Vaccine adjuvant lipid compounds based on Toll-like receptor agonists, and their use

Abstract

This invention provides vaccine adjuvant lipid compounds based on Toll-like receptor agonists and their uses. [Solution] Specifically, the present invention discloses a compound represented by formula (I), its stereoisomer, its N-oxide compound, its solvate, or a pharmaceutically acceptable salt thereof. The mRNA-TLP composition produced with the adjuvant lipid compound of the present invention can significantly improve the targeting of mouse spleen antigen-presenting cells and significantly inhibit tumor growth. TIFF2026109612000078.tif38170

Description

[Technical Field]

[0001] This invention relates to vaccine adjuvant lipid compounds based on Toll-like receptor agonists, and to the use thereof. [Background technology]

[0002] In recent years, messenger RNA (mRNA)-based tumor immunotherapy has attracted widespread attention due to its significant clinical potential. Compared to DNA vaccines, mRNA can induce transient expression of tumor antigens while simultaneously avoiding the possibility of insertional mutations. Furthermore, mRNA can enhance therapeutic effects by inducing stronger humoral and cellular responses. Compared to conventional protein-based vaccines, mRNA vaccines offer many advantages, including biocompatibility, non-toxicity, simplicity, and scalable manufacturing processes. Encouraged by the advantages of mRNA cancer vaccines, more than 20 mRNA-based immunotherapies have demonstrated antitumor efficacy in preclinical and clinical studies.

[0003] To reduce high innate immunogenicity and improve tolerance and translation efficiency, modified nucleosides such as 1-methylpsoiduridine (m1ψ) have been incorporated into in vitro transcribed mRNA sequences. However, a problem with such modifications is that they can impair innate immunogenicity. Immunogenicity is crucial for the activation of dendritic cells (DCs, the primary antigen-presenting cells).

[0004] Toll-like receptors (TLRs), a family of transmembrane proteins, are innate immune receptors that play a role in directly or indirectly detecting pathogen-associated molecular patterns (PAMPs). They activate DCs cells, triggering a series of biosynthetic reactions, including the production of specific cytokines (e.g., tumor necrosis factor-α, TNF-α), which enhances the expression of costimulatory molecules and improves antigen-presenting ability. These molecular mechanisms are crucial for activating both innate and adaptive immune responses and can effectively stimulate the transition from innate to adaptive immunity. Increasing research is being conducted to incorporate lipid-modified TLRs into mRNA delivery systems to enhance the scale and duration of adaptive immune responses. Therefore, the development of a new generation of vaccine adjuvant lipids based on Toll-like receptor 7 and 8 agonists is of great significance for improving mRNA oncology. [Overview of the project]

[0005] The technical problem that this invention aims to solve is to provide a vaccine adjuvant lipid compound based on a Toll-like receptor agonist and its use, in order to overcome the limitations of conventional vaccine adjuvant lipid compounds based on Toll-like receptors 7 and 8, which have a single structure and insufficient targeting of the prepared mRNA composition to antigen-presenting cells. The mRNA-TLP composition prepared with the adjuvant lipid compound of this invention can significantly improve the targeting of mouse spleen antigen-presenting cells and significantly inhibit tumor growth.

[0006] The present invention provides a compound represented by formula (I), its stereoisomer, its N-oxide compound, its solvate, or a pharmaceutically acceptable salt thereof.

[0007] [ka]

[0008] L1 is C 1-10 Alkylene or C 1-4 Alkylene-C 6-10Aryl-C 1-4 is an alkylene, M1 is -NH-, -OC(O)HN-, -C(O)O- or -O-, L2 is C 1-8 alkylene, -R 3a C(O)OR 4a -, -R 3b OR 4b -,

[0009] [Chemical formula] where R 3a , R 3b , R 3c , R 3d , R 4a , R 4b , R 4c , R 4d and R5 are independently C 1-8 alkylene,

[0010] R1 is C 1-5 alkyl or C 1-6 alkyl substituted by C 1-5 alkoxy, R2 is,

[0011] [Chemical formula] where L3 and L4 are independently C

[0012] alkylene, 1-7 where R6 and R7 are independently C alkyl, 7-28 where R8 is C alkylene, R9 and R 1-7 are independently C 10 alkyl or C 10-20 alkynyl. 10-20 In some embodiments, L1 is C

[0013] linear alkylene, L2 is C 1-6 linear alkylene, 1-6Linear alkylene C(O)OC 1-4 Linear alkylenes a or

[0014] [ka] And the a-terminus is R 2 It is connected to R 3d , R 4d And R5 stands independently C 1-4 It is a linear alkylene,

[0015] Alternatively, L1 is C 1-4 Linear alkylene-C 6-10 Aryl-C 1-4 It is a linear alkylene, and L2 is C 1-4 Linear alkylene or C 1-6 Linear alkylene C(O)OC 1-4 Linear alkylenes a And the a-terminus is R 2 It is connected to.

[0016] In some embodiments, R1 is C 1-5 It is a linear alkyl group. In some embodiments, L3 is C 1-4 It is a linear alkylene. In some embodiments, L4 is C 4-6 It is a linear alkylene.

[0017] In some embodiments, R6 is C 8-12 It is a linear alkyl group. In some embodiments, R7 is C 16-20 It is a branched-chain alkyl group. In some embodiments, R8 is C 1-4 It is a linear alkylene.

[0018] In some embodiments, R9 and R 10 C is independent 16-20 It is Alkenil. In some embodiments, L1 is -(CH2)4-, -CH2C((CH3)2)-b or

[0019] [ka] The b-terminus is ligated to M1.

[0020] In some embodiments, M1 is -NH- or -OC(O)HN-, and its nitrogen terminus is connected to L2. In some embodiments, M1 is -NH-.

[0021] In some embodiments, L2 is -(CH2)2-, -(CH2)3C(O)O(CH2)2- a ,-(CH2)5C(O)O(CH2)2- a ,

[0022] [ka] Therefore, the a-terminus is ligated to R2.

[0023] In some embodiments, L1 is -(CH2)4- and L2 is -(CH2)5C(O)O(CH2)2- a or

[0024] [ka] And the a-terminus is connected to R2,

[0025] Alternatively, L1 is

[0026] [ka] Therefore, L2 is -(CH2)2-, -(CH2)3C(O)O(CH2)2- a Or -(CH2)5C(O)O(CH2)2- a Therefore, the a-terminus is ligated to R2.

[0027] In some embodiments, R1 is -(CH2)3CH3 or -CH2OCH2CH3. In some embodiments, R2 is

[0028] [ka] That is the case.

[0029] In some embodiments, the compound represented by (I) is one of the compounds of the following formulas.

[0030] [ka] TIFF2026109612000011.tif101170

[0031] The present invention provides a lipid composition comprising substance Z, wherein substance Z is a compound represented by the above formula (I), its stereoisomer, its N-oxide compound, its solvate, or a pharmaceutically acceptable salt thereof.

[0032] In some embodiments, the molar percentage of substance Z in the lipid composition is 2.5 to 20%. In some embodiments, the molar percentage of substance Z in the lipid composition is 5 to 15%.

[0033] In some embodiments, the molar percentage of substance Z in the lipid composition is 10%. In some embodiments, the lipid composition comprises substance Z, a permanent anionic lipid, a permanent cationic lipid, and a neutral lipid. The molar percentage ratio of substance Z, the permanent anionic lipid, the permanent cationic lipid, and the neutral lipid is preferably (2.5-20):(10-33):(20-60):(20-40).

[0034] In some embodiments, the permanent anionic lipid is selected from one or at least two combinations of the group consisting of 2-acetamidoethyl((R)-2,3-bis(oleoyloxy)propyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylthioureido)ethyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylureido)ethyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-propylureido)ethyl) phosphate, and (R)-2,3-bis(oleoyloxy)propyl-(2-(3-butylureido)ethyl) phosphate and salts thereof.

[0035] In some embodiments, the permanent anionic lipid is 2-acetylaminoethyl((R)-2,3-bis(oleoyloxy)propyl) sodium phosphate.

[0036] In some embodiments, the molar percentage ratio of the permanent anionic lipid in the lipid mixture is 25%. In some embodiments, the permanent cationic lipid is selected from one or at least two combinations of the group consisting of 1,2-dioctadeceneoxy-3-methylammoniumpropane, (2,3-dioleyloxypropyl)trimethylammonium, and salts thereof.

[0037] In some embodiments, the permanent cationic lipid is 1,2-dioctadeceneoxy-3-methylammoniumpropane (chloride salt). In some embodiments, the molar percentage ratio of the permanent cationic lipid in the lipid mixture is 30-47.5%, for example, 35%, 40%, or 45%.

[0038] In some embodiments, the neutral lipid is selected from one or at least two combinations of the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphocholine, and salts thereof.

[0039] In some embodiments, the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine. In some embodiments, the molar percentage ratio of the neutral lipid in the lipid mixture is 25%.

[0040] In some embodiments, the lipid mixture comprises substance Z, 2-acetamidoethyl((R)-2,3-bis(oleoyloxy)propyl) phosphate, 1,2-dioctadecene-3-methylammoniumpropane (chloride salt), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, wherein the molar percentages of substance Z, 2-acetamidoethyl((R)-2,3-bis(oleoyloxy)propyl) phosphate, 1,2-dioctadecene-3-methylammoniumpropane (chloride salt), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine are (2.5~20):25:(30~47.5):25, preferably (5~15):25:(35~45):25, and more preferably 10:25:40:25.

[0041] In some embodiments, the lipid composition comprises substance Z, cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids. The ratio of the mole percentages of substance Z, cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids is preferably (2.5-20):(25-75):(5-25):(15-65):(0.5-10).

[0042] In some embodiments, the cationic lipid is selected from any one or at least two combinations of the group consisting of the compounds (1) to (7) below. (1) A compound represented by formula (II), its stereoisomer, its N-oxide compound, its solvate, or a pharmaceutically acceptable salt thereof, where G1 is C 1~6 It is alkylene, and G2 is C 2~8 It is alkylene, and G3 is C 1~3 It is alkylene, and L1 is C 6~15 It is a linear alkyl group, and L2 is C 12~25 A branched-chain alkyl compound,

[0043] [ka]

[0044] (2) A compound represented by formula (III), its stereoisomer, its N-oxide compound, its solvate, or a pharmaceutically acceptable salt thereof, where G1 is C 2~8 It is alkylene, and G2 is C 2~8 It is an alkylene, L1 is -C(O)O- or -OC(O)-, L2 is -C(O)O- or -OC(O)-, and R1 is C 6~25 It is a linear or branched alkyl group, and R2 is C 6~25 A compound that is linear or branched alkyl, where G3 is HO(CH2)2- or HO(CH2)3-, G4 is HO(CH2)2- or HO(CH2)3-, and L is (CH2)2-, -(CH2)3-, or -(CH2)4-.

[0045] [ka]

[0046] (3) A compound represented by formula (IV), its stereoisomer, its N-oxide compound, its solvate, or a pharmaceutically acceptable salt thereof, where G1 is C 1~6 It is alkylene, and G2 is C 2~8 It is alkylene, and R1 is C 6~20 It is a linear or branched alkyl group, and R2 is C 12~25A branched alkyl compound in which G3 is HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2-, or CH3CH2NH(CH2)2-.

[0047] [ka]

[0048] (4) A compound represented by formula (V), its stereoisomer, its N-oxide compound, its solvate, or a pharmaceutically acceptable salt thereof, where G1 is C 1~8 It is alkylene, and G2 is C 2~8 It is alkylene, and R1 is C 6~25 It is a linear or branched alkyl group, and R2 is C 12~25 A linear or branched alkyl compound, where G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, and R3 is -CH3, -CH2CH3, or -CH2CH2OH.

[0049] [ka]

[0050] (5) A compound represented by formula (VI), its stereoisomer, its N-oxide compound, its solvate, or a pharmaceutically acceptable salt thereof, where G 1 and G 2 Each is independently C 6~10 It is alkylene, G 3 is C 1~12 It is alkylene, R 1 and R 2 Each is independently C 6~24 Alkyl or C 6~24 It is an alkenyl, R 3 is OR 5, N, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 where R 4 is C 1~12 hydrocarbyl, and R 5 is H or C 1~6 hydrocarbyl, a compound

[0051] [Chemical formula]

[0052] (6) A compound represented by formula (VII), its stereoisomer, its N-oxide compound, its solvate, or its pharmaceutically acceptable salt, where R4 is -(CH2) n Q or -(CH2) n CHQR, where Q is -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R or heterocyclyl, n is 1, 2 or 3, and R is C 1-8 alkyl, and X is H or C 1-8 alkyl, a compound

[0053] [Chemical formula]

[0054] (7) A compound represented by formula (VIII), its stereoisomer, its N-oxide compound, its solvate, or its pharmaceutically acceptable salt.

[0055] [Chemical formula]

[0056] In some embodiments, the cationic lipid is selected from one or at least two combinations of the group consisting of YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA.

[0057] [ka]

[0058] In some embodiments, the cationic lipid is YK-009. In some embodiments, the molar percentage of the cationic lipid in the lipid composition is 30-47.5%, for example, 35%, 40%, or 45%.

[0059] In some embodiments, the neutral lipid is selected from one or at least two combinations of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0060] In some embodiments, the neutral lipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine The following are selected from the group consisting of lysophosphate (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE): one or at least two combinations thereof.

[0061] In some embodiments, the neutral lipid is DOPE and / or DSPC, preferably DSPC. In some embodiments, the molar percentage ratio of the neutral lipid in the lipid mixture is 10%.

[0062] In some embodiments, the structural lipid is selected from one or at least two combinations of the group consisting of cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

[0063] In some embodiments, the structural lipid is cholesterol. In some embodiments, the molar percentage of the structural lipid in the lipid composition is 38.5%.

[0064] In some embodiments, the polymer-conjugated lipid is selected from one or at least two combinations of the group consisting of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), and methoxypoly(ethylene glycol) ditetradecylacetamide (ALC-0159).

[0065] In some embodiments, the polymer-conjugated lipid is DMG-PEG2000. In some embodiments, the molar percentage of the polymer-conjugated lipid in the lipid composition is 1.5%.

[0066] In some embodiments, the lipid composition comprises substance Z, YK-009, DSPC, cholesterol, and DMG-PEG2000, wherein the molar percentages of substance Z, YK-009, DSPC, cholesterol, and DMG-PEG2000 are preferably (2.5~20):(30~47.5):10:38.5:1.5, and more preferably 10:40:10:38.5:1.5.

[0067] In some embodiments, the lipid composition further comprises one or more cell-penetrating peptides. The present invention relates to a therapeutic and / or prophylactic agent comprising (A) one or at least two combinations of nucleic acid molecules, small molecule compounds, polypeptides, or proteins, (B) Provides a pharmaceutical composition comprising the above-mentioned lipid composition.

[0068] In some embodiments, the amount of the therapeutic agent and / or prophylactic agent used and the lipid composition is such that the charge ratio of positive charge to negative charge in the pharmaceutical composition is 1:(2~5), for example, 1:2.

