Liposomes containing phosphorylated tau peptides for inducing sustained immune responses

Abstract

A method for inducing a sustained immune response against phosphorylated tau in humans is described. The method includes administering to a subject an effective amount of liposomes comprising a toll-like receptor 4 agonist, a helper T cell epitope, a lipidated CpG oligonucleotide, and a tau phosphopeptide displayed on the surface of the liposome, thereby obtaining a sustained immune response.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is an international application claiming priority to U.S. Provisional Patent Application No. 63 / 260,227, filed August 12, 2021, U.S. Provisional Patent Application No. 63 / 263,541, filed November 4, 2021, and U.S. Provisional Patent Application No. 63 / 267,975, filed February 14, 2022, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (SequenceListing_7WO1.xml; size: 42,600 bytes; and creation date: July 8, 2022) are incorporated herein by reference in their entirety.

[0003] FIELD OF THEINVENTION The present invention is in the field of medicine. In particular, the present invention relates to liposomes containing phosphorylated tau peptides for inducing a sustained antibody response against phosphorylated tau protein (p-tau) in a subject in need of preventing or treating tauopathies such as Alzheimer's disease.

[0004] background Alzheimer's disease (AD) is a progressive, debilitating neurodegenerative disorder that affects an estimated 44 million people worldwide (Alzheimers.net). Current commercialized AD therapies aim to affect clinical symptoms but do not target the pathogenic processes underlying the disease (disease-modifying effects). Unfortunately, current therapies are only minimally effective, and therefore there is an urgent need to develop and test additional preventive and therapeutic measures.

[0005] The pathology characteristic of Alzheimer's disease is the accumulation of extracellular plaques containing grossly aggregated amyloid beta protein, and intracellular "tangles" or aggregates of hyperphosphorylated tau protein. The molecular events that cause the accumulation of these proteins are poorly characterized. With regard to amyloid, it has been hypothesized that abnormal cleavage of the amyloid precursor protein leads to the accumulation of an aggregation-prone fragment containing amino acids 1-42. With regard to tau, it has been hypothesized that dysregulation of kinases, phosphatases, or both, leads to the abnormal phosphorylation of tau. When tau becomes hyperphosphorylated, it loses its ability to effectively bind and stabilize microtubules, and instead accumulates in the cytoplasm of affected neurons. Unbound hyperphosphorylated tau appears to form first oligomers and then higher order aggregates, the presence of which appears to negatively affect the function of the neurons in which they are formed, possibly via interruption of normal axonal transport.

[0006] In developed countries, individuals diagnosed with Alzheimer's disease or other dementing tauopathies are commonly treated with cholinesterase inhibitors (e.g., Aricept®) or memantine (e.g., Namenda™). These drugs are reasonably well tolerated but have very modest efficacy. For example, Aricept® delays the worsening of symptoms by 6-12 months in approximately 50% of treated individuals. The remaining treatments are non-pharmacological and focus on enabling patients to better handle everyday tasks while their cognitive abilities decline.

[0007] Results of ADAMANT (EudraCT 2015-000630-30), a 24-month double-blind, parallel-group, randomized, phase 2, multicenter, placebo-controlled study of AADvac1, an active peptide vaccine designed to target pathological tau in Alzheimer's disease, were recently published (Novak et al., Nature Aging vol 1: 521-534, 2021). AADvac1 contains a synthetic peptide derived from amino acids 294-305 of the tau sequence coupled to keyhole limpet hemocyanin (KLH) via an N-terminal cysteine. Patients with mild AD dementia received 11 doses of 40 μg of AADvac1 per dose over the course of the study. The vaccine induced high levels of IgG antibodies, but no significant effects were found in cognitive and functional tests for the entire study sample (ibid.).

[0008] ACI-35, a vaccine using synthetic peptides based on human p-tau396 / 404, was shown to improve motor performance and prolong survival in mice carrying the P301L mutation (Theunis et al., PLOS ONE. 2013. 8(8): e72301). In a phase 1b study, ACI-35 was well tolerated and induced antibody responses, but with limited booster response potential.

[0009] There is a need for safe and effective treatments for neuronal degenerative diseases such as Alzheimer's disease. Summary of the Invention

[0010] The present invention is based on findings from clinical studies of an improved liposomal vaccine comprising phosphorylated tau peptides displayed on the surface of liposomes, which induced a strong and sustained immune response, including a sustained antibody response against p-tau, that could be boosted by booster shots.

[0011] Thus, in one general aspect, the present invention provides a method of inducing an antibody response against phosphorylated tau protein (pTau) in a human subject in need thereof, comprising the steps of: (1) a tau phosphopeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:27 to SEQ ID NO:29 and SEQ ID NO:31 to SEQ ID NO:38 in an amount of 25 to 750 nmole, for example 300 μg to 1800 μg per dose; (2) a toll-like receptor 4 agonist comprising monophosphoryl lipid A; (3) a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 17, SEQ ID NO: 23 to SEQ ID NO: 26, and SEQ ID NO: 39 to SEQ ID NO: 44; (4) A CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22 administering to a subject an effective amount of a liposome comprising Tau phosphopeptides are displayed on the surface of liposomes, Methods are provided, wherein the antibody response persists for at least 6 weeks, such as at least 6, 7, 8, 9, 10 weeks, following initial administration of an effective amount of the liposomes to the human subject.

[0012] In some embodiments, an effective amount of liposomes comprises: (1) a tau phosphopeptide having the amino acid sequence of SEQ ID NO: 28 in an amount of 300 μg to 1800 μg per dose; (2) a toll-like receptor 4 agonist in an amount of 100 μg to 585 μg per dose; and (3) a helper T cell epitope in an amount of 75 μg to 550 μg per dose; and (4) CpG oligonucleotide in an amount of 100 μg to 1000 μg per dose Includes:

[0013] In some embodiments, the CpG oligonucleotide has one or more phosphorothioate internucleotide linkages, and the CpG oligonucleotide is covalently linked to at least one lipophilic group, optionally via a PEG linker.

[0014] In some embodiments, the tau phosphopeptide is administered in an amount of about 25 nmole to about 750 nmole per dose, such as about 29.7 nmole to about 742.5 nmole per dose, preferably about 90 nmole to about 715 nmole per dose, such as about 89.1 nmole to about 712.8 nmole per dose, or about 90 nmole to about 535 nmole per dose, such as about 89.1 nmole to about 534.6 nmole per dose, or about 90 nmole to about 275 nmole per dose, such as about 89.1 nmole to about 267.3 nmole per dose. In certain embodiments, the tau phosphopeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:27 to SEQ ID NO:29 and SEQ ID NO:31 to SEQ ID NO:38, preferably the amino acid sequence of SEQ ID NO:28. In one embodiment, the tetrapalmitoylated tau phosphopeptide is administered in an amount of 100 μg to 2500 μg per dose, corresponding to 29.7 nmole to 742.5 nmole per dose, preferably 300 μg to 2400 μg per dose, corresponding to 89.1 nmole to 712.8 nmole per dose, for example 300 μg, 900 μg, 1800 μg or 2400 μg per dose, corresponding to 89.1 nmole, 267.3 nmole, 534.6 nmole or 712.8 nmole per dose.

[0015] In certain embodiments, an effective amount of liposomes comprises a toll-like receptor 4 agonist in an amount of 30 μg to 900 μg, preferably 100 μg to 585 μg per dose. In certain embodiments, an effective amount of liposomes comprises a toll-like receptor agonist, monophosphoryl hexaacyl lipid A, 3-desacylated, in an amount of 30 μg to 900 μg, preferably 100 μg to 585 μg per dose.

[0016] In certain embodiments, the effective amount of liposomes comprises a T helper cell epitope in an amount of 25 μg to 625 μg, preferably 75 μg to 550 μg, for example 75 μg to 450 μg per dose. In certain embodiments, the effective amount of liposomes comprises a T50 helper T cell epitope consisting of the amino acid sequence of SEQ ID NO: 13 in an amount of 25 μg to 625 μg, preferably 75 μg to 450 μg per dose. In certain embodiments, an effective amount of liposomes comprises helper T cell epitopes in an amount of about 2 nmole to about 110 nmole per dose, e.g., about 4.02 nmole to about 100.44 nmole per dose, or about 4 nmole to about 75 nmole per dose, e.g., about 4.02 nmole to about 72.32 nmole per dose, or about 10 nmole to about 105 nmole per dose, e.g., about 12.06 nmole to about 100.44 nmole per dose, or about 70 to about 105 nmole per dose, e.g., about 72.32 nmole to about 100.44 nmole per dose. In certain embodiments, an effective amount of liposomes comprises a T50 helper T cell epitope consisting of the amino acid sequence of SEQ ID NO: 13 in an amount of about 3 nmole to about 105 nmole per dose, preferably about 10 nmole to about 105 nmole per dose, for example, about 12.06 nmole to about 100.44 nmole per dose. In one embodiment, an effective amount of liposomes comprises a helper T cell epitope in an amount of 2 to 5 nmole per dose, for example, 2, 3, 4, or 5 nmole per dose, or any value therebetween, for example, about 3.82, 3.92, 4.02, or 4.12 nmole per dose. In another embodiment, an effective amount of liposomes comprises helper T cell epitopes in an amount of 10-15 nmoles per dose, for example 10, 11, 12, 13, 14, or 15 nmoles per dose, or any value therebetween, for example 11.86, 11.96, 12.06, 12.16 nmoles per dose.In another embodiment, an effective amount of liposomes comprises helper T cell epitopes in an amount of 70-75 nmoles per dose, such as 70, 71, 72, 73, 74, or 75 nmoles per dose, or any value therebetween, such as 72.02, 72.12, 72.22, 72.32, 72.42 nmoles per dose. In yet another embodiment, an effective amount of liposomes comprises helper T cell epitopes in an amount of 98-103 nmoles per dose, such as 98, 99, 100, 101, 102, or 103 nmoles per dose, or any value therebetween, such as 100.24, 100.34, 100.44, 100.54, or 100.64 nmoles per dose.

[0017] In certain embodiments, the effective amount of liposomes comprises a lipidated CpG oligonucleotide in an amount of 50 μg to 1250 μg, preferably 100 μg to 1000 μg, for example 150 μg to 800 μg per dose. In certain embodiments, the effective amount of liposomes comprises a CpG oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 18 in an amount of 50 μg to 1250 μg, preferably 150 μg to 800 μg per dose.

[0018] In certain embodiments, the liposomes are administered subcutaneously.

[0019] In certain embodiments, the liposomes are administered intramuscularly.

[0020] In certain embodiments, the liposome further comprises one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol.

[0021] In certain embodiments, the liposome comprises: (1) a tau phosphopeptide having the amino acid sequence of SEQ ID NO: 28; (2) a toll-like receptor 4 agonist comprising monophosphoryl hexaacyl lipid A, 3-deacylated; (3) a helper T cell epitope comprising the amino acid sequence of SEQ ID NO: 39; and (4) a lipidated CpG oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 18; and (5) at least one lipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol; Includes:

[0022] In an embodiment of the application, the antibody response comprises a specific IgG antibody response directed against pTau. Preferably, the specific IgG antibody response has an anti-pTau IgG titer that is at least 50, 60, 70, 80, 90, 100 or more times higher than that of a placebo control.

[0023] In another embodiment of the present application, the antibody response comprises a specific IgM antibody response directed to pTau, and class switching of the specific IgM antibody response to a specific IgG antibody response directed to pTau.

[0024] In yet another embodiment of the present application, the antibody response comprises an IgG antibody response that preferentially recognizes pTau over non-phosphorylated Tau protein. Preferably, the ratio of anti-pTau IgG titer to anti-Tau IgG titer is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 or more.

[0025] In another embodiment of the present application, the antibody response comprises an IgG antibody response to concentrated paired helical fibrils (ePHF). Preferably, the IgG antibody response has an anti-ePHF IgG titer that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times higher than that of a placebo control. More preferably, the anti-ePHF IgG has an increased binding avidity to pathological ePHF tau for at least 6 weeks after a first dose or a boosting dose of an effective amount of liposomes, when measured at least 2 weeks after the boosting dose, and preferably the anti-ePHF IgG has an avidity index of at least 0.3, 0.4, 0.5, 0.6, or 0.7.

[0026] In an embodiment of the application, the antibody response may be boosted by a booster administration.

[0027] In one embodiment, the method of the present application further comprises administering to the subject a second dose of an effective amount of liposomes 4-12 weeks, e.g., 8 weeks, after the initial administration of the effective amount of liposomes. The antibody response is boosted when measured at least 2 weeks after administration of the second dose of an effective amount of liposomes. Preferably, the antibody response is boosted by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more when measured at least 2 weeks after administration of the second dose of an effective amount of liposomes.

[0028] In another embodiment, the method of the present application further comprises administering to the subject a third dose of an effective amount of liposomes 20-28 weeks, e.g., 24 weeks, after the initial administration of the effective amount of liposomes. The antibody response is boosted when measured at least 2 weeks after administration of the third dose of an effective amount of liposomes, preferably the antibody response is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more when measured at least 2 weeks after administration of the third dose of an effective amount of liposomes.

[0029] In yet another embodiment, the method of the present application further comprises administering a fourth dose of an effective amount of liposomes to the subject 44-52 weeks, e.g., 48 weeks, after the initial administration of the effective amount of liposomes. The antibody response is boosted at least 2 weeks after administration of the fourth dose of an effective amount of liposomes, preferably the antibody response is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more upon administration of the fourth dose of an effective amount of liposomes.

[0030] In certain embodiments, human subject needs to eliminate tau aggregates.In certain embodiments, subject needs to prevent or treat Alzheimer's disease, for example, preclinical Alzheimer's disease, early stage Alzheimer's disease, mild cognitive impairment (MCI) due to Alzheimer's disease, mild Alzheimer's disease, or mild to moderate Alzheimer's disease.In other embodiments, subject is amyloid positive in brain but does not yet show significant cognitive impairment.

[0031] The present invention also relates to a vaccine combination for use in inducing an immune response, such as an antibody response against phospho-tau protein (pTau), in a human subject in need thereof, comprising a priming vaccine and a booster vaccine of an embodiment of the invention, wherein the immune response persists for at least 10 weeks, such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50 weeks or more following administration of the priming vaccine to the human subject.

[0032] Further aspects, features, and advantages of the present invention will be better understood from a reading of the following detailed description of the invention and the appended claims. [Brief description of the drawings]

[0033] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

[0034] [Figure 1] Overview of the study design for Cohort 1 (ACI-35.030 or placebo) in the Phase 1b / 2a study (NCT04445831). [Diagram 2] 1 shows the geometric mean anti-pTau IgG titers in patients with early Alzheimer's disease treated with placebo or 300 μg or 900 μg doses of ACI-35.030 at weeks 0, 8, and 24. [Diagram 3] 1 shows the geometric mean anti-tau (non-phosphorylated) IgG titers in patients with early Alzheimer's disease treated with placebo or ACI-35.030 at doses of 300 μg or 900 μg at weeks 0, 8, and 24. [Figure 4] 1 shows the geometric mean anti-ePHF IgG titers in patients with early Alzheimer's disease treated with placebo or 300 μg or 900 μg doses of ACI-35.030 at weeks 0, 8, and 24. [Diagram 5] 1 shows the geometric mean anti-tau (non-phosphorylated) IgG titers in patients with early Alzheimer's disease treated with placebo or ACI-35.030 at doses of 300 μg, 900 μg, or 1800 μg. [Figure 6] 1 shows the geometric mean anti-pTau IgG titers in patients with early Alzheimer's disease treated with placebo or ACI-35.030 at doses of 300 μg, 900 μg or 1800 μg. [Figure 7] 1 shows the geometric mean anti-ePHF IgG titers in patients with early Alzheimer's disease treated with placebo or ACI-35.030 at doses of 300 μg, 900 μg, or 1800 μg. [Figure 8] (Figures 8A and 8B) Epitope recognition profiles of antibodies induced in patients with early Alzheimer's disease treated with placebo or a 900 μg dose of ACI-35.030 at weeks 0, 8, and 24. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Various publications, articles, and patents are cited or described in the Background and throughout this specification, and each of these references is incorporated herein by reference in its entirety. Any discussion of documents, acts, materials, devices, articles, and the like which has been included in this specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention(s) disclosed or claimed.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms used herein have the meanings described herein.

