Immunogenic composition comprising lipid nanoparticles having immunogenic peptide encapsulated therein

Abstract

The purpose of the present invention is to provide a new means for simply and efficiently encapsulating an immunogenic peptide in LNPs. Provided is a method for producing an immunogenic composition comprising LNPs having an immunogenic peptide encapsulated therein, the method comprising: a step for selecting a lipid component to be contained in LNPs in accordance with the charge of the immunogenic peptide in a buffer solution; and a step for mixing the immunogenic peptide dissolved in the buffer solution with the lipid component selected in the aforementioned step and the like to form the LNPs having the immunogenic peptide encapsulated therein.

Description

An immunogenic composition comprising lipid nanoparticles encapsulating an immunogenic peptide

[0001] The present invention relates to a method for producing an immunogenic composition comprising lipid nanoparticles encapsulating an immunogenic peptide.

[0002] Lipid nanoparticles (Lipid nanoparticle (LNP)) are nanoparticles with a diameter of about 10 nm to 1000 nm mainly composed of multiple types of lipids, and today, as a system (DDS) for delivering nucleic acid drugs to cells, they are widely used in various fields. For example, an mRNA vaccine formed by encapsulating mRNA encoding a part (antigen) of a viral protein in LNP has been put into practical use as a preventive measure against coronavirus disease (COVID-19) that caused a pandemic in 2020, and in the future, it is expected to contribute as a preventive measure against other infectious diseases.

[0003] In the production of this mRNA vaccine, generally, an LNP encapsulating mRNA is formed by the interaction between the mRNA encoding the antigen and the lipids constituting the LNP. That is, in a buffer solution, negatively charged mRNA and positively charged lipids (cationic lipids) constituting the LNP interact to form a complex in which the mRNA is on the inside and the positively charged lipids are on the outside, and by the interaction of this complex with other lipids (neutral phospholipids, cholesterol, PEG-modified lipids, etc.), an LNP in which the lipids encapsulate the mRNA with a predetermined orientation is formed.

[0004] On the other hand, peptides are also used as immunogens (Patent Document 1), and the delivery of peptides to cells using LNP as a vaccine has been studied.

[0005] WO2024 / 111633

[0006] However, peptides have different charging patterns depending on the difference in their amino acid sequences in a buffer solution, and the charging pattern is not determined like mRNA, so it has not been easy to encapsulate them in LNP. For this reason, in this field, a simple and efficient means for encapsulating an immunogenic peptide in LNP has been eagerly desired.

[0007] The present invention aims to provide a novel method for simply and efficiently encapsulating immunogenic peptides in LNPs.

[0008] As a result of diligent research to solve the above problems, the inventors have found that by selecting the lipid component to be included in the LNP according to the charge of the immunogenic peptide in the buffer solution, more specifically, by selecting anionic lipids when the charge of the immunogenic peptide in the buffer solution is positive and cationic lipids when the charge is negative, and by including these selected lipids in the lipids constituting the LNP, immunogenic peptides can be encapsulated in LNPs in a simple and efficient manner.

[0009] The present invention is based on these novel findings and encompasses the following inventions: [1] A method for producing an immunogenic composition comprising lipid nanoparticles (LNPs) encapsulating an immunogenic peptide, comprising: (1) a step of selecting a lipid component to be contained in the LNP according to the charge of the immunogenic peptide in a buffer, (a) selecting an anionic lipid if the charge is positive, and (b) selecting a cationic lipid if the charge is negative; and (2) a step of forming an LNP encapsulating the immunogenic peptide by mixing the immunogenic peptide dissolved in a buffer with the lipid component selected in step (1), PEG-modified lipid, cholesterol, and neutral phospholipids and / or ionized lipids. [2] The method of [1], wherein the pH value of the buffer (at room temperature, preferably 4 to 35°C, more preferably 20 to 30°C, and even more preferably 25°C) is 3 to 9.6. [3] The method of [1] or [2], wherein the buffer solution is selected from sucrose aqueous solution, physiological saline, phosphate-buffered physiological saline, HEPES-buffered physiological saline, acetate buffer, and carbonate buffer. [4] The lipid component selected in step (1) is an anionic lipid, and is selected from the group consisting of dipalmitoylphosphatidylglycerol (DPPG), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), dipalmitoylphosphatidic acid (DPPA), dimyristoylphosphatidylglycerol, distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), distearoylphosphatidic acid (DSPA), and dimyristoylphosphatidic acid, according to any of the methods [1] to [3].[5] The lipid component selected in step (1) is a cationic lipid, and is selected from the group consisting of O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride (DC-6-14), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleyl-3-dimethylaminopropane (DODMA), ALC-0315, cKK-E12, SM-102, DLin-KC2-DMA, and heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), by any of the methods [1] to [3]. [6] In step (2), the PEG-modified lipid is selected from the group consisting of DMG-PEG and DSPE-PEG, according to any of the methods [1] to [5]. [7] In step (2), the neutral phospholipid is selected from the group consisting of hydrogenated soybean phosphatidylcholine (HSPC), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), palmitoyl oleoyl phosphatidylcholine (POPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, and distearoyl phosphatidylethanolamine, according to any of the methods [1] to [6]. [8] Any method of [1] to [7] wherein in step (2), the ionized lipid is selected from the group consisting of Dlin-MC3-DMA, DODMA, ALC-0315, SM-102, DLin-KC2-DMA, DODAP, and cKK-E12 (however, the ionized lipid is different from the lipid component). [9] Any method of [1] to [8] wherein in step (2), the mixing is performed using a microfluidic device.

[10] Any method of [1] to [9] further comprising the step of subjecting the LNP containing the immunogenic peptide formed in step (2) to a dialysis treatment using an aqueous sucrose solution as the dialysate.

[11] The method of any one of [1] to

[10] wherein the immunogenic peptide comprises 3 to 50 amino acids.

[12] The method of any one of [1] to

[11] wherein the immunogenic peptide is a peptide that induces an anti-human TNF-α antibody, an anti-human amyloid-β antibody, an anti-human PD-L1 antibody, an anti-human PD-1 antibody, an anti-mouse PD-1 antibody, or an anti-mouse TNF-α antibody.

[13] The method of any one of [1] to

[12] wherein the immunogenic composition is a pharmaceutical composition.

[14] The method of any one of [1] to

[13] wherein the immunogenic composition is a vaccine preparation.

[0010]

[15] An immunogenic composition comprising LNPs encapsulating an immunogenic peptide.

[16] The immunogenic composition of

[15] , wherein the immunogenic peptide comprises 3 to 50 amino acids.

[17] The immunogenic composition of

[15] or

[16] , wherein the immunogenic peptide is a peptide that induces an anti-human TNF-α antibody, an anti-human amyloid-β antibody, an anti-human PD-L1 antibody, an anti-human PD-1 antibody, an anti-mouse PD-1 antibody, or an anti-mouse TNF-α antibody.

[18] The immunogenic composition of any of

[15] to

[17] , wherein the LNP comprises a lipid component, PEG-modified lipid, cholesterol, and neutral phospholipids and / or ionized lipids depending on the charge of the immunogenic peptide in the buffer, and the lipid component is (a) an anionic lipid when the charge is positive, and (b) a cationic lipid when the charge is negative.

[19] The immunogenic composition according to

[18] , wherein the lipid component corresponding to the charge is an anionic lipid, and is selected from the group consisting of dipalmitoylphosphatidylglycerol (DPPG), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), dipalmitoylphosphatidic acid (DPPA), dimyristoylphosphatidylglycerol, distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), distearoylphosphatidic acid (DSPA), and dimyristoylphosphatidic acid.

[20] The immunogenic composition according to

[18] or

[19] , wherein the lipid component corresponding to the charge is a cationic lipid, selected from the group consisting of O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride (DC-6-14), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleyl-3-dimethylaminopropane (DODMA), ALC-0315, cKK-E12, SM-102, DLin-KC2-DMA, and heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA).

[21] An immunogenic composition according to any one of

[18] to

[20] , wherein the PEG-modified lipid is selected from the group consisting of DMG-PEG and DSPE-PEG.

[22] An immunogenic composition according to any one of

[18] to

[21] , wherein the neutral phospholipid is selected from the group consisting of hydrogenated soybean phosphatidylcholine (HSPC), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), palmitoyl oleoyl phosphatidylcholine (POPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, and distearoyl phosphatidylethanolamine.

[23] An immunogenic composition according to any of

[18] to

[22] , wherein the ionized lipid is selected from the group consisting of Dlin-MC3-DMA, DODMA, ALC-0315, SM-102, DLin-KC2-DMA, DODAP, and cKK-E12 (provided that the ionized lipid and the lipid component are different).

[24] An immunogenic composition according to any one of

[18] to

[22] , comprising the lipid component, the PEG-modified lipid, the cholesterol, and the neutral phospholipid and / or the ionized lipid in a mass ratio of (lipid component: PEG-modified lipid: cholesterol: neutral phospholipid: ionized lipid) of 30-55% by mass: 0.3-3% by mass: 35-50% by mass: 0-30% by mass: 0-50% by mass, wherein the total of the lipid component, the PEG-modified lipid, the cholesterol, and the neutral phospholipid and / or the ionized lipid is 100% by mass.

[25] An immunogenic composition according to any one of

[15] to

[24] , wherein the LNP has an average particle size of 40-200 nm.

[26] An immunogenic composition according to any one of

[15] to

[25] , which is a pharmaceutical composition.

[27] An immunogenic composition according to any one of

[15] to

[26] , which is a vaccine preparation.

[28] Any immunogenic composition of

[15] to

[27] , administered subcutaneously or intramuscularly. This specification includes the contents of the specification of Japanese Patent Application No. 2024-198892, filed on 14 November 2024, which is the basis of the priority of this application. All publications, patents and patent applications referenced herein are incorporated herein by reference in their entirety.

[0011] According to the present invention, a new means can be provided for simply and efficiently encapsulating immunogenic peptides in LNPs.

