Peptide conjugate vaccine composition and method for the treatment of Alzheimer's disease

Abstract

The present invention provides a composition and a method for treating a disease associated with amyloid deposits of Aβ in a patient's brain, such as Alzheimer's disease. Such a method involves administering a pharmaceutical composition comprising an immunogenic fragment of Aβ capable of inducing a beneficial immune response in the form of an antibody against Aβ. The immunogenic fragment comprises a linear or polyvalent peptide of Aβ. The pharmaceutical composition comprises an immunogenic fragment chemically linked to a carrier molecule that can be administered together with an ajuvant.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims the benefit of U.S. provisional patent application serial number 63 / 599,885 filed on November 16, 2023, the whole of which is incorporated herein by reference.

[0003] Reference to the electronically submitted sequence list

[0004] The present application contains a list of sequences submitted electronically in XML format, the full text of which is incorporated herein by reference. The XML file was created on October 1, 2024, named 25864-WO-PCT_SL.xml, and is 59,964 bytes in size.

[0005] field

[0006] The present disclosure generally relates to peptide-based pharmaceutical compositions and conjugate vaccines, as well as methods for using said compositions and vaccines for the prevention and treatment of neurodegenerative diseases, such as Alzheimer's disease. Background Technology

[0007] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by dementia and pathological changes in the brain, such as the accumulation of amyloid plaques and neurofibrillary tangles. [JA Hardy, et al., Alzheimer's Disease: the amyloid cascade hypothesis. Science (1992) 256:184-185]. According to the "amyloid cascade hypothesis," increased accumulation of amyloid β (Aβ) peptides in the brain leads to the formation of Aβ oligomers and plaques, which in turn leads to neurite damage, the formation of neurofibrillary tangles, neuronal cell death, and cognitive decline. [JA Hardy, et al., The amyloid hypothesis of Alzheimer's Disease: progress and problems on the road to therapeutics. Science (2002) 297:353-356].

[0008] The Aβ peptide consists of a sequence of 40 to 42 amino acids derived from the amyloid precursor protein (APP). Aβ is an N-terminal truncated version. pE3 is Aβ 1-42 It is generated from the cleavage of two N-terminal amino acids and the cyclization of the glutamate side chain to form pyroglutamate. Reference [T. Iwatsubo, et al., Full-length amyloid-beta (1-42(43)) and amino-terminally modified and truncated amyloid-beta 42(43) deposit in diffuse plaques. Am J Pathol. (1996) 149(6):1823-30]. Aβ pE3Peptides tend to aggregate highly and accumulate early in the plaque-forming amyloid cascade. References [TC Saido, et al., Dominant and differential deposition of distinct beta-amyloid peptide species, Aβ N3(pE), in senile plaques. Neuron (1995)14:457-466. T. Iwatsubo, et al., Full-length amyloid-beta (1-42(43)) and amino-terminally modified and truncated amyloid-beta 42(43) deposit in diffuse plaques. Am. J. Pathol. (1996) 149:1823-1830. YM Kuo, et al., Isolation, chemical characterization, and quantitation of Aβ 3-pyroglutamyl peptide from neuritic plaques and vascular amyloid deposits. Biochem. Biophys. Res. Commun., (1997) 237:188-191. S. Schilling, et al., On the seeding and oligomerization of pGlu-amyloid peptides (in vitro). Biochemistry (2006) 45:12393-12399. Z. Schlenzig, et al., Pyroglutamate formation influences solubility and amyloidogenicity of amyloid peptides. Biochemistry (2009) 48:7072-7078.].

[0009] Clearance of Aβ plaques was posited as a means to slow the progression of AD, and Aβ pE3Passive immunotherapy approaches targeting [the substance] have demonstrated plaque clearance and the slowing of cognitive decline in clinical trials. [JR Sims, et al., Donanemab in early symptomatic Alzheimer Disease: the TRAILBLAZER-ALZ 2 randomized clinical trial. JAMA. (2023) 330(6):512-527]. Active immunotherapy (i.e., vaccine) regimens have also been tested for the treatment of Alzheimer's disease. [F. Mantile, et al., Vaccination against β-amyloid as a strategy for the prevention of Alzheimer's Disease. Biology (Basel). (2020) 9(12):425]. Aβ with QS-21 Azuvant 1-42 The vaccine candidate AN1792 was tested in a Phase II clinical trial, and evidence of post-mortem plaque reduction was revealed. Nevertheless, AN1792 induced meningoencephalitis in a subset of patients attributable to T-cell responses to the vaccine. Reference [JAR Nicoll, et al., Persistent neuropathological effects 14 years following amyloid-β immunization in Alzheimer's Disease. Brain (2019) 142(7):2113-2126].

[0010] Currently, there is no specific and highly effective preventive or curative treatment for Alzheimer's disease. Therefore, there is a strong need for additional therapeutic measures.

[0011] The object of the present invention is to provide a pharmaceutical composition for the preventive and curative treatment of Alzheimer's disease. In particular, an aspect of the present invention provides an Alzheimer's disease vaccine that can be administered to mammals for the prevention and / or treatment of Alzheimer's disease.

[0012] The present disclosure provides a composition and a method for treating a disease associated with amyloid deposits of Aβ in the brain of a patient, such as Alzheimer's disease. In one aspect, the present disclosure provides a pharmaceutical composition comprising an immunogenic peptide of at least six adjacent amino acids of SEQ ID NO: 1 and an immunogenic carrier protein.

[0013] In some embodiments, the immunogenic peptide is missing 1 to 10 amino acids at the N-terminus or 1 to 33 amino acids at the C-terminus of sequence identification number: 1. In other embodiments, the immunogenic peptide is missing 1 to 10 amino acids at the N-terminus and 1 to 33 amino acids at the C-terminus of sequence identification number: 1.

[0014] In some embodiments, the immunogenic peptide comprises at least 10 adjacent amino acids of sequence identification number: 1.

[0015] In some embodiments, the immunogenic peptide comprises 1 to 5 modified amino acids. In certain embodiments, the immunogenic peptide comprises a pyroglutamate residue at amino acid positions 3 and / or 11 of SEQ ID NO: 1.

[0016] In some embodiments, the immunogenic peptide is selected from the group consisting of sequence identification numbers: 2-13.

[0017] In some embodiments, the immunogenic carrier protein is selected from the group consisting of CRM197; diphtheria toxin fragment B (DTFB); DTFB C8; diphtheria toxoid (DT); tetanus toxoid (TT); fragment C of TT; pertussis toxoid; cholera toxoid; E. coli LT; E. coli ST; Neisseria meningitidis outer membrane protein complex (OMPC); exotoxin A from Pseudomonas aeruginosa; methylculture keyhole limpet hemocyanin (mcKLH); and bacteriophage AP205 coat protein.

[0018] In some embodiments, the immunogenic carrier protein is conjugated at the N-terminus or C-terminus of the immunogenic peptide. In certain embodiments, the immunogenic carrier protein is conjugated at the C-terminus of the immunogenic peptide.

[0019] In some embodiments, the immunogenic carrier protein is conjugated to the immunogenic peptide by a linker. In some embodiments, the linker is selected from the group consisting of N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS); polyethylene glycol (PEG); aminohexanoic acid (Ahx); sulfhydryl-reactive crosslinker; maleimide (MA) linker; oligopeptide; dendrimer; cyclodextrin; and glycine-rich peptide.

[0020] In some embodiments, the pharmaceutical composition further comprises a spacer comprising 1 to 10 amino acids adjacent to the linker.

[0021] In a specific embodiment, the immunogenic peptide comprises sequence identification number: 2, and the immunogenic carrier protein is CRM197.

[0022] In another specific embodiment, the immunogenic peptide is linked to CRM197 via a GMBS linker, and the composition further comprises a glycine-cysteine ​​spacer between the immunogenic peptide and the GMBS linker.

[0023] In some embodiments, the immunogenic peptide is a monomer. In some embodiments, the immunogenic peptide is a polymer.

[0024] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable ajuvant. In some embodiments, the pharmaceutically acceptable ajuvant is selected from the group consisting of glucopyranosol lipid ajuvant (GLA); AVT1; AVT2; AVT3, AVT4; AVT5; AVT6; AVT7; QS-21; aluminum-based ajuvant; saponin-based ajuvant; and TLR7 / 8 agonists.

[0025] In some embodiments, the pharmaceutically acceptable ajuvant is AVT1, AVT5, or AVT7.

[0026] In another aspect, the present disclosure provides a method for preventing or treating a disease associated with amyloid deposits of Aβ in the brain of a patient who requires prevention or treatment of a disease associated with amyloid deposits of Aβ in the brain, which comprises administering an effective dose of a pharmaceutical composition comprising an immunogenic peptide of at least six adjacent amino acids of SEQ ID No. 1 and an immunogenic carrier protein.

[0027] In some embodiments, the immunogenic peptide is missing 1 to 10 amino acids at the N-terminus or 1 to 33 amino acids at the C-terminus of sequence identification number: 1. In other embodiments, the immunogenic peptide is missing 1 to 10 amino acids at the N-terminus and 1 to 33 amino acids at the C-terminus of sequence identification number: 1.

[0028] In some embodiments, the immunogenic peptide comprises at least 10 adjacent amino acids of sequence identification number: 1.

[0029] In some embodiments, the immunogenic peptide comprises 1 to 5 modified amino acids. In certain embodiments, the immunogenic peptide comprises a pyroglutamate residue at amino acid positions 3 and / or 11 of SEQ ID NO: 1.

[0030] In some embodiments, the immunogenic peptide is selected from the group consisting of sequence identification numbers: 2-13.

[0031] In some embodiments, the immunogenic carrier protein is selected from the group consisting of CRM197; diphtheria toxin fragment B (DTFB); DTFB C8; diphtheria toxoid (DT); tetanus toxoid (TT); fragment C of TT; pertussis toxoid; cholera toxoid; E. coli LT; E. coli ST; Neisseria meningitidis outer membrane protein complex (OMPC); exotoxin A from Pseudomonas aeruginosa; methylculture keyhole limpet hemocyanin (mcKLH); and bacteriophage AP205 coat protein.

[0032] In some embodiments, the immunogenic carrier protein is conjugated at the N-terminus or C-terminus of the immunogenic peptide. In certain embodiments, the immunogenic carrier protein is conjugated at the C-terminus of the immunogenic peptide.

[0033] In some embodiments, the immunogenic carrier protein is conjugated to the immunogenic peptide by a linker. In some embodiments, the linker is selected from the group consisting of N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS); polyethylene glycol (PEG); aminohexanoic acid (Ahx); sulfhydryl-reactive crosslinker; maleimide (MA) linker; oligopeptide; dendrimer; cyclodextrin; and glycine-rich peptide.

[0034] In some embodiments, the pharmaceutical composition further comprises a spacer comprising 1 to 10 amino acids adjacent to the linker.

[0035] In a specific embodiment, the immunogenic peptide comprises sequence identification number: 2, and the immunogenic carrier protein is CRM197.

[0036] In another specific embodiment, the immunogenic peptide is linked to CRM197 via a GMBS linker, and the composition further comprises a glycine-cysteine ​​spacer between the immunogenic peptide and the GMBS linker.

[0037] In some embodiments, the immunogenic peptide is a monomer. In some embodiments, the immunogenic peptide is a polymer.

[0038] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable ajuvant. In some embodiments, the pharmaceutically acceptable ajuvant is selected from the group consisting of glucopyranosol lipid ajuvant (GLA); AVT1; AVT2; AVT3, AVT4; AVT5; AVT6; AVT7; QS-21; aluminum-based ajuvant; saponin-based ajuvant; and TLR7 / 8 agonists.

[0039] In some embodiments, the pharmaceutically acceptable ajuvant is AVT1, AVT5, or AVT7.

[0040] In another aspect, the present disclosure provides a use of a pharmaceutical composition described in any aspect or embodiment of the present disclosure in the manufacture of a medicine for preventing or treating a disease associated with amyloid deposits of Aβ in the brain of a patient who requires prevention or treatment of a disease associated with amyloid deposits of Aβ in the brain.

[0041] In another aspect, the present disclosure provides a method for inducing an immune response to an Aβ peptide in a patient requiring induction of an immune response to an Aβ peptide, which comprises administering to said patient an immunologically effective dose of a pharmaceutical composition described in any aspect or embodiment of the present disclosure.

[0042] In some embodiments, the disease associated with amyloid deposits of Aβ in the brain is a disease selected from the group consisting of Alzheimer's disease (AD); cerebral amyloid angiopathy (CAA); inflammatory CAA; and cerebral amyloidoma. In certain embodiments, the disease associated with amyloid deposits of Aβ in the brain is AD.

[0043] The summary of the technology described above is non-limiting, and other features and benefits of the technology will become apparent from the following detailed description and claims. Brief explanation of the drawing

[0044] The above and other purposes, features, and advantages will become apparent from the following description of specific embodiments of the invention, as illustrated in the accompanying drawings, where similar reference characters throughout the drawings refer to the same parts. The drawings are not necessarily in scale, but are instead highlighted to illustrate the principles of various embodiments of the invention. Figure 1 shows Aβ conjugated to the CRM197 immunogenic carrier protein via a GMBS linker. pE3_9-G-C Shows the chemical structure of an immunogenic peptide. Sequence identification numbers 42 and 2 are disclosed in the order of appearance, respectively. FIGS. 2aa to 2af are Aβ pE3-9_G_C This is a multi-panel schematic diagram showing the synthesis of a tetramer. Sequence identification numbers 32 and 43 are disclosed in the order of appearance, respectively. FIGS. 2ba to 2bg are Aβ pE3-9_G_C This is a multi-panel schematic diagram showing the synthesis of an octamer. Sequence identification number: 35 is disclosed. Figure 3a shows the total anti-Aβ generated in non-human primates vaccinated with the pE3-9-CRM197 conjugate vaccine. pE3-42 This is a line graph showing antibodies. Figure 3b shows the total anti-Aβ generated in non-human primates vaccinated with the pE3-14-CRM197 conjugate vaccine. pE3-42 This is a line graph showing antibodies. Figures 4a to 4d show Aβ in vaccinated non-human primates. 1-42 - and Aβ pE3-42 This is a data plot showing the measurement of -specific CD4 and CD8 T cell responses. Figure 4a is Aβ 1-42 It shows the expression of cytokines by CD4 T cells after stimulation with a peptide. Figure 4b shows Aβ pE3-42 It shows the expression of cytokines by CD4 T cells after stimulation with a peptide. Figure 4c shows Aβ 1-42 It shows the expression of cytokines by CD8 T cells after stimulation with a peptide. Figure 4d shows Aβ pE3-42 Shows cytokine expression by CD8 T cells after peptide stimulation. The dotted line indicates the test-defined positive limit. IL4 = Interleukin 4, IL5 = Interleukin 5, IFNg = Interferon Gamma, IL2 = Interleukin 2, TNFa = Tumor Necrosis Factor Alpha. N = 5 individual animals per vaccine group. Figures 5a to 5d show Aβ in vaccinated mice. 1-42 - and Aβ pE3-42 This is a data plot showing the measurement of -specific CD4 and CD8 T cell responses. Figure 5a is Aβ 1-42 It shows the expression of cytokines by CD4 T cells after stimulation with a peptide. Figure 5b shows Aβ pE3-42 It shows the expression of cytokines by CD4 T cells after stimulation with a peptide. Figure 5c shows Aβ 1-42 It shows the expression of cytokines by CD8 T cells after stimulation with a peptide. Figure 5d shows Aβ pE3-42 Shows cytokine expression by CD8 T cells after peptide stimulation. The dotted line indicates the test-defined positive limit. IL4 = Interleukin 4, IL10 = Interleukin 10, IL5 = Interleukin 5, IFNg = Interferon Gamma, IL2 = Interleukin 2, TNFa = Tumor Necrosis Factor Alpha. N = 5 individual mice per vaccine group. Figure 6 is a bar graph showing the immunoreactivity of human cerebral cortex tissue from Alzheimer's disease patients stained with serum from mice vaccinated with pE3-9-C, pE3-9-G_C, pE3-10-C, pE3-11-C, pE3-12-C, pE3-13-C, pE3-14-C, pE3-15-C, pE3-15_PEG4_C, pE3-16-C, and pE3-17-C peptide vaccines at increasing dilutions. The amyloid plaque area (positive immunohistochemical staining) for the vaccinated serum is plotted as a percentage (y-axis) compared to different vaccines (x-axis) of serum percentage. Dilutions ranging from 1:500 to 1:40,500 were converted to serum percentage (x-axis). Figure 7 is a bar graph showing the phagocytic activity of microglia upon the addition of vaccinated serum, plotted as the number of phagocytic Aβ spots per cell (Y-axis) for each treatment group (X-axis) serially diluted (1:2) starting from 2% serum. Figure 8 is a plot of a nanotemper differential scanning fluorescence experiment demonstrating the intrinsic fluorescence of pE3-CRM conjugates containing various linkers when applied to increasing temperatures. Figure 9 is a melting temperature plot of a pE3-CRM conjugate containing various linkers. Figure 10 is a plot of dynamic light scattering experiments demonstrating the melting temperature of pE3-CRM197 conjugates containing various linkers. Figure 11 is a plot of the dynamic light scattering diameter analysis of pE3-CRM conjugates containing various linkers after the application of stirring stress. Figure 12 is a plot of a stirring-stress applied pE3-CRM conjugate containing various linkers that demonstrated stability as a function of fluorescence. Figure 13 shows a plot of an experiment demonstrating that pE3-9-CRM samples formulated with polysorbate 80 (PS80) exhibit reduced aggregation. Figure 14 shows a plot and results of a freeze-thaw experiment demonstrating that a sample formulated with 10% sucrose exhibits reduced aggregation. Figures 15a and 15b are data plots of a microglia phagocytosis assay to evaluate the functional activity of non-human primate (NHP) vaccine serum. The plots show phagocytic Aβ per cell plotted for each treatment group over time in time units (X-axis). pE3-42 Shows the number of spots (Y-axis). Figure 16 is a data plot of the microglia phagocytosis assay in HMC3 cells to evaluate the functional activity of NHP vaccine serum on day 42. Specific details for implementing the invention

[0045] The singular forms used herein refer to one or more than one (i.e., at least one) grammatical object. For example, "element" means one element or more than one element. Furthermore, the use of the term "containing" as well as other forms, such as "include," "includes," and "included," is not restrictive.

