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

Abstract

The present invention discloses a vaccine adjuvant lipid compound based on Toll-like receptor agonists and its application, and specifically discloses the compound shown in formula (I), its stereoisomers, its N-oxides, its solvates, or its pharmaceutically acceptable salts. The mRNA-TLP composition prepared from the adjuvant lipid compound of the present invention significantly improves the targeting to mouse spleen antigen-presenting cells and can significantly inhibit tumor growth.

Description

Technical Field

[0001] The present invention relates to a vaccine adjuvant lipid compound based on Toll-like receptor agonists and its application. Background Art

[0002] In recent years, messenger ribonucleic acid (mRNA)-based cancer immunotherapy has attracted extensive attention due to its remarkable clinical potential. Compared with DNA vaccines, mRNA can induce transient expression of tumor antigens while avoiding the possibility of insertional mutagenesis. In addition, mRNA can enhance the therapeutic effect by inducing stronger humoral and cellular responses. Compared with traditional protein-based vaccines, mRNA vaccines have many advantages, such as biocompatibility, non-toxicity, and a simple and scalable production process. Inspired by the advantages of mRNA cancer vaccines, more than twenty mRNA-based immunotherapies have shown anti-tumor potential in preclinical and clinical studies.

[0003] To reduce the high innate immunogenicity and improve tolerance and translation efficiency, modified nucleosides such as 1-methylpseudouridine (m1ψ) are incorporated into the in vitro transcribed mRNA sequences. However, the problem with this modification is that it may impair innate immunogenicity. And immunogenicity is crucial for activating dendritic cells (DCs, the main cells of antigen-presenting cells).

[0004] Toll-like receptors (TLRs), as a family of transmembrane proteins, are a type of innate immune receptor that directly or indirectly responsible for detecting pathogen-associated molecular patterns (PAMPs). It can trigger a series of biosynthetic reactions by activating DCs cells, including the production of specific cytokines (such as tumor necrosis factor-α, TNF-α), which will enhance the expression of co-stimulatory molecules and the ability of antigen presentation. These molecular mechanisms are crucial for activating innate and adaptive immune responses and can effectively stimulate the transition from innate immunity to adaptive immunity. Incorporating lipid-modified TLRs into mRNA delivery systems to enhance the amplitude and duration of adaptive immune responses has been increasingly widely studied. Therefore, developing a new generation of vaccine adjuvant lipids based on Toll-like receptor 7 and 8 agonists is of great significance for improving mRNA cancer therapy. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the disadvantages that the vaccine adjuvant lipid compounds based on Toll-like receptor 7 and 8 agonists have a single structure in the prior art, and the prepared mRNA composition has poor targeting to antigen-presenting cells. Therefore, a vaccine adjuvant lipid compound based on Toll-like receptor agonists and its application are provided. The mRNA-TLP composition prepared from the adjuvant lipid compound in the present invention significantly improves the targeting to mouse spleen antigen-presenting cells and can significantly inhibit tumor growth.

[0006] The present invention provides a compound represented by formula (I), its stereoisomers, its N-oxides, its solvates, or its pharmaceutically acceptable salts:

[0007] ,

[0008] L1 is C 1-10 alkylene or C 1-4 alkylene-C 6-10 aryl-C 1-4 alkylene;

[0009] M1 is -NH-, -OC(O)HN-, -C(O)O- or -O-;

[0010] L2 is C 1-8 alkylene, -R 3a C(O)OR 4a -, -R 3b OR 4b -, or , 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;

[0011] R1 is C 1-5 alkyl or C 1-6 alkyl substituted by C 1-5 alkoxy;

[0012] R2 is or ,

[0013] wherein, L3 and L4 are independently C 1-7 alkylene;

[0014] R6 and R7 are independently C 7-28 alkyl;

[0015] R8 is C 1-7 alkylene, and R9 and R 10 are independently C 10-20 alkyl or C 10-20 alkenyl.

[0016] In some embodiments, L1 is C 1-6 linear alkylene, L2 is C 1-6 linear alkylene C(O)OC 1-4 linear alkylene a or , with the a end connected to R 2 ; R 3d , R 4d and R5 are independently C 1-4 linear alkylene;

[0017] Alternatively, L1 is C 1-4 linear alkylene-C 6-10 aryl-C 1-4 linear alkylene, L2 is C 1-4 linear alkylene or C 1-6 linear alkylene C(O)OC 1-4 linear alkylene a , with the a end connected to R 2 .

[0018] In some embodiments, R1 is C 1-5 linear alkyl.

[0019] In some embodiments, L3 is C 1-4 linear alkylene.