[0012] Figure 1 is a graph showing (A) the encapsulation rate of the peptide in LNPs (%), (B) the particle size (nm) of the LNPs containing the peptide, and (C) the polydispersity index (PDI) when the extraneous fluid used during dialysis was changed from PBS to physiological saline (0.9% NaCl), HEPES (HBS), or 10% sucrose, after encapsulating ovalbumin (OVA)-derived peptides in LNPs using a microfluidic channel. Figure 2 is a graph showing the results of the analysis of antipeptide serum in mice that received human amyloid-β peptide-encapsulated LNPs intravenously (i.v.), intramuscularly (i.m.), intraperitoneally (i.p.), and subcutaneously (s.c.). For the control group (free amyloid-β peptide), amyloid-β peptide was mixed with complete Freund's adjuvant (CFA) and administered subcutaneously. Unimmunized mouse serum was used as a control. A, B, and C in the figure represent different individual mice. Figure 3 is a graph showing the results of antipeptide serum analysis in mice that received intramuscular (IM) and intravenous (IV) administration of two types of LNPs containing human TNFα peptide. For the control group (CFA / IFA), human TNFα peptide was mixed with CFA and administered subcutaneously. Immunized mouse serum was used as a control. Figure 4 is a graph showing the results of antipeptide serum analysis in mice that received intramuscular administration of four types of LNPs containing human TNFα peptide. Immunized mouse serum was used as a control. Figure 5 is a graph showing the results of antipeptide serum analysis in mice that received intramuscular administration of four types of LNPs containing human TNFα peptide. Immunized mouse serum was used as a control. A, B, C, and D in the figures represent different individual mice. Figure 6 is a graph showing the results of antipeptide serum analysis in mice that received intramuscular administration of seven types of LNPs containing human TNFα peptide. As a control, unimmunized mouse serum was used. Figure 7 is a graph showing the results of the analysis of antipeptide serum in rabbits that were intramuscularly administered LNPs containing three types of human TNFα peptides. Unimmunized rabbit serum was used as a negative control for the analysis. A, B, and C in the figure represent different rabbit individuals. Figure 8 shows the dilution curves obtained by serially diluting the serum described in Figure 7 from 25-fold.Figure 9 is a graph showing the results of antipeptide serum analysis in mice that received intramuscular administration (i.m.) of LNPs containing ionized lipids encapsulating human amyloid-beta peptide. For the control group (free amyloid-beta peptide), amyloid-beta peptide was mixed with complete Freund's adjuvant (CFA) and administered subcutaneously (s.c.). Unimmunized mouse serum was used as a control. Figure 10 is a graph showing the results of antipeptide serum analysis in mice that received intramuscular administration of four types of LNPs (1) to (4) encapsulating human TNFα peptide, respectively, after a) two immunizations, b) three immunizations, and c) final immunization. For the control group, human TNFα peptide was mixed with CFA and administered subcutaneously. Unimmunized mouse serum was used as a control. A, B, C, and D in the figure represent different individual mice. Figure 11 is a graph showing the results of antipeptide serum analysis in mice that received intramuscular administration of LNPs containing four different human TNFα peptides. Unimmunized mouse serum was used as a control. A, B, C, and D in the figure represent different individual mice. Figure 12 is a graph showing the results of antipeptide serum analysis in mice that received intramuscular administration (i.m.) of LNPs containing human TNFα peptides. The control group received subcutaneous injection (s.c.) of human TNFα peptide mixed with CFA. Unimmunized mouse serum was used as a control. Figure 13 is a graph showing the results of antipeptide serum analysis in mice that received intramuscular administration (i.m.) of LNPs containing mouse PD-1 peptides. The control group received subcutaneous injection (s.c.) of mouse PD-1 peptide mixed with CFA. Unimmunized mouse serum was used as a control. Figure 14 is a graph showing the results of the analysis of antipeptide serum in mice that received intramuscular administration (i.m.) of LNPs containing mouse PD-1 peptide. For the control group, mouse PD-1 peptide was mixed with CFA and administered subcutaneously (s.c.). Unimmunized mouse serum was used as the control.

[0013] In this invention, "immunogenic peptide" refers to a partial peptide in a target protein that has the ability to induce antibodies against the target protein. In this invention, any peptide can be used as the immunogenic peptide, as long as it has the ability to induce antibodies against the target protein, and is not particularly limited.

[0014] Immunogenic peptides can be selected and prepared according to conventionally known methods (e.g., WO2024 / 111633), and are not particularly limited. For example, multiple types of partial peptides can be prepared from the amino acid sequence of a target protein, a mixture containing multiple types of these partial peptides can be used to immunize mammals, it can be determined whether or not antibodies specific to the target protein are induced in the mixture, and the desired immunogenic peptide can be obtained by identifying the partial peptide that induces the specific antibody from the mixture in which antibodies are determined to have been induced.

[0015] Here, the multiple types of partial peptides selected from the amino acid sequence of the target protein consist of 3 to 50 amino acid residues, preferably 5 to 40 amino acid residues, more preferably 8 to 45 amino acid residues, even more preferably 10 to 30 amino acid residues, and most preferably 15 to 30 amino acid residues, and it is preferable to prepare them so as to cover the entire region of the amino acid sequence of the target protein. Each partial peptide may overlap with another adjacent partial peptide by a specific number of amino acid residues at its N-terminus or C-terminus, and the sequences of each partial peptide may be selected so that 50% or more, preferably 60% or more, and even more preferably 70% or more of the sequences overlap with each other. For the selection of partial peptides, openly available and well-known immunoepitope databases (e.g., IEDB or SYFPEITHI) can be used.

[0016] The mixture of partial peptides can generally contain 4 to 20 types, preferably 5 to 20 types, more preferably 7 to 15 types, even more preferably 8 to 12 types, and typically 10 types of partial peptides.

[0017] Mammalian immunization using a mixture of partial peptides can be performed using mammals other than humans (e.g., mice, rats, rabbits, dogs, monkeys, etc., with rabbits or monkeys being preferred). Adjuvants can be used during immunization, and precipitating adjuvants (e.g., aluminum compounds, etc.) and oily adjuvants (e.g., incomplete Freund's adjuvants) can be appropriately selected and used.

[0018] Determining whether specific antibodies have been induced, i.e., detecting induced peptide antibodies, can be performed using any means, including various immunoassays such as immunoassays on immobilized peptides or target proteins (peptide ELISA), Western blotting (WB) using nitrocellulose, immunoprecipitation (IP), receptor assays (RRA), or cell-based neutralization activity (L929, etc.) measurements, CDC activity measurements, ADCC activity measurements, evaluation of whether there is a decrease in the actual blood concentration of the soluble target protein or an increase in clearance in animal models, or immunohistochemistry (IHC) using target protein-expressing cells or tissues, or flow cytometry (FCM) of cells in suspension (but not limited to these).

[0019] From a mixture in which a specific antibody has been determined to have been induced, the identification of the partial peptide that induces the antibody can be performed by using a plate with the source protein immobilized on it, and identifying the peptide that inhibits the binding of the source protein to the antiserum obtained by immunization. The amino acid sequence of the identified peptide can be determined using conventionally known amino acid sequence analysis methods such as the Sanger method or the Edman method.

[0020] In the present invention, the "target protein" can be any protein and is not particularly limited, but examples include proteins directly or indirectly involved in the onset of disease or the manifestation of symptoms, components or production components of pathogens (e.g., viruses, bacteria, fungi, parasitic microorganisms, mycoplasma, etc.), and components of cancer cells (e.g., cancer markers, etc.). Furthermore, examples of target proteins include proteins recognized by antibodies that are used in treatment or marketed as pharmaceuticals, and examples of such proteins include, but are not limited to, EGF receptor protein, CD20 protein, TNFα protein, IL6 receptor protein, VEGF protein, IL-1β protein, CCR4 protein, PD-1 protein, PD-L1 protein, amyloid-β protein, etc.

[0021] In the present invention, immunogenic peptides may include peptides known to have antibody-inducing ability against target proteins. Examples of such immunogenic peptides include those disclosed in WO2024 / 111633, but are not limited to these.

[0022] For example, examples of immunogenic peptides usable in the present invention include (but are not limited to) the following partial peptides or their mutant peptides.

[0023]

[0024] In the present invention, a "mutant peptide" of an immunogenic peptide (or partial peptide) means: • An amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence of an immunogenic peptide, and which has antibody-inducing ability against the target protein of the immunogenic peptide, or

[0025] This refers to a peptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of an immunogenic peptide, and having the ability to induce antibodies against the target protein of the immunogenic peptide.

[0026] Here, "one or more amino acids" means 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 amino acids. Furthermore, "sequence identity" means exhibiting sequence identity of 80% or more, preferably 85% or more, more preferably 90%, even more preferably 95%, even more preferably 97% or more, particularly preferably 98% or more, and especially preferably 99% or more, relative to the total length of the amino acid sequence of the immunogenic peptide. Sequence identity can be determined using well-known programs such as BLAST, FASTA, CLUSTALW, etc.

[0027] In the present invention, immunogenic peptides may be produced by conventionally known genetic recombination methods or chemical synthesis methods (solid-phase synthesis), or they may be obtained using a synthesis contract service (for example, GenScript, Inc.). Furthermore, immunogenic peptides may be modified to have d-amino acids, acetylated / amidated at the N / C terminal amino acids to enhance serum stability, polymerized or dendrimed peptides to increase antigenicity, or peptides to which adjuvant peptides have been added, as long as they have antibody-inducing ability against the target protein of the immunogenic peptide.

[0028] The immunogenic composition of the present invention comprises lipid nanoparticles (hereinafter referred to as "LNPs") encapsulating immunogenic peptides.

[0029] In the present invention, LNP comprises (i) a lipid component, (ii) a PEG-modified lipid, (iii) cholesterol, and (iv) a neutral phospholipid and / or (v) an ionized lipid, and has a structure in which a vesicle (outer shell) made of a lipid membrane is formed in which the hydrophilic head of the lipid molecule is oriented outward facing the aqueous medium and the hydrophobic tail is oriented inward, and a vesicle (inner shell) made of a lipid molecule is contained in an inverse micelle. The LNP contains an immunogenic peptide on the inside of its inner shell.

[0030] In the present invention, the "(i) lipid component" constituting the LNP can be selected according to the charge of the immunogenic peptide in the buffer solution. In the present invention, "buffer solution" refers to an aqueous buffer solution having a pH value capable of dissolving the immunogenic peptide and imparting a desired charge, and any buffer solution can be used, but for example, a buffer solution with a pH value of about 3 to 9.6 can be preferably used. Suitable buffer solutions include, for example, sucrose aqueous solution, physiological saline, phosphate-buffered physiological saline (PBS), acetate buffer, HEPES-buffered physiological saline (HBS), and carbonate buffer (but are not limited to these), and in particular, 10% sucrose aqueous solution (pH 7.4), physiological saline (pH 7.4), PBS (pH 7.4), 100 mM acetate buffer (pH 4), HBS (pH 7.4), and carbonate buffer (pH 9.6) can be preferably used. Note that the pH value is the value at which the buffer solution temperature is room temperature, preferably 4 to 35°C, more preferably 20 to 30°C, and even more preferably 25°C. Unless otherwise specified herein, pH values ​​are those at 25°C.

[0031] In this invention, the "charge of the immunogenic peptide in the buffer solution" can be calculated based on the amino acid sequence and the following formula, which applies the Henderson-Hasselbalch equation.

[0032] (In the formula, "Z" is the net charge of the peptide sequence, "Ni" is the number of arginine, lysine, and histidine residues and the N-terminus, "pKai" is the pKa value of the N-terminus and the arginine, lysine, and histidine residues, "Nj" is the number of aspartic acid, glutamic acid, cysteine, and tyrosine residues and the C-terminus, "pKaj" is the pKa value of the C-terminus and the aspartic acid, glutamic acid, cysteine, and tyrosine residues, and "pH" is the pH value of the buffer solution.)

[0033] The pKa values ​​(in parentheses) for cysteine ​​(8.33), aspartic acid (3.86), glutamic acid (4.25), histidine (6.0), lysine (10.53), arginine (12.48), tyrosine (10.07), N-terminus (9.69), and C-terminus (2.34) are based on Lehninger's *The Principles of Biochemistry* (1982). Microsoft Excel® software (Microsoft Corporation, Washington, US) can be used for calculations.

[0034] In the present invention, (i) the lipid component is selected as follows: (a) an anionic lipid is selected when the immunogenic peptide in the buffer solution has a positive charge, and (b) a cationic lipid is selected when the immunogenic peptide in the buffer solution has a negative charge.

[0035] The "anionic lipids" selected here are lipids that are negatively charged under the mixing of the aqueous and oil phases as detailed below, and any of the lipids commonly used to constitute conventionally known lipid nanoparticles can be used, and are not particularly limited. Examples include dipalmitoylphosphatidylglycerol (DPPG), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), dipalmitoylphosphatidic acid (DPPA), dimyristoylphosphatidylglycerol, distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), distearoylphosphatidic acid (DSPA), and dimyristoylphosphatidic acid. The anionic lipid used may be a single type or a combination of several different types. Preferably, the anionic lipid is DPPG, DPPS, or DSPS.

[0036] The "cationic lipids" selected here are lipids that are positively charged in a mixture of the aqueous and oil phases, as detailed below, and any of the lipids commonly used to constitute conventionally known lipid nanoparticles can be used; there are no particular limitations. Furthermore, the "cationic lipids" in this invention may also include so-called "ionized lipids" that have no charge at neutral pH and are positively charged at low pH. Examples of "cationic lipids" in the present invention include O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride (DC-6-14), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleyl-3-dimethylaminopropane (DODMA), ALC-0315, cKK-E12, SM-102, DLin-KC2-DMA, and heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (Dlin-MC3-DMA (which may be referred to as "D-Lin-MC3" or "MC3" in this specification)). The cationic lipid used may be a single type or a combination of several different types. Preferably, the cationic lipid is Dlin-MC3-DMA, DC-6-14, or DOTAP.

[0037] Furthermore, in the production of the immunogenic composition of the present invention, (i) the step of selecting lipid components does not need to be performed again once the lipid components have been selected, and the production method can be carried out using the selected lipid components by omitting / skipping this step.

[0038] In the present invention, the "(ii) PEG-modified lipid" constituting the LNP can be any of the conventionally known lipid nanoparticles commonly used to constitute lipid nanoparticles, and is not particularly limited, but examples include DMG-PEG, DSPE-PEG, etc. The PEG-modified lipid used may be a single type or a combination of multiple different types. The size of the PEG is not particularly limited, but for example, those with a molecular weight of about 750 to 5000 can be suitably used, and those with a molecular weight of about 2000 can be more suitably used. The end of the PEG can be any functional group, for example, -OMe, -OH, -COOH, -NH ２ They may also have -SH, etc. (but are not limited to these). Preferably, the PEG-modified lipid is DSPE-PEG, and by including these PEG-modified lipids in LNP, higher antibody induction against immunogenic peptides can be obtained.

[0039] In the present invention, the "(iii) cholesterol" constituting the LNP can be any of the conventionally known cholesterol commonly used to constitute lipid nanoparticles, and is not particularly limited. In this specification, "cholesterol" may be written as "Chol".

[0040] In the present invention, the “(iv) neutral phospholipid” constituting the LNP is a lipid that exhibits charge neutrality under the mixing of the aqueous phase and the oil phase detailed below at least, and any one generally used to constitute lipid nanoparticles can be used, and it is not particularly limited. For example, hydrogenated soy phosphatidylcholine (HSPC), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), palmitoyl oleoyl phosphatidylcholine (POPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, etc. may be mentioned. The neutral phospholipid used may be of a single type or a combination of a plurality of different types. Preferably, the neutral phospholipid is HSPC, DSPC, or DOPE.

[0041] In the present invention, the “(v) ionizable lipid” constituting the LNP is a lipid that has no charge at neutral pH and is positively charged at low pH. For example, Dlin-MC3-DMA, DODMA, ALC-0315, SM-102, DLin-KC2-DMA, DODAP, cKK-E12, etc. may be mentioned, but it is not limited thereto. However, the (v) ionizable lipid is different from the cationic lipid selected as the (i) lipid component.

[0042] In the present invention, the formulation of (i) a lipid component, (ii) a PEG-modified lipid, (iii) cholesterol, and (iv) a neutral phospholipid and / or (v) an ionizable lipid that constitute the LNP is in a mass ratio ((i):(ii):(iii):(iv):(v)) when the total of all components of (i), (ii), (iii), and (iv) and / or (v) is 100% by mass, 30 to 55% by mass: 0.3 to 3% by mass: 35 to 50% by mass: 0 to 30% by mass: 0 to 50% by mass, preferably 35 to 50% by mass: 0.5 to 3% by mass: 35 to 45% by mass: 3 to 25% by mass: 10 to 50% by mass, more preferably 35 to 50% by mass: 0.5 to 2.5% by mass: 35 to 45% by mass: 5 to 20% by mass: 20 to 50% by mass, still more preferably 40 to 50% by mass: 1 to 2.5% by mass: 38 to 45% by mass: 5 to 10% by mass: 30 to 50% by mass. By setting the formulation of each component in the LNP within the above ranges, it is possible to encapsulate an immunogenic peptide and deliver it in vivo, and an LNP capable of efficiently inducing an antibody against the peptide can be obtained.

[0043] In the present invention, the formation of LNP encapsulating an immunogenic peptide is carried out by mixing an immunogenic peptide (aqueous phase) dissolved in a buffer solution with a mixture (oil phase) of (i) a lipid component, (ii) a PEG-modified lipid, (iii) cholesterol, and (iv) a neutral phospholipid and / or (v) an ionizable lipid that constitute the LNP and are dissolved in an organic solvent such as chloroform or ethanol. By this mixing, the components constituting the LNP form a structure having the outer shell and inner shell described above, and the immunogenic peptide dissolved in the buffer solution is encapsulated inside the inner shell by electrostatic interaction with the (i) lipid component (charged positively or negatively) selected according to its charge. This mixing can be carried out at room temperature, preferably 4 to 35°C, more preferably 20 to 30°C, still more preferably 25°C.

[0044] The aqueous phase is prepared by dissolving the immunogenic peptide in a buffer having a predetermined pH value used in the calculation of the charge of the immunogenic peptide described above. The amount of immunogenic peptide dissolved in the buffer is not particularly limited, but is preferably 50 to 1000 μg / mL, more preferably 100 to 500 μg / mL. By setting the amount of immunogenic peptide dissolved in the buffer within this range, it is possible to efficiently encapsulate the immunogenic peptide in the LNP, which is preferable. The immunogenic peptide dissolved in the buffer may be a single type of immunogenic peptide, or multiple types of different immunogenic peptides may be dissolved in combination.

[0045] The oil phase is prepared by separating and blending (i) lipid components, (ii) PEG-modified lipids, (iii) cholesterol, and (iv) neutral phospholipids and / or (v) ionized lipids, each dissolved in an organic solvent such as chloroform or ethanol, so that each component is present in the above-mentioned proportions. The amount of each component contained in the oil phase is not particularly limited as long as the above-mentioned proportions are satisfied, but it is preferable that (i) lipid components are present in the oil phase in an amount of 10 to 1000 mg / mL, more preferably 10 to 100 mg / mL, and the other components can be appropriately determined based on the above-mentioned proportions. It is preferable that the amount of each component dissolved in the oil phase be within this range so that immunogenic peptides can be efficiently encapsulated in LNPs.

[0046] In this mixing process, the amount of the aqueous phase and the oil phase mixed can be any amount that allows for the encapsulation of immunogenic peptides in the LNPs, and is not particularly limited, but is preferably (aqueous phase:oil phase) 2 to 10:1 by mass ratio, more preferably 2 to 6:1, and even more preferably 2 to 4:1. By setting the amounts of each phase within this range, immunogenic peptides can be efficiently encapsulated in the LNPs, and by setting the amount of the aqueous phase and oil phase mixed within this range, the size of the LNPs containing the immunogenic peptides can be efficiently adjusted to a desired size, which is preferable.

[0047] The formation of LNPs encapsulating immunogenic peptides can be carried out by means commonly used in the formation of conventionally known lipid nanoparticles, and is not particularly limited. For example, it can be carried out by combining one or more means such as thin-film shaking, ethanol injection, homogenization, solvent dialysis, forced hydration, reverse-phase evaporation, microfluidic devices, microemulsification, sonication, chelate dialysis, solvent injection, spontaneous formation, solvent vaporization, controlled surfactant dialysis, extrusion, and filtration. Preferably, in the present invention, the formation of LNPs encapsulating immunogenic peptides is carried out using a microfluidic device. A "microfluidic device" is a device equipped with a substrate made of resin, glass, etc., through which a liquid flows (generally with a diameter of several tens to several hundred μm), and is also called a "microfluidic device" or "microfluidic chip." Microfluidic devices can move very small amounts of liquid or mix them on a microscale through their fine channels. In the present invention, the formation of LNPs containing immunogenic peptides can be carried out by mixing an aqueous phase and an oil phase using a microfluidic device, which is preferable because it allows for the uniform, highly reproducible, rapid, and efficient formation of LNPs containing immunogenic peptides.