[0046] In quantitative terms, the term "about" as used herein refers to plus or minus 10% of the value it modifies (where the value is not subdividable, such as the number of molecules or amino acid residues, rounded to the nearest integer).

[0047] All ranges disclosed herein include the mentioned endpoints and can be independently combined (e.g., ranges of “50 mg to 500 mg” or “50-500 mg” include the endpoints, 50 mg and 500 mg, and any intermediate values). The endpoints and any values ​​of the ranges disclosed herein are not limited to exact ranges or values; they are sufficiently inaccurate to include values ​​that are approximations to these ranges and / or values.

[0048] As used herein, the term “comprising” may include embodiments that are “composed” and “essentially composed”. As used herein, the terms “comprising,” “comprising,” “having,” “having,” “may,” “containing,” and variations thereof are intended to be open-ended connecting phrases, terms, or words that require the presence of the named component / step and allow for the presence of other components / steps. However, such descriptions should also be interpreted as describing a composition or process that is “composed” and “essentially composed” of the listed components, which allows for the presence of only the named component or compound together with any permissible carrier or fluid, and excludes other components or compounds.

[0049] The present disclosure relates to a composition and a method for the treatment or prevention of a disease associated with amyloid deposits of Aβ in the brain of a patient requiring treatment or prevention of a disease associated with amyloid deposits of Aβ in the brain, e.g., Alzheimer's disease. Such a method is capable of inducing a beneficial immune response with Aβ 1-42 It involves administering a pharmaceutical composition comprising an immunogenic fragment of a peptide (i.e., "immunogenic peptide") in the form of an antibody against Aβ. Aβ 1-42 The amino acid sequence of the peptide is presented below.

[0050] Aβ 1-42 : DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (Sequence Identification Number: 1)

[0051] In one aspect, the present disclosure provides a pharmaceutical composition comprising an immunogenic peptide comprising adjacent amino acids of sequence identification number: 1, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 adjacent amino acids. In some embodiments, the immunogenic peptide comprises at least 6 adjacent amino acids of sequence identification number: 1. In other embodiments, the immunogenic peptide comprises at least 10 adjacent amino acids of sequence identification number: 1. In another embodiment, the immunogenic peptide is selected from the group consisting of sequence identification numbers: 2-13. In a specific embodiment, the immunogenic peptide is sequence identification number: 2.

[0052] Immunogenic peptide

[0053] In some embodiments, the immunogenic peptide includes gene products, naturally occurring peptides, synthetic peptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs thereof. The peptide may be a single molecule (i.e., a monomer) or a multimolecular complex (i.e., a multimer), such as a dimer, trimer, or tetramer. In some embodiments, the immunogenic peptide is a monomer. In other embodiments, the immunogenic peptide is a multimer.

[0054] Peptides may also include single-stranded or multi-stranded peptides and may be associated or linked to one another. Most commonly, disulfide linkages are found in multi-stranded peptides. The term "peptide" may also be applied to amino acid polymers in which at least one amino acid residue is an artificial chemical analog of a corresponding naturally occurring amino acid.

[0055] A "peptide variant" is a molecule whose amino acid sequence differs from that of a natural sequence or a reference sequence. An amino acid sequence variant may have substitutions, deletions, insertions, or any combination of two or three of the above at specific positions within the amino acid sequence compared to the natural sequence or the reference sequence. Typically, the variant has at least 50% identity with respect to the natural sequence or the reference sequence. In some embodiments, the variant shares at least 80% identity or at least 90% identity with the natural sequence or the reference sequence.

[0056] "An analogue" is intended to include peptide variants that differ by one or more amino acid changes, such as substitution, addition, or deletion of amino acid residues, which still retain one or more characteristics of the parent or starting peptide.

[0057] The present disclosure provides various types of peptide-based compositions, including variants and derivatives. These include, for example, substitutions, insertions, deletions, and covalent variants and derivatives. The term “derivative” is synonymous with the term “variant” and generally refers to a molecule that is “modified” and / or altered in any way relative to a reference molecule or starting molecule. In some embodiments, the immunogenic peptide comprises 1 to 5 modified amino acids. In certain embodiments, the immunogenic peptide comprises a pyroglutamate residue at amino acid positions 3 and / or 11 of SEQ ID NO: 1.

[0058] Peptides containing substitutions, insertions and / or additions, deletions and covalent modifications in relation to reference sequences, particularly the peptide sequences disclosed herein, are included within the scope of this disclosure. For example, amino acid residues located in the carboxyl and amino-terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted to provide a truncated sequence. Certain amino acids (e.g., C-terminal residues or N-terminal residues) may alternatively be deleted depending on the use of the sequence. In some embodiments, the immunogenic peptide lacks 1 to 10 amino acids at the N-terminus or 1 to 33 amino acids at the C-terminus of Sequence No. 1. In other embodiments, the immunogenic peptide lacks 1 to 10 amino acids at the N-terminus and 1 to 33 amino acids at the C-terminus of Sequence No. 1.

[0059] When referring to a peptide, a “substituted variant” is one in which at least one amino acid residue in the natural or starting sequence is removed and a different amino acid is inserted at the same position. Substitutions may be single when only one amino acid is substituted in the molecule, or multiple when two or more amino acids (e.g., three, four, or five) are substituted in the same molecule.

[0060] As used herein, the term “conservative amino acid substitution” refers to the substitution of an amino acid normally present in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue, such as isoleucine, valine, and leucine, with another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue with another, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue, such as lysine, arginine, or histidine, with another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid, with another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of non-polar (hydrophobic) amino acid residues, e.g. isoleucine, valine, leucine, alanine, and methionine, with polar (hydrophilic) residues, e.g. cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of polar residues with non-polar residues.

[0061] When referring to peptides, the term “domain” as used herein refers to a motif of a peptide having one or more identifiable structural or functional features or characteristics (e.g., binding ability, acting as a site for protein-protein interactions).

[0062] As recognized by a person skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the category of peptides of interest. For example, any protein fragment of a reference protein (meaning a peptide sequence that is at least one amino acid residue shorter than the reference peptide sequence but otherwise identical) is provided herein. In another example, a stretch of two or more adjacent amino acids that are 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the sequences described herein (in a specific embodiment, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42) Any protein comprising may be used in accordance with the present disclosure. In some embodiments, the peptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations as provided in or presented in any of the sequences mentioned herein.

[0063] The peptides of the present disclosure may share a certain degree of sequence similarity or identity with reference molecules (e.g., reference peptides), e.g., molecules described in the art (e.g., engineered or designed molecules or wild-type molecules). The term “identity,” as known in the art, refers to the relationship between the sequences of two or more peptides, as determined by comparing the sequences. In the art, identity also refers to the degree of sequence relatedness between two sequences, as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the equal percentage of matches between the smaller of the two or more sequences by gap alignment (if present), which is handled by a specific mathematical model or computer program (e.g., “algorithm”). The identity of the related peptides can be easily calculated by known methods. When applied to peptide sequences, "% identity" is defined as the percentage of candidate amino acids or residues in a nucleic acid sequence (amino acid residues or nucleic acid residues) that are identical to residues in the amino acid sequence or nucleic acid sequence of the second sequence after achieving maximum percentage identity by aligning the sequences and introducing gaps if necessary. Methods and computer programs for alignment are widely known in the relevant art. Identity depends on the calculation of percentage identity, but the value may differ due to gaps and penalties introduced in the calculation. Generally, variants of a specific peptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity with respect to a specific reference peptide, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such alignment tools include those of the BLAST suite (Stephen F.Altschul, et al., (1997)." Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147:195-197). A common global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48:443-453). More recently, a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, reported to generate global alignment of protein sequences faster than other optimal global alignment methods, including the Needleman-Bunsch algorithm. Other tools are described herein, specifically in the definition of "identity" below.

[0064] As used herein, the term “homology” refers to the overall relationship between polymer molecules, for example, between peptide molecules. Polymer molecules (e.g., peptide molecules) that share a threshold level of similarity or identity determined by the alignment of matching residues are named as homology. Homology is a qualitative term describing the relationship between molecules and may be based on quantitative similarity or identity. Similarity or identity is a quantitative term defining the degree of sequence matching between two compared sequences. In some embodiments, polymer molecules are considered “homology” of each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar. The term "homologous" must refer to a comparison between at least two sequences (peptide sequences).

[0065] Homology implies that the sequences being compared diverged in evolution from a common origin. The term "homologous" refers to a first amino acid sequence or nucleic acid sequence (e.g., a gene (DNA or RNA) or protein sequence) derived from a common ancestral sequence and associated with a second amino acid sequence or nucleic acid sequence. The term "homologous" may be applied to the relationship between genes and / or proteins separated by speciation events or between genes and / or proteins separated by gene duplication events. An "ortholog" is a gene (or protein) of a different species that evolved from a common ancestral gene (or protein) through speciation. Typically, orthologs retain the same function during evolution. A "paralog" is a gene (or protein) related by duplication within the genome. While orthologs retain the same function during evolution, paralogs evolve new functions, even if they are related to the original.

[0066] The term "identity" refers to the overall correlation between polymer molecules, for example, between peptide molecules. The calculation of the percentage identity of two peptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (for example, a gap may be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). In certain embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The comparison of sequences and the determination of the percentage identity between two sequences can be achieved using a mathematical algorithm. For example, percentage identity between two nucleic acid sequences is [Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.It can be determined using methods such as those described in [M Stockton Press, New York, 1991]; each of these is incorporated herein by reference. For example, the percentage identity between two nucleic acid sequences can be determined using the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4 using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) incorporated into the ALIGN program (version 2.0). Alternatively, the percentage identity between two nucleic acid sequences can be determined using the NWSgapdna.CMP matrix using the GAP program within the GCG software package. Methods commonly used to determine the percentage identity between sequences include, but are not limited to, those disclosed in the literature incorporated herein by reference [Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988)]. Techniques for determining identity are systematized in publicly available computer programs. Exemplary computer software for determining homology between two sequences includes, but is not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, SF et al., J. Molec. Biol., 215, 403 (1990)).

[0067] When referring to peptides, the term "site" as used herein is used synonymously with "residue" and "amino acid side chain" when relating to amino acid-based embodiments. A site indicates a peptide or a location within a peptide that may be modified, manipulated, altered, derivatized, or changed within a peptide-based molecule.

[0068] When referring to peptides, the terms "terminals" or "terminal" as used herein refer to the end of the peptide. Such end is not limited to the initial or final site of the peptide but may include additional amino acids in the terminal region. Peptide-based molecules may be characterized by having both an N-terminus (terminated by an amino acid having a free amino group (NH2)) and a C-terminus (terminated by an amino acid having a free carboxyl group (COOH)). Proteins consist of multiple peptide chains held together in some cases by disulfide bonds or non-covalent forces (multimers, oligomers). These proteins have multiple N- and C-terminals.

[0069] Alternatively, the ends of the peptide may be modified to start or end with a non-peptide-based moiety, such as a carrier protein, depending on the case. Before conjugation with the carrier protein, the immunogenic peptide may be chemically activated using any activation or coupling chemistry known in the relevant art so that the peptide can react with the carrier protein to form a conjugate molecule. As used herein, the term "activated peptide" refers to a peptide that has been chemically modified to enable conjugation to a linker or an immunogenic carrier protein.

[0070] Immunogenic carrier protein

[0071] The immunogenic peptide disclosed herein may be conjugated to a carrier protein to enhance immunogenicity in human subjects. In some embodiments of the present invention, CRM197 is used as a carrier protein. CRM197 is a non-toxic variant of diphtheria toxin (DT). The CRM197 carrier protein is a mutant form of DT that has been made non-toxic by a single amino acid substitution at residue 52 in fragment A. In one embodiment, the CRM197 carrier protein is isolated from a culture of Corynebacterium diphtheria strain C7 (β197) grown in a casamino acid and yeast extract-based medium. In another embodiment, CRM197 is recombinantly prepared according to the method described in U.S. Patent No. 5,614,382. Typically, CRM197 is purified by a combination of ultrafiltration, ammonium sulfate precipitation, and ion-exchange chromatography. In some embodiments, CRM197 is manufactured from Pseudomonas fluorescens using Pfenex Expression Technology™ (Pfenex Inc., San Diego, California).

[0072] Other suitable carrier proteins include additional inactivated bacterial toxins, such as DT, diphtheria toxoid fragment B (DTFB), TT (tetanus toxoid) or fragment C of TT, pertussis toxoid, cholera toxoid (e.g., as described in International Patent Application Publication No. WO 2004 / 083251), E. coli LT (heat-unstable enterotoxin), E. coli ST (heat-stable enterotoxin), and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane proteins, e.g., outer membrane complex c (OMPC), porin, transferrin-binding protein, Streptococcus pneumoniae surface protein A (PspA; see International Patent Application Publication No. WO 02 / 091998), Streptococcus pneumoniae adhesin protein (PsaA), C5a peptidase from Group A or Group B Streptococcus, or Haemophilus influenzae protein D, Streptococcus pneumomorisin (Kuo et al., 1995, Infect Immun 63; 2706-13), e.g., ply decoded in some way, e.g., dPLY-GMBS (see International Patent Application Publication No. WO 04 / 081515) or dPLY-formol, PhtX, e.g., fusions of PhtA, PhtB, PhtD, PhtE, and Pht proteins, e.g., PhtDE fusions, PhtBE fusions (International Patent Application Publication No. WO (See 01 / 98334 and WO 03 / 54007) other proteins may also be used. Purified protein derivatives of other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tuberculin (PPD), PorB (N.Those from *Meningitidis*), PD (protein D of *Haemophilus influenzae*; see, e.g., European Patent No. EP 0 594 610 B) or their immunological functional equivalents, synthetic peptides (see European Patent Nos. EP0378881 and EP0427347), heat shock proteins (see International Patent Application Publication Nos. WO 93 / 17712 and WO 94 / 03208), pertussis proteins (see International Patent Application Publication No. WO 98 / 58668 and European Patent No. EP0471177), cytokines, lymphokines, growth factors, or hormones (see International Patent Application Publication No. WO 91 / 01146), artificial proteins comprising multiple human CD4+ T cell epitopes from antigens derived from various pathogens (see [Falugi et al., 2001, Eur J Immunol 31:3816-3824]), e.g., N19 proteins (See reference [Baraldoi et al., 2004, Infect Immun 72:4884-7]), iron absorption protein (see International Patent Application Publication No. WO 01 / 72337), toxin A or B of C. difficile (see International Patent Publication No. WO 00 / 61761) and flagellin (see reference [Ben-Yedidia et al., 1998, Immunol Lett 64:9]) can also be used as carrier proteins.