[0020] In some embodiments, L4 is C 4-6 linear alkylene.

[0021] In some embodiments, R6 is C 8-12 linear alkyl.

[0022] In some embodiments, R7 is C 16-20 branched alkyl.

[0023] In some embodiments, R8 is C 1-4 linear alkylene.

[0024] In some embodiments, R9 and R 10 are independently C 16-20 alkenyl.

[0025] In some embodiments, L1 is -(CH2)4-, -CH2C((CH3)2)- b or , with the b end connected to M1.

[0026] In some embodiments, M1 is -NH- or -OC(O)HN-, and the nitrogen terminus is connected to L2.

[0027] In some embodiments, M1 is -NH-.

[0028] In some embodiments, L2 is -(CH2)2-, -(CH2)3C(O)O(CH2)2- a , -(CH2)5C(O)O(CH2)2- a , or , and the a-terminus is connected to R2.

[0029] In some embodiments, L1 is -(CH2)4- and L2 is -(CH2)5C(O)O(CH2)2- a or , and the a-terminus is connected to R2;

[0030] or, L1 is , and L2 is -(CH2)2-, -(CH2)3C(O)O(CH2)2- a or -(CH2)5C(O)O(CH2)2- a , and the a-terminus is connected to R2.

[0031] In some embodiments, R1 is -(CH2)3CH3 or -CH2OCH2CH3.

[0032] In some embodiments, R2 is or .

[0033] In some embodiments, the compound of formula (I) is any one of the following compounds:

[0034] , , , , , , or .

[0035] The present invention provides a lipid composition comprising a substance Z, wherein the substance Z is a compound of formula (I) as described above, its stereoisomers, its N-oxides, its solvates, or its pharmaceutically acceptable salts.

[0036] In some embodiments, the molar percentage of the substance Z in the lipid composition is 2.5-20%.

[0037] In some embodiments, the molar percentage of the substance Z in the lipid composition is 5-15%.

[0038] In some embodiments, the molar percentage of the substance Z in the lipid composition is 10%.

[0039] In some embodiments, the lipid composition comprises the substance Z, a permanent anionic lipid, a permanent cationic lipid, and a neutral lipid. The ratio of the molar percentages of the 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).

[0040] In some embodiments, the permanent anionic lipid is selected from any one or a combination of at least two of the group consisting of 2-acetamidoethyl ((R)-2,3-bis(oleyloxy)propyl) phosphate, (R)-2,3-bis(oleyloxy)propyl-(2-(3-ethylthioureido)ethyl) phosphate, (R)-2,3-bis(oleyloxy)propyl-(2-(3-ethylureido)ethyl) phosphate, (R)-2,3-bis(oleyloxy)propyl-(2-(3-propylureido)ethyl) phosphate, (R)-2,3-bis(oleyloxy)propyl-(2-(3-butylureido)ethyl) phosphate, and their salts.

[0041] In some embodiments, the permanent anionic lipid is sodium 2-acetamidoethyl ((R)-2,3-bis(oleyloxy)propyl) phosphate.

[0042] In some embodiments, the molar percentage of the permanent anionic lipid in the lipid composition is 25%.

[0043] In some embodiments, the permanent cationic lipid is selected from any one or a combination of at least two of the group consisting of 1,2-dioctadecyloxy-3-methylammonium propane, (2,3-dioleyloxypropyl)trimethylammonium, and their salts.

[0044] In some embodiments, the permanent cationic lipid is 1,2-dioctadecyloxy-3-methylammonium propane (chloride).

[0045] In some embodiments, the molar percentage of the permanent cationic lipid in the lipid composition is 30-47.5%, such as 35%, 40%, or 45%.

[0046] In some embodiments, the neutral lipid is selected from any one or a combination of at least two of the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, distearoyl phosphatidylcholine, and their salts.

[0047] In some embodiments, the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0048] In some embodiments, the molar percentage of the neutral lipid in the lipid composition is 25%.

[0049] In some embodiments, the lipid composition includes substance Z, 2-acetamidoethyl ((R)-2,3-bis(oleyloxy)propyl) phosphate, 1,2-bis(octadecyloxy)-3-methylammonium propane (chloride), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; the molar percentages of substance Z, 2-acetamidoethyl ((R)-2,3-bis(oleyloxy)propyl) phosphate, 1,2-bis(octadecyloxy)-3-methylammonium propane (chloride), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine are (2.5-20):25:(30-47.5):25; preferably (5-15):25:(35-45):25, more preferably 10:25:40:25.

[0050] In some embodiments, the lipid composition includes substance Z, cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid. The ratio of the molar percentages of substance Z, cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid is preferably (2.5-20):(25-75):(5-25):(15-65):(0.5-10).