[0048] The prepared LNPs containing immunogenic peptides are subjected to a dialysis device according to a conventional method to remove organic solvents derived from the oil phase. This dialysis device is preferable because it allows for the purification and concentration of the LNPs containing immunogenic peptides while maintaining the integrity of the LNPs and the encapsulation of the immunogenic peptides. Dialysis can be performed using physiological saline, PBS, glucose aqueous solution, sucrose aqueous solution, etc., with sucrose aqueous solution being preferred. Performing dialysis with sucrose aqueous solution is preferable because it removes organic solvents derived from the oil phase and allows for a high encapsulation rate of immunogenic peptides, preferably 70-100%, more preferably 80-100%. The sucrose aqueous solution can be used with a concentration of 5-15% by mass, preferably 10% by mass. Dialysis can be performed at room temperature, preferably 4-35°C, more preferably 20-30°C, and even more preferably 25°C.

[0049] In the present invention, the size of the LNPs containing immunogenic peptides is such that the average particle diameter is 40 nm to 200 nm, preferably 60 nm to 150 nm, and more preferably 80 nm to 120 nm. By setting the average particle diameter of the LNPs containing immunogenic peptides within this range, sterilization by filtration becomes possible, and an effective amount of immunogenic peptide can be efficiently delivered, which is preferable. In the present invention, "average particle diameter" refers to the number-average particle diameter measured by dynamic light scattering (DLS), and this measurement can be performed using commercially available equipment such as the ZETASIZER NANO (Malvern, Worcestershire, UK). The size of the LNP containing the immunogenic peptide can be adjusted by adjusting the mixing ratio of the aqueous phase and the oil phase as described above, and / or by a combination of one or more means such as extrusion, filtration, sonication, and homogenization of the formed LNP containing the immunogenic peptide, for example, by extruding it through pores of a predetermined size under pressure.

[0050] In the present invention, the polydispersity index (PDI) of LNPs containing immunogenic peptides is 0.05 to 0.5, preferably 0.05 to 0.3, and more preferably 0.05 to 0.25. By setting the PDI of the immunogenic peptides within this range, the particle size distribution of LNPs containing immunogenic peptides can be reduced. The PDI of LNPs containing immunogenic peptides can be measured using a commercially available device such as a ZETASIZER NANO (Malvern, Worcestershire, UK), and its value can be adjusted by the type and amount of each component constituting the LNP, the mixing ratio of the aqueous phase and the oil phase, etc.

[0051] The immunogenic composition of the present invention can be provided as a pharmaceutical composition, and more particularly as a vaccine formulation. The pharmaceutical composition and vaccine formulation of the present invention can be used in methods for preventing and / or treating diseases associated with a target protein, and can induce antibody production against the target protein in the administered animal, thereby preventing and / or treating diseases associated with the target protein.

[0052] In this invention, "prevent" means reducing the likelihood of developing a disease related to the target protein, delaying the onset of an infectious disease, or delaying or preventing the progression (worsening / severity) of symptoms / lesions after onset.

[0053] In this invention, "to treat" means not only that the symptoms / lesions of the disease related to the target protein completely disappear, but also that the symptoms / lesions are temporarily or permanently reduced or absent, or that they are stable without worsening. For example, this includes one or more of the following: reduction of symptoms / lesions, decrease in the level of markers indicating symptoms / lesions, improvement of symptoms / lesions, and extension of measures such as overall survival, progression-free survival, and median survival.

[0054] In the pharmaceutical composition and vaccine formulation of the present invention, the content of LNPs containing the immunogenic peptide may vary depending on factors such as the type and severity of the disease related to the target protein, the type, size and age of the animal to which the pharmaceutical composition and vaccine formulation are administered, and the dosage and administration of the pharmaceutical composition and vaccine formulation. However, any amount capable of obtaining the above-described effects can be included. Typically, the amount of immunogenic peptide can be 1 μg to 1000 mg, preferably 10 μg to 100 mg, particularly 100 μg to 10 mg, or alternatively, for example, 1 nmol to 1 mmol, preferably 10 nmol to 100 μmol, particularly an amount appropriately selected from the range of 100 nmol to 10 μmol.

[0055] The pharmaceutical compositions and vaccine formulations of the present invention may, in addition to LNPs containing the immunogenic peptide, optionally include excipients, binders, disintegrants, lubricants, diluents, solubilizers, suspending agents, isotonic agents, pH adjusters, buffers, stabilizers, and other additives commonly used in the manufacture of pharmaceuticals, and can be manufactured in a dosage form suitable for the intended route of administration.

[0056] The administration route of the pharmaceutical composition and vaccine preparation of the present invention is limited to obtaining the above-described effects and may be oral or parenteral administration, but parenteral administration is preferred. In particular, the administration methods that are common for vaccine preparations, such as intramuscular, intradermal, subcutaneous, intraocular, intratracheal, intranasal, nasal, pulmonary, and intravenous administration, are preferred, with intramuscular and subcutaneous administration being especially preferred. The form of the pharmaceutical composition and vaccine preparation of the present invention can be a dosage form suitable for these administration routes, for example, a solution, emulsion, injection, suspension, etc. (but is not limited to these). These can each be compounded, molded, or prepared according to methods commonly used in the art. Furthermore, the pharmaceutical composition and vaccine preparation of the present invention can be freeze-dried to a storable state, and then, at the time of use, dissolved in a buffer containing water or biosaline, etc., to adjust to an appropriate concentration before use.

[0057] The dosage of the pharmaceutical composition and vaccine formulation of the present invention is not particularly limited, as long as the above-described effects are obtained, but is preferably an amount of immunogenic peptide per dose that is appropriately selected from the range of 100 μg to 10 mg, more preferably 300 μg to 3 mg. The number of doses of the pharmaceutical composition and vaccine formulation of the present invention may be once, two or more times.

[0058] The subjects to whom the pharmaceutical composition and vaccine formulation of the present invention are administered include mammals (e.g., humans, non-human primates (chimpanzees, etc.), livestock (cattle, horses, pigs, sheep, goats, camels, alpacas, llamas, etc.), pets (dogs, cats, rabbits, hamsters, ferrets, etc.), laboratory animals (mice, rats, guinea pigs, etc.)), poultry (chickens, quail, turkeys, ducks, geese, longtailed chickens, bantams, etc.), reptiles, fish, etc., which are at risk of or already suffering from diseases related to the target protein, but are preferably mammals, and particularly preferably humans.

[0059] When the pharmaceutical compositions and vaccine formulations of the present invention are administered to a target, they induce antibody production against the target protein in the administered animal body, thereby preventing and / or treating diseases associated with the target protein. The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0060] Example 1: Encapsulation of peptides into LNPs Example 1-1: Effect of lipid composition and pH of peptide aqueous solution on encapsulation rate Using OVA-derived peptides (OVA323-339; SEQ ID NO: 38) as a model, they were dissolved in buffers with pH values ​​listed in Table 2 (acetic acid buffer at pH 4, HBS buffer at pH 7.4). They were mixed with an oil phase containing phospholipids, cholesterol, and PEG lipids in a microfluidic channel and attempted to be encapsulated into LNPs. The prepared LNPs were dialyzed using PBS at pH 7.4. The peptide concentration in the sample after dialyzing was measured by HPLC and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO.

[0061] Regarding encapsulation rates, the highest results were obtained when the peptide was dissolved in a pH 4 buffer with the F15 composition (DPPG / DOPE / CHOL / PEG-DSPE = 50:10:38.5:1.5) (Table 2).

[0062]

[0063] Examples 1-2: Effect of Dialysis Solution Solvent on Inclusion Rate Based on F15 in Table 2, LNPs were prepared using OVA-derived peptides (OVA323-339) as a model in the same manner as in Example 1, using a microfluidic channel. Figure 1 shows the evaluation of the inclusion rate of the model peptides into LNPs when the extracellular solution during subsequent dialysis was changed from PBS (pH 7.4) to physiological saline (pH 7.4), HEPES (pH 7.4), and 10% sucrose (pH 7.4). It was found that the inclusion rate was highest when dialysis was performed using 10% sucrose.

[0064] Examples 1-3: Effect of peptide charge on encapsulation rate in different pH buffers pH 4 acetate buffer (82 mM CH4) ３ COOH, 18mM CH ３ To investigate the effect of peptide charge on encapsulation rate when dissolved in COONa and pH 7.4 HBS buffer (25 mM HEPES, 140 mM NaCl; pH adjusted with HCl), six TNFα-derived peptides with nearly identical hydrophobic and hydrophilic properties, as listed in Table 3, were selected. The charges of these peptides when dissolved in pH 4 acetate buffer and pH 7.4 HBS buffer are as shown in Table 3.

[0065]

[0066] Six types of TNFα-derived peptides listed in Table 3 were dissolved in a pH 4 buffer and mixed with an oil phase containing DPPG / DOPE / CHOL / PEG-DSPE (50:10:38.5:1.5) via a microfluidic channel, and attempt was made to encapsulate them into LNPs. The obtained LNPs were dialyzed with 10% sucrose. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated (Table 4). In addition, the particle size and PDI of the obtained LNPs were evaluated using a ZETASIZER NANO.

[0067] Regarding each encapsulation rate, it was found that peptides with a positive charge at pH 4 are more easily encapsulated in compositions containing anionic phospholipids (DPPG / DOPE / CHOL / PEG-DSPE).

[0068]

[0069] Six types of TNFα-derived peptides listed in Table 3 were dissolved in a pH 7.4 buffer and mixed with an oil phase containing DC-6-14 / DSPC / CHOL / PEG-DSPE (50:10:38.5:1.5) via a microfluidic channel, and attempt was made to encapsulate them into LNPs. The obtained LNPs were dialyzed with 10% sucrose. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated (Table 5). In addition, the particle size and PDI of the obtained LNPs were evaluated using a ZETASIZER NANO.

[0070] Regarding each encapsulation rate, it was found that peptides with a negative charge at pH 7.4 are more easily encapsulated in a composition containing cationic phospholipids (DC-6-14 / DSPC / CHOL / PEG-DSPE).

[0071]

[0072] Example 2: Encapsulation of Amyloid-β Peptide into LNPs and Immunization of Mice Example 2-1: Encapsulation of Amyloid-β Peptide into LNPs Amyloid-β derived peptide (SEQ ID NO: 18) was used as a model. Each lipid component contained in the LNPs was selected according to the charge of the immunogenic peptide in the buffer, as described herein. The peptide was dissolved in HBS buffer at pH 7.4 and mixed with an oil phase containing DC-6-14:DSPC:PEG-DSPE:Chol (50:10:1.5:38.5) in a microfluidic channel and encapsulated in LNPs. The prepared LNPs were dialyzed using HBS at pH 7.4. The peptide concentration in the sample after dialyzing was measured by HPLC and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using ZETASIZER NANO (Table 6). An encapsulation rate of approximately 44% was obtained.

[0073] Furthermore, the peptide was dissolved in an acetate buffer at pH 4, mixed with an oil phase containing DPPG:DOPE:PEG-DSPE:Chol (50:10:1.5:38.5) via a microfluidic channel, and encapsulated in LNPs. The prepared LNPs were dialyzed using HBS at pH 7.4. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 6). An encapsulation rate of approximately 61% was achieved. This method confirmed that amyloid-beta derived peptides (SEQ ID NO: 18) can be encapsulated in LNPs.

[0074]

[0075] Example 2-2: Antibody induction by administration of amyloid-beta peptide-encapsulated LNPs. Amyloid-beta peptide-encapsulated LNPs prepared in Example 2-1 were administered to mice every two weeks at a dose of 50 μg peptide / mouse, using 1) intravenous administration, 2) intramuscular administration, 3) intraperitoneal administration, and 4) subcutaneous administration for eight doses. For the control group, amyloid-beta peptide was mixed with complete Freund's adjuvant (CFA) and administered subcutaneously at a dose of 50 μg peptide / mouse following the same schedule. Blood was collected 7 days after the final immunization to obtain serum. Unimmunized mouse serum was used as a control.

[0076] The obtained antipeptide serum was analyzed as follows: (Analysis of antipeptide serum) For the peptide immobilization solution, 10 μL of the antigen, amyloid-β peptide, was added to each 96-well microplate to a concentration of 20 μg / mL. Then, 90 μL of 0.05 M carbonic acid-bicarbonate buffer was added, and the mixture was allowed to stand at room temperature for 2 hours to immobilize each antigen peptide. Next, after removing the antigen solution, 350 μL each of 0.1% BSA / PBS was added to each well, and the mixture was allowed to stand at room temperature for 90 minutes to block the peptides. After removing the 0.1% BSA / PBS, 100 μL of antipeptide serum from the group administered with 100-fold diluted peptide-encapsulated LNP or peptide mixed with CFA was added to each antigen peptide immobilized well, and incubated overnight at 4°C. After this, the plates were washed three times with PBS / 0.05% Tween-20. Next, 100 μL / well of HRP (horseradish peroxidase)-labeled protein G was added and incubated at room temperature for 2 hours. After this, the plates were washed five times with PBS / 0.05% Tween-20. Finally, 100 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB) was added, and after sufficient color development, 100 μL of 1N sulfuric acid solution was added to each well to stop the reaction. The color development of the substrate solution was measured by measuring the absorbance at 450 nm using a plate reader (Titertec).

[0077] The results are shown in Figure 2. In the case of LNPs based on cationic lipids (DC-6-14), antibody induction was observed with both intramuscular and subcutaneous administration.

[0078] Furthermore, in the case of LNPs based on anionic lipids (DPPG), antibody induction was observed with both intramuscular and subcutaneous administration. These results demonstrate that antibody induction is possible by administering amyloid-beta peptide-encapsulated LNPs.

[0079] Example 3: Encapsulation of TNFα peptide into LNPs and immunization of mice Example 3-1: Encapsulation of TNFα peptide into LNPs TNFα-derived peptide (H128-157; SEQ ID NO: 15) was used as a model. Each lipid component contained in the LNP was selected according to the charge of the immunogenic peptide in the buffer, as described herein. The peptide was dissolved in acetate buffer at pH 3.5 and 4, mixed with an oil phase containing DPPG / DOPE / CHOL / PEG-DSPE (51.07:5:43.43:0.5) in a microfluidic channel, and encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and surface charge of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 7).

[0080] Similarly, peptides were dissolved in acetate buffer at pH 3.5 and 4, mixed with an oil phase containing DPPG / DOPE / CHOL / PEG-DSPE (29.96:25:42.54:2.5) via a microfluidic channel, and encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 7).

[0081] This method confirmed that TNFα-derived peptide (H128-157) can be encapsulated in LNPs.

[0082]

[0083] Example 3-2: Antibody induction by administration of TNFα-derived peptide-encapsulated LNPs. From the TNFα-derived peptide-encapsulated LNPs prepared in Example 3-1, #2 and #4 in Table 7, which showed relatively high encapsulation rates, were selected. #2 and #4 were designated as NP-A and NP-B, respectively.

[0084] Each of the two drugs was administered to mice at a dose of 20 μg peptide / mouse every week for six weeks, either 1) intramuscularly (IM) or 2) intravenously (IV). For the control group, TNFα-derived peptide was mixed with CFA and administered subcutaneously at a dose of 20 μg peptide / mouse following the same schedule. Blood was collected 7 days after the final immunization to obtain serum. Unimmunized mouse serum was used as a control. Experiments were conducted with 6 mice in each group.

[0085] The obtained antiserum was analyzed as follows: (Analysis of antiserum) Human TNFα was found in PBS (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na) ２ HPO ４ , 2.0 mM NaH ２ PO ４ The pH was adjusted to 7.4 with HCl. The solution was adjusted to 2 μg / mL, and 100 μL was added to each of the 96-well microplates. The plates were then allowed to stand overnight at 4°C to solidify. After this, the plates were washed three times with TBS (50 mM Tris, 140 mM NaCl; pH adjusted to 8.0 with HCl) / 0.05% Tween-20. 350 μL each of 0.1% BSA / PBS was added to each of the TNFα solid-phase wells and allowed to stand at room temperature for at least 1.5 hours. After this, the plates were washed three times with TBS / 0.05% Tween-20. 100 μL of 50-fold diluted antiserum was added to each of the TNFα solid-phase wells and incubated overnight at room temperature. After this, the plates were washed three times with TBS / 0.05% Tween-20. Next, 100 μL / well of HRP (horseradish peroxidase)-labeled anti-rabbit IgG antibody (Bio-Rad) was added and incubated at room temperature for 2 hours. After this, the plates were washed five times with TBS / 0.05% Tween-20. Finally, 100 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB) was added and allowed to develop sufficiently. Then, 100 μL of 2M sulfuric acid solution was added to each well to stop the reaction. The color development of the substrate solution was measured by measuring the absorbance at 450 nm using a plate reader (Tecan).

[0086] The results are shown in Figure 3. Intravenous administration of NP-A induced antibodies to a similar degree as administration of the positive control CFA (control group).

[0087] Furthermore, intramuscular and intravenous administration of NP-B induced stronger antibodies than administration with the positive control CFA (control group). These results demonstrate that antibody induction is possible by administration of TNFα peptide-encapsulated LNPs.

[0088] Example 4 Effect of Adding Cationic Lipids to TNFα Peptide-Encapsulated LNPs Example 4-1: Encapsulation of TNFα Peptide in LNPs Similar to Example 3, a TNFα-derived peptide (H128-157) was used as a model. Each lipid component contained in the LNP was selected according to the charge of the immunogenic peptide in the buffer, as described herein. The peptide was dissolved in acetate buffer at pH 3.5 and mixed with an oil phase containing DPPG / DOPE / CHOL / PEG-DSPE (29.96:25:42.54:2.5) in a microfluidic channel and encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. This was designated as NP-B. The peptide concentration in the sample after dialyzing was measured by HPLC and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 8).

[0089] Similarly, the peptide was dissolved in acetate buffer at pH 3.5 and mixed with an oil phase containing DPPG / DOPE / CHOL / PEG-DMG (29.96:25:42.54:2.5) via a microchannel, and then encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. This was designated as NP-B DMG. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 8).

[0090] The peptide was dissolved in HBS buffer at pH 7.4 and mixed with an oil phase containing DPPG / MC3 / CHOL / PEG-DMG (29.96:25:42.54:2.5) via a microchannel, and then encapsulated in LNPs. This was designated as DPPG MC3. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 8).

[0091] Similarly, peptides were dissolved in HBS buffer at pH 7.4 and mixed with an oil phase containing MC3 / DOPE / CHOL / PEG-DMG (29.96:25:42.54:2.5) via a microchannel, and encapsulated in LNPs. This was designated as MC3 DOPE. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 8).

[0092] As shown in Table 8, it was confirmed that TNFα-derived peptides (H128-157 peptide) can be encapsulated in LNPs using this method.

[0093]

[0094] Example 4-2: Antibody induction by administration of TNFα-derived peptide-encapsulated LNPs. TNFα-derived peptide-encapsulated LNPs prepared in Example 4-1 were administered intramuscularly to mice at a dose of 20 μg peptide / mouse every week for six doses. Blood was collected 7 days after the final immunization to obtain serum. Serum from unimmunized mice was used as a control. Experiments were conducted with 6 mice in each group.

[0095] The obtained antiserum was analyzed in the same manner as described in Example 3-2 (Analysis of Antiserum). The results are shown in Figure 4. NP-B induced anti-human TNFα antibody, similar to Figure 3. On the other hand, when PEG-DSPE was changed to PEG-DMG in the composition of NP-B (NP-B DMG), antibody induction decreased significantly. Meanwhile, in DPPG-MC3, where DOPE was changed to a cationic lipid (MC-3) in the composition of NP-B DMG, approximately three times higher induction of anti-human TNFα antibody was observed compared to NP-B. Furthermore, in MC3-DOPE, where DPPG was changed to a cationic lipid (MC-3) in the composition of NP-B DMG, approximately three times higher induction of anti-human TNFα antibody was observed compared to NP-B, similar to the case of DPPG-MC3. These results indicate that the addition of cationic lipids to the composition of LNP further increases antibody induction.

[0096] Example 5 Effect of PEG-modified lipid changes on TNFα peptide-encapsulated LNPs Example 5-1: Encapsulation of TNFα peptide into LNPs A TNFα-derived peptide (H128-157) was used as a model. Each lipid component contained in the LNP was selected according to the charge of the immunogenic peptide in the buffer, as described herein. The peptide was dissolved in HBS buffer at pH 7.4 and mixed with an oil phase containing D-Lin-MC3:HSPC:methoxyPEG-DSPE:Chol (50:9:2.5:38.5), D-Lin-MC3:HSPC:hydroxyPEG-DSPE:Chol (50:9:2.5:38.5), or D-Lin-MC3:HSPC:methoxyPEG-DMG:Chol (50:9:2.5:38.5) via a microfluidic channel and encapsulated into LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 9). It was confirmed that model peptides could be encapsulated in these lipid compositions as well.