[0073] Other DT mutants, e.g., CRM176, CRM228, CRM45 (Uchida et al., 1973, J Biol Chem 218:3838-3844); CRM9, CRM45, CRM102, CRM103, and CRM107 and other mutants described in the literature [Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992]; deletion of Glu-148 or mutation to Asp, Gln, or Ser and / or deletion of Ala 158 or mutation to Gly and other mutants disclosed in U.S. Patent No. 4,709,017 or U.S. Patent No. 4,950,740; At least one mutation of at least one residue of Lys 516, Lys 526, Phe 530 and / or Lys 534 and other mutations disclosed in U.S. Patent No. 5,917,017 or U.S. Patent No. 6,455,673; or a fragment disclosed in U.S. Patent No. 5,843,711 may also be used as a carrier protein.

[0074] In some embodiments, the immunogenic carrier protein is selected from the group consisting of CRM197; diphtheria toxin fragment B (DTFB); DTFB C8; diphtheria toxoid (DT); tetanus toxoid (TT); fragment C of TT; pertussis toxoid; cholera toxoid; E. coli LT; E. coli ST; Neisseria meningitidis outer membrane protein complex (OMPC); exotoxin A from Pseudomonas aeruginosa; mariculture keyhole limpet hemocyanin (mcKLH); and virus-like particles derived from bacteriophage AP205 coat protein.

[0075] In certain embodiments, the immunogenic peptide comprises sequence identification number: 2, and the immunogenic carrier protein is CRM197. In some embodiments, the immunogenic carrier protein is conjugated at the N-terminus or C-terminus of the immunogenic peptide. In certain embodiments, the immunogenic carrier protein is conjugated at the C-terminus of the immunogenic peptide.

[0076] In some embodiments, the immunogenic carrier protein is conjugated to the immunogenic peptide by a linker. In some embodiments, the linker is selected from the group consisting of N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS); polyethylene glycol (PEG); aminohexanoic acid (Ahx); sulfhydryl-reactive crosslinker; maleimide (MA) linker; oligopeptide; dendrimer; cyclodextrin; and glycine-rich peptide. In some embodiments, the pharmaceutical composition of the present disclosure further comprises a spacer comprising 1 to 10 amino acids adjacent to the linker. In certain embodiments, the immunogenic peptide is connected to CRM197 via a GMBS linker, and the composition further comprises a glycine-cysteine ​​spacer between the immunogenic peptide and the GMBS linker.

[0077] After conjugation, the peptide-conjugate may be purified from the unconjugated material by various techniques known to those skilled in the art. These techniques include dialysis, concentration / dialysis filtration, tangential flow filtration, ultrafiltration, precipitation / elution, column chromatography (ion exchange chromatography, multimode ion exchange chromatography, DEAE, or hydrophobic interaction chromatography), and deep filtration. For example, refer to U.S. Patent No. 6,146,902. In one embodiment, the sugar conjugate is purified by dialysis filtration, ion exchange chromatography, or size exclusion chromatography.

[0078] Azuvant

[0079] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable ajuvant. As defined herein, an "ajuvant" is a substance that serves to enhance the immunogenicity of the immunogenic composition of the present invention. An immunoajuvant can enhance an immune response to an antigen that is weakly immunogenic when administered alone, e.g., does not or weakly induces antibody titers or cell-mediated immune responses, and / or can increase antibody titers against the antigen, and / or reduce the dose of the antigen effective in achieving an immune response in an individual. Accordingly, ajuvants are often provided to boost an immune response, which is widely known to those skilled in the art.

[0080] Pharmaceutically acceptable ajuvants for enhancing the efficacy of the disclosed immunogenic peptide composition include, but are not limited to, those described in Example 7 and below.

[0081] Aluminum salts (alum), e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc. Aluminum salt azurants may be alum-precipitated vaccines or alum-adsorbed vaccines. Aluminum-salt azurants are widely known in the relevant art and are described, for example, in the literature [Harlow, E. and D. Lane (1988; Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992; Aluminum salts. Research in Immunology 143:489-493)]. Aluminum salts include, but are not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, Alhydrogel®, Superphos, Amphogel®, aluminum (III) hydroxyphosphate, aluminum hydroxyphosphate (aluminum phosphate ajuvant (APA)), amorphous alumina, trihydrated alumina, or trihydroxyaluminum.

[0082] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is prepared by precipitating aluminum hydroxyphosphate by blending aluminum chloride and sodium phosphate in a 1:1 volume ratio. After the blending process, the material is reduced in size using a high-shear mixer to achieve a monodisperse particle size distribution. Subsequently, the product is dialyzed through physiological saline and steam sterilized.

[0083] In some embodiments, commercially available Al(OH)3 (e.g., Alhydrogel® or Superforce from Denmark / Accurate Chemical and Scientific Co., Westbury, New York) is used to adsorb proteins. In another embodiment, the adsorption of proteins depends on the pI (isoelectric pH) of the protein and the pH of the medium. Proteins with a lower pI are adsorbed to strongly positively charged aluminum ions more than proteins with a higher pI. Aluminum salts can establish a deposit of antigens that are slowly released over a period of 2–3 weeks, accompany non-specific activation of macrophages and complement activation, and / or stimulate innate immune mechanisms (possibly through stimulation by uric acid). For example, see the literature [Lambrecht et al., 2009, Curr Opin Immunol 21:23].

[0084] Oil-in-water emulsion formulations (with or without other specific immunostimulators, e.g., muramyl peptide or bacterial cell wall components), e.g., (a) MF59 (International Patent Application Publication No. WO 90 / 14837) containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (with optionally varying amounts of MTP-PE), formulated into submicrometer particles using a microfluidizer, e.g., a Model 110Y microfluidizer (Microfluidics, Newton, Massachusetts); (b) SAF containing 10% squalene, 0.4% Tween 80, 5% Pluronic-blocked polymer L121, and thr-MDP, which is microfluidized into a submicrometer emulsion or vortexed to form an emulsion of larger particle sizes; (c) 2% squalene, Ribi™ Azuvant System (RAS) (Corixa, Hamilton, Montana) containing 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of 3-O-deacylated monophosphorylipid A (MPL™), trehalose dimicholate (TDM), and cell wall skeleton (CWS) as described in U.S. Patent No. 4,912,094, preferably MPL+CWS (Detox™); and (d) Montanid ISA. Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nomuramyl-L-alanine-2-(1',2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE). In certain embodiments, AVT7 is an exemplary oil-in-water emulsion ajuvant comprising squalene, SPAN-85, and PS-20 components stabilized in an aqueous buffer at pH 5.8.

[0085] Saponin ajuvants, such as Quill A or Stimulon™ QS-21 (Antigenics, Framingham, Massachusetts) (see, for example, U.S. Patent No. 5,057,540) or particles derived therefrom, such as ISCOM (an immunostimulatory complex formed by a combination of cholesterol, saponin, phospholipid, and amphiphilic protein) and Iscomatrix® (which has essentially the same structure as ISCOM but lacks protein) may be used.

[0086] A bacterial lipopolysaccharide, synthetic lipid A analog, such as an aminoalkyl glucosamine phosphate compound (AGP) or its derivatives or analogs, available from Corixa and described in U.S. Patent No. 6,113,918; one such AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl 2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-bD-glucopyranoside, which is also known as 529 (previously known as RC529), which is formulated as an aqueous form or a stable emulsion.

[0087] Synthetic polynucleotides, e.g., oligonucleotides containing CpG motif(s) (U.S. Patent No. 6,207,646).

[0088] A stable nanoemulsion or SNE refers to an emulsifier and / or solubilizer and / or surfactant and / or lipid preparation having augant properties in a conjugate vaccine. In exemplary embodiments, SNE refers to an SNE augant preparation comprising 1) sorbitan trioleate (SPAN-85); 2) polysorbate-20 (PS-20); 3) squalene; and optionally 4) cationic lipids, as described in WO2022169789A1, the full contents of which are incorporated herein. In certain embodiments, SNE comprises 6 μg / mL - 14 mg / mL SPAN-85, 6 μg / mL - 14 mg / mL PS-20 or PS-80, and 60 μg / mL - 34 mg / mL squalene. In some embodiments, SNE further comprises 30 μg / mL - 2.4 mg / mL of cationic lipid. In some embodiments, the cationic lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine.

[0089] Cytokines, e.g., interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), co-stimulatory molecules B7-1 and B7-2, etc.

[0090] CpG-containing nucleotide sequences, e.g., CpG-containing oligonucleotides, particularly CpG-containing oligodeoxynucleotides (CpG ODN). In another embodiment, the ajuvant is ODN 1826, which is available from Coley Pharmaceutical Group. "CpG-containing nucleotide," "CpG-containing oligonucleotide," "CpG oligonucleotide," and similar terms refer to nucleotide molecules of length 6 to 50 nucleotides containing a nonmethylated CpG moiety. For example, refer to the literature [Wang et al., 2003, Vaccine 21:4297]. In another embodiment, definitions of terms accepted in any other relevant art are intended. CpG-containing oligonucleotides include any synthetic nucleoside linkages, modified bases, and / or modified sugars, which are modified oligonucleotides. Methods for using CpG oligonucleotides are widely known in the relevant technical field and are described, for example, in the literature [Sur et al., 1999, J Immunol. 162:6284-93; Verthelyi, 2006, Methods Mol Med. 127:139-58; and Yasuda et al., 2006, Crit Rev Ther Drug Carrier Syst. 23:89-110].

[0091] Complement, for example, a trimer of complement component C3d.

[0092] Liquid nanoparticle (LNP) ajuvants are a class of ajuvants containing cationic lipids / cholesterol / DSPC / PEG-DMG as described in WO2015 / 130584A2, the full contents of which are incorporated herein. Exemplary LNP ajuvants may contain cationic lipids / cholesterol / DSPC / PEG-DMG in approximately the following molar ratios: 59 / 30 / 10 / 1; 58 / 30 / 10 / 2; 43 / 41 / 15 / 1; 42 / 41 / 15 / 2; 40 / 48 / 10 / 2; 39 / 41 / 19 / 1; 38 / 41 / 19 / 2; 34 / 41 / 24 / 1; and 33 / 41 / 24 / 2. In certain embodiments, AVT1 is an exemplary LNP ajuvant containing lipid, cholesterol, DSPC, and PEG-DMG components.

[0093] In one embodiment, the ajuvant is a mixture of two, three, or more of the ajuvants, for example, SBAS2 (an oil-in-water emulsion also containing 3-deacylated monophosphoryl lipid A and QS21).

[0094] Prevention and treatment methods

[0095] Compositions (e.g., pharmaceutical compositions including immunogenic peptides, conjugates, and vaccines), methods, kits, and reagents for the prevention, treatment, or diagnosis of neurodegenerative diseases, such as Alzheimer's disease, associated with amyloid deposits of Aβ in the brain of a patient in humans are provided herein. In some embodiments, the method of the present disclosure comprises administering an effective dose of a pharmaceutical composition, e.g., a vaccine, comprising an immunogenic peptide of at least six adjacent amino acids of SEQ ID NO: 1 and an immunogenic carrier protein, to a patient in need thereof.

[0096] In some embodiments, the immunogenic peptide is missing 1 to 10 amino acids at the N-terminus or 1 to 33 amino acids at the C-terminus of sequence identification number: 1. In other embodiments, the immunogenic peptide is missing 1 to 10 amino acids at the N-terminus and 1 to 33 amino acids at the C-terminus of sequence identification number: 1.

[0097] In some embodiments, the immunogenic peptide comprises at least 10 adjacent amino acids of sequence identification number: 1.

[0098] In some embodiments, the immunogenic peptide comprises 1 to 5 modified amino acids. In certain embodiments, the immunogenic peptide comprises a pyroglutamate residue at amino acid positions 3 and / or 11 of SEQ ID NO: 1.

[0099] In some embodiments, the immunogenic peptide is selected from the group consisting of sequence identification numbers: 2-13.

[0100] In some embodiments, the immunogenic carrier protein is selected from the group consisting of CRM197; diphtheria toxin fragment B (DTFB); DTFB C8; diphtheria toxoid (DT); tetanus toxoid (TT); fragment C of TT; pertussis toxoid; cholera toxoid; E. coli LT; E. coli ST; Neisseria meningitidis outer membrane protein complex (OMPC); exotoxin A from Pseudomonas aeruginosa; methylculture keyhole limpet hemocyanin (mcKLH); and bacteriophage AP205 coat protein.

[0101] In some embodiments, the immunogenic carrier protein is conjugated at the N-terminus or C-terminus of the immunogenic peptide. In certain embodiments, the immunogenic carrier protein is conjugated at the C-terminus of the immunogenic peptide.

[0102] In some embodiments, the immunogenic carrier protein is conjugated to the immunogenic peptide by a linker. In some embodiments, the linker is selected from the group consisting of N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS); polyethylene glycol (PEG); aminohexanoic acid (Ahx); sulfhydryl-reactive crosslinker; maleimide (MA) linker; oligopeptide; dendrimer; cyclodextrin; and glycine-rich peptide.

[0103] In some embodiments, the pharmaceutical composition further comprises a spacer comprising 1 to 10 amino acids adjacent to the linker.

[0104] In a specific embodiment, the immunogenic peptide comprises sequence identification number: 2, and the immunogenic carrier protein is CRM197.

[0105] In another specific embodiment, the immunogenic peptide is linked to CRM197 via a GMBS linker, and the composition further comprises a glycine-cysteine ​​spacer between the immunogenic peptide and the GMBS linker.

[0106] In some embodiments, the immunogenic peptide is a monomer. In some embodiments, the immunogenic peptide is a polymer.

[0107] As used herein, the terms “treat” or “treating” mean administering, either internally or externally, a composition containing a therapeutic moiety, such as any of the immunogenic peptide, conjugate, or vaccine of the present invention, to a subject or patient having one or more disease symptoms in which the therapeutic moiety has therapeutic activity, or to a subject or patient diagnosed with the disease. In specific embodiments, the immunogenic peptide, conjugate, or vaccine may be administered topically, subcutaneously, intramuscularly, intradermally, intravenously, or systemically. Typically, the moiety is administered in an amount effective to (i) induce regression of one or more disease symptom(s) to any clinically measurable degree in the treated subject or population, or to alleviate them by inhibiting their progression, or (ii) to inhibit or reduce the severity of the disease in the individual. The amount of therapeutic moiety effective in alleviating any specific disease symptoms in an individual and / or, in specific embodiments, suppressing or reducing the severity of the disease, including neurodegenerative diseases, may depend on factors such as the individual's impairment or disease status, age and / or body weight, and the therapeutic moiety's ability to elicit a desired response in the individual. Whether one or more disease symptoms have been alleviated or the severity of the disease has been suppressed or reduced may be evaluated by any clinical measurement typically used by a physician or other skilled healthcare provider to assess the symptom(s) or the severity or progression of the disease. Treatment using immunogenic peptides, conjugates, or vaccines may also be combined with other interventions (in specific embodiments, antibodies, nucleic acids, additional vaccines, and small molecule compounds) to treat other symptoms or diseases.

[0108] As used herein, the terms “subject, individual” or “patient in need of him / her” refer to a human or animal subject, individual, or patient who is a subject of treatment and is diagnosed with or exhibiting symptoms of a disease associated with amyloid deposits of Aβ in the patient’s brain, such as Alzheimer’s disease.

