEQ ID NO: 140), and KGKG (SEQ ID NO: 142).

[0076] [Cys] is optionally present and can serve to conjugate the polypeptide to a carrier. If present, Cys can be at the C-terminal portion of the polypeptide or at the N-terminal portion of the polypeptide.

[0077] Examples of multiple Aβ / tau / alpha-synuclein polypeptides of the present disclosure include: [Table 1] [Table 2]

[0078] Polypeptide Immunogens

[0079] According to the present disclosure, Aβ peptides, tau peptides, alpha-synuclein, and multiple Aβ / tau / alpha-synuclein polypeptides are immunogens. In some embodiments, peptides and multiple Aβ-tau / alpha-synuclein polypeptides can be linked to a suitable carrier to help induce an immune response. Thus, one or more peptides and multiple Aβ / tau / alpha-synuclein polypeptides of the present disclosure can be linked to a carrier. For example, each of the Aβ peptides, tau peptides, alpha-synuclein peptides, and Aβ / tau / alpha-synuclein polypeptides may be linked to a carrier with or without spacer amino acids (e.g., Gly-Gly, Gly-Gly-Gly, Ala-Ala, Ala-Ala-Ala, Lys-Lys, Lys-Lys-Lys, Ser-Ser, Ser-Ser-Ser, Gly-Ala-Gly-Ala (SEQ ID NO: 139), Ala-Gly-Ala-Gly (SEQ ID NO: 140), and Lys-Gly-Lys-Gly (SEQ ID NO: 141)). In certain embodiments, multiple Aβ / tau / alpha-synuclein polypeptides can be linked to a suitable carrier using a C-terminal cysteine ​​to provide a linker between the peptide(s) and the carrier or between the multiple Aβ / tau / alpha-synuclein polypeptides and the carrier. In certain embodiments, the multiple Aβ / tau / alpha-synuclein polypeptides can be linked to a suitable carrier using an N-terminal cysteine ​​to provide a linker between the peptide(s) and the carrier. In some embodiments, when the C-terminal residue in the immunogen is IVYKPV (SEQ ID NO: 194), VYKPV (SEQ ID NO: 195), YKPV (SEQ ID NO: 196), KPV, or PV, the linker is an amino acid linker that does not have an N-terminal glycine (e.g., GG, GAGA (SEQ ID NO: 139)).

[0080] 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 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 immunogens of the invention to immunostimulatory polymer molecules (e.g., tripalmitoyl-S-glyceryl cysteine ​​(Pam3Cys), mannan (mannose polymer), or glucan (β1-2 polymer)), cytokines (e.g., IL-1, IL-1 alpha and beta peptides, IL-2, gamma-INF, IL-10, GM-CSF), and chemokines (e.g., MIP1-α and β, and RANTES). Additional carriers include virus-like particles. In some compositions, immunogenic peptides can also be linked to carriers by chemical crosslinking. Techniques for linking immunogens to carriers 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 adding a cysteine ​​residue). These reagents create disulfide linkages between themselves and peptide cysteine ​​residues on certain proteins, and amide linkages through the epsilon-amino on lysine or other free amino groups in other amino acids. In some embodiments, chemical crosslinking can involve the use of SBAP (succinimidyl 3-(bromoacetamido)propionate), a short (6.2 angstrom) crosslinker for amine-to-sulfhydryl conjugation via N-hydroxysuccinimide (NHS) ester and bromoacetyl reactive groups.A variety of 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 linkages. Many of these thioether-forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, and 2-iodoacetic acid, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid. 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 known as pseudovirions or virus-derived particles, represent subunit structures composed of multiple copies of viral capsid and / or envelope proteins that can self-assemble into defined spherically symmetric VLPs in vivo (Powilleit, et al., (2007) PLoS ONE 2(5):e415). Alternatively, peptide immunogens can be linked to at least one artificial T cell epitope, such as a pan-DR epitope ("PADRE"), that can bind to most MHC class II molecules. Pan-DR binding peptides (PADREs) are described in US5,736,142, WO95 / 07707, and Alexander, et al., Immunity, 1:751-761 (1994).

[0081] The active immunogen may exist in a multimeric form, in which multiple copies of the immunogen (peptide of the polypeptide) are present on the carrier as a single covalently linked molecule. In some embodiments, the carrier comprises multiple Aβ / tau / alpha-synuclein polypeptides in various forms. For example, the immunogenic multiple Aβ / tau / alpha-synuclein polypeptides may include polypeptides with Aβ antigens, tau antigens, and alpha-synuclein antigens in different orders, or may exist with or without intrapeptide linkers and / or linkers to the carrier.

[0082] In some compositions, the immunogenic peptide can also be expressed as a fusion protein with a carrier. In certain compositions, the immunogenic peptide can be linked to the carrier at the amino terminus, carboxyl terminus, or internally. In some compositions, the carrier is CRM197. In some compositions, the carrier is diphtheria toxoid.

[0083] nucleic acid

[0084] The present disclosure further provides nucleic acids encoding any of the amyloid-beta (Aβ) peptides, tau peptides, and / or alpha-synuclein peptides disclosed herein. The nucleic acid immunotherapy compositions disclosed herein comprise, consist of, or consist essentially of a first nucleic acid sequence encoding an amyloid-beta (Aβ) peptide, a second nucleic acid sequence encoding a tau peptide, and / or a third nucleic acid sequence encoding an alpha-synuclein peptide disclosed herein. For example, the Aβ peptide is a sequence 3-10 amino acid residues in length derived from the first 10 or 12-25 N-terminal residues of SEQ ID NO: 01; the tau peptide is a sequence 3-13 amino acids in length derived from residues 244-400 of SEQ ID NO: 02; and the alpha-synuclein peptide is a sequence 3-10 amino acids in length derived from residues 81-140 of SEQ ID NO: 58. Thus, nucleic acids encoding any of SEQ ID NOS: 3-38 or 1002-1057 may be combined with nucleic acids encoding any of SEQ ID NOS: 39-57 or 142-1000, and / or nucleic acids encoding any of SEQ ID NOS: 59-129 to provide components of the immunogens and pharmaceutical compositions of the present disclosure. Similarly, one or more nucleic acids encoding any of the Aβ, tau, and alpha-synuclein sequences may contain codons for an RR-N-terminal or -RR-C-terminal dipeptide or polypeptide. In certain embodiments, the Aβ, tau, 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 and / or a C-terminal cysteine ​​as described herein. Additionally, if a single nucleic acid sequence encodes both peptides, the sequence may also encode an intrapeptide linker as described herein. The nucleic acid compositions (pharmaceutical compositions) described herein can be used in methods for treating or effecting prophylaxis and / or prevention of Alzheimer's disease.In another embodiment, the nucleic acid immunotherapy compositions disclosed herein provide compositions for reducing pathogenic forms of Aβ and / or tau and / or alpha-synuclein in a subject and / or in a subject's tissue. In some embodiments, the Aβ and / or tau and / or alpha-synuclein reduced by the immunotherapy composition is pathological form(s) of Aβ (e.g., extracellular plaque deposits of β-amyloid peptide (Aβ), neuritic amyloid plaques), tau (e.g., tau frame-shaped neurofibrillary tangles, tau neurofibrillary tangles), and / or alpha-synuclein (e.g., oligomeric or fibrillar alpha-synuclein aggregates, and protofibrillar intermediates of alpha-synuclein oligomers). In yet other embodiments, pathological indicators of neurodegenerative diseases and / or synucleinopathies are reduced by the nucleic acid immunotherapy compositions. In another embodiment, the nucleic acid immunotherapy compositions disclosed herein provide compositions for reducing brain Aβ, brain tau and brain alpha-synuclein.