[0097]

[0098] Example 5-2: Antibody induction by administration of TNFα-derived peptide-encapsulated LNPs. TNFα-derived peptide-encapsulated LNPs prepared in Example 5-1 were administered intramuscularly to mice at a dose of 20 μg peptide / mouse every week for four times. Blood was collected 7 days after the final immunization to obtain serum. Similarly, TNFα-derived peptide-encapsulated LNPs (DC-6-14:HSPC:methoxyPEG-DSPE:Chol=50:9:2.5:38.5) prepared in the same manner as in Example 5-1 were administered to mice, and blood was collected 7 days after the final immunization to obtain serum. Unimmunized mouse serum was used as a control.

[0099] The analysis of the obtained serum was carried out in the same manner as described in Example 3-2 (analysis of antiserum), except that 100 μL of 100-fold diluted antiserum was added to each TNFα solid-phase well and incubated overnight at room temperature, and 100 μL / well of HRP (horseradish peroxidase) labeled protein G (Bio-Rad) was added.

[0100] The results are shown in Figure 5. Compared to the use of LNPs containing D-Lin-MC3, LNPs modified with methoxyPEG-DSPE and hydroxyPEG-DSPE, LNPs containing DC-6-14 (DC-6-14:HSPC:methoxyPEG-DSPE:Chol = 50:9:2.5:38.5), and LNPs modified with methoxyPEG-DMG (D-Lin-MC3:HSPC:methoxyPEG-DMG:Chol (50:9:2.5:38.5)), induction of anti-human TNFα antibodies was observed at a higher probability. These results do not indicate that LNPs containing DC-6-14 or LNPs modified with methoxyPEG-DMG cannot induce anti-human TNFα antibodies, but rather that the induction efficiency of anti-human TNFα antibodies was lower in this immunization method compared to LNPs with other configurations. Even when using LNPs containing DC-6-14 or LNPs modified with methoxyPEG-DMG, anti-human TNFα antibodies can be induced by general adjustments to the immunization method, including changes in antigen dose and number of administrations. Note that A, B, C, and D in Figure 5 represent different mouse individuals.

[0101] Example 6 Antibody induction when different TNFα peptides were encapsulated in different LNPs and administered to mice Example 6-1: Encapsulation of TNFα peptides in LNPs Seven TNFα-derived peptides listed in Table 10 were used as models. Each lipid component contained in the LNP was selected according to the charge of the immunogenic peptide in the buffer, as described herein. TNFα-derived peptides (H1-30 and H8-37) were dissolved in acetate buffer at pH 4 and mixed with an oil phase containing D-Lin-MC3:DPPG:HSPC:mPEG-DSPE:Chol (10:40:9:2.5:38.5) via a microfluidic channel and encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC and the encapsulation rate (EE%) was calculated. Furthermore, the particle size and PDI of the obtained LNPs were evaluated using ZETASIZER NANO (Table 10). It was confirmed that peptides could be encapsulated in LNPs under these conditions as well.

[0102] Furthermore, TNFα-derived peptide (H31-60) was dissolved in a pH 10 carbonate buffer and mixed with an oil phase containing D-Lin-MC3:DC-6-14:mPEG-DSPE:Chol (50:9:2.5:38.5) via a microfluidic channel, and then encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 10). It was confirmed that peptides can be encapsulated in LNPs under these conditions as well.

[0103] Furthermore, TNFα-derived peptide (H91-120) was dissolved in HBS at pH 7.4, mixed with an oil phase containing D-Lin-MC3:HSPC:mPEG-DSPE:Chol (50:9:2.5:38.5) via a microfluidic channel, and encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 10). It was confirmed that peptides can be encapsulated in LNPs under these conditions as well.

[0104] Furthermore, TNFα-derived peptide (H98-127) was dissolved in a pH 10 carbonate buffer and mixed with an oil phase containing D-Lin-MC3:HSPC:mPEG-DSPE:Chol (50:9:2.5:38.5) via a microfluidic channel, and then encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 10). It was confirmed that peptides can be encapsulated in LNPs under these conditions as well.

[0105] Furthermore, TNFα-derived peptides (H121-150) were dissolved in HBS at pH 7.4, mixed with an oil phase containing D-Lin-MC3:HSPC:mPEG-DSPE:Chol (50:9:2.5:38.5) via a microfluidic channel, and encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 10). It was confirmed that peptides can be encapsulated in LNPs under these conditions as well.

[0106] Furthermore, TNFα-derived peptide (H128-157) was dissolved in HBS at pH 7.4 and mixed with an oil phase containing D-Lin-MC3:DC-6-14:mPEG-DSPE:Chol (50:9:2.5:38.5) via a microfluidic channel, and then encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 10). It was confirmed that peptides can be encapsulated in LNPs under these conditions as well.

[0107]

[0108] Example 6-2: Antibody induction by intramuscular administration of TNFα-derived peptide-encapsulated LNPs. TNFα-derived peptide-encapsulated LNPs prepared in Example 6-1 were administered intramuscularly to mice at a dose of 20 μg peptide / mouse every two weeks for six doses. Blood was collected seven days after the final immunization to obtain serum. Serum from unimmunized mice was used as a control. Experiments were conducted with six mice in each group.

[0109] The analysis of the obtained antiserum was carried out in the same manner as described in Example 3-2 (analysis of antiserum), except that 100 μL of 100-fold diluted antiserum was added to each TNFα solid phase well and incubated overnight at room temperature, and 100 μL / well of HRP (horseradish peroxidase) labeled protein G (Bio-Rad) was added.

[0110] The results are shown in Figure 6. Of the seven LNPs administered, the following were observed: D-Lin-MC3:DPPG:HSPC:mPEG-DSPE:Chol (10:40:9:2.5:38.5) containing hTNAα (H1-30), D-Lin-MC3:HSPC:mPEG-DSPE:Chol (50:9:2.5:38.5) containing hTNAα (H91-120), and D-Lin-MC3:HSPC:mPE (50:9:2.5:38.5) containing hTNAα (H98-127). Induction of anti-human TNFα antibodies was confirmed in G-DSPE:Chol (50:9:2.5:38.5), D-Lin-MC3:HSPC:mPEG-DSPE:Chol (50:9:2.5:38.5) containing hTNAα (H121-150), and D-Lin-MC3:DC-6-14:mPEG-DSPE:Chol (50:9:2.5:38.5) containing hTNAα (H128-157). Among these, D-Lin-MC3:DC-6-14:mPEG-DSPE:Chol (50:9:2.5:38.5) containing hTNAα (H128-157) showed the strongest induction. These results do not indicate that anti-human TNFα antibodies cannot be induced when using D-Lin-MC3:DPPG:HSPC:mPEG-DSPE:Chol (10:40:9:2.5:38.5) containing hTNAα (H8-37) or D-Lin-MC3:DC-6-14:mPEG-DSPE:Chol (50:9:2.5:38.5) containing hTNAα (H31-60). Rather, they merely indicate that the induction efficiency of anti-human TNFα antibodies was lower in this immunization method compared to LNPs with other compositions. Even when using these LNPs, anti-human TNFα antibodies can be induced by general adjustments to the immunization method, including changes in antigen dose and number of administrations.

[0111] Example 7 Antibody induction when different TNFα peptides were encapsulated in different LNPs and administered intramuscularly to rabbits Example 7-1: Encapsulation of TNFα peptides in LNPs Three TNFα-derived peptides listed in Table 11 were used as models. Each lipid component contained in the LNP was selected according to the charge of the immunogenic peptide in the buffer, as described herein. The TNFα-derived peptide (H8-37) was dissolved in acetate buffer at pH 4 and mixed with an oil phase containing D-Lin-MC3:DPPG:HSPC:mPEG-DSPE:Chol (10:40:9:2.5:38.5) in a microfluidic channel and encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC and the encapsulation rate (EE%) was calculated. Furthermore, the particle size and PDI of the obtained LNPs were evaluated using ZETASIZER NANO (Table 11). It was confirmed that peptides could be encapsulated in LNPs under these conditions as well.

[0112] Furthermore, TNFα-derived peptide (H91-120) was dissolved in a pH 10 carbonate buffer and mixed with an oil phase containing D-Lin-MC3:HSPC:mPEG-DSPE:Chol (50:9:2.5:38.5) via a microfluidic channel, and then encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 11). It was confirmed that peptides can be encapsulated in LNPs under these conditions as well.

[0113] Furthermore, TNFα-derived peptides (H128-157) were dissolved in HBS at pH 7.4, mixed with an oil phase containing D-Lin-MC3:HSPC:mPEG-DSPE:Chol (50:9:2.5:38.5) via a microfluidic channel, and encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 11). It was confirmed that peptides can be encapsulated in LNPs under these conditions as well.

[0114]

[0115] Example 7-2: Antibody induction by administration of TNFα-derived peptide-encapsulated LNPs. The TNFα-derived peptide-encapsulated LNPs prepared in Example 7-1 were administered intramuscularly to rabbits at a dose of 300 μg peptide / rabbit every two weeks for four doses. Blood was collected 7 days after the final immunization to obtain serum. Serum from unimmunized rabbits was used as a control.

[0116] The analysis of the obtained serum was carried out in the same manner as described in Example 3-2 (analysis of antiserum), except that 100 μL of 10-fold diluted antiserum was added to each TNFα solid-phase well and incubated overnight at room temperature, and 100 μL / well of HRP (horseradish peroxidase) labeled protein G (Bio-Rad) was added.

[0117] The results are shown in Figure 7. It was found that LNP (D-Lin-MC3:HSPC:mPEG-DSPE:Chol(50:9:2.5:38.5)) containing the TNFα-derived peptide (H128-157) induced anti-human TNFα antibodies with the highest probability and strongest effect. Furthermore, when LNPs containing TNFα-derived peptide (H8-37) (D-Lin-MC3:DPPG:HSPC:mPEG-DSPE:Chol (10:40:9:2.5:38.5)) and LNPs containing TNFα-derived peptide (H91-120) (D-Lin-MC3:HSPC:mPEG-DSPE:Chol (50:9:2.5:38.5)) were administered, anti-human TNFα antibodies were induced with almost the same efficiency (2 / 3 of the rabbits), but the strength of the antibodies tended to be slightly higher with LNPs containing TNFα-derived peptide (H8-37) (D-Lin-MC3:DPPG:HSPC:mPEG-DSPE:Chol (10:40:9:2.5:38.5)). A, B, and C in Figure 7 represent different rabbit individuals.

[0118] Figure 8 shows the dilution curves obtained by serially diluting the serum from Figure 7 starting from 25 times. Comparing the average values, LNPs containing TNFα-derived peptide (H128-157) (D-Lin-MC3:HSPC:mPEG-DSPE:Chol (50:9:2.5:38.5)) induced the largest amount of anti-human TNFα antibody. LNPs containing TNFα-derived peptide (H8-37) (D-Lin-MC3:DPPG:HSPC:mPEG-DSPE:Chol (10:40:9:2.5:38.5)) and LNPs containing TNFα-derived peptide (H91-120) (D-Lin-MC3:HSPC:mPEG-DSPE:Chol (50:9:2.5:38.5)) induced less antibody than LNPs containing TNFα-derived peptide (H128-157), indicating little difference between the two.

[0119] Example 8: Encapsulation of Amyloid-β Peptide into LNPs and Immunization of Mice Example 8-1: Encapsulation of Amyloid-β Peptide into LNPs Amyloid-β derived peptide (SEQ ID NO: 18) was used as a model. Each lipid component contained in the LNPs was selected according to the charge of the immunogenic peptide in the buffer, as described herein. The peptide was dissolved in HBS buffer at pH 7.4 and mixed with an oil phase containing D-Lin-MC3:DC-6-14:mPEG-DSPE:Chol (20:39:2.5:38.5) in a microfluidic channel and encapsulated in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 12). This method confirmed that amyloid-beta derived peptides (SEQ ID NO: 18) can be encapsulated in LNPs.

[0120]

[0121] Example 8-2: Antibody induction by administration of amyloid-beta peptide-encapsulated LNPs. Amyloid-beta peptide-encapsulated LNPs prepared in Example 8-1 were administered intramuscularly to mice at a dose of 20 μg peptide / mouse every two weeks for five doses (i.m.). As a control group, amyloid-beta peptide was mixed with CFA and administered subcutaneously at a dose of 20 μg peptide / mouse on the same schedule (s.c.). Blood was collected 7 days after the final immunization to obtain serum. Unimmunized mouse serum was used as a control. Experiments were conducted with 4 mice in each group.

[0122] The obtained antipeptide serum was analyzed in the same manner as described in Example 2-2 (Analysis of antipeptide serum).

[0123] The results are shown in Figure 9. Strong induction of anti-amyloid-beta peptide antibodies was observed with only three intramuscular administrations using amyloid-beta peptide-encapsulated LNPs. In contrast, in the control group, almost no induction of anti-amyloid-beta peptide antibodies was observed even after four administrations.

[0124] In this example, it was shown that by adding ionized lipids with immunostimulatory properties, significantly higher antibody induction ability can be obtained even with a smaller antigen dose and fewer administrations compared to the antibody induction shown in Example 2 (Figure 2).

[0125] Example 9 Antibody induction when mice were immunized with LNPs having different lipid compositions and encapsulated with TNFα peptide (H128-157) Example 9-1: Encapsulation of TNFα peptide into LNPs TNFα-derived peptide (H128-157) was used as a model. Each lipid component contained in the LNP was selected according to the charge of the immunogenic peptide in the buffer, as described herein. TNFα-derived peptide (H128-157) was dissolved in HBS at pH 7.4 and mixed with four oil phases containing D-Lin-MC3:HSPC:mPEG-DSPE:Chol (50:9:2.5:38.5), D-Lin-MC3:DC-6-14:mPEG-DSPE:Chol (20:39:2.5:38.5), DC-6-14:HSPC:mPEG-DSPE:Chol (50:9:2.5:38.5), and D-Lin-MC3:DC-6-14:mPEG-DSPE:Chol (50:9:2.5:38.5) via microchannels, and then encapsulated in four types of LNPs. The prepared LNPs were dialyzed using a 10% sucrose aqueous solution. The peptide concentration in the dialysis-treated sample was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 13). It was confirmed that peptides could be encapsulated in LNPs under these conditions.

[0126]

[0127] Example 9-2: Antibody induction by administration of TNFα-derived peptide-encapsulated LNPs. The TNFα-derived peptide-encapsulated LNPs prepared in Example 9-1 were administered intramuscularly to mice at a dose of 20 μg peptide / mouse every two weeks for seven doses. As a control group, TNFα-derived peptide (H128-157) and CFA (IFA was used from the second immunization onward) were emulsified in a 1:1 ratio and administered subcutaneously to Balb / c mice at a dose of 20 μg peptide / mouse every two weeks for seven doses (s.c.).

[0128] Blood samples were collected and serum obtained from mice seven days after the second immunization, the third immunization, and the final immunization. Serum from unimmunized mice was used as a control.

[0129] The analysis of the obtained antiserum was carried out in the same manner as described in Example 3-2 (analysis of antiserum), except that 100 μL of 100-fold diluted antiserum was added to each TNFα solid phase well and incubated overnight at 4°C, and 100 μL / well of HRP (horseradish peroxidase) labeled protein G (Bio-Rad) was added.

[0130] The results are shown in Figure 10. It was confirmed that immunization with LNP(4) (D-Lin-MC3:DC-6-14:mPEG-DSPE:Chol(50:9:2.5:38.5)) induced anti-human TNFα IgG with the highest probability and strongest response from the early stages of immunization. It was also confirmed that LNP(3) (DC-6-14:HSPC:mPEG-DSPE:Chol(50:9:2.5:38.5)), which does not contain D-Lin-MC3, had a lower ability to induce anti-human TNFα IgG compared to the other LNPs. Furthermore, it was confirmed that anti-human TNFα IgG was not induced in the control group that was subcutaneously immunized with TNFα peptide mixed with CFA. In the control group, the reason for the low anti-human TNFα IgG induction ability may be that the peptide used aggregated within the adjuvant due to its hydrophobic properties, thus hindering antigen presentation.

[0131] Example 10 Antibody induction by administration of LNPs containing TNFα protein C-terminal peptide Example 10-1: Encapsulation of TNFα peptide into LNP Four types of TNFα-derived peptides (in order from closest to the C-terminus) (H91-120, H98-127, H121-150, H128-157) were used as models. Each lipid component contained in the LNP was selected according to the charge of the immunogenic peptide in the buffer, as described herein. The four types of TNFα-derived peptides (H91-120, H98-127, H121-150, H128-157) were each dissolved in HBS at pH 7.4, and each was mixed with an oil phase containing D-Lin-MC3:DC-6-14:mPEG-DSPE:Chol (50:9:2.5:38.5) via a microfluidic channel and encapsulated into LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC, and the encapsulation rate (EE%) was calculated. The particle size and PDI of the obtained LNPs were also evaluated using a ZETASIZER NANO (Table 14). It was confirmed that peptides could be encapsulated in LNPs under these conditions.

[0132]

[0133] Example 10-2: Antibody induction by administration of TNFα-derived peptide-encapsulated LNPs Four types of TNFα-derived peptide-encapsulated LNPs prepared in Example 10-1 were administered intramuscularly to mice at a dose of 20 μg peptide / mouse every two weeks for six doses. Blood was collected from each mouse seven days after the final immunization to obtain serum. Serum from unimmunized mice was used as a control.

[0134] The analysis of the obtained antiserum was carried out in the same manner as described in Example 3-2 (analysis of antiserum), except that 100 μL of 100-fold diluted antiserum was added to each TNFα solid phase well and incubated overnight at 4°C, and 100 μL / well of HRP (horseradish peroxidase) labeled protein G (Bio-Rad) was added.

[0135] The results are shown in Figure 11. It was confirmed that immunization with two types of LNPs containing TNFα-derived peptides H98-127 and H128-157 resulted in the highest probability and strongest induction of anti-human TNFα IgG. These results do not indicate that anti-human TNFα IgG cannot be induced when using LNPs containing TNFα-derived peptides H91-120 and H121-150, but rather that the induction efficiency of anti-human TNFα IgG was lower in this immunization method compared to LNPs with other configurations. Even when using these LNPs, anti-human TNFα IgG can be induced by general adjustments to the immunization method, including changes in antigen dose and number of administrations.

[0136] Example 10-3: Comparison of immunization of TNFα-derived peptide (H98-127)-encapsulated LNPs and subcutaneous immunization of TNFα-derived peptide (H98-127) TNFα-derived peptide (H98-127)-encapsulated LNPs prepared in Example 10-1 were administered intramuscularly to mice at a dose of 20 μg peptide / mouse every two weeks for seven doses. In addition, as a control group, TNFα peptide (H98-127) was mixed with CFA and administered subcutaneously at a dose of 20 μg peptide / mouse on the same schedule (s.c.). Blood was collected seven days after the final immunization to obtain serum. Serum from unimmunized mice was used as a control. Experiments were conducted with nine mice in each group.

[0137] The analysis of the obtained antiserum was carried out in the same manner as described in Example 3-2 (analysis of antiserum), except that 100 μL of 100-fold diluted antiserum was added to each TNFα solid phase well and incubated overnight at 4°C, and 100 μL / well of HRP (horseradish peroxidase) labeled protein G (Bio-Rad) was added.

[0138] The results are shown in Figure 12. Subcutaneous immunization with TNFα-derived peptide (H98-127) failed to induce anti-human TNFα IgG, whereas immunization with LNPs carrying TNFα-derived peptide (H98-127) was confirmed to induce anti-human TNFα IgG with a high probability and strongly. In the control group, the reason for the low anti-human TNFα IgG induction ability may be due to factors such as the peptide's hydrophobic properties causing aggregation within the adjuvant and hindering antigen presentation.

[0139] Example 11 Antibody induction when mice were immunized with PD-1 peptide (m50-64) embedded LNPs Example 11-1: Embedding of PD-1 peptide into LNPs Mouse PD-1 derived peptide (m50-64: SEQ ID NO: 34) was used as a model antigen. Each lipid component contained in the LNP was selected according to the charge of the immunogenic peptide in the buffer, as described herein. The PD-1 derived peptide (m50-64) was dissolved in HBS at pH 7.4 and mixed with an oil phase containing D-Lin-MC3:DC-6-14:mPEG-DSPE:Chol = 50:9:2.5:38.5 in a microfluidic channel and embedded in LNPs. The prepared LNPs were dialyzed with a 10% sucrose aqueous solution. The peptide concentration in the sample after dialyzing was measured by HPLC and the embedding rate (EE%) was calculated. Furthermore, the particle size and PDI of the obtained LNPs were evaluated using ZETASIZER NANO (Table 15). It was confirmed that peptides could be encapsulated in LNPs under these conditions as well.

[0140]

[0141] Example 11-2: Antibody induction by administration of LNPs encapsulated with mPD-1-derived peptide (m50-64) The mPD-1-derived peptide (m50-64) encapsulated LNPs prepared in Example 11-1 were administered intramuscularly to C57BL / 6N mice at a dose of 20 μg peptide / mouse every two weeks for four doses (i.m.). As a control group, mPD-1-derived peptide (m50-64) and CFA (IFA was used from the second immunization onwards) were emulsified in a 1:1 ratio and administered subcutaneously to C57BL / 6N mice at a dose of 20 μg peptide / mouse every two weeks for four doses (s.c.). Experiments were conducted with 5 mice in each group. Blood was collected and serum was obtained 7 days after the second immunization, third immunization, and final immunization. Serum from unimmunized mice was used as a control.

[0142] The obtained serum was analyzed as follows: (Analysis of antiserum) mPD-1 derived peptide (m50-64) was adjusted to 20 μg / mL with DMSO, 10 μL was added to each of the m50-64 solid phase wells, and 90 μL of PBS was added. The mixture was then immobilized at room temperature for 2 hours. After this, the plates were washed three times with TBS / 0.05% Tween-20. 350 μL each of 0.1% BSA / PBS was added to each of the m50-64 solid phase wells and allowed to stand at room temperature for at least 1.5 hours. After this, the plates were washed three times with TBS / 0.05% Tween-20. 100 μL of 100-fold diluted antiserum was added to each of the m50-64 solid phase wells and incubated overnight at 4°C. After this, the plates were washed three times with TBS / 0.05% Tween-20. Next, 100 μL / well of HRP (horseradish peroxidase)-labeled protein G (Bio-Rad) was added and incubated at room temperature for 1 hour. After this, the plates were washed five times with TBS / 0.05% Tween-20. Finally, 100 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB) was added, and after sufficient color development, 100 μL of 2M sulfuric acid solution was added to each well to stop the reaction. The color development of the substrate solution was measured by measuring the absorbance at 450 nm using a plate reader (Tecan).

[0143] The results are shown in Figure 13. It was confirmed that intramuscular administration of mPD-1 peptide encapsulated in LNPs could induce anti-mPD-1 peptide IgG at a similar level to that of mPD-1 peptide mixed with CFA and administered subcutaneously (control group).

[0144] Example 12 Antibody induction when LNPs containing PD-1 peptide (m50-64) were immunized to different strains of mice. LNPs containing mPD-1 derived peptides prepared in Example 11-1 were administered intramuscularly to C3H / He mice at a dose of 20 μg peptide / mouse every two weeks for four doses. As a control group, mPD-1 derived peptide (m50-64) and CFA (IFA was used from the second immunization onward) were emulsified in a 1:1 ratio and administered subcutaneously to C3H / He mice at a dose of 20 μg peptide / mouse every two weeks for four doses (s.c.). Experiments were conducted with eight mice in each group. Blood was collected seven days after the final immunization to obtain serum. Serum from unimmunized mice was used as a control. The analysis of the obtained serum was performed in the same manner as described in Example 11-2 (analysis of antiserum).

[0145] The results are shown in Figure 14. Intramuscular administration of mPD-1 peptide encapsulated in LNPs resulted in significantly higher anti-mPD-1 peptide antibody titers compared to subcutaneous administration of mPD-1 peptide mixed with CFA (control group). Furthermore, while 0 out of 8 mice in the control group developed antibodies, 6 out of 8 mice in the group administered mPD-1 peptide encapsulated in LNPs developed antibodies. These results demonstrate that antibodies can be induced using LNPs encapsulated with antigen peptides, without being limited to specific mouse strains or administration methods.

Claims

1. A method for producing an immunogenic composition comprising lipid nanoparticles (LNPs) encapsulating an immunogenic peptide, comprising: (1) a step of selecting a lipid component to be contained in the LNP according to the charge of the immunogenic peptide in a buffer solution, (a) selecting an anionic lipid if the charge is positive, and (b) selecting a cationic lipid if the charge is negative; and (2) a step of forming LNPs encapsulating the immunogenic peptide by mixing the immunogenic peptide dissolved in a buffer solution with the lipid component selected in step (1), PEG-modified lipids, cholesterol, and neutral phospholipids and / or ionized lipids.

2. The method according to claim 1, wherein the pH value of the buffer solution is 3 to 9.

6.

3. The method according to claim 1, wherein the buffer solution is selected from sucrose aqueous solution, physiological saline, phosphate-buffered physiological saline, HEPES-buffered physiological saline, acetate buffer solution, and carbonate buffer solution.

4. The method according to claim 1, wherein the lipid component selected in step (1) is an anionic lipid, and is selected from the group consisting of dipalmitoylphosphatidylglycerol (DPPG), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), dipalmitoylphosphatidic acid (DPPA), dimyristoylphosphatidylglycerol, distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), distearoylphosphatidic acid (DSPA), and dimyristoylphosphatidic acid.

5. The method according to claim 1, wherein the lipid component selected in step (1) is a cationic lipid, selected from the group consisting of O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride (DC-6-14), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleyl-3-dimethylaminopropane (DODMA), ALC-0315, cKK-E12, SM-102, DLin-KC2-DMA, and heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA).

6. The method according to claim 1, wherein in step (2), the PEG-modified lipid is selected from the group consisting of DMG-PEG and DSPE-PEG.

7. The method according to claim 1, wherein in step (2), the neutral phospholipid is selected from the group consisting of hydrogenated soybean phosphatidylcholine (HSPC), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), palmitoyl oleoyl phosphatidylcholine (POPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, and distearoyl phosphatidylethanolamine.

8. The method according to claim 1, wherein in step (2), the ionized lipid is selected from the group consisting of Dlin-MC3-DMA, DODMA, ALC-0315, SM-102, DLin-KC2-DMA, DODAP, and cKK-E12 (however, the ionized lipid is different from the lipid component).

9. The method according to claim 1, wherein the mixing in step (2) is performed using a microfluidic device.

10. The method according to claim 1, further comprising the step of subjecting the LNP containing the immunogenic peptide formed in step (2) to a dialysis treatment using an aqueous sucrose solution as the dialysate.

11. The method according to claim 1, wherein the immunogenic peptide comprises 3 to 50 amino acids.

12. The method according to claim 1, wherein the immunogenic peptide is a peptide that induces an anti-human TNF-α antibody, an anti-human amyloid-β antibody, an anti-human PD-L1 antibody, an anti-human PD-1 antibody, an anti-mouse PD-1 antibody, or an anti-mouse TNF-α antibody.

13. The method according to claim 1, wherein the immunogenic composition is a pharmaceutical composition.

14. The method according to claim 1, wherein the immunogenic composition is a vaccine preparation.

15. An immunogenic composition comprising LNPs encapsulating immunogenic peptides.

16. The immunogenic composition according to claim 15, wherein the immunogenic peptide comprises 3 to 50 amino acids.

17. The immunogenic composition according to claim 15, wherein the immunogenic peptide is a peptide that induces an anti-human TNF-α antibody, an anti-human amyloid-β antibody, an anti-human PD-L1 antibody, an anti-human PD-1 antibody, an anti-mouse PD-1 antibody, or an anti-mouse TNF-α antibody.

18. The immunogenic composition according to claim 15, wherein the LNP comprises a lipid component, PEG-modified lipid, cholesterol, and neutral phospholipids and / or ionized lipids, depending on the charge of the immunogenic peptide in the buffer, and the lipid component is (a) an anionic lipid when the charge is positive, and (b) a cationic lipid when the charge is negative.

19. The immunogenic composition according to claim 18, wherein the lipid component corresponding to the charge is an anionic lipid, selected from the group consisting of dipalmitoylphosphatidylglycerol (DPPG), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), dipalmitoylphosphatidic acid (DPPA), dimyristoylphosphatidylglycerol, distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), distearoylphosphatidic acid (DSPA), and dimyristoylphosphatidic acid.

20. The immunogenic composition according to claim 18, wherein the lipid component corresponding to the charge is a cationic lipid, selected from the group consisting of O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride (DC-6-14), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleyl-3-dimethylaminopropane (DODMA), ALC-0315, cKK-E12, SM-102, DLin-KC2-DMA, and heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA).

21. The immunogenic composition according to claim 18, wherein the PEG-modified lipid is selected from the group consisting of DMG-PEG and DSPE-PEG.

22. The immunogenic composition according to claim 18, wherein the neutral phospholipid is selected from the group consisting of hydrogenated soybean phosphatidylcholine (HSPC), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), palmitoyl oleoyl phosphatidylcholine (POPC), dimyristril phosphatidylcholine (DMPC), dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, and distearoyl phosphatidylethanolamine.

23. The immunogenic composition according to claim 18, wherein the ionized lipid is selected from the group consisting of Dlin-MC3-DMA, DODMA, ALC-0315, SM-102, DLin-KC2-DMA, DODAP, and cKK-E12 (provided that the ionized lipid and the lipid component are different).

24. The immunogenic composition according to claim 18, comprising the lipid component, the PEG-modified lipid, the cholesterol, and the neutral phospholipid and / or the ionized lipid in a mass ratio of (lipid component: PEG-modified lipid: cholesterol: neutral phospholipid: ionized lipid) of 30-55% by mass: 0.3-3% by mass: 35-50% by mass: 0-30% by mass: 0-50% by mass, wherein the total of the lipid component, the PEG-modified lipid, the cholesterol, and the neutral phospholipid and / or the ionized lipid is 100% by mass.

25. The immunogenic composition according to claim 15, wherein the LNP has an average particle size of 40 to 200 nm.

26. The immunogenic composition according to claim 15, which is a pharmaceutical composition.

27. The immunogenic composition according to claim 15, which is a vaccine preparation.

28. The immunogenic composition according to claim 15, which is administered subcutaneously or intramuscularly.

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