[0037] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0038] Unless otherwise stated, any numerical value, e.g., concentration or concentration range, described herein should be understood to be modified in all cases by the term "about". Thus, numerical values ​​typically include ±10% of the indicated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all possible subranges, i.e., all individual numerical values ​​within such ranges, including integers within such ranges and fractional parts of the values, unless the context clearly indicates otherwise.

[0039] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0040] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variations thereof, can be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers, and are intended to be non-exclusive or non-limiting. For example, a composition, mixture, process, method, article, or device that comprises listed elements is not necessarily limited to only those elements, but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or device. Further, unless expressly stated to the contrary, "or" refers to a non-exclusive or, not an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0041] It should also be understood that the terms "about," "approximately," "generally," "substantially," and similar terms used herein when referring to dimensions or characteristics of preferred inventive components indicate that the described dimensions / characteristics are not precise boundaries or parameters, as would be understood by one of ordinary skill in the art, but do not exclude minor variations therefrom that are functionally the same or similar. At a minimum, such references including numerical parameters may include variations without altering the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0042] The present invention provides a method for inducing anti-phosphorylated tau antibodies in a human subject in need thereof without inducing severe adverse events, such as encephalitis, that are somewhat or significantly likely to be associated with an investigational vaccine. In certain embodiments, the method comprises administering to the subject an effective amount of liposomes comprising a tau phosphopeptide and a toll-like receptor 4 agonist, the liposomes being presented on the surface of the liposome.

[0043] As used herein, the term "anti-phosphorylated tau antibody" refers to an antibody that binds to tau in which an amino acid residue at one or more positions in the amino acid sequence of tau is phosphorylated. The phosphorylated amino acid residue may be, for example, serine (Ser), threonine (Thr), or tyrosine (Tyr). The site of phosphorylated tau to which the anti-phosphorylated tau antibody binds is preferably a site that is specifically phosphorylated in neurodegenerative diseases such as Alzheimer's disease. Examples of sites of phosphorylated tau to which the anti-phosphorylated tau antibody binds include, for example, Tyr18, Ser199, Ser202, Thr205, Thr212, Ser214, Ser396, Ser404, Ser409, Ser422, and Thr427. As used throughout this application, amino acid positions are obtained with reference to the sequence of human microtubule-associated protein tau isoform 2, whose amino acid sequence is represented in GenBank Accession No. NP_005901.2.

[0044] Whether anti-phosphorylated tau antibodies can be induced upon administration can be determined by testing a biological sample from a subject (e.g., blood, plasma, serum, PBMC, urine, saliva, feces, CSF, or lymph) for the presence of antibodies, e.g., IgG or IgM antibodies, directed against the immunogenic tau peptide administered in the pharmaceutical composition (see, e.g., Harlow, 1989, Antibodies, Cold Spring Harbor Press). For example, the titer of antibodies produced in response to administration of a composition providing an immunogen can be measured by enzyme-linked immunosorbent assay (ELISA), other ELISA-based assays (e.g., MSD-Meso Scale Discovery), dot blots, SDS-PAGE gels, ELISPOT, or antibody-dependent cellular phagocytosis (ADCP) assays.

[0045] As used herein, the term "adverse event" (AE) refers to any untoward medical occurrence in a patient to whom a pharmaceutical formulation is administered, not necessarily having a causal relationship with treatment. In an embodiment of the present invention, AEs are rated on a three-level scale of increasing severity, using the following definitions: mild (Grade 1), referring to an AE that is easily tolerated by the subject, causes minimal discomfort, and does not interfere with daily activities; moderate (Grade 2), referring to an AE that is sufficiently unpleasant to interfere with normal daily activities and may require intervention; severe (Grade 3), referring to an AE that interferes with normal daily activities and usually requires treatment or other intervention. A serious AE (SAE) may be any AE occurring at any dose that results in any of the following outcomes: death, which is an outcome, not an event; life-threatening, which refers to an event where the patient is at risk of death at the time of the event's occurrence; life-threatening does not refer to an event that may have caused death if it had been more severe; hospitalization for treatment, i.e., unplanned overnight hospitalization, or extension of an existing hospitalization; permanent or significant impairment or substantial destruction of the ability to perform normal life functions; congenital anomaly / birth defect; a significant medical event (as determined by the investigator) that may endanger the patient or require medical or surgical intervention to prevent one of the other outcomes listed above (e.g., emergency room or intensive treatment at home for allergic bronchospasm, or blood disorders or seizures that do not result in hospitalization). Hospitalization is a formal admission to a hospital. Hospitalization or extended hospitalization constitutes the criteria for an AE to be serious, but is not considered an SAE in itself. In the absence of an AE, hospitalization or extended hospital stay should not be reported as an SAE by the participating investigator. This may be the case in the following situations: the hospitalization or extended hospital stay is necessary for a procedure required by the protocol; or the hospitalization or extended hospital stay is part of a routine procedure followed by the central site (e.g., stent removal after a surgical procedure). This should be documented in the study file. Hospitalization for elective procedures for a pre-existing condition that did not worsen during the study is not considered an AE.

[0046] A complication occurring during hospitalization is an AE. If the complication prolongs the hospital stay or meets any of the other SAE criteria, the event is an SAE.

[0047] As used herein, the term "encephalitis" refers to inflammation of the brain, which may be due to infectious and non-infectious causes.As used herein, the term "meningoencephalitis" refers to a condition characterized by infection or inflammation of the brain meninges and brain.The diagnosis of encephalitis or meningoencephalitis can be determined by techniques known to those skilled in the art in view of the present disclosure, for example, clinical, neurological, and psychiatric examinations, biological sample collection, including blood and CSF sample collection, MRI scanning, and electroencephalography (EEG).

[0048] As used herein, the term "liposome" generally refers to lipid vesicles made from materials with high lipid content, e.g., phospholipids, cholesterol. The lipids in these vesicles are generally organized in the form of lipid bilayers. The lipid bilayers generally encapsulate a volume that is interspersed between multiple onion-like frameworks of lipid bilayers forming multilamellar lipid vesicles (MLVs) or contained within a central amorphous cavity. Lipid vesicles with a central amorphous cavity are unilamellar lipid vesicles, i.e., lipid vesicles with a single peripheral bilayer surrounding the cavity. Large unilamellar vesicles (LUVs) generally have a diameter of 100 nm to several micrometers, e.g., 100 to 200 nm or more, and small unilamellar lipid vesicles (SUVs) generally have a diameter of less than 100 nm, e.g., 20 to 100 nm, typically 15 to 30 nm.

[0049] As used herein, the term "tau" or "tau protein", also known as microtubule-associated protein tau, MAPT, neurofibrillary tangle protein, paired helical fibril-tau, PHF-tau, MAPTL, MTBT1, refers to an abundant central and peripheral nervous system protein with multiple isoforms. In the human central nervous system (CNS), six major tau isoforms exist, ranging in size from 352 to 441 amino acids long, due to alternative splicing (Hanger et al., Trends Mol Med. 15:112-9, 2009). Examples of tau include, but are not limited to, tau isoforms in the CNS, such as the longest tau isoform of 441 amino acids (4R2N), also named microtubule-associated protein tau isoform 2, with four repeats and two inserts, such as human tau isoform 2, whose amino acid sequence is represented in GenBank accession number NP_005901.2. Other examples of tau include the shortest (fetal) isoform (3R0N) of 352 amino acids long, also named microtubule-associated protein tau isoform 4, with three repeats and no insertion, such as human tau isoform 4, whose amino acid sequence is represented in GenBank Accession No. NP_058525.1. Examples of tau also include the "big tau" isoform expressed in peripheral nerves, which contains 300 additional residues (exon 4a). Friedhoff et al., Biochimica et Biophysica Acta 1502 (2000) 122-132. Examples of tau include human big tau, a 758 amino acid long protein encoded by a 6762 nucleotide long mRNA transcript (NM_016835.4), or its isoforms. The amino acid sequence of the exemplary human big tau is represented in GenBank Accession No. NP_058519.3. As used herein, the term "tau" includes homologs of tau from non-human species, such as Macaca Fascicularis (cynomolgus monkey), rhesus monkey, or Pan troglodytes (chimpanzee).As used herein, the term "tau" includes proteins that include mutations of full-length wild-type tau, such as point mutations, fragments, insertions, deletions, and splice variants. The term "tau" also includes post-translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, phosphorylation.

[0050] As used herein, the term "peptide" or "polypeptide" refers to a polymer composed of amino acid residues linked via peptide bonds, related naturally occurring structural variants, and non-naturally occurring synthetic analogs thereof. The term refers to peptides of any size, structure, or function. Typically, peptides are at least three amino acids in length. Peptides can be naturally occurring, recombinant, or synthetic peptides, or any combination thereof. Synthetic peptides can be synthesized, for example, using an automated polypeptide synthesizer. Examples of tau peptides include any peptide of tau protein that is about 5 to about 30 amino acids in length, preferably about 10 to about 25 amino acids in length, and more preferably about 16 to about 21 amino acids in length. In this disclosure, peptides are listed from N-terminus to C-terminus using standard three- or one-letter amino acid abbreviations, with the phosphate residue indicated by "p". Examples of tau peptides useful in the present invention include, but are not limited to, tau peptides comprising any of the amino acid sequences of SEQ ID NOs: 1-12, or tau peptides having an amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to any of the amino acid sequences of SEQ ID NOs: 1-12.

[0051] As used herein, the term "phosphopeptide" or "phosphoepitope" refers to a peptide that is phosphorylated at one or more amino acid residues. Examples of tau phosphopeptides include any tau peptide that contains one or more phosphorylated amino acid residues.

[0052] The tau peptides of the present invention can be synthesized by solid-phase peptide synthesis or recombinant expression systems. Automated peptide synthesizers are commercially available from a number of suppliers, such as Applied Biosystems (Foster City, Calif.). Recombinant expression systems can include bacteria, such as E. coli, yeast, insect cells, or mammalian cells. Recombinant expression procedures are described by Sambrook et al., Molecular Cloning: A Laboratory Manual (CSHP Press, NY 2d ed., 1989).

[0053] In certain embodiments, the liposome comprises one or more tau peptides. In certain embodiments, the tau peptides in the liposome may be the same or different. In view of the present disclosure, any suitable tau peptide known to one of skill in the art may be used in the present invention. In certain embodiments, the one or more tau peptides comprise an amino acid sequence of one of SEQ ID NOs: 1-12. In other embodiments, the one or more tau peptides comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to an amino acid sequence of one of SEQ ID NOs: 1-12, and none of the amino acid residues are phosphorylated or one or more amino acid residues are phosphorylated.

[0054] In certain embodiments, the one or more tau peptides are tau phosphopeptides. In certain embodiments, the one or more tau phosphopeptides comprise an amino acid sequence of one of SEQ ID NOs: 1-3 or 5-12, or an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to an amino acid sequence of one of SEQ ID NOs: 1-3 or 5-12, and one or more of the indicated amino acid residues are phosphorylated. Preferably, the tau phosphopeptide comprises an amino acid sequence of one of SEQ ID NOs: 1-3. The tau peptide may be C-terminally amidated.

[0055] In an embodiment of the present application, tau peptide is presented on the surface of liposome. Tau peptide, preferably tau phosphopeptide, can be presented on the surface of liposome using methods known in the art in view of the present disclosure. For example, see the relevant disclosures in U.S. Patent Nos. 8,647,631 and 9,687,447, and International Patent Application No. PCT / US18 / 57286, the contents of which are incorporated herein by reference. In certain embodiments, one or more tau peptides, including phosphopeptides, further comprise one or more modifications, such as palmitoylation or dodecyl modification, that allow the tau peptide to be presented on the surface of liposome. Additional amino acid residues, such as Lys, Cys, or optionally Ser or Thr, can be added to the tau peptide to facilitate modification. It has been reported that the position of the lipid anchor induces different conformations of the peptide sequence (Hickman et al., J. Biol. Chem. vol. 286, No. 16, pp. 13966-13976, April 22, 2011). Without wishing to be bound by theory, it is believed that adding hydrophobic moieties to both ends may increase the pathological beta-sheet conformation of the tau peptide. Thus, one or more tau peptides further comprise hydrophobic moieties at both ends. The modified tau peptide may be amidated at the C-terminus. Preferably, the tau peptide presented on the surface of the liposome consists of one of the amino acid sequences of SEQ ID NO:27 to SEQ ID NO:29 and SEQ ID NO:31 to SEQ ID NO:38.

[0056] Examples of tau liposomes useful in the present invention include, but are not limited to, tau liposomes described in U.S. Pat. Nos. 8,647,631 and 9,687,447, and International Patent Application No. PCT / US18 / 57286, the disclosures of each of which are incorporated herein by reference in their entireties.

[0057] As used herein, the term "effective amount" refers to the amount of an active ingredient or component that induces a desired biological or medical response in a subject. The selection of a specific effective dose can be determined (e.g., through clinical trials) by a person skilled in the art based on the consideration of several factors, including the disease to be treated or prevented, the symptoms involved, the patient's weight, the patient's immune status, and other factors known by a person skilled in the art. The exact dose to be used in the formulation may also depend on the method of administration, the route of administration, the target site, the physiological state of the patient, other medications administered, and the severity of the disease, and should be determined according to the judgment of the practitioner and the circumstances of each patient. For example, the effective amount of tau phosphopeptide also depends on whether an adjuvant is also administered, with higher dosages being required in the absence of an adjuvant. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0058] In an embodiment of the present application, the effective amount of liposomes comprises a sufficient amount of tau phosphopeptide to increase the level of anti-phosphorylated tau antibodies without inducing severe adverse events such as encephalitis. In a particular embodiment, the effective amount of liposomes comprises an amount of tau phosphopeptide of about 25 nmole to about 750 nmole per dose, for example, about 29.7 nmole to about 742.5 nmole per dose, preferably about 90 nmole to about 715 nmole per dose, for example, about 89.1 nmole to about 712.8 nmole per dose, or about 90 nmole to about 535 nmole per dose, for example, about 89.1 nmole to about 534.6 nmole per dose, or about 90 nmole to about 275 nmole per dose, for example, about 89.1 nmole to about 267.3 nmole per dose. The amount of tau phosphopeptide administered can also be expressed by weight. For example, 29.7 nmoles per dose corresponds to 100 μg of tetrapalmitoylated tau phosphopeptide consisting of the amino acid sequence of SEQ ID NO: 28 per dose, 742.5 nmoles per dose corresponds to 2500 μg of tetrapalmitoylated tau phosphopeptide consisting of the amino acid sequence of SEQ ID NO: 28 per dose, 89.1 nmoles per dose corresponds to 300 μg of tetrapalmitoylated tau phosphopeptide consisting of the amino acid sequence of SEQ ID NO: 28 per dose, 712.8 nmoles per dose corresponds to 2400 μg of tetrapalmitoylated tau phosphopeptide consisting of the amino acid sequence of SEQ ID NO: 28 per dose, and 534.6 nmoles per dose corresponds to 1800 μg of tetrapalmitoylated tau phosphopeptide consisting of the amino acid sequence of SEQ ID NO: 28 per dose. The tetrapalmitoylated tau phosphopeptide has four lipid chains that allow the presentation of the tau phosphopeptide to the surface of the liposome. Doses of 300, 900 and 1800 μg of tetrapalmitoylated tau phosphopeptide consisting of the amino acid sequence of SEQ ID NO: 28 correspond to 169, 508 and 1016 μg, respectively, of the corresponding "naked" peptide without any lipid chains.

