WO2023145749A1 - Adjuvant - Google Patents

Abstract

To provide an adjuvant containing lipid particles having a higher immune response inducing ability (preferably, Th1-type immune response inducing ability). This adjuvant contains lipid particles containing a cationic lipid represented by general formula (1).

Description

Immunostimulant

The present invention relates to immunostimulators and the like.

In the development of vaccines against infectious diseases, there are vaccines that require the combined use of an immunostimulant (hereinafter also referred to as an adjuvant) such as aluminum hydroxide. However, aluminum hydroxide has many problems, such as being inflammatory and being unable to induce a Th1-type immune response at all. For example, when the influenza split vaccine is combined with aluminum hydroxide, no protective ability against influenza viruses of different subtypes is observed, whereas when combined with an adjuvant capable of inducing a Th1-type immune response, strong infection is observed. Show defense. In this way, the induction of Th1-type immune responses is becoming essential in the development of vaccines against viral infections.

Japanese Patent Application Laid-Open No. 2020-090445

Patent Document 1 describes an immunostimulant using 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-containing lipid particles. The present inventor focused on further improving the ability of lipid particles to induce a Th1-type immune response in the course of their research.

An object of the present invention is to provide an immunostimulant containing lipid particles with higher immune response-inducing ability (preferably Th1-type immune response-inducing ability).

As a result of intensive research, the present inventor found that the above problems could be solved by an immunostimulator containing lipid particles containing cationic lipids represented by general formula (1). Based on this finding, the inventors have further studied and completed the present invention. That is, the present invention includes the following aspects.

Section 1. General formula (1):

[In the formula: R ¹ and R ² are the same or different and represent an unsaturated chain hydrocarbon group. R ³ , R ⁴ and R ⁵ are the same or different and represent an alkyl group. p is an integer from 1 to 3; q is an integer from 0 to 3. r represents an integer from 1 to 3; ]
An immunostimulant containing lipid particles containing a cationic lipid represented by

Section 2. Item 1, wherein the unsaturated chain hydrocarbon group has only one double bond and has 10 to 30 carbon atoms.

Section 3. Item 3. The immunostimulant according to item 1 or 2, wherein the p is 1, the q is 0, and the r is 1.

Section 4. Items 1 to 3, wherein the cationic lipid is 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA).

Item 5. Items 1 to 4, wherein the content of the cationic lipid is 10 to 75 mol% relative to 100 mol% of the lipid constituting the lipid particles.

Item 6. Items 1 to 5, wherein the lipid particles contain at least one selected from the group consisting of phospholipids, sterols, and water-soluble polymer-modified lipids.

Item 7. Items 1 to 6, wherein the lipid particles contain a phospholipid, a sterol, and a water-soluble polymer-modified lipid.

Item 8. Items 1 to 7, which do not contain CpG oligodeoxynucleotides and aluminum compounds.

Item A. Items 1 to 8, wherein the lipid particles are lipid nanoparticles.

Item 9. A pharmaceutical containing the immunostimulant according to any one of Items 1 to 8 and A.

Item 10. Item 9, which is a vaccine composition.

Item 11. Item 9 or 10, further comprising an antigen.

Item 12. Items 9 to 11, which are for antiviral or antibacterial purposes.

Item B. Item 12, wherein the antigen comprises at least one selected from the group consisting of influenza vaccine antigens and SARS-CoV-2 vaccine antigens.

According to the present invention, it is possible to provide an immunostimulant containing lipid particles with a higher ability to induce immune responses.

1 shows the evaluation results of the ability to induce antibody production in Test Example 1. FIG. The vertical axis indicates the ELISA measurement value of the antibody shown above each graph. In the horizontal axis, Cont. indicates the case without drug administration, SV indicates the case of antigen alone administration, and others indicate the case of administration of lipid particles together with antigen. 1 shows the evaluation results of T cell response-inducing ability of Test Example 1. FIG. The vertical axis indicates the concentration value measured by ELISA of the molecule shown above each graph. In the horizontal axis, Cont. indicates the case without drug administration, SV indicates the case of antigen alone administration, and others indicate the case of administration of lipid particles together with antigen. 1 shows the evaluation results of the ability to induce antibody production in Test Example 1. FIG. The vertical axis indicates the ELISA measurement value of the antibody shown above each graph. In the horizontal axis, Cont. indicates the case without drug administration, SV indicates the case of antigen alone administration, and others indicate the case of administration of lipid particles together with antigen. 1 shows the evaluation results of T cell response-inducing ability of Test Example 1. FIG. The vertical axis indicates the concentration value measured by ELISA of the molecule shown above each graph. In the horizontal axis, Cont. indicates the case without drug administration, SV indicates the case of antigen alone administration, and others indicate the case of administration of lipid particles together with antigen. 2 shows the evaluation results of the ability to induce antibody production in Test Example 2. FIG. The vertical axis indicates the ELISA measurement value of the antibody shown above each graph. In the horizontal axis, Cont. indicates the case of no drug administration, SV indicates the case of antigen alone administration, and others indicate the case of administration of lipid particles or an existing adjuvant together with the antigen. 2 shows the evaluation results of the ability to induce T cell responses in Test Example 2. FIG. The vertical axis indicates the concentration value measured by ELISA of the molecule shown above each graph. In the horizontal axis, Cont. indicates the case of no drug administration, SV indicates the case of antigen alone administration, and others indicate the case of administration of lipid particles or an existing adjuvant together with the antigen. FIG. 10 shows changes in body weight (upper graph) and measurement results of survival rate (lower graph) in Test Example 2. FIG. In the legend, Cont. indicates the case without drug administration, SV indicates the case of antigen alone administration, and others indicate the case of administration of lipid particles or an existing adjuvant together with antigen. FIG. 2 shows changes in body weight (upper graph) and measurement results of survival rate (lower graph) when DOTMA-containing LNP was administered in Test Example 2. FIG. The legend indicates the antibody administered. 3 shows the evaluation results of the ability to induce antibody production in Test Example 3. FIG. The vertical axis indicates the ELISA measurement value of the antibody shown above each graph. In the horizontal axis, Cont. indicates the case of no drug administration, S indicates the case of antigen alone administration, and others indicate the case of administration of lipid particles or an existing adjuvant together with the antigen. 3 shows the evaluation results of the ability to induce T cell responses in Test Example 3. FIG. The vertical axis indicates the concentration value measured by ELISA of the molecule shown above each graph. In the horizontal axis, Cont. indicates the case of no drug administration, S indicates the case of antigen alone administration, and others indicate the case of administration of lipid particles or an existing adjuvant together with the antigen. 4 shows the measurement results of spleen weight and body weight ratio of spleen weight in Test Example 4. FIG. In the horizontal axis, SV indicates the case of administration of antigen alone, and others indicate the case of administration of lipid particles or an existing adjuvant together with antigen. 4 shows the measurement results of inflammatory cytokines in Test Example 4. FIG. In the legend, SV indicates administration of antigen alone, and others indicate administration of lipid particles or an existing adjuvant together with antigen. The horizontal axis indicates the elapsed time after drug administration. 4 shows the evaluation results of the ability to induce antibody production in Test Example 5. FIG. The vertical axis indicates the ELISA measurement value of the antibody shown above each graph. In the horizontal axis, Cont. indicates the case without drug administration, SV indicates the case of antigen alone administration, and others indicate the case of administration of lipid particles together with antigen. 5 shows the evaluation results of the ability to induce T cell responses in Test Example 5. FIG. The vertical axis indicates the concentration value measured by ELISA of the molecule shown above each graph. In the horizontal axis, Cont. indicates the case without drug administration, SV indicates the case of antigen alone administration, and others indicate the case of administration of lipid particles together with antigen. 1 shows the evaluation results of the ability to induce antibody production in Test Example 6. FIG. The vertical axis indicates the ELISA measurement value of the antibody shown above each graph. In the horizontal axis, Cont. indicates the case of no drug administration, PspA indicates the case of antigen alone administration, and others indicate the case of administration of lipid particles or an existing adjuvant together with the antigen. 1 shows the evaluation results of the ability to induce T cell responses in Test Example 6. FIG. The vertical axis indicates the concentration value measured by ELISA of the molecule shown above each graph. In the horizontal axis, Cont. indicates the case of no drug administration, PspA indicates the case of antigen alone administration, and others indicate the case of administration of lipid particles or an existing adjuvant together with the antigen.

In this specification, the expressions "contain" and "include" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

1. Immunostimulant In one aspect of the present invention, a lipid particle containing a cationic lipid represented by general formula (1) (herein also referred to as "the lipid particle of the present invention") is provided. Contained immunostimulant (in this specification, it may be indicated as "immunostimulator of the present invention"). This will be explained below.

General formula (1) is the formula shown below.

wherein R ¹ and R ² are the same or different and represent an unsaturated chain hydrocarbon group; R ³ , R ⁴ and R ⁵ are the same or different and represent an alkyl group. p is an integer from 1 to 3; q is an integer from 0 to 3. r represents an integer from 1 to 3;

The unsaturated chain hydrocarbon group is a monovalent chain hydrocarbon group containing a double bond, and is not particularly limited as long as it is. The unsaturated chain hydrocarbon group includes both straight-chain and branched-chain, and particularly preferably straight-chain. The number of carbon atoms in the unsaturated chain hydrocarbon group is not particularly limited as long as it is capable of forming lipid particles. 22, more preferably 16-20, particularly preferably 17-19, particularly preferably 18. The number of double bonds contained in the unsaturated chain hydrocarbon group is not particularly limited as long as it is the number capable of forming lipid particles, for example 1 to 6, preferably 1 to 4, more preferably 1 to 3, and further Preferably 1 to 2, particularly preferably 1.

The unsaturated hydrocarbon group preferably includes a group represented by general formula (2).

　s is an integer greater than or equal to 0. s is preferably 2 to 26, more preferably 3 to 20, even more preferably 4 to 14, even more preferably 5 to 10, especially preferably 6 to 8, particularly preferably 7.

　t is an integer greater than or equal to 1. t is preferably 3 to 27, more preferably 4 to 21, even more preferably 5 to 15, even more preferably 6 to 11, especially preferably 7 to 9, particularly preferably 8.

The sum of s and t is, for example, 5 to 27, preferably 7 to 27, more preferably 9 to 23, still more preferably 11 to 19, even more preferably 13 to 17, particularly preferably 14 to 16, particularly preferably is 15.

p is preferably 1 to 2, particularly preferably 1. q is particularly preferably 0. r is preferably 1 to 2, particularly preferably 1. In a preferred embodiment of the invention, p is 1, q is 0 and r is 1.

A preferred embodiment of general formula (1) is general formula (1A).

A particularly preferred embodiment of general formula (1) is general formula (1AA).

In general formula (1AA), two s may be the same or different, and two t may be the same or different.

The cationic lipid represented by general formula (1) is particularly preferably 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA).

The cationic lipid represented by general formula (1) can also be in the form of a salt. Cationic lipids represented by the general formula (1) include, for example, inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; acetate, propionate, tartrate, fumaric acid It can be a salt, an organic acid salt such as maleate, malate, citrate, methanesulfonate, paratoluenesulfonate.

The cationic lipid represented by general formula (1) may be used alone or in combination of two or more.

The lipid particles of the present invention are not particularly limited as long as they contain the cationic lipid represented by general formula (1). The particle size of the lipid particles of the present invention is not particularly limited. The particle size is preferably nano-sized, specifically for example 10-700 nm, 10-400 nm, 10-200 nm, 10-150 nm or less than 100 nm. The lipid particles of the present invention are preferably lipid nanoparticles (LNP: Lipid NanoParticles) having a nano-sized particle size.

The lipid particles of the present invention may contain lipids other than the cationic lipid represented by general formula (1) as lipids constituting the particles, or may contain no other lipids. can be. The content of the cationic lipid represented by general formula (1) is not particularly limited as long as lipid particles can be formed. From the viewpoint of the immune response-inducing action of the lipid particles, the content is, for example, 10 to 100 mol%, preferably 10 to 85 mol%, more preferably 100 mol% of the lipid constituting the lipid particles of the present invention. 10 to 75 mol %, more preferably 20 to 70 mol %, even more preferably 30 to 65 mol %, particularly preferably 40 to 60 mol %, particularly preferably 45 to 55 mol %. In addition, from the viewpoint of the immune response-inducing action of the lipid particles, the content is, for example, 10 mol% or more, preferably 20 mol% or more, more preferably 100 mol% of the lipid constituting the lipid particles of the present invention. It is 30 mol % or more, more preferably 40 mol % or more, and particularly preferably 45 mol % or more. In one aspect of the present invention, the content is preferably 80 mol% or less, more preferably 70 mol% or less, even more preferably 65 mol% or less, still more preferably 60 mol%, from the viewpoint of lipid particle formation. Below, particularly preferably 55 mol % or less, particularly preferably 50 mol % or less.

Other lipids include, but are not particularly limited to, phospholipids, sterols, water-soluble polymer-modified lipids, cationic lipids other than the cationic lipids represented by general formula (1), glycolipids, and the like. In a preferred embodiment of the present invention, the lipid particles of the present invention preferably contain at least one selected from the group consisting of phospholipids, sterols, and water-soluble polymer-modified lipids, all of which (phospholipids, sterols, , and water-soluble polymer-modified lipids).

Specific examples of phospholipids include phosphatidylcholines such as dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilinoleoylphosphatidylcholine, myristoylpalmitoylphosphatidylcholine, myristoylstearoylphosphatidylcholine, palmitoylstearoylphosphatidylcholine; Phosphatidyl such as lauroyl phosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dilinoleoylphosphatidylglycerol, myristoylpalmitoylphosphatidylglycerol, myristoylstearoylphosphatidylglycerol, palmitoylstearoylphosphatidylglycerol glycerol; dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, dilinoleoylphosphatidylethanolamine, myristoylpalmitoylphosphatidylethanolamine, myristoylstearoylphosphatidylethanolamine, phosphatidylethanolamines such as palmitoylstearoylphosphatidylethanolamine; phosphatidylserine; phosphatidic acid; phosphatidylinositol; sphingomyelin; cardiolipin;

Phospholipids may be used alone or in combination of two or more.

When the lipid particles of the present invention contain phospholipids, the content is not particularly limited as long as the lipid particles can be formed. %, preferably 10 to 30 mol %, more preferably 15 to 25 mol %, still more preferably 17 to 23 mol %.

Specific examples of sterols include cholesterol, cholesteryl hemisuccinate, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, phytosterol, phytosterol, stigmasterol, zymosterol, ergosterol, sitosterol, campesterol, and brassicasterol. be. In particular, the sterol has the effect of stabilizing the lipid particle membrane and adjusting the fluidity of the lipid particle membrane, so it is desirable that the sterol is contained as a constituent lipid of the lipid particle membrane.

The sterols may be of one type alone or in combination of two or more types.

When the lipid particles of the present invention contain sterol, the content is not particularly limited as long as the lipid particles can be formed. , preferably 20 to 40 mol %, more preferably 25 to 35 mol %, still more preferably 27 to 33 mol %.

A water-soluble polymer-modified lipid is a lipid to which a water-soluble polymer has been added, and is not particularly limited as long as it is. Examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG) chains. The molecular weight of the water-soluble polymer is not particularly limited, but is, for example, 200-10000, preferably 500-7000, more preferably 500-4000, still more preferably 1000-3000, still more preferably 1500-2500. Lipids modified with water-soluble polymers preferably include amphipathic lipids, more preferably phospholipids.

The water-soluble polymer-modified lipid may be used singly or in combination of two or more.

When the lipid particles of the present invention contain a water-soluble polymer-modified lipid, the content is not particularly limited as long as the lipid particles can be formed. 0 to 10 mol%, preferably 0 to 5 mol%, more preferably 0 to 2 mol%, still more preferably 0 to 1 mol%, even more preferably 0.2 to 1 mol%, particularly preferably 0.3 to 0.7 mol% is.

The lipid particles of the present invention may contain other components (other than the solvent) in addition to the lipid. Examples of other components include various components known to be incorporated into lipid particles, specifically, for example, membrane stabilizers, charged substances, antioxidants, membrane proteins, polyethylene glycol (PEG), antibodies, peptides. , sugar chains, and the like.

Antioxidants can be contained to prevent oxidation of the film, and are used as necessary as a component of the film. Antioxidants used as membrane constituents include, for example, butylated hydroxytoluene, propyl gallate, tocopherols, tocopherol acetate, concentrated mixed tocopherols, vitamin E, ascorbic acid, L-ascorbic stearate, palmitic acid. Examples include ascorbic acid, sodium hydrogen sulfite, sodium sulfite, sodium edetate, erythorbic acid, citric acid and the like.

Membrane proteins can be contained for the purpose of adding functions to membranes or stabilizing membrane structures, and are used as necessary as membrane constituents. Membrane proteins include, for example, surface membrane proteins, integral membrane proteins, albumin, recombinant albumin and the like.

The content of other components is, for example, 10% by mass or less, preferably 5% by mass or less, more preferably 2% by mass or less, and still more preferably 1% by mass or less, relative to 100% by mass of the lipid particles of the present invention. .

The other ingredients may be used singly or in combination of two or more.

The lipid particles of the present invention can exhibit a high ability to induce Th1-type immune responses without relying on other adjuvants. Therefore, from the viewpoint of reducing side effects caused by other adjuvants, the lipid particles of the present invention preferably do not contain other adjuvants or their content is less. Other adjuvants include CpG oligodeoxynucleotide (CpG nucleic acid: CpG ODN: e.g. A type, B type, C type, P type etc.), aluminum compounds (e.g. aluminum hydroxide, aluminum phosphate etc.), oil emulsions and the like. mentioned. The content of other adjuvants is, for example, 10% by mass or less, preferably 5% by mass or less, more preferably 2% by mass or less, more preferably 1% by mass or less, with respect to 100% by mass of the lipid particles of the present invention. Preferably it is 0% by mass.

The lipid particles may be of one type alone or in combination of two or more types.

The lipid particles of the present invention are usually formed in an aqueous solution. Examples of aqueous solutions include various buffers (eg, acetate buffer, phosphate buffer, formate buffer, histidine buffer, etc.).

The lipid particles of the present invention can be produced according to or according to known methods for lipid particles. The lipid particles of the present invention can be preferably produced by a method including a step of mixing a lipid-containing alcohol solution and an aqueous solution (Step 1).

The alcohol that is the solvent for the alcohol solution is not particularly limited as long as it can dissolve lipids. Ethanol is preferably mentioned as the alcohol.

The lipid concentration in the alcohol solution is, for example, 0.1-20% by mass, preferably 0.1-10% by mass, more preferably 0.5-3.0% by mass.

The mixing ratio of the aqueous solution and the alcohol solution (aqueous solution/alcohol solution, v/v) is, for example, 20/1 to 1/1, preferably 4/1 to 2/1.

The mode of mixing is not particularly limited as long as it is a mode in which lipid particles can be formed, but it is usually a mode of vigorously stirring with a vortex or the like. Alternatively, when it is performed in a reaction system using a microchannel, mixing occurs within the reaction system.

Step 1 is usually performed at room temperature or under heating.

The immunostimulant of the present invention may contain other substances besides the lipid particles of the present invention. Other substances include, but are not particularly limited to, adjuvants other than the lipid particles of the present invention.

The content of other substances is, for example, 10% by mass or less, preferably 5% by mass or less, more preferably 2% by mass or less, and still more preferably 1% by mass or less with respect to 100% by mass of the immunostimulant of the present invention. be.

The other substance may be of one type alone or may be a combination of two or more types.

The lipid particles of the present invention can exhibit a high ability to induce Th1-type immune responses without relying on other adjuvants. Therefore, from the viewpoint of reducing side effects caused by other adjuvants, the immunostimulant of the present invention preferably does not contain other adjuvants or contains them in a smaller amount. Other adjuvants are the same as above. The content of other adjuvants is, for example, 10% by mass or less, preferably 5% by mass or less, more preferably 2% by mass or less, more preferably 1% by mass or less, relative to 100% by mass of the immunostimulant of the present invention, Particularly preferably, it is 0% by mass.

The immunostimulant of the present invention can be obtained from the lipid particles of the present invention as they are or by adding the above-described components/substances to the particles.

2. Use The immunostimulant of the present invention can exhibit a higher ability to induce immune responses. The immunostimulant of the present invention is capable of exhibiting higher cell-mediated immunity induction (in particular, Th1-type immune response induction) (for example, IFN-γ induction) and higher antibody induction. can also be demonstrated. In addition, the immunostimulant of the present invention can exert the above-mentioned various inducing abilities while suppressing side reactions such as inflammation as compared with other existing adjuvants. Therefore, the immunostimulant of the present invention can be used in various applications utilizing its immunostimulatory action, such as medicines, reagents, etc. Specifically, it can be used for vaccine compositions, antiviral agents, antibacterial agents, and the like.

The drug of the present invention is not particularly limited as long as it contains the immunostimulant of the present invention, and may further contain other ingredients as necessary. Other ingredients are not particularly limited as long as they are pharmaceutically acceptable ingredients. Other components include additives as well as components having pharmacological action. Examples of additives include various components known to be blended with immunostimulants, and specific examples include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, and binders. , disintegrants, lubricants, thickeners, humectants, coloring agents, fragrances, chelating agents and the like.

The lipid particles of the present invention can exhibit a higher ability to induce immune responses without relying on other adjuvants. Therefore, from the viewpoint of reducing side effects caused by other adjuvants, the drug of the present invention preferably does not contain other adjuvants or contains less adjuvants. Other adjuvants are the same as above. The content of other adjuvants is, for example, 10% by mass or less, preferably 5% by mass or less, more preferably 2% by mass or less, still more preferably 1% by mass or less, particularly preferably 1% by mass or less, relative to 100% by mass of the drug of the present invention. is 0% by mass.

The agent of the present invention preferably contains an antigen. An antigen is any agent (e.g., protein , peptides, polysaccharides, glycoproteins, glycolipids, nucleic acids or combinations thereof). As defined herein, an antigen-induced immune response may be humoral, cellular, or both. An agent is said to be "antigenic" when it is capable of specifically interacting with an antigen-recognition molecule of the immune system, such as an immunoglobulin (antibody) or T-cell antigen receptor (TCR).

In some embodiments, one or more antigens are protein-based antigens. In other embodiments, one or more antigens are peptide-based antigens. In various embodiments, the one or more antigens are selected from the group consisting of cancer antigens, viral antigens, bacterial antigens (bacterial antigens, fungal antigens), and pathogen antigens. A "microbial antigen," as used herein, is an antigen of a microorganism and includes, but is not limited to, infectious viruses, infectious bacteria, infectious parasites, and infectious fungi. Microbial antigens are intact microorganisms as well as natural isolates, fragments or derivatives thereof, as well as the same or similar to naturally occurring microbial antigens, preferably specific to the corresponding microorganism (from which the naturally occurring microbial antigen is derived). It may also be a synthetic compound that induces a potent immune response. In one embodiment the antigen is a cancer antigen. In one embodiment the antigen is a viral antigen. In another embodiment, the antigen is a fungal antigen. In various embodiments, the antigen is a pathogen antigen. In some embodiments, pathogen antigens are synthetic or recombinant antigens.

Antigens preferably include viral antigens. Viruses from which antigens are derived are not particularly limited, but examples include influenza viruses (e.g., type A, type B, etc.), rubella virus, Ebola virus, coronavirus, measles virus, varicella-zoster virus, herpes simplex virus, mumps virus, Enveloped viruses such as arbovirus, respiratory syncytial virus, SARS virus, hepatitis virus (e.g., hepatitis B virus, hepatitis C virus, etc.), yellow fever virus, AIDS virus, rabies virus, hantavirus, dengue virus, Nipah virus, lyssa virus viruses); non-enveloped viruses (viruses without envelopes) such as adenovirus, norovirus, rotavirus, human papillomavirus, poliovirus, enterovirus, coxsackievirus, human parvovirus, encephalomyocarditis virus, rhinovirus, etc. be done. Among these, envelope viruses are preferred, and influenza viruses, coronaviruses and the like are more preferred.

Recently, the SARS coronavirus 2 (SARS-CoV-2) has become a global epidemic, and due to the lack of established treatment technology, it continues to cause enormous damage in many areas, including medical care and the economy. The immunostimulants of the present invention are also effective against SARS-CoV-2. SARS-CoV-2 is not particularly limited, and includes not only known strains such as Wuhan strain, alpha strain, delta strain, lambda strain, and Omicron strain, and their substrains, but also various unknown strains that will be discovered in the future. mentioned.

Antigens preferably include bacterial antigens. Bacteria from which antigens are derived are not particularly limited. acnes, faecalis, difficile, pneumococcus, haemophilus influenzae, moraxella, pneumoniae, coynebacterium, hemolytic streptococcus, pseudomonas aeruginosa, staphylococcus, mycoplasma, candida, and aspergillus.

The mode of use of the drug of the present invention is not particularly limited, and an appropriate mode of use can be adopted according to its type. The agent of the present invention can be used, for example, in vitro (e.g., added to the culture medium of cultured cells) or in vivo (e.g., administered to an animal), depending on its use. can also

The application target of the agent of the present invention is not particularly limited, but examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. Moreover, animal cells etc. are mentioned as a cell. The types of cells are not particularly limited, such as blood cells, hematopoietic stem cells/progenitor cells, gametes (sperm, ovum), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells. , epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells and the like.

The agent of the present invention can be in any dosage form, such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), pills, granules, fine granules, powders, Oral dosage forms such as hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), jelly, and injection preparations (e.g., drip injections (e.g., intravenous drip preparations, etc.), intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (e.g., ointments, poultices, lotions), suppository inhalers, eye drops, ophthalmic ointments, drops Parenteral formulations such as nose drops, ear drops, and liposomes can be used.

The administration route of the agent of the present invention is not particularly limited as long as the desired effect is obtained, and oral administration; enteral administration such as tube feeding and enema administration; intravenous administration, transarterial administration, intramuscular administration, Examples include parenteral administration such as intracardiac administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and nasal administration.

The content of the immunostimulant of the present invention in the drug of the present invention depends on the mode of use, the subject of application, the state of the subject of application, etc., and is not limited, but is, for example, 0.0001 to 100% by weight, preferably It can be from 0.001 to 50% by weight.

When the agent of the present invention is administered to an animal, the dosage is not particularly limited as long as it is an effective amount that exhibits efficacy. 0.1 to 1000 mg/kg body weight per day, preferably 0.5 to 500 mg/kg body weight per day, and 0.01 to 100 mg/kg body weight per day, preferably 0.05 to 50 mg/day for parenteral administration. kg body weight. The above dosage can be adjusted appropriately depending on age, disease state, symptoms and the like.