[0069] In some embodiments, the mass ratio of the lipid composition to the therapeutic or prophylactic agent is (12.5-25):1. In some embodiments, the mass ratio of the lipid composition to the therapeutic or prophylactic agent is 15:1.

[0070] In some embodiments, the pharmaceutical composition is used to deliver the therapeutic and / or prophylactic agent to antigen-presenting cells in a target organ or tissue. In some embodiments, the target organ or tissue is selected from one or at least two combinations of the group consisting of the spleen, liver, lymph, muscle, and lung, preferably the spleen or lymph.

[0071] In some embodiments, the antigen-presenting cells are selected from one or at least two combinations of the group consisting of B cells, NK cells, cDC cells, pDC cells, and macrophages.

[0072] In some embodiments, the therapeutic and / or prophylactic agent is a nucleic acid molecule capable of encoding one or more antigens. In some embodiments, the antigen is a disease-related antigen, or the nucleic acid molecule or antigen can induce an immune response against a disease-related antigen or a cell expressing a disease-related antigen.

[0073] In some embodiments, the nucleic acid molecule is RNA encoding one or more antigens. In some embodiments, the pharmaceutical composition further comprises at least one auxiliary component, which may be a pharmaceutical carrier, diluent, or excipient.

[0074] In some embodiments, the pharmaceutical composition further comprises one or more hydrophobic small molecules, permeability-enhancing molecules, carbohydrates, polymers, surface modifiers, functionalized lipids, or cytokines.

[0075] The present invention also provides the use of substance Z, the lipid composition, or the pharmaceutical composition in the production of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides, or protein drugs.

[0076] The present invention also provides the use of substance Z, the lipid composition described above, or the pharmaceutical composition described above in the manufacture of a pharmacopoeia for treating a disease or symptom. The disease or symptom is preferably characterized by the activity of a dysfunctional or abnormal protein or polypeptide.

[0077] In some embodiments, the disease or symptom is selected from one or at least two combinations of the group consisting of infectious diseases, tumors, proliferative disorders, genetic disorders, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal diseases, and metabolic diseases. In some embodiments, the infection is a disease caused by a coronavirus, influenza virus or HIV virus, childhood pneumonia, Rift Valley fever, yellow fever, rabies, or multiple herpes.

[0078] In some forms, the tumors are breast cancer, ovarian cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer, lymphoma, colon cancer, liver cancer, melanoma, bladder cancer, cervical cancer, or prostate cancer.

[0079] In some embodiments, the pharmacopoeia is used to treat a disease or condition in a mammal in need. The mammal is selected from one or at least two combinations of the group consisting of humans, non-human primates, companion animals, exotic species, domesticated animals, and food animals.

[0080] In some embodiments, the route of administration of the drug is intravenous, intramuscular, intradermal, subcutaneous, intranasal, or inhalation. In some embodiments, the route of administration of the drug is intravenous or intramuscular.

[0081] In some preferred embodiments, the dosage of the pharmaceutical agent is 0.001 to 10 mg / kg. The adjuvant lipid compounds and lipid compositions provided by the present invention can be used to encapsulate nucleic acids (e.g., mRNA, etc.) to form corresponding nucleic acid drugs.

[0082] Definition of Terms All publications and patents referenced herein are incorporated herein by reference in their entirety. In the event of any conflict between any usage or terminology used in any of the incorporated publications or patents and any usage or terminology used in the present invention, the usage and terminology of the present invention shall prevail.

[0083] The section headings used herein are for organizational purposes and should not be construed as restricting the subject matter. Unless otherwise defined, all technical and scientific terms used herein have the common meaning in the art to which the claimed subject matter pertains. If a term has multiple definitions, the definition of this invention shall prevail.

[0084] Unless otherwise stated in the examples or elsewhere, all numerical values ​​in the specification and claims, such as those for dosages, are understood to be modified in all cases by the term "approximately." Furthermore, it should be understood that all numerical ranges enumerated in this invention are intended to include all subranges within that range and any combination of endpoints within that range or subrange.

[0085] In the present invention, the fragment is

[0086] [ka] " indicates that the aforementioned fragment is linked to other fragments within the molecule via this bond. For example,

[0087] [ka] teeth"

[0088] [ka] This refers to being linked to the rest of the molecule via a link.

[0089] In this invention, the term "one or more" refers to one, two, three, four, five, or six, for example, one, two, or three. In this invention, the term "alkyl" refers to a specified number of carbon atoms (e.g., C1, C2, C3, C4, C5, C6, C9, C 10 , C 11 , C 12 , C 16 , C 17 , C 18 , C 19 , C 20 Alkyl refers to a linear or branched saturated monovalent hydrocarbon group having ). Alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl,

[0090] [ka] This includes, but is not limited to, the following.

[0091] In this invention, the term "alkylene" means a saturated divalent hydrocarbon group obtained by removing two hydrogen atoms from a saturated linear or branched hydrocarbon group. Alkylenes include methylene (-CH2-), ethylene (including -CH2CH2- or -CH(CH3)-), isopropylene (including -CH(CH3)CH2- or -C(CH3)2-),

[0092] [ka] This includes, but is not limited to, the following.

[0093] In this invention, the term "alkoxy" is R Y -O- group refers to R Y The definition is the same as the term "alkyl". Alkoxy includes, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, n-hexoxy, etc.

[0094] The term "alkenyl" refers to a linear or branched hydrocarbon group that has at least one double bond, consists only of carbon and hydrogen atoms, has, for example, 10 to 20 (or, for example, 16, 17, 18, or 19) carbon atoms, and is bonded to the rest of the molecule by single bonds. Alkenyls are vinyl,

[0095] [ka] This includes, but is not limited to, the following.

[0096] In this invention, the term "aryl" refers to a specific number of carbon atoms (for example, C6-C6). 10 The term refers to a monocyclic or polycyclic (e.g., two) cyclic, unsaturated, monovalent hydrocarbon group having aryl rings, where, if polycyclic, the monocyclic rings share two atoms and one bond, and each ring is aromatic. The aryl is linked to the rest of the molecule via the aromatic ring. Aryls include, but are not limited to, phenyl and naphthyl.

[0097] Furthermore, when referring to a number or range of numbers, the term "approximately" means that the number or range referred to is an approximation within the range of general tolerance, experimental variation, or statistical experimental error in the art, and therefore the number or range may vary, for example, between 1% and 15% of the number or range. For example, "approximately" can be understood as approximately 2 standard deviations from the mean, and if "approximately" precedes a series of numbers or ranges, it should be understood that "approximately" may modify each number within the series of numbers or ranges.

[0098] Furthermore, in the present invention, when a numerical range is used in the general formula and / or structural formula of a compound, it means that the number of corresponding groups within the numerical range may be any one natural number within the numerical range, for example, "C A-B " means that the number of carbon atoms is any integer within the range from the starting point to the ending point, and that both A and B are integers, and also, for example, C 1-5 This means that the number of carbon atoms is 1, 2, 3, 4, or 5, i.e., when combined with other groups in the general formula and / or structural formula of a compound, C A-B It can be used in combination with any group containing carbon atoms to limit the number of carbon atoms, for example, C 1-5 Alkyl / alkylene represents various possibilities such as alkyl / alkylene having one C, alkyl / alkylene having two C, alkyl / alkylene having three C, alkyl / alkylene having four C, and / or alkyl / alkylene having five C.

[0099] Similar words used in this invention, such as "include," "contain," or "equip," mean that the element preceding the word includes the element described after the word and their equivalents, and do not exclude elements not described. The terms "contain" or "include (equip)" used in this invention may be open, semi-closed, or closed. In other words, the terms also include "basically consist of" or "consist of."

[0100] In this invention, the term "pharmaceutically acceptable" means that the compound or composition is chemically and / or toxicologically compatible with the other components of the formulation and / or with the human or mammal to whom the disease or condition is to be prevented or treated.

[0101] In the present invention, the terms "subject" or "patient" may include mammalian subjects. For example, the mammalian subjects may be selected from any one or at least two combinations of the group consisting of humans, non-human primates, companion animals, exotic species, livestock, and food animals.

[0102] As used herein, the term “treatment” means administering one or more medicinal substances to a patient or subject suffering from a disease or symptoms of said disease in order to cure, alleviate, reduce, improve, or influence said disease or symptoms of said disease. In the context of the present invention, unless otherwise stated, the term “treatment” may also include prevention.

[0103] In the present invention, the term “antigen” includes any molecule comprising an epitope and / or an epitope that can induce at least one immune response and / or is the target of an immune response, preferably a peptide or protein. Preferably, in the context of the present invention, an antigen is a molecule that, after being optionally treated, preferably induces a specific immune response to the antigen or cells expressing the antigen. In particular, “antigen” refers to a molecule that, after being optionally treated, is presented by an MHC molecule and specifically reacts with T lymphocytes (T cells).

[0104] Therefore, the antigen or fragment can be recognized by a T cell receptor. Preferably, once the antigen or fragment is recognized by a T cell receptor, it can induce clonal proliferation of T cells having a T cell receptor that specifically recognizes the antigen or fragment in the presence of appropriate costimulatory signals. In the context of embodiments of the present invention, the antigen or fragment is preferably presented in the context of MHC molecules by cells, preferably antigen-presenting cells and / or lesion cells, resulting in an immune response against the antigen or antigen-expressing cells.

[0105] According to the present invention, any suitable antigen can be considered as a candidate for an immune response, where the immune response is preferably a cellular immune response. The antigen is preferably a natural antigen or a product derived from a natural antigen. The natural antigen may include allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen may be a tumor antigen. According to the present invention, the antigen may correspond to a naturally occurring product, such as a viral protein or a part thereof.

[0106] The term "pathogen" refers to pathogenic microorganisms, including viruses, bacteria, fungi, single-celled organisms, and parasites. Examples of pathogenic viruses include, but are not limited to, human immunodeficiency virus (HIV), cytomegalovirus (CMV), herpes simplex virus (HSV), hepatitis A virus (HAV), HBV, HCV, papillomavirus, and human T-lymphotropic virus (HTLV). Examples of single-celled organisms include, but are not limited to, malaria parasites, trypanosomes, and amoebas.

[0107] The term "disease-related antigen" refers to all antigens with pathogenic significance, including "tumor antigens." According to the present invention, it is desirable to induce or express disease-related antigens, and preferably to induce an immune response against cells that present disease-related antigens in the context of MHC molecules. Preferably, the disease-related antigen is a naturally occurring antigen. In one embodiment, the disease-related antigen is expressed in diseased cells and preferentially presented by the cell's MHC molecules.

[0108] The antigen encoded by the RNA (i.e., therapeutic and / or prophylactic agent) of the (lipid composition) nanoparticles described in the present invention needs to induce an immune response against a target disease-related antigen or cells expressing a target disease-related antigen. Therefore, the antigen encoded by the RNA contained in the nanoparticles of the present invention may correspond to or contain a disease-related antigen or one or more immunogenic fragments thereof (such as one or more MHC-binding peptides of the disease-related antigen). Accordingly, the antigen encoded by the RNA contained in the nanoparticles of the present invention may be a recombinant antigen.

[0109] Therapeutic and / or prophylactic agents The lipid compositions of the present invention can be used to deliver pharmaceutically active ingredients such as therapeutic and / or prophylactic agents. Based on this, the present invention further provides (pharmaceutical) compositions used for delivering active pharmaceutical ingredients, comprising the lipid compositions provided by the present invention. The compositions of the present invention may contain one or more therapeutic and / or prophylactic agents (as pharmaceutically active ingredients). The pharmaceutically active ingredients may be encapsulated within the lipid composition or bound to the lipid composition.

[0110] The therapeutic and / or prophylactic agent comprises, but is not limited to, one or more nucleic acid molecules, small molecule compounds, polypeptides, and proteins. Preferably, it is a nucleic acid molecule.

[0111] For example, the therapeutic and / or prophylactic agent is a vaccine or compound that can induce an immune response. Therefore, in some preferred embodiments, the therapeutic and / or prophylactic agent may be a nucleic acid molecule capable of encoding one or more antigens.

[0112] The lipid compositions of the present invention can deliver therapeutic and / or prophylactic agents (as carriers) to target cells and / or target organs of a subject (such as a mammal), and therefore the present invention also provides a method for treating a disease or symptom of a subject requiring such treatment, comprising administering a composition containing therapeutic and / or prophylactic agents to a subject in need, and / or bringing the cells of the subject into contact with the composition.

[0113] Therapeutic and / or prophylactic agents are biologically active substances and may also be called “activators,” “active ingredients,” etc. A therapeutic and / or prophylactic agent may be a substance that, when delivered to a cell or organ, induces a desired change in that cell or organ, or in other body tissues or systems. Such types can be used to treat one or more diseases, symptoms, or conditions. In some embodiments, therapeutic and / or prophylactic agents are small molecule drugs that can be used to treat specific diseases, symptoms, or conditions. Examples of drugs usable in the composition include antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), and antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside).Arabinoside, anthracycline, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs (such as methotrexate), purines and pyrimidine analogs), antiinfective agents, local anesthetics (e.g., dibucaine, chlorpromazine), β-adrenergic receptor blockers (e.g., propranolol, timolol, labetalol), antihypertensive agents (e.g., clonidine, hydralazine), antidepressants (e.g., imipramine, amitriptyline, doxepin), antispasmodics (e.g., phenytoin), antihistamines Antibiotics (e.g., diphenhydramine, chlorpheniramine, promethazine), antibiotics / antimicrobials (e.g., gentamycin, ciprofloxacin, cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, amphotericin B) B)) Includes, but is not limited to, antiparasitic drugs, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma drugs, vitamins, sedatives, and contrast agents.

[0114] In some embodiments, therapeutic and / or prophylactic agents are cytotoxins, radioactive ions, chemotherapeutic agents, vaccines, compounds that induce an immune response, and / or other therapeutic and / or prophylactic agents. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emidine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, and dihydroxyanthracine dione. This includes, but is not limited to, dione, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids (such as maytansinol), rachelmycin (CC-1065), and their analogs or congeners. Radioactive ions include iodine (e.g., iodine 125 or iodine 131 ),strontium 89 Phosphorus, palladium, cesium, iridium, phosphate group, cobalt, yttrium 90 ,samarium 153Vaccines include, but are not limited to, praseodyms and other compounds. Vaccines include compounds and formulations that can provide immunity to one or more pathological conditions associated with infectious diseases (such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, epidemic, hepatitis, and pulmonary tuberculosis), which may contain nucleic acid molecules (e.g., mRNA) encoding infectious disease antigens and / or epitopes. Vaccines may further include compounds and formulations that induce an immune response against cancer cells, which may contain nucleic acid molecules (e.g., mRNA) encoding tumor cell antigens, epitopes and / or neoepitopes. Compounds that induce an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other substances. Other therapeutic and / or prophylactic agents include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, rashelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromamine) This includes, but is not limited to, cinnitol, streptozotocin, mitomycin C, and cis-dichlorodiammineplatinum(II) (DDP, cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC)), and mitotic inhibitors (e.g., vincristine, vinblastine, taxol, mytansinoids).

[0115] In other embodiments, the therapeutic and / or prophylactic agent is a protein. Therapeutic proteins available for use in the nanoparticles of the present invention include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), VIR factor, luteinizing hormone-releasing hormone (LHRH) analogues, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0116] In some embodiments, the therapeutic and / or prophylactic agent may be a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term “polynucleotide” in its broadest sense includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used in the present invention include, but are not limited to, one or more deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) (e.g., messenger mRNA (mRNA) or its hybrids, RNAi inducers, RNAi factors, siRNA, shRNA, miRNA, antisense RNA, ribozyme, catalytic DNA, RNA induced from a triple helix, aptamers, etc.). In some preferred embodiments, the therapeutic and / or prophylactic agent is RNA. The RNA available for use in the compositions and methods described in the present invention may be selected from, but is not limited to, the group consisting of shortmer, antagomir, antisense RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the RNA is mRNA.

[0117] In some embodiments, the therapeutic and / or prophylactic agent is mRNA. mRNA can encode polypeptides of any use, including any polypeptide that is naturally occurring, unnaturally occurring, or otherwise modified. The polypeptide encoded by mRNA may have any size, and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by mRNA may have a therapeutic effect when expressed in a cell.

[0118] In some embodiments, RNA is mRNA. siRNA can selectively reduce or downregulate the expression of a target gene. For example, due to the selectivity of siRNA, a composition containing the siRNA may silence genes associated with a specific disease, condition, or pathological state after administration to a subject in need. siRNA may contain a sequence complementary to the mRNA sequence encoding the target gene or protein. In some embodiments, siRNA may be immunomodulatory siRNA.

[0119] In some embodiments, the therapeutic and / or prophylactic agent is sgRNA and / or cas9 mRNA. sgRNA and / or cas9 mRNA may be used as a tool for gene editing. For example, the sgRNA-cas9 complex affects the translation of mRNA of a cell's genes.

[0120] In some embodiments, the therapeutic and / or prophylactic agent is shRNA or a carrier or plasmid encoding it. The shRNA may be produced within the target cell after a suitable construct has been delivered into the nucleus. Constructs and mechanisms associated with shRNA are well known in the relevant fields.

[0121] Disease or medical condition The compositions / carriers of the present invention can deliver therapeutic and / or prophylactic agents to subjects or patients to achieve the treatment and / or prevention of diseases or symptoms. The therapeutic and / or prophylactic agents include, but are not limited to, one or more nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Accordingly, the compositions of the present invention may be used to manufacture nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides, or protein drugs. Due to the wide variety of therapeutic and / or prophylactic agents, the compositions of the present invention can be used to treat or prevent a variety of diseases or conditions.

[0122] In one embodiment, the disease or condition is characterized by dysfunction or abnormal activity of a protein or polypeptide. The reagents, compositions, and methods described in the present invention can be used to treat subjects suffering from a disease (e.g., a disease characterized by the appearance of an expressed antigen and diseased cells that present an antigenic peptide) or to prevent subjects from suffering from a disease. Examples of treatable and / or preventable diseases cover all diseases expressing one of the antigens described in the present invention. Particularly preferred diseases are infectious diseases (e.g., viral diseases) and cancer. The reagents, compositions, and methods described in the present invention may also be used for immunization or vaccination to prevent the diseases described in the present invention.

[0123] According to the present invention, the term "disease" refers to all pathological conditions, including infectious diseases and cancer, and in particular, to the forms thereof of infectious diseases and diseases described in the present invention. According to the present invention, the diseases to be treated are preferably antigen-related diseases. According to the present invention, “antigen-related diseases” or similar descriptions mean that the antigen is expressed within the cells of diseased tissue or organ. Expression in the cells of diseased tissue or organ may be elevated compared to the state in healthy tissue or organ. In one embodiment, expression occurs only in tissue with pathological changes, and expression is inhibited in healthy tissue. According to the present invention, antigen-related diseases include infectious diseases and cancer, where the disease-related antigens are preferably infectious antigens and tumor antigens, respectively. Preferably, antigen-related diseases are diseases relating to cells that express an antigen and present the antigen in an environment with MHC molecules (particularly MHC class I).

[0124] For example, the disease or condition is selected from the group consisting of infectious diseases, cancer and proliferative disorders, genetic disorders, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renovascular diseases, and metabolic diseases.

[0125] Examples of the aforementioned infectious diseases include (1) viral infections (e.g., AIDS (HIV), hepatitis A, hepatitis B or C, herpes zoster (varicella), rubella (rubella virus), yellow fever, dengue fever, etc., flavivirus, coronavirus, influenza virus, rabies virus, hemorrhagic infections (Marburg virus or Ebola virus)), (2) bacterial infections (e.g., Legionnaires' disease) (1) Infections caused by disease (Legionella), gastric ulcer (Helicobacter), cholera (Vibrio), Escherichia coli, Staphylococcus, Salmonella, or Streptococcus (tetanus)), (2) Infections caused by prokaryotic pathogens (e.g., malaria, African sleeping sickness, leishmaniasis, toxoplasmosis, i.e., infections caused by Plasmodium, Trypanosoma, Leishmania, or Toxoplasma), or (3) Fungal infections (e.g., Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides imitis) This includes immitis (caused by Blastomyces dermatitidis or Candida albicans).

[0126] Cancer, or malignant tumor in medical terms, is a disease in which some cells exhibit uncontrolled proliferation (dividing beyond the normal limit), invasiveness (invading and destroying adjacent tissues), and, in some cases, metastasis (spreading to other parts of the body via the lymphatic system or bloodstream). These three harmful characteristics of cancer distinguish it from benign tumors, which are self-contained and do not invade or metastasize. Most cancers form tumors, i.e., swellings or pathological changes formed by the abnormal proliferation of cells (called malignant neoplasm cells or tumor cells), but some (such as leukemia) are not of this type. According to the present invention, the term "cancer" includes leukemia, seminomas, melanoma, teratoma, lymphoma, sarcoma, blastoma, neuroblastoma, glioma, glioblastoma, kidney cancer, adrenal cancer, renal cell carcinoma, thyroid cancer, hematological cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, lung cancer, intestinal cancer, head and neck cancer, digestive tract cancer, multiple myeloma, lymph node cancer, esophageal cancer, colon cancer, rectal cancer, bladder cancer, prostate cancer, endometrial cancer, pancreatic cancer, ear, nose, and throat (ENT) cancer, breast cancer, uterine cancer, breast cancer, prostate cancer, ovarian cancer, and their metastases.

[0127] Malignant melanoma is a type of severe skin cancer. It arises from the uncontrolled proliferation of pigment cells called melanocytes. According to the present invention, “epithelial cancer” is a malignant tumor derived from epithelial cells. This includes the most common cancers, such as breast cancer, prostate cancer, lung cancer, and common forms of colon cancer.

[0128] Lymphoma and leukemia are malignant tumors that originate from hematopoietic (blood-forming) cells. Sarcoma is a type of cancer that arises from a single transformed cell in some tissues that originate from the fetal mesoderm. Therefore, sarcomas include bone tumors, chondroma, lipoma, muscle tumor, vascular tumor, and hematopoietic tissue tumor.

[0129] A blastic tumor, or blastocyte tumor, is a tumor that resembles immature or fetal tissue (and is generally malignant). Many of these tumors occur in children.

[0130] Gliomas are a type of tumor that originates in the brain or spinal cord. They are called gliomas because they originate from glial cells. The brain is the most common site for gliomas. Other ingredients The pharmaceutical composition of the present invention may contain one or more components other than those described in the above section. For example, the composition may contain one or more hydrophobic small molecules such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0131] The composition may further contain one or more permeability-enhancing molecules, carbohydrates, polymers, surface modifiers, or other components. The permeability-enhancing molecules may be, for example, those described in U.S. Patent Application Publication No. 2005 / 0222064. The carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0132] Surface modifiers include anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants (such as dimethyldistearylammonium bromide)), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocysteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, thiopronin, gelsolin, thymosin β4, dornase α) The composition may include, but is not limited to, alfa, neltenexine, erdosteine, and DNase (e.g., rhDNase). The surface modifier may be positioned within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent bonding, or other means).

[0133] The composition may further contain one or more functionalized lipids. For example, the lipids may be functionalized with alkynyl groups that can undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. More precisely, the lipid bilayer may be functionalized with one or more groups that can effectively promote membrane permeability, cell recognition, or contrast enhancement in this manner. The surface of the composition may be coupled with one or more useful antibodies. Functional groups and conjugates used for targeted cell delivery, contrast enhancement, and membrane permeability are well known in the art.

[0134] The composition may contain any substance used in pharmaceutical compositions other than these components. For example, the composition may contain, but is not limited to, one or more pharmaceutically acceptable (i.e., pharmaceutically acceptable) excipients or helper components, such as one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, flow accelerators, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, and colorants.

[0135] The term "pharmaceutical-grade" refers to materials that are non-toxic and do not affect the action of the active ingredients in a pharmaceutical composition. Non-pharmaceutical-grade ingredients may be used to manufacture pharmaceutical ingredients and are included in this invention.

[0136] Suitable buffering agents for use in the compositions of the present invention include salts of acetic acid, citric acid, boric acid, and phosphoric acid. As used in the present invention, the term "excipient" means all substances that are not active ingredients and may be present in the pharmaceutical composition of the present invention, such as carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents, or colorants. Examples of excipients include starch, lactose, or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st ed., ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0137] Suitable preservatives for use in the compositions of the present invention include benzalkonium chloride, chlorobutanol, parahydroxybenzoic acid ester, and thimerosal. Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, corn starch, powdered sugar, and / or combinations thereof.

[0138] Dosage form and administration The compositions of the present invention may be manufactured as solid, semi-solid, liquid, or gaseous formulations, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, or aerosols. The compositions of the present invention may be manufactured by methods well known in the pharmaceutical field. For example, a sterile solution for injection may be manufactured by adding a predetermined amount of the therapeutic or prophylactic agent and each of the other specified components described above to a suitable solvent, such as sterile distilled water, followed by filtration and sterilization. Furthermore, a surfactant may be added to promote the formation of a homogeneous solution or suspension.

[0139] For example, the compositions of the present invention may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In one embodiment, the composition is administered intravenously or subcutaneously.

[0140] Therapeutic effective dose The "therapeutic dose" is the amount of therapeutic agent that, when administered to a patient, can improve a disease or symptom. The "preventive dose" is the amount of preventive agent that, when administered to a subject, can prevent a disease or symptom. The amount of therapeutic agent constituting the "therapeutic dose" or the amount of preventive agent constituting the "preventive dose" varies depending on the therapeutic agent and / or preventive agent, the state and severity of the disease, the age and weight of the patient and / or subject receiving treatment and / or prevention, etc. A person skilled in the art may generally determine the therapeutic dose and the preventive dose based on their knowledge and the present invention.

[0141] The compositions of the present invention are administered in therapeutically effective doses, the amount of which varies not only depending on the specific agent selected, but also on the route of administration, the characteristics of the disease being treated, and the age and condition of the patient, and may ultimately be determined at the discretion of the attending physician or clinician. For example, the therapeutic or prophylactic agent may be administered to a mammal (e.g., a human) in doses of about 0.0001 to about 10 mg / kg.

[0142] antigen presenting cells Antigen-presenting cells (APCs) are cells that present (i.e., display) antigens in an environment containing the major histocompatibility complex (MHC) on their surface. This includes cases where they present one or more fragments of the antigen. T cells can recognize this complex with their T cell receptor (TCR). Antigen-presenting cells process the antigen and then present it to T cells.

[0143] Specialized antigen-presenting cells are adept at taking up antibodies (by phagocytosis or receptor-mediated endocytosis) and then displaying antigen fragments that bind to MHC class II molecules on their membranes. T cells recognize and interact with the antigen-MHC class II molecule complex on the membrane of the antigen-presenting cell. The antigen-presenting cell then produces additional costimulatory signals to activate the T cell. The expression of costimulatory molecules is a typical characteristic of specialized antigen-presenting cells.

[0144] The main types of specialized antigen-presenting cells are dendritic cells (which have the broadest antigen-presenting range and may be the most important antigen-presenting cells), macrophages, B cells, and some activated epithelial cells.

[0145] Dendritic cells are a group of white blood cells that include plasmacytoid dendritic cells (pDC cells) and classical dendritic cells (cDC cells). They present antigens captured in peripheral tissues to T cells via two antigen presentation pathways: MHC class II and class I. Dendritic cells are potent inducers of the immune response, and their activation is a crucial step in inducing anti-tumor immunity.

[0146] The peptide presented by MHC may be loaded onto antigen-presenting cells by transduction of nucleic acids encoding peptides or proteins containing the presented peptide (e.g., nucleic acids encoding antigens (e.g., RNA)). Transfection of dendritic cells with mRNA is a promising antigen loading technique that stimulates potent anti-tumor immunity.

[0147] The term "immunogenicity" refers to the relative efficiency of an antigen in inducing an immune response. The terms "T cell" and "T lymphocyte" may be used interchangeably in this invention, and helper T cells (CD4 + T cells), cytolytic T cells (CTL, CD8 + Includes T cells.

[0148] T cells belong to a group of white blood cells called lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other types of lymphocytes (e.g., B cells, natural killer cells) by the presence of a special receptor called the T cell receptor (TCR) located on their surface. The thymus is a major organ involved in T cell maturation. Several different T cell subsets, each with distinct functions, have been discovered.

[0149] Helper T cells synchronize with other white blood cells in the immune process, such as maturing B cells into plasma cells and activating cytotoxic T cells and macrophages. These cells express the CD4 protein on their surface, hence the CD4 + Also known as T cells, helper T cells are activated when MHC class II molecules expressed on the surface of antigen-presenting cells (APCs) present peptide antigens to them. After activation, they immediately divide and secrete small proteins called cytokines that regulate or support the active immune response.

[0150] Cytotoxic T cells destroy diseased cells (e.g., infected cells (such as virus-infected cells)) and cancer cells, and are also involved in transplant rejection. These cells express the CD8 glycoprotein on their surface, hence the name CD8. + These cells are also called T cells. They recognize their targets by binding to antigens associated with MHC class I, and MHC class I is present on the surface of almost all cells in the body.

[0151] Most T cells possess a T cell receptor (TCR), which exists as a complex of multiple proteins. The actual T cell receptor consists of two independent peptide chains, called the α-TCR chain and the β-TCR chain, produced by independent T cell receptor α and β (TCRα and TCRβ) genes. γδ T cells are a small subtype of T cells that have a unique T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR consists of one γ chain and one δ chain. This group of T cells is rarer than αβ T cells (2% of all T cells).