[0051] In some embodiments, the cationic lipid is selected from any one or a combination of at least two of the compounds consisting of the following (1) to (7):

[0052] (1) The compound shown in formula (II), its stereoisomer, its N-oxide, its solvate, or its pharmaceutically acceptable salt, wherein G1 is C 1～6 alkylene; G2 is C 2～8 alkylene; G3 is C 1～3 alkylene; L1 is C 6～15 straight-chain alkyl; L2 is C 12～25 branched-chain alkyl;

[0053] (II)

[0054] (2) The compound represented by formula (III), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts, wherein G1 is C 2~8 alkylene; G2 is C 2~8 alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6~25 straight-chain or branched alkyl; R2 is C 6~25 straight-chain or branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2-, -(CH2)3- or -(CH2)4-;

[0055] (III)

[0056] (3) The compound represented by formula (IV), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts, wherein: G1 is C 1~6 alkylene; G2 is C 2~8 alkylene; R1 is C 6~20 straight-chain or branched alkyl; R2 is C 12~25 branched alkyl; 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-;

[0057] (IV)

[0058] (4) The compound represented by formula (V), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts, wherein G1 is C 1~8 alkylene; G2 is C 2~8 alkylene; R1 is C 6~25 straight-chain or branched alkyl; R2 is C 12~25 straight-chain or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3, -CH2CH3 or -CH2CH2OH ;

[0059] (V)

[0060] (5) A compound represented by formula (VI), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts, wherein G 1 and G 2 are each independently C 6~10 alkylene; G 3 is C 1~12 alkylene; R 1 and R 2 are each independently C 6~24 alkyl or C 6~24 alkenyl; R 3 is OR 5 , N, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 is C 1~12 hydrocarbyl; R 5 is H or C 1~6 hydrocarbyl;

[0061] (VI)

[0062] (6) A compound represented by formula (VII), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts, wherein R4 is -(CH2) n Q and -(CH2) n CHQR; 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 a heterocycle; n is 1, 2 or 3; R is C 1-8 alkyl; X is H or C 1-8 alkyl;

[0063] (VII)

[0064] (7) A compound represented by formula (VIII), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts,

[0065] (VIII).

[0066] In some embodiments, the cationic lipid is selected from any one or a combination of at least two of the group consisting of YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA:

[0067] , ,

[0068] , , , 。

[0069] In some embodiments, the cationic lipid is YK-009.

[0070] In some embodiments, the molar percentage of the cationic lipid in the lipid composition is 30-47.5%, such as 35%, 40%, or 45%.

[0071] In some embodiments, the neutral lipid is selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and its derivatives.

[0072] In some embodiments, the neutral lipid is selected from any one or a combination of at least two of the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-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-doundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 DietherPC), 1-oleoyl-2-cholesteryl succinyl-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-diphytanoyl-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), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

[0073] In some embodiments, the neutral lipid is DOPE and / or DSPC, preferably DSPC.

[0074] In some embodiments, the molar percentage of the neutral lipid in the lipid composition is 10%.

[0075] In some embodiments, the structural lipid is selected from any one or a combination of at least two of the group consisting of cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

[0076] In some embodiments, the structural lipid is cholesterol.

[0077] In some embodiments, the molar percentage of the structural lipid in the lipid composition is 38.5%.

[0078] In some embodiments, the polymer-conjugated lipid is selected from any one or a combination of at least two of the group consisting of distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

[0079] In some embodiments, the polymer-conjugated lipid is DMG-PEG2000.

[0080] In some embodiments, the molar percentage of the polymer-conjugated lipid in the lipid composition is 1.5%.

[0081] In some embodiments, the lipid composition comprises the substance Z, the YK-009, DSPC, cholesterol, and DMG-PEG2000; the molar percentages of the substance Z, the YK-009, DSPC, cholesterol, and DMG-PEG2000 are preferably (2.5-20) : (30-47.5) : 10 : 38.5 : 1.5; more preferably 10 : 40 : 10 : 38.5 : 1.5.

[0082] In some embodiments, the lipid composition further comprises one or more cell-penetrating peptides.

[0083] The present invention provides a pharmaceutical composition, comprising: (A) a therapeutic agent and / or a prophylactic agent, which comprises any one or a combination of at least two of the group consisting of nucleic acid molecules, small molecule compounds, polypeptides, or proteins;

[0084] (B) the lipid composition as described above.

[0085] In some embodiments, the amounts of the therapeutic agent and / or the prophylactic agent and the lipid composition are such that the charge ratio of positive charge to negative charge in the pharmaceutical composition is 1 : (2-5), such as 1 : 2.

[0086] In some embodiments, the mass ratio of the lipid composition to the therapeutic or prophylactic agent is (12.5 - 25) : 1.

[0087] In some embodiments, the mass ratio of the lipid composition to the therapeutic or prophylactic agent is 15 : 1.

[0088] 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.

[0089] In some embodiments, the target organ or tissue is selected from any one or a combination of at least two of the group consisting of spleen, liver, lymph, muscle, and lung; preferably spleen or lymph.

[0090] In some embodiments, the antigen - presenting cell is selected from any one or a combination of at least two of the group consisting of B cells, NK cells, cDC cells, pDC cells, and macrophages.

[0091] In some embodiments, the therapeutic and / or prophylactic agent is a nucleic acid molecule capable of encoding one or more antigens.

[0092] In some embodiments, the antigen is a disease - related antigen, or the nucleic acid molecule or antigen is capable of eliciting an immune response against a disease - related antigen or cells expressing a disease - related antigen.

[0093] In some embodiments, the nucleic acid molecule is RNA encoding one or more antigens.

[0094] In some embodiments, the pharmaceutical composition further comprises at least one auxiliary component. The auxiliary component can be a pharmaceutical carrier, diluent, or excipient.

[0095] In some embodiments, the pharmaceutical composition further comprises one or more hydrophobic small molecules, permeability - enhancing molecules, carbohydrates, polymers, surface - modifying agents, functionalized lipids, or cytokines.

[0096] The present invention also provides the use of substance Z as described above, the lipid composition as described above, or the pharmaceutical composition as described above in the preparation of nucleic acid drugs, gene vaccines, small - molecule drugs, polypeptides, or protein drugs.

[0097] The present invention also provides the use of substance Z as described above, the lipid composition as described above, or the pharmaceutical composition as described above in the preparation of a drug for treating a disease or disorder. The disease or disorder is preferably characterized by a malfunction or abnormal activity of a protein or polypeptide.

[0098] In some embodiments, the disease or disorder is any one or a combination of at least two selected from the group consisting of infectious diseases, tumors, proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0099] In some embodiments, the infectious disease is a disease caused by a coronavirus, influenza virus or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies or multiple herpes.

[0100] In some embodiments, the tumor is breast cancer, ovarian cancer, lung cancer, pancreatic cancer, kidney cancer, gastric cancer, lymphoma, colon cancer, liver cancer, melanoma, bladder cancer, cervical cancer or prostate cancer.

[0101] In some embodiments, in the use, the drug is a drug for treating a disease or disorder in a mammal in need thereof. The mammal can be any one or a combination of at least two selected from the group consisting of humans, non-human primates, companion animals, exotic species, livestock animals and food animals.

[0102] In some embodiments, the administration route of the drug is intravenous, intramuscular, intradermal, subcutaneous, intranasal or inhalation.

[0103] In some embodiments, the administration route of the drug is intravenous or intramuscular.

[0104] In some embodiments, the administration dose of the drug is 0.001 to 10 mg / kg.

[0105] The adjuvant lipid compound and lipid composition provided by the present invention can be used for encapsulating nucleic acids (such as mRNA, etc.) to form corresponding nucleic acid drugs.

[0106] Term Definitions

[0107] All publications and patents mentioned in the present invention are hereby incorporated by reference in their entirety into the present invention. If there are conflicts between the uses or terms used in any incorporated publications and patents and those used in the present invention, then the uses and terms of the present invention shall prevail.

[0108] The chapter headings used in the present invention are only for the purpose of organizing the article and should not be construed as limiting the subject matter.

[0109] Unless otherwise specified, all technical terms and scientific terms used in the present invention have the ordinary meanings in the field to which the claimed subject matter belongs. If there are multiple definitions for a certain term, then the definition of the present invention shall prevail.

[0110] Except as otherwise indicated in the Examples or elsewhere, all numbers expressing quantities such as amounts in the specification and claims are to be understood as being modified in all instances by the term "about". It should also be understood that any numerical range recited in this invention is intended to include all sub-ranges within that range and any combination of the endpoints of that range or sub-ranges.

[0111] In the present invention, " " in the structural fragment means that this structural fragment is connected to the rest of the molecule through this bond. For example, means that it is connected to the rest of the molecule through " ".

[0112] In the present invention, the term "one or more" means 1, 2, 3, 4, 5, or 6, such as 1, 2, or 3.

[0113] In the present invention, the term "alkyl" means a straight-chain or branched-chain, saturated monovalent hydrocarbