Liposome constructs

Abstract

The liposome construct comprises a liposome, at least one adjuvant, and a peptide containing at least one T cell epitope, the peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4, or an analog thereof that retains the alanine at position 2 and does not contain a methionine residue. The peptide preferably has the amino acid sequence of SEQ ID NO: 6. This liposome construct can be used to produce a liposome vaccine composition further comprising at least one antigenic peptide displayed on the liposome surface. This liposome vaccine composition is useful for therapy. A method of manufacture is also described.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to liposomal constructs suitable for use in vaccine compositions, liposomal vaccine compositions, and their preparation. [Background technology]

[0002] Background of the Invention The immune system is a complex, interactive system within the body that involves diverse components, including cells and cellular factors, that interact with internal and external stimuli. One of the best-known aspects of the immune system is its ability to respond to foreign antigens presented by invading organisms, internal cellular changes, or vaccinations.

[0003] The immune system's response to antigens includes both humoral and cellular immune responses. Humoral immune responses are mediated by noncellular factors released by cells, which may or may not be present free in plasma or intracellular fluids. The primary component of the humoral immune response of the immune system is mediated by antibodies produced by B lymphocytes. Cellular immune responses arise from interactions between antigen-presenting cells and cells such as B lymphocytes (B cells) and Th15 lymphocytes (T cells). Stimulating the immune response through vaccination has been shown to be an effective strategy for treating or preventing a variety of diseases and disorders in animals and humans, including infectious diseases, allergies, neurodegenerative diseases, and cancer.

[0004] Liposomal vaccines have attracted increasing attention in recent years. The idea of ​​using liposomes as a vehicle for antigen presentation was validated more than 30 years ago (Allison and Gregoriadis, Nature, 1974, 252, 252). For example, liposomally incorporated diphtheria toxoid has been shown to be more immunogenic than the free form. Antigens presented via liposomes can induce not only cellular but also humoral immune responses. Liposomes are artificial vesicles composed primarily of (phospho)lipids and may contain drugs or soluble antigens in the internal aqueous volume or amphiphilic antigens such as membrane proteins in the bilayer. Many microbial and tumor cell-derived antigens have been incorporated into such liposomes after extensive characterization and in vivo testing. Clinical studies using antigen-containing liposomes have shown them to be safe and generally do not cause serious side effects (Kersten and Crommelin, Biochimica et Biophysica Acta 1995, 1241, 117-138).

[0005] WO2005 / 081872 describes a supramolecular antigen structure comprising an antigen peptide PEGylated at each terminus and reconstituted within a liposome, thereby enhancing peptide presentation. WO2010 / 106127 describes an antigen composition having a highly repetitive modified antigen sequence on the surface of a liposome. This antigen composition is suitable for inducing T-cell-independent immune responses in the treatment of diseases. WO2012 / 055933 relates to a liposome-based antigen structure having an antigen peptide of interest modified via a hydrophobic moiety, and a method for preparing the liposome-based structure.

[0006] WO2012 / 020124 describes methods and compositions that allow for control of peptide conformation by adjusting the lipidation pattern of the peptide, the spacer, and the liposome composition, or by co-administration with a small molecule compound.

[0007] WO2019 / 197414 relates to a liposomal vaccine composition comprising an amyloid beta (Abeta)-derived peptide antigen, a peptide containing a T cell epitope, and an adjuvant, presented on the surface of a liposome, for use in treating, preventing, or alleviating symptoms associated with Abeta-related diseases.

[0008] However, challenges remain in this field, not only in disease targeting and treatment, but also in vaccine manufacturing and product stability. Vaccine instability can be caused by light, heat, oxidation, radiation, environmental changes, or reactions with other components in the vaccine mixture. This can lead to a gradual loss of product potency or degradation, making the product unsuitable for use.

[0009] Recent pandemics have made it clear that countering rapidly mutating viruses requires faster ways to produce new vaccines, including "plug-and-play" technologies that allow antigens in vaccine compositions to be switched as viruses mutate, facilitating the rapid development of new vaccines.

[0010] It would be desirable to address or mitigate at least some of the above problems. Summary of the Invention

[0011] In a first aspect, the present invention provides a liposome construct comprising a liposome, at least one adjuvant, and at least one peptide containing at least one T cell epitope. The at least one T cell epitope may comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 4 or an analog thereof. The liposome of the liposome construct may be an at least partially spherical vesicle and may comprise at least one lipid bilayer defining a core.

[0012] In some embodiments, the liposome may have a negative surface charge, meaning that the liposome may be anionic. Preferably, the liposome comprises a phospholipid, more preferably, the phospholipid comprises dimylcitoylphosphatidylcholine (DMPC) and dimylcitoylphosphatidylglycerol (DMPG). The liposome may further comprise cholesterol. In some embodiments, the molar ratio of these three components may be 9:1:7.

[0013] The liposome constructs of the present invention comprise a peptide containing at least one T-cell epitope.

[0014] Preferably, the peptide containing at least one T-cell epitope is at least partially encapsulated within the liposome.

[0015] In some embodiments, the peptide may further be at least partially displayed on the inner and / or outer surface of the lipid bilayer of the liposome, and / or the peptide may be at least partially incorporated into at least one layer of the lipid bilayer of the liposome. The peptide may be associated with the lipid bilayer of the liposome. For example, the peptide may be associated with the liposome so as to be displayed on the surface of the liposome. For example, the peptide may be associated with the liposome through electrostatic interactions between the peptide and the lipid bilayer of the liposome.

[0016] In a preferred embodiment, a peptide containing at least one T cell epitope is encapsulated within a liposome. In another preferred embodiment, the peptide is at least partially encapsulated within the liposome and further at least partially displayed on the inner and / or outer surface of the liposome.

[0017] The peptides contained in the liposome constructs of the present invention contain at least one T cell epitope. Therefore, the peptides can activate T cells, particularly helper T cells. Each of the at least one T cell epitope is typically a universal T cell epitope. "Universal T cell epitope" refers to an epitope specific for T cells present in the majority of the human population. These epitopes are usually derived from antigens to which humans are commonly exposed throughout their lives. Examples include antigens contained in routinely administered vaccines. Specific examples include T cell epitopes of tetanus, influenza, diphtheria, keyhole limpet hemocyanin (KLH), and Epstein-Barr virus (EBV). The "universal" ability of a T cell epitope to activate T cells is the result of at least two complementary properties: i) binding affinity to the HLA groove (meaning binding strength), and ii) the ability to indiscriminately bind to different HLA haplotypes (meaning the ability to cover a highly diverse human population with respect to differences in HLA molecule expression). A universal T cell epitope may bind to a majority of MHC class II alleles present in the human population. Therefore, the universal T cell epitope contained in the liposome construct and liposome vaccine composition of the present invention may be capable of stimulating a CD4 T cell response. The universal T cell epitope contained in the liposome construct and liposome vaccine composition of the present invention may be capable of stimulating a helper T cell response that enhances antibody production by B cells, for example, in response to an antigenic peptide (e.g., amino acids 1-15 of Abeta) displayed on the surface of the liposome, as further described herein.

[0018] The peptide containing at least one T cell epitope comprises an epitope derived from influenza hemagglutinin modified to substitute alanine for the second methionine in SEQ ID NO:2 (SEQ ID NO:4), or an analog of SEQ ID NO:4 as defined herein. In some embodiments, the peptide containing at least one T cell epitope may further comprise one or more epitopes derived from one or more of tetanus, influenza, diphtheria, and keyhole limpet hemocyanin (KLH) and Epstein-Barr virus (EBV).

[0019] In a preferred embodiment, the peptide containing at least one T-cell epitope may further comprise an epitope derived from tetanus toxin.

[0020] Peptides containing at least one T cell epitope can be synthesized by solid-phase synthesis. In some embodiments, the peptides containing at least one T cell epitope can contain up to 85 amino acids. Optionally, the peptides containing at least one T cell epitope can have a length of up to 80, 75, or 70 amino acids.

[0021] In some embodiments, a peptide containing at least one T cell epitope may contain at least 10 amino acids. Using a peptide with at least 10 amino acids ensures that a sufficiently immunogenic T cell epitope is generated. Optionally, the peptide containing at least one T cell epitope may contain more than 10 amino acids. For example, the peptide containing at least one T cell epitope may contain at least 20, 30, or 40 amino acids. In other embodiments, the peptide may contain 30 to 60 amino acids. This is based on the preferred minimum length per universal T cell epitope and the preferred range for peptides containing at least two, three, or four T cell epitopes.

[0022] In some embodiments, the peptide containing at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 4. Alternatively, the peptide containing at least one T cell epitope may consist essentially of or consist of the amino acid sequence of SEQ ID NO: 4.

[0023] In some embodiments, the peptide containing at least one T cell epitope can include at least two T cell epitopes, at least one of which can comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO:4.

[0024] Analogs are sequences that are functionally equivalent with respect to their ability to stimulate T helper cells and may contain one or more modifications compared to the listed sequences. In the context of SEQ ID NO: 4, analogs must additionally retain the alanine at position 2 of the sequence. Analogs must not contain a methionine residue. Modifications may include one or more (preferably one or two) additions, deletions, or substitutions, as long as the function as a (universal) T-cell epitope is maintained. The minimum and maximum lengths of peptide analogs are defined above and apply mutatis mutandis to analogs. If a peptide contains multiple T-cell epitopes, the minimum length of the peptide is adjusted accordingly to maintain the function of each T-cell epitope.

[0025] In a preferred embodiment, a peptide containing at least one T cell epitope comprises a first epitope comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:4 or an analog thereof, and may further comprise a further T cell epitope comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3, or an analog of SEQ ID NO:1 or SEQ ID NO:3. In this context, an analog of SEQ ID NO:1 or SEQ ID NO:3 is as defined above, i.e., a functionally equivalent sequence with respect to its ability to stimulate T helper cells, and may contain one or more modifications compared to the recited sequence. The modifications may include one or more (preferably one or two) additions, deletions, or substitutions, as long as the function as a (universal) T cell epitope is maintained. These modifications do not introduce a methionine residue into the peptide. The minimum and maximum lengths of peptide analogs are described above and apply mutatis mutandis to analogs.

[0026] In a preferred embodiment, the peptide containing at least one T cell epitope comprises at least three T cell epitopes, at least one of which comprises, consists of, or consists essentially of SEQ ID NO: 4 or an analog thereof. In a preferred embodiment, the at least three T cell epitopes may further comprise, consist of, or consist essentially of an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 1 and / or SEQ ID NO: 3, or analogs thereof.

[0027] Thus, the peptide can comprise at least three T cell epitopes, wherein the at least three T cell epitopes comprise, consist of, or consist essentially of the amino acid sequences of SEQ ID NO:4, SEQ ID NO:1, and SEQ ID NO:3, or analogs of these sequences as defined herein.

[0028] In a preferred embodiment, the at least three T cell epitopes comprise, consist of, or consist essentially of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4. In a particularly preferred embodiment, the peptide containing at least one T cell epitope comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6, or an analog thereof. An analog of SEQ ID NO: 6 is as defined above, considering that the peptide contains four separate (universal) T cell epitopes. Thus, the minimum length of an analog herein is 40 amino acids to ensure that functionality of each T cell epitope is retained (minimum length per T cell epitope is 10 amino acids).

[0029] In some embodiments, a peptide containing at least one T cell epitope may include one or more linkers. The linkers may be configured to link one or more T cell epitopes of the peptide to one another. In other words, a peptide may include multiple T cell epitopes linked to one another by linkers.

[0030] Linkers are used to physically link T cell epitopes together in a manner that does not impair the immunogenicity of each linked epitope. Preferred linkers are themselves amino acid-based linkers, i.e., peptide linkers, and thus can link T cell epitopes together via peptide bonds.

[0031] Thus, a peptide containing at least one T cell epitope may further comprise one or more linkers, for example, a peptide having at least two T cell epitopes may comprise at least one linker to connect the two T cell epitopes.

[0032] In some preferred embodiments, the peptide containing at least one T cell epitope comprises at least three T cell epitopes linked by at least two linkers, and thus the peptide may be a linear peptide in which the terminal epitopes are not linked to each other.

[0033] The linker preferably comprises a substrate for a lysosomal cysteine ​​protease of the papain family. The linker may comprise one or more substrates for cathepsin S, cathepsin B, and cathepsin L. The linker may comprise, consist essentially of, or consist of at least two or at least three amino acids. In some embodiments, the linker may comprise, consist essentially of, or consist of the amino acids VVR, TVGLR, KVSVR, PMGAP, or PMGLP.

[0034] In a preferred embodiment, at least one linker comprises, consists of, or consists essentially of the amino acid sequence VVR. In a preferred embodiment, all linkers included in a peptide containing at least one T-cell epitope comprise, consist of, or consist essentially of the amino acid sequence VVR.

[0035] In a preferred embodiment, the peptide containing at least one T cell epitope may comprise the amino acid sequence of SEQ ID NO:6.

[0036] In some embodiments, a peptide having a universal T-cell epitope can consist of or consist essentially of the amino acid sequence of SEQ ID NO:6.

[0037] A peptide comprising at least three T cell epitopes and having the amino acid sequence of SEQ ID NO: 6 is also referred to interchangeably herein as a SAT58 peptide.

[0038] Advantageously, the T cell epitope-containing peptides described herein are stable to oxidation. The stability of the T cell epitope-containing peptides to oxidation advantageously results in a reduction or absence of oxidative degradation products of the T cell epitope peptides, which may adversely affect the toxicity profile and / or shelf life of the liposomal construct or liposomal vaccine. Thus, the present invention advantageously enables the provision of stable liposomal constructs and vaccine compositions, as demonstrated in the examples included herein.

[0039] By reducing the oxidation of T cell epitope-containing peptides, the production of unwanted degradation products in products in which liposome constructs are used, such as liposome vaccines, is reduced, thereby eliminating or minimizing the risk of toxicity caused by peptide degradation products.By reducing the oxidation of T cell epitope-containing peptides, the shelf life of liposome constructs or products in which liposome constructs are used can be extended.For example, by using liposome constructs in vaccine compositions, the risk of oxidation during storage of vaccines can be reduced, thereby extending the shelf life and usefulness of vaccines.

[0040] These and further advantages and benefits of the liposomal constructs of the present invention are illustrated with reference to the following examples.

[0041] The liposome construct comprises at least one adjuvant. Optionally, the liposome construct may comprise multiple adjuvants. In some embodiments, the adjuvant may form part of the liposome. For example, the adjuvant may form part of the lipid bilayer of the liposome. Optionally, the adjuvant may be at least partially incorporated within the lipid bilayer of the liposome. In some embodiments, the adjuvant may be at least partially presented on the surface of the liposome and / or at least partially incorporated within the lipid bilayer of the liposome.

[0042] Adjuvants, when used in pharmaceutical compositions such as vaccines, serve to enhance the immune response. Adjuvants typically stimulate the immune system to induce a stronger and / or longer-lasting immune response.

[0043] Optionally, the adjuvant for the liposome construct may be selected from one or more of monophosphoryl lipid A (MPLA), diphosphoryl lipid A (DPLA), alum, Pam2CSK4, Pam3CSK4, Pam3CAG, saponin, CpG, lipidated CpG (e.g., CpG cholesterol), cationic lipid, phosphorothioated PS-CpG-ODN, CpG oligodeoxynucleotide (CpG-ODN), CpG-A, CpG-B, or CpG-C.

[0044] Further adjuvants that can be used in accordance with the present invention include aluminum phosphate or hydroxide (Al(OH)3, AlPO4), calcium, iron, zirconium salts, QuilA, QS-21, trehalose dimycolate (TDM), lipoteichoic acid (purified from Staphylococcus aureus), DDAB (dimethyldioctadecylammonium (bromide salt)), MF59, L18-MDP and B30-MDP (hydrophobic muramyl dipeptide derivatives), C12-iE-DAP (diaminopimelic acid).

[0045] In a preferred embodiment, at least one adjuvant may be a lipid-based adjuvant.

[0046] In some embodiments, the adjuvant may be a Toll-like receptor (TLR) agonist, particularly a TLR4 agonist or a TLR9 agonist.

[0047] As used herein, the term "Toll-like receptor 4 agonist" refers to any compound that acts as an agonist of TLR4. Examples of TLR4 agonists useful in the present invention include, but are not limited to, monophosphoryl lipid A (MPLA). 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.

[0048] Monophosphoryl lipid A, or MPLA, refers to a modified form of lipid A, the biologically active moiety of the lipopolysaccharide (LPS) endotoxin of Gram-negative bacteria. MPLA has immunostimulatory activity but is less toxic than LPS.

[0049] The term MPLA includes MPLA derivatives such as monophosphoryl hexaacyl lipid A, 3-deacyl (synthetic) (3D-(6-acyl)PHAD®), PHAD® (phosphorylated hexaacyl disaccharide), PHAD®-504, 3D-PHAD® (Avanti Polar Lipids, Alabama, USA), or MPL. Thus, according to certain embodiments, the composition further comprises MPLA. MPLA is typically added during liposome formation (as further described herein). In one embodiment, the adjuvant for the liposome construction may be 3D-(6-acyl)PHAD®.

[0050] For example, a preferred liposome construct of some embodiments may comprise dimylcitoylphosphatidylcholine (DMPC), dimylcitoylphosphatidylglycerol (DMPG), cholesterol, and MPLA, and the molar ratio of these four components may be 9:1:7:0.05 in some embodiments.

[0051] As used herein, the term "Toll-like receptor 9 agonist" (TLR9 ​​agonist) refers to any compound that acts as an agonist of TLR9. Examples of suitable TLR9 agonists include, but are not limited to, CpG oligonucleotides. As used herein, the terms "CpG oligonucleotide," "CpG oligodeoxynucleotide," or "CpG ODN" refer to an oligonucleotide containing at least one CpG motif. As used herein, "oligonucleotide," "oligodeoxynucleotide," or "ODN" refers to a polynucleotide formed from multiple linked nucleotide units. Such oligonucleotides can be obtained from existing nucleic acid sources or produced by synthetic methods. As used herein, the term "CpG motif" refers to a nucleotide sequence containing an unmethylated cytosine-phosphate guanine (CpG) dinucleotide (i.e., cytosine (C) followed by guanine (G)) linked by a phosphate bond, a phosphodiester backbone, or other internucleotide bond. Examples of synthetic CpG oligonucleotides include, but are not limited to, CpG2006 (also known as CpG 7909), CpG 1018, CpG2395, CpG2216, or CpG2336.

[0052] In one embodiment, the adjuvant of the liposome construct may include CpG.

[0053] In one embodiment, the CpG oligonucleotide may be covalently linked to the lipid bilayer of the liposome.

[0054] In another aspect, the present invention relates to a liposomal vaccine composition comprising the liposomal construct of the present invention and at least one antigenic peptide. The term "liposomal vaccine composition" is used throughout this disclosure. This term can be used interchangeably with "liposomal composition," "liposomal vaccine," and "vaccine composition." These compositions are immunogenic and therefore can also be referred to as "liposomal immunogenic compositions."

[0055] An antigenic peptide is understood to include any peptide that can induce an immune response in a mammal, particularly a human, when administered to the mammal. The antigenic peptide may be derived from a foreign (external) antigen, such as a virus or an allergen, or may be derived from an autoantigen, such as a tumor antigen, a cytokine such as IL-17 or IL-27, or a protein involved in a proteinopathy, such as Abeta, tau, or a-syn.

[0056] The antigenic peptide is at least partially displayed on the surface of the liposome. Thus, the liposome can serve as a carrier of the antigenic peptide. If desired, the antigenic peptide can be at least partially bound to the lipid bilayer of the liposome. For example, the antigenic peptide can be at least partially inserted into the lipid bilayer of the liposome, or fixed to the lipid bilayer, and / or form part of the outer surface of the liposome.

[0057] The antigenic peptide is presented on the surface of the liposome. As understood by those skilled in the art, "presented on the surface of the liposome" means that the peptide is at least partially presented on the outer surface of the (intact) liposome (see, e.g., Muhs, 2007; Pihlgren, 2013). This is usually achieved by insertion into the outer surface of the liposome or by other methods of immobilization. The antigenic peptide preferably contains at least one lipophilic or hydrophobic moiety to facilitate this. Optionally, the antigenic peptide may be modified with multiple lipophilic or hydrophobic moieties. For example, the antigenic peptide may contain two, three, or four lipophilic or hydrophobic moieties. The lipophilic or hydrophobic moiety can bind the antigenic molecule to the liposome. One or more lipophilic or hydrophobic moieties may be at least partially inserted into the outer surface of the liposome, i.e., into the lipid bilayer of the liposome. The one or more lipophilic or hydrophobic moieties are preferably hydrophobic moieties to facilitate insertion into the lipid bilayer. In some embodiments, the one or more moieties may be one or more of a fatty acid, a triglyceride, a diglyceride, a steroid, a sphingolipid, a glycolipid, or a phospholipid.

[0058] At least one lipophilic or hydrophobic moiety can be incorporated into the lipid bilayer of the liposome to facilitate binding of the antigenic peptide, which can act as an anchor for the peptide in the liposome bilayer, positioning and / or stabilizing the antigenic peptide in close proximity to the liposome surface.

[0059] Preferably, at least one lipophilic or hydrophobic moiety is a fatty acid. The fatty acid may comprise a carbon backbone having at least 3 carbon atoms. Optionally, the fatty acid may comprise a carbon backbone having 4, 6, 8, 12, or 14 carbon atoms.

[0060] In some embodiments, the fatty acid may comprise a carbon backbone having up to 24 carbon atoms. The fatty acid may comprise at least 14 carbon atoms. Alternatively, the fatty acid may comprise a carbon backbone having at least 16 carbon atoms.

[0061] Hydrophobic moieties include, but are not limited to, palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, itnolenic acid, cholesterol, or 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE).

[0062] In a preferred embodiment, the moiety(s) may include a palmitoyl residue. Thus, the antigenic peptide may be palmitoylated, i.e., monopalmitoylated or multipalmitoylated. For example, the antigenic molecule may be modified with at least two palmitoyl residues. Alternatively, the antigenic molecule may be modified with at least four palmitoyl residues (i.e., tetrapalmitoylated). The antigenic peptide may contain additional residues, such as lysine residues, to facilitate palmitoylation. These residues are typically located at the N- and / or C-terminus of the peptide. In some embodiments, one to four lysine residues may be added to the N- and / or C-terminus. In one embodiment, a preferred configuration includes an antigenic peptide bound to two palmitoyl residues in the N- and / or C-terminal regions of the peptide. Thus, the antigenic peptide is dipalmitoylated or tetrapalmitoylated. This is facilitated by incorporating two lysine residues in the N- and / or C-terminal regions of the peptide antigen and palmitoylating the lysine residues.

[0063] In some embodiments, two different antigenic peptides are introduced into the liposome. The two different antigenic peptides can be derived from the same protein or different proteins. If the proteins are different, they typically target the same disease.

[0064] The liposome constructs of the present invention are applicable to a variety of antigenic peptides and can ultimately be used in therapeutic formulations and vaccines for diseases and disorders including, but not limited to, neurodegenerative diseases, cancer, autoimmune diseases, and infectious diseases.

[0065] In some embodiments, the antigenic peptide may be derived from a foreign antigen. As used herein, the term "foreign antigen" refers to any molecule not naturally produced by an individual (human). Generally, an individual's immune system recognizes the foreign antigen and generates an immune response against the antigen. For example, the foreign antigen may be a viral antigen or an allergen. Examples of viral antigens include, but are not limited to, antigens derived from rhinovirus, coronavirus, enterovirus, adenovirus, parainfluenza virus, and respiratory syncytial virus. In some embodiments, the antigen may be a viral fusion protein.

[0066] In some embodiments, antigen peptides may be autoantigens. As used herein, the term "autoantigen" refers to any peptide derived from an antigen naturally produced by an individual. Generally, an individual's immune system will show tolerance to autoantigen molecules, so no immune reaction will occur. In some cases, the immune system may not show tolerance to autoantigens, resulting in autoimmune diseases.

[0067] The use of self-antigens allows one to target molecules to which the immune system shows tolerance, which may help induce an immune response that would not otherwise occur.

[0068] In a preferred embodiment, the antigenic peptide is an autoantigenic peptide.

[0069] In some embodiments, the antigenic peptide is a peptide derived from an amyloid protein. In some embodiments, the antigenic peptide may be derived from an amyloid-like protein. Amyloid-like proteins include, but are not limited to, prion protein, tau protein, alpha-synuclein (a-syn), huntingtin, amylin, or Abeta.

[0070] In a preferred embodiment, the autoantigen peptide may be derived from at least one protein selected from Abeta, tau protein, alpha-synuclein, huntingtin, prion protein, or amylin. Thus, the autoantigen peptide may be Abeta peptide, alpha-synuclein peptide, tau peptide, huntingtin peptide, prion peptide, or amylin peptide. Thus, the antigen peptide may target one or more of Abeta-related diseases or disorders, tau-related diseases or disorders, alpha-synuclein-related diseases or disorders, Huntington's disease-related diseases or disorders, prion-related diseases or disorders, and amylin-related diseases or disorders.

[0071] In some embodiments, the liposomal constructs or liposomal vaccine compositions of the present invention can be used therapeutically, i.e., as a pharmaceutical, for example, to prevent, treat, or alleviate an infectious disease, an exoantigenic disease or disorder, such as an autoantigenic disease or disorder, or a symptom thereof.

[0072] In one embodiment, the autoantigen-associated disease or disorder may be a neurological or neurodegenerative disease or disorder.

[0073] In a preferred embodiment, the autoantigenic peptide may be an Abeta peptide or a fragment thereof. The amyloid-beta-derived peptide antigen may comprise amino acids 1-15 of amyloid-beta. Alternatively, the amyloid-beta-derived peptide antigen may consist of amino acids 1-15 of Abeta or consist essentially of Abeta. In some embodiments, the liposome construct or liposome vaccine composition according to the present invention can be used to prevent, treat, or alleviate symptoms of Abeta-related diseases or disorders. For example, Abeta-related diseases or disorders may include Alzheimer's disease, mild cognitive impairment (MCI), Down's syndrome (OS), Down's syndrome-associated Alzheimer's disease, cardiac amyloidosis, cerebral amyloid angiopathy (CAA), multiple sclerosis, Parkinson's disease, dementia with Lewy bodies, amyotrophic lateral sclerosis (ALS), adult-onset diabetes, inclusion body myositis (IBM), ocular amyloidosis, glaucoma, macular degeneration, lattice dystrophy, and optic neuritis.

[0074] Thus, in a preferred embodiment, the present invention provides a liposomal vaccine composition comprising: liposomes, MPLA adjuvant, A peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6, and An amyloid-beta-derived peptide antigen presented on the surface of said liposome, comprising, consisting essentially of, or consisting of amino acids 1-15 of Abeta.

[0075] In a preferred embodiment, the liposomal vaccine composition comprises: liposomes, an MPLA adjuvant incorporated into said liposome; A peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6, and An amyloid-beta-derived peptide antigen comprising or consisting essentially of amino acids 1-15 of Abeta, wherein the antigen is palmitoylated and palmitic acid residues are incorporated into the outer layer of the liposome.

[0076] In a preferred embodiment, the liposomal vaccine composition comprises: Liposomes MPLA adjuvant incorporated into the liposomes A peptide consisting of the amino acid sequence of SEQ ID NO: 6 and An amyloid-beta-derived peptide antigen consisting of amino acids 1-15 of amyloid-beta, which is tetrapalmitoylated via N-terminal and C-terminal lysine residues (two at each end) added to the peptide, and palmitic acid residues are incorporated into the outer layer of the liposome.

[0077] Tetrapalmitoylated Abeta 1-15 is represented by SEQ ID NO: 7 below: H-Lys(palmitoyl)-Lys(palmitoyl)-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys(palmitoyl)-Lys(palmitoyl)-OH

[0078] Abeta 1-15 is represented by SEQ ID NO: 8 below: H-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-OH

[0079] In a preferred embodiment, a peptide comprising, consisting essentially of, or consisting of, preferably consisting of, the amino acid sequence of SEQ ID NO: 6 is at least partially encapsulated within a liposome.

[0080] Preferably, the liposome comprises a phospholipid, and more preferably, the phospholipid comprises dimylcitoylphosphatidylcholine (DMPC) and dimylcitoylphosphatidylglycerol (DMPG). The liposome may further comprise cholesterol. The molar ratio of these three components may be 9:1:7 in some embodiments. A more particularly preferred liposome construct of the present invention comprises dimylcitoylphosphatidylcholine (DMPC), dimylcitoylphosphatidylglycerol (DMPG), cholesterol, and MPLA. The molar ratio of these four components may be 9:1:7:0.05.

[0081] A preferred liposomal vaccine composition of the present invention is designated and referenced herein as ACI-24.060.

[0082] In another preferred embodiment, the autoantigenic peptide may be an alpha-synuclein (a-syn)-derived peptide or a fragment thereof. The term alpha-synuclein-derived peptide is intended to encompass both native alpha-synuclein and its variants. Examples of suitable variants include, but are not limited to, the alpha-synuclein-derived antigenic peptides described in WO 2022 / 029181 (incorporated herein by reference). In particular, variants of the a-syn peptide derived from amino acids 111-124 of the full-length human a-syn amino acid sequence (GILEDMPVDPDNEA (SEQ ID NO: 10)) are included. Such variant peptides can elicit potent anti-a-syn antibody responses, and the induced antibodies exhibit high cross-reactivity with human a-syn, despite the peptides having sequences different from the native sequence. Thus, in one embodiment, the alpha-synuclein-derived peptide antigen may comprise the amino acid sequence of SEQ ID NO: 9 (GG-KESMPVDPDNEA), or a version lacking the two glycine residues. SEQ ID NO: 9 is a mutant α-synuclein peptide consisting of amino acids 111 to 124 of the full-length human α-synuclein amino acid sequence (GILEDMPVDPDNEA (SEQ ID NO: 10)). In another embodiment, the α-synuclein-derived peptide antigen may comprise the amino acid sequence of SEQ ID NO: 10.

[0083] The native sequence of human alpha-synuclein is: MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (SEQ ID NO: 11).

[0084] In a preferred embodiment, the α-synuclein-derived antigenic peptides described herein may be conjugated to one or two palmitoyl residues in the N-terminal region and / or one or two palmitoyl residues in the C-terminal region of the peptide. Thus, the antigenic peptide is monopalmitoylated or dipalmitoylated at one or, optionally, both, of the peptide's terminal regions. This may be facilitated by incorporating lysine residues in the N-terminal and / or C-terminal regions of the α-synuclein-derived peptide antigen, where the lysine residues are palmitoylated. In one embodiment, the antigenic peptide may be dipalmitoylated via lysine residues at the N-terminus or C-terminus, where the lysine residues are palmitoylated. In another embodiment, the antigenic peptide may be tetrapalmitoylated via lysine residues at the N-terminus and C-terminus (two at each end). In a preferred embodiment, the alpha-synuclein-derived peptide antigen may comprise, consist of, or consist essentially of an alpha-synuclein-derived antigenic peptide having the amino acid sequence of SEQ ID NO: 9, dipalmitoylated via two lysine residues in the N-terminal region.

[0085] In some embodiments, liposomal constructs or liposomal vaccine compositions according to the present invention can be used to prevent, treat, or alleviate the symptoms of an alpha-synuclein-related disease or disorder.

[0086] Thus, in one aspect, the present invention provides a liposomal vaccine composition comprising: liposomes, MPLA adjuvant, CpG cholesterol, A peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, and An alpha-synuclein-derived antigenic peptide displayed on the surface of the liposome, comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

[0087] In a preferred embodiment, the liposomal vaccine composition comprises: liposomes, an MPLA adjuvant incorporated into said liposome; CpG cholesterol incorporated into the liposome, A peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 6, and An alpha-synuclein-derived antigenic peptide presented on the surface of the liposome and consisting essentially of the amino acid sequence of SEQ ID NO: 9, wherein the antigenic peptide is palmitoylated and palmitic acid residues are incorporated into the outer layer of the liposome.

[0088] In a preferred embodiment, the liposomal vaccine composition comprises: liposomes, MPLA adjuvant incorporated into liposomes, CpG cholesterol incorporated into liposomes, A peptide consisting of the amino acid sequence of SEQ ID NO: 6, and An alpha-synuclein-derived peptide antigen displayed on the surface of the liposome comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 9, wherein the antigenic peptide comprises one or two palmitic acid residues in the N-terminal region of the peptide or one or two palmitic acid residues in the C-terminal region of the peptide, and the palmitic acid residues are incorporated into the outer layer of the liposome.

[0089] In a preferred embodiment, the alpha-synuclein-derived peptide antigen displayed on the surface of a liposome comprises or consists essentially of the amino acid sequence of SEQ ID NO: 9, and the antigenic peptide comprises two palmitic acid residues in the N-terminal region of the peptide, which palmitic acid residues are incorporated into the outer layer of the liposome. Preferably, palmitoylation is achieved via lysine residues added to the peptide, as described herein.

[0090] Preferably, the liposome comprises a phospholipid and cholesterol. The phospholipid may comprise dimylcitoylphosphatidylcholine (DMPC) and dimylcitoylphosphatidylglycerol (DMPG). In some preferred embodiments, the Abeta-related disease may be Alzheimer's disease. Additionally or alternatively, the Abeta-related disease may be Down's syndrome.

[0091] In another embodiment, the autoantigenic peptide may be a peptide derived from tau protein or a fragment thereof. In some embodiments, the liposome construct or liposome vaccine composition of the present invention can be used to prevent, treat, or alleviate symptoms of a tau protein-related disease or disorder.

[0092] Examples of tau-associated diseases or disorders include Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, dementia vogelii, Down's syndrome, Gerstmann-Straussler-Scheinker disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis, Guam-type Parkinsonism-Dementia Complex, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic granular dementia, corticobasal degeneration, Lewy-type amyotrophic lateral sclerosis, and diffuse neurogenic tau with calcifications. Examples of the dementia include fibrotic tangles, frontotemporal dementia, preferably frontotemporal dementia with chromosome 17-linked Parkinsonism (FTDP-17), frontotemporal lobar dementia, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle dementia, postencephalitic Parkinson's disease, myotonic dystrophy, chronic traumatic encephalopathy (CTE), primary age-related tauopathy (PART), or dementia with Lewy bodies (LBD).

[0093] In another embodiment, the autoantigenic peptide may be an a-syn protein-derived peptide or a fragment thereof. In some embodiments, the liposome constructs or liposome vaccine compositions according to the present invention may be used to prevent, treat, or alleviate the symptoms of a-syn-related disease or disorder (synucleopathy).

[0094] Alpha-synuclein-associated diseases or disorders include Lewy body diseases (LBD), particularly Parkinson's disease (PD), Parkinson's disease with dementia (PDD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) or neurodegeneration with brain iron accumulation type I (NBIA type I).

[0095] The liposomal vaccine compositions of the present invention optionally comprise at least one pharmaceutically acceptable carrier, which term expressly includes diluents and excipients. Such agents are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, 15th or 18th Ed. (Alfonso R. Gennaro, ed.; Mack Publishing Company, Easton, PA, 1990); Remington: the Science and Practice of Pharmacy 19th Ed. (Lippincott, Williams & Wilkins, 1995); Handbook of Pharmaceutical Excipients, 3rd Ed. (Arthur H. Kibbe, ed.; Amer. Pharmaceutical Assoc, 1999); Pharmaceutical Codex: Principles and Practice of Pharmaceutics 12th Ed. (Walter Lund ed.; Pharmaceutical Press, London, 1994); The United States Pharmacopeia: The National Formulary (United States Pharmacopeial Convention); Fiedler's "Lexikon der Hilfstoffe" 5th Ed., Edition Cantor Verlag, the disclosures of which are incorporated herein by reference. See Aulendorf 2002; "The Handbook of Pharmaceutical Excipients", 4th Ed., American Pharmaceuticals Association, 2003, and Goodman and Gilman's: The Pharmacological Basis of Therapeutics (Louis S. Goodman and Lee E. Limbird, eds.; McGraw Hill, 1992).Carriers, diluents, and excipients can be selected taking into consideration the intended route of administration and standard pharmaceutical practice. These compounds must be acceptable in the sense that they are not harmful to the recipient. Preferred examples of usable carriers include buffer solutions.

[0096] In another aspect, the present invention relates to a method for producing or manufacturing a liposomal construct of the present invention. The method may include providing a peptide containing at least one T cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:4, or an analog thereof that retains the alanine at position 2 and does not contain a methionine residue. The method may be adapted to produce any liposomal construct described herein. Thus, for example, this step of the method may include providing a peptide containing at least three universal T cell epitopes. The at least three universal T cell epitopes may comprise the amino acid sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or analogs thereof defined herein. Alternatively, the at least three universal T cell epitopes may consist of or essentially consist of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or analogs thereof defined herein. The method may further include providing a liposome containing at least one adjuvant.

[0097] The method includes a step of conjugating a peptide containing at least one T cell epitope with a liposome to produce a liposome construct. The conjugation step preferably results in at least partial encapsulation of the peptide containing at least one T cell epitope in the liposome. The peptide containing at least one T cell epitope preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6 or an analog thereof. This is effectively the only essential step of the method, considering that other steps are related to providing a product for conjugation. Therefore, the providing steps of the method for generating or manufacturing a liposome construct can be performed in any order to enable the essential step of conjugating the two components.

[0098] In a further aspect, the present invention provides a method for producing or manufacturing a liposomal vaccine composition, which method comprises, following the preparation of a liposomal construct (outlined below), the further step of incorporating at least one peptide antigen (as described herein), optionally an autoantigenic peptide, into the liposome. The antigenic peptide is incorporated into the liposome such that the at least one antigen is at least partially displayed on the surface of the liposome. Thus, the key step here is the incorporation of at least one peptide antigen into the liposomal construct of the present invention to form the liposomal vaccine composition of the present invention.

[0099] According to these methods, the at least one adjuvant may include MPLA and may further include a CpG adjuvant.

[0100] In methods comprising incorporating at least one peptide antigen, the peptide antigen may be palmitoylated. In such embodiments, palmitic acid residues are incorporated into the outer layer of the liposome.

[0101] Suitable peptide antigens are described herein.Accordingly, in some embodiments, the peptide antigen is an amyloid-beta-derived peptide.In a preferred embodiment, the peptide antigen is an amyloid-beta-derived peptide antigen that comprises, consists of, or essentially consists of amino acids 1-15 of Abeta.

[0102] In other embodiments, the peptide antigen is an alpha-synuclein-derived peptide. In a preferred embodiment, the peptide antigen is an alpha-synuclein-derived antigenic peptide displayed on the surface of a liposome comprising, consisting of, or consisting of the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. [Brief explanation of the drawings]

[0103] Non-limiting embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Figure 1] FIG. 1 is a graph of an oxidative stability study using vaccine compositions ACI-24.043 (containing SAT47) and ACI-24.060 (containing SAT58 peptide). [Figure 2] Figure 2 is a graph of SAT peptide recovery using a liposomal construct (ACI-24.060) at different stages of the same manufacturing process. The recovery of ACI-24.060 is compared to the peptide recovery of a different liposomal product (ACI-24.043) using the same manufacturing process. [Figure 3] Figure 3 shows the kinetics and amplitude of the plasma anti-Abeta 1-42 IgG profile (AU / mL) in C57BL / 6 mice following three immunizations with ACI-24.043 (combined with SAT47) or ACI-24.060 (combined with SAT58). Arrows indicate the days of immunization. Data are presented as geometric mean ± 95% CI, n = 10 per group.

[0104] Non-limiting embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: DETAILED DESCRIPTION OF THE INVENTION [Example]

[0105] Example 1: Design of T cell epitopes The design of the universal T cell epitope of the present invention is similar to the design of the T cell epitope described in WO2019 / 197414.

[0106] The T cell activation potential (immunogenicity score) of a universal T cell epitope is the result of two complementary properties: i) affinity for HLA and ii) the ability to bind promiscuously to different HLA haplotypes. Aiming to select peptides with the highest immunogenicity scores, we performed an in silico evaluation (Epivax) of multiple T cell epitopes from different sources. In a preliminary phase, we evaluated 10 peptides from different sources (keyhole limpet hemocyanin (KLH), diphtheria toxin, influenza virus, Epstein-Barr virus, and herpes virus).

[0107] Peptides with the highest immunogenicity scores (≥10) were selected because they were likely to be highly immunogenic in humans based on predicted HLA affinity and HLA haplotype coverage (selected peptide sequences are shown in Table 1).

[0108] [Table 1]

[0109] Based on the results of screening the individual peptides, the promiscuous peptide SAT58 was designed (Table 2). Peptide SAT47 is described in WO2019 / 197414 and is reproduced in the following examples for direct comparison.

[0110] Due to constraints in the peptide synthesis and vaccine encapsulation processes, the goal was to improve the immunogenicity score without increasing the size of the final promiscuous peptide. Simply put, the longer the peptide, the lower the yield and success rate of peptide synthesis. This effect is particularly pronounced for peptides longer than 30 amino acids and those composed primarily of hydrophobic residues.

[0111] Furthermore, peptide encapsulation rates decrease with peptide length, as longer peptides become less likely to be able to accommodate the peptide within the liposome lumen. The in silico immunogenicity scores of these two promiscuous T cell epitopes were very high and, importantly, higher than those of the individual component peptides, confirming that combining peptides of different origins can improve HLA affinity and HLA haplotype coverage (the sequences of the promiscuous T cell epitopes are shown in Table 2).

[0112] [Table 2] [Example]

[0113] Example 2: Preparation of liposomal constructs and liposomal vaccine composition ACI-24.060 The manufacturing process involves two main parts: i) preparation of a SAT58 liposomal construct containing an adjuvant (here, 3D-(6-acyl)PHAD®) and SAT58 peptide; and ii) Insertion of palmitoylated antigen peptide (here Abeta 1-15 (Pal1-15)) into preformed SAT58 liposomes. The detailed procedure is as follows:

[0114] Step 1: Synthesis and purification of T cell epitope peptides The SAT58 T cell epitope-containing peptide was prepared by linear solid-phase peptide synthesis (SPPS) using 2-chlorotrityl resin with standard Fmoc methods. The standard coupling procedure was carried out using 3.0 equivalents of amino acid (SAT58 peptide) and coupling reagent in the presence of 3.0 equivalents of base in DMF for at least 1 hour at room temperature. For difficult coupling sequences, double coupling was implemented with extended reaction times. After completion of amino acid coupling, an acetylation capping step was introduced using 5.0 equivalents of Ac20 (acetic anhydride) in pyridine to avoid undesired peptide chain elongation. The resin was washed with DMF, and the Fmoc group was removed using 20% ​​piperidine in DMF for 5 minutes. Upon completion of SPPS, global deprotection and peptide cleavage from the resin were carried out using a standard cleavage cocktail (TFA / TIS / water / TBMTP) for at least 2 hours at room temperature. The crude product was then precipitated with a 10-fold excess of cold isopropyl ether / heptane, washed with IPE, and the solid was filtered using a glass frit and dried under vacuum. The crude peptide was purified on a reverse-phase C18 column using a preparative HPLC system with a gradient of solvent A (water, 0.1% TFA) and solvent B (acetonitrile, 0.1% TFA). HPLC fractions containing the desired peptide with a purity of 90% or higher were pooled, diluted with water, and then subjected to ion exchange. The desired ion-exchanged fraction was lyophilized to obtain a powder. The identity and purity of the final peptide were confirmed by HPLC-MS analysis.

[0115] Step 2: Preparation of ACI-24.060 vaccine containing SAT58 (cross-flow injection) Lipids (DMPG, DMPC, cholesterol, and 3D-(6-acyl)PHAD® (Avanti Polar Lipids, USA)) were dissolved in 96% EtOH (ethanol) in a heating cabinet at 60 °C. After the lipids were completely dissolved, the solution was filtered through a 0.2 μm pore size filter and injected into an injection system heated to 60 °C. Specifically, an appropriate amount of (SAT58) was dispersed in EtOH at room temperature by sonication (the EtOH concentration was typically 2% v / v of the final SAT58 solution) to form a peptide slurry, which was then solubilized by dilution with His-Sucrose buffer (10 mM histidine, 250 mM sucrose). The SAT58 solution was filtered through a 0.2 μm pore size filter, placed in an injection vessel, and heated to 40 °C. Upon mixing of the lipid / EtOH solution and the SAT58 solution, liposomes were formed at the injection site. Immediately after liposome formation, an online dilution step was performed with 10 mM histidine and 250 mM sucrose to reduce the EtOH concentration. The intermediate liposomes were extruded through a 100 nm pore-size polycarbonate membrane at room temperature. EtOH was removed by ultrafiltration / diafiltration (UDF) using hollow fiber membranes (MWCO: 500 kD), and the buffer was exchanged with PBS pH 6.9. Next, SAT58 liposomes were diluted to a total lipid concentration of 1 mg / mL with dispersion buffer (PBS pH 6.9) and warmed to 35°C. Pal1-15 was dissolved in a 10% w / v beta-OG solution in 10 mM NaHPO pH 11.4 buffer at 60°C and further diluted to a final concentration of 1 mg / mL with the same buffer. After mixing these two solutions using a cross-flow injection module, the liposome suspension was further incubated at 35°C with stirring to allow complete insertion of Pal1-15. A second UDF step using hollow fiber membranes (MWCO: 500 kD) was performed to remove β-OG and exchange the buffer for 10 mM histidine and 250 mM sucrose. The product was filtered through a 0.2 μm Acrodisc mPES syringe filter. [Example]

[0116] Example 3: Peptide oxidation analysis ACI-24.060 (SAT58) was prepared as described above (Example 2).

[0117] ACI-24.043 (SAT47) was prepared according to the method described in WO2019 / 197414.

[0118] Samples were stored for 12 months at 2-8°C. Oxidative stability over time was measured using reverse-phase HPLC, and the results are shown in Table 3 and Figure 1.

[0119] The SAT47 and RRT (relative retention time) 0.98 content (oxidized SAT47) were measured by reverse-phase HPLC using a HPLC Ultimate 3000 system (Dionex / Thermo Fisher) with UV detection at 207 nm. Samples were diluted with isopropanol and water to a final composition of 70% isopropanol and 30% water. The samples were stored at 8 °C until injection into the HPLC system (injection volume = 4 μL). SAT47 and RRT0.98 were separated from other components using a C18 column (Acquity UPLC BEH C18 1.7 μm, 2.1 x 100 mm (Waters, part number 186002352) with a column temperature setting of 80 °C) with a gradient of water and acetonitrile (both mobile phases containing 0.1% TFA) varying from 10% to 90% acetonitrile over 6.2 min. The column was then washed with 0.1% TFA in isopropanol for 2.1 minutes and re-equilibrated at the starting conditions for 2.5 minutes. All chromatographic steps were performed at a flow rate of 0.5 mL / min. The concentration and RRT of SAT47 (0.98) were calculated from the standard curve of SAT47.

[0120] SAT58 content was measured by reverse-phase HPLC using a HPLC Ultimate 3000 system (Dionex / Thermo Fisher) with UV detection (207 nm). Samples were prepared by dilution with 10 mM histidine, 250 mM sucrose, and 20% ethanol. The samples were stored at 8°C until injection into the HPLC system (injection volume = 4 μL). SAT58 was separated from other components using a C18 column (Acquity UPLC BEH C18 1.7 μm, 2.1 x 100 mm (Waters, part number 186002352) with a column temperature setting of 80°C) using a gradient of water and acetonitrile (both mobile phases containing 0.1% TFA) from 10% acetonitrile to 90% acetonitrile over 6.2 minutes. The column was then washed with isopropanol containing 0.1% TFA for 2.1 minutes and re-equilibrated to the starting conditions for 2.5 minutes. All chromatographic steps were performed at a flow rate of 0.5 mL / min. No peak corresponding to an RRT of 0.98 was observed in the SAT58 chromatogram. The concentration of SAT58 was calculated from a standard curve of SAT58.

[0121] [Table 3]

[0122] The decrease in SAT47 and the increase in RRT0.98 values ​​confirm the continuous production of oxidized SAT47 over time. No comparable production of SAT58 was observed.

[0123] As shown in Figure 1 and Table 3, SAT58 concentrations remained stable over time, whereas SAT47 concentrations decreased significantly during the 12-month storage period.

[0124] Advantageously, ACI-24.060 containing SAT58 was observed to have improved oxidative stability compared to SAT47 in comparable liposomal vaccine compositions. Without being bound by theory, this improved oxidative profile is believed to be due to the change of a methionine residue in the amino acid sequence of the T cell epitope containing peptide SAT47 to an alanine residue in the T cell epitope containing peptide SAT58. [Example]

[0125] Example 4: Calculated recovery of SAT peptides in different manufacturing processes The peptide recovery of SAT58 throughout the entire manufacturing process was compared with that of SAT47 at the same manufacturing stage. Measurements were performed at three time points during the manufacturing process and analyzed using HPLC. HPLC was performed as in Example 3.

[0126] Peptide recovery: The recovery (or mass balance) at a process step is defined as the total amount of peptide in the solution or product at that step compared to the initial peptide input. The recovery of SAT58 and SAT47 was tested in liposome preparations, bulk product preparations, and the final product. The liposome preparation corresponds to the preparation before the antigen molecules are incorporated. The bulk product corresponds to the preparation after the antigen molecules are incorporated. The final product corresponds to the preparation obtained after performing all steps of the process, i.e., ACI-24.060.

[0127] Recovery is calculated by multiplying the SAT content (concentration) measured by HPLC by the volume of solution or product in the process. It is usually expressed as a percentage of the initial SAT input and is an indicator of process loss.

[0128] Surprisingly, SAT58 was observed to have a higher peptide recovery in the final product compared to SAT47.

[0129] As shown in Figure 2, about 70% of the initial amount of SAT47 was recovered in the SAT47 liposome formulation, whereas the recovery rate in the SAT58 formulation was about 78%.

[0130] In bulk product preparation, SAT47 had a recovery rate of approximately 55%, while SAT58 had a recovery rate of approximately 68%.

[0131] In the final product, only about 45% of SAT47 was recovered in the SAT47 final formulation, whereas about 65% of SAT58 was recovered in the SAT58 final formulation.

[0132] Thus, the recovery rate of SAT58 is higher than that of SAT47 throughout the entire production process, including before the addition of the antigen peptide Pal1-15. [Example]

[0133] Example 5: Liposome constructs and liposomal vaccine compositions containing alpha-synuclein-derived antigenic peptides The vaccine was produced using a three-step approach: preparation of intermediate SAT58 liposomes, followed by incorporation of CpG-Chol to generate fully adjuvanted liposomes, and finally insertion of a palmitoylated α-synuclein (a-syn)-derived antigenic peptide containing amino acid sequence SEQ ID NO: 9 (GG-KESMPVDPDNEA) as described in WO2022 / 029181.

[0134] Intermediate liposomes: First, lipids (DMPG, DMPC, cholesterol, and monophosphorylhexaacyl lipid A (3D-(6-acyl)PHAD® (Avanti Polar Lipids, USA)), a first adjuvant) were dissolved in ethanol at 60°C. After complete dissolution, the lipid / ethanol solution was filtered through a 0.2 μm pore size filter and injected into an injection system preheated to 60°C. In a separate container, a peptide containing the T cell epitope SAT58 was dispersed in ethanol by sonication at room temperature and solubilized by dilution with 10 mM histidine and 250 mM sucrose. The SAT58 solution was filtered through a 0.2 μm pore size filter and heated to 40°C. Next, the lipid / ethanol solution and the SAT58 solution were mixed using a cross-flow injection module to form intermediate liposomes. Active cooling was then performed and size reduction was achieved using repeated extrusion cycles. Finally, ultrafiltration / diafiltration (UDF) was performed to remove ethanol. The intermediate SAT58 liposomes were filtered through a 0.2 μm pore size filter and stored at 4°C until use.

[0135] Incorporation of CpG-Chol: The intermediate SAT58 liposomes were diluted to a lipid concentration of 1 mg / mL with 20 mM histidine and 145 mM NaCl and heated to 60°C. A second adjuvant, CpG-cholesterol, was added dropwise to the liposomes. The liposome dispersion was further incubated at 60°C for 30 minutes with stirring. The fully adjuvanted liposomes were purified by UDF, filtered through 0.45 μm and then 0.2 μm pore filters, and stored at 4°C.

[0136] Insertion of α-synuclein (a-syn)-derived antigenic peptide: Fully adjuvanted liposomes were diluted with 20 mM histidine and 145 mM NaCl to a total lipid concentration of 1 mg / mL. Simultaneously, N-terminally dipalmitoylated α-synuclein-derived antigenic peptide (SEQ ID NO: 9) was dissolved in 20 mM histidine and 145 mM NaCl at 60°C to a final concentration of 1 mg / mL and filtered through a 0.2 μm pore size filter. Finally, the liposomes and peptide solution were mixed using a cross-flow module, and the liposome dispersion was stirred at 60°C for 30 minutes. A UDF step was performed, and the buffer was exchanged into the final formulation system, i.e., 10 mM histidine, 250 mM sucrose buffer. The product was concentrated to the final volume and filtered through a 0.45 μm pore size filter followed by a 0.2 μm pore size filter. The product was stored at 4°C.

[0137] Stability analysis of T cell epitope peptides Samples of the liposomal vaccine containing the α-synuclein-derived antigen peptide containing amino acid sequence SEQ ID NO: 9 (GG-KESMPVDPDNEA) prepared as described above were stored at 2-8°C for 6 months. The stability of the T cell epitope peptide SAT58 over time was measured using reverse-phase HPLC as described in Example 3 above. The results are shown in Table 4.

[0138] [Table 4]

[0139] As shown in Table 4, SAT58 concentrations were stable over time and no degradation products were observed. [Example]

[0140] Example 6: Immunogenicity of SAT58-containing liposome constructs To evaluate the immunogenicity of liposomal vaccine compositions ACI-24.043 (containing the T cell epitope peptide SAT47) and ACI-24.060 (containing the T cell epitope peptide SAT58), an in vivo study was conducted using mice. Two groups of C57BL / 6 mice (n = 10) received a total of three subcutaneous injections each. Either ACI-24.043 or ACI-24.060 was administered at a target dose of 80 μg of Pa1-15 (SEQ ID NO: 7) on days 1, 15, and 29. Plasma samples were collected before administration (day -7) and one week after each immunization (days 8, 22, and 36), and antibody responses to Abeta 1-42 were analyzed using ELISA.

[0141] Plates were coated overnight at 4°C with 10 μg / ml human Abeta 1-42 peptide film (Bachem, Switzerland). After washing with 0.05% Tween 20 / PBS and blocking with 1% BSA / 0.05% Tween / PBS, serial dilutions of plasma were added to the plates and incubated for 2 hours at 37°C. After washing, the plates were incubated with alkaline phosphatase (AP)-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, USA) for 2 hours at 37°C. After a final wash, the plates were incubated with AP substrate (pNPP) for 2.5 hours and measured at 405 nm using an ELISA plate reader. Anti-Abeta 1-42 antibody concentrations were back-calculated using a standard curve constructed using a serial dilution series of the commercially available antibody 6E10 (Biolegend, UK, catalog no. 803002).

[0142] The results in Figure 3 show that animals immunized with ACI-24.043 and ACI-24.060 exhibited similar anti-Abeta 1-42 antibody profiles, indicating that liposomal vaccine composition ACI-24.060 (containing SAT58) exhibited an excellent immunogenicity profile, comparable to that obtained with liposomal vaccine composition ACI-24.043 (containing SAT47).

[0143] References TIFF2026505011000005.tif238164 TIFF2026505011000006.tif83164

[0144] 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. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes related to the present invention.

[0145] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the present invention described herein are construed as broadly applicable and combinable with any other consistent embodiments, including those appropriately cited (including alone) from other aspects of the present invention.

Claims

1. liposomes, at least one adjuvant, and A peptide containing at least one T-cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4, or an analog thereof that retains an alanine at position 2 and does not contain a methionine residue. A liposome construct comprising:

2. 2. The liposome construct of claim 1, wherein the peptide containing at least one T cell epitope comprises at least one additional T cell epitope, and the at least one additional T cell epitope comprises, consists essentially of, or consists of an amino acid sequence selected from SEQ ID NO: 1 and / or SEQ ID NO: 3, or an analog thereof.

3. 3. The liposome construct of claim 1 or 2, wherein the peptide contains at least three T cell epitopes comprising, consisting essentially of, or consisting of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4, or analogs thereof.