[0152] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells reside in the thymus and proliferate through cell division, producing a large number of immature thymocytes. Early thymocytes do not express either CD4 or CD8, and are therefore double-negative (CD4). - CD8 - They are classified as ) cells. As they develop, they become double-positive thymocytes (CD4 + CD8 + ) becomes, and finally matures into a single positive (CD4 + CD8 - or CD4 - CD8 + They become thymocytes and are later released from the thymus into peripheral tissues.

[0153] The initial signal for T cell activation is provided when the T cell receptor binds to a short peptide presented by the major histocompatibility complex (MHC) on another cell. This ensures that only T cells with a TCR specific to that peptide are activated. The partner cell is generally a specialized antigen-presenting cell (APC), typically a dendritic cell in the primary response, but B cells and macrophages can also be important APCs. CD8 is activated by MHC class I molecules. + The peptide presented to T cells is 8-10 amino acids long and is transmitted to CD4 by MHC class II molecules. + Peptides presented to T cells are longer because the end of the binding cleft of the MHC class II molecule is open.

[0154] As long as it does not violate common sense in the art, the above preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention. The reagents and raw materials used in this invention are commercially available.

[0155] Positive and progressive effects of the present invention: Compared to the prior art, mRNA compositions prepared using the compound represented by formula (I) provided in the present invention have one or more of the following advantages.

[0156] 1. The particle size is good and the particle distribution is uniform. 2. Significantly increase the protein expression of the antigen in the muscle or spleen of a subject (e.g., a mouse) in vivo.

[0157] 3. Significantly increases the percentage of antigen-presenting cells expressing the antigen in the spleen. 4. Significantly improves the maturation of antigen-presenting cells in the spleen. 5. Significantly improves the activation state of T cells in the spleen.

[0158] 6. Tumor volume is significantly reduced, and mouse survival rate is significantly improved. [Brief explanation of the drawing]

[0159] To more clearly explain the technical solutions of the embodiments of the present invention, the drawings of the present invention will be briefly described below. Needless to say, the drawings mentioned in the following description relate to some specific embodiments of the present invention and are not limiting to the present invention.

[0160] [Figure 1] These are fluorescence images of live mice and mouse organs (liver, spleen) 6 hours after intravenous injection of Fluc-mRNA-TLP compositions prepared with YK-1202, YK-1204, YK-1205, YK-1206, or YK-1208, and Fluc-mRNA-TLP compositions without adjuvant lipids. (Left: Live mouse, Right: Mouse organ) [Figure 2] These are flow cytometry images (eGFP) of mouse spleen cells 24 hours after intravenous injection of a blank, an eGFP-mRNA-TLP composition containing YK-1202 or YK-1204, and an eGFP-mRNA-TLP composition without adjuvant lipids into mice. [Figure 3] This is a flow cytometry diagram (CD86) of mouse spleen cells 24 hours after intravenous injection of a blank, an eGFP-mRNA-TLP composition containing YK-1205, or TMX-201 into mice. [Figure 4] This is a flow cytometry diagram (CD69) of mouse spleen cells 24 hours after intravenous injection of a TLP composition containing a blank, YK-1204, YK-1205, or TMX-201 into mice. [Figure 5] This describes the stimulation status of cytokines IFN-γ and IL-12 in the serum of B16F10-OVA mice inoculated with an OVA mRNA-TLP composition containing YK-1202, YK-1204, YK-1205, or YK-1208. [Figure 6] This shows the tumor growth in B16F10-OVA mice inoculated with an OVA mRNA-TLP composition containing YK-1202, YK-1204, YK-1205, or YK-1208. [Figure 7]The survival rate of B16F10-OVA mice inoculated with an OVA mRNA-TLP composition added with YK-1202, YK-1204, YK-1205 or YK-1208. [Figure 8] The stimulation status of cytokines IFN-γ and IL-12 in the serum of B16F10-OVA mice inoculated with an OVA mRNA-TLP composition added with YK-1202, YK-1204, YK-1206 or YK-1208. [Figure 9] The tumor growth status in the body of MC38-OVA mice inoculated with an OVA mRNA-LNP composition added with YK-1202, YK-1204, YK-1206 or YK-1208. [Figure 10] The survival rate of MC38-OVA mice inoculated with an OVA mRNA-LNP composition added with YK-1202, YK-1204, YK-1206 or YK-1208.

Mode for Carrying Out the Invention

[0161] Hereinafter, the present invention will be further described using examples, but the present invention is not limited to the following examples. The implementation conditions used in the examples may be further adjusted based on specific different requirements in their use. When the implementation conditions are not specified, they are the normal conditions in the art. All raw materials used in the specific examples of the present invention can be obtained through commercial routes. Unless otherwise specified, the percentages mentioned in the context are weight percentages, and all temperatures are shown in degrees Celsius. The technical features according to each embodiment of the present invention may be combined with each other as long as they do not conflict.

[0162] The following abbreviations and letters represent the following reagents respectively. ADOPE: 2-Aminoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate sodium salt (

[0163]

Chemical formula

[0164] DOTMA: 1,2-dioctadecenyloxy-3-trimethylammonium propane (chloride salt) DOPE: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine DOPG: 1,2-Dioleoyl-sn-glycero-3-phospho-rac-glycerol sodium salt DSPC:1,2-distearoyl-sn-glycero-3-phosphocholine DMG-PEG2000:1,2-Dimyristoyl-rac-Glycero-Methoxypolyethylene Glycol-2000 Boc2O: Ditert-butyl dicarbonate DMAP: 4-dimethylaminopyridine EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride TsOH: p-toluenesulfonic acid TFA: Trifluoroacetic acid DCM: Dichloromethane DMF: N,N-dimethylformamide THF: Tetrahydrofuran TrCl: Triphenylchloromethane.

[0165] Example 1: Synthesis of lipid-bound TLR adjuvants 1. Synthesis of YK-1201 The synthesis route for YK-1201 is as follows:

[0166] [ka]

[0167] Step 1: Synthesis of YK-1201-PM1 YK-009 (500 mg, 0.765 mmol) was dissolved in dichloromethane (10 mL), and thionyl chloride (180 mg, 1.530 mmol) was added dropwise under an ice bath. The mixture was stirred and reacted at room temperature for 6 hours. The reaction was stopped, the solvent was rotated dry under reduced pressure, and the product was purified by silica gel chromatography (0% to 15% dichloromethane / methanol) to obtain product YK-1201-PM1 (500 mg, 0.743 mmol, 97.1%).

[0168] Step 2: Synthesis of YK-1201 YK-1201-PM1 (150 mg, 0.223 mmol) and YK-1201-SM1 (84 mg, 0.270 mmol) were dissolved in DMF (3 mL), potassium carbonate (93 mg, 0.673 mmol) and potassium iodide (3 mg, 0.018 mmol) were added at room temperature, and the mixture was stirred and reacted at 70°C for 6 hours. After the reaction was complete, the temperature was lowered, and ethyl acetate and water were sequentially added to the reaction solution for separation. The aqueous phase was back-extracted twice with ethyl acetate, the organic phases were combined, and the solvent was rotated dry to obtain the crude product. The crude product was purified by silica gel chromatography (0%~15% dichloromethane / methanol) to obtain product YK-1201 (120 mg, 0.127 mmol, 57.0%). 58 H 102 N6O4, MS (ES): m / z (1 / 2 M + Na + ) = 496.63.

[0169] YK-1201: 1H NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.43 - 7.35 (m, 1H), 7.26 - 7.19 (m, 1H), 6.42 (s, 2H), 4.52 - 4.44 (m, 2H), 3.99 - 3.86 (m, 6H), 3.07 - 2.96 (m, 2H), 2.94 - 2.86 (m, 2H), 2.40 - 2.30 (m, 4H), 2.29 - 2.21 (m, 4H), 1.85 - 1.74 (m, 4H), 1.62 - 1.40 (m, 12H), 1.29 - 1.17 (m, 45H), 1.13 - 1.05 (m, 2H), 0.96 (t, J = 7.2 Hz, 3H), 0.88 - 0.79 (m, 9H). 2. Synthesis of YK-1202 The synthesis route for YK-1202 is as follows:

[0170] [ka]

[0171] Using YK-1201-PM1 (100 mg, 0.149 mmol) and YK-1202-SM1 (64 mg, 0.178 mmol) as raw materials, product YK-1202 (34 mg, 0.0342 mmol, 22.9%) was obtained according to the synthesis method for YK-1201. 62 H 102 N6O4, MS (ES): m / z (1 / 2 M + Na + ) = 520.58.

[0172] YK-1202: 11H NMR (400 MHz, DMSO-d6) δ 7.79 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.38 - 7.33 (m, 1H), 7.25 - 7.20 (m, 2H), 7.10 - 7.00 (m, 3H), 6.82 - 6.67 (m, 2H), 5.86 (s, 2H), 4.36 - 4.29 (m, 3H), 4.15 - 3.86 (m, 3H), 3.19 - 3.15 (m, 2H), 2.94 - 2.65 (m, 5H), 2.41 - 2.14 (m, 4H), 2.03 - 1.89 (m, 2H), 1.75 - 1.65 (m, 4H), 1.63 - 0.90 (m, 54H), 0.89 - 0.79 (m, 12H). 3. Synthesis of YK-1203 The synthetic route of YK-1203 is as follows:

[0173] [Chemical formula]

[0174] Step 1: Synthesis of YK-1203-PM1 YK-009 (500 mg, 0.764 mmol) and 4-bromobutyric acid (612 mg, 3.665 mmol) were dissolved in dichloromethane (5 mL). EDCI (440 mg, 2.295 mmol) and DMAP (38 mg, 0.311 mmol) were added at room temperature, and the mixture was stirred at 35 °C for 16 hours. The reaction was stopped, and the solvent was rotary evaporated under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% - 15% dichloromethane / methanol) to obtain the product YK-1203-PM1 (480 mg, 0.598 mmol, 78.2%).

[0175] Step 2: Synthesis of YK-1203 Using YK-1203-PM1 (150 mg, 0.187 mmol) and YK-1201-SM1 (70 mg, 0.224 mmol) as raw materials, product YK-1203 (24 mg, 0.0232 mmol, 12.4%) was obtained according to the synthesis method for YK-1201. 62 H 108 N6O6, MS (ES): m / z (1 / 2 M + Na + ) = 539.68.

[0176] YK-1203: 1 H NMR (400 MHz, DMSO-d6) δ 8.01 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.48 - 7.40 (m, 1H), 7.31 - 7.24 (m, 1H), 6.94 - 6.56 (m, 2H), 4.55 - 4.45 (m, 2H), 4.02 - 3.87 (m, 6H), 3.07 - 2.96 (m, 2H), 2.94 - 2.86 (m, 2H), 2.63 - 2.54 (m, 2H), 2.40 - 2.30 (m, 4H), 2.29 - 2.21 (m, 4H), 1.85 - 1.70 (m, 6H), 1.62 - 1.40 (m, 10H), 1.29 - 1.17 (m, 50H), 1.13 - 1.05 (m, 2H), 0.96 (t, J = 7.2 Hz, 3H), 0.88 - 0.79 (m, 9H). 4. Synthesis of YK-1204 The synthesis route for YK-1204 is as follows:

[0177] [ka]

[0178] Step 1: Synthesis of YK-1204-PM1 Using YK-009 (500 mg, 0.764 mmol) and 6-bromohexanoic acid (596 mg, 3.058 mmol) as raw materials, the product YK-1204-PM1 (520 mg, 0.626 mmol, 81.9%) was obtained according to the synthesis method for YK-1203-PM1.

[0179] Step 2: Synthesis of YK-1204 Using YK-1204-PM1 (150 mg, 0.180 mmol) and YK-1201-SM1 (84 mg, 0.271 mmol) as raw materials, product YK-1204 (60 mg, 0.0565 mmol, 31.4%) was obtained according to the synthesis method for YK-1201. 64 H 112 N6O6, MS (ES): m / z (1 / 2 M + Na + ) = 553.57.

[0180] YK-1204: 1 H NMR (400 MHz, DMSO-d6) δ 8.04 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.45 - 7.36 (m, 1H), 7.26 - 7.20 (m, 1H), 6.42 (s, 2H), 4.57 - 4.47 (m, 2H), 4.03 - 3.93 (m, 4H), 3.92 - 3.86 (m, 2H), 2.95 - 2.87 (m, 2H), 2.72 - 2.53 (m, 4H), 2.41 - 2.31 (m, 4H), 2.30 - 2.20 (m, 6H), 1.89 - 1.75 (m, 4H), 1.63 - 1.39 (m, 14H), 1.29 - 1.17 (m, 50H), 1.13 - 1.05 (m, 2H), 0.96 (t, J = 7.2 Hz, 3H), 0.87 - 0.80 (m, 9H). 5. Synthesis of YK-1205 The synthesis route for YK-1205 is as follows:

[0181] [ka]

[0182] Using YK-1203-PM1 (100 mg, 0.125 mmol) and YK-1202-SM1 (66 mg, 0.184 mmol) as raw materials, product YK-1205 (60 mg, 0.0555 mmol, 44.4%) was obtained according to the synthesis method for YK-1201. 66 H 108 N6O6, MS (ES): m / z (1 / 2 M + Na + ) = 563.66.

[0183] YK-1205: 1 H NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.40 - 7.33 (m, 1H), 7.18 (d, J = 8.4 Hz, 2H), 7.11 - 7.04 (m, 1H), 7.18 (d, J = 8.4 Hz, 2H), 5.84 (s, 2H), 4.13 - 4.08 (m, 2H), 4.01 - 3.89 (m, 8H), 2.93 - 2.86 (m, 2H), 2.38 - 2.32 (m, 4H), 2.30 - 2.24 (m, 4H), 1.78 - 1.68 (m, 4H), 1.59 - 1.47 (m, 10H), 1.41 - 1.30 (m, 8H), 1.29 - 1.23 (m, 42H), 0.89 - 0.80 (m, 12H). 6. Synthesis of YK-1206 The synthesis route for YK-1206 is as follows:

[0184] [ka]

[0185] Using YK-1204-PM1 (100 mg, 0.120 mmol) and YK-1202-SM1 (52 mg, 0.144 mmol) as raw materials, product YK-1205 (40 mg, 0.0360 mmol, 30.0%) was obtained according to the synthesis method for YK-1201. 68 H 112 N6O6, MS (ES): m / z (1 / 2 M + Na + ) = 577.72.