[0109] vaccine

[0110] The vaccine of the present disclosure may be administered to healthy individuals as part of an active immunization regimen or early after diagnosis of a neurodegenerative disease, either prophylactically or therapeutically. In some embodiments, the amount of the vaccine of the present disclosure provided to a cell, tissue, or subject may be an amount effective for immune prevention or treatment.

[0111] The vaccine may be administered together with other prophylactic or therapeutic compounds. By non-limiting example, the prophylactic or therapeutic compound may be an ajuvant or a booster. When referring to a prophylactic composition, e.g., a vaccine, the term "booster" as used herein refers to an additional administration of the prophylactic (vaccine) composition. The booster (or booster vaccine) may be provided after an earlier administration of the prophylactic composition. The administration time between the initial administration of the prophylactic composition and the booster is 1 min, 2 mins, 3 mins, 4 mins, 5 mins, 6 mins, 7 mins, 8 mins, 9 mins, 10 mins, 15 mins, 20 mins, 25 mins, 30 mins, 35 mins, 40 mins, 45 mins, 50 mins, 55 mins, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, It may be 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or exceed 99 years, but is not limited thereto. In some embodiments, the time between the initial administration of the preventive composition and the booster may be 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year, but is not limited thereto.

[0112] In some embodiments, the vaccine may be administered orally, intramuscularly, intradermally, or nasally, similar to the administration of an inactivated vaccine known in the art. In some embodiments, the vaccine is administered intramuscularly.

[0113] Vaccines may be used in various settings depending on the prevalence of underlying diseases, family history, or the degree or level of unmet medical needs. As a non-limiting example, vaccines may be used to treat and / or prevent a class of neurodegenerative diseases associated with amyloid deposits of Aβ in the patient's brain. Vaccines possess superior characteristics in that they generate much higher antibody titers and induce an early response compared to commercially available non-immunogenic compositions.

[0114] A pharmaceutical composition comprising a vaccine combined with one or more pharmaceutically acceptable excipients is provided herein.

[0115] The vaccine may be formulated or administered alone or in combination with one or more other components. For example, the vaccine (vaccine composition) may include, but is not limited to, other components that include a pharmaceutically acceptable ajuvant. In some embodiments, the pharmaceutically acceptable ajuvant is selected from the group consisting of glucopyranosol lipid ajuvant (GLA); AVT1; AVT2; AVT3, AVT4; AVT5; AVT6; AVT7; QS-21; aluminum-based ajuvants; saponin-based ajuvants; and TLR7 / 8 agonists.

[0116] In some embodiments, the vaccine does not contain ajuvant (this is the absence of ajuvant).

[0117] The vaccine may be formulated or administered in combination with one or more pharmaceutically acceptable adjuvants. In some embodiments, the vaccine composition comprises at least one additional active substance, e.g., a therapeutic-active substance, a prophylactic-active substance, or a combination of both. The vaccine composition may be sterile, pyrogen-free, or both sterile and pyrogen-free. General considerations regarding the formulation and / or manufacture of pharmaceutical agents, such as vaccine compositions, can be found, for example, in the literature [Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005] (the full text of which is incorporated herein by reference). In some embodiments, the vaccine is administered to a human, e.g., a human patient or subject.

[0118] The formulations of the vaccine compositions described herein may be prepared by any method known in the pharmacological field or to be subsequently developed. Generally, such preparation methods include the step of associating an active ingredient (e.g., an immunogenic peptide conjugate) with an excipient and / or one or more other auxiliary ingredients, and then, if necessary or preferred, dividing, molding, and / or packaging the product into desired single- or multiple-dose units.

[0119] The relative amounts of the active ingredient, pharmaceutically acceptable excipients and / or any additional ingredients in the pharmaceutical composition according to the present disclosure will vary depending on the identity, size and / or condition of the subject being treated and additionally depending on the route by which the composition is administered. For example, the composition may contain 0.1% to 100%, e.g. 0.5% to 50%, 1-30%, 5-80%, or at least 80% (w / w) of the active ingredient.

[0120] Vaccine administration method

[0121] Vaccines may be administered by any route that yields a therapeutically effective result. These include, but are not limited to, oral, intradermal, intramuscular, intranasal, and / or subcutaneous administration. The present disclosure provides a method comprising administering a vaccine to a subject who requires it. The exact amount required will vary from subject to subject depending on the subject's species, age and general condition, severity of the disease, specific composition, mode of administration, mode of action, etc. Vaccine compositions are typically formulated in the form of dosing units for ease of administration and uniformity of dosage. However, it will be understood that the total daily dose of the vaccine composition may be determined by the attending physician within the scope of reasonable medical judgment. Specific therapeutically effective, prophylactically effective, or appropriate imaging dose levels for any specific patient are determined by the disorder to be treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and elimination rate of the specific compound used; the duration of treatment; and drugs used in combination with or simultaneously with the specific vaccine used. It will be influenced by various factors, including other factors widely known in the field of medical technology.

[0122] In some embodiments, the vaccine composition may be administered at a dosage level sufficient to obtain the desired therapeutic, diagnostic, preventive, or imaging effect (e.g., see the range of unit doses described in International Publication No. WO2013078199, the full contents of which are incorporated herein by reference). The desired dosage may be delivered three times a day, twice a day, once a day, every other day, every three days, every four days, every five days, weekly, every two weeks, every three weeks, every four weeks, every two months, every three months, every four months, every five months, every six months, etc. In some embodiments, the desired dosage may be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations). When multiple administrations are used, a split-dose regimen as described herein may be used.

[0123] The vaccine pharmaceutical compositions described herein may be formulated into the administration forms described herein, such as oral, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, intranasal, and subcutaneous).

[0124] Vaccine preparations and methods of use

[0125] Some aspects of the present disclosure provide a vaccine formulation, wherein the vaccine is formulated at an effective dose to induce an antigen-specific immune response in a subject (e.g., the production of antibodies specific to an Aβ antigen peptide). An "effective dose" is a dose of the vaccine effective in inducing an antigen-specific immune response. Additionally, a method for inducing an antigen-specific immune response in a subject is provided herein.

[0126] In some embodiments, the antigen-specific immune response is characterized by measuring the titer of anti-Aβ antigen peptide antibodies produced in subjects administered a vaccine as provided herein. The antibody titer is a measure of the amount of antibodies in the subject that are, for example, specific to a particular antigen (e.g., Aβ antigen peptide) or an epitope of the antigen. The antibody titer is typically expressed as the reciprocal of the maximum dilution that yields a positive result. For example, the enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers.

[0127] In some embodiments, antibody titers are used to assess whether a subject has a neurodegenerative disease or to determine whether immunization is required. In some embodiments, antibody titers are used to determine the intensity of an autoimmune response, to determine whether booster immunization is required, to determine whether a previous vaccine was effective, and to confirm the onset or progression of the disease. According to the present disclosure, antibody titers may be used to determine the intensity of an immune response induced in a subject by a vaccine.

[0128] The summary of the technology described above is non-limiting, and other features and benefits of the technology will become apparent from the following detailed description and claims.

[0129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the relevant technical field. In general, the nomenclature and laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry used herein are widely known and commonly used in the relevant technical field.

[0130] Examples

[0131] The following examples are intended to be exemplary and should not be interpreted as further limiting. The drawings and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0132] The abbreviations used in the following examples may include, but are not limited to, those presented in Table 1 below.

[0133] Table 1. Abbreviations

[0134]

[0135]

[0136] Example 1: Synthesis of AB Peptide

[0137] Aβ pE3 To evaluate the immunogenicity of peptide antigens, natural Aβ pE3 Synthetic peptide sequences of various lengths were prepared from the sequence. Aβ pE3-9 to Aβ pE3-17 Twelve peptide sequences were synthesized and are presented in Table 2 below. The peptide sequences can also be linked together to form multimeric peptides as described in Example 2. Each peptide or multimeric peptide contained a linker and a chemically reactive group at the C-terminus to enable chemical conjugation to a carrier protein or particle. The linker and the chemically reactive group may vary depending on the peptide sequence and the carrier protein or particle used.

[0138] Aβ peptide synthesis was completed in three steps unless otherwise indicated. The following protected natural amino acids were used: Fmoc-Arg(Pbf)-OH; Fmoc-Asp(OMpe)-OH, Fmoc-Cys(Trt)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-Glu(OtBu)-OH; Fmoc-His(Trt)-OH; Fmoc-Phe-OH; Fmoc-Ser(t-Bu)-OH; Fmoc-Tyr(t-Bu)-OH; and Fmoc-Val-OH.

[0139] Step 1: Synthesis. Peptides were synthesized on a linked amide resin (0.25 mmol, 0.35-0.58 mmol / g) using Fmoc / t-Bu chemistry. Amino acids were sequentially incorporated into the resin via single or double coupling cycles. Synthesis was performed manually or automatically as described in Protocol A and Protocol B, respectively.

[0140] For Protocol A, the linked amide MBHA resin was loaded into a glass sintered vessel fitted with a frit. The reaction was carried out at room temperature on an orbital shaker. Typical deprotection and coupling reaction conditions were as follows. For deprotection, the reaction mixture was allowed to react in 20% (v / v) piperidine for 20 minutes. The mixture was filtered, and the peptidyl resin was washed with DMF (5 x 15 mL), DCM (5 x 15 mL), and DMF (5 x 15 mL). For coupling, a pre-activated DMF solution of 3 equivalents of amino acid, 3 equivalents of DIC, and 3 equivalents of HOBt, or a DMF solution of 3 equivalents of HATU, 3 equivalents of HOBt, and 6 equivalents of DIPEA was added to the resin, and the mixture was allowed to react at room temperature for 3 hours. After the Kaiser test indicated the completion of the reaction, the mixture was filtered, and the peptidyl resin was washed with DMF (5x15 mL), DCM (5x15 mL), and DMF (5x15 mL).

[0141] For Protocol B, the linked amide MBHA resin was loaded onto CEM’s Liberty Blue microwave-assisted synthesizer (CEM Corporation (CEM Corp), Matthews, North Carolina, USA). Typical deprotection and coupling reaction conditions were as follows. For deprotection, the reaction mixture was reacted in 20% (v / v) piperidine in DMF plus 0.1 M HOBt (25 seconds at 75°C; followed by 65 seconds at 90°C), followed by 3-4 cycles of DMF washing. For each coupling cycle, 4 equivalents of amino acid in 5 ml of 0.2 M DMF stock solution were delivered to the resin, followed by the addition of 4 equivalents of DIC in 1 ml of 1 M DMF stock solution and 4 equivalents of Oxima in 1 ml of 1 M DMF stock solution. All residues except His were reacted at 90°C for 2 minutes, followed by 3-4 cycles of DMF washing. His was reacted at 50°C for 10 minutes, and then 3-4 cycles of DMF washing were performed.

[0142] Step 2: Cleaving. The peptidyl resins were washed with DMF and DCM (3 x 15 ml), respectively, followed by MeOH (15 ml) and diethyl ether (2 x 15 ml). Finally, the resins were dried overnight under vacuum. Once dried, the resins were treated with 10 ml of a reagent cocktail (v / v) (95% TFA, 2.5% TIPS, 2.5% DODT) at room temperature for 3 hours. The cleavage mixture was collected by filtration, and the resins were washed with TFA. The combined filtrate containing the peptides was precipitated with a sufficient amount of cold (0°C) diethyl ether (approximately 9 x filtrate volume). The precipitated peptides were centrifuged (4000 rpm), and the supernatant was removed. Fresh diethyl ether was added to the peptides, and the mixture was re-centrifuged. This process was repeated three times. Subsequently, the precipitated peptides were dried overnight under high vacuum to obtain a crude product as a white solid. The quality and identity of the target peptide were confirmed using HPLC and LCMS analysis. The total crude weight obtained was 0.35 g.

[0143] Step 3: Purification. The crude peptide was dissolved in water and MeCN and purified using a Shimadzu Prominence LC-20AP (Shimadzu Corp., Japan) equipped with an SPD-M20A diode array detector. The HPLC conditions were as follows: Column: Phenomenex Luna C18 5u 100A 250*21.2 mm; Buffer A: 0.01% TFA in H2O; Buffer B: 100% MeCN; Flow rate: 15 ml / min; Gradient: 2-25% within 30 minutes. The HPLC fraction containing the pure peptide product was pooled and freeze-dried to obtain the final peptide product.

[0144] Table 2. Pyroglutamate-modified Aβ peptide

[0145]

[0146] Example 2: Synthesis of Multimeric AB Peptide

[0147] The design of this antigen aimed to mimic pathogenic amyloid beta plaques to elicit a target-specific immune response. Antigen Aβ peptides, e.g., pE3-X, can be further modified into multimers to better capture the structural characteristics of amyloid beta plaques, which could potentially improve the immunogenicity and specificity of the vaccine. These multimer formulations can be achieved through chemical synthesis by crosslinking Aβ peptide monomers using linkers, such as PEG, oligopeptides, dendrimers, cyclodextrins, etc.

[0148] THPTA (tris(3-hydroxypropyltriazolylmethyl)amine, 18.6 mg, 42.8 μM), copper sulfate (1.37 mg, 8.6 μM), and a stirring bar were added to a 5 mL glass vial containing 450 μL of water. After stirring the reaction mixture at room temperature for 2 minutes, sodium ascorbate (42.4 mg, 214 μM) was added, followed by Ac-SH-alkyne (18.6 mg, 64 μM, prepared as a solution in 250 μL of acetonitrile). After stirring for 5 minutes, azido-bis-amine (30 mg, 42.8 μM) was added to the water-acetonitrile reaction mixture. After a 1-hour reaction at room temperature, all starting materials were consumed and the product Ac-SH-bis-amine was formed, which was confirmed by LC-MS. The mixture was subjected to column chromatography purification using a combi-flash system (C18 reverse phase, 0–60% acetonitrile over 10 min, RT= 8 min). The collected product fractions were combined and freeze-dried overnight. A colorless oil-like product was obtained (yield= 10%). Calculated [M+H]+= 991.58; observed [M+H]+= 991.26.

[0149] NHS-bis-mal (2 mg, 2 μM), Ac-SH-bis-amine (1 mg, 1 μM), and 120 μL of 50 mM sodium phosphate buffer (pH 8) were added to a 0.6 mL microtube. The reaction mixture was incubated overnight at room temperature with rotation. Subsequently, pE3-9 peptide (4 mg, 4 μM) was added to the reaction mixture, followed by 4 hours of incubation at room temperature with rotation. The formation of the target product was confirmed by LC-MS. Product purification was performed using a 3K MWCO (molecular weight cutoff) column and centrifugation. The solvent was then removed by lyophilization to obtain the product as a pale pink solid. Calculated [M+4H] 4+ = 1677.75; Observation [M+4H] 4+ = 1677.46.

[0150] For example, examples of Aβ monomeric peptides that can be conjugated to a carrier protein as monomers or polymers are listed in Table 3 below. A specific pE 3-9_GC peptide (Fig. 1) tetramerized using a PEG linker is presented in Figs. 2aa to 2af. This peptide (Sequence Identification No.: 32) was treated to generate free thiol groups immediately prior to conjugation. The peptide (5.3 mg) was dissolved in 100 μl of DMSO and then added to 500 μl of 1N hydroxylamine solution, pH 7.4. A deacylated peptide with a m / z of 1668.4 (+4) was detected immediately after mixing. The peptide solution was further diluted 5x with conjugation buffer so that the hydroxylamine concentration was less than 0.1 N during conjugation.

[0151] Another multimeric peptide (sequence identification number: 35) shown in Figures 2ba to 2bg was synthesized via SPPS on a linked amide MBHA resin (0.2 mmol, 0.33 mmol / g) according to the manual synthesis (protocol A) procedure described above. A K4K2K core was constructed using Fmoc-Lys (Fmoc). Eight Aβ peptide sequences were independently assembled on each chain of the core. The completed resin was cleaved according to the procedure described above. HPLC purification of the crude peptide was performed on a YMC-gel 10 μm C4-HG column (160 mm x 25 mm) (GX-281) at a flow rate of 20 mL / min using a linear gradient of buffer B in A (A: H2O (0.1% TFA in H2O), mobile phase B: B: CH3CN:IPA = 1:2 (0.1% TFA)). The gradient was 24% B-45% B within 40 minutes. The product was eluted at 26 minutes. The fraction containing the product was pooled and freeze-dried to obtain a white solid (34 mg, 2.18% yield), with observed m / z values ​​of 1524.3 (+4); 1089.4 (+6).