The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

Abbreviations Abbreviations used in the examples and their official names are shown below.
DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane
DPPC: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
NG-DOPE: N-glutaryl-L-α-dioleoylphosphatidylethanolamine
DODAP: 1,2-dioleoyloxy-3-dimethylaminopropane
DOTMA: 1,2-di-O-octadecenyl-3-trimethylammonium propane
DSPE-PEG-Ome: N-(methylpolyoxyethyleneoxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG chain molecular weight: 2000)

Production example 1. Production of lipid particles 1
An ethanol solution (lipid concentration: 1.0% by mass) containing lipids with the composition and molar ratio shown in Table 1 and 25 mM Sodium Acetate (pH 4.0) were prepared using NanoAssemblr (manufactured by Precision NanoSystems) at a ratio of 1:3 (ethanol solution : 25 mM sodium acetate aqueous solution (pH 4.0, mixing ratio), mixed at 15 mL / min to form lipid nanoparticles, dialyzed (5% glucose aqueous solution), and each lipid particle with the name shown in Table 1 (hereinafter referred to as It is sometimes indicated as “LNP”.) was obtained. The lipid particles of this production example were used in Test Examples 1 to 4 below.

Test example 1. Assessment of immunostimulatory capacity1
<Experimental animals, reagents>
- Mice: 6-week-old male C57BL6 mice - Antigen: A split vaccine (SV) derived from H1N1 influenza A virus (strain: A/California/7/2009 (Cal7)) was administered at 0.5 μg/mouse.
- Lipid nanoparticles (LNP): Each LNP listed in Table 1 was administered at 100 µg/mouse.

<Method (Evaluation of Ability to Induce Antibody Production: Figures 1 and 3)>
Mice were immunized subcutaneously on days 0 and 21 after SV alone or SV mixed with various LNPs. On day 28, blood was collected and plasma SV-specific total IgG, IgG1, IgG2b, and IgG2c were evaluated by ELISA.

<Method (Evaluation of ability to induce T cell responses: Figures 2 and 4)>
Immune cells were harvested from the spleens on day 31 and cultured with SV (10 μg/mL). After 1 or 3 days, the culture supernatant was collected and IL-2, IFN-γ and IL-13 levels were measured by ELISA.

<Results>
The results are shown in Figures 1-4.

　Figures 1 and 2 used LNPs created by varying the content of DOTAP, which is used as a cationic lipid. In addition, instead of DOTAP, LNP using the anionic lipid NG-DOPE was used. In addition, it is clarified that the surface charge of the LNP used becomes close to neutral by decreasing the amount of DOTAP, and the negative charge increases by increasing the amount of NG-DOPE. As a result, it was found that when SV was used as an antigen, the higher the DOTAP content, the higher the antibody-producing ability. For T cell responses, IFN-γ production, an indicator of Th1 immunity, was observed in some LNPs with DOTAP and NG-DOPE. IL-13 production, an indicator of Th2 immunity, was observed in LNPs with NG-DOPE.

In Figures 3 and 4, DOTAP was used as the cationic lipid, and LNPs with varying contents of polyethylene glycol (PEG)-modified lipids were used. Also, instead of DOTAP, LNPs using DODAP or DOTMA, which are cationic lipids, were used. A downward trend was observed for antibody production and T-cell responses with increasing PEG-modified lipid content. DODAP-containing LNPs also tended to induce lower immune responses than DOTAP-containing LNPs. On the other hand, increased antibody production (particularly IgG2c) was observed for DOTMA-containing LNPs compared to DOTAP-containing LNPs. Furthermore, both Th1 and Th2 immunity were found to be strongly induced by DOTMA-containing LNP. These results demonstrate that DOTMA-containing LNPs can strongly induce antibody production and T cell responses.

Test example 2. Comparison with existing adjuvants <laboratory animals, reagents>
- Mice: 6-week-old male C57BL6 mice - Antigen: A split vaccine (SV) derived from H1N1 influenza A virus (strain: A/California/7/2009 (Cal7)) was administered at 0.5 μg/mouse.
• Adjuvant: Aluminum hydroxide (alum) was administered at 50 μg/mouse, or B-type CpG nucleic acid (CpG K3) was administered at 50 μg/mouse.
• Lipid nanoparticles (LNPs): DOTAP-containing LNPs were administered at 100 μg/mouse, or DOTMA-containing LNPs were administered at 100 μg/mouse.

<Method (Evaluation of Ability to Induce Antibody Production: FIG. 5)>
Mice were immunized subcutaneously on days 0 and 21 after SV alone or mixed with alum, CpG K3, DOTAP-containing LNPs or DOTMA-containing LNPs. On day 28, blood was collected and plasma SV-specific total IgG, IgG1, IgG2b, and IgG2c were evaluated by ELISA.

<Method (Evaluation of ability to induce T cell response: Fig. 6)>
Immune cells were harvested from the spleens on day 31 and cultured with SV (10 μg/mL). After 1 or 3 days, the culture supernatant was collected and IL-2, IFN-γ and IL-13 levels were measured by ELISA.

<Method (body weight change, survival rate: Fig. 7)>
On the 31st day, the mice were nasally infected with H1N1 influenza A virus (strain: A/Puerto Rico/8/1934 (PR8)) different from the vaccine strain, and body weight changes and survival rates were measured over the course of the day.