[0085] Nucleic acids, such as DNA, that encode immunogens and are used as vaccines can be referred to as "DNA immunogens" or "DNA vaccines" because the encoded polypeptides are expressed in vivo after administration of the DNA. DNA vaccines are designed to induce antibodies against the encoded protein of interest in a subject by incorporating DNA encoding the protein into a vector (plasmid or virus), administering the vector to the subject, and stimulating the subject's immune system to express the protein. DNA vaccines remain in the subject's body for a long period of time after administration, slowly producing the encoded protein. In this way, excessive immune responses can be avoided. DNA vaccines can also be modified using genetic engineering techniques. If necessary, such nucleic acids can further encode a signal peptide and be expressed with the signal peptide linked to the peptide. The coding sequence of the nucleic acid can be operably linked to regulatory sequences, such as a promoter, enhancer, ribosome binding site, or transcription termination signal, to ensure expression of the coding sequence. Nucleic acids encoding Aβ, tau, and / or alpha-synuclein can occur in isolated form or can 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, tau and / or alpha-synuclein peptides and polypeptides, with or without linkers or cleavable linkers, and with or without protein-based carriers, can be joined as one contiguous nucleic acid, for example, in an expression vector.

[0086] Although DNA is more stable than RNA, it poses some potential safety risks, such as the induction of anti-DNA antibodies. Therefore, in some embodiments, the nucleic acid may be RNA. RNA nucleic acids encoding immunogens and used as vaccines may be referred to as "RNA immunogens," "RNA vaccines," or "mRNA vaccines" because the encoded polypeptides are expressed in vivo after administration of the RNA. Ribonucleic acid (RNA) vaccines can safely induce the subject's cellular machinery to produce one or more polypeptides of interest. In some embodiments, RNA vaccines may be non-replicating mRNA (messenger RNA) or self-amplifying viral RNA. While mRNA-based vaccines encode the antigen of interest and contain 5' and 3' untranslated regions (UTRs), self-amplifying RNA encodes not only the antigen but also the viral replication machinery, allowing for intracellular RNA amplification and abundant protein expression. In vitro-transcribed mRNA can be produced from a linear DNA template using T7, T3, or Sp6 phage RNA polymerase. The resulting product may contain an open reading frame encoding a peptide of interest disclosed herein, flanked by 5'- and 3'-UTR sequences, a 5'-cap, and a poly(A) tail. In some embodiments, the RNA vaccine may comprise a trans-amplifying RNA (see, e.g., Beissert et al., Molecular Therapy January 2020 28(1):119-128). In certain embodiments, the RNA vaccine encodes the Aβ and tau peptides disclosed herein and, particularly when transferred into cells such as immature antigen-presenting cells, may express the Aβ and tau peptides. 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 RNA that does not naturally occur in RNA. For example, modified RNA may refer to RNA with a 5'-cap; however, the RNA may contain additional modifications.The 5'-cap can be modified to have the ability to stabilize RNA when bound to it. In certain embodiments, further modifications can be extension or truncation of the naturally occurring poly(A) tail, 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 generate an antigen-specific immune response in a subject. For example, the RNA vaccine formulation is administered to a subject to stimulate the subject's humoral and / or cellular immune system against Aβ, tau, and alpha-synuclein antigens. Therefore, the RNA vaccine formulation may further contain one or more adjuvants, diluents, carriers, and / or excipients, and is applied to a subject by any suitable route to induce a protective and / or therapeutic immune response against Aβ, tau, and alpha-synuclein antigens.

[0087] Basic texts disclosing general methods of molecular biology, all of which are incorporated herein by reference, include Sambrook, J et al., Molecular Cloning: A Laboratory Manual, 2002; 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).

[0088] Techniques for manipulating nucleic acids, such as, for example, generating mutations in sequences, subcloning, labeling probes, sequencing, hybridization, etc., are well described in the scientific and patent literature. See, e.g., 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).

[0089] Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of several common means well known to those of skill in the art, including, for example, NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC) and hyperdiffusion chromatography, various immunological methods such as fluid or gel precipitin reactions, immunodiffusion, immunoelectrophoresis, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), immunofluorescence assays, 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.

[0090] Pharmaceutical Composition

[0091] Each of the peptides and immunogens described herein can be present in a pharmaceutical composition to be administered together with a pharmaceutically acceptable adjuvant and a pharmaceutically acceptable excipient. The adjuvant increases the titer and / or binding affinity of the induced antibodies compared to when the peptide is used alone. Various adjuvants can be used in combination with the immunogens of the present disclosure to induce an immune response. Some adjuvants enhance the intrinsic response to the immunogen without causing conformational changes in the immunogen that affect the qualitative form of the response. The adjuvant can be a natural compound, a modified version or derivative of a natural compound, or a synthetic compound.

[0092] Some adjuvants include aluminum salts, such as aluminum hydroxide and aluminum phosphate, 3 De-O-acylated monophosphoryl lipid A (MPL™) (see GB 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Montana, now part of Corixa)). As used herein, MPL refers to natural and synthetic versions of MPL. Examples of synthetic versions include PHAD®, 3D-PHAD®, and 3D(6A)-PHAD® (Avanti Polar Lipids, Alabaster, Alabama).

[0093] 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 is disclosed, characterized, and evaluated in U.S. Pat. Nos. 5,057,540 and 8,034,348, the disclosures of which are incorporated herein by reference. Additionally, 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 the 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, which contains 50 mcg of QS-21. In certain embodiments, the amount of QS-21 is from about 10 μg to about 500 μg.

[0094] TQL1055 is an analog of QS-21 (Adjuvance Technologies, Lincoln, NE). Semisynthetic TQL1055 is characterized as having high purity, increased stability, reduced local 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-fold 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 TQL1055 dose. WO2018200656A1 teaches that an optimal amount of TQL1055 can reduce the amount of antigen and achieve excellent titers. In certain embodiments, the amount of TQL1055 is about 10 μg to about 500 μg.

[0095] Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil), optionally combined with immunostimulants such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)), Pluronic® polymers, and killed mycobacteria. Ribi adjuvant is an oil-in-water emulsion. Ribi contains a metabolizable oil (squalene) emulsified with saline containing Tween® 80. Ribi also contains purified mycobacterial products and bacterial monophosphoryl lipid A, which act as immunostimulants. Other adjuvants can be CpG oligonucleotides (see WO 98 / 40100), cytokines (e.g., IL-1, IL-1 alpha and beta peptides, IL-2, gamma-INF, IL-10, GM-CSF), chemokines (e.g., MIP1-alpha and beta, and RANTES), saponin, RNA, and / or TLR agonists (e.g., TLR4 agonists such as MPL and synthetic MPL molecules), aminoalkyl glucosaminide phosphates, and other TLR agonists. The adjuvant can be administered as a component of a therapeutic composition with the active agent, or can be administered separately, before, together with, or after administration of the therapeutic agent.

[0096] In various embodiments of the present 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 formulated in a liposome.