[0152] Table 3. Aβ peptides for conjugation

[0153]

[0154] Example 3: Preparation of AB Peptide-CRM197 Conjugate

[0155] Purified recombinant CRM197 (detoxified diphtheria toxin) was diluted to a total protein concentration of 1 mg / mL using 25 mM HEPES, pH 7.3, 150 mM NaCl, and 5 mM EDTA. CRM197 was activated with the heterofunctional crosslinking agent N-maleimidobutyryloxysuccinimide ester, GMBS (TCI America, Portland, Oregon, USA). A fresh stock solution of 30 mg / mL GMBS was prepared in DMSO (Sigma Aldrich, D2650), and 50 equivalents of GMBS were added to the 1 mg / mL CRM197 solution. The reaction mixture was incubated at ambient temperature for 2 hours. Excess GMBS was removed by gel filtration using a disposable Sephadex® G25 desalting column (Cytiva). The level of maleimide in GMBS-activated CRM197 was measured by the consumption of N-acetylcysteine ​​by CRM197-bound maleimide, and N-acetylcysteine ​​thiol was determined using Elman's reagent (5,5-dithio-bis-(2-nitrobenzoic acid)).

[0156] Synthetic Aβ peptides, e.g., the Aβ peptides listed in Table 3 above, were dissolved in 20% DMSO at a concentration of 10 mg / mL. The volume of the peptide solution was added to GMBS-activated CRM197 to achieve a final conjugation reaction thiol-to-maleimide molar ratio equivalent to 1.5. The reaction mixture was incubated at 4°C for 16 hours. Unreacted maleimide groups were capped by the addition of an excess amount of N-acetylcysteine. A GMBS-activated CRM197-only control was performed in parallel via the conjugation protocol. The conjugate was purified by five consecutive rounds of centrifugation filtration on a 30,000 Dalton molecular weight cutoff filter using 25 mM HEPES, pH 7.3, and 150 mM NaCl. The purified conjugate was sterile filtered through a 13 mm 0.2 μm Durapore® PVDF filter unit.

[0157] Analysis of Aβ peptide-CRM197 conjugates. The molecular weight of the conjugates was determined by size exclusion chromatography, multiple-angle light scattering (MALS), differential refractive index (dRI), and UV detection at 280 nm. 50 μL of peptide conjugate or control (GMBS-activated CRM197 quenched with N-acetylcysteine) was injected at a flow rate of 0.5 mL / min onto a Superose® 6-increase 10 / 300 GL column (Citiba, part number GE29-0915-96) equilibrated with PBS + 0.02% sodium azide. The molecular weight of the eluted peaks was calculated using Wyatt Astra® software. The peptide loading ratio (peptide moles / CRM197 moles) was calculated by dividing the difference in molecular weight between the peptide-CRM197 conjugates and the CRM197-alone control by the molecular weight of the peptides. The peptide loading ratio for the conjugate ranged from 8.5 to 13.9 mol / CRM197 mol for peptide, and the average loading ratio was 11.4 mol / mol for peptide.

[0158] Alternatively, the molar ratio of peptide to protein was calculated by quantitatively comparing the experimentally determined amino acid composition of the conjugate and the control (no peptide) using a modified least-squares algorithm described in the literature [Shuler, et al., (Shuler, KR, Dunham, RG, and Kanda, PJ Immunol. Methods, 156 (1992), 137-149)]. The mass loading of the peptide can be calculated from this ratio using the peptide molecular weight and the amino acid sequence-derived molecular weight for CRM197 (58,408 Da).

[0159] Example 4: Preparation of AB Peptide-OMPC Conjugate

[0160] Maleimide Chemistry. Outer membrane protein complex (OMPC) purified from Neisseria meningitidis was suspended at 5 mg / mL in 50 mM NaHCO3, pH 8.5. 30 mg / mL of freshly prepared GMBS in 20% DMSO was added to the OMPC to achieve a GMBS-to-lysine molar ratio of approximately 1.2 (assuming 0.42 μmol lysine / mg OMPC Lowry protein [Leanza, WJ, Chupak, LS, Tolman, RL, and Marbug, S. Bioconjug Chem 3(1992), 514-518]). The reaction mixture was incubated at room temperature for 2 hours. The reaction mixture was desalted by dialysis against 25 mM HEPES, pH 7.3, 150 mM NaCl, and 5 mM EDTA using a 100,000 Dalton molecular cutoff dialysis membrane. The level of maleimide in GMBS-activated OMPC was measured by the consumption of N-acetylcysteine ​​by OMPC-bound maleimide, and N-acetylcysteine ​​thiol was determined using Elman reagent (5,5-dithiobis-(2-nitrobenzoic acid)).

[0161] The synthetic Aβ peptide was dissolved in 20% DMSO at a concentration of 10 mg / mL. The volume of the peptide solution was added to GMBS-activated OMPC to achieve a final conjugation reaction thiol / maleimide molar ratio equivalent to 1.5. The reaction mixture was incubated at 4°C for 16 hours. Unreacted maleimide groups were capped by the addition of an excess of N-acetylcysteine. The conjugate was purified by dialysis against 25 mM HEPES, pH 7.3, and 150 mM NaCl using a 100,000 Da molecular weight cutoff dialysis membrane.

[0162] The molar ratio of peptide to protein was calculated by quantitatively comparing the experimentally determined amino acid composition of the conjugate and the control (no peptide) using a modified least-squares algorithm described in the literature [Shuler, et al., (Shuler, KR, Dunham, RG, and Kanda, PJ Immunol. Methods, 156 (1992), 137-149)]. The peptide loading ratio of the conjugate was 4464 moles of peptide per mole of OMPC. The peptides conjugated to the maleimide-activated OMPC are listed in Table 4 below.

[0163] Table 4. Peptides conjugated to maleimide-activated OMPC

[0164]

[0165] Thiol-derivatived OMPC. An alternative chemistry for conjugation involves reacting an Aβ peptide containing a terminal bromoacetyl functional group with the thiol-derivatived OMPC. Purified sterile OMPC was thiolized on a portion of its surface-accessible lysine residues using the reagent N-acetylhomocysteinethiolactone, NAHT (Sigma Aldrich, St. Louis, Missouri). The OMPC in water was centrifuged at approximately 197,000 xg for 60 minutes at 4°C to pellet, and the supernatant was discarded. N2-aerated activation buffer (0.11 M sodium borate, pH 11) was added to the centrifuge tube, and the pellet was removed with a glass stirring rod. The suspension was transferred to a glass Downs homogenizer and resuspended for 30 strokes. The centrifuge tube was washed, and the wash was downwashed for 30 strokes. The resuspended pellet and wash were combined in a clean container to obtain an OMPC concentration of approximately 7.8 mg / mL. Solid EDTA and DTT were dissolved in N2-aerated activation buffer and added to the reactants at a ratio of 0.106 mg DTT / mg OMPC and 0.57 mg EDTA / mg OMPC. After gentle mixing, NAHT was dissolved in N2-aerated water and added to the reaction at a ratio of 0.89 mg NAHT / mg OMPC. The reaction was carried out at ambient temperature for 3 hours while protected from light. Upon completion, the OMPC was pelleted as described above and resuspended in N2-aerated conjugation buffer (25 mM sodium borate, pH 8.5, 0.15 M NaCl) by Downs homogenization. The aliquot was removed for the determination of free thiols by Elman assay, and the bulk product was stored on ice in a dark room until use.

[0166] The bromoacetylated peptide was dissolved at 10 mg / mL in N2-aerated conjugation buffer and slowly added to the thiolized OMPC solution. The reaction mixture was protected from light and incubated at ambient temperature for approximately 16 hours. Residual free OMPC thiol groups were quenched with a 5-fold molar excess of N-ethylmaleimide at ambient temperature for 1 hour. A thiolized OMPC-alone control was performed in parallel using the conjugation protocol. Upon completion of quenching, the conjugate and control were transferred to a 100,000 Da molecular weight cutoff dialysis unit and thoroughly dialyzed for at least 5 changes of conjugation buffer. The final purified conjugate was stored in sterile polypropylene tubes at 4°C.

[0167] Analysis of Aβ peptide-OMPC conjugates. Total protein was determined by a modified Lowry assay, and conjugate and control samples were analyzed by quantitative amino acid analysis (AAA). OMPC-specific concentrations were determined from hydrolysis-stable residues that are absent in the peptide sequence and are therefore unique to the OMPC protein. The molar ratio of peptide to protein was calculated by quantitatively comparing the experimentally determined amino acid composition of the conjugate and control (no peptide) using a modified least-squares algorithm described in the literature [Shuler, et al. (Shuler, KR, Dunham, RG, and Kanda, PJ Immunol. Methods, 156 (1992), 137-149)]. The peptide loading ratio of the conjugate was 3963 moles of peptide per mole of OMPC. The mass loading of the peptide can be calculated from this ratio using the peptide molecular weight and the average OMPC mass of 40,000,000 Da. Peptides conjugated to thiol-activated OMPC are listed in Table 5 below.

[0168] Table 5. Peptides conjugated to thiol-activated OMPC

[0169]

[0170] Example 5: Preparation of AB peptide-KLH conjugate

[0171] A 4 mg / ml suspension of mariculture keyhole limpet hemocyanin (mcKLH) was prepared by adding water to Imject PEGylated maleimide-activated mcKLH (ThermoFisher Scientific, Waltham, Massachusetts, USA). 0.5 ml of the mcKLH suspension was added directly to 2 mg of pE3_9-GC solid peptide, the peptide and activated mcKLH were gently mixed, and the mixture was allowed to react overnight at room temperature. The next day, the mixture was centrifuged at 3000 rpm to remove any precipitates formed during the reaction. The supernatant was transferred to a Slide-A-Riser 20k MWCO dialysis cassette (ThermoFisher Scientific, Waltham, Massachusetts, USA) for dialysis against PBS buffer. The retained material was collected and sterilized by filtration through a 0.2 μm MILLEX filter unit (Millipore Corp, Bedford, Massachusetts, USA). The amount of peptide incorporated into the conjugate was estimated by amino acid analysis after 70 hours of acid hydrolysis. The peptide concentration was determined to be 0.17 mg / ml.

[0172] A negative control was prepared by adding 5 μl of 0.5 M N-acetylcysteine ​​solution to 0.5 ml of the above-mentioned activated mcKLH suspension. After reacting overnight at room temperature, the mixture was centrifuged at 3000 rpm, and the supernatant was transferred to a 20 k MWCO dialysis cassette for dialysis against PBS buffer. The retained sample was collected and sterilized by filtering through a 0.2 u filter unit. Peptides conjugated to maleimide-activated KLH are listed in Table 6 below.

[0173] Table 6. Peptides conjugated to maleimide-activated KLH

[0174]

[0175] Example 6: Preparation of AB Peptide-VLP Conjugate

[0176] Aβ peptides were conjugated to virus-like particles (VLPs) of the bacteriophage AP205 coat protein using the SpyTag / SpyCatcher system. A 1 mg / mL solution of Aβ peptide containing the SpyTag sequence (Sequence Identification No.: 36) was prepared in 25 mM HEPES, pH 7.3, and 150 mM NaCl. A 0.5 mg sample of AP205-SpyCatcher VLP (Sequence Identification No.: 37) was added to the peptide at a 1:1 molar ratio, and the mixture was incubated at 4°C for 4 hours. The SpyTag and SpyCatcher sequences are presented in Table 7 below.

[0177] Table 7. SpyTag and SpyCatcher sequences

[0178]

[0179] The conjugate was separated from the unreacted peptide by buffer exchange using a 30 kDa Amicon spin filter (Millipore Corporation, Bedford, Massachusetts, USA). The conjugate was sterilized by filtration through a 0.2 μm filter unit (Millipore Corporation, Bedford, Massachusetts, USA). The amount of peptide incorporated into the conjugate was estimated by time-of-flight mass spectrometry in the reduced state. The peptide concentration was determined to be between 20 and 40 μg / mL. The peptides conjugated to the VLP are listed in Table 8 below.

[0180] Table 8. VLP-based junction

[0181]

[0182] Mass Spectrometry. For mass spectrometry in the reduced state, 0.1 mg / mL Aβ peptide-VLP conjugates were prepared in a 25 mM 1,4-dithiothreitol (DTT) solution. Samples were incubated at 50°C for 10 minutes. Samples (0.5 μg / sample) were injected onto an Agilent 6230 LC-QToF-MS system (capillary voltage: 5K; desolvation temperature: 400°C) equipped with an Agilent PLRP-S column (1000A 5 μm, 2.1 x 50 mm). Water containing 0.1% (v / v) formic acid and acetonitrile containing 0.1% (v / v) formic acid were used as mobile phases A and B, respectively. Analysis was performed at 60°C using the gradients shown in Table 9 below.

[0183] Table 9. Mass Spectrometry Gradient Conditions

[0184]

[0185] Example 7: Preparation of Azuvant

[0186] Amorphous aluminum hydroxyphosphate sulfate azuvant (AAHS) was prepared by the precipitation of alum using aluminum hydroxide and sodium hydroxide. The azuvant concentration was aluminum (Al 3+ It was calculated based on the content. The aluminum concentration in each vaccine formulation was 450 μg / ml. In animal studies, the final aluminum dose per injection was 45 μg in a volume of 100 μl.

[0187] Alum + CpG (Type B ODN 1018). Amorphous aluminum hydroxyphosphate sulfate azuvant (AAHS) was prepared by the precipitation of alum using sodium hydroxide. The azuvant concentration was aluminum (Al 3+It was calculated based on the content. CpG (Type B ODN 1018, Trilink) was dissolved in 20 mM histidine, 100 mM NaCl, pH 6.5 buffer. Subsequently, the stock CpG solution was filtered through a 0.22 µm PVDF membrane filter (4 mm diameter). AAHS was buffered to 20 mM histidine, 100 mM NaCl, pH 6.5 and concentrated by centrifugation. CpG 1018 was added to AAHS in the formulation buffer and mixed by pipetting. The aluminum concentration in each vaccine formulation was 450 μg / ml. In animal studies, the final aluminum dose per injection was 45 μg Al per 100 μl volume. 3+ and 5 μg CpG or 30 μg CpG.

[0188] Liposomes. Liposomes containing QS-21, a plant extract from Quillaja saponaria (Desert King International, Product 120-2-177), 1,2-dioleoyl-sn-glycero-3-phosphocholine (Avanti™, Product 850375), cholesterol (Avanti™, Product 700100), and GLA were formulated using thin-film evaporation. DOPC, cholesterol, and GLA were dissolved in ethanol. The ethanol was evaporated using a RotoVap™ in a round-bottom flask. The lipid film was resuspended in 50 mM Na / K phosphate, 100 mM NaCl pH 6.2 buffer in a water bath set at 50°C. The resulting liposomes were extruded six times through a 0.1 μm polycarbonate filter at 50°C. QS-21 and an additional 50 mM Na / K phosphate, 100 mM NaCl pH 6.2 buffer were added to the formulation and subsequently sterile-filtered through a PES 0.2 μm pore size membrane. In a specific embodiment, AVT5 is an exemplary liposomal ajuvant comprising DOPC, cholesterol, GLA, and QS-21 components.

[0189] AS04-like; TLR4 + alum. The TLR4 agonist GLA was dissolved in 0.2% triethylamine. This was heated, homogenized, and sonicated to completely dissolve the solution and produce particles of uniform size. Subsequently, the particles were filtered through a 0.22 μm PES sterile filter. The filtered GLA was then added to AAHS in 10 mM histidine, 325 mM NaCl, 0.01% PS80, and pH 6.2. To ensure complete adsorption of GLA to AAHS, the formulation was allowed to equilibrate at 2–8°C for at least 4 hours. A final buffer exchange was performed using 10 mM histidine, 325 mM NaCl, 0.01% PS80, and pH 6.2. The supernatant and wash solution were collected, and if unbound GLA was present, its amount was quantified.

[0190] Matrix-M analog. QS-21 was dissolved in 150 mM NaCl, 10 mM K / Na Pi pH 6.2. DPPC and cholesterol were dissolved in 20% N-decanoyl-N-methylglucarmine and pipette-mixed. The dissolved QS-21 was added to a lipid vial and mixed by pipetting and gentle vial vortexing. 150 mM NaCl, 10 mM K / Na Pi pH 7.4 buffer was added and mixed. Stirred overnight at 30°C. After dialyzing for 24 hours against 150 mM NaCl, 10 mM K / Na Pi pH 7.4, the buffer was exchanged 3x at 24-hour intervals against 150 mM NaCl, 10 mM K / Na Pi pH 6.2. Sterile filtration was performed through a 0.22 μm pore size PES filter. In animal studies, the final dose per injection was 0.2 μg or 5 μg of GLA in a volume of 100 μL.

[0191] SNE + L608. As described in WO2017070623A1, the full contents of which are incorporated herein, squalene, PS-20, SPAN-85, and L608 ((12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-diene-1-amine) were combined to produce oil phases at target concentrations of 15, 1.5, 1.5, and 15 mg / mL, respectively. An aqueous buffer (20 mM histidine, pH 5.8) was added to the oil phase and mixed on a stirred plate. After using a two-step high-pressure homogenization process, the mixture was sterile filtered through a PES 0.22 μm pore filter (Catalog 12992). The filtered preparation was diluted with a pH 5.8 buffer combination of 20 mM histidine, 300 mM NaCl, and 0.2% PS-20 to obtain a final SNE ajuvant preparation targeting L608 at a concentration of 2.4 mg / mL.

[0192] SNE + TLR7 / 8. TLR7 / 8 agonist compounds of chemical formulas I, Ia, II, IIa, III, IIIa, IV, or Iva, or pharmaceutically acceptable salt(s) thereof, are described in International Patent Application No. PCT / US2024 / 029596, the full text of which is incorporated herein. In some embodiments, the TLR7 / 8 agonist compound is N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidine-2-yl)methyl)-3,5-dimethoxyphenyl)piperazine-1-yl)-5-oxopentyl)stearamide, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidine-2-yl)methyl)-3-methoxyphenyl)piperazine-1-yl)-5-oxopentyl)stearamide, or N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidine-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide. In a specific exemplary embodiment, the TLR7 / 8 agonist compound is N-(5-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidine-2-yl)methyl)-3-methoxyphenyl)piperazine-1-yl)-5-oxopentyl)stearamide. The TLR7 / 8 agonist compound was combined with squalene, PS-20 or PS-80, and SPAN-85, and diluted with a pH 5.8 buffering combination of histidine, PS-20, NaCl, L-met, and EDTA to produce an SNE ajuvant formulation.

[0193] LNPs containing L608, DSPC, cholesterol, and PEG-DMG were formed by T-mix precipitation. The lipids were dissolved in ethanol at a molar ratio of 58 / 10 / 30 / 2 to produce the exemplary LNP ajuvant, AVT1. Using a syringe pump, the lipid / ethanol stream was introduced into the aqueous stream (10 mM citrate buffer, pH 5) within the t-mix composition. Two downstream buffer dilutions were introduced into the product stream (20 mM sodium citrate, 300 mM NaCl, pH 6.0, and 1X Dulbecco PBS). The resulting LNPs were annealed at room temperature for 30 minutes, followed by buffer exchange (20 mM Tris, 10% sucrose, pH 7.5) and tangential film filtration for concentration. The fractions were stored at -20℃ until the day of the animal study, allowed to thaw at room temperature, and diluted with 20 mM Tris, 10% sucrose, and pH 7.5.

[0194] Example 8: In vivo immunogenicity study

[0195] A vaccine formulation comprising a peptide antigen conjugated to a carrier protein / particle co-formulated with a vaccine ajuvant was prepared, and the immunogenicity of the peptide was tested. The peptide antigen was conjugated to a carrier protein or particle selected from CRM197, KLH, OMPC, or AP205 virus-like particles (VLP). The conjugated peptide was co-formulated with the ajuvant described in Example 7.

[0196] Mouse In Vivo Study Protocol. Female C57Bl6 / NTac mice (Taconic Biosciences) were arrived at the facility and housed in groups of four per cage in corn cob bedding under a normal light cycle (lighting at 0700 h, turning off at 1900 h) with free access to food and water. Mice reached 8 weeks of age and were acclimatized to the facility for at least one week prior to the start of the study, when the mice were 9–11 weeks of age. On Day 0, mice were vaccinated intramuscularly with 0.05 mL of vaccine per quadriceps, with a total injection volume of 0.1 mL per mouse. The treatment group consisted of 8 mice, and the animals were tagged on the ears for identification purposes. On day 15, mice were placed in a warming box to promote tail vasodilation, tail blood was collected, and the blood was gathered into a serum separator tube (SST). The blood was then centrifuged at 10,000 rpm for 6 minutes, and approximately 100 μL of serum was transferred to a 96-well plate according to the plate map. On day 28, mice were administered a second vaccination with the same preparation as that administered on day 0 (0.05 mL IM per quadriceps, with a total infusion volume of 0.1 mL per mouse). On day 42, mice were euthanized with CO2, CSF was collected, and transferred to a 96-well plate according to the plate map. The thoracic cavity was opened, blood was collected into the SST (processed and plated as described on day 15), the spleen was dissected and placed into a collection vial maintained on moist ice, and the mice were subsequently perfused perforated with ice-cold phosphate-buffered saline via a syringe. The brain was removed, halved, and frozen fresh on dry ice. Subsequently, the tissue was processed as described in the present application.

[0197] Rhesus Macaque In Vivo Study Protocol. All animal studies were conducted at the New Iberian Primate Research Center (NIRC) (New Iberiana, Louisiana, USA) and were approved by the Institutional Animal Care and Use Committee (IACUC). Healthy adult rhesus macaques of Indian origin of one sex were used in these studies. To minimize differences based on animal age, sex, body weight, and pre-existing immunity to vaccine components, animals were pre-screened prior to study enrollment and assigned to the experimental group.

[0198] Animals were vaccinated according to established NIRC procedures, including sedation, shaving of the injection site, and injection of the test vaccine material. The vaccine was delivered via the intramuscular (IM) route of the deltoid muscle, and each arm received 0.5 ml of vaccine (total volume of 1.0 ml per animal). The vaccine was administered to animals on Day 0 and Day 28 after immunization, and reactogenicity was determined by monitoring the injection site for Draize scores. To evaluate the immunogenicity of the test vaccine material, venous blood was collected from breeding stock at appropriate times, and serum, plasma, and peripheral blood mononuclear cells (PBMCs) were isolated. Serum was isolated from whole blood using an appropriately sized non-additive serum separator Vacutainer-type tube (SST), allowed to coagulate, centrifuged for serum collection, and transferred to a long-term storage vial. Plasma was isolated from whole blood by collection into an EDTA-containing tube, the sample was centrifuged, and the plasma was collected for long-term storage. PBMC was isolated by collecting 30 ml of whole blood into a suitable EDTA-containing vacuum container tube, processed by density gradient centrifugation according to NIRC standard operating procedures, and frozen before long-term storage.

[0199] Example 9: Binding of vaccine-treated animal serum to AB peptide

[0200] To determine the immunogenicity of the vaccine formulation, mice were immunized twice at a 4-week interval. Two weeks after each immunization, blood samples were collected, and serum was analyzed for Aβ by ELISA. pE3-42 and Aβ 1-42 The antibody titers against were tested. These titers were Aβ pE3-42 and Aβ 1-42 Regarding this, the results after the first administration (PD1) and the second administration (PD2) are presented in Table 11 below.

[0201] To further evaluate the immunogenicity of the vaccine formulation, non-human primates were immunized twice at a 4-week interval. Two weeks after each immunization, blood samples were collected, and serum was analyzed for Aβ by ELISA. pE3-42 and Aβ 1-42 The antibody titers against were tested. These titers were Aβ pE3-42 and Aβ 1-42 Table 12 presents the details.

[0202] Aβ pE3-42 Immunogenicity was evaluated using titer, and Aβ pE3-42 vs. Aβ 1-42 Aβ by comparing titers pE3-42 Specificity for was evaluated. Aβ peptide, Aβ pE3-42 and Aβ 1-42 Evaluation of the binding of mouse or non-human primate (NHP) serum to [the substance] was performed by enzyme-linked immunosorbent assay (ELISA) according to the protocol described in Example 3 of International Patent Application Publication WO2010005858A, the full contents thereof of which are incorporated herein. 25 μL of Aβ per well was placed in black 384-well immunoplates (Thermo Fisher Scientific, Rochester, NY). pE3-42 or Aβ 1-42The samples were coated with peptide at a concentration of 0.25 μg per mL in PBS and stored overnight at 4°C. Vaccinated animal serum was prepared in milk-PBST by an initial 1:50 serum dilution and subsequent 4-fold serial dilutions in milk-PBST to achieve 10-point, 4-fold titrations of animal serum in milk-PBST across serum block plates. Each individual animal serum sample was plated across the serum block plates in this manner.

[0203] Next, the assay plate was washed 6 times with PBS (PBST) containing 0.05% Tween-20 and blocked for 1 hour with 3% skim milk (milk-PBST) in 80 μL of PBST per well. Subsequently, the plate was washed once, and 20 μL of serum was stamped from the serum block plate onto the assay plate. The assay plate containing serum in the wells was incubated in a humidified incubator at 22°C for 2 hours. Subsequently, the assay plate was washed 6 times with PBS (PBST) containing 0.05% Tween-20. Then, 20 μL of the freshly prepared HRP-conjugated secondary antibody was applied to each well and incubated in a humidified incubator at 22°C for 1 hour. The antibodies used for this ELISA assay are presented in Table 10 below.

[0204] Table 10. ELISA Antibodies

[0205]

[0206] Next, the black plate was moved to an open atmosphere and incubated at room temperature for 10 minutes. The black plate was washed 6 times with PBS (PBST) containing 0.05% Tween-20. West Pico Plus chemiluminescent substrates were prepared according to the manufacturer's protocol (Thermo Fisher Scientific, Rochester, New York). 20 μL of the freshly prepared chemiluminescent substrate was added to each well of the black plate and incubated at room temperature in an open atmosphere for 15 minutes. Luminescence was read on an Envision multimode plate reader (Perkin Elmer, Waltham, Massachusetts) set to read ultra-high sensitivity luminescence at 0.1 sec / well.

[0207] Next, the interpolated ELISA titer was calculated from the plate reader raw data using a threshold of 50,000 relative luminescence unit (RLU) counts. The titer was calculated as follows.

[0208] Titer = (Starting Dilution / Series of Dilutions) x (Series of Dilutions^t)

[0209] Here, t = x - [(cutoff - L) / (H - L)]

[0210] H = High well count = Count of the first well exceeding the cutoff

[0211] L = Low well count = Count of the first well below the cutoff

[0212] x = Low well number = Column of "Low well count" #

[0213] Cutoff = 50,000 counts

[0214] The interpolated titer is the calculated x dilution where the line between data points exceeding and below the 50,000 RLU threshold is the point where the line exceeds 50,000 RLU. Samples where the dilution does not exceed the threshold are given a placeholder titer of "25". The results of this test are summarized in Tables 11 and 12 and Figures 3a and 3b below. The graphs plot individual animal data, the geometric mean of data points per vaccine treatment group, and the 95% CI.

[0215] Table 11. Summary of mouse titers for all vaccine conjugates.

[0216]

[0217]

[0218]

[0219] Table 12. Geometric mean of interpolated ELISA titers in NHP animal groups

[0220]

[0221]

[0222]

[0223] Analysis of CRM197 conjugated antigens using AVT1 ajuvant (a lipid nanoparticle ajuvant prepared as described in WO2015 / 130584A2, the full contents of which are incorporated herein by reference, containing cationic lipids / cholesterol / DSPC / PEG-DMG in a molar ratio of 40 / 48 / 10 / 2) was Aβp E3-42 vs. Aβ 1-42 It was shown that the immunogenicity and specificity for can vary depending on the antigen sequence and linker. Further analysis of the data in Tables 11 and 12 showed that carrier proteins or particles and ajuvants can also affect immunogenicity and specificity. The pE3-9-CRM197+AVT1 preparation was Aβ 1-42 Contrast Aβ pE3-42It represents a preparation having the desired immunogenicity and selectivity for.

[0224] Example 10: Rhesus PBMC Restimulation Assay and Intracellular Cytokine Staining

[0225] Aβ pE3-42 or Aβ 1-42 By evaluating the T cell response after stimulation with the vaccine, the Aβ pE3-42 or Aβ 1-42 It was determined whether a T cell response to was induced. Cryopreserved Rhesus macaque PBMCs were rapidly thawed in a 37°C water bath and washed with R10 medium (RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES buffer (pH 7.2–7.5), 2 mM L-glutamine, 1x penicillin-streptomycin, 1 mM sodium pyruvate, and 50 μM 2-mercaptoethanol). Cells were incubated overnight at 37°C, 5% CO2. After overnight harvesting, cells were counted, distributed into 96-well round-bottom plates, and human Aβ 1-42 Sequence (decomer, overlapped by 14 amino acids), human Aβ pE3-42 The plates were incubated with 2 µg / ml of peptide across the sequence (pentamer, overlapping by 14 amino acids) or CRM197 protein (pentamer, overlapping by 11 amino acids) and 1.25 µg / ml of CD28 / CD49d mouse anti-human co-stimulatory antibody. The plates were incubated at 37°C for 60 minutes. After incubation, freshly diluted Brefeldin A was added to a final concentration of 12.5 µg / ml, and the plates were incubated for an additional 5 hours. DMSO was added to the mock control wells instead of the peptides to determine background cytokine expression levels.

[0226] After stimulation, PBMCs were washed with phosphate-buffered saline (PBS) and stained with mouse anti-human CCR7-BV650 (clone G043H7, Biolegend) at 37°C for 10 minutes. Live / dead fixable aqua stain (Invitrogen) was added to each well, and samples were incubated at room temperature for 15 minutes. Cells were washed with FACS buffer (PBS containing 1% FBS and 0.01% sodium azide) and stained with a cocktail of fluorescently labeled antibodies targeting surface markers at room temperature for 30 minutes. Antibodies targeting surface markers included mouse anti-human CD14-BV711 (clone M5E2, BioLegend), mouse anti-human CD20-BV711 (clone 2H7, BioLegend), mouse anti-human CD3-APC-Cy7 (clone SP34.2, BD Biosciences), mouse anti-human CD4-BV605 (clone L200, BD Biosciences), mouse anti-human CD8-BUV395 (clone RPA-T8, BD Biosciences), and mouse anti-human CD95-PE-Cy5 (clone DX2, BD Biosciences). An antibody cocktail for surface staining was prepared in FACS buffer supplemented with Brilliant Stain Buffer Plus (BD Biosciences). After surface staining incubation, the samples were washed with FACS buffer, fixed, and permeated with BD Cytopix / Cytoperm (BD Biosciences) at 4°C for 25 minutes. The cells were washed twice with BD perm / wash buffer (BD Biosciences) and then stained with a cocktail of fluorescently labeled antibodies and intracellular cytokines at room temperature for 60 minutes.Antibodies targeting intracellular cytokines included rat anti-human IL-2-PE (clone MQ1-17H12, BD Biosciences), mouse anti-human TNF-PE-Cy7 (clone Mab11, BD Biosciences), rat anti-human IL-4-PE-CF594 (clone MP4-25D2, BD Biosciences), rat anti-human IL-5-Vio515 (clone JES1-39D10, Miltenyi Biotec), and mouse anti-human IFNg-R718 (clone B27, BD Biosciences). An antibody cocktail for intracellular cytokine staining was prepared in BD perm / wash buffer supplemented with Brilliant Stain Buffer Plus. After incubation, the samples were washed with BD perm / wash buffer, fixed overnight at 4°C with BD stabilizing fixative (BD Biosciences), and then samples were obtained on a flow cytometer.

[0227] All samples were acquired on a Symphony A5 flow cytometer (Vidi Biosciences) using FACSDiva (Vidi Biosciences) software. Data analysis was performed using custom R scripts utilizing OMIQ (Dotmatics) and RStudio (Posit). Data cleaning prior to compensation, scaling, and gating was performed using the OMIQ implementation in flowAI. The frequency of cells expressing each cytokine in mock-stimulated samples was Aβ 1-42 , Aβ pE3-42 The percentage of cells that specifically responded to peptide stimulation was calculated by subtracting those stimulated by the peptide pool for , and CRM197. The data presented in graphs in Figures 4a-4d demonstrated that no significant T cell response was observed in cells isolated from any vaccinated animals.

[0228] Example 11: Mouse Splenocyte Restimulation Assay and Intracellular Cytokine Staining

[0229] To determine the magnitude of the T cell response to the test vaccine material, spleens were collected from vaccinated mice, processed into a single-cell suspension, and Aβ 1-42 , Aβ pE3-42 ..., and were restimulated with a peptide pool targeting CRM197. Briefly, spleen and R10 medium were placed in a 60 mm tissue culture dish (S-3770, Sigma) containing a steel mesh circle and mechanically dissociated using the plunger of a sterile 10 ml syringe (Vidi Biosciences). The cell suspension was collected in a 15 ml conical container, centrifuged to pellet the cells, and then resuspended in ACK lysis buffer (Gibco) to lyse the erythrocytes. The reaction mixture was quenched with R10 medium, and the cells were washed with R10 medium a total of two times. The cell suspension was resuspended to a final volume of 2 ml and filtered over a 70 μM cell filter (Cellart) to obtain the final single-cell splenocyte suspension. Cells were counted, dispensed into 96-well round-bottom plates, and mouse Aβ 1-42 Sequence (decomer, overlapped by 14 amino acids), mouse Aβ pE3-42 2 µg / ml of peptide across the sequence (pentamer, overlapping by 14 amino acids) or CRM197 protein (pentamer, overlapping by 11 amino acids) and 1.25 µg / ml of hamster anti-mouse CD28 (clone 37.51, Vidi Biosciences) and rat anti-mouse CD49d (clone R1-2, Vidi Biosciences) co-stimulating antibodies were incubated. The plates were incubated at 37°C for 60 minutes. After incubation, freshly diluted Brefeldin A was added to a final concentration of 12.5 µg / ml, and the plates were incubated for an additional 5 hours. DMSO was added to the mock control wells instead of the peptide to determine background cytokine expression levels.

[0230] After stimulation, splenocytes were washed with phosphate-buffered saline (PBS) and stained with live / dead fixable aqua stain (Invitrogen) at room temperature for 15 minutes. Samples were washed with FACS buffer (PBS containing 1% FBS and 0.01% sodium azide) and stained with mouse Fc block in FACS buffer (Vidi Biosciences) at 4°C for 5 minutes. Subsequently, a cocktail of fluorescently labeled antibodies targeting surface markers was added, and samples were incubated at 4°C for 30 minutes. Antibodies targeting surface markers included hamster anti-mouse CD3-APC-Cy7 (clone 145-2C11, BD Biosciences), rat anti-mouse CD4-BUV737 (clone RM4-5, BD Biosciences), and rat anti-mouse CD8a-BUV395 (clone 53-6.7, BD Biosciences). An antibody cocktail for surface staining was prepared in FACS buffer supplemented with Brilliant Stain Buffer Plus (BD Biosciences). After surface staining incubation, samples were washed with FACS buffer, fixed, and permeated with BD Cytopix / Cytoperm (BD Biosciences) at 4°C for 25 minutes. Cells were washed with BD perm / wash buffer (BD Biosciences) and then stained with mouse Fc block in BD perm / wash buffer (BD Biosciences) at 4°C for 5 minutes. Subsequently, a cocktail of fluorescently labeled antibodies targeting surface markers was added, and the samples were incubated at 4°C for 35 minutes. The antibody targeting intracellular cytokines was rat anti-mouse IFNγ-APC (clone XMG1.It included 2, BD Biosciences), rat anti-mouse IL-2-PE-CF594 (clone JES6-5H4, BD Biosciences), rat anti-mouse TNF-PE-Cy7 (clone MP6-XT22, BD Biosciences), rat anti-mouse IL-4-AF488 (clone 11B11, BD Biosciences), rat anti-mouse IL-5-PE (clone TRFK5, BD Biosciences), and rat anti-mouse IL-10-BV421 (clone JES5-16E3, BD Biosciences). An antibody cocktail for intracellular cytokine staining was prepared in BD perm / wash buffer supplemented with Brilliant Stain Buffer Plus. After incubation, the samples were washed twice with BD perm / wash buffer, fixed overnight at 4°C with BD stabilizing fixative (BD Biosciences), and then samples were obtained on a flow cytometer.

[0231] All samples were acquired on a Symphony A5 flow cytometer (Vidi Biosciences) using FACSDiva (Vidi Biosciences) software. Data analysis was performed using custom R scripts utilizing OMIQ (Domatix) and RStudio (Posit). Data cleaning prior to compensation, scaling, and gating was performed using the OMIQ implementation in FlowAI. The frequency of cells expressing each cytokine in mock-stimulated samples was Aβ 1-42 , Aβ pE3-42 The percentage of cells that specifically responded to peptide stimulation was calculated by subtracting from those stimulated by the peptide pool for , and CRM197.

[0232] Example 12: AD tissue IHC

[0233] To demonstrate that the vaccine formulation induces antibodies associated with human AD, serum was tested for binding to human AD brain tissue. Serum collected at 2 weeks PD2 from animals treated with pE3-9-GC-CRM + AVT1 was tested for reactivity with human AD tissue using immunohistochemistry.

[0234] Anti-Aβ from NHP serum pE3 Antibody affinity purification. Anti-Aβ pE3 For antibody affinity purification, pyroglutamate Aβ 3-42 Protein (Aβ pE3-42 ; Anaspec) was coupled to superparamagnetic Dynabeads M280 tosyl-activated (ThermoFisher Scientific) according to the manufacturer's instructions. The synthesized Aβ pE3-42 The beads were incubated with vaccinated NHP serum at room temperature for 30 minutes while rotating. The conjugated antibodies were eluted with 0.2 M glycine at pH 2.7 and immediately neutralized with 1 M Tris-HCl at pH 8.0.

[0235] Immunohistochemistry (IHC) and imaging analysis. Brain cortical blocks from individuals diagnosed with advanced AD and Braak stage 5 or 6 were purchased from Analytical Biological Services Inc. (ABS). These samples were rapidly autopsied and frozen within 4 hours post-mortem. To obtain frozen sections, brain tissue was mounted on a cryostat (Thermo Cryostar NX70, CT=-18°C; OT=-14°C) and sectioned to a thickness of 10 μm. IHC staining using vaccinated mouse serum or positive control antibodies was performed on a Leyca Bond RX automated staining machine using the Bond polymer precision detection system IHC protocol F (DS9800; Leyca Biosystems, UK). Briefly, after treatment with a Background Punisher-containing protein blocker (BP974M, Biocare Medical) and a peroxide blocker, sections were immunostained with the primary antibody / vaccinated animal serum diluted in Da Vinci Green diluent (PD900M, Biocare Medical). 8-point 3X serial dilutions of serum were performed, starting from an initial serum or a 500-fold dilution of 1 mg / ml antibody. After washing with the primary antibody, a primary follow-up (rabbit anti-mouse IgG in 10% (v / v) animal serum in Tris-buffered saline / 0.1% ProClin™ 950) was applied. After further rinsing, a polymer (anti-rabbit poly-HRP-IgG containing 10% (v / v) animal serum in Tris-buffered saline / 0.1% ProClin™ 950) was used. The sections were rinsed again and treated with diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide to produce visible reaction products. Hematoxylin was used as a nuclear counterstain at the end of the staining process. After staining, the slides were dehydrated with graded alcohol, mounted on DPX mounting medium (06522; Sigma, USA), and covered with coverslips.Slides were imaged using a digital pathology slide scanner—ZEISS Axioscan Z1 (ZEISS)—and image analysis was performed using HALO v3.4 software provided by Indica Labs. The experiments were analyzed in batches. A deep learning algorithm was trained to classify images into amyloid plaques and background regions. For this purpose, representative selections of each class were annotated, and the algorithm (HALO AI DensNet V2) was subsequently trained using these annotations. Gray matter was annotated as a Region of Interest (ROI) for analysis. The image analysis results were exported from HALO as a .csv file and analyzed using pivot charts / tables in Excel. The percentage of amyloid plaques was quantified, and statistical analysis was performed using GraphPad Prism 9. As shown in Figure 6, serum from pE3-9-G_C-CRM197 immunized animals was highly reactive with AD plaques in brain tissue.

[0236] Immunohistochemistry (IHC) procedure for NHP serum. Cortical blocks were purchased from PrecisionMed from individuals diagnosed with advanced AD and having Brach stage 5 or 6. These samples were rapidly autopsied within 4 hours post-mortem and frozen. To obtain frozen sections, brain tissue was mounted on a cryostat (Thermo Cryostar NX70, CT=-18°C; OT=-14°C) and sectioned to a thickness of 10 μm. Anti-Aβ pE3 IHC staining with antibodies was performed on the Leyca Bond RX automated staining instrument using the modified Bond Polymer Precision Detection System IHC Protocol F (DS9800; Leyca Biosystems, UK). Briefly, prior to staining, the Human-on-Human HRP-Polymer Kit (BRR4056KG; Biocare Medical) was used to test for anti-Aβ according to the manufacturer's instructions. pE3The antibody was labeled with digoxigenin. After treatment with a background punisher-containing protein blocker (BP974M; Biocare Medical) and a peroxide blocker, the digoxigenin-labeled anti-Aβ fragments were diluted in Da Vinci Green diluent (PD900M; Biocare Medical). pE3 Immunostaining was performed with antibodies. Starting with 2 μg / ml antibody, 8-point 2X serial dilutions of serum were performed. After washing with the primary antibody, a primary follow-up (mouse anti-digoxigenin secondary (BRR4055G; Biocare Medical)) was applied. After further rinsing, a polymer (MACH 2 mouse HRP-polymer (MHRP520G; Biocare Medical)) was used. The sections were rinsed again and treated with DAB and hydrogen peroxide to produce visible reaction products. Hematoxylin was used as a nuclear counterstain at the end of the staining process. After staining, the slides were dehydrated with graded alcohol, mounted on DPX mounting medium (06522; Sigma, USA), and covered with coverslips. Slides were imaged using a digital pathology slide scanner—ZEISS Axioscan Z1—and image analysis was performed using HALO v3.6 software provided by Indica Labs. The experiment was analyzed as a batch. A deep learning algorithm was trained to classify images into amyloid plaques and background regions. For this purpose, representative selections of each class were annotated, and an algorithm (HALO AI DensNet V2) was trained using these annotations. Gray matter was annotated as a Region of Interest (ROI) for analysis. Image analysis results were exported from HALO as a .csv file and analyzed using pivot charts / tables in Excel. The percentage of amyloid plaques was quantified, and the results are presented in Table 13 below. Statistical analysis was performed using GraphPad Prism 9.

[0237] Table 13. Summary of Natural Plaque Binding Profiles

[0238]

[0239] Example 13: Microglia phagocytosis

[0240] The functional activity of vaccine-induced serum antibodies was further evaluated using mouse and human microglia phagocytosis assays.

[0241] Mouse Microglia Phagocytosis Assay. The functional activity of the Aβ vaccine candidate was evaluated by performing a mouse microglia phagocytosis assay. Mouse microglia from 2-day-old mice were obtained from Transnetyx tissue (C57PMWB). Cells were plated at a cell density of 10,000 per well in PDL-coated 384-well plates (PerkinElmer, PEMSD-6057500) using a liquid handler in NB MicroPro medium (NBMicroPro500). The medium was replaced every 2-3 days by removing 50% of the growth medium and adding an equivalent volume of fresh medium. Cells were allowed to proliferate for 4-5 days, and the experiment was performed within one week of plating.

[0242] β-amyloid (pE3-42) peptide (Anaspec; AS-29907) was reconstituted with 100 μl of 1% NH4OH (Anaspec; AS-61322) and diluted to 0.1 mg / ml by adding an additional 900 μl of PBS buffer. The solution was heated in a 37°C water bath for 10–15 minutes to ensure complete dissolution, aliquoted, and stored as a 20 μM stock solution at -20°C. On the day of the assay, the 20 μM stock solution was mixed with 0.5 mM Aβ in NB MicroPro medium. pE3-42The solution was diluted and added to a master block plate (Griener; 781270). Vaccine serum samples were pooled for each test group, starting at a 2% (final) concentration, and 2-fold serial dilutions were performed to achieve a 10-point curve. This serum and peptide complex was incubated at 37°C for 1 hour. Subsequently, the microglia medium was replaced with the peptide and vaccine serum complex, and the cells were incubated at 37°C for 1 hour. Afterward, the cells were washed with PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences; 1224SK) for 15 minutes. Subsequently, the cells were washed with PBS, and immunocytochemistry was performed.

[0243] The plates were blocked for 1 hour with 10% normal goat serum (Sigma; G9023) and 0.3% Triton-x-100 (Sigma; T8787) in PBS. Subsequently, the cells were incubated overnight at 4°C with 5% NGS primary antibody rabbit Iba-1 (Wako; 019-19741; 1:500) and mouse 6E10 antibody (BioLegend; 803003; 1:1000) in PBS. On the next day, the cells were washed three times with PBS for 5 minutes each. Next, secondary antibodies alexafluoride were added to rabbit 488 (Molecular probes; A32731; 1:1000) and mouse 555 (Molecular probes; A32727, 1:1000) in 5% BSA, 0.3% Tx-100 / PBS at room temperature for 1 hour. Subsequently, cells were washed twice with PBS and incubated with Hoechst (Anaspec; 83218; 1:5000) for 5 minutes. After a final wash with PBS, cells were imaged on an Operetta CLS high-content plate scanner. For vaccine seroeval, multiple fields (15–20) per well and n=3 per treatment group were imaged.

[0244] Next, the built-in Operetta module was used to analyze the images to determine the phagocytic Aβ per microglia cell pE3-42 The number of spots was evaluated. Subsequently, this data was exported and statistical analysis of the final data was performed using GraphPad Prism software. As shown in Figure 7, the pE_3-9_G_C-CRM197 vaccinated group exhibited the most robust level of phagocytosis, even at lower serum dilutions.

[0245] Induced human microglia (iMGL) phagocytosis assay. On day 42, a human phagocytosis assay was performed using human microglia derived from human induced pluripotent stem cells (iPSCs) to evaluate the functional activity of different ajuvant non-human primate serums. The non-human primate serums are presented in Table 14 below.

[0246] Table 14. Non-human primate serum

[0247]

[0248] Human BX-0900-CS-2M iPS cell lines (BrainXell) were cultured, and differentiation into human microglia (iMGL) was induced according to the vendor's protocol. The reagents and media preparations for the culture and differentiation of BX-0900-CS-2M are presented in Tables 15 and 16 below.

[0249] Table 15. List of Reagents

[0250]

[0251] Table 16. Basic and Differentiated Media

[0252]

[0253] Phagocytosis of HF-488-labeled Aβ peptides by human iMGL was observed over 24 hours by live cell imaging. On the day of the experiment, half (50 μl) of the medium was removed from a multi-well culture plate and replaced with 2X erythrocyte tracer (Thermo Fisher; C34552) at a final concentration of 1:4000 and 1 drop / ml Nucblue (Thermo Fisher; R37605), and incubated for 1 hour. During this time, 0.5 μM of labeled Aβ pyro E3-42 peptide (Anaspec; 83960-3) was prepared in microglia complete differentiation medium and incubated at 37°C for 1 hour in a first plate containing 2% NHP serum and a second plate containing 1 μg / ml of purified NHP Day 42 serum. Microglia conditioning culture medium was replaced with 100 μl of Aβ peptide and serum or antibody complex, and incubated at 37°C for 24 hours while performing live imaging of the cells. Plates were imaged at 40x magnification on an Operetta CSL High Content Imaging System. For vaccine serology evaluation, multiple fields per well and n=3 per treatment group were imaged. Images were analyzed using Harmony software, and the number of phagocytic Aβ spots per cell was determined as the outcome measure. Data were exported and further analyzed on GraphPad Prism software. A significant increase in phagocytosis was noted in the Day 42 serum compared to the Day 0 serum (Fig. 15a). Similarly, when purified NHP serum was added to all groups at a concentration of 1 μg / ml on Day 42, labeled Aβ pE3-42 Phagocytosis was visualized over 24 hours (Fig. 15b). Data were presented as follows: phagocytic Aβ per cell for each treatment group on a time scale (X-axis) over time. pE3-42 It was plotted as the number of spots (Y-axis).

[0254] HMC3 Human Microglia Phagocytosis Assay. On day 42, a human phagocytosis assay was performed using the human microglia cell line HMC3 to evaluate the functional activity of different Azuvant non-human primate serums. HMC3 cells were spin-down at 280 g for 5 minutes at room temperature (RT), washed, and 1 x 10⁶ cells were placed in Maxcyte electroporation buffer. 8 The cells were resuspended at a final cell density of 10 cells / ml. The resuspended cells were added to 2 μl of 1 μg / μl FCGR1A DNA (100 μl of aliquoted cells, 10 6Electroporation was performed using 2 μg per cell. The electroporated cells were transferred to warmed medium and plated in 96-well culture plates at a density of 1:5000 cells / well. The plated cells were incubated for 24 hours at 37°C, 5% CO2, and high humidity. On the day of the experiment, half of the medium (50 μl) was removed and replaced with 2X erythrocyte tracer and Nuke Blue (1 drop / ml) at a final concentration of 1:4000, and incubated for 1 hour. During this time, 0.5 μM of labeled Aβ-pyro E3-42 peptide was prepared in HMC3 medium and incubated at 37°C for 1 hour with 1 μg / ml of purified NHP Day 42 serum. Microglia conditioning culture medium was replaced with 100 μl of Aβ peptide and serum or antibody complex, and incubated at 37°C for 24 hours while performing live imaging of the cells. Cells were live-imaging for Aβ HF488 at 20X using cell tracers, and the number of phagocytic spots was examined by analysis using Harmony software. Subsequently, cells were fixed in 4% PFA paraformaldehyde (Electron Microscopy Sciences; 1224SK) for 15 minutes. Plates were imaged at 40x magnification on an Operetta CSL High Content Imaging System. For vaccine seroeval, multiple fields per well and n=3 per treatment group were imaged. Images were analyzed using Harmony software, and the number of phagocytic Aβ spots per cell was measured as a result. Data were exported and further analyzed on GraphPad Prism software. Data were presented as the number of phagocytic Aβ spots per cell for each treatment group over time (X-axis). pE3-42 It was plotted as the number of spots (Y-axis) (Fig. 16).

[0255] Example 14: Analysis of Antigen-CRM197 Linker

[0256] The physical stability of pE3-9-CRM197 samples containing different linkers was evaluated by applying them to various analyses. Samples were prepared in 364-well plates, and their thermal stability was evaluated using a Prometheus nanotemper differential scanning fluorescence (nanoDSF) instrument. In nanoDSF, proteins in solution were exposed to a temperature gradient that induces protein unfolding. The intrinsic fluorescence of the proteins, originating mainly from the aromatic side chains of tyrosine and tryptophan residues, was investigated, and the data are shown in Fig. 8. Additionally, the melting temperature of the pE3-9-CRM197 conjugate and the first derivative of the fluorescence signal at 350 nm relative to the melting temperature for each linker type were determined as presented in Figs. 9 and 10, respectively.

[0257] In addition, the linker was examined for its potential to induce aggregation in experiments applying tumble stirring stress. To perform the experiment, samples were diluted to a concentration of 0.18 mg / mL in a buffer containing 25 mM HEPES, 150 mM NaCl, and pH 7.4. Subsequently, the diluted samples were placed on a magnetic tumble stirring plate set to 60% power. Precipitates were taken at specific time intervals (0, 4, 6, and 24 hours) and transferred to a 96-well plate for dynamic light scattering analysis to measure the intensity of scattered light to determine particle size and aggregation status. These data are presented in Figure 11.

[0258] Additionally, the stressed samples were plated in black-walled 96-well plates and treated with proteostat dye. These dyes selectively bind to aggregated proteins and can be used as indicators of protein aggregation. Fluorescence was measured on a SpectraMax M5 plate reader with excitation and emission set to 550 nm and 600 nm, respectively. These data are presented in Fig. 12.

[0259] The effects of polysorbate 80 and sucrose on formulation stability were evaluated using similar assays. To perform these experiments, samples were diluted to a concentration of 0.18 mg / mL in buffers containing 25 mM HEPES, 150 mM NaCl, and pH 7.4, and 0, 0.01, 0.02, 0.05, 0.1, or 0.2% PS80. These samples were then placed on magnetic tumble-stirred plates set to 60% output. Precipitates were taken at specific time intervals (0, 4, 6, and 24 hours) and transferred to 96-well plates for dynamic light scattering analysis, which measures the intensity of scattered light to determine particle size and aggregation status.

[0260] Stressed samples were also plated in black-walled 96-well plates and treated with proteostat dyes. These dyes selectively bind to aggregated proteins and can be used as indicators of protein aggregation. Fluorescence was measured on a Spectramax M5 plate reader with excitation and emission set to 550 nm and 600 nm, respectively. These data, presented in Figure 13, demonstrate that pE3-9_G_C-CRM197, formulated with polysorbate 80, inhibits aggregation.

[0261] The effect of sucrose on formulation stability was evaluated using a similar assay. To perform these experiments, samples were diluted to a concentration of 0.18 mg / mL in a buffer containing 25 mM HEPES, 150 mM NaCl, 10% sucrose, and pH 7.4. Samples underwent 0, 1, 2, or 3 freeze-thaw cycles. After each freeze-thaw cycle, samples were plated in 96-well plates containing proteostat dye. These data are presented in Figure 14.

[0262] The scope of the disclosed subject matter should not be limited by the specific embodiments and examples described herein. In fact, various variations of the disclosure in addition to those described will be apparent to those skilled in the art from the description and the accompanying drawings. Such variations are intended to fall within the scope of the appended claims.

[0263] All references cited herein (e.g., publications or patents or patent applications) are incorporated herein by reference in their entirety for all purposes to the same extent that each individual reference (e.g., publications or patents or patent applications) is specifically and individually indicated as being incorporated by reference in its entirety for all purposes. Other embodiments are within the scope of the following claims.

Claims

Claim 1 A pharmaceutical composition comprising an immunogenic peptide of at least 6 adjacent amino acids of sequence identification number 1 and an immunogenic carrier protein. Claim 2 A pharmaceutical composition according to claim 1, wherein the immunogenic peptide lacks 1 to 10 amino acids at the N-terminus or 1 to 33 amino acids at the C-terminus of sequence identification number:

1. Claim 3 A pharmaceutical composition according to claim 1, wherein the immunogenic peptide lacks 1-10 amino acids at the N-terminus and 1-33 amino acids at the C-terminus of sequence identification number:

1. Claim 4 A pharmaceutical composition according to any one of claims 1 to 3, wherein the immunogenic peptide comprises at least 10 adjacent amino acids of sequence identification number:

1. Claim 5 A pharmaceutical composition according to any one of claims 1 to 4, wherein the immunogenic peptide comprises 1 to 5 modified amino acids. Claim 6 A pharmaceutical composition according to claim 5, wherein the immunogenic peptide comprises a pyroglutamate residue at amino acid positions 3 and / or 11 of sequence identification number:

1. Claim 7 A pharmaceutical composition according to any one of claims 1 to 6, wherein the immunogenic peptide is selected from the group consisting of sequence identification number: 2-13. Claim 8 In any one of claims 1 to 7, the immunogenic carrier protein is CRM197; diphtheria toxin fragment B (DTFB); DTFB C8; diphtheria toxoid (DT); tetanus toxoid (TT); fragment C of TT; pertussis toxoid; cholera toxoid; E. coli ( E. coli ) LT; E. coli ST; Neisseria meningitidis( Neisseria meningitidis ) Outer membrane protein complex (OMPC); Pseudomonas aeruginosa( Pseudomonas aeruginosa A pharmaceutical composition selected from the group consisting of exotoxin A from ); mariculture keyhole limpet hemocyanin (mcKLH); and bacteriophage AP205 coat protein. Claim 9 A pharmaceutical composition according to any one of claims 1 to 8, wherein the immunogenic carrier protein is conjugated at the N-terminus or C-terminus of an immunogenic peptide. Claim 10 A pharmaceutical composition according to claim 9, wherein the immunogenic carrier protein is conjugated at the C-terminus of an immunogenic peptide. Claim 11 A pharmaceutical composition according to any one of claims 1 to 10, wherein an immunogenic carrier protein is conjugated to an immunogenic peptide by a linker. Claim 12 A pharmaceutical composition according to claim 11, wherein the linker is selected from the group consisting of N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS); polyethylene glycol (PEG); aminohexanoic acid (Ahx); sulfhydryl-reactive crosslinking agent; maleimide (MA) linker; oligopeptide; dendrimer; cyclodextrin; and glycine-rich peptide. Claim 13 A pharmaceutical composition according to claim 12, further comprising a spacer comprising 1 to 10 amino acids adjacent to the linker. Claim 14 A pharmaceutical composition according to any one of claims 1 to 13, wherein the immunogenic peptide comprises sequence identification number: 2 and the immunogenic carrier protein is CRM197. Claim 15 A pharmaceutical composition according to claim 14, wherein the immunogenic peptide is connected to CRM197 via a GMBS linker, and the composition further comprises a glycine-cysteine ​​spacer between the immunogenic peptide and the GMBS linker. Claim 16 A pharmaceutical composition according to any one of claims 1 to 15, wherein the immunogenic peptide is a monomer. Claim 17 A pharmaceutical composition according to any one of claims 1 to 16, wherein the immunogenic peptide is a multimer. Claim 18 A pharmaceutical composition comprising, in any one of claims 1 to 17, an additionally pharmaceutically acceptable ajuvant. Claim 19 A pharmaceutical composition according to claim 18, wherein the pharmaceutically acceptable ajuvant is selected from the group consisting of glucopyranosol lipid ajuvant (GLA); AVT1; AVT2; AVT3, AVT4; AVT5; AVT6; AVT7; QS-21; aluminum-based ajuvant; saponin-based ajuvant; and TLR7 / 8 agonists. Claim 20 In paragraph 19, a pharmaceutical composition in which the pharmaceutically acceptable ajuvant is AVT1, AVT5, or AVT7. Claim 21 A method for preventing or treating a disease associated with amyloid deposits of Aβ in the brain of a patient requiring prevention or treatment of a disease associated with amyloid deposits of Aβ in the brain, comprising administering an effective dose of a pharmaceutical composition comprising an immunogenic peptide of at least six adjacent amino acids and an immunogenic carrier protein of SEQ ID No.

1. Claim 22 A method according to claim 21, wherein the immunogenic peptide is missing 1-10 amino acids at the N-terminus or 1-33 amino acids at the C-terminus of sequence identification number:

1. Claim 23 A method according to claim 21, wherein the immunogenic peptide is missing 1-10 amino acids at the N-terminus and 1-33 amino acids at the C-terminus of sequence identification number:

1. Claim 24 A method according to any one of claims 21 to 23, wherein the immunogenic peptide comprises at least 10 adjacent amino acids of sequence identification number:

1. Claim 25 A method according to any one of claims 21 to 24, wherein the immunogenic peptide comprises 1 to 5 modified amino acids. Claim 26 The method of claim 25, wherein the immunogenic peptide comprises a pyroglutamate residue at amino acid positions 3 and / or 11 of sequence identification number:

1. Claim 27 A method according to any one of claims 21 to 26, wherein the immunogenic peptide is selected from the group consisting of sequence identification number: 2-13. Claim 28 A method according to any one of claims 21 to 27, wherein the immunogenic carrier protein is selected from the group consisting of CRM197; diphtheria toxin fragment B (DTFB); DTFB C8; diphtheria toxoid (DT); tetanus toxoid (TT); fragment C of TT; pertussis toxoid; cholera toxoid; E. coli LT; E. coli ST; Neisseria meningitidis outer membrane protein complex (OMPC); exotoxin A from Pseudomonas aeruginosa; methylculture keyhole limpet hemocyanin (mcKLH); and bacteriophage AP205 coat protein. Claim 29 A method according to any one of claims 21 to 28, wherein the immunogenic carrier protein is conjugated at the N-terminus or C-terminus of the immunogenic peptide. Claim 30 In paragraph 29, a method in which the immunogenic carrier protein is conjugated at the C-terminus of an immunogenic peptide. Claim 31 A method according to any one of claims 21 to 29, wherein the immunogenic carrier protein is conjugated to an immunogenic peptide by a linker. Claim 32 A method according to claim 31, wherein the linker is selected from the group consisting of N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS); polyethylene glycol (PEG); aminohexanoic acid (Ahx); sulfhydryl-reactive crosslinker; maleimide (MA) linker; oligopeptide; dendrimer; cyclodextrin; and glycine-rich peptide. Claim 33 A method according to claim 32, further comprising a spacer containing 1 to 10 amino acids adjacent to the linker. Claim 34 A method according to any one of claims 21 to 33, wherein the immunogenic peptide comprises sequence identification number: 2 and the immunogenic carrier protein is CRM197. Claim 35 A method according to claim 34, wherein the immunogenic peptide is connected to CRM197 via a GMBS linker, and the composition further comprises a glycine-cysteine ​​spacer between the immunogenic peptide and the GMBS linker. Claim 36 A pharmaceutical composition in which the immunogenic peptide is a monomer in any one of claims 21 to 35. Claim 37 A pharmaceutical composition in which the immunogenic peptide is a multimer, in any one of claims 21 to 35. Claim 38 A method comprising additionally including a pharmaceutically permissible ajuvant in any one of paragraphs 21 to 37. Claim 39 A method according to paragraph 38, wherein the pharmaceutically acceptable ajuvant is selected from the group consisting of glucopyranosol lipid ajuvant (GLA); AVT1; AVT2; AVT3, AVT4; AVT5; AVT6; AVT7; QS-21; aluminum-based ajuvant; saponin-based ajuvant; and TLR7 / 8 agonists. Claim 40 In paragraph 39, the method in which the pharmaceutically permissible ajuvant is AVT1, AVT5, or AVT7. Claim 41 A method according to any one of claims 21 to 40, wherein the disease associated with amyloid deposits of Aβ in the brain is a disease selected from the group consisting of Alzheimer's disease (AD); cerebral amyloid angiopathy (CAA); inflammatory CAA; and cerebral amyloidoma. Claim 42 In paragraph 41, a method in which the disease associated with amyloid deposits of Aβ in the brain is AD. Claim 43 Use of any one of claims 1 to 20 in the manufacture of a medicine for preventing or treating a disease associated with amyloid deposits of Aβ in the brain of a patient who requires prevention or treatment of a disease associated with amyloid deposits of Aβ in the brain. Claim 44 In paragraph 43, the use is for a disease associated with amyloid deposits of Aβ in the brain selected from the group consisting of Alzheimer's disease (AD); cerebral amyloid angiopathy (CAA); inflammatory CAA; and cerebral amyloidoma. Claim 45 In Clause 44, use for a disease associated with amyloid deposits of Aβ in the brain, which is AD. Claim 46 A method for inducing an immune response to an Aβ peptide in a patient requiring induction of an immune response to an Aβ peptide, comprising administering to the patient an immunologically effective dose of a pharmaceutical composition according to any one of claims 1 to 20. Claim 47 In claim 46, the method wherein the disease associated with amyloid deposits of Aβ in the brain is a disease selected from the group consisting of AD; CAA; inflammatory CAA; and brain amyloidoma. Claim 48 In paragraph 47, a method in which the disease associated with amyloid deposits of Aβ in the brain is AD.

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