[0186] YK-1206: 1 H NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.40 - 7.33 (m, 1H), 7.18 (d, J = 8.4 Hz, 2H), 7.11 - 7.04 (m, 1H), 7.18 (d, J = 8.4 Hz, 2H), 5.84 (s, 2H), 4.01 - 3.96 (m, 4H), 3.95 - 3.86 (m, 6H), 2.93 - 2.87 (m, 2H), 2.40 - 2.34 (m, 4H), 2.27 - 2.24 (m, 4H), 1.76 - 1.63 (m, 4H), 1.56 - 1.45 (m, 14H), 1.38 - 1.30 (m, 8H), 1.27 - 1.23 (m, 42H), 0.88 - 0.81 (m, 12H). 7. Synthesis of YK-1207 The synthesis route for YK-1207 is as follows:

[0187] [ka]

[0188] Step 1: Synthesis of YK-1207-PM1 YK-1207-SM1 (830 mg, 2.64 mmol) was dissolved in tetrahydrofuran (10 mL), cooled to 0°C, and triethylamine (1.33 g, 13.15 mmol) was slowly added. After stirring for 5 minutes, anhydrous Boc (2.88 g, 13.20 mmol) was added, the mixture was allowed to return to room temperature, stirred overnight, and the reaction was monitored by LC-MS. The reaction was stopped, water and ethyl acetate were sequentially added to the reaction solution, and the mixture was separated. The aqueous phase was back-extracted twice with ethyl acetate, the organic phases were combined, and the solvent was rotated dry to obtain the crude product. The crude product was purified by silica gel chromatography (0%~10% dichloromethane / methanol) to obtain product YK-1207-PM1 (830 mg, 2.00 mmol, 75.8%). 22 H 30 N4O4, MS (ES): m / z (M + H + ) = 415.38.

[0189] Step 2: Synthesis of YK-1207-PM2 YK-1207-PM1 (300 mg, 0.72 mmol) was dissolved in dichloromethane (3.5 mL), cooled to 0°C, DMAP (531 mg, 4.30 mmol) was added and the mixture was stirred for 5 minutes. Then YK-1207-SM2 (437 mg, 2.16 mmol) was added, and the mixture was allowed to return to room temperature and stirred for 4 hours. The reaction was monitored by TLC. The reaction was stopped, and the product was purified by silica gel chromatography (0%~50% dichloromethane / ethyl acetate) to obtain product YK-1207-PM2 (105 mg, 0.181 mmol, 25.2%). 29 H 33 N5O8, MS (ES): m / z (M + H + ) = 580.75.

[0190] Step 3: Synthesis of YK-1207-PM3 YK-1207-PM2 (105 mg, 0.18 mmol) was dissolved in dichloromethane (3 mL), and DIEA (94 mg, 0.73 mmol) and DOPE (539 mg, 0.73 mmol) were added at room temperature. The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. The reaction was stopped, and the solvent was rotated dry to obtain the crude product. The crude product was purified by silica gel chromatography (0%~10% dichloromethane / methanol) to obtain product YK-1207-PM3 (100 mg, 0.084 mmol, 46.9%). 64 H 106 N5O 13 P,MS (ES): m / z (M + H + ) = 1185.17.

[0191] Step 4: Synthesis of YK-1207 YK-1207-PM3 (100 mg, 0.084 mmol) was dissolved in 1 mL of ethyl acetate solution of 4 M hydrogen chloride, stirred at room temperature for 4 hours, and the reaction was monitored by LC-MS. The reaction was stopped, the solvent was rotated dry to obtain the crude product, and the product was purified by silica gel chromatography (0%~10% dichloromethane / methanol) to obtain product YK-1207 (20 mg, 0.018 mmol, 21.9%). 59 H 98 N5O 11 P,MS (ES): m / z (M + H + ) = 1085.13.

[0192] YK-1207: 1H NMR (400 MHz, DMSO-d6) δ 8.44 (d, J = 8.0 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 5.34 - 5.24 (m, 4H),5.07 - 4.99 (m, 1H), 4.78 - 4.68 (m, 1H), 4.25 - 4.18 (m, 1H), 3.99 - 3.94 (m, 1H), 3.87 - 3.76 (m, 4H), 3.56 - 3.48 (m, 6H), 2.95 - 2.93 (m, 10H), 2.16 - 2.12 (m, 2H), 2.04 - 1.92 (m, 8H), 1.44 - 1.32 (m, 9H), 1.27 - 1.21 (m, 48H), 0.88 - 0.81 (m, 9H). 8. Synthesis of YK-1208 The synthesis route for YK-1208 is as follows:

[0193] [ka]

[0194] Step 1: Synthesis of YK-1208-PM1 5-Chlorovalanal (77 mg, 0.639 mmol) was dissolved in dichloromethane (10 mL), DOPG (500 mg, 0.645 mmol) and p-toluenesulfonic acid (62 mg, 0.33 mmol) were added at room temperature, and the mixture was stirred overnight at room temperature. The reaction was monitored by TLC. After the reaction of the starting materials was complete, the reaction was stopped, the reaction solution was poured into a saturated aqueous sodium bicarbonate solution, extracted with dichloromethane, the organic phases were combined, and the solvent was rotated dry to obtain the crude product. The crude product was purified by silica gel chromatography (0%~10% dichloromethane / methanol) to obtain product YK-1208-PM1 (420 mg, 0.479 mmol, 74.9%). 47 H 86 ClO 10 P,MS (ES): m / z (M - H +) = 875.84.

[0195] Step 2: Synthesis of YK-1208 YK-1208-PM1 (320 mg, 0.364 mmol) and YK-1201-SM1 (114 mg, 0.366 mmol) were dissolved in DMF (3.5 mL), and potassium carbonate (150 mg, 1.10 mmol) and potassium iodide (6 mg, 0.040 mmol) were added at room temperature. The mixture was then stirred and reacted at 70°C for 16 hours. After the reaction was complete, the temperature was lowered, the reaction solution was poured into a saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, the organic phases were combined, and the solvent was rotated dry to obtain the crude product. The crude product was purified by silica gel chromatography (0%~20% dichloromethane / methanol) to obtain product YK-1208 (105 mg, 0.091 mmol, 24.6%). 65 H 110 N5O 10 P,MS (ES): m / z (M + H + ) = 1153.26.

[0196] 1 H NMR (400 MHz, CDCl3) δ 7.94 - 8.00 (m, 2H), 7.48 - 7.54 (m, 2H), 5.27 - 5.33 (m, 8H), 4.88 - 5.03 (m, 2H), 4.52 (s, 1H), 4.41 - 4.44 (m, 2H), 4.20 (s, 3H), 4.00 - 4.08 (m, 6H), 3.49 (t, J = 8.0, 2H), 2.91 (t, J = 8.0, 4H), 2.24 - 2.32 (m, 8H), 2.00 (s, 16H), 1.85 - 1.88 (m, 6H), 1.76 - 1.79 (m, 5H), 1.58 (s, 8H), 1.27 (s, 23H), 1.00 (t, J = 8.0, 3H), 0.86 - 0.88 (m, 9H). 9. Synthesis of TMX201

[0197] [ka]

[0198] The synthesis of TMX201 was carried out by referring to the synthesis route of compound A in WO2011134669A, yielding 25 mg of TMX201. 10. Synthesis of Compound 23

[0199] [ka]

[0200] The synthesis of compound 23 was carried out by referring to the synthesis route of compound 23 in WO2021237055A1, and 30 mg of compound 23 was obtained. Synthesis of 11.63-15

[0201] [ka]

[0202] The synthesis of 63-15 was carried out by referring to the synthesis route of compound number 63-15 in WO2019040491A1, yielding 42 mg of 63-15. Example 2: Preparation of mRNA lipid composition A) Preparation of Fluc DNA, eGFP DNA, and OVA DNA templates 1) Restriction enzymes consisting of luciferase (Luciferase protein CDS), green fluorescent protein (GFP), and ovalbumin (OVA) circular plasmids were constructed by ligating them into the EcoRVpVAX1 vector (purchased from Thermo Fisher Scientific).

[0203] 2) The plasmid constructed with the pVAX1 vector in step 1) was homogeneously mixed with 50 μL of E. coli competent cells Stbl2 (purchased from Thermo Fisher Scientific), then bathed in ice for 30 minutes, subjected to a heat shock at 42°C for 90 seconds, immediately returned to ice, and bathed in ice for 2 minutes.

[0204] 3) Add 400 μL of LB medium (purchased from Thermo Fisher Scientific) and incubate at 30°C in a shaker for 45-60 minutes with gentle shaking.

[0205] 4) 50-100 μL of bacterial suspension was spread onto LB solid medium containing the antibiotic kanamycin (100 μg / mL, Yisheng Biotechnology Co., Ltd), and incubated inverted at 37°C overnight.

[0206] 5) The accuracy of the sequencing was verified by performing sequencing on the obtained monoclonal colony plates. Monoclonal colonies with correct sequencing results were selected and cultured overnight in a shaker at 30°C with gentle shaking.

[0207] 6) Plasmid extraction was performed using an endotoxin-free plasmid extraction kit (purchased from Yeasen Biotechnology Co., Ltd.). 7) Using restriction enzymes, digest the extracted plasmid into a linear plasmid, which is then used as a transcription template. For specific steps of the digestion process, refer to steps (1) to (3).

[0208] Step (1): A linear DNA transcription template was obtained by digesting 1 mg of luciferase circular plasmid at 37°C for 4 hours (using BspQ I enzyme, purchased from Yeasen Biotechnology Co., Ltd.) (see Table 1 for digestion system details).

[0209] Scleavage reaction system

[0210] [Table 1]

[0211] Step (2): After the reaction is complete, add anhydrous ethanol and sodium acetate in sequence, V 消化反応生成物 :V 無水エタノール :V 3Mの酢酸ナトリウムAnhydrous ethanol and 3M sodium acetate were added in a volume ratio of 1:3:1, and allowed to stand at -20°C for 1 hour to precipitate. The precipitate was then collected by centrifugation at 12000 rpm.

[0212] Step (3): The precipitate from Step (2) was washed twice with 70% ethanol, and the resulting substance was dried at 55°C for 10 minutes. Then, 1.7 mL of sterile water for injection was added and dissolved.

[0213] The concentration of linear plasmids in the lysis solution was 500 ng / μL, the linearization rate was over 90%, and the purification and recovery efficiency was 85%. B) Production of Fluc mRNA, eGFP mRNA, and OVA mRNA 1) Co-transcription and capping reaction Using the Fluc DNA, eGFP DNA, and OVA DNA prepared in A) as templates, mRNA was synthesized by T7 RNA polymerase transcription using NTP solution (NTPs) and Cap1 cap analog (catalog number: 10678ES80, purchased from Yeasen Biotechnology Co., Ltd.) as starting materials. Details of the reaction system can be found in Table 2. The prepared reaction system was placed in a 37°C incubator and cultured with shaking for 3 hours to allow the reaction to proceed. The Cap1 cap analog is Cap1-GAG having an m7G(5')ppp(5')(2'-OMeA)pG structure, and its molecular formula is C 32 H 43 N 15 O 24 It was P4.

[0214] [Table 2]

[0215] 2) Digestion of template DNA DNase I (purchased from Yeasen Biotechnology Co., Ltd.) was added to the co-transcription capping reaction system after completing the reaction in step 1) above to obtain a linear plasmid with a final concentration of 1 U / μg. After homogeneous mixing, the system was centrifuged and digested at 37°C for 1 hour to obtain the co-transcription capping product.

[0216] 3) Purification by lithium chloride precipitation method The co-transcription capping product obtained in step 2) above was purified by lithium chloride precipitation, and the method was as follows:

[0217] Step (1): Addition of lithium chloride: A lithium chloride solution (purchased from Thermo Fisher Scientific) was added to the product from Step (2) above, resulting in a final concentration of 2.8 M. The mixture was then allowed to precipitate at low temperature for 2 hours.

[0218] Step (2): Sedimentation: The precipitate was held by high-speed centrifugation at 12000 rpm for 15 minutes. Step (3): Washing: The mRNA solution was obtained by washing twice with 75% ethanol and then dissolving it in sterile water for injection. The purified mRNA solution was stored at -80°C.

[0219] Example 3: Effects of various adjuvant lipid addition amounts on TLP-mRNA compositions ADOPE, DOTMA, DOPE, and YK-1202 were weighed in the proportions shown in Table 3 and dissolved in ethanol to prepare an ethanol-lipid complex solution (total lipid concentration: 266 mM). The ethanol-lipid complex solution was quickly added to sterile enzyme-free water being stirred at a rotational speed of 120 rpm using the ethanol injection method, stirred at room temperature for 30 minutes, and the resulting mixture was filtered through a polycarbonate membrane with a pore size of 450 nm to obtain a lipid composition solution, which was stored at 4°C to 8°C. mRNA was diluted with buffer to obtain an aqueous mRNA solution (mRNA concentration: 0.5 mg / mL, buffer: 10 mM HEPES buffer containing 0.1 mM EDTA). An aqueous sodium chloride solution (0.9% w / w) was taken with a syringe and injected into the prepared mRNA aqueous solution to obtain a mixed solution of mRNA and sodium chloride. The lipid complex solution was collected using a syringe, injected into the above-mentioned mixture of mRNA and sodium chloride, vortexed for 30 seconds, and incubated at room temperature for 10 minutes to obtain a TLP pharmaceutical composition (final RNA concentration: 100 μg / mL) with or without adjuvant lipids, which was then stored at 4°C to 8°C. Here, the amount of mRNA and lipid complex solution used was such that the charge ratio of the TLP-mRNA composition was 1:2, and the charge ratio was calculated using the following formula.

[0220] The charge ratio of the mRNA-TLP composition = (moles of positive charge carried by permanent cationic lipids - molars of negative charge carried by permanent anionic lipids) / (weight of mRNA (g) ÷ average molecular weight of bases 330 (g / mol)).

[0221] mRNA-TLP compositions were diluted with an aqueous sodium chloride solution (0.9% w / w) in a 1:5 volume ratio, and the particle size and dispersion index (PDI) were measured using a Malvern laser particle size analyzer utilizing dynamic light scattering.

[0222] [Table 3]

[0223] The results showed that when the permanent cationic lipid DOTMA component in the TLP composition was partially substituted with YK-1202 at concentrations of 2.5%, 5.0%, 10.0%, 15.0%, and 20.0%, the particle size and PDI of the resulting TLP pharmaceutical composition were both within the acceptable range (particle size: 220-500 nm, PDI value < 0.5). In the following examples, a 10% adjuvant lipid ratio is adopted.

[0224] Example 4: Effects of adding various adjuvant lipids to TLP-mRNA compositions

[0225] [Table 4] TIFF2026109612000042.tif217170

[0226] Following the manufacturing method of Example 3, the adjuvant lipid compounds in Table 4 were used to partially replace the proportion of DOTMA in the TLP composition by 10 mol%, i.e., the molar ratio of the permanent anionic lipid ADOPE, the permanent cationic lipid DOTMA, the neutral lipid DOPE, and the adjuvant lipid was 5:8:5:2 (i.e., in the lipid composition, the molar percentages of the permanent anionic lipid, the permanent cationic lipid, the neutral lipid, and the adjuvant lipid were 25%, 40%, 25%, and 10%, respectively), and an mRNA-TLP pharmaceutical composition was produced, the results of which are shown in Table 5.

[0227] [Table 5] TIFF2026109612000044.tif51170

[0228] The results showed that adjuvant lipids YK-1201 to YK-1208 and compounds 23, 63 to 15, and TMX-201 in Table 4 can all be used to produce mRNA compositions with suitable particle size and PDI (particle size controlled within 220 to 500 nm and PDI less than 0.5) according to the manufacturing method in Example 3.