[0097] Additionally, some embodiments of the present disclosure can include a multiple antigen presentation system (MAP). Multiple antigen presentation peptide vaccine systems have been developed to avoid the adverse effects associated with traditional vaccines (i.e., live-attenuated, killed, or inactivated pathogens), carrier proteins, and cytotoxic adjuvants. Multiple antigen presentation peptide vaccine systems have been developed using two major approaches: (1) the addition of functional components, such as T cell epitopes, cell-penetrating peptides, and lipophilic moieties; and (2) a synthetic approach using size-defined nanomaterials, such as self-assembling peptides, nonpeptidic dendrimers, and gold nanoparticles, as antigen presentation platforms. The use of a multiple antigen peptide (MAP) system can improve the sometimes poor immunogenicity of subunit peptide vaccines. In the MAP system, multiple copies of an antigen peptide are simultaneously attached to the α- and ε-amino groups of a non-immunogenic Lys-based dendritic scaffold, helping to confer stability from degradation and thus enhancing molecular recognition by immune cells and the induction of stronger immune responses compared to small antigen peptides alone. In some compositions, 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.

[0098] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the selected route of administration. For injection, the peptides of the present disclosure can be formulated in aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the injection site). The solution can contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the peptide compositions can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0099] The peptide (and, if necessary, a carrier fused to the peptide(s)) can also be administered in the form of a nucleic acid encoding the peptide(s) and expressed in situ in the subject. The nucleic acid segment encoding the immunogen is typically linked to regulatory elements, such as a promoter and enhancer, that allow expression of the DNA segment in the intended target cells of the subject. For expression in blood cells, as desired for induction of an immune response, promoter and enhancer elements from, for example, light or heavy chain immunoglobulin genes, or the CMV major intermediate-early promoter and enhancer are suitable for directing expression. The linked regulatory elements and coding sequence are often cloned into a vector.

[0100] DNA and RNA can be delivered in naked form (i.e., without colloidal or encapsulating materials). Alternatively, retroviral systems (see, e.g., Boris-Lawrie and Teumin, Cur. Opin. Genet. Develop. 3(1):102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol. 67(10);5911-21 (1993)); adeno-associated viral vectors (see, e.g., Zhou et al., J. Exp. Med. 179(6):1867-75 (1994)); viral vectors from the pox family, including vaccinia virus and avian poxvirus, viral vectors from the alphavirus genus, such as those derived from Sindbis virus and Semliki Forest virus (see, e.g., Dubensky et al., J. Virol. 70(1):508-519 (1994)). Several viral vector systems can be used, including rhabdoviruses such as rabdovirus (see U.S. Pat. No. 5,643,576; see U.S. Pat. No. 5,643,576; see U.S. Pat. No. 5,643,576), Venezuelan equine encephalitis virus (see U.S. Pat. No. 5,643,576), and vesicular stomatitis virus (see WO 96 / 34625), as well as papillomaviruses (see WO 94 / 12629; Ohe et al., Human Gene Therapy 6(3):325-333 (1995); and Xiao & Brandsma, Nucleic Acids. Res. 24(13):2620-2622 (1996)).

[0101] The DNA and RNA encoding immunogen, or the vector containing it, can be packaged in liposome, nanoparticle or lipoprotein complex.Suitable other polymers include, for example, protamine liposome, polysaccharide particle, cationic nanoemulsion, cationic polymer, cationic polymer liposome, cationic lipid nanoparticle, cationic lipid, cholesterol nanoparticle, cationic lipid-cholesterol, PEG nanoparticle or dendrimer nanoparticle.Other 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 its entirety. Vectors and DNA encoding immunogens can also be adsorbed to or associated with particulate carriers, examples of which include polymethyl methacrylate polymers, and polylactide and poly(lactide-co-glycolide) (see, e.g., McGee et al., J. Micro Encap. Mar-Apr 1997; 14(2):197-210).

[0102] Pharmaceutically acceptable carrier compositions may include, but are not limited to, water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, 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 acceptable as pharmaceutical excipients.

[0103] Suitable subjects for treatment

[0104] The presence of Aβ plaques and / or neurofibrillary tangles is associated with Alzheimer's disease, Down's syndrome, mild cognitive impairment, cerebral amyloid angiopathy, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, and globular glial tauopathy. It has been found in several diseases, including amyotrophic lateral sclerosis / parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), glioglobular tauopathy (GGT), Parkinson's disease, progressive supranuclear palsy (PSP), dry age-related macular degeneration (AMD), and inclusion body myositis.

[0105] The compositions and methods of the present disclosure can be used in the treatment or prevention of any of these diseases.Due to the widespread association between neurological diseases and Aβ and / or tau and / or alpha-synuclein, the compositions and methods of the present disclosure can be used in the treatment or prevention of any subject who shows elevated levels of Aβ and / or tau and / or alpha-synuclein (e.g., in CSF) compared to the average level in individuals without neurological diseases.The compositions and methods of the present disclosure can also be used in the treatment or prevention of neurological diseases in individuals with mutations in Aβ and / or tau and / or alpha-synuclein associated with neurological diseases.The methods are particularly suitable for the treatment or prevention of Alzheimer's disease.

[0106] Subjects suitable for treatment include individuals who are at risk of disease but do not show symptoms, and patients who currently show symptoms, including treatment-naive subjects who have not been previously treated for the disease.Subjects at risk of disease include subjects in the elderly population, asymptomatic subjects with Aβ and / or tau and / or alpha-synuclein pathology and known genetic risk of disease.Such individuals include individuals with relatives who have experienced this disease, and individuals whose risk has been determined by analyzing genetic or biochemical markers.Genetic markers of risk include mutations in Aβ and / or tau and / or alpha-synuclein, and mutations in other genes related to neurological diseases.For example, heterozygous ApoE4 alleles, let alone homozygous ApoE4 alleles, are associated with the risk of Alzheimer's disease (AD). Other markers of Alzheimer's disease risk include mutations in the APP gene, particularly mutations at position 717 and mutations at positions 670 and 671, known as the Hardy and Swedish mutations, respectively; mutations in the presenilin genes PS1 and PS2; and a family history of AD, hypercholesterolemia, or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be recognized by PET imaging due to the presence of characteristic dementia and the risk factors described above. In addition, several diagnostic tests are available to identify individuals with AD. These include measuring tau or phosphorylated tau levels and Aβ42 levels in CSF or blood. Elevated tau or phosphorylated tau levels and decreased Aβ42 levels indicate the presence of AD. Some mutations, such as Ala30Pro or Ala53Thr, or mutations in other genes associated with Parkinson's disease, such as leucine-rich repeat kinase (LRRK2 or PARK8), are associated with Parkinson's disease. The subject may also be diagnosed with any of the above-mentioned neurological disorders according to DSM IV TR criteria.

[0107] In asymptomatic subjects, treatment can be initiated at any age (e.g., 10, 20, 30, or older). However, it is usually not necessary to begin 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 decreases, booster doses are indicated. In the case of potential Down syndrome patients, treatment can be initiated before birth or shortly after birth by administering a therapeutic agent to the mother.

[0108] Methods of Treatment and Use

[0109] The present disclosure provides a method for inhibiting or reducing the aggregation of Abeta and / or tau and / or alpha-synuclein in a subject having or at risk of developing a neurodegenerative disease (e.g., Alzheimer's disease). The method includes administering a composition disclosed herein to the subject. A therapeutically effective amount is a dosage that, when administered for an effective period of time, achieves a desired immunological or clinical effect. Dosage regimens 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 exigencies of the therapeutic situation.

[0110] In prophylactic applications, the compositions described herein can be administered to subjects susceptible to or otherwise at risk of a disease (e.g., Alzheimer's disease) in a regimen (dosage, frequency, and route of administration) effective to reduce the risk of the disease, reduce the severity of the disease, or delay the onset of at least one sign or symptom of the disease. In particular, the regimen is effective to inhibit or delay Aβ plaque formation, and / or inhibit or delay tau or phosphorylated tau and paired filaments formed therefrom in the brain and / or alpha-synuclein synucleinopathy, and / or inhibit or delay the toxic effects thereof, and / or inhibit or delay the development of behavioral deficits. In therapeutic applications, the compositions described herein are administered to a subject suspected of having a disease (e.g., Alzheimer's disease) or a patient already suffering from the disease in a regimen (dosage, frequency, and route of administration) effective to improve at least one sign or symptom of the disease or at least inhibit its further worsening. In particular, the regimen is preferably effective to reduce or at least inhibit further increases in the levels of Aβ plaques and / or tau, phosphorylated tau, or paired filaments formed therefrom and / or alpha synuclein synucleinopathy, associated toxicity, and / or behavioral deficits.

[0111] A regimen is considered therapeutically or prophylactically effective if the treated individual achieves an outcome that is more favorable than the average outcome in a control population of comparable subjects not treated by the methods of the invention, or if a better outcome is demonstrated in treated versus control subjects in a controlled clinical trial (e.g., a Phase II, Phase II / III, or Phase III trial) at a p<0.05 or 0.01, or even 0.001 level.

[0112] The effective dose will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is an ApoE carrier, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic.

[0113] In some embodiments, the effective amount is a total dose of 25 μg to 1000 μg, or 50 μg to 1000 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a 25 μg dose administered to the subject a total of two times. In some embodiments, the effective amount is a 100 μg dose administered to the subject a total of two times. In some embodiments, the effective amount is a 400 μg dose administered to the subject a total of two times. In some embodiments, the effective amount is a 500 μg dose administered to the subject a total of two times. In some embodiments, the RNA (e.g., mRNA) vaccine is administered to the subject by intradermal injection, intramuscular injection, or intranasal administration.

[0114] In some embodiments, the amount of agent for active immunotherapy varies from 1 to 1,000 micrograms (μg) per patient, or 0.1 to 500 μg, or 10 to 500 μg, or 50 to 250 μg, and may be 1 to 100 μg or 1 to 10 μg per injection for human administration. The timing of injections can vary widely, from once daily to once weekly, once monthly, once yearly, or once every 10 years. A typical regimen consists of an immunization followed by booster injections at time intervals such as 6 weeks or 2 months. Another regimen consists of an 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 entails lifelong injections every 2 months. Alternatively, booster injections can be irregular, as indicated by monitoring the immune response. The frequency of administration may be one or more times, as long as the side effects are within a clinically acceptable range.

[0115] In some embodiments, a composition or method disclosed herein includes administering to a subject a nucleic acid vaccine comprising one or more DNA or RNA polynucleotides having open reading frames encoding a first peptide and a second peptide, wherein a dosage of 10 μg / kg to 400 μg / kg of the nucleic acid vaccine is administered to the subject. In some embodiments, the dosage of the RNA polynucleotide 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, or 80-200 μg per dose. , 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, a second dose of nucleic acid is administered to the subject on the 7th day, or the 14th day, or the 21st day.

[0116] The compositions described herein are preferably administered via a peripheral route (i.e., a route in 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 administration can be performed at a single site or multiple sites. Intramuscular injections are most typically performed in the arm or leg muscles. In some methods, the agent is injected directly into a specific tissue where deposits have accumulated.

[0117] The number of doses administered can be adjusted to produce a more robust immune response (e.g., higher titers). For acute disorders or acute exacerbations of chronic disorders, 1 to 10 doses are often sufficient. Sometimes, for acute disorders or acute exacerbations of chronic disorders, a single bolus dose, divided as needed, is sufficient. For chronic disorders, the vaccines / immunotherapies disclosed herein can be administered at regular intervals, for example, weekly, biweekly, monthly, every three months, or every six months, for at least 1, 5, or 10 years, or for the patient's lifetime.

[0118] An effective amount of DNA or RNA encoding an immunogen can be 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. Dosage forms suitable for internal administration preferably contain about 0.1 μg to 100 μg of 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, an effective dose of antigen-loaded dendritic cells can be about 10 4 ~10 8 Those skilled in the art of immunotherapy will be able to adjust these doses without undue experimentation.

[0119] The nucleic acid composition can be administered in a convenient manner, for example, by injection through a convenient and effective route. Routes can include, but are not limited to, intradermal "gene gun" delivery or intramuscular injection. Modified dendritic cells can be administered subcutaneously, intravenously, or intramuscularly. Other possible routes include oral administration, intrathecal administration, inhalation, transdermal application, or rectal administration.

[0120] Depending on the route of administration, the composition may be coated with a material that protects the compound from the effects of enzymes, acids, and other natural conditions that may inactivate the compound.Therefore, it may be necessary to coat the composition with a material that prevents its inactivation, or to co-administer the composition with that material.For example, in a suitable carrier such as an enzyme inhibitor of nuclease or protease (e.g., pancreatic trypsin inhibitor, diisopropyl fluorophosphate, and trasylol), or liposomes (including water-in-oil-in-water emulsions) and conventional liposomes (Strejan et al., J. Neuroimmunol 7(1):27-41, 1984).

[0121] The immunotherapeutic compositions disclosed herein may also be used in combination with other treatments for diseases associated with the accumulation of Aβ and / or tau and / or alpha-synuclein, e.g., anti-Aβ antibodies, such as antibodies that specifically bind to any of the Aβ epitopes disclosed herein. For example, aducanumab, or any of the antibodies disclosed, for example, in U.S. Patent Publication No. 20100202968 and U.S. Patent No. 8,906,367, and / or anti-tau antibodies, e.g., antibodies that specifically bind to any of the tau epitopes disclosed herein, ABBV-8E12, goslanemab, zagotenemab, RG-6100, BIIB076, or antibodies disclosed in WO2014 / 165271, US10,501,531, WO2017 / 191560, US2019 / 0330314, WO2017 / 191561, US2019 / 0330316, W and any of the antibodies disclosed in WO2017 / 191559, and WO2018 / 204546, and / or anti-alpha-synuclein antibodies, e.g., antibodies that specifically bind to any of the alpha-synuclein epitopes disclosed herein, or antibodies and / or other alpha-synuclein binding compounds such as PRX002 / RO7046015, PRX002 / RG7935 (prasinezumab), NPT200-11 / UCB0599, NPT088, BIIB054 (cinpanemab), ABBV-0805, MEDI-1341, NPT088, Lu AF82422, etc. In some combination therapy methods, the patient undergoes passive immunotherapy prior to the active immunotherapy methods disclosed herein. In other methods, patients receive passive immunotherapy and active immunotherapy during the same period of treatment. Alternatively, patients may receive active immunotherapy before passive immunotherapy. Combinations may also include small molecule therapy and non-immunogenic therapy, such as RAZADYNE® (galantamine), EXELON® (rivastigmine), and ARICEPT® (donepezil), as well as other compositions that improve neuronal function in the brain.

[0122] The compositions of the present disclosure may be used in the manufacture of a medicament for the treatment regimens described herein.

[0123] Treatment regimen

[0124] The desired outcome of the treatment methods disclosed herein varies according to the disease and patient profile and can be determined by those skilled in the art. Desired outcome includes the improvement of the patient's health status. Generally, desired outcome includes measurable indicators, such as the reduction or elimination of pathogenic amyloid fibrils, reduced or inhibited amyloid aggregation and / or amyloid fibril deposition, reduced or elimination of Aβ plaque formation, and / or inhibited or delayed tau or phosphorylated tau and paired filaments, and / or reduced alpha-synuclein synucleinopathy, and increased immune response to pathogenic and / or aggregated amyloid fibrils. Desired outcome also includes the alleviation of specific symptoms of amyloid disease. As used herein, relative terms such as "improve," "increase," or "reduce" refer to values ​​compared to a control, for example, a measurement value in the same individual before the start of the treatment described herein, or a measurement value in a control individual or control group. The control individual is an individual who has not been treated with the disclosed immunotherapy / vaccine formulation, but is of approximately the same age as the treated individual (to ensure that the stage of disease in the treated individual and the control individual is comparable), and suffers from the same amyloid disease as the treated individual. Alternatively, the control individual is a healthy individual who is approximately the same age as the treated individual. The change or improvement in response to treatment is generally described by a p-value of 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 can be considered statistically significant and significant.

[0125] The effective dose of the compositions disclosed herein for treating a subject will vary depending on many factors, including the means of administration, the target site, the patient's physiological condition, whether the patient is human or animal, other drugs, if any, administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages can be titrated to optimize safety and efficacy. The amount of immunogen may also depend on whether an adjuvant is also administered; higher dosages are required in the absence of an adjuvant. The amount of immunogen for administration sometimes varies from 1 to 500 μg per patient, 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 dosing can vary widely, from once daily to once per year to once per decade. On any given day when a dose of immunogen is given, the dosage is greater than 1 μg / patient, usually greater than 10 μg / patient if an adjuvant is also administered, greater than 10 μg / patient in the absence of an adjuvant, and usually greater than 100 μg / patient. A typical regimen consists of immunization followed by booster dose(s) at 6-week intervals. Another regimen consists of immunization followed by booster dose(s) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months later. Another regimen involves lifelong dose(s). Alternatively, booster dose(s) can be irregular as indicated by monitoring the immune response.

[0126] When administered in combination with a second treatment for Alzheimer's disease, such as Razadyne® (galantamine), Exelon® (rivastigmine), and Aricept® (donepezil), the second treatment can be administered according to the product label or as needed in light of treatment with the compositions of the present disclosure.

[0127] kit

[0128] The present disclosure further provides a kit (e.g., a container) containing the compositions disclosed herein and related materials, such as instructions for use (e.g., a package insert). The instructions for use may include, for example, instructions for administering the composition and, optionally, one or more additional agents. The container of the peptide and / or nucleic acid composition may be a unit dose, a bulk package (e.g., a multi-dose package), or a sub-unit dose.

[0129] Package insert refers to instructions customarily included in commercial packaging of therapeutic products containing information about the indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. The kit may also include a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0130] use

[0131] Each of the peptides, polypeptides, immunogens and pharmaceutical compositions described herein may be used in 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 in a method for treating 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 medicament for use in treating one or more of the diseases described herein.

[0132] The following is provided for illustrative purposes only and is not intended to limit the scope of the invention, which has been described in broad terms above.

[0133] All US and international patent applications identified herein are hereby incorporated by reference in their entirety. [Example]

[0134] Example 1 animal immunity

[0135] Female Swiss Webster mice are injected subcutaneously at two sites with 100 μl (200 μl total) of immunogen on days 0, 14, and 28. The immunogen is prepared by combining 25 μg of test immunogen and 25 μg of QS21 adjuvant in 200 μl of phosphate-buffered saline (PBS). At least on days 21 and 35, mice are bled by tail nick and 50 μl of blood is collected and subsequently processed into serum. The immunogens tested are as follows: [Table 3]

[0136] The immunogen comprises an Aβ / tau / alpha-synuclein peptide, a C-terminal linker and a C-terminal cysteine, linked to CRM-197 via a maleimide bond via the C-terminal cysteine.

[0137] Guinea pigs were injected intramuscularly with 50 μg of test immunogen and 25 μg of QS21 in 200 μl of Addavax on days 0, 21, 49, and 77. Bleeding began 7 days after immunization. The test immunogens are listed in Table 3. The immunogen peptide is linked to CRM-197 via a maleimide bond through the C-terminal cysteine.

[0138] Female guinea pigs were at least 5 weeks old and weighed approximately 350–500 g at the start of the study. Appropriate animal housing and research procedures for animal care and maintenance were performed in an accredited facility in accordance with United States Department of Agriculture (USDA) and Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) guidelines.

[0139] The concentration of the immunogen was 0.5 mg / ml. Before each administration of the test immunogen, approximately 3 cm of each hind limb was cut to visualize the injection site. 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, each with 100 μl per injection (i.e., animals received 50 μg immunogen in 100 μl PBS + 25 μg QS21 in 100 μl MF59). A 25G-27G needle was inserted intramuscularly in the hind limb approximately 0.25-0.5 cm deep and injected at 100 μl per site. Injection sites were rotated between four separate sites per hind limb, with each dose spaced at least 2 cm apart.

[0140] Example 2 Measurement of antibody titers

[0141] Whole blood samples were collected into clot activator tubes via the jugular vein of guinea pigs at weeks 1, 4, 8, and 12 (250–350 μl per collection) and via tail nick in mice at weeks 1, 3, 7, and 11 (50 μl per collection). The maximum volume of whole blood was collected into clot activator tubes by cardiac puncture at the end of the final collection week. All blood samples were allowed to clot for >30 minutes at room temperature, centrifuged at 3,000 RPM for 10–15 minutes at ambient temperature (approximately 20–25°C), and serum supernatants were individually transferred to clean cryovials. Serum supernatants were stored frozen at -80°C (±12°C).

[0142] Aβ titer (mouse)

[0143] 2 μg / ml Aβ1-28 monomer was coated onto the plate at 100 μl / well in PBS and incubated overnight at room temperature. The plate was blocked with 1% BSA in PBS for 1 hour. The plate was aspirated, and 200 μl of 0.1% BSA in PBS Tween was added to row A. Column 1 contained a 1:100 dilution of negative mouse serum, while the rest of the row contained 1:100 dilutions of test serum. The rows were serially diluted 1:2 down the plate, ranging from 1:100 to 12,800. The wells were incubated for 2 hours at room temperature and then washed. A 5:5000 dilution of anti-mouse IgG HRP in 0.1% BSA in PBS Tween was prepared, and 100 μl was added to the washed wells. This was incubated for 1 hour and then washed. Thermo-Fisher OPD tablets were used to prepare OPD substrate, one tablet per 10 mL. Thermo Fisher substrate buffer was added at 10-fold dilutions, 100 μl per 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 on a Molecular Devices Spectromax. Titers were defined as the dilution giving 50% of the maximum OD and extrapolated if they fell between dilutions.

[0144] Tau titer (mouse)

[0145] Plates were coated with 2 μg / ml recombinant WT Tau4R2N in PBS using 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 200 μl of 0.1% BSA in PBS Tween was added to row A. Column 1 contained a 1:100 dilution of negative mouse serum, while the rest of the rows contained 1:100 dilutions of test serum. Rows were serially diluted 1:2 down the plate, ranging from 1:100 to 12,800. The wells were incubated for 2 hours at room temperature and then washed. A 5:5000 dilution of anti-mouse IgG HRP in 0.1% BSA in PBS Tween was prepared, and 100 μl was then added to the washed wells. The reaction mixture was incubated for 1 hour and washed. Thermo-Fisher OPD tablets were used to prepare OPD substrate, one tablet per 10 mL. Thermo Fisher substrate buffer was added at 10-fold dilutions, 100 μl per 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 on a Molecular Devices Spectromax. The titer was defined as the dilution giving 50% of the maximum OD reading and extrapolated if it fell between dilutions.

[0146] Alpha-synuclein titer (mouse)

[0147] Plates were coated with 2 μg / ml recombinant human alpha-synuclein in PBS at 100 μl / well and incubated overnight at room temperature. The plates were blocked with 1% BSA in PBS for 1 hour. The plates were aspirated, and 200 μl of 0.1% BSA in PBS Tween was added to row A. Column 1 contained a 1:100 dilution of negative mouse serum, while the rest of the rows contained 1:100 dilutions of test serum. Rows were serially diluted 1:2 down the plate, ranging from 1:100 to 12,800. The wells were incubated for 2 hours at room temperature, then washed. A 5:5000 dilution of anti-mouse IgG HRP was prepared in PBS Tween containing 0.1% BSA, and 100 μl was added to the washed wells. This was incubated for 1 hour and washed. Thermo-Fisher OPD tablets were used to prepare OPD substrate, one tablet per 10 mL. Thermo Fisher substrate buffer was added at 10-fold dilutions, 100 μl per 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 on a Molecular Devices Spectromax. The titer was defined as the dilution giving 50% of the maximum OD and extrapolated if it fell between dilutions.

[0148] Aβ titer (guinea pig)

[0149] Plates were coated with 2 μg / ml Aβ1-28 monomer at 100 μl / well in PBS and incubated overnight at room temperature. Plates were blocked with 1% BSA in PBS for 1 hour. The plates were re-aspirated, and 200 μl of 0.1% BSA in PBS Tween was added to row A. Column 1 contained negative guinea pig serum at a 1:100 dilution, while the rest of the rows contained test serum at a 1:100 dilution. Rows were serially diluted 1:2 down the plate, ranging from 1:100 to 12,800. Wells were incubated for 2 hours at room temperature, then washed. A 5:5000 dilution of anti-guinea pig IgG HRP was prepared in PBS Tween containing 0.1% BSA, and 100 μl was added to the washed wells. This was incubated for 1 hour and washed. OPD substrate was prepared using Thermo-Fisher OPD tablets, one tablet per 10 mL. Thermo Fisher substrate buffer was added at 10-fold dilutions, 100 μl per 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 on a Molecular Devices Spectromax. The titer was defined as the dilution giving 50% of the maximum OD and extrapolated if it fell between dilutions.

[0150] Tau titer (guinea pig)

[0151] Plates were coated with 2 μg / ml recombinant wild-type tau 4R2N in PBS at 100 μl / well and incubated overnight at room temperature. The plates were blocked with 1% BSA in PBS for 1 hour. The plates were aspirated, and 200 μl of 0.1% BSA in PBS Tween was added to row A. Column 1 contained a 100x dilution of negative rabbit serum, while the rest of the rows contained 100x dilutions of test serum. Rows were serially diluted 1:2 down the plate, ranging from 100x to 12,800x dilutions. The wells were incubated for 2 hours at room temperature and then washed. A 5,000x dilution of anti-rabbit IgG HRP in 0.1% BSA in PBS Tween was prepared, and 100 μl was then added to the washed wells. This mixture was incubated for 1 hour and washed. Thermo-Fisher OPD tablets were used to prepare OPD substrate, one tablet per 10 mL. Thermo Fisher substrate buffer was added in 10-fold dilutions (100 μl added to each well) and incubated for 15 minutes. The reaction was stopped by adding 50 μl of 2N H2SO4 and the plate was read at 490 nm on a Molecular Devices Spectromax. The titer was defined as the dilution giving 50% of the maximum OD and extrapolated if it fell between dilutions. [Table 4]

[0152] Alpha-synuclein titer (guinea pig)

[0153] Plates were coated with 2 μg / ml recombinant human alpha-synuclein in PBS at 100 μl / well and incubated overnight at room temperature. The plates were blocked with 1% BSA in PBS for 1 hour. The plates were re-aspirated, and 200 μl of 0.1% BSA in PBS Tween was added to row A. Column 1 contained a 1:100 dilution of negative guinea pig serum, while the rest of the rows contained 1:100 dilutions of test serum. Rows were serially diluted 1:2 down the plate, ranging from 1:100 to 12,800. The wells were incubated for 2 hours at room temperature and then washed. A 5:5000 dilution of anti-guinea pig IgG HRP in 0.1% BSA in PBS Tween was prepared, and 100 μl was then added to the washed wells. This mixture was incubated for 1 hour and washed. Thermo-Fisher OPD tablets were used to prepare OPD substrate, one tablet per 10 mL. Thermo Fisher substrate buffer was added in a 10-fold dilution (100 μl added to each well) and the mixture was incubated for 15 minutes. The reaction was stopped by adding 50 μl of 2N H2SO4 and the plate was read at 490 nm on a Molecular Devices Spectromax. The titer was defined as the dilution giving 50% of the maximum OD and was extrapolated if it fell between dilutions.

[0154] Example 3 Staining of Alzheimer's brain tissue with serum from animals immunized with the immunogens disclosed herein.

[0155] Autopsy blocks (approximately 0.5 g) of fresh-frozen human brain tissue were embedded in optimal cutting temperature compound (OCT compound) and cut using a cryostat to generate 10 μm sections. Sections were placed in a solution of glucose oxidase and beta-D-glucose in the presence of sodium azide to block endogenous peroxidase. Once the tissue sections were prepared, staining with designated animal sera from animals immunized with the vaccines disclosed herein was performed using appropriate species secondary antibodies at two dilutions (1:300 and 1:1500) and the DAKO DAB Detection Kit according to the manufacturer's instructions. Staining was performed using an automated Leica Bond Stainer. The results indicate whether sera from animals immunized with the vaccines disclosed herein contain antibodies specific to Aβ, tau, and / or alpha-synuclein in human brain tissue from Alzheimer's patients.

[0156] Example 4 Serum from vaccinated animals blocks soluble Aβ aggregates from binding to neurons.

[0157] E18 primary rat hippocampal neurons were cultured as previously described (Zago, et al. "Neutralization of Soluble, Synaptotoxic Amyloid β Species by Antibodies Is Epitope Specific," J Neurosci. 2012 Feb 22;32(8):2696-2702). To block soluble Aβ aggregates from binding to neurites, soluble Aβ aggregates were preincubated with or without animal vaccine serum at culture days 14-21. Animal serum was isolated from animals vaccinated with immunogen peptides as shown in Table 3. Fresh unlabeled, biotinylated, or (9:1) soluble Aβ was prepared 1 day in advance and incubated overnight at 4°C. Each diluted serum sample (1:1000, 1:300, and 1:100) and soluble Aβ solution were prepared in NeuroBasal-phenol red-free (NB-NPR) medium at twice the final concentration in half the final treatment volume. This was combined with half the final volume of soluble Aβ at twice the final concentration and half the final volume of twice the final diluted animal vaccine serum to create a total final treatment volume of 1x final concentration. This mixture was mixed well and pre-incubated at 37°C for 30 minutes. E18 neurons were rinsed with 150 μL / well of NB-NPR before adding the binding treatment. Animal serum from vaccinated animals / Aβ treatment was added to E18 neurons at 60 μL / well, followed by incubation at 37°C for 30 minutes under normal incubator conditions (5% CO2; 9% O2). Cells were rinsed twice with 150 μL / well of NB-NPR and then fixed with 4% paraformaldehyde in 1x DPBS for 20 minutes. Cells were permeabilized with 0.1% TX-100 for 5 minutes and blocked with 10% normal goat serum (NGS) for 1 hour at room temperature (RT). Cells were incubated overnight at 4°C with MAP2 and NeuN primary antibodies in 100 μL / well of 1x DPBS containing 1% BSA and 1% NGS. The next day, cells were rinsed twice with 150 μL / well of 1x DPBS for 5 minutes per wash.Secondary antibody was added at 100 μL / well in 1x DPBS + 1% BSA + 1% NGS for 1 hour at room temperature. To quantify neurite-associated soluble aggregate Aβ, high-content imaging (HCI) analysis was performed using an Operetta HCI CLS instrument (Perkin Elmer; modified Neuroite Outgrowth algorithm: 40x H2O objective; 40 fields per well; n = 3 per condition; data are shown as mean (±) SD). MAP2 and NeuN (Abcam) neuronal markers were used to trace dendritic neurites, and the number of cell bodies per optical field was counted. Aβ soluble aggregate spots in the neurites were detected using streptavidin 488 or a polyclonal Aβ antibody (Thermo; Millipore). Data were reported as Aβ soluble aggregate spots / neuron (or as integrated intensity).

[0158] Approximately 80-150 neurons were observed per well for each condition tested. The results indicate whether animal sera from animals vaccinated with the immunogenic peptides disclosed herein contain Aβ-specific antibodies that can block the binding of soluble Aβ aggregates to neurons.

[0159] Example 5 Serum from vaccinated animals stains Aβ plaques and / or tau pathology and / or synucleinopathy in human brain tissue.

[0160] Fresh-frozen human brain tissue from autopsied Alzheimer's disease donors or non-diseased controls was embedded in OCT and cut on a cryostat to generate 10 μm frozen sections. Tissue sections were incubated in a solution of glucose oxidase and beta-D-glucose in the presence of sodium azide to block endogenous peroxidase. Staining with serum from vaccinated animals (animals vaccinated with the immunogenic peptides disclosed in Table 3) or control animals was then performed at a 1:1000 dilution in an automated Leica Bond Rx Stainer (Leica Biosystems). Antibody binding was detected using the Bond Polymer Refine Detection Kit (DS9800, Leica Biosystems), which is based on anti-mouse polymer detection, DAB visualization, and hematoxylin nuclear counterstaining. After coverslipping, stained tissue slides were digitally imaged using a Hamamatsu NanoZoomer 2.0HT slide scanner (Hamamatsu Corporation) with NDP.Scan 2.5.85 software. Digitized images were visually inspected and analyzed using NDP.view 2.7.43.0 software.

[0161] The results demonstrate that Aβ plaques and / or tau neurofibrillary tangles and / or alpha-synuclein-based synucleinopathies were identified based on their typical histopathological features. Such lesions were not present in tissues incubated with control animal serum. Non-diseased tissues did not have such lesion staining after incubation with serum from animals vaccinated with the immunogen peptides disclosed herein. The immunogens disclosed herein produce Aβ antibody titers capable of inducing Aβ phagocytosis, and synuclein titers can prevent the internalization of soluble synuclein aggregates into cells.

[0162] 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 may be made thereto by those skilled in the art without departing from the scope and spirit of the present invention. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] During the ceremony, Xaa1 is I or C, Xaa2 is G, Xaa3 is T, K, or L; Xaa4 is E, D, or G; Xaa5 is L or I, Xaa6 is K, H or T. (Q / E)IVYK(S / P) (SEQ ID NO: 996) Alpha-synuclein isoform NACP140 [Homo sapiens] (SEQ ID NO: 58) NCBI reference sequence: NP_000336.1 [ka] [ka] [ka]

Claims

1. (a) a first peptide comprising 5 to 10 amino acids from residues 1 to 10 of SEQ ID NO: 01; (b) a second peptide comprising 6 to 13 amino acids from residues 244 to 372 of SEQ ID NO: 02; and (c) a polypeptide comprising a third peptide comprising 6 to 10 amino acids from residues 81 to 140 of SEQ ID NO:58, A polypeptide, wherein the first peptide, second peptide and third peptide are arranged in any order in the polypeptide.

2. (a) a peptide comprising 5 to 10 amino acids from residues 1 to 10 of SEQ ID NO: 01; (b) a peptide comprising 6 to 13 amino acids from residues 244 to 372 of SEQ ID NO: 02, and (c) a peptide comprising 6 to 10 amino acids from residues 81 to 140 of SEQ ID NO:

58. and further comprising a fourth peptide selected from one of: The polypeptide of claim 1 , wherein the first peptide, the second peptide, the third peptide, and the fourth peptide are arranged in any order in the polypeptide.

3. A polypeptide described in any of claims 1 to 2, wherein the 5 to 10 amino acids of the first polypeptide comprise the amino acid sequence EFR, the 6 to 13 amino acids of the second peptide comprise the amino acid sequence NLK, and the 6 to 10 amino acids derived from the third peptide comprise the amino acid sequence DNE. 【Request 4】 【Chemical 14】 The polypeptide of claim 1, comprising the amino acid sequence:

5. (a) the first peptide is 【Chemistry 15】 and comprising an amino acid sequence selected from the group consisting of: (b) the second peptide is 【Chemistry 16-1】 【Chemistry 16-2】 and comprising an amino acid sequence selected from the group consisting of: (c) the third peptide is 【Chemistry 17】 and comprising an amino acid sequence selected from the group consisting of: (d) the fourth peptide, if present, is any one of the amino acid sequences of SEQ ID NOs: 3-8, 11-16, 18-23, 59-63, 67-71, 74-79, 80-84, 696, 697, 702, 703, 707, 708, 711, and 712; A polypeptide according to any one of claims 1 to 3.

6. 6. The polypeptide of any one of claims 1 to 3 and 5, wherein two or more of the first peptide, the second peptide, the third peptide, and, if present, the fourth peptide, are each linked by a cleavable linker.

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

8. 8. The polypeptide of claim 7, wherein the amino acid sequence of each cleavable linker is independently selected from the group consisting of arginine-arginine (Arg-Arg), arginine-valine-arginine-arginine (Arg-Val-Arg-Arg; SEQ ID NO: 138), 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: 139), alanine-glycine-alanine-glycine (Ala-Gly-Ala-Gly; SEQ ID NO: 140), and lysine-glycine-lysine-glycine (Lys-Gly-Lys-Gly; SEQ ID NO: 141).

9. The polypeptide of any one of claims 1 to 3 and 5 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 to the carrier comprises an amino acid sequence selected from the group consisting of GG, GGG, AA, AAA, KK, KKK, SS, SSS, GAGA (SEQ ID NO: 139), AGAG (SEQ ID NO: 140) and KGKG (SEQ ID NO: 141).

11. The polypeptide of any one of claims 1 to 3 and 5 to 9, 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, further comprising a blocked amine at the N-terminus.

13. The polypeptide of any one of claims 2 to 3 and 5 to 11, wherein any one of the first peptide, the second peptide, the third peptide, and the fourth peptide comprises 5 to 10 amino acids.

14. The polypeptide of any one of claims 1 to 3 and 5 to 12, wherein the first peptide is DAEFRHD (SEQ ID NO: 06).

15. The polypeptide of any one of claims 1 to 3 and 5 to 12, wherein the third peptide comprises the amino acid sequence PDNEAYE (SEQ ID NO: 75).

16. The polypeptide of any one of claims 1 to 3 and 5 to 12, wherein the third peptide comprises the amino acid sequence DPDNEAY (SEQ ID NO: 69).

17. 17. An immunotherapeutic composition comprising the polypeptide of any one of claims 1 to 16, wherein the polypeptide is linked to a carrier, optionally comprising 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.

18. 18. A pharmaceutical formulation comprising: (a) a polypeptide according to any one of claims 1 to 16 or the immunotherapy composition of claim 17; and (b) at least one adjuvant, optionally 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), an oil-in-water emulsion (such as squalene or peanut oil), CpG, polyglutamic acid, polylysine, AddaVax™, MF59®, and combinations thereof; and optionally wherein the adjuvant comprises a liposomal formulation.

19. 20. The pharmaceutical formulation of claim 18, wherein the composition comprises at least one pharmaceutically acceptable diluent.

20. 20. The pharmaceutical formulation of claim 18 or 19, comprising a multiple antigen-presenting system (MAP), optionally comprising 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, self-assembled nanoparticles and gold nanoparticles as antigen-presenting platforms.

21. (a) a first peptide sequence comprising 5 to 10 amino acid residues derived from the first 10 N-terminal residues of SEQ ID NO: 01; (b) a second peptide sequence comprising 6 to 13 amino acids from residues 244 to 400 of SEQ ID NO: 02; and (c) a third peptide sequence comprising 6 to 10 amino acids from residues 81 to 140 of SEQ ID NO:

58.

10. An immunotherapeutic composition comprising:

22. (a) a peptide comprising 5 to 10 amino acids from residues 1 to 10 of SEQ ID NO: 01; (b) a peptide comprising 6 to 13 amino acids from residues 244 to 400 of SEQ ID NO: 02, and (c) a peptide comprising 6 to 10 amino acids from residues 81 to 140 of SEQ ID NO:

58.

22. The immunotherapy composition of claim 21, further comprising a fourth peptide sequence selected from one of:

23. (a) the first peptide sequence is 【Chemistry 18-1】 【Chemistry 18-2】 and comprising an amino acid sequence selected from the group consisting of: (b) the second peptide sequence is 【Chemistry 19】 and comprising an amino acid sequence selected from the group consisting of: (c) the third peptide sequence is 【Chemistry 20】 and comprising an amino acid sequence selected from the group consisting of: (d) the fourth peptide sequence, if present, is any one of the amino acid sequences of SEQ ID NOs: 3-8, 11-16, 18-23, 59-63, 67-71, 74-79, 80-84, 696, 697, 702, 703, 707, 708, 711, and 712; 23. The immunotherapy composition of claim 21 or claim 22, wherein each of the first peptide sequence, the second peptide sequence, the third peptide sequence and, if present, the fourth peptide sequence, can optionally include a C-terminal cysteine.

24. 24. The immunotherapy composition of any one of claims 21-23, wherein at least one of the first peptide sequence, the second peptide sequence, the third peptide sequence, and the fourth peptide sequence further comprises a linker to a carrier at either the C-terminal portion of the polypeptide or the N-terminal portion of the polypeptide.

25. 25. The immunotherapy composition of claim 24, 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: 139), AGAG (SEQ ID NO: 140) and KGKG (SEQ ID NO: 141), and optionally the linker to the carrier comprises a C-terminal cysteine (C).

26. 26. The immunotherapy composition of any one of claims 24-25, 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.

27. The immunotherapeutic composition of any one of claims 24 to 26, further comprising at least one pharmaceutically acceptable diluent.

28. 28. The immunotherapy composition of any one of claims 17-27, further comprising a multiple antigen-presenting system (MAP), optionally comprising 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, self-assembled nanoparticles and gold nanoparticles as antigen-presenting platforms.

29. 29. A pharmaceutical formulation comprising the immunotherapeutic composition of any of claims 21-28 and at least one adjuvant, optionally 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.

30. A nucleic acid comprising a nucleic acid sequence encoding a polypeptide according to any one of claims 1 to 16 of an immunotherapeutic composition according to claims 17 and 21 to 28.

31. A nucleic acid immunotherapy composition comprising the nucleic acid of claim 30 and at least one adjuvant.

32. An immunotherapeutic composition according to any of claims 17 and 21 to 28 or a pharmaceutical formulation according to any of claims 18 to 20 and 29 for treating or effecting the prevention of Alzheimer's disease in a subject.

33. 30. An immunotherapeutic composition according to any of claims 17 and 21-28 or a pharmaceutical formulation according to any of claims 18-20 and 29 for inhibiting or reducing aggregation of at least one of Aβ, tau and alpha-synuclein in a subject having or at risk of developing Alzheimer's disease.

34. 32. The nucleic acid immunotherapy composition of claim 31 for treating or effecting prevention of Alzheimer's disease in a subject.

35. 32. The nucleic acid immunotherapy composition of claim 31 for inhibiting or reducing aggregation of at least one of Aβ, tau, and alpha-synuclein in a subject having or at risk of developing Alzheimer's disease.

36. A composition comprising a polypeptide according to claims 1 to 16, an immunotherapy composition according to claims 17 and 21 to 28, a pharmaceutical formulation according to claims 18 to 20 and 29, or a nucleic acid immunotherapy composition according to claim 31, for inducing an immune response comprising antibodies that specifically bind to Aβ, tau and / or alpha-synuclein in an animal.

37. 37. The composition or formulation of claim 36, wherein the immune response comprises antibodies that specifically bind to Aβ, antibodies that specifically bind to tau, and antibodies that specifically bind to alpha-synuclein.

38. 38. The composition or formulation of any of claims 36-37, wherein said inducing said immune response comprises an antibody that specifically binds to the N-terminal region of Aβ, the microtubule region of tau, and / or the C-terminal region of alpha-synuclein.

39. 29. An immunotherapy kit comprising the immunotherapeutic composition of any one of claims 17 and 21-28, optionally further comprising an adjuvant, and further optionally wherein the immunotherapeutic composition is in a first container and the adjuvant is in a second container.

40. 32. A kit comprising the nucleic acid immunotherapy composition of claim 31, optionally further comprising an adjuvant, and optionally wherein the nucleic acid is in a first container and the adjuvant is in a second container.

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