<Method (body weight change, survival rate: Fig. 8)>
We examined the role of Th1 immunity in protecting against infection in the DOTMA-containing LNP combination group. Anti-IFN-γ antibody or isotype control antibody was administered intraperitoneally on day 30 (one day before infection). After that, on the 31st day, they were infected nasally with H1N1 influenza A virus (strain: A/Puerto Rico/8/34 (PR8)) different from the vaccine strain, and body weight changes and survival rate were measured over time. .

<Results>
The results are shown in Figures 5-8.

In Figure 5, together with DOTAP-containing LNP, which is widely used as LNP, we verified the immune-inducing effect of the originally discovered DOTMA-containing LNP. All of the alum, CpG K3, DOTAP-containing LNPs, and DOTMA-containing LNPs were used in combination with SV to increase the antibody titer. Especially IgG1 was improved with alum, and IgG2c was improved with CpG nucleic acid. DOTMA-containing LNPs showed high antibody titers for all of IgG1, IgG2b, and IgG2c.

When T cell responses were evaluated in Figure 6, stronger production of IFN-γ, an indicator of Th1 immunity, was observed in the DOTMA-containing LNP group than in the CpG K3 group. IL-13, an index of Th2 immunity, was produced in alum.

In Fig. 7, when heterozygous strains were infected after vaccination, significant weight loss and decreased survival rate were observed in the SV alone, alum combined group, and DOTAP-containing LNP combined group. On the other hand, in the CpG K3 combination group and the DOTMA-containing LNP combination group, no weight loss was observed, and survival was observed in all mice. 

In Figure 8, a decrease in body weight and survival rate was observed in the anti-IFN-γ antibody group compared to the isotype control antibody group. That is, it was shown that strong Th1 immunity induction in the DOTMA-containing LNP group observed in FIG. 7 is important for infection protection against heterozygous strains.

Test example 3. Evaluation of immunostimulatory ability when using SARS-CoV-2 antigen <laboratory animal, reagent>
・Mice: 6-week-old male BALB/c mice ・Antigen: Recombinant S protein derived from SARS-CoV-2 was produced in mammalian cells. S protein was administered at 1 μg/mouse.
• Adjuvant: Aluminum hydroxide (alum) was administered at 50 μg/mouse, or B-type CpG nucleic acid (CpG K3) was administered at 50 μg/mouse.
• Lipid nanoparticles (LNPs): DOTAP-containing LNPs were administered at 100 μg/mouse, or DOTMA-containing LNPs were administered at 100 μg/mouse.

<Method (Evaluation of Ability to Induce Antibody Production: FIG. 9)>
Mice were immunized subcutaneously on days 0 and 21 after S protein alone or mixed with alum, CpG K3, DOTAP-containing LNPs or DOTMA-containing LNPs. Blood was collected on day 28, and SV-specific total IgG, IgG1, IgG2a, and IgG2b in plasma were evaluated by ELISA.

<Method (Evaluation of ability to induce T cell response: Fig. 10)>
Immune cells were harvested from the spleen on day 31 and cultured with S protein (10 μg/mL). One day later, the culture supernatant was collected and the IFN-γ level was measured by ELISA.

<Results>
The results are shown in Figures 9-10.

　In Figure 9, an improvement in antibody titer was observed in all of alum, CpG K3, DOTAP-containing LNP, and DOTMA-containing LNP.

In Figure 10, when T cell responses were evaluated, stronger production of IFN-γ, an indicator of Th1 immunity, was observed in the DOTMA-containing LNP group than in the CpG K3 group. That is, DOTMA-containing LNP was found to be capable of strongly inducing Th1 immunity not only in SV but also in S protein.

Test example 4. Evaluation of side reactions <laboratory animals, reagents>
- Mice: 6-week-old male C57BL6 mice - Antigen: A split vaccine (SV) derived from H1N1 influenza A virus (strain: A/California/7/2009 (Cal7)) was administered at 0.5 μg/mouse.
• Adjuvant: B-type CpG nucleic acid (CpG K3) was administered at 50 μg/mouse.
- Lipid nanoparticles (LNP): DOTMA-containing LNP was administered at 100 μg/mouse.

<Method (measurement of spleen weight: Fig. 11)>
After mixing SV with CpG K3 or DOTMA-containing LNPs, mice were immunized subcutaneously twice every two days. Spleen weights were assessed 6 days after the first dose.

<Method (measurement of inflammatory cytokine: FIG. 12)>
Mice were immunized subcutaneously with SV alone or after SV was mixed with CpG K3 or DOTMA-containing LNPs. Blood inflammatory cytokine level (IL-12 p40 level) was measured daily by ELISA.

<Results>
The results are shown in Figures 11-12.

In FIG. 11, a significant increase in spleen weight was observed in the CpG K3 combination group, but not in the DOTMA-containing LNP combination group.

In Figure 12, a significant increase in IL-12 p40 was observed at 72 hours and 120 hours after administration in the CpG K3 combination group, while it was not observed in the DOTMA-containing LNP combination group.

From the above results, it was shown that CpG K3 at this dose may induce systemic side effects, while it was shown that DOTMA-containing LNP can be used safely.