[0229] Example 5: Protein expression experiment in mice of mRNA-TLP composition prepared with adjuvant lipids. Fluc-mRNA-TLP compositions with or without adjuvant lipids prepared in Example 4 were injected via tail vein into 4-6 week old, 17-19 g female BALB / c albino mice (dosage: approximately 20 μg of Fluc-mRNA / mouse). Six hours after administration, the mice were intraperitoneally injected with a fluorescent imaging substrate. The mice were allowed to move freely for 5 minutes, after which the total radiation intensity of the proteins expressed by the mRNA carried by the mRNA composition (corresponding to the expression intensity of the fluorescent protein, i.e., the amount of protein expressed) was detected using the IVIS Spectrum small animal in vivo imaging system. After sample collection, the mice were killed by cervical dislocation and dissected to accurately isolate the internal organs of the mice: liver and spleen. The total radiation intensity of proteins expressed by Fluc-mRNA in various organs of the mice was detected using the IVIS Spectrum small animal in vivo imaging system (corresponding to the expression intensity of the fluorescent protein, i.e., the amount of protein expressed). The results of in vivo imaging of mice and detection of protein expression in various organs are shown in Table 6 and Figure 1.

[0230] [Table 6] TIFF2026109612000046.tif83170

[0231] By adding the adjuvant lipid of the present invention to the mRNA-TLP composition, mRNA can be efficiently delivered to the spleen of animals, and it was found that the delivery effect is significantly improved compared to the TLP composition without the adjuvant lipid.

[0232] Compared to the case in which conventional adjuvant lipids (compounds 23, 63-15, and TMX-201) were added, the compositions containing YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 of the present invention significantly improved the total radiation intensity of the spleen and the total radiation intensity of the living body. For example, the composition containing YK-1204 showed total radiation intensity of the spleen that was 4.4 times, 4.7 times, and 4.3 times higher than the compositions containing compounds 23, 63-15, and TMX-201, respectively, and radiation intensity of the living body that was 3.3 times, 3.5 times, and 2.9 times higher than the compositions containing compounds 23, 63-15, and TMX-201.

[0233] Compositions containing different adjuvant lipids having similar structures to those of the present invention showed significant differences in total radiation intensity in the spleen and total radiation intensity in the living body. For example, the composition containing YK-1206 showed total radiation intensity in the spleen and total radiation intensity in the living body that were 2.7 times and 3.3 times higher, respectively, than the composition containing YK-1201.

[0234] Example 6: Targeting study of mRNA-TLP composition with added adjuvant lipids in mouse spleen cells 1. The eGFP-mRNA-TLP compositions prepared in Example 3, with or without the adjuvant lipid, were injected via tail vein into female C57BL / 6 mice aged 4-6 weeks and weighing 17-19 g (dosage: approximately 80 μg of eGFP-mRNA / mouse). 24 hours after administration, the mice were killed by cervical vertebral dislocation, and their spleens were accurately isolated by dissection.

[0235] 2. Production of single cells 1) The separated spleen tissue was crushed, and the spleen tissue was subjected to a cell strainer to obtain single cells.

[0236] 2) Ten times the volume (4 mL) of erythrocyte lysate was added to dissolve and remove the erythrocytes from the tissue. 3) Count the cells, 5 × 10 6 The cells were collected in flow cytometry tubes (ensuring that each sample had the same number of cells).

[0237] 3. Detection of spleen tissue immune cells 1) 100 μL of surface antibody mix (details of the surface antibody mix components are shown in Table 7) was added to each single-cell suspension and incubated in the dark at room temperature for 15 minutes (a portion of the negative controls).

[0238] [Table 7]

[0239] 2) Add 2 mL of PBS, centrifuge at 500 g for 5 minutes, and discard the supernatant. 3) Cells were resuspended in 200 μL of PBS (after filtration through a 200-mesh nylon mesh) and loaded into a Cytoflex S flow cytometer for detection. The percentage of GFP content in each cell was analyzed. The detection order for each cell line is as follows:

[0240] GFP ratio in T cells: CD45 + →CD3 + →GFP + GFP ratio in B cells: CD45 + →CD3 - CD19 + →GFP + GFP ratio in NK cells: CD45 + →NK1.1 + →GFP + GFP ratio in cDC cells: CD45 + →F4 / 80 - CD11c + →GFP + GFP ratio in pDC cells:CD45 + →F4 / 80 - CD11c int CD317 + →GFP + GFP ratio in macrophages: CD45 + →F4 / 80 + →GFP + Experimental results: The percentage of eGFP-positive cells in mouse spleen cells is shown in Table 8.

[0241] [Table 8] TIFF2026109612000049.tif72170

[0242] Antigen-presenting cells (APCs) are a type of immune cell that can absorb and process antigens and present the processed antigens to lymphocytes. They include dendritic cells (DC cells), B lymphocytes, and macrophages, and play important roles in the human body, mainly in immune recognition, immune responses, and immunomodulation.

[0243] From the data in Table 8 and Figure 2, it was found that the mRNA-TLP composition produced with the adjuvant lipid of the present invention can significantly increase the proportion of antigen-presenting cells expressing antigens in the spleen, and can significantly increase the proportion of antigen-presenting cells expressing antigens in the spleen compared to the TLP-mRNA composition without adjuvant. For example, the mRNA-TLP composition produced with YK-1204 increased the percentage of eGFP-positive antigen-presenting cells in the spleen by 2.1 times, 1.7 times, 1.7 times, and 1.7 times, respectively, for B cells, cDC cells, pDC cells, and macrophages.

[0244] Compared to conventional adjuvants, the mRNA-TLP composition produced with the adjuvant lipid of the present invention significantly improved targeting of mouse spleen antigen-presenting cells. For example, the proportion of eGFP-positive B cells, cDC cells, pDC cells, and macrophages in the mouse spleen of YK-1204 was 1.9 times, 1.5 times, 1.5 times, and 1.7 times, respectively, compared to TMX-201.

[0245] The mRNA-TLP compositions prepared with different adjuvant lipids in this invention showed significant differences in targeting of mouse spleen antigen-presenting cells, with data for YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 being significantly higher than those for the other compounds. For example, the mRNA-TLP composition prepared with YK-1206 showed eGFP-positive cell percentages of B cells, cDC cells, pDC cells, and macrophages that were 2.2 times, 1.8 times, 1.7 times, and 1.9 times higher, respectively, than the mRNA-TLP composition prepared with YK-1203.

[0246] Example 7: Study on the maturation state of mouse spleen antigen-presenting cells using mRNA-TLP compositions prepared with adjuvant lipids. CD86 (differentiation cluster 86) is a molecule expressed on antigen-presenting cells that provides a co-stimulatory signal necessary for T cell activation and survival, and can indicate the maturation state of spleen antigen-presenting cells through the upregulation of CD86. In this example, based on the flow cytometry experiment of Example 5, an experiment to detect the upregulation of CD86 was added, and the surface antibody used was Brilliant Violet 510. TM The detection procedure for each cell line is as follows:

[0247] B cell CD86 ratio: CD45 + →CD3 - CD19 + →CD86 + NK cell CD86 ratio: CD45 + →NK1.1 + →CD86 + cDC cell CD86 ratio: CD45 + →F4 / 80 - CD11c + →CD86 + pDC cell CD86 percentage: CD45 + →F4 / 80 - CD11c int CD317 + →CD86 + Macrophage CD86 ratio: CD45 + →F4 / 80 + →CD86 + Experimental results: The percentage of CD86-positive cells in mouse spleen cells is shown in Table 9.

[0248] [Table 9]

[0249] mRNA-TLP compositions prepared with the adjuvant lipids of the present invention can significantly increase the percentage of CD86-positive antigen-presenting cells in the spleen. mRNA-TLP compositions prepared with the adjuvant lipids YK-1202, YK-1204~YK-1206, and YK-1208 of the present invention were found to significantly improve the maturation state of antigen-presenting cells in the mouse spleen compared to mRNA-TLP compositions without adjuvants. For example, mRNA-TLP compositions with YK-1205 added showed percentages of CD86-positive B cells, cDC cells, pDC cells, and macrophages in the mouse spleen to be 1.1 times, 1.5 times, 1.6 times, and 1.7 times, respectively, compared to the corresponding percentages of TLP without adjuvant lipids.

[0250] mRNA-TLP compositions prepared with the adjuvant lipids YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 of the present invention significantly improved the maturation state of mouse spleen antigen-presenting cells compared to adjuvants of the conventional technology. For example, the mRNA-TLP composition prepared with YK-1205 resulted in percentages of CD86-positive B cells, cDC cells, pDC cells, and macrophages in mouse spleens that were 1.3, 1.9, 1.7, and 1.4 times, respectively, compared to the corresponding percentages for TMX-201. [Figure 3] mRNA-TLP compositions prepared with various adjuvant lipids provided in this invention showed significant differences in the percentage of CD86-positive cells in spleen antigen-presenting cells. For example, the mRNA-TLP composition prepared with YK-1206 resulted in a CD86-positive cell percentage in mouse spleen B cells that was 1.8 times higher than the corresponding percentage for YK-1207.

[0251] Example 8: Study on T cell activation in mouse spleen using mRNA-TLP composition prepared with adjuvant lipids. CD69 is a marker of T cell activation and one of the earliest markers to be upregulated after T cell activation. Splenic T cell activation is indicated by the upregulation of CD69. In this example, based on the flow cytometry experiment of Example 5, an additional detection experiment for CD69 upregulation was performed. The surface antibody used was APC anti-mouse CD69, and the detection procedure for each cell line is as follows.

[0252] Percentage of T cell CD69: CD45 + →CD3 + →CD69 + Experimental results: The percentage of CD86-positive cells in mouse spleen T cells is shown in Table 10.

[0253] [Table 10]

[0254] The mRNA-TLP compositions prepared with the adjuvant lipids of the present invention were all able to significantly increase the proportion of CD69-positive cells in spleen T cells, and compared to mRNA-TLP compositions without adjuvant lipids, they significantly increased the percentage of CD69-positive cells in spleen T cells. For example, compared to TLP without adjuvant, mRNA-TLP compositions prepared with the adjuvant lipids YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 of the present invention showed significantly improved percentages of CD69-positive cells in spleen T cells, with the percentages being 1.5 times, 1.7 times, 1.7 times, 1.6 times, and 1.6 times, respectively, compared to TLP without adjuvant.

[0255] Compared to conventional adjuvant lipids, the mRNA-TLP composition produced with the adjuvant lipid of the present invention significantly improved the activation state of T cells in mouse spleens. For example, the mRNA-TLP composition produced with YK-1205 showed a 2.5-fold increase in the percentage of CD69-positive T cells in mouse spleens compared to the mRNA-TLP composition produced with TMX-201. [Figure 4] The mRNA-TLP compositions produced with various adjuvant lipids according to the present invention showed significant differences in the percentage of CD69-positive T cells in mouse spleens. For example, the percentage of CD69-positive T cells in mouse spleens produced with mRNA-TLP compositions YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 was significantly higher than that of the other compositions. Among these, the percentage of CD69-positive T cells was highest after administration of the mRNA-TLP composition produced with YK-1205, which was 1.4 times higher than that of the lowest-performing composition, YK-1207.

[0256] Example 9: Stimulating effect of mRNA-TLP composition containing adjuvant lipids on cytokines IFN-γ and IL-12 Interferon-γ (IFN-γ) is an important cytokine primarily produced by activated T cells and natural killer (NK) cells. It plays a crucial role in immune responses, possessing various functions such as antiviral, antitumor, immunomodulatory, and pro-inflammatory responses, and can be used to treat a variety of diseases. Interleukin-12 (IL-12) is a multifunctional cytokine produced by dendritic cells, macrophages, B lymphocytes, and other antigen-presenting cells, playing a vital role in regulating immune responses, promoting Th1 immunity, enhancing cytotoxicity, and tumor immunity. By detecting the stimulation of cytokines IFN-γ and IL-12 by mRNA-TLP compositions containing adjuvant lipids, the ameliorative effect of the mRNA-TLP composition on innate immunity can be reflected.

[0257] Experimental process: Six hours after injecting the OVA-mRNA-TLP (40 μg) composition prepared in Example 4 into the tail vein of eight-week-old female C57BL / 6J mice, the mice were euthanized by removing their eyeballs, and their blood was collected to obtain as much serum as possible. The IFN-γ and IL-12 content in the serum was measured by ELISA, and mice injected with the same volume of blank lipid solution were simultaneously designated as a blank group.

[0258] ELISA measurement: Standard ELISA measurement was performed according to the manufacturer's instructions to detect mouse IFN-γ and IL-12 in mouse serum.

[0259] [Table 11]

[0260] Experimental results: As shown in Table 11 and Figure 5, compared to mRNA-TLP compositions without adjuvant lipids, mRNA-TLP compositions containing adjuvant lipids significantly increased serum IFN-γ and IL-12 cytokines 6 hours after injection. Among these, IFN-γ and IL-12 stimulated by YK-1202-TLP were 1.8 times and 1.5 times, respectively, compared to mRNA-TLP compositions without adjuvant lipids; YK-1204-TLP was 2.7 times and 3.1 times, respectively; YK-1205-TLP was 2.0 times and 1.8 times, respectively; and YK-1208-TLP was 2.1 times and 1.9 times, respectively.

[0261] Experimental results demonstrated that the adjuvant lipids of the present invention can initiate an immune stimulation program and significantly improve the innate immunity of the mRNA-TLP composition by detecting IFN-γ and IL-12 cytokines stimulated by the mRNA-TLP composition containing the adjuvant lipids.

[0262] Example 10: Therapeutic effect of an mRNA-TLP composition containing adjuvant lipids on a tumor-bearing mouse model. Experimental process: 1) Construction of the B16F10-OVA mouse model: Female C57BL / 6J mice 6-8 weeks old were acclimatized for at least 3 days prior to the study. The mice had free access to food and sterile water and were housed in an environment of 22°C ± 2°C and 55% ± 15% relative humidity with a 12-hour light-dark cycle. B16F10-OVA cells (MeisenCTCC) were cultured in complete medium at 5% CO2 and 37°C according to the instructions for B16F10-OVA (CTCC-001-0727). The cells were collected with 0.25% trypsin-EDTA, resuspended in Dulbecco's phosphate-buffered saline (DPBS), and then 2 × 10⁶ cells were collected. 5 Cells / 100μL / mouse (B16F10-OVA) were transplanted into Scalpel C57BL / 6J mice by subcutaneous (SC) injection to construct a subcutaneous B16F10-OVA tumor model, with a tumor volume of approximately 100mm².3 Vaccination was started when the target was reached.

[0263] 2) Vaccination: For the B16F10-OVA mouse model, C57BL / 6J mice selected for the group were vaccinated by tail vein injection on days 3, 7, 10, 12, 14, and 17, respectively, after cell injection (day 0), with OVA mRNA YK-1202-TLP, YK-1204-TLP, YK-1205-TLP, and YK-1208-TLP compositions (40 μg) containing TLR adjuvant lipids prepared according to Example 3 (each mouse received a single injection containing 40 μg of the therapeutic mRNA-OVA vaccine). Simultaneously, the same volume of OVA mRNA-TLP mice without adjuvant were set up as a control group, and eight mice were placed in each parallel group.

[0264] 3) Measurement of tumor size and mouse survival rate: From day 7 after tumor inoculation, the diameter of the tumor was measured three times a week. Formula: V(mm) 3 ) = x × y 2 The tumor volume of C57BL / 6J mice was calculated according to the formula / 2, in units of mm, where V represents the tumor volume, x represents the major axis of the tumor, and y represents the minor axis of the tumor. Simultaneously, the body weight changes of C57BL / 6J mice were recorded three times a week using an electronic balance, and survival rates were statistically analyzed.

[0265] Experimental results: As shown in Figure 6 and Table 12, on day 10 after tumor inoculation, mice in the blank lipid control group and the OVA mRNA-TLP composition group without adjuvant entered a phase of rapid tumor growth, but the growth rate of the TLP group was much lower than that of the blank control group. Correspondingly, the OVA mRNA pharmaceutical composition groups with added adjuvant lipids all showed a significant delay in tumor growth. From day 14 onwards, compared to the TLP group, the mRNA-TLP pharmaceutical compositions with added adjuvant lipids showed stronger tumor growth inhibition in terms of tumor volume, and among them, the mRNA-TLP groups produced with YK-1204 and YK-1208 showed relatively superior tumor inhibitory effects.

[0266] [Table 12]

[0267] Regarding survival rates, as shown in Figure 7, the blank lipid control group died from day 17 after tumor inoculation, with all mice dying on day 19. The adjuvant-free TLP group died from day 19, with all mice dying on day 27. The mRNA-TLP drug compositions with adjuvant lipids YK-1202, YK-1204, YK-1205, and YK-1208 added resulted in death from days 23, 25, 25, and 30, respectively, with all mice dying on days 31, 43, 37, and 45, respectively. It was found that the addition of adjuvant lipids significantly extended the survival time of the mice.

[0268] Example 11: Effects of various amounts of adjuvant lipids on LNP-mRNA compositions This example investigated the effect of adjuvant lipids on LNP compositions.

[0269] Experimental process: YK-009, YK-1202, DSPC, cholesterol, and DMG-PEG2000 were weighed according to the ratios in Table 12 and dissolved in ethanol to prepare an ethanol-lipid solution. eGFP-mRNA was diluted with citrate buffer (pH=4~5) to obtain an aqueous mRNA solution. Using a microfluidic device, the ethanol-lipid solution was mixed with the Fluc mRNA aqueous solutions prepared with the different cap structures described above at a flow rate of 10 mL / min in a 1:3 volume ratio to produce LNPs with a total lipid to mRNA weight ratio of approximately 15:1. The obtained liposomes were diluted 10-fold with PBS and ultrafiltered through a 300 kDa ultrafiltration tube to remove ethanol. The solution was immobilized to a predetermined volume with PBS, and finally the lipid nanoparticles were filtered through a 0.2 μm sterile filter to obtain LNP pharmaceutical compositions with or without adjuvant lipids. Particle size and polydispersity index (PDI) were measured using a Malvern laser particle size analyzer with dynamic light scattering. A 10 μL liposome solution was collected, diluted to 1 mL with RNase-free deionized water, and added to the sample pool. Measurements were repeated three times for each sample. Measurement conditions: scattering angle was 90° and temperature was 25°C. The encapsulation rate of lipid nanoparticles was determined using the Quant itRibogreenRNA quantitative measurement kit (ThermoFisherScientific, UK) as instructed by the manufacturer, and the results are shown in Table 13.

[0270] [Table 13]

[0271] Results: When the cationic lipid component YK-009 of the LNP composition was partially substituted with YK-1202 at concentrations of 2.5%, 5.0%, 10.0%, 15.0%, and 20.0%, the resulting LNP pharmaceutical compositions showed that the particle size, PDI, and encapsulation efficiency were all within the acceptable range (particle size < 200 nm, PDI < 0.3, encapsulation rate > 85%). When the adjuvant content was 10 mol%, the particle size, PDI, and encapsulation rate all reached their optimal state, and then the adjuvant lipids were studied at an adjuvant lipid ratio of 10%.

[0272] Example 12: Effects of various adjuvant lipids on LNP-mRNA compositions Experimental process: According to the manufacturing method of Example 11, the adjuvant lipid compounds in Table 4 were used to partially replace the proportion of YK-009 in the LNP composition by 10 mol%, i.e., the Fluc-mRNA-LNP pharmaceutical composition was prepared using YK-009, DSPC, cholesterol, DMG-PEG2000, and adjuvant lipids in a molar ratio of 40:10:38.5:1.5:10. Evaluation Method: First, particle size, polydispersity index (PDI), and encapsulation rate were measured according to the method of Example 11. Then, the drug composition was added to the cell culture medium of a 96-well plate, and incubation was continued for 24 hours. The corresponding reagents were added according to the instructions of the Gaussia Luciferase Assay Kit, and the fluorescence expression intensity of each well was detected using an IVIS fluorescence detection system. Finally, 10 μL of CCK-8 solution was added to each well of the well plate that had been incubated for 24 hours, and the culture plate was incubated in an incubator for 1 hour. Cell viability was then detected by measuring the absorbance at 450 nm with a microplate reader, and the results are shown in Table 14.

[0273] [Table 14]

[0274] Results: The adjuvant lipids YK-1201 to 1208 of this application, as well as the adjuvant lipids 63-15 and TMX-201 disclosed in the prior art, all demonstrated that good mRNA-LNP compositions could be produced by replacing YK-009 at a ratio of 10%. All lipid nanoparticles had a particle size of 67 to 88 nm, a PDI value of 0.08 to 0.13, and an encapsulation rate of 90% or higher for all.

[0275] However, there were significant differences in relative fluorescence intensity (mRNA translation efficiency) and cell viability (cytotoxicity) of the LNP compositions produced as described above. The mRNA-LNP compositions produced with YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 showed significantly higher relative fluorescence intensity than the YK-1201, YK-1203, and YK-1207 groups, and simultaneously compared to the adjuvant lipid-free LNP group and 63-15. The mRNA-LNP compositions produced with YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 showed significantly higher cytotoxicity than the mRNA-LNP group with TMX-201 added, and significantly lower cytotoxicity than the YK-1201, YK-1203, and YK-1207 groups. This level of toxicity was comparable to the mRNA-LNP group without adjuvant lipids and the mRNA-LNP group with 63-15 and TMX-201 added.

[0276] Example 13: Expression of LNP-mRNA compositions with various adjuvant lipids in animals Experimental process: A 10 μg LNP preparation of Fluc-mRNA containing various adjuvant lipids, prepared according to Example 11, was injected via the tail muscle into 4-6 week old, 17-19 g female BALB / C mice. At a specific time point (6 hours) after administration, the mice were intraperitoneally injected with a fluorescent imaging substrate. The mice were allowed to move freely for 5 minutes, and then the average emission intensity (corresponding to fluorescence expression intensity) of the protein expressed by the mRNA carried by the mouse LNP was detected using the IVIS Spectrum small animal in vivo imaging system.

[0277] After sample collection, the mice were euthanized with carbon dioxide and dissected to accurately isolate internal organs such as the liver, spleen, and lungs. The total radiation intensity (equivalent to fluorescence intensity) of proteins expressed by mRNA carried by LNPs in various organs of the mice was detected by IVIS Spectrum small animal in vivo imaging, and the results of in vivo imaging detection in mice are shown in Table 15.

[0278] [Table 15]

[0279] Experimental results: The mRNA carried by LNP preparations manufactured with YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 showed significantly increased mRNA expression levels in the injection site, abdominal cavity, liver, and spleen of mice compared to LNP without adjuvant lipids and LNP with TMX-201, a representative adjuvant lipid of conventional technology. For example, in the mRNA-LNP group with YK-1206, the expression level at the injection site of mice reached 1.7 times that of LNP without adjuvant lipids, and the expression in the spleen of mice reached 1.8 times that of LNP with TMX-201.

[0280] The presence of a large number of APC cells in the spleen and muscles (injection site) and the resulting increase in mRNA expression in these areas allowed the mRNA vaccine to rapidly induce an immune response and generate antibodies in the body. This significantly improved the preventive and therapeutic effects without altering the vaccine's components, which has clinical significance.

[0281] Furthermore, the expression of Fluc mRNA-containing LNP preparations with added adjuvant lipids varied significantly in various organs of mice. All LNP preparations, including YK-1202, YK-1204, YK-1205, YK-1206, YK-1208, and TMX-201, which were produced without the addition of adjuvant lipids, were expressed in the liver and spleen, but not in the lungs.

[0282] Compared to the structurally similar adjuvant lipid YK-1201, mRNA-LNP preparations produced with YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 significantly increased mRNA expression levels in the liver and spleen of mice. For example, the LNP preparation containing YK-1206 showed mRNA expression levels at the injection site to be 2.8 times higher than that of the LNP preparation containing YK-1201, and expression levels in the spleen to be 3.8 times higher.

[0283] Example 14: Stimulating effect of mRNA-LNP composition containing adjuvant lipids on cytokines IFN-γ and IL-12 Experimental process: Eight-week-old female C57BL / 6J mice were intramuscularly injected with an OVA-mRNA-LNP (5 μg) composition prepared according to Example 11. Six hours later, the mice's eyes were removed, they were euthanized, and as much serum as possible was collected. The IFN-γ and IL-12 content in the serum was measured by ELISA. Simultaneously, mice injected with the same volume of blank lipid solution were designated as a blank group.

[0284] ELISA measurement: Standard ELISA measurement was performed according to the manufacturer's instructions to detect mouse IFN-γ and IL-12 in mouse serum.

[0285] [Table 16]

[0286] Experimental results: As shown in Table 16 and Figure 8, mRNA-LNP compositions containing adjuvant lipids increased both IFN-γ and IL-12 cytokines in serum 6 hours after injection compared with mRNA-LNP compositions without adjuvant lipids. Of these, the serum content of the cytokine IL-12 stimulated by mRNA-LNP compositions containing YK-1202-LNP, YK-1204-LNP, YK-1206-LNP, and YK-1208-LNP was 1.3 times, 1.6 times, 2.1 times, and 1.5 times higher than that of mRNA-LNP compositions without adjuvant lipids. The serum content of the cytokine IFN-γ stimulated by mRNA-LNP compositions containing YK-1202-LNP, YK-1204-LNP, YK-1206-LNP, and YK-1208-LNP was 1.5 times, 2.9 times, 1.8 times, and 5.3 times higher than that of mRNA-LNP compositions without adjuvant lipids.

[0287] The experimental results, by detecting IFN-γ and IL-12 cytokines stimulated by the mRNA-LNP composition containing the adjuvant lipid, demonstrated that the adjuvant lipid of the present invention can initiate an immune stimulation program and significantly improve the innate immunity of the mRNA-LNP composition.

[0288] Example 15: Therapeutic effect of an mRNA-LNP composition containing adjuvant lipids on a tumor-bearing mouse model. Experimental process: 1) Construction of the MC38-OVA mouse model: Female C57BL / 6J mice aged 6-8 weeks were acclimated for at least 3 days prior to the study. The mice had free access to food and sterile water and were housed in an environment of 22°C ± 2°C and 55% ± 15% relative humidity with a 12-hour light-dark cycle. MC38-OVA cells were cultured in complete medium (RPMI-1640 medium with 10% fetal bovine serum (FBS) added) at 37°C and 5% CO2 according to the manufacturer's instructions. Cells were collected with 0.25% trypsin-EDTA, resuspended in Dulbecco's phosphate-buffered saline (DPBS), and then 1.5 × 10⁶ cells. 6 Cells / 100μL / mouse (MC38-OVA) were subcutaneously injected (SC) into C57BL / 6J mice to construct a subcutaneous MC38-OVA tumor model, with a tumor volume of approximately 50mm². 3 Vaccination was started when the target was reached.

[0289] 2) Vaccination: C57BL / 6J mice registered in the group were vaccinated by intramuscular injection of OVA mRNA-YK-1202-LNP, YK-1204-LNP, YK-1206-LNP, and YK-1208-LNP compositions containing the adjuvant lipids prepared in Example 11, on days 3, 7, 10, 12, 15, and 19, respectively, after cell injection (day 0). (Each mouse was injected with a vaccine containing 5 μg of the therapeutic mRNA-OVA each time.) Simultaneously, mice inoculated with the same volume of OVA mRNA-LNP without the adjuvant lipids were set up as a control group, and eight mice were placed in each group in parallel.

[0290] 3) Tumor size and mouse survival rate: The diameter of the tumor was measured three times a week starting 7 days after tumor inoculation. Formula: V(mm) 3 ) = x × y 2 The tumor volume of C57BL / 6J mice was calculated using the formula / 2, in units of mm, where V represents the tumor volume, x represents the major axis of the tumor, and y represents the minor axis of the tumor. Simultaneously, the body weight changes of C57BL / 6J mice were recorded three times a week using an electronic balance, and the survival rate was calculated.

[0291] Experimental results: As shown in Figure 9 and Table 17, on day 12 after tumor inoculation, mice in the blank lipid control group (Control Group) and those receiving OVA mRNA-LNP compositions without adjuvant lipids entered a rapid tumor growth phase. However, the difference between the adjuvant-free LNP group and the blank control group gradually widened over time. All OVA mRNA-LNP compositions with added adjuvant lipids significantly slowed tumor growth. From day 14 onwards, the mRNA-LNP groups with YK-1202, YK-1204, YK-1206, and YK-1208 showed stronger tumor growth inhibition in tumor volume compared to the adjuvant-free LNP group, with the YK-1204 and YK-1208 groups exhibiting superior tumor inhibitory effects.

[0292] [Table 17]

[0293] In terms of survival rates, as shown in Figure 10, the blank lipid control group began dying from day 17 after tumor inoculation and all died by day 19. The adjuvant-free mRNA-LNP group began dying from day 21 and all died by day 30. The adjuvant-treated mRNA-LNP groups YK-1202, YK-1204, YK-1206, and YK-1208 began dying from days 25, 27, 31, and 29, respectively, and all died on days 33, 40, 41, and 42, respectively. It was found that the addition of the adjuvant lipids of the present invention significantly extended the survival time of mice.

[0294] The present invention designs a series of novel vaccine adjuvant lipids based on Toll-like receptor 7 and 8 agonists such as YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208, which have at least one of the following advantages compared to adjuvant lipids disclosed in the prior art.

[0295] 1. The chemical structure of the adjuvant lipid designed in this invention is different from the chemical structure of adjuvant lipids disclosed in the prior art and is a completely novel compound. 2. When the adjuvant lipid of the present invention is added to an mRNA-TLP composition, it significantly increases protein expression in mice and simultaneously exhibits significant spleen targeting. Compared to conventional adjuvants, the mRNA-TLP composition produced with the adjuvant lipid of the present invention can significantly increase protein expression in live mice and the spleen.

[0296] 3. The mRNA-TLP composition produced with the adjuvant lipid of the present invention significantly increases the percentage of antigen-presenting cells (e.g., B cells, pDC cells, cDC cells, and macrophages) expressing the antigen in the spleen. Compared with conventional adjuvant lipids, the mRNA-TLP composition produced with the adjuvant lipid of the present invention significantly improves targeting of mouse spleen antigen-presenting cells.

[0297] 4. The mRNA-TLP composition produced with the adjuvant lipid of the present invention can significantly increase the percentage of CD86-positive antigen-presenting cells in the spleen. Compared with conventional adjuvant lipids, the mRNA-TLP composition produced with the adjuvant lipid of the present invention significantly improves the maturation state of mouse spleen antigen-presenting cells.

[0298] 5. The mRNA-TLP composition produced with the adjuvant lipid of the present invention can significantly increase the percentage of CD69-positive T cells in the spleen. Compared with conventional adjuvant lipids, the mRNA-TLP composition produced with the adjuvant lipid of the present invention significantly improves the activation state of mouse spleen T cells.

[0299] 6. The mRNA-TLP composition prepared by adding the adjuvant lipid of the present invention significantly inhibits tumor growth and significantly extends the survival period of tumor-bearing experimental animals in animal experiments compared to mRNA-TLP compositions that do not contain the adjuvant lipid.

[0300] 7. The mRNA-LNP composition prepared by adding the adjuvant lipid of the present invention (2.5% to 20 mol%) has good particle size (<200 nm) and uniform particle distribution (PDI (<0.3)). The mRNA-LNP composition prepared by adding the adjuvant lipid of the present invention significantly improves mRNA translation efficiency, significantly reduces cytotoxicity, significantly increases protein expression in the injection site, abdominal cavity, liver, and spleen of mice, significantly inhibits tumor growth, and significantly extends the survival period of tumor-bearing experimental animals in animal experiments.

[0301] The present invention has been described in detail above, but this is intended to enable those skilled in the art to understand and implement the present invention, and this does not limit the scope of protection of the present invention. All equivalent changes or modifications in the spirit and essence of the present invention should be included within the scope of protection of the present invention.

Claims

1. A compound represented by formula (I), or a pharmaceutically acceptable salt thereof. 【Chemistry 1】 (L １ C １－１０ Alkylene or C １－４ Alkylene-C ６－１０ Aryl-C １－４ It is alkylene, M １ is -NH- or -OC(O)HN-, L ２ is C １－８ alkylene, -R ３ａ C(O)OR ４ａ -, 【Chemistry 2】 And here, R ３ａ , R ３ｃ , R ３ｄ , R ４ａ , R ４ｃ , R ４ｄ and R ５ C is independent １－８ It is alkylene, R １ C １－５ Alkyl or C １－６ C substituted with alkoxy １－５ It is alkyl, R ２ teeth, 【Transformation 3】 And, Here, L ３ and L ４ C is independent １－７ It is alkylene, R ６ and R ７ C is independent ７－２８ It is alkyl, R ８ C １－７ It is alkylene, R ９ and R １０ C is independent １０－２０ Alkyl or C １０－２０ It is alkinyl.

2. The compound represented by formula (I) is (1) L １ C １－６ It is a linear alkylene, L ２ C １－６ Linear alkylene C(O)OC １－４ Linear alkylenes ａ or 【Chemistry 4】 And the a-terminus is R ２ It is connected to R ３ｄ , R ４ｄ and R ５ C is independent １－４ It is a linear alkylene, Or, L １ C １－４ Linear alkylene-C ６－１０ Aryl-C １－４ It is a linear alkylene, L ２ C １－４ Linear alkylene or C １－６ Linear alkylene C(O)OC １－４ Linear alkylenes ａ And the a-terminus is R ２ Conditions linked to, (2) R １ C １－５ Conditions for being a linear alkyl group, (3) L ３ C １－４ Conditions for being a linear alkylene, (4) L ４ C ４－６ Conditions for being a linear alkylene, (5) Caution ６ C ８－１２ Conditions for being a linear alkyl group, (6) R ７ C １６－２０ Conditions for being a branched-chain alkyl group, (7) R ８ C １－４ Conditions for being a linear alkylene, (8) R ９ and R １０ C is independent １６－２０ Conditions for being an Alkenil, A compound represented by formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that it satisfies the condition of being selected from any one or at least two combinations of the group consisting of the following.

3. The compound represented by formula (I) is (1) L １ is, -(CH ２ ) ４ -ien-CH ２ C((CH ３ ) ２ ) - ｂ or 【Transformation 5】 The b-terminus is M １ It is connected to, (2) M １ It is -NH-, (3)L ２ は、-(CH ２ ) ２ -、-(CH ２ ) ３ C(O)O(CH ２ ) ２ - ａ 、-(CH ２ ) ５ C(O)O(CH ２ ) ２ - ａ 、 【Transformation 6】 And the a-terminus is R ２ It is connected to, (4) R １ is -(CH ２ ) ３ CH ３ or -CH ２ OCH ２ CH ３ and (5) Caution ２ teeth, 【Transformation 7】 The condition, A compound represented by formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that it satisfies the condition of being selected from any one or at least two combinations of the group consisting of the following.

4. L １ is, -(CH ２ ) ４ - and L ２ is, -(CH ２ ) ５ C(O)O(CH ２ ) ２ - ａ or 【Transformation 8】 And the a-terminus is R ２ It is connected to, Or, L １ teeth, 【Chemistry 9】 And L ２ is, -(CH ２ ) ２ -, - (CH ２ ) ３ C(O)O(CH ２ ) ２ - ａ or - (CH ２ ) ５ C(O)O(CH ２ ) ２ - ａ And the a-terminus is R ２ A compound represented by formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, characterized by being linked to a compound.

5. The compound represented by formula (I) is one of the following compounds, as described in claim 1, or a pharmaceutically acceptable salt thereof. 【Chemistry 10】 【change】

6. A lipid composition comprising substance Z, wherein substance Z is a compound represented by formula (I) as described in claim 1, or a pharmaceutically acceptable salt thereof.

7. The lipid composition according to claim 6, characterized in that the molar percentage of substance Z in the lipid composition is 2.5 to 20%.

8. The lipid composition according to claim 7, characterized in that the molar percentage of substance Z in the lipid composition is 5%, 10%, or 15%.

9. The lipid composition according to claim 6, characterized by comprising the substance Z, a permanent anionic lipid, a permanent cationic lipid, and a neutral lipid.

10. The lipid composition is (1) The permanent anionic lipid is a condition selected from one or at least two combinations of the group consisting of 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylthioureido)ethyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylureido)ethyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-propylureido)ethyl) phosphate, and (R)-2,3-bis(oleoyloxy)propyl-(2-(3-butylureido)ethyl) phosphate and salts thereof, (2) The permanent cationic lipid is a condition selected from one or at least two combinations of the group consisting of 1,2-bisoctadeceneoxy-3-methylammoniumpropane, (2,3-dioleyloxypropyl)trimethylammonium, and salts thereof. (3) The neutral lipid is selected from one or at least two combinations of the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphocholine, and salts thereof. (4) The condition that the ratio of the molar percentages of substance Z, permanent anionic lipids, permanent cationic lipids and neutral lipids is (2.5-20):(10-33):(20-60):(20-40), The lipid composition according to claim 9, characterized in that it satisfies the condition of being selected from any one or at least two combinations of the group consisting of the following.

11. The lipid composition is (1) The permanent anionic lipid is 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) sodium phosphate, (2) The condition that the molar percentage of the permanent anionic lipid in the lipid composition is 25%, (3) The condition that the permanent cationic lipid is a chloride salt of 1,2-dioctadeceneoxy-3-methylammoniumpropane, (4) The condition that the molar percentage of the permanent cationic lipid in the lipid composition is 30%, 35%, 40%, 45%, or 47.5%, (5) The neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, (6) The condition that the molar percentage of the neutral lipid in the lipid composition is 25%, The lipid composition according to claim 9, characterized in that it satisfies the condition of being selected from any one or at least two combinations of the group consisting of the following.

12. The lipid composition according to claim 6, characterized in that it comprises the substance Z, a cationic lipid, a neutral lipid, a structural lipid, and a polymer-conjugated lipid.

13. The lipid composition according to claim 12, characterized in that the molar ratio of substance Z, cationic lipid, neutral lipid, structural lipid and polymer-conjugated lipid is (2.5-20):(25-75):(5-25):(15-65):(0.5-10).

14. The cationic lipid is (1) A compound represented by formula (II), or a pharmaceutically acceptable salt thereof, where G １ is C １～６ It is alkylene, G ２ is C ２～８ It is alkylene, G ３ is C １～３ It is alkylene, L １ is C ６～１５ It is a linear alkyl group, L ２ is C １２～２５ A branched-chain alkyl compound, 【Chemistry 11】 (2) A compound represented by formula (III), or a pharmaceutically acceptable salt thereof, where G １ is C ２～８ It is alkylene, G ２ is C ２～８ It is alkylene, L １ is -C(O)O- or -OC(O)-, L ２ is -C(O)O- or -OC(O)-, R １ is C ６～２５ It is a linear or branched alkyl group, R ２ is C ６～２５ It is a linear or branched alkyl group, G ３ is HO(CH ２ ) ２ - or HO(CH ２ ) ３ - and G ４ is HO(CH ２ ) ２ - or HO(CH ２ ) ３ - and L is (CH ２ ) ２ -, - (CH ２ ) ３ - or - (CH ２ ) ４ - A compound, 【Chemistry 12】 (3) A compound represented by formula (IV), or a pharmaceutically acceptable salt thereof, where G １ is C １～６ It is alkylene, G ２ is C ２～８ It is alkylene, R １ is C ６～２０ It is a linear or branched alkyl group, R ２ is C １２～２５ It is a branched alkyl group, G ３ is HO(CH ２ ) ２ N(CH ３ ) (CH ２ ) ２ -, HO(CH ２ ) ２ N(CH ２ CH ３ ) (CH ２ ) ２ -, (HO(CH ２ ) ２ ) ２ N(CH ２ ) ２ - , CH ３ O(CH ２ ) ２ N(CH ３ ) (CH ２ ) ２ -, (CH ３ ) ２ N(CH ２ ) ３ SC(O)O(CH ２ ) ２ -, (CH ３ ) ２ N(CH ２ ) ３ SC(O)-, CH ３ NH(CH ２ ) ２ N(CH ３ ) (CH ２ ) ２ - or CH ３ CH ２ NH(CH ２ ) ２ - A compound, 【Chemistry 13】 (4) A compound represented by formula (V), or a pharmaceutically acceptable salt thereof, where G １ is C １～８ It is alkylene, G ２ is C ２～８ It is alkylene, R １ is C ６～２５ It is a linear or branched alkyl group, R ２ is C １２～２５ It is a linear or branched alkyl group, G ３ is HO(CH ２ ) ２ N(R) ３ )CH ２ CH(OH)CH ２ - and here, R ３ ha-CH ３ ien-CH ２ CH ３ or -CH ２ CH ２ Compounds that are OH groups, 【Chemistry 14】 (5) A compound represented by formula (VI), or a pharmaceutically acceptable salt thereof, where G １ and G ２ Each is independently C ６～１０ It is alkylene, G ３ is C １～１２ It is alkylene, R １ and R ２ Each is independently C ６～２４ Alkyl or C ６～２４ It is an alkenyl, R ３ is OR ５ , N, -C(=O)OR ４ -OC(=O)R ４ or -NR ５ C(=O)R ４ And R ４ is C １～１２ Hydrocarbil, R ５ is H or C １～６ A compound that is hydrocarbyl, 【Chemistry 15】 (6) A compound represented by formula (VII), or a pharmaceutically acceptable salt thereof, where R ４ ha- (CH ２ ) ｎ Q or - (CH ２ ) ｎ CHQR, where Q is -OR, -OH, -O(CH ２ ) ｎ N(R) ２ , -OC(O)R, -CX ３ , -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O) ２ R, -N(H)S(O) ２ R, -N(R)C(O)N(R) ２ , -N(H)C(O)N(R) ２ , -N(H)C(O)N(H)(R), -N(R)C(S)N(R) ２ , -N(H)C(S)N(R) ２ , -N(H)C(S)N(H)(R), -N(R)S(O) ２ R is a heterocyclyl, n is 1, 2, or 3, and R is C １－８ It is an alkyl group, and X is H or C １－８ Alkyl compounds, 【Chemistry 16】 (7) Compounds represented by formula (VIII), or compounds that are pharmaceutically acceptable salts thereof, 【Chemistry 17】 The lipid composition according to claim 12, characterized in that it is selected from any one or at least two combinations of the group consisting of each of the compounds.

15. The lipid composition according to claim 12, characterized in that the neutral lipid is selected from any one or at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, and sterol.

16. The lipid composition is (1) The cationic lipid is selected from one or at least two combinations of the group consisting of YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA. [Chemistry 18] (2) The neutral lipids are 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diundecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl Lu-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,Conditions selected from any one or at least two combinations of the group consisting of 2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoylphosphatidylglycerol, palmitoylphosphatidylethanolamine, distearoyl-phosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine, (3) The structural lipid is selected from one or at least two combinations of the group consisting of cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids. (4) The polymer-conjugated lipid is selected from any one or at least two combinations of the group consisting of distearoylphosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristoyl-rac-glycero-methoxypolyethylene glycol-2000, and methoxypoly(ethylene glycol)ditetradecylacetamide. The lipid composition according to claim 12, characterized in that it satisfies the condition of being selected from any one or at least two combinations of the group consisting of the following.

17. The lipid composition is (1) The cationic lipid is YK-009, (2) The condition that the molar percentage of the cationic lipid in the lipid composition is 30%, 35%, 40%, 45%, or 47.5%, (3) The neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), (4) The condition that the molar percentage of the neutral lipid in the lipid composition is 10%, (5) The condition that the structural lipid is cholesterol, (6) The condition that the molar percentage of the structural lipid in the lipid composition is 38.5%, (7) The polymer-conjugated lipid is 1,2-dimyristoyl-rac-glycero-methoxypolyethylene glycol-2000 (DMG-PEG2000), (8) The condition that the mole percentage of the polymer-conjugated lipid in the lipid composition is 1.5%, The lipid composition according to claim 16, characterized in that it satisfies the condition of being selected from any one or at least two combinations of the group consisting of the following.

18. The lipid composition according to claim 6, further comprising one or more cell-permeable peptides.

19. (A) A therapeutic or prophylactic agent comprising one or at least two combinations from the group consisting of nucleic acid molecules, small molecule compounds, polypeptides, or proteins, (B) Lipid composition according to any one of claims 9 to 17 A pharmaceutical composition characterized by containing the following.

20. The pharmaceutical composition according to claim 19, wherein the therapeutic or prophylactic agent is a nucleic acid molecule capable of encoding one or more antigens.

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