4. The liposome construct according to claim 2 or 3, wherein the peptide further comprises at least one linker between at least two T cell epitopes.

5. 5. The liposome construct of claim 4, wherein the at least one linker comprises, consists essentially of, or consists of the amino acid sequence VVR, TVGLR, KVSVR, PMGAP, or PMGLP.

6. 6. The liposome construct of claim 4 or 5, wherein the at least one linker comprises, consists essentially of, or consists of the amino acid sequence VVR.

7. 7. The liposome construct of claim 1, wherein the peptide containing at least one T-cell epitope comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6 or an analog thereof.

8. The liposome construct according to any one of claims 1 to 7, wherein the peptide containing at least one T-cell epitope is at least partially encapsulated within the liposome.

9. The liposome construct according to any one of claims 1 to 8, wherein the at least one adjuvant is at least partially presented on the surface of the liposome.

10. The liposome construct according to any one of claims 1 to 9, wherein the at least one adjuvant is at least partially incorporated within the lipid bilayer of the liposome.

11. 11. The liposome construct of claim 1, wherein the at least one adjuvant is selected from the group consisting of monophosphoryl lipid A (MPLA), diphosphoryl lipid A (DPLA), alum, Pam2CSK4, Pam3CSK4, Pam3CAG, saponin, CpG, lipidated CpG, preferably CpG cholesterol; cationic lipids; phosphorothioated PS-CpG-ODN; CpG oligodeoxynucleotide (CpG-ODN); CpG-A; CpG-B or CpG-C.

12. The liposome construct according to any one of claims 1 to 11, wherein the at least one adjuvant is a TLR4 agonist, preferably monophosphoryl lipid A (MPLA).

13. The liposome construct according to any one of claims 1 to 11, wherein the at least one adjuvant is a TLR9 agonist, preferably a CpG oligonucleotide.

14. A liposomal construct according to any one of claims 1 to 13 for use in the preparation of a liposomal vaccine composition.

15. A liposome construct according to any one of claims 1 to 13, and At least one antigenic peptide, and optionally two different antigenic peptides, displayed on the surface of the liposome.

1. A liposomal vaccine composition comprising:

16. 16. The liposomal vaccine composition of claim 15, wherein the at least one antigenic peptide is modified with one or more lipophilic or hydrophobic moieties, optionally wherein the one or more lipophilic or hydrophobic moieties are selected from fatty acids, triglycerides, diglycerides, steroids, sphingolipids, glycolipids, or phospholipids.

17. 17. The liposomal vaccine composition of claim 16, wherein the one or more lipophilic or hydrophobic moieties comprise a palmitoyl residue (palmitic acid).

18. 18. The liposomal vaccine composition of claim 17, wherein the at least one antigenic peptide is tetrapalmitoylated.

19. The liposomal vaccine composition according to any one of claims 15 to 18, wherein the at least one antigenic peptide is an autoantigen.

20. 20. The liposomal vaccine composition of claim 19, wherein the autoantigen is derived from IL-17, IL-27, A beta, tau, alpha-synuclein, huntingtin, prion, or amylin protein.

21. The liposomal vaccine composition of claim 19 or 20, wherein the at least one antigenic peptide is an A beta peptide or a fragment thereof, the A beta peptide or a fragment thereof preferably comprising, consisting essentially of, or consisting of amino acids 1-15 of A beta.

22. liposomes, MPLA adjuvant, A peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6, and an amyloid beta-derived peptide antigen presented on the surface of said liposome, comprising, consisting essentially of, or consisting of amino acids 1-15 of A beta; A liposomal vaccine composition comprising:

23. liposomes, an MPLA adjuvant incorporated into said liposomes; A peptide consisting essentially of, or consisting of, the amino acid sequence of SEQ ID NO: 6, and Amyloid beta-derived peptide antigen consisting of or consisting essentially of amino acids 1-15 of A beta wherein the antigen is palmitoylated and palmitic acid residues are incorporated into the outer layer of the liposome.

24. liposomes, an MPLA adjuvant incorporated into said liposomes; A peptide consisting of the amino acid sequence of SEQ ID NO: 6, and Amyloid beta-derived peptide antigen consisting of amino acids 1-15 of A beta wherein the antigen is tetrapalmitoylated via N-terminal and C-terminal lysine residues (two at each end) added to the peptide, and palmitic acid residues are incorporated into the outer layer of the liposome.

25. liposomes, MPLA adjuvant. CpG cholesterol, A peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, and an alpha-synuclein-derived antigenic peptide displayed on the surface of said liposome, comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; A liposomal vaccine composition comprising:

26. 26. The liposomal vaccine composition of any one of claims 15 to 25, wherein the peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6 is at least partially encapsulated within the liposome.

27. The liposomal vaccine composition according to any one of claims 15 to 26, wherein the liposome comprises a phospholipid and cholesterol.

28. 28. The liposomal vaccine composition of claim 27, wherein the phospholipid comprises dimylcitoylphosphatidylcholine (DMPC) and dimylcitoylphosphatidylglycerol (DMPG), optionally in a molar ratio of DPMC:DPMG:cholesterol of 9:1:

7.

29. 29. The liposomal vaccine composition of claim 28, wherein the molar ratio of DPMC:DPMG:cholesterol:MPLA in the liposome is 9:1:7:0.

05.

30. The liposomal vaccine composition according to any one of claims 15 to 18, wherein the at least one antigenic peptide is a foreign antigen.

31. The liposomal vaccine composition of claim 30, wherein the foreign antigen is a viral antigen or an allergen.

32. 32. The liposomal vaccine composition of claim 31, wherein the viral antigen is derived from a virus selected from rhinovirus, coronavirus, enterovirus, adenovirus, parainfluenza virus, and respiratory syncytial virus.

33. A liposomal vaccine composition according to any one of claims 15 to 32 for use in therapy.

34. 34. The liposomal vaccine composition of any one of claims 15 to 33 for use in a method for the prevention or treatment of symptoms of a neurodegenerative disease or disorder, or symptoms associated with a neurodegenerative disease or disorder.

35. The liposomal vaccine composition of claim 34, wherein the neurodegenerative disease is Alzheimer's disease or Down's syndrome.

36. 14. A method for producing a liposome construct according to any one of claims 1 to 13, comprising the steps of: a) providing a peptide containing at least one T-cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4, or an analog thereof that retains the alanine at position 2 and does not contain a methionine residue; b) providing liposomes comprising at least one adjuvant; and c) combining said peptide containing at least one T cell epitope with said liposome to produce a liposome construct.

37. 37. The method of claim 36, wherein the peptide containing at least one T-cell epitope comprises, consists of, or consists essentially of: a) a combination of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4; or b) The amino acid sequence of SEQ ID NO: 6, or an analog thereof.

38. 33. A method for producing the liposomal vaccine composition of any one of claims 15 to 32, comprising the steps of: a) providing a peptide containing at least one T-cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4, or an analog thereof that retains the alanine at position 2 and does not contain a methionine residue; b) providing liposomes comprising at least one adjuvant; c) combining said peptide containing at least one T cell epitope with said liposome; and d) incorporating at least one peptide antigen into said liposomes such that said at least one peptide antigen is displayed on the surface of said liposomes.

39. 39. The method of claim 38, wherein the peptide containing at least one T-cell epitope comprises, consists of, or consists essentially of: a) a combination of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4; or b) The amino acid sequence of SEQ ID NO: 6, or an analog thereof.

40. 40. The method of claim 38 or 39, wherein the at least one adjuvant comprises MPLA.

41. 41. The method of claim 40, wherein the at least one adjuvant further comprises a CpG adjuvant.

42. 42. The method of any one of claims 38 to 41, wherein the peptide antigen is palmitoylated and palmitic acid residues are incorporated into the outer layer of the liposome.

43. 43. The method of any one of claims 38 to 42, wherein the peptide antigen is an amyloid beta-derived peptide, preferably an amyloid beta-derived peptide antigen comprising, consisting of, or consisting essentially of amino acids 1-15 of A beta.

44. 43. The method of any one of claims 38 to 42, wherein the peptide antigen is an alpha-synuclein-derived peptide, preferably an alpha-synuclein-derived antigenic peptide displayed on the surface of the liposome comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

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