Production example 2. Production of lipid particles 2
An ethanol solution (lipid concentration 1.0% by mass) containing lipids in the composition and molar ratio shown in Table 2 and 25 mM Sodium Acetate (pH 4.0) were prepared using NanoAssemblr (manufactured by Precision NanoSystems) at a ratio of 1:3 (ethanol solution: 25 mM sodium acetate aqueous solution (pH 4.0, mixing ratio), mixed at 15 mL/min to form lipid nanoparticles, and dialyzed (5% glucose aqueous solution) to obtain each lipid particle with the name shown in Table 2. DOTMA50 in Table 2 has the same composition as DOTMA in Table 1. The lipid particles of this production example were used in Test Examples 5 and 6 below.

Test example 5. Evaluation of immunostimulatory capacity 2
<Experimental animals, reagents>
- Mice: 6-week-old male C57BL6 mice - Antigen: A split vaccine (SV) derived from H1N1 influenza A virus (strain: A/California/7/2009 (Cal7)) was administered at 0.5 μg/mouse.
- Lipid nanoparticles (LNP): DOTMA-containing LNP was administered at 100 μg/mouse.

<Method (Evaluation of Ability to Induce Antibody Production: FIG. 13)>
Mice were immunized subcutaneously on days 0 and 21 after SV alone or SV mixed with DOTMA-containing LNPs. On day 28, blood was collected and plasma SV-specific total IgG, IgG1, IgG2b, and IgG2c were evaluated by ELISA.

<Method (Evaluation of ability to induce T cell response: Fig. 14)>
Immune cells were harvested from the spleens on day 31 and cultured with SV (10 μg/mL). After 1 or 3 days, the culture supernatant was collected and IL-2, IFN-γ and IL-13 levels were measured by ELISA.

<Results>
The results are shown in Figures 13-14.

In Fig. 13, it was revealed that DOTMA50 had the highest antibody titers. In particular, IgG2b and IgG2c were remarkable.

In FIG. 14, when T cell responses were evaluated, all cytokines were highest at DOTMA50, and cytokine production decreased as the DOTMA content decreased. Especially for IFN-γ production, DOTMA50 was the highest, indicating that DOTMA is essential for Th1 immune response.

Test example 6. Evaluation of immunostimulatory ability when using pneumococcal antigen <laboratory animal, reagent>
・Mouse: 6-week-old male C57BL6 mouse ・Antigen: Recombinant PspA protein derived from Streptococcus pneumoniae was produced in Escherichia coli. PspA protein was administered at 1 μg/mouse.
・Adjuvant: Aluminum hydroxide (alum) was administered at 50 μg/mouse, and B-type CpG nucleic acid (CpG K3) was administered at 50 μg/mouse.
- Lipid nanoparticles (LNP): DOTMA-containing LNP was administered at 100 μg/mouse.

<Method (Evaluation of Ability to Induce Antibody Production: FIG. 15)>
Mice were immunized subcutaneously on days 0 and 21 after PspA protein alone or mixed with LNP containing alum, CpG K3 and DOTMA. Blood was collected on the 28th day, and PspA-specific total IgG in plasma was evaluated by ELISA.

<Method (Evaluation of ability to induce T cell response: Fig. 16)>
Immune cells were harvested from the spleens on day 31 and cultured with PspA protein (10 μg/mL). One day later, the culture supernatant was collected and the IFN-γ level was measured by ELISA.

<Results>
The results are shown in Figures 15-16.

In FIG. 15, an improvement in antibody titer was observed in all of alum, CpG K3, and DOTMA-containing LNPs. Especially strong antibody titers were observed in DOTMA-containing LNPs.

In Figure 16, when T cell responses were evaluated, stronger production of IFN-γ, an indicator of Th1 immunity, was observed in the DOTMA-containing LNP group than in the CpG K3 group. That is, DOTMA-containing LNP was found to be capable of strongly inducing Th1 immunity not only in the SV and S proteins but also in the PspA protein.

Claims (12)

1. General formula (1):
   

   

   [In the formula: R ¹ and R ² are the same or different and represent an unsaturated chain hydrocarbon group. R ³ , R ⁴ and R ⁵ are the same or different and represent an alkyl group. p is an integer from 1 to 3; q is an integer from 0 to 3. r represents an integer from 1 to 3; ]
   An immunostimulant containing lipid particles containing a cationic lipid represented by
2. The immunostimulant according to claim 1, wherein the unsaturated chain hydrocarbon group has only one double bond and has 10 to 30 carbon atoms.
3. 3. The immunostimulator according to claim 1 or 2, wherein said p is 1, said q is 0 and said r is 1.
4. The immunostimulant according to any one of claims 1 to 3, wherein said cationic lipid is 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA).
5. The immunostimulant according to any one of claims 1 to 4, wherein the content of said cationic lipid is 10 to 75 mol% with respect to 100 mol% of lipid constituting said lipid particles.
6. The immunostimulant according to any one of claims 1 to 5, wherein the lipid particles contain at least one selected from the group consisting of phospholipids, sterols, and water-soluble polymer-modified lipids.
7. The immunostimulant according to any one of claims 1 to 6, wherein the lipid particles contain phospholipids, sterols, and water-soluble polymer-modified lipids.
8. The immunostimulant according to any one of claims 1 to 7, which does not contain CpG oligodeoxynucleotides and aluminum compounds.
9. A medicament containing the immunostimulant according to any one of claims 1 to 8.
10. The medicament according to claim 9, which is a vaccine composition.
11. The medicament according to claim 9 or 10, further comprising an antigen.
12. The medicament according to any one of claims 9 to 11, which is for antiviral or antibacterial use.

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