[0059] In an embodiment of the present application, an effective amount of liposomes comprises tau phosphopeptide in an amount of about 25 nmole to about 750 nmole per dose, e.g., about 25 nmole, about 30 nmole, about 35 nmole, about 40 nmole, about 45 nmole, about 50 nmole, about 55 nmole, about 60 nmole, about 65 nmole, about 70 nmole, about 75 nmole, about 80 nmole, about 85 nmole, about 90 nmole, about 95 nmole, about 100 nmole, about 125 nmole, about 150 nmole, about 175 nmole, about 200 nmole, about 225 nmole, about 260 nmole, about 280 nmole, about 300 nmole, about 350 nmole, about 40 nmole, about 45 nmole, about 50 nmole, about 55 nmole, about 60 nmole, about 65 nmole, about 70 nmole, about 75 nmole, about 80 nmole, about 85 nmole, about 90 nmole, about 95 nmole, about 100 nmole, about 125 nmole, about 150 nmole, about 175 nmole, about 200 nmole, about 225 nmole, about 265 nmole, about 285 nmole, about 300 nmole, about 350 nmole, about 350 nmole, about 350 nmole, about 400 nmole, about 450 nmole, about 450 nmole, about 50 nmole, about 55 nmole, about 60 nmole, about 65 nmole, about 70 nmole, about 75 nmole, about 80 nmole, about 85 nmole, about 90 nmole, about 95 nmole, about The tau phosphopeptide comprises one of the amino acid sequences of SEQ ID NOs: 1-3 or 5-12, about 250 nmole, about 275 nmole, about 300 nmole, about 325 nmole, about 350 nmole, about 375 nmole, about 400 nmole, about 425 nmole, about 450 nmole, about 475 nmole, about 500 nmole, about 525 nmole, about 550 nmole, about 575 nmole, about 600 nmole, about 625 nmole, about 650 nmole, about 675 nmole, about 700 nmole, about 725 nmole, about 750 nmole. Preferably, the tau phosphopeptide comprises one of the amino acid sequences of SEQ ID NOs: 27-29 and SEQ ID NOs: 31-38. More preferably, the tau phosphopeptide comprises the amino acid sequence of SEQ ID NO: 28.

[0060] In an embodiment of the present application, the effective amount of liposomes is from 100 μg to 2500 μg, 300 μg to 2400 μg, 300 μg to 1800 μg, or 300 μg to 900 μg per dose, for example, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 80 μg ... 0 μg, 900 μg, 1000 μg, 1100 μg, 1200 μg, 1300 μg, 1400 μg, 1500 μg, 1600 μg, 1700 μg, 1800 μg, 1900 μg, 2000 μg, 2100 μg, 2200 μg, 2300 μg, 2400 μg, or 2500 μg of tetrapalmitoylated tau phosphopeptide.

[0061] In an embodiment of the present application, the tau phosphopeptide is presented on the surface of the liposome. In an embodiment of the present application, the tau phosphopeptide comprises one of the amino acid sequences of SEQ ID NOs: 1 to 3 or 5 to 12. Preferably, the tau phosphopeptide consists of one of the amino acid sequences of SEQ ID NOs: 27 to 29 and SEQ ID NOs: 31 to 38. More preferably, the tau phosphopeptide consists of the amino acid sequence of SEQ ID NO: 28.

[0062] In another embodiment of the present application, the effective amount of liposomes further comprises a toll-like receptor 4 agonist in an amount of 30 μg to 900 μg, preferably 100 μg to 585 μg per dose. For example, the effective amount of liposomes can comprise a toll-like receptor 4 agonist in an amount of 30 μg, 50 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 330 μg, 360 μg, 390 μg, 420 μg, 450 μg, 480 μg, 500 μg, 520 μg, 540 μg, 560 μg, 580 μg, 600 μg, 700 μg, 800 μg, or 900 μg per dose.

[0063] In an embodiment of the present application, toll-like receptor 4 comprises 3D-(6-acyl)PHAD®.

[0064] In other embodiments of the present application, the effective amount of liposomes further comprises 25 μg to 625 μg, preferably 75 μg to 550 μg, for example 75 μg to 450 μg, 80 μg to 540 μg, 82.5 μg to 535 μg, 85 μg to 530 μg, 87.5 μg to 525 μg, or 90 μg to 520 μg of helper T cell epitopes per dose. For example, an effective amount of liposomes can contain helper T cell epitopes in an amount of 25 μg, 50 μg, 70 μg, 72.5 μg, 75 μg, 77.5 μg, 80 μg, 82.5 μg, 85 μg, 87.5 μg, 90 μg, 100 μg, 125 μg, 150 μg, 175 μg, 200 μg, 225 μg, 250 μg, 275 μg, 300 μg, 325 μg, 350 μg, 375 μg, 400 μg, 425 μg, 450 μg, 475 μg, 500 μg, 525 μg, 550 μg, 575 μg, 600 μg, or 625 μg per dose.

[0065] In other embodiments of the present application, the effective amount of liposomes further comprises a helper T cell epitope in an amount of about 3 nmole to about 105 nmole per dose, e.g., about 4 nmole, about 5 nmole, about 6 nmole, about 7 nmole, about 8 nmole, about 9 nmole, about 10 nmole, about 15 nmole, about 20 nmole, about 25 nmole, about 30 nmole, about 35 nmole, about 40 nmole, about 45 nmole, about 50 nmole, about 55 nmole, about 60 nmole, about 65 nmole, about 70 nmole, about 75 nmole, about 80 nmole, about 85 nmole, about 90 nmole, about 95 nmole, about 100 nmole, or about 105 nmole per dose.

[0066] In an embodiment of the present application, the helper T cell epitope is a T50 helper T cell epitope consisting of the amino acid sequence of SEQ ID NO: 13, a T46 helper T cell epitope consisting of the amino acid sequence of SEQ ID NO: 14, a T48 helper T cell epitope consisting of the amino acid sequence of SEQ ID NO: 15, a T51 helper T cell epitope consisting of the amino acid sequence of SEQ ID NO: 16, or a T52 helper T cell epitope consisting of the amino acid sequence of SEQ ID NO: 17, and preferably, the helper T cell epitope is a T50 helper T cell epitope consisting of the amino acid sequence of SEQ ID NO: 13.

[0067] In certain embodiments, the effective amount of liposomes further comprises lipidated CpG oligonucleotide in an amount of 50 μg to 1250 μg, preferably 100 μg to 1000 μg, e.g., 150 μg to 800 μg, 150 to 900 μg, 125 μg to 950 μg, or 150 μg to 850 μg per dose. For example, an effective amount of liposomes can contain lipidated CpG oligonucleotides in an amount of 50 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 1000 μg, 1050 μg, 1100 μg, 1200 μg, or 1250 μg per dose.

[0068] In an embodiment of the present application, the lipidated CpG oligonucleotide is a CpG oligonucleotide comprising the nucleotide sequence of one of SEQ ID NOs: 18-22, preferably the lipidated CpG oligonucleotide is a CpG oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 18. In an embodiment of the present application, the lipidated CpG oligonucleotide is a CpG oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 18, having one or more phosphorothioate internucleotide linkages and covalently linked to cholesterol via a linker comprising polyethylene glycol (PEG).

[0069] In embodiments, an effective amount of liposomes comprises 50 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 1000 μg, 1050 μg, 1100 μg, 1200 μg, or 1250 μg of CpG oligonucleotides covalently linked to cholesterol via a PEG linker per dose.

[0070] As used herein, a "sustained immune response" or a "sustainable immune response" refers to an immune response that lasts for at least 6 weeks after the initial administration of an effective amount of liposomes. In an embodiment of the present application, a "sustained immune response" is a sustained antibody response that lasts for at least 6 weeks, at least 12 weeks, at least 24 weeks, at least 36 weeks, at least 48 weeks, at least 60 weeks, at least 72 weeks or more, and the antibody response can be characterized by the presence of anti-phosphorylated tau IgG, anti-phosphorylated tau IgM, or anti-ePHF. Anti-phosphorylated tau IgG, anti-phosphorylated tau IgM, and anti-ePHF can be detected and measured by any method known to those skilled in the art, including those described herein.

[0071] As used herein, a "sustained antibody response" refers to an antibody response that is maintained at a level equal to or higher than a defined threshold level for a certain period of time after the first administration of an effective amount of liposomes, the defined threshold level being higher than the baseline level measured before the first administration of an effective amount of liposomes. In some embodiments, the baseline level is determined based on the average measured level of antibody titer before the first administration, preferably two measurements are taken. In one embodiment, the antibody response comprises a specific IgG antibody response directed against p-tau, and the defined threshold level is at least 1.5 times or more than the baseline level, e.g., at least 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 times or more than the baseline level. In another embodiment, the antibody response comprises an IgG immune response against ePHF, and the defined threshold level is at least 2.0 times or more than the baseline level, e.g., at least 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 times or more than the baseline level.

[0072] In certain embodiments, the human subject is in need of treatment for a neurodegenerative disease, disorder, or condition.

[0073] As used herein, "neurodegenerative disease, disorder, or condition" includes any neurodegenerative disease, disorder, or condition known to one of skill in the art in light of the present disclosure. Examples of neurodegenerative diseases, disorders, or conditions include neurodegenerative diseases or disorders caused by or associated with the formation of neurofibrillary lesions, such as tau-associated diseases, disorders, or conditions referred to as tauopathies. In certain embodiments, the neurodegenerative disease, disorder, or condition includes Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, Pugilist dementia, Down's syndrome, Gerstmann-Straussler-Scheinker disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis, Parkinson's dementia complex of Guam, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, amyotrophic lateral sclerosis with Lewy dementia, and the like. The neurodegenerative disease, disorder, or condition may be any disease or disorder that exhibits coexistence of tau and amyloid pathology, including, but not limited to, cerebrospinal fluid (CSF) sclerosis, diffuse neurofibrillary tangles with calcification, frontotemporal dementia, preferably frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar dementia, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle only dementia, postencephalitic parkinsonism, myotonic dystrophy, chronic traumatic encephalopathy (CTE), primary age-related tauopathy (PART), cerebrovascular disease, or dementia with Lewy bodies (LBD). In certain embodiments, the neurodegenerative disease, disorder, or condition is Alzheimer's disease or another tauopathy. In a preferred embodiment, the neurodegenerative disease, disorder, or condition is Alzheimer's disease.

[0074] The clinical course of Alzheimer's disease can be divided into multiple stages according to the progression pattern of cognitive and functional impairment.Stages can be defined using assessment scales known in the art, including, for example, the NIA-AA research framework.See, for example, Dubois et al., Alzheimer's & Dementia 12 (2016) 292-323; Dubois et al., Lancet Neurol 2014; 13: 614-29; Jack et al., Alzheimer's & Dementia 14 (2018) 535-562, the contents of each of which are incorporated herein by reference in their entirety.

[0075] In preferred embodiments, the neurodegenerative disease, disorder, or condition is early Alzheimer's disease, mild cognitive impairment (MCI) due to Alzheimer's disease, mild Alzheimer's disease, or mild to moderate Alzheimer's disease.

[0076] In some embodiments, the subject in need of treatment is amyloid positive in the brain, but has not yet shown significant cognitive impairment.Amyloid deposits in the brain can be detected using methods known in the art, such as PET scanning, immunoprecipitation mass spectrometry, or other methods.

[0077] As used herein, the term "toll-like receptor" or "TLR" refers to a class of pattern recognition receptor (PRR) proteins that play an important role in the innate immune response. TLRs recognize pathogen-associated molecular patterns (PAMPs) from microbial pathogens, e.g., bacteria, fungi, parasites, and viruses, which can be distinguished from host molecules. TLRs are transmembrane proteins that typically function as dimers and are expressed by cells involved in the innate immune response, including antigen-presenting dendritic cells and phagocytic macrophages. There are at least ten human TLR family members, namely TLR1-TLR10, and at least twelve murine TLR family members, namely TLR1-TLR9 and TLR11-TLR13, which differ in the types of antigens they recognize. For example, TLR4 recognizes lipopolysaccharide (LPS), a component present in many Gram-negative bacteria, as well as viral proteins, polysaccharides, and endogenous proteins, such as low-density lipoproteins, beta-defensins, and heat shock proteins, while TLR9 is a nucleotide-sensing TLR that is activated by unmethylated cytosine-phosphate-guanine (CpG) single- or double-stranded dinucleotides, which are abundant in prokaryotic genomes but rare in vertebrate genomes. Activation of TLRs triggers a series of signaling events, leading to the production of type I interferons (IFNs), inflammatory cytokines, and chemokines, and the induction of immune responses. Ultimately, this inflammation also activates the adaptive immune system, which then leads to the elimination of invading pathogens and infected cells.

[0078] As used herein, the term "agonist" refers to a molecule that binds to one or more TLRs and induces a receptor-mediated response. For example, an agonist can induce, activate, elevate, activate, facilitate, enhance, or upregulate the activity of the receptor. Such activity is referred to as "agonist activity." For example, a TLR4 or TLR9 agonist can activate or increase cell signaling through the bound receptor. Agonists include, but are not limited to, nucleic acids, small molecules, proteins, carbohydrates, lipids, or any other molecules that bind to or interact with a receptor. Agonists can mimic the activity of natural receptor ligands. Agonists can match these natural receptor ligands in terms of sequence, conformation, charge, or other features that allow the agonist to be recognized by the receptor. This recognition can result in physiological and / or biochemical changes in the cell such that the cell responds to the presence of the agonist in the same way as if the natural receptor ligand were present. In certain embodiments, the toll-like receptor agonist is at least one of a toll-like receptor 4 agonist and a toll-like receptor 9 agonist.

[0079] As used herein, the terms "induce" and "stimulate" and variations thereof refer to any measurable increase in cellular activity. Induction of immune response can include, for example, activation, proliferation, or maturation of a population of immune cells, increasing cytokine production, and / or other indicators of increased immune function. In certain embodiments, induction of immune response can include increasing B cell proliferation, producing antigen-specific antibodies, increasing antigen-specific T cell proliferation, improving dendritic cell antigen presentation, and / or increasing the expression of certain cytokines, chemokines, and costimulatory markers.

[0080] As used herein, the term "toll-like receptor 4 agonist" refers to any compound that acts as an agonist of TLR4. In view of the present disclosure, any suitable toll-like receptor 4 agonist known to one of skill in the art can be used in the present invention. Examples of toll-like receptor 4 ligands useful in the present invention include TLR4 agonists, including but not limited to monophosphoryl lipid A (MPLA). As used herein, the term "monophosphoryl lipid A" or "MPLA" refers to a modified form of lipid A, the biologically active portion of the gram-negative bacterial lipopolysaccharide (LPS), an endotoxin. MPLA is less toxic than LPS, but maintains immunostimulatory activity. As a vaccine adjuvant, MPLA stimulates both cellular and humoral responses to vaccine antigens. Examples of MPLA include, but are not limited to, 3-O-desacyl-4'-monophosphoryl lipid A, monophosphoryl hexaacyl lipid A, 3-deacylated (synthetic) (also referred to as 3D-(6-acyl)PHAD®), monophosphoryl 3-deacylated lipid A, and structurally related variants thereof.MPLA useful in the present invention can be obtained using methods known in the art or from commercial sources, such as 3D-(6-acyl)PHAD®, PHAD®, PHAD®-504, 3D-PHAD® from Avanti Polar Lipids (Alabaster, Alabama, USA), or MPL® from various commercial sources.In certain embodiments, the toll-like receptor 4 agonist is MPLA. In certain embodiments, the liposomes comprising the tau phosphopeptide and the toll-like receptor 4 agonist also contain a helper T cell epitope capable of binding to most or all HLA DR (human leukocyte antigen-antigen D related) molecules. The helper T cell epitope then binds to the CD4 +It can activate T cells and provide essential maturation and survival signals to tau-specific B cells. Tau liposomes can be used to generate high quality antibodies against p-tau antigen in allogeneic or heterogeneous immunization schemes where liposomes are used in prime and / or boost.

[0081] As used herein, the term "helper T cell epitope" refers to a polypeptide that contains an epitope capable of recognition by a helper T cell. Examples of helper T cell epitopes include, but are not limited to, tetanus toxoid (e.g., the P2 and P30 epitopes, also designated T2 and T30, respectively), Hepatitis B surface antigen, cholera toxin B, diphtheria toxoid, measles virus F protein, Chlamydia trachomatis major outer membrane protein, Plasmodium falciparum circumsporozoite T, P. falciparum CS antigen, Schistosoma mansoni triosephosphate isomerase, Bordetella pertussis, Clostridium tetani, Pertusaria trachythallina, Escherichia coli TraT, and influenza virus hemagglutinin (HA).

[0082] In view of the present disclosure, any suitable helper T cell epitope known to one of skill in the art can be used in the present invention. In certain embodiments, the helper T cell epitope comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 23 to SEQ ID NO: 26. Preferably, the helper T cell epitope comprises two or more of the amino acid sequences of SEQ ID NO: 23 to SEQ ID NO: 26 fused together via a linker, e.g., a peptide linker comprising one or more amino acids, e.g., Val (V), Ala (A), Arg (R), Gly (G), Ser (S), Lys (K). The length of the linker can vary, preferably 1 to 5 amino acids. Preferably, the helper T cell epitope comprises three or more of the amino acid sequences of SEQ ID NO: 23 to SEQ ID NO: 26 fused together via one or more linkers selected from the group consisting of VVR, GS, RR, RK. The helper T cell epitope may be amidated at its C-terminus.

[0083] In an embodiment of the present application, the helper T cell epitope can be immobilized by incorporating, for example covalently binding, a hydrophobic moiety to the liposome surface, where the hydrophobic moiety is an alkyl group, a fatty acid, a triglyceride, a diglyceride, a steroid, a sphingolipid, a glycolipid, or a phospholipid, particularly an alkyl group or a fatty acid, particularly having a carbon backbone of at least 3 carbon atoms, particularly at least 4 carbon atoms, particularly at least 6 carbon atoms, particularly at least 8 carbon atoms, particularly at least 12 carbon atoms, particularly at least 16 carbon atoms. In one embodiment of the present invention, the hydrophobic moiety is palmitic acid. Alternatively, the helper T cell epitope can be encapsulated in a liposome. In a particular embodiment, the helper T cell epitope is encapsulated in a liposome.

[0084] In view of the present disclosure, the helper T cell epitope can be modified using methods known in the art for the desired location in the liposome. In certain embodiments, the helper T cell epitope useful in the present invention comprises the amino acid sequence of one of SEQ ID NO: 39 to SEQ ID NO: 44. Preferably, the helper T cell epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 17.

[0085] In certain embodiments, the liposomes comprising tau phosphopeptides and toll-like receptor 4 agonists also comprise toll-like receptor 9 agonists. As used herein, the term "toll-like receptor 9 agonists" refers to any compound that acts as an agonist of TLR9. In view of the present disclosure, any suitable toll-like receptor 9 agonists known to those skilled in the art can be used in the present invention. Examples of toll-like receptor 9 ligands useful in the present invention include TLR9 agonists, including but not limited to CpG oligonucleotides.

[0086] As used herein, the term "CpG oligonucleotide", "CpG oligodeoxynucleotide", or "CpG ODN" refers to an oligonucleotide that contains at least one CpG motif. As used herein, "oligonucleotide", "oligodeoxynucleotide" or "ODN" refers to a polynucleotide formed from a plurality of linked nucleotide units. Such oligonucleotides can be obtained from existing nucleic acid sources or can be produced by synthetic methods. As used herein, the term "CpG motif" refers to a nucleotide sequence that contains unmethylated cytosine-phosphate-guanine (CpG) dinucleotides (i.e., cytosine (C) followed by guanine (G)) linked by phosphate bonds or phosphodiester backbones or other internucleotide linkages.

[0087] In certain embodiments, the CpG oligonucleotide is lipidated, ie, conjugated (covalently linked) to a lipid moiety.

[0088] As used herein, "lipid moiety" refers to a moiety that contains a lipophilic structure. Lipid moieties, such as alkyl groups, fatty acids, triglycerides, diglycerides, steroids, sphingolipids, glycolipids, or phospholipids, especially sterols such as cholesterol or fatty acids, when bound to highly hydrophilic molecules, such as nucleic acids, can substantially improve plasma protein binding and, as a result, the circulatory half-life of the hydrophilic molecule. In addition, binding to certain plasma proteins, such as lipoproteins, has been shown to increase uptake into certain tissues that express the corresponding lipoprotein receptors (e.g., LDL receptor, HDL receptor, or scavenger receptor SR-B1). In particular, lipid moieties conjugated with phosphopeptides and / or CpG oligonucleotides allow the peptides and / or oligonucleotides to be fixed to the membrane of liposomes via the hydrophobic moiety.

[0089] In certain embodiments, in view of the present disclosure, the CpG oligonucleotide can include any suitable internucleotide linkage.

[0090] As used herein, the term "internucleotide linkage" refers to a chemical linkage consisting of a phosphorus atom and a charged or neutral group between adjacent nucleosides that connects two nucleotides through their sugars. Examples of internucleotide linkages include phosphodiester (po), phosphorothioate (ps), phosphorodithioate (ps2), methylphosphonate (mp), and methylphosphorothioate (rp). Phosphorothioate, phosphorodithioate, methylphosphonate, and methylphosphorothioate are stabilized internucleotide linkages, and phosphodiester is a naturally occurring internucleotide linkage. Oligonucleotide phosphorothioates are typically synthesized as a random racemic mixture of Rp and Sp phosphorothioate linkages.

[0091] In view of the present disclosure, any suitable CpG oligonucleotide known to those skilled in the art can be used in the present invention.Examples of such CpG oligonucleotides include, but are not limited to, CpG2006 (also known as CpG7909) (SEQ ID NO: 18), CpG1018 (SEQ ID NO: 19), CpG2395 (SEQ ID NO: 20), CpG2216 (SEQ ID NO: 21) or CpG2336 (SEQ ID NO: 22).

[0092] The CpG oligonucleotide can be lipidated using methods known in the art in view of the present disclosure. In some embodiments, the CpG oligonucleotide is directly covalently linked to a cholesterol molecule. In some embodiments, the 3' end of the CpG oligonucleotide is covalently linked to a cholesterol molecule by a phosphate bond, optionally via a PEG linker. In some embodiments, the 5' end of the CpG oligonucleotide is covalently linked to a cholesterol molecule by a phosphate bond, optionally via a PEG linker. Other lipophilic moieties can also be covalently linked to the 5' or 3' end of the CpG oligonucleotide. For example, the CpG oligonucleotide can be covalently linked to a lipid anchor of the same length as the phospholipid from the liposome, such as a palmitic acid chain (using activated Pal-OH or analogs for coupling), or two palmitic acids (using activated 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) or analogs for coupling), optionally via a PEG linker. See, for example, the relevant disclosure in US Patent No. 7,741,297, the contents of which are incorporated herein by reference. The length of the PEG can vary, for example, from 1 to 5 PEG units.

[0093] Other linkers can also be used to covalently link CpG oligonucleotides to lipophilic moieties (e.g., cholesterol molecules), including, but not limited to, alkyl spacers having 3-12 carbons. A short linker compatible with oligonucleotide chemistry is required as an aminodiol. In some embodiments, no linker is used for covalent binding. See, for example, Ries et al., "Convenient synthesis and application of versatile nucleic acid lipid membrane anchors in the assembly and fusion of liposomes," Org. Biomol. Chem., 2015, 13, 9673, the relevant disclosure of which is incorporated herein by reference.

[0094] In a particular embodiment, the lipidated CpG oligonucleotide useful in the present invention comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22, the nucleotide sequence comprises one or more phosphorothioate internucleotide linkages, and the nucleotide sequence is covalently linked to at least one cholesterol via a linker. In a preferred embodiment, the lipidated CpG oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 18, has one or more phosphorothioate internucleotide linkages, and is covalently linked to cholesterol. Any suitable linker can be used to covalently link the CpG oligonucleotide to the cholesterol molecule. Preferably, the linker comprises polyethylene glycol (PEG).

[0095] In certain embodiments, the liposome further comprises one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol.

[0096] In certain embodiments, the liposome further comprises a buffer. In view of the present disclosure, any suitable buffer known to those skilled in the art can be used in the present invention. In one embodiment, the liposome comprises phosphate buffered saline. In certain embodiments, the buffer comprises histidine and sucrose.

[0097] An exemplary liposome for use in the present invention comprises a tau tetrapalmitoylated phosphopeptide (pTau peptide T3, SEQ ID NO: 28) that is presented on the surface of the liposome via two palmitic acids at each end of the tau peptide; a TLR-9 ligand comprising lipidated CpG (adjuvant CpG7909 (CpG2006), SEQ ID NO: 18) that is incorporated into the liposome membrane via a cholesterol molecule, where the cholesterol molecule is covalently linked to the CpG via a PEG linker. a TLR-9 ligand that is bound to the liposome; a TLR-4 ligand that is incorporated into the membrane (monophosphoryl lipid A (e.g., 3D-(6-acyl)PHAD®)); an encapsulated helper T cell epitope (PAN-DR binding site T50, SEQ ID NO: 13); and 1,2-dimyristoyl-sn-glycero-3-phospho-choline (DMPC), 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DMPG), and cholesterol as lipid components of the liposome.

[0098] The liposomes of the present invention can be made using methods known in the art in view of the present disclosure. The optimal ratio of each component of the liposome can be determined by techniques known to those of skill in the art in view of the present disclosure.

[0099] The liposomes can be administered by any suitable means for prophylactic and / or therapeutic treatment. In a preferred embodiment, the liposomes are administered by subcutaneous or intramuscular injection. Intramuscular injections are most typically performed into the arm or leg muscles.

[0100] In one general aspect, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of liposomes together with a pharma- ceutically acceptable excipient and / or carrier. Pharmaceutically acceptable excipients and / or carriers are well known in the art (see Remington's Pharmaceutical Science (15th ed.), Mack Publishing Company, Easton, Pa., 1980). The preferred method of making the pharmaceutical composition depends on the intended method of administration and therapeutic application. The composition may contain a pharma- ceutically acceptable non-toxic carrier or diluent, defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also contain other carriers, adjuvants, or non-toxic, non-therapeutic, and non-immunogenic stabilizers, and the like. It will be understood that the characteristics of the carrier, excipient, or diluent may depend on the route of administration for a particular application.

[0101] The target antigens of vaccines are located in the brain, which is separated from the circulation by a specialized cellular structure called the blood-brain barrier (BBB). The BBB limits the passage of substances from the circulation to the brain. This prevents the entry of toxins, microorganisms, etc. into the central nervous system. The BBB also has the potentially more undesirable effect of preventing the efficient entry of immune mediators (e.g., antibodies) into the interstitial and cerebrospinal fluids that surround the brain.

[0102] Approximately 0.1% of antibodies present in the systemic circulation cross the BBB and enter the brain. This suggests that the systemic titer induced by a vaccine targeting a CNS antigen must be at least 1000 times greater than the minimum effective titer to be effective in the brain. The minimum titer of antibodies in serum required to be effective is not readily apparent. In addition, not only the quantity but also the quality (e.g., avidity) of the immune response must be considered for safe and effective immunotherapy targeting CNS disorders, such as neurodegenerative diseases, disorders, or conditions.

[0103] The avidity of an antibody can be measured by an avidity index using methods known in the art in view of the present disclosure. The titer of an antibody against a particular antigen is measured at two different concentrations of coated antigen: one at a saturation concentration where all antibodies can bind to the antigen, and the other at a low concentration where only antibodies with very high binding capacity can bind to the antigen. As used herein, "avidity index" refers to the ratio of the levels of antibody titer measured at low and high density coatings of antigen. For example, the avidity of an antibody against an antigen, such as ePHF or pTau, can be measured at multiple different time points after one immunization or after multiple immunizations to assess whether the avidity (as measured by the avidity index) increases over time. As used herein, an antibody with "increased avidity" or "increased binding avidity" against an antigen refers to an antibody with an increased avidity index against the antigen over time during the course of treatment or immunization. An increase in avidity suggests possible affinity maturation of the antibody.

[0104] Therefore, in certain embodiments, the pharmaceutical composition of the present invention further comprises one or more suitable adjuvants to achieve a desired immune response in the subject. A suitable adjuvant can be administered before, after, or simultaneously with the administration of liposomes. A preferred adjuvant enhances the intrinsic response to the immunogen without causing conformational changes in the immunogen that affect the qualitative form of the response. Examples of adjuvants are aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate. Such adjuvants can be used with or without other specific immunostimulants, such as MPLAs (3 de-O-acylated monophosphoryl lipid A (MPL™), monophosphoryl hexaacyl lipid A 3-deacylated synthetic (3D-(6-acyl)PHAD™, PHAD™, PHAD™-504, 3D-PHAD™) lipid A), polymeric or monomeric amino acids, such as polyglutamic acid or polylysine. Such adjuvants can be used with or without other specific immunostimulants, such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™), or other bacterial cell wall components.Oil-in-water emulsions include MF59, containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE), which is formulated into submicrometer particles using a microfluidizer (see WO 90 / 14837); SAF, containing 10% squalene, 0.4% Tween 80, 5% pluronic block polymer L121, and thr-MDP, which is microfluidized into a submicrometer emulsion or vortexed to generate a larger particle size emulsion; and Ribi™ Adjuvant System (RAS) (Ribi ImmunoChem, Hamilton, Mont.) 0.2% Tween 80, and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL™), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL™+CWS (Detox™). Other adjuvants include complete Freund's adjuvant (CFA), and cytokines such as interleukins (IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF), and tumor necrosis factor (TNF).

[0105] As used herein, the term "in combination" refers to the use of more than one therapy in the context of administering two or more therapies to a subject.The use of the term "in combination" does not limit the order in which the therapies are administered to a subject.For example, a first therapy (e.g., a composition described herein) can be administered to a subject before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks), simultaneously, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) the administration of a second therapy. In some embodiments of the present invention, the pharmaceutical composition of the present invention can be used in combination with biologically active substances, such as known compounds, used in medicine for tauopathy and / or amyloidosis, a group of diseases and disorders related to amyloid or amyloid-like proteins, such as amyloid β proteins, involved in Alzheimer's disease.Other biologically active compounds can include neurotransmitter enhancers, psychotherapeutic drugs, acetylcholinesterase inhibitors, calcium channel blockers, biogenic amines, benzodiazepine tranquilizers, acetylcholine synthesis, storage or release enhancers, acetylcholine postsynaptic receptor agonists, monoamine oxidase-A or -B inhibitors, N-methyl-0-aspartic acid glutamate receptor antagonists, nonsteroidal anti-inflammatory drugs, antioxidants, and serotonergic receptor antagonists.In particular, the other biologically active compound may be selected from the group consisting of compounds that fight against oxidative stress, anti-apoptotic compounds, metal chelators, inhibitors of DNA repair, such as pirenzepine and metabolites, 3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonic acid (1,3PDS), secretase activators and v-secretase inhibitors, tau protein, neurotransmitters, β-sheet breakers, anti-inflammatory molecules, or cholinesterase inhibitors (ChEIs), such as tacrine, rivastigmine, donepezil, and / or galantamine, and other drugs and nutritional supplements, together with the therapeutic vaccine of the present invention, and optionally with a pharma-ceutically acceptable carrier and / or diluent and / or excipient. In a further embodiment, the other biologically active compound may include niacin or memantine, together with the liposome of the present invention, and optionally with a pharma-ceutically acceptable carrier and / or diluent and / or excipient. In yet another embodiment of the present invention, other compounds may be included with the liposomes of the present invention, such as "atypical antipsychotics" that treat positive or negative psychotic symptoms, including hallucinations, delusions, thought disorder (manifested by prominent scattered, derailed, and off-topic thoughts), and bizarre or disorganized behavior, as well as anhedonia, affective flattening, apathy, and social withdrawal, such as clozapine, ziprasidone, risperidone, aripiprazole, or olanzapine.Other compounds which may be suitably used in combination with the pharmaceutical compositions of the invention are described, for example, in WO 2004 / 058258 (see in particular pages 16 and 17), including therapeutic drug targets (pages 36-39), alkanesulfonic acids and alkanol sulfates (pages 39-51), cholinesterase inhibitors (pages 51-56), NMDA receptor antagonists (pages 56-58), estrogens (pages 58-59), non-steroidal anti-inflammatory drugs (pages 60-61), antioxidants (pages 61-62), peroxisome proliferator-activated receptors. These include PPAR agonists (pages 63-67), cholesterol lowering agents (pages 68-75); amyloid inhibitors (pages 75-77), amyloid formation inhibitors (pages 77-78), metal chelators (pages 78-79), antipsychotics and antidepressants (pages 80-82), nutritional supplements (pages 83-89), as well as compounds that increase the availability of biologically active substances in the brain (see pages 89-93) and prodrugs (pages 93 and 94), this document is incorporated herein by reference, with particular reference to the compounds mentioned on the pages indicated above.

[0106] The timing of administration can vary widely, from once a day to once a year to once every ten years. A typical regimen consists of an immunization followed by booster injections spaced at time intervals, for example 1-24 weeks apart. Another regimen consists of an immunization followed by booster injections 1, 2, 4, 6, 8, 10, and 12 months later. Another regimen involves lifelong injections every two months. Alternatively, booster injections may be irregular, as indicated by monitoring of the immune response.

[0107] It is easily understood by those skilled in the art that the regimen for priming and boosting administration can be adjusted based on the immune response measured after administration.For example, the boosting composition is generally administered several weeks or months after the administration of the priming composition, for example, about 1 week, or 2 weeks, or 3 weeks, or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks, or 36 weeks, or 40 weeks, or 44 weeks, or 48 weeks, or 52 weeks, or 56 weeks, or 60 weeks, or 64 weeks, or 68 weeks, or 72 weeks, or 76 weeks, or 1 to 2 years after the administration of the priming composition.

[0108] In certain embodiments, one or more boosts may be administered. The antigens in each priming and boosting composition need not be identical, no matter how many boosting compositions are used, but should have common antigenic determinants or be substantially similar to each other.

[0109] As known to those skilled in the art, immunogenicity, boostability and sustainability are important considerations for the efficacy of a vaccine. It has been discovered in the present invention that administration of an effective amount of liposomes described herein can induce a strong antibody response against p-tau in a patient in need thereof, for example, a patient in need of treating Alzheimer's disease (e.g., mild to moderate Alzheimer's disease or early Alzheimer's disease) or mild cognitive impairment (MCI) due to Alzheimer's disease. The antibody response is sustainable, for example, lasting at least six weeks after the first administration of liposomes. The antibody response is also boosted by one or more subsequent boosting administrations. As used herein, "boosted" in the context of an antibody response refers to an antibody response that is maintained or strengthened after a subsequent administration, when measured at least two weeks after administration of the subsequent administration. For example, an antibody response is "boosted" by a subsequent administration if the antibody titer measured two weeks after the subsequent administration is increased compared to the antibody titer before the subsequent administration.

[0110] The pharmaceutical compositions of the present invention can be formulated according to methods known in the art in view of the present disclosure. The optimal ratio of each component in the composition can be determined by techniques known to those skilled in the art in view of the present disclosure.

[0111] In a preferred embodiment of the invention, administration of tau peptides via administration of a pharmaceutical composition according to an embodiment of the invention induces an active immune response in a subject, e.g., an antibody response against the tau peptides and pathological forms of tau, thereby facilitating clearance of associated tau aggregates, slowing the progression of tau pathology-related behaviors, and / or treating the underlying tauopathy.

[0112] Tau is a human "self" protein. This means that, in principle, all lymphocytes bearing receptors specific for tau should have been eliminated during development (central tolerance) or rendered refractory by peripheral tolerance mechanisms. This problem has proven to be a major obstacle in the development of vaccines against self or "altered self" proteins (e.g. tumor antigens). Generating high-quality antibodies against an antigen (self or infectious) requires the development of a large number of immune cells, not only antibody-producing B lymphocytes, but also CD4 + It also requires the action of T "helper" lymphocytes. CD4 + T cells provide vital survival and maturation signals to B lymphocytes and CD4 + T cell deficient animals are severely immunosuppressed. + T cells are also subject to immune tolerance mechanisms, and a further obstacle in generating strong anti-self (e.g., anti-tau) antibody responses is the tau-reactive CD4 + T cells are also likely to be rare or absent in the human / animal repertoire.

[0113] In this aspect of the invention, the immune response involves the generation of a beneficial humoral (antibody-mediated) response directed to the tau peptide, and a cellular (mediated by antigen-specific T cells or their secreted products) response directed to the T cell epitope or immunogenic carrier.

[0114] As used herein, tau pathology associated behavioral phenotypes include, but are not limited to, cognitive impairment, early personality changes and disinhibition, apathy, apraxia, mutism, perseveration, stereotyped movements / behaviors, lip-smacking, disorganization, inability to plan or organize sequential tasks, selfish behavior / callousness, antisocial features, lack of empathy, lack of consistency, agrammatic speech with frequent erroneous errors but relatively preserved comprehension, impaired comprehension and word finding, slowly progressive gait unsteadiness, retropulsion, freezing, frequent falls, non-levodopa responsive axial rigidity, supranuclear gaze palsy, square wave eye movements, slow vertical saccadic eye movements, pseudobulbar palsy, limb apraxia, dystonia, cortical sensory loss, and tremor.

[0115] When carrying out the method of the present invention, in certain embodiments of the present invention, it is preferable to select subjects who have or are at risk of having Alzheimer's disease or other tauopathies, who have tau aggregates in their brains, or who exhibit tangle-associated behavioral phenotypes before administering the immunogenic peptide or antibody of the present invention.Treatable subjects include individuals who are at risk of disease but do not show symptoms, and patients who currently show symptoms.In the case of Alzheimer's disease, virtually everyone is at risk of suffering from Alzheimer's disease.Therefore, the method can be administered prophylactically to the general population without the need for any evaluation of the risk of the subject patient.The method is particularly useful for individuals with known genetic risk of Alzheimer's disease to prevent or treat the disease.Such individuals include individuals who have relatives who have experienced the disease, and individuals whose risk is determined by analysis of genetic or biochemical markers. In a preferred embodiment, the subject needs to treat Alzheimer's disease, preferably early stage Alzheimer's disease, mild cognitive impairment (MCI) due to Alzheimer's disease, mild Alzheimer's disease, or mild to moderate Alzheimer's disease.In another preferred embodiment, the subject needs to prevent Alzheimer's disease, preferably preclinical stage Alzheimer's disease, early stage Alzheimer's disease, mild cognitive impairment (MCI) due to Alzheimer's disease, mild Alzheimer's disease, or mild to moderate Alzheimer's disease.Preclinical stage Alzheimer's disease is the stage before early stage Alzheimer's disease.

[0116] In asymptomatic patients, treatment can be started at any age (e.g., 10, 20, 30 years old). However, it is usually not necessary to start treatment until the patient reaches 40, 50, 60, or 70 years old. Treatment typically involves multiple dosages over a period of time. Treatment can be monitored over time by assaying antibody, or activated T cell or B cell response to the therapeutic agent. If response declines, booster dosages are indicated.

[0117] In preventative applications, pharmaceutical compositions containing tau peptides are administered to patients susceptible to or otherwise at risk of Alzheimer's disease or other tauopathies in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of the disease, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and pathological endophenotypes manifested during the development of the disease. In therapeutic applications, pharmaceutical compositions containing tau peptides are administered to patients suspected of or already suffering from such diseases in an amount sufficient to treat or at least partially halt the symptoms of the disease (biochemical, histological, and / or behavioral), including the complications of the disease, and pathological endophenotypes in the development of the disease.

[0118] Composition can be provided in a kit, container or dispenser that can contain one or more unit dosage forms containing active ingredient if desired.Kit can contain metal or plastic foil, such as blister pack.Kit, container or dispenser can be provided with instructions for administration.

[0119] Embodiment The present invention also provides the following non-limiting embodiments.

[0120] Embodiment 1. A method of inducing an immune response, such as an antibody response against phosphorylated tau protein (pTau), in a human subject in need thereof, comprising: (1) tau phosphopeptides consisting of the amino acid sequences of SEQ ID NO:27 to SEQ ID NO:29 and SEQ ID NO:31 to SEQ ID NO:38 in an amount of 300 μg to 1800 μg per dose; (2) a toll-like receptor 4 agonist comprising monophosphoryl lipid A; (3) a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 17, SEQ ID NO: 23 to SEQ ID NO: 26, and SEQ ID NO: 39 to SEQ ID NO: 44; (4) A CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22 administering to a subject an effective amount of a liposome comprising Tau phosphopeptides are displayed on the surface of liposomes, The method, wherein the antibody response is sustained for at least six weeks following initial administration of an effective amount of the liposomes to the human subject.

[0121] Embodiment 2: An effective amount of liposomes is (1) a tau phosphopeptide having the amino acid sequence of SEQ ID NO: 28 in an amount of 300 μg to 1800 μg per dose; (2) a toll-like receptor 4 agonist in an amount of 100 μg to 585 μg per dose; and (3) a helper T cell epitope in an amount of 75 μg to 450 μg per dose; and (4) CpG oligonucleotide in an amount of 150 μg to 800 μg per dose 2. The method of embodiment 1, comprising:

[0122] Embodiment 3. The method of embodiment 1 or 2, wherein the effective amount of liposomes comprises 300 μg, 900 μg or 1800 μg of tau phosphopeptide per dose.

[0123] Embodiment 3a. The method of embodiment 1 or 2, wherein the effective amount of liposomes comprises 300 μg of tau phosphopeptide per dose.

[0124] Embodiment 3b. The method of embodiment 1 or 2, wherein the effective amount of liposomes comprises 900 μg of tau phosphopeptide per dose.

[0125] Embodiment 3c. The method of embodiment 1 or 2, wherein the effective amount of liposomes comprises 1800 μg of tau phosphopeptide per dose.

[0126] Embodiment 4. The method of any one of embodiments 1-3c, wherein an effective amount of the liposomes is administered subcutaneously.

[0127] Embodiment 5. The method of any one of embodiments 1-3c, wherein an effective amount of the liposomes is administered intramuscularly.

[0128] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the CpG oligonucleotide has one or more phosphorothioate internucleotide linkages, and the CpG oligonucleotide is covalently linked to at least one lipophilic group, optionally via a PEG linker.

[0129] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the tau phosphopeptide comprises the amino acid sequence of SEQ ID NO: 28, the toll-like receptor 4 agonist comprises monophosphoryl hexaacyl lipid A, 3-desacylated, the helper T cell epitope comprises the amino acid sequence of SEQ ID NO: 39, the CpG oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 18, and the liposome further comprises at least one lipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol.

[0130] Embodiment 8. The method of any one of embodiments 1-7, wherein the antibody response has conformational specificity against pathological tau protein and which increases over time following initial administration of an effective amount of the liposome to the human subject, preferably the antibody response comprises a specific IgG antibody response directed against p-tau, preferably the specific IgG antibody response has an anti-p-tau IgG titer that is at least 50, 60, 70, 80, 90, 100 or more times higher than that of a placebo control.

[0131] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the antibody response induces a class switch of a specific IgM antibody response to a specific IgG antibody response directed against pTau, with indications of memory formation.

[0132] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the antibody response comprises an IgG immune response that preferentially recognizes pTau over non-phosphorylated Tau protein, and preferably has a ratio of anti-pTau IgG titer to anti-Tau IgG titer of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70.

[0133] Embodiment 10a.p The method of any one of embodiments 1-10, wherein the IgG immune response against tau is maintained over time.

[0134] Embodiment 10b. The method of any one of embodiments 1-10a, wherein the IgG immune response to non-phosphorylated tau protein decreases over time.

[0135] Embodiment 11. The method of any one of embodiments 1 to 10b, wherein the antibody response comprises an IgG immune response against enriched paired helical fibrils (ePHFs).

[0136] Embodiment 12. The method of embodiment 11, wherein the IgG immune response has an anti-ePHF IgG titer that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times higher than that of a placebo control.

[0137] Embodiment 12a. The method of embodiment 11 or 12, wherein the IgG immune response matures towards a stronger preference for binding to ePHF while simultaneously reducing antibody titers against non-phosphorylated tau.

[0138] Embodiment 12b. The method of embodiment 11 or 12, wherein the IgG immune response has a higher IgG titer against ePHF than against non-phosphorylated tau.

[0139] Embodiment 13. The method of embodiment 11, 12, 12a or 12b, wherein the anti-ePHF IgG has increased binding avidity for pathological ePHF tau for at least 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or more weeks after initial administration of an effective amount of liposomes, preferably the anti-ePHF IgG has an avidity index of at least 0.3, 0.4, 0.5, 0.6, or 0.7.

[0140] Embodiment 14. The method of any one of embodiments 1 to 13, further comprising administering to the subject a second dose of an effective amount of liposomes 4 to 12 weeks, such as 8 weeks, after the initial administration of the effective amount of liposomes.

[0141] Embodiment 14a. The method of embodiment 14, wherein the effective amount of liposomes comprises 300 μg of tau phosphopeptide per dose for each of the first dose and the second dose, and the second dose is administered to the subject 8 weeks after the first dose.

[0142] Embodiment 14b. The method of embodiment 14, wherein the effective amount of liposomes comprises 900 μg of tau phosphopeptide per dose for each of the first dose and the second dose, and the second dose is administered to the subject 8 weeks after the first dose.

[0143] Embodiment 14c. The method of embodiment 14, wherein the effective amount of liposomes comprises 1800 μg of tau phosphopeptide per dose for each of the first dose and the second dose, and the second dose is administered to the subject 8 weeks after the first dose.

[0144] Embodiment 15. The method of any one of embodiments 14-14c, wherein the antibody response, including an IgG immune response against p-tau, is boosted following administration of the second dose of an effective amount of liposomes, preferably the antibody response is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more when measured at least two weeks after administration of the second dose of an effective amount of liposomes.

[0145] Embodiment 15a. The method of embodiment 15, wherein the anti-ePHF IgG response is boosted following administration of an effective amount of liposomes when measured at least two weeks after administration of the second dose of an effective amount of liposomes.

[0146] Embodiment 16. The method of any one of embodiments 14-15a, further comprising administering a third dose of an effective amount of liposomes to the subject 20-28 weeks, such as 24 weeks, after the initial administration of the effective amount of liposomes.

[0147] Embodiment 16a. The method of embodiment 16, wherein the effective amount of liposomes comprises 300 μg of tau phosphopeptide per dose for each of the first dose, the second dose, and the third dose, and the second dose and the third dose are administered to the subject 8 weeks and 24 weeks, respectively, after the first dose.

[0148] Embodiment 16b. The method of embodiment 16, wherein the effective amount of liposomes comprises 900 μg of tau phosphopeptide per dose for each of the first dose, the second dose, and the third dose, and the second dose and the third dose are administered to the subject 8 weeks and 24 weeks, respectively, after the first dose.

[0149] Embodiment 16c. The method of embodiment 16, wherein the effective amount of liposomes comprises 1800 μg of tau phosphopeptide per dose for each of the first dose, the second dose, and the third dose, and the second dose and the third dose are administered to the subject 8 weeks and 24 weeks, respectively, after the first dose.

[0150] Embodiment 17. The method of any one of embodiments 16-16c, wherein an antibody response, including an IgG immune response, against p-tau is boosted following administration of the third dose of an effective amount of liposomes, preferably the antibody response is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more when measured at least two weeks after administration of the third dose of an effective amount of liposomes.

[0151] Embodiment 17a. The method of embodiment 17, wherein the anti-ePHF IgG response is boosted following administration of a third dose of an effective amount of liposomes when measured at least two weeks after administration of the second dose of an effective amount of liposomes.

[0152] Embodiment 18. The method of any one of embodiments 16-17a, further comprising administering to the subject a fourth dose of an effective amount of liposomes 44-52 weeks, such as 48 weeks, after the initial administration of the effective amount of liposomes.

[0153] Embodiment 18a. The method of embodiment 18, wherein the effective amount of liposomes comprises 300 μg of tau phosphopeptide per dose for each of the first dose, the second, third and fourth doses, and the second, third and fourth doses are administered to the subject 8 weeks, 24 weeks and 48 weeks, respectively, after the first dose.

[0154] Embodiment 18b. The method of embodiment 18, wherein the effective amount of liposomes comprises 900 μg of tau phosphopeptide per dose for each of the first dose, the second, third and fourth doses, and the second, third and fourth doses are administered to the subject 8 weeks, 24 weeks and 48 weeks, respectively, after the first dose.

[0155] Embodiment 18c. The method of embodiment 18, wherein the effective amount of liposomes comprises 1800 μg of tau phosphopeptide per dose for each of the first dose, the second, third and fourth doses, and the second, third and fourth doses are administered to the subject 8 weeks, 24 weeks and 48 weeks, respectively, after the first dose.

[0156] Embodiment 19. The method of embodiment 18, wherein an antibody response, including an IgG immune response against p-tau, is boosted following administration of the fourth dose of an effective amount of liposomes, preferably the antibody response is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more when measured at least two weeks by administration of the fourth dose of an effective amount of liposomes.

[0157] Embodiment 19a. The method of embodiment 19, wherein the anti-ePHF IgG response is boosted following administration of a fourth dose of an effective amount of liposomes when measured at least two weeks after administration of the second dose of an effective amount of liposomes.

[0158] Embodiment 20. A method for inducing a sustained immune response against phosphorylated tau protein (pTau) in a human subject in need thereof, comprising: i. administering intramuscularly to a subject a priming vaccine comprising an effective amount of a liposome; and ii. administering to the subject intramuscularly a first booster vaccine containing an effective amount of liposomes 6 to 10 weeks after administration of the priming vaccine; Including, A sustained immune response is produced that persists for at least about 20 weeks after administration of the priming vaccine; The liposome is (1) a tau phosphopeptide having the amino acid sequence of SEQ ID NO: 28, which is presented on the surface of a liposome; (2) a toll-like receptor 4 agonist comprising monophosphoryl lipid A; (3) a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 24, 25, and 26; (4) A CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22 and An effective amount of liposomes (1) a tau phosphopeptide in an amount of 300 μg to 1800 μg per dose; (2) a toll-like receptor 4 agonist in an amount of 100 μg to 585 μg per dose; and (3) a helper T cell epitope in an amount of 75 μg to 550 μg per dose, e.g., 75 μg to 450 μg, 80 μg to 540 μg, 82.5 μg to 535 μg, 85 μg to 530 μg, 87.5 μg to 525 μg, or 90 μg to 520 μg per dose; (4) CpG oligonucleotide in an amount of 100 μg to 1000 μg per dose, e.g., 150 μg to 800 μg, 125 μg to 950 μg, 150 μg to 900 μg, or 150 μg to 850 μg. and

[0159] Embodiment 20a. The method of embodiment 20, wherein the helper T cell epitope has an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 40, 41, 42, and 43.

[0160] Embodiment 20b. The method of embodiment 20, wherein the helper T cell epitope has an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 44.

[0161] Embodiment 20c. The method of any one of embodiments 20 to 20b, wherein the lipidated CpG oligonucleotide has the nucleotide sequence of SEQ ID NO: 18, and the CpG oligonucleotide is covalently linked to at least one cholesterol via a linker.

[0162] Embodiment 20d. The method of any one of embodiments 20-20c, wherein the first booster vaccine is administered 8 weeks after administration of the priming vaccine, and the sustained immune response persists for at least about 24 weeks after administration of the priming vaccine.

[0163] Embodiment 20e. The method of any one of embodiments 20-20d, further comprising intramuscularly administering to the subject a second booster vaccine comprising an effective amount of liposomes 22-26 weeks after administration of the priming vaccine, wherein the sustained immune response persists for at least about 36 weeks after administration of the priming vaccine.

[0164] Embodiment 20f. The method of embodiment 20e, wherein the second booster vaccine is administered 24 weeks after administration of the priming vaccine, and the sustained immune response persists for at least about 48 weeks after administration of the priming vaccine.

[0165] Embodiment 20g. The method of embodiment 20e or 20f, further comprising intramuscularly administering to the subject a third booster vaccine comprising an effective amount of liposomes 45 to 50 weeks after administration of the priming vaccine, wherein the sustained immune response persists for at least about 60 weeks after administration of the priming vaccine.

[0166] Embodiment 20h. The method of embodiment 20g, wherein the third booster vaccine is administered 48 weeks after administration of the priming vaccine, and the sustained immune response persists for at least about 72 weeks after administration of the priming vaccine.

[0167] Embodiment 20i. The method of any one of embodiments 20-20h, wherein the effective amount of liposomes comprises 300 μg of tau phosphopeptide per dose.

[0168] Embodiment 20j. The method of any one of embodiments 20-20h, wherein the effective amount of liposomes comprises 900 μg of tau phosphopeptide per dose.

[0169] Embodiment 20k. The method of any one of embodiments 20-20h, wherein the effective amount of liposomes comprises 1800 μg of tau phosphopeptide per dose.

[0170] Embodiment 20k1. The method of any one of embodiments 20i to 20k, wherein the effective amount of liposome comprises 80 μg to 540 μg of helper T cell epitope per dose and 125 μg to 950 μg of CpG oligonucleotide per dose.

[0171] Embodiment 20k2. The method of any one of embodiments 20i to 20k, wherein the effective amount of liposome comprises 82.5 μg to 535 μg of helper T cell epitopes per dose and 125 μg to 950 μg of CpG oligonucleotides per dose.

[0172] Embodiment 20k3. The method of any one of embodiments 20i to 20k, wherein the effective amount of liposome comprises 87.5 μg to 525 μg of helper T cell epitopes per dose and 150 μg to 900 μg of CpG oligonucleotides per dose.

[0173] Embodiment 20k4. The method of any one of embodiments 20i to 20k, wherein the effective amount of liposome comprises 85 μg to 530 μg of helper T cell epitopes per dose and 150 μg to 900 μg of CpG oligonucleotides per dose.

[0174] Embodiment 20l. The method of any one of embodiments 20 to 20k4, wherein the sustained immune response comprises an IgG immune response that preferentially recognizes p-tau over non-phosphorylated tau protein, preferably with a ratio of anti-p-tau IgG titer to anti-tau IgG titer of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70.

[0175] Embodiment 20m. The method of any one of embodiments 20-20l, wherein the sustained immune response comprises an IgG immune response to enriched paired helical fibrils (ePHF) having an anti-ePHF IgG titer that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times higher than that of a placebo control.

[0176] Embodiment 21. The method of any one of embodiments 1 to 20m, wherein the subject is in need of clearance of tau aggregates.

[0177] Embodiment 22. The method of any one of embodiments 1 to 21, wherein the subject is in need of treatment for Alzheimer's disease, e.g., preclinical Alzheimer's disease, mild to moderate Alzheimer's disease or early Alzheimer's disease, mild cognitive impairment (MCI) due to Alzheimer's disease.

[0178] Embodiment 23. The method of any one of embodiments 1 to 21, wherein the subject is in need of prevention of Alzheimer's disease, e.g., preclinical Alzheimer's disease, mild to moderate Alzheimer's disease or early Alzheimer's disease, mild cognitive impairment (MCI) due to Alzheimer's disease.

[0179] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the immune response comprises anti-phosphorylated tau antibodies that specifically bind to an epitope comprising phosphorylated Ser396.

[0180] Embodiment 25 The method of embodiment 24, wherein the epitope further comprises phosphorylated Ser404. EXAMPLES

[0181] The following examples of the present invention are intended to further illustrate the nature of the present invention. It should be understood that the following examples do not limit the present invention, the scope of which should be determined by the appended claims.

[0182] All experimental methods used in the following examples are conventional unless otherwise indicated. All reagents used in the following embodiments are purchased from conventional reagent suppliers unless otherwise indicated.

[0183] In all of the following examples, ACI-35.030 refers to a liposomal formulation according to an embodiment of the present invention that contains a phosphorylated tau peptide having the amino acid sequence of SEQ ID NO:28, MPLA (3D-(6-acyl)PHAD (registered trademark)), DMPC, DMPG, cholesterol, a helper T cell epitope of SEQ ID NO:13, a CpG2006 oligonucleotide covalently linked to a cholesterol group via a PEG linker, and a buffer solution, and ACI-35 refers to a liposomal formulation according to an embodiment of the present invention that contains a phosphorylated tau peptide having the amino acid sequence of SEQ ID NO:28, MPLA, DMPC, DMPG, cholesterol, and a buffer solution.

[0184] [Example 1] Clinical Study of the Safety and Efficacy of ACI-35.030 in Humans The safety, tolerability, and immunogenicity of the ACI-35.030 vaccine (ACI-35.030) will be evaluated in a Phase 1b / 2a multicenter, double-blind, randomized, placebo-controlled clinical study in patients with early AD (mild cognitive impairment (MCI) due to AD or mild AD) in Europe (ACI-35-1802 study). See also study number NCT04445831 at clinicaltrials.gov. The study design is outlined in Figure 1.

[0185] OBJECTIVE: To evaluate ACI-35.030 at up to three dosages in patients with early AD (e.g., mild cognitive impairment due to AD (MCI or mild AD)) over a 74 week time frame for safety and tolerability in patients with early AD, and for induction of antibody responses against abnormal forms of tau protein, including induction of anti-phospho-tau antibodies (e.g., those that bind anti-p-tau and ePHF-tau) in the serum.

[0186] Secondary objectives: to further evaluate the immunogenicity of the study vaccine over a 74 week time frame, e.g., by assessing serum induction of IgG titers against tau and IgM titers against ptau and tau, as well as to evaluate the avidity of antibodies induced by immunization.

[0187] Exploratory Objectives: To explore the effect of the study vaccine on putative biomarkers of AD progression, e.g., blood and / or CSF concentrations of total tau and p-tau protein, respectively, in a 74-week time frame; to explore the effect of the study vaccine on T cell activation in the blood; to explore the effect of the study vaccine on blood inflammatory cytokines (e.g., IL-1β, IL-2, IL-6, IL-8, IL-10, IFN-γ, and TNF-α); to explore the effect of the study vaccine on behavioral, cognitive and functional activities.

[0188] Methods: Each of the three sub-cohorts consisted of patients receiving placebo or various dosages of ACI-35.030, referred to by the amount of pTau Peptide T3 in the composition (300 μg, 900 μg or 1800 μg of tetrapalmitoylated phosphopeptide pTau Peptide T3, SEQ ID NO: 28), followed for 48 weeks (dosed at weeks 0, 8, 24 and 48), followed by a 24-week follow-up period.

[0189] Forty-one patients were randomized into three subcohorts, each of which received either ACI-35.030 or placebo (active / placebo ratio 3:1). Doses were administered intramuscularly.

[0190] Safety assessments will be performed / will be conducted for all study patients by telephone immediately after each dose and 48-72 hours later. In each subcohort, the first dose of the first 4 patients will be administered / will be administered after a safety assessment 48-72 hours after the previous patient has been performed to confirm the absence of clinically relevant safety issues associated with the study vaccine by the site principal investigator.

[0191] All treated patients will have a 24-week follow-up period after the end of the treatment period. During this period, patients will be asked to attend a first follow-up visit 19 weeks after the last dose and a final visit at the end of the follow-up period (26 weeks after the last dose). Patient safety will be monitored throughout the study by regular review of safety data by an independent Data Safety Monitoring Board (DSMB).

[0192] Interim analyses have been / will be performed as follows:

[0193] The first interim analysis was conducted in all subcohorts of Cohort 1 after all subjects in their respective subcohorts had completed Visit 4 (Week 10), i.e., 2 weeks after the second injection. The objective was to review safety, tolerability, and immunogenicity data for ACI-35.030 to this point.

[0194] A second interim analysis will be performed in subcohorts 1.1, 1.2, and 1.3 after all subjects in their respective subcohorts have completed Visit 6 (Week 26), i.e., 2 weeks after the third injection. The objective is to determine the possibility of extending Cohort 1 to collect additional safety / tolerability data at the dose that presents the most favorable profile in terms of immunogenicity, safety, and tolerability.

[0195] The third interim analysis was performed at the end of the treatment period (i.e., 2 weeks after the fourth injection). The objective is to review safety / tolerability and immunogenicity data to this point, including data from patients in subcohort expansion, if applicable. Biomarker results can be included as supportive exploratory data. Results will be compared with results subsequently obtained for other cohorts to select the best strategy for further clinical development from all study cohorts.

[0196] The fourth interim analysis has been / will be performed at the end of the follow-up period, i.e. after all Cohort 1 patients have completed Visit 11 (Week 74). The objectives will be the same as for the fourth interim analysis, after which the results will be compared across all cohorts.

[0197] The study population was 50–75 years of age (males and females) with a diagnosis of mild AD or MCI due to AD according to NIA-AA criteria.

[0198] The selection criteria were as follows: 1. Male or female, between 50 and 75 years old. 2. Mild cognitive impairment (MCI) due to AD or mild AD according to NIA-AA criteria and a Clinical Dementia Rating (CDR) total score of 0.5 or 1. Mini-Mental State Examination (MMSE) score of 3.22 or higher. 4. CSF amyloid beta 42 (Aβ42) and phosphorylated tau levels at screening consistent with the NIA-AA2018 criteria for AD pathology. In cases that are borderline with respect to CSF ​​Aβ42 levels, other results that help determine amyloid positivity may be considered, such as Aβ42 / Aβ40 ratio, and a case-by-case history of a positive amyloid PET scan or positive CSF Aβ42 levels. Results of CSF sampling performed within 3 months prior to screening are acceptable on a case-by-case basis, provided that they are consistent with the presence of amyloid pathology and that the corresponding CSF sample can be used in the study for testing. 5. Patients not receiving any marketed treatment for AD or receiving stable doses of acetylcholinesterase inhibitors and / or memantine for at least 3 months prior to baseline. 6. Patients cared for by a reliable informant or caregiver who ensures compliance, assists in clinical evaluations, and reports safety issues. 7. Women must be at least 1 year postmenopausal and / or surgically sterilized. Women of childbearing potential or who are not postmenopausal must have a negative pregnancy test at screening and be willing to use highly effective contraceptive methods from the screening visit until the end of participation. Urine pregnancy retests will be performed throughout the treatment period to determine if subjects can continue to receive the study vaccine. Male patients with partners of childbearing potential must be willing to use appropriate contraceptive measures during the study. 8. Patients who, in the investigator's opinion, are able to understand and give written informed consent. 9. Patients and informants or caregivers must be fluent in one of the study languages ​​and able to comply with all study procedures, including lumbar puncture.

[0199] The exclusion criteria were as follows: 1. Participation in previous clinical trials for AD and / or neuropathy using active immunization, except where patients were treated with placebo only and there is documented evidence that the placebo formulation is not expected to induce any specific immune response. 2. Participation within the past 6 months (or 5 half-lives of the test antibody, whichever is longer) prior to Screening in a previous clinical trial for AD and / or neuropathy using any passive immunization, unless subjects were treated with placebo only and there is documented evidence that the placebo is not expected to induce any specific immune response. 3. Participation in a previous clinical trial for AD and / or neurological disorders using any small molecule drug, including a BACE-1 inhibitor, within the past 3 months prior to screening. 4. Concurrent participation in any other clinical trial using an experimental or approved drug or therapy. 5. The presence of positive antinuclear antibody (ANA) titers at a dilution of at least 1 / 160 in patients without clinical symptoms of autoimmune disease. 6. Current or past history of autoimmune disease, or clinical symptoms consistent with the presence of an autoimmune disease. 7. Immunosuppression, including but not limited to use of immunosuppressants or systemic steroids, unless temporarily prescribed for >3 months prior to screening. 8. History of severe allergic reactions (e.g., anaphylaxis), including but not limited to severe allergic reactions to previous vaccines and / or medications. 9. Prior history of clinically significant hypoglycemic episodes. 10. Drug or alcohol abuse or dependence currently met or met within the past 5 years according to Diagnostic and Statistical Manual of Mental Disorders V (DSM-V) criteria for any condition. 11. Any clinically significant medical condition likely to interfere with the assessment of safety and tolerability of study treatment and / or compliance with all scheduled study visits. 12. Any clinically significant medical condition in the patient that is likely to affect the immune system and / or is expected to potentially impair the immunization potential of the study vaccine (e.g., any history of an acquired or innate immune suppression disorder). 13. Use of hydralazine, procainamide, quinidine, isoniazid, TNF inhibitors, or minocycline within the 12 months immediately prior to screening. 14. Use of diltiazem for at least 3 months prior to screening, except on a stable dose. 15. Overt suicide risk, defined as subject responding "yes" to suicidal ideation questions 4 or 5 or "yes" to suicidal behavior within the past 12 months using the Columbia Suicide Severity Scale. 16. Concurrent psychiatric or neurological disorders other than those considered related to AD (e.g., head injury with loss of consciousness, symptomatic stroke, Parkinson's disease, severe carotid artery occlusive disease, TIA). 17. History or presence of uncontrolled epileptic seizures. In case of history of epileptic seizures, the seizures must be well controlled such that their occurrence was not present within 2 years prior to baseline. Use of antiepileptic medications is acceptable if on a stable dose for at least 3 months prior to screening. 18. History of meningoencephalitis within the past 10 years prior to screening. 19. Patients with a history of hemorrhagic and / or non-hemorrhagic stroke. 20. Presence or history of peripheral neuropathy. 21. History of inflammatory neuropathy with possible CNS involvement. 22. Screening MRI scan showing structural evidence of an alternative pathology inconsistent with AD that could be causing the patient's symptoms. Evidence of a space-occupying lesion other than a benign meningioma less than 1 cm in diameter, 3 or more lacunar infarcts or a single infarct greater than 1 cm in diameter, or any single area of ​​superficial hemosiderosis, or evidence of a previous major hemorrhage greater than 10 mm. T2 * Microbleeds on MRI can be seen up to 10 regardless of location. 23. Unable to undergo MRI examination for any reason including but not limited to metal implants and / or severe claustrophobia that contraindicate MRI studies. 24. Significant hearing or vision impairment, or other problems judged by the investigator to be relevant to preventing following the protocol and conducting outcome measures. 25. Clinically significant infection or major surgical procedure within 3 months prior to screening. Planned surgical procedures anticipated to be performed during participation in the study must be reviewed and approved by the medical monitor at the time of screening. 26. Any vaccine administered within the past 2 weeks prior to baseline, including influenza vaccine. 27. Clinically significant arrhythmias or other clinically significant abnormalities on ECG at screening. 28. Myocardial infarction, unstable angina, or significant coronary artery disease within 1 year prior to baseline. 29. Patients with a history of cancer within the past 5 years other than treated squamous cell carcinoma, basal cell carcinoma, and intraepidermal melanoma, or non-invasive prostate cancer or non-invasive breast cancer that has been completely removed and is considered to have been treated. 30. Clinically significant deviations from normal values ​​for hematological parameters, liver function tests, and other biochemical measurements that, in the opinion of the investigators at the site, are judged to be clinically significant. 31. Female subject who is pregnant, planning pregnancy, or breastfeeding, as confirmed by serology at screening. 32. Patients receiving any anticoagulant or antiplatelet agent except aspirin at a dose less than 100 mg daily (to avoid the risk of bleeding during scheduled or unscheduled lumbar puncture). 33. Patients receiving antipsychotics, except for stable low doses for the treatment of insomnia. 34. Patient has donated blood or blood products within 30 days prior to screening or plans to donate blood while participating in the study. 35. Positive VDRL (Venereal Disease Laboratory) consistent with active syphilis at screening. 36. Patients with a positive HIV test result at screening. 37. Patients with active Hepatitis B and / or C as measured by laboratory testing at screening. 38. Patients with creatinine >1.5 times the upper limit of normal, abnormal thyroid function test results, or clinically significant decreases in serum B12 or folate levels (Note: all oral doses of thyroid replacement medications, B12, or folate must be stable for at least 3 months prior to screening).

[0200] Patient background: The study is ongoing. Patient demographics for subcohorts 1.1 and 1.2 (as of the cutoff date of the end of September 2021) are summarized in Table 1.

[0201] [Table 1]

[0202] Results / Conclusion: The following primary endpoints were / will be assessed: Safety and tolerability - adverse events, immediate and delayed reactogenicity (e.g. local and systemic reactogenicity including anaphylaxis, pain, redness, immune complex disease, swelling, fever); global assessment of tolerability; suicidal ideation (C-SSRS); behavior (NPI); cognitive and functional assessments to evaluate safety (RBANS, CDR-SB); vital signs; MRI imaging; electrocardiogram; routine hematology and biochemistry assessments of blood and urine; assessment of autoimmune antibodies including anti-DNA antibodies in blood; inflammatory markers in blood and CSF. Immune response - anti-p-tau IgG titers in serum (geometric mean, change from baseline, responder rate, peak, and area under the curve).

[0203] The following secondary endpoints were / will be assessed: Immune response - serum anti-tau IgG, anti-p-tau IgM, anti-ePHF IgG and anti-tau IgM titers (geometric mean, change from baseline, responder rate, peak and area under the curve), determination of IgG response profile by avidity testing.

[0204] The following exploratory endpoints were / will be evaluated: Change from baseline in biomarker titers in blood and / or CSF (e.g., total tau and p-tau protein), change from baseline in T cell activation levels in blood, change from baseline in inflammatory cytokine (e.g., IL-1B, IL-2, IL-6, IL-8, IL-10, IFN-γ, and TNF-α) titers in blood, change from baseline in suicidal ideation (C-SSRS), behavior (NPI), cognitive and functional activities (RBANS, CDR-SB) scores.

[0205] The study is ongoing. As shown in Table 2, three subcohorts received ACI-35.030 at dosage levels of 300 μg, 900 μg, or 1800 μg of the tetrapalmitoylated phosphopeptide pTau peptide T3 (SEQ ID NO: 28), and placebo.

[0206] [Table 2]

[0207] Interim safety and tolerability results as of the cutoff date of June 20, 2022, showed that no safety or tolerability issues were identified for ACI-35.030. There were no discontinuations due to adverse events in subcohorts 1.1, 1.2, and 1.3 of cohort 1. Additionally, none of the patients studied to date had CNS inflammation or other notable changes reported on MRI.

[0208] Following an initial dose of 300 μg, 900 μg or 1800 μg of the tetrapalmitoylated phosphopeptide pTau peptide T3 (SEQ ID NO: 28), an increase in anti-pTau specific IgG titers was observed in the serum of 100% of subjects 2 weeks after administration of ACI-35.030 compared to baseline. This anti-pTau IgG response showed a preference for pTau over non-pTau peptides in all early AD subjects treated with the active drug, and the antibody response was boosted by an additional dose of ACI-35.030, as indicated by an increase in anti-pTau specific IgG titers and / or an increase in anti-ePHF IgG titers measured 2 weeks after an additional dose of ACI-35.030. No antibody responses were observed in subjects receiving placebo, except for one subject in subcohort 1.2 who received placebo, who had a single, limited increase in IgM and IgG anti-p-tau titers after the 67-week study treatment period, and whose response was near the threshold established to define responders.

[0209] Anti-p-tau IgG response of ACI-35.030 in humans Specific IgG antibody responses directed against phosphorylated tau peptide (p-tau) induced by the ACI-35.030 vaccine in the three subcohorts in Table 2 were measured by MSD.

[0210] Table 3 shows anti-pTau IgG titers and responder rates following immunization with either ACI-35.030 at a dosage level of 300 μg of the tetrapalmitoylated phosphopeptide pTau Peptide T3 (ACI-35.030 300 μg) or placebo in subcohort 1.1 (ITT population).

[0211] [Table 3-1]

[0212] [Table 3-2]

[0213] Anti-pTau IgG titers and responder rates following immunization with either ACI-35.030 at a dosage level of 900 μg of the tetrapalmitoylated phosphopeptide pTau Peptide T3 (ACI-35.030 900 μg) or placebo in subcohort 1.2 (ITT population) are summarized in Table 4.

[0214] [Table 4-1]

[0215] [Table 4-2]

[0216] Anti-pTau IgG titers and responder rates following immunization with either ACI-35.030 at a dosage level of 1800 μg of the tetrapalmitoylated phosphopeptide pTau Peptide T3 (ACI-35.030 1800 μg) or placebo in subcohort 1.3 (ITT population) are summarized in Table 5.

[0217] [Table 5-1]

[0218] [Table 5-2]

[0219] As shown by the results in Tables 3, 4 and 5, and Figures 2 and 6, immunization with ACI-35.030 at 300 μg, 900 μg and 1800 μg dose levels, respectively, induced anti-p-tau IgG responses directed against the T3.5 peptide having the amino acid sequence of SEQ ID NO: 28. All subjects treated with ACI-35.030 at the 300 μg dose level were responders from week 2 to week 10, of which 66.7% were also responders at week 36, although 7 out of 8 subjects in this subcohort did not receive a prior injection at week 24 due to the Covid-19 pandemic. To avoid possible unblinding of the study, the percentage of responders at week 36 and beyond in subcohort 1 will not be reported for the time being. All subjects treated with ACI-35.030 at the 900 μg dose level were responders at all time points between weeks 2 and 74, while one subject receiving placebo developed a limited anti-p-tau IgG response at week 67 (after the treatment period) (1.9-fold above baseline), slightly higher than the threshold established to define a responder (1.81-fold above baseline). All subjects treated with ACI-35.030 at the 1800 μg dose level were responders at all time points between weeks 2 and 50, while no subject receiving placebo developed an anti-p-tau IgG response by week 42.

[0220] In subjects treated with either 300 μg or 900 μg of ACI-35.030, high responder rates were observed as early as 2 weeks after vaccination. Overall, high responder rates were observed after the first vaccination and after all vaccinations. With the 900 μg dose of ACI-35.030, the responder rate for phosphorylated tau was 100% when analyzed at any study time point between weeks 2 and 74. The responder rate for pathological ePHF with the 900 μg dose of ACI-35.030 ranged from 66.7% to 100% at any time point during the treatment period between weeks 2 and 48, and ranged from 50% to 100% at any time point after the treatment period between weeks 50 and 74. Moreover, rapid class switching from IgM to IgG was observed in patients treated with either 300 μg or 900 μg of ACI-35.030. A summary of the overall response rates for subcohorts 1.1, 1.2 and 1.3 treated with 300 μg, 900 μg or 1800 μg of ACI-35.030, respectively, is shown in Table 6.

[0221] [Table 6]

[0222] In general, each booster immunization at weeks 8, 24 or 48 at both the 300 and 900 μg dose levels resulted in a boosting of the anti-p-tau IgG response, as indicated by the increase in anti-p-tau specific IgG titers measured 2 weeks after administration of the booster immunization at weeks 10, 26 and 50, respectively (FIGS. 2 and 6). Booster immunizations at weeks 8, 24 and 48 with the 1800 μg dose level also resulted in a boosting of the anti-p-tau IgG response, as indicated by the increase in anti-p-tau specific IgG titers measured 2 weeks after administration of the booster immunization at weeks 10, 26 and 50 (FIGS. 4 and 7).

[0223] Anti-p-tau IgM responses of ACI-35.030 in humans Specific IgM antibody responses directed against phosphorylated tau peptides induced by the ACI-35.030 vaccine were measured by MSD in the three subcohorts in Table 2. Anti-pTau IgM titers and responder rates after immunization with either 300 μg ACI-35.030 or placebo in subcohort 1.1 (ITT population) are shown in Table 7.

[0224] [Table 7-1]

[0225] [Table 7-2]

[0226] Anti-pTau IgM titers and responder rates following immunization with either ACI-35.030 900 μg or placebo in subcohort 1.2 (ITT population) are shown in Table 8.

[0227] [Table 8-1]

[0228] [Table 8-2]

[0229] Anti-pTau IgM titers and responder rates following immunization with either ACI-35.030 1800 μg or placebo in subcohort 1.3 (ITT population) are shown in Table 9.

[0230] [Table 9-1]

[0231] [Table 9-2]

[0232] As shown by the results in Tables 7 to 9, immunization of early AD subjects with 300 μg, 900 μg and 1800 μg doses of ACI-35.030 induced anti-p tau IgM responses directed against the T3.5 peptide having the amino acid sequence of SEQ ID NO: 28 by week 10. All subjects treated with ACI-35.030 were responders, whereas subjects treated with placebo did not develop anti-p tau IgM responses, except for one subject at week 67 (after the treatment period) who had a limited anti-p tau IgM response slightly higher than the threshold set to define a responder. Taken together, the decline in anti-p tau IgM responses by week 8 and the anti-p tau IgG antibody responses measured from week 2 onwards suggest that ACI-35.030 induced a class switch from IgM to IgG.

[0233] Specificity for p-tau over tau (non-p-tau) See Figures 3 and 5 for IgG titers against non-phosphorylated tau (anti-tau IgG titers) induced by various dosages of ACI-35.030 or placebo over time. The specificity of the IgG antibody response induced by immunization with ACI-35.030 for binding to p-tau over that to tau (where the response to tau represents the response to non-p-tau) in the three subcohorts of Table 2 was measured over time as the ratio of anti-p-tau IgG / anti-tau IgG response. Anti-tau IgG titers and responder rates after immunization with either ACI-35.030 300 μg or placebo (ITT population) in subcohort 1.1 are shown in Table 10.

[0234] [Table 10-1]

[0235] [Table 10-2]

[0236] Anti-tau IgG titers and responder rates following immunization with either ACI-35.030 900 μg or placebo in subcohort 1.2 (ITT population) are shown in Table 11.

[0237] [Table 11-1]

[0238] [Table 11-2]

[0239] Anti-tau IgG titers and responder rates following immunization with either ACI-35.030 1800 μg or placebo in sub-cohort 1.3 (ITT population) are shown in Table 12.

[0240] [Table 12-1]

[0241] [Table 12-2]

[0242] [Table 12-3]

[0243] The ratio of anti-pTau IgG / anti-Tau IgG titers following immunization with 300 μg of ACI-35.030 in sub-cohort 1 (ITT population) is shown in Table 13.

[0244] [Table 13]

[0245] Table 14 shows the ratio of anti-pTau IgG / anti-Tau IgG titers following immunization with 900 μg ACI-35.030 in subcohort 1.2 (ITT population).

[0246] [Table 14]

[0247] Table 15 shows the ratio of anti-pTau IgG / anti-Tau IgG titers following immunization with 1800 μg of ACI-35.030 in subcohort 1.3 (ITT population).

[0248] [Table 15]

[0249] As shown by the results in Tables 13 to 15, immunization with 300 μg, 900 μg and 1800 μg doses of ACI-35.030 induced IgG antibody responses that preferentially recognized p-tau peptides over non-p-tau peptides, and this preference persisted over time.

[0250] Recognition of pathological p-tau (enriched paired helical filaments - ePHF) from human AD brains The ability of IgG polyclonal antibodies induced by immunization with ACI-35.030 to bind ePHF derived from human AD brains was measured by MSD over time in the three subcohorts of Table 2. Table 16 shows anti-ePHF IgG titers and responder rates (ITT population) following immunization with either 300 μg ACI-35.030 or placebo in subcohort 1.1.

[0251] [Table 16-1]

[0252] [Table 16-2]

[0253] Table 17 shows anti-ePHF IgG titers and responder rates following immunization with either ACI-35.030 900 μg or placebo in subcohort 1.2 (ITT population).

[0254] [Table 17-1]

[0255] [Table 17-2]

[0256] Table 18 shows anti-ePHF IgG titers and responder rates following immunization with either ACI-35.030 1800 μg or placebo in subcohort 1.3 (ITT population).

[0257] [Table 18-1]

[0258] [Table 18-2]

[0259] The results in Tables 16 to 18 and Figures 4 and 7 indicate that immunization with 300 μg, 900 μg and 1800 μg doses of ACI-35.030 can induce IgG antibody responses that recognize pathological ePHF tau derived from human AD brain. Furthermore, anti-ePHF IgG titers could be boosted. The geometric mean of anti-ePHF IgG titers increased 2 weeks after each immunization. The responder rates for IgG anti-ePHF in subjects treated with 300 μg of ACI-35.030 were 66.7% at week 2, 83.3% at weeks 8 and 10, and 33.3% at week 36, respectively, although 7 out of 8 subjects in this subcohort did not receive a prior injection at week 24 due to the Covid-19 pandemic. To avoid possible unblinding of the study, the percentage of responders at week 36 and beyond in this subcohort is not reported at this time. The responder rates for anti-ePHF IgG in subjects treated with ACI-35.030 900 μg were 100% at weeks 2, 10 and 26, 66.7% at weeks 8 and 24, 83.3% at weeks 36 and 48, and 100% at week 50. The same two subjects treated with active drug were non-responders at these two time points (weeks 8 and 24), while they were considered responders at weeks 2, 10 and 26 (e.g., 2 weeks after the injections given at weeks 0, 8 and 24, respectively). The responder rates for IgG anti-ePHF IgG in subjects treated with ACI-35.030 1800 μg were 66.7%, 33.3% and 83.3% at weeks 2, 8 and 10, respectively, followed by 50% at week 24, 66.7% at week 26, and 80% at week 50.

[0260] Avidity for pathological p-tau (enriched versus helical filaments - ePHF) from human AD brain The IgG antibody response induced by immunization with ACI-35.030 in subcohort 1.1, subcohort 1.2, and subcohort 1.3 was measured over time by MSD using both low-density and high-density coating of ePHF on plates. Antibody concentration is measured on low-density coating (only antibodies with high binding capacity can bind) and high-density coating (all antibodies can bind). Avidity index is calculated by the ratio of antibody concentration on low / high-density coating. Table 19 shows the avidity index for ePHF after immunization with ACI-35.030 at 300 μg in subcohort 1.1.

[0261] [Table 19]

[0262] Table 20 shows the avidity index for ePHF following immunization with ACI-35.030 at 900 μg in subcohort 1.2.

[0263] [Table 20]

[0264] Table 21 shows the avidity index for ePHF following immunization with ACI-35.030 at 1800 μg in subcohort 1.3.

[0265] [Table 21]

[0266] The results in Tables 19, 20, and 21 indicate that in most patients, immunization with the 300 μg, 900, and 1800 μg doses of ACI-35.030, respectively, induced IgG immune responses that showed increasing binding avidity between weeks 2 and 10.

[0267] To date, preliminary results have shown, for example, that ACI-35.030 induces highly specific and sustained antibody responses directed against pathological species of tau (phospho-tau and ePHF), with a clear dose response between low and medium doses and evidence of immunoglobulin class switching from IgM to IgG. Individual responder rates were high and consistent, especially for anti-p-tau and ePHF antibodies. Administration of ACI-35.030 did not appear to raise any specific safety concerns for the study vaccine at the time of data analysis, providing support for the favorable safety and tolerability profile of ACI-35.030, which was able to induce sustained antibody responses above baseline values ​​in immunized patients. Over time, the data demonstrate that the IgG response matures toward a stronger preference for binding to the more pathological species, ePHF, while simultaneously decreasing antibody titers against non-pathological non-phosphorylated tau. Preliminary results support further development of this vaccine as an effective AD disease-modifying treatment, as well as an approach for the potential prevention of AD.

[0268] [Example 2] Vaccination with ACI-35.030 induces relatively homogenous antibody responses with broad epitope coverage To further profile the antibody response for breadth and selectivity against pathological p-tau, epitope mapping was performed on the sera of human subjects. A study was conducted to determine the epitope recognition profile of the antibodies induced by the liposomal vaccine in human subjects. In a phase 1 / 2 clinical trial, seven Alzheimer's disease (AD) patients were intramuscularly immunized with the liposomal vaccine with 900ug per dose of acetate tetrapalmitoylated phosphorylated tau peptide T3 (SEQ ID NO: 28) or placebo (subcohort 1.2) at weeks 0, 8, 24, and 48. The epitope recognition profile of the antibody was determined by epitope mapping ELISA using a library of N-terminally biotinylated 8-mer peptides covering the entire sequence of phospho-tau peptide T3.30 (SEQ ID NO: 45) and the corresponding sequence of non-phosphorylated tau peptide T3.56 (SEQ ID NO: 46) with a one amino acid shift before the first immunization (V1, week 0) and after the third immunization (V6, week 26). In addition, binding of the antibody to full-length phospho-tau peptide T3.30 (and tau peptide T3.56, as well as another N-terminally biotinylated phospho-tau peptide T3.85 (SEQ ID NO: 47) and the corresponding non-phosphorylated tau peptide T3.86 (SEQ ID NO: 48) (with an additional C-terminal amino acid) was also determined.

[0269] Data are expressed as pretreatment subtracted optical density (OD) values, obtained for each peptide and each patient by subtracting the OD obtained after the third immunization (V6, week 26) from that obtained before the first immunization (V1, week 0). Negative values ​​after subtraction were set to 0.000.

[0270] Tables 22 and 23 show the epitope recognition profile of antibodies induced by vaccination with ACI-35.030 as determined by epitope mapping ELISA on short overlapping 8-mer peptides covering phospho-peptides T3.30 and T3.85 and non-phospho-peptides T3.56 and T3.86.

[0271] [Table 22]

[0272] [Table 23]

[0273] Table 22 and Figure 8A show that two AD patients essentially did not produce any IgG antibodies against the phospho-tau peptide T3.30 and T3.85 sequences or sequences therein after three immunizations at week 26 (patients #1 and #2), whereas the other five AD patients developed IgG antibodies against the phospho-tau peptide T3.30 and T3.85 sequences or sequences therein, with overall similar binding to the phospho-tau peptide T3.30 and T3.85 sequences. The OD values ​​obtained for the 8-mer peptides show that the IgG antibodies induced after three immunizations bound primarily to the N-terminal portion of the phospho-tau peptide T3.30 (SEQ ID NO: 45) and T3.85 (SEQ ID NO: 47) sequences, including the phosphorylated serine at position 396. Overall lower binding was observed to the C-terminal portions of the sequences of phospho-tau peptides T3.30 (SEQ ID NO: 45) and T3.85 (SEQ ID NO: 47), which contain phosphorylated serine at position 404.

[0274] Table 23 and FIG. 8B show that two AD patients did not produce essentially any IgG antibodies against the non-phosphorylated tau peptide T3.56 and T3.86 sequences at week 26 (patients #1 and #2). Four AD patients produced IgG antibodies that weakly recognized the non-phosphorylated tau peptide T3.56 and T3.86 sequences or sequences within them, and their binding appeared to be associated with binding of the C-terminal 8-mer peptide (tau 401-408). One AD patient (patient #3) produced IgG antibodies against the non-phosphorylated tau peptide T3.56 and T3.86 sequences or sequences within them, and their binding appeared to be primarily associated with the N-terminal portion of the sequences.

[0275] For ACI-35.030, subject IgG responses were relatively homogeneous and binding occurred across the pTau sequences examined, indicating broad epitope coverage, and importantly, with neither substantial specificity for the termini of the peptide sequence nor substantial binding to non-phosphorylated sequences.

[0276] [ka]

[0277] [ka]

[0278] [ka]

[0279] [ka]

Claims

1. 1. A pharmaceutical composition for use in a method for inducing an antibody response against phosphorylated tau protein (p-tau) in a human subject in need thereof, comprising: the pharmaceutical composition comprises liposomes; The method comprises: (1) a tau phosphopeptide consisting of the amino acid sequence of SEQ ID NO: 28 in an amount of 300 μg to 1800 μg per dose; (2) a toll-like receptor 4 agonist comprising monophosphoryl lipid A; (3) a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 17, 23 to 26, and 39 to 44; (4) A CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 18 to 22. administering to the subject an effective amount of liposomes comprising the tau phosphopeptide is displayed on the surface of the liposome; The pharmaceutical composition, wherein the antibody response persists for at least six weeks after initial administration of the effective amount of liposomes to the human subject. (a) the effective amount of liposomes is (1) the tau phosphopeptide in an amount of 300 μg to 1800 μg per dose; (2) the toll-like receptor 4 agonist in an amount of 100 μg to 585 μg per dose; (3) the helper T cell epitope in an amount of 75 μg to 550 μg per dose; (4) The CpG oligonucleotide in an amount of 150 μg to 900 μg per dose. and / or (b) the effective amount of liposomes comprises 300 μg, 900 μg, or 1800 μg of the tau phosphopeptide per dose. (a) the effective amount of liposomes is administered subcutaneously; or (b) the effective amount of the liposome is administered intramuscularly; The pharmaceutical composition of claim 1. (a) the CpG oligonucleotide has one or more phosphorothioate internucleotide linkages, and the CpG oligonucleotide is covalently linked to at least one lipophilic group, optionally via a PEG linker; (b) the tau phosphopeptide comprises the amino acid sequence of SEQ ID NO: 28, the toll-like receptor 4 agonist comprises monophosphoryl hexaacyl lipid A, 3-desacylated, the helper T cell epitope comprises the amino acid sequence of SEQ ID NO: 39, the CpG oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 18, and the liposome further comprises at least one lipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol; (c) the antibody response comprises a specific IgG antibody response directed against the p-tau, preferably the specific IgG antibody response having an anti-p-tau IgG titer that is at least 50, 60, 70, 80, 90, 100 or more times higher than that of a placebo control. (d) the antibody response induces class switching of a specific IgM antibody response to a specific IgG antibody response directed against the p-tau; and / or (e) the antibody response comprises an IgG immune response that preferentially recognizes the p-tau over non-phosphorylated tau protein, and preferably the ratio of the anti-p-tau IgG titer to the anti-tau IgG titer is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70; The pharmaceutical composition of claim 1. (a) the antibody response comprises an IgG immune response to enriched paired helical fibrils (ePHFs); and / or (b) the IgG immune response has an anti-ePHF IgG titer that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times higher than that of a placebo control; Optionally, the anti-ePHF IgG has increased binding avidity to pathological ePHF tau for at least 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or more weeks after initial administration of the effective amount of liposomes, preferably wherein the anti-ePHF IgG has an avidity index of at least 0.3, 0.4, 0.5, 0.6, or 0.

7. The pharmaceutical composition of claim 1. (a) the method further comprises administering to the subject a second dose of the effective amount of liposomes 4 to 12 weeks, e.g., 8 weeks, after the initial administration of the effective amount of liposomes. Optionally, the antibody response, preferably the anti-ePHF IgG titer, is boosted after administration of the second dose of the effective amount of liposomes, preferably the antibody response increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more when measured at least two weeks after administration of the second dose of the effective amount of liposomes; and / or (b) the method further comprises administering to the subject a third dose of the effective amount of liposomes 20 to 28 weeks, e.g., 24 weeks, after the initial administration of the effective amount of liposomes; Optionally, the antibody response, preferably the anti-ePHF IgG titer, is boosted after administration of a third dose of the effective amount of liposomes, preferably the antibody response increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more when measured at least two weeks after administration of a third dose of the effective amount of liposomes. Preferably, the method further comprises administering to the subject a fourth dose of said effective amount of liposomes 44 to 52 weeks, e.g., 48 weeks, after the initial administration of said effective amount of liposomes. More preferably, the anti-ePHF IgG titer is boosted after administration of a fourth dose of the effective amount of liposomes, preferably the antibody response increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more as measured at least two weeks after administration of a fourth dose of the effective amount of liposomes. The pharmaceutical composition according to any one of claims 1 to 5.

7. 1. A priming vaccine and / or first booster vaccine for use in a method for inducing a sustained immune response against phosphorylated tau protein (p-tau) in a human subject in need thereof, comprising: the priming vaccine and the first booster vaccine each comprise an effective amount of liposomes; The method comprises: i. intramuscularly administering to the subject a priming vaccine comprising the effective amount of liposomes; and ii. Administering the first booster vaccine comprising the effective amount of liposomes intramuscularly to the subject 6 to 10 weeks after administration of the priming vaccine. Including, the sustained immune response persists for at least about 20 weeks after administration of the priming vaccine; The liposome is (1) A tau phosphopeptide consisting of the amino acid sequence of SEQ ID NO: 28, wherein the tau phosphopeptide is present on the surface of the liposome; and (2) a toll-like receptor 4 agonist comprising monophosphoryl lipid A; (3) a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 24, 25, and 26; (4) A CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 18 to 22. and The effective amount of liposomes (1) the tau phosphopeptide in an amount of 300 μg to 1800 μg per dose; (2) the toll-like receptor 4 agonist in an amount of 100 μg to 585 μg per dose; (3) the helper T cell epitope in an amount of 85 μg to 525 μg per dose; (4) The CpG oligonucleotide in an amount of 150 μg to 900 μg per dose. A priming vaccine and / or a first booster vaccine comprising: (a) the helper T cell epitope has an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 40, 41, 42, and 43; or (b) the helper T cell epitope has an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 44; The priming vaccine and / or first booster vaccine according to claim 7. (a) a lipidated CpG oligonucleotide having the nucleotide sequence of SEQ ID NO: 18, wherein the CpG oligonucleotide is covalently linked to at least one cholesterol via a linker; (b) the first booster vaccine is administered 8 weeks after administration of the priming vaccine, and the sustained immune response persists for at least about 24 weeks after administration of the priming vaccine; and / or (c) the method further comprises intramuscularly administering to the subject a second booster vaccine comprising the effective amount of liposomes 22 to 26 weeks after administration of the priming vaccine, wherein the sustained immune response persists for at least about 36 weeks after administration of the priming vaccine. Optionally, the second booster vaccine is administered 24 weeks after administration of the priming vaccine, and the sustained immune response persists for at least about 48 weeks after administration of the priming vaccine. Preferably, the method further comprises intramuscularly administering to the subject a third booster vaccine comprising the effective amount of liposomes 45 to 50 weeks after administration of the priming vaccine, wherein the sustained immune response persists for at least about 60 weeks after administration of the priming vaccine. For example, the third booster vaccine is administered 48 weeks after administration of the priming vaccine, and the sustained immune response persists for at least about 72 weeks after administration of the priming vaccine. The priming vaccine and / or first booster vaccine according to claim 7. (a) the effective amount of liposomes comprises 300 μg of tau phosphopeptide per dose; (b) the effective amount of liposomes comprises 900 μg of tau phosphopeptide per dose; or (c) the effective amount of liposomes comprises 1800 μg of tau phosphopeptide per dose; The priming vaccine and / or first booster vaccine according to claim 7. (a) the sustained immune response comprises an IgG immune response that preferentially recognizes the p-tau over non-phosphorylated tau protein, preferably with a ratio of the anti-p-tau IgG titer to the anti-tau IgG titer of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70; and / or (b) the sustained immune response comprises an IgG immune response to concentrated paired helical fibrils (ePHF) having an anti-ePHF IgG titer that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times higher than that of a placebo control; The priming vaccine and / or first booster vaccine according to claim 7.

12. the subject is in need of clearance of tau aggregates; Optionally, the subject is in need of prevention or treatment of Alzheimer's disease, such as preclinical Alzheimer's disease, mild to moderate Alzheimer's disease or early Alzheimer's disease, or mild cognitive impairment (MCI) due to Alzheimer's disease; A priming vaccine and / or first booster vaccine according to any one of claims 7 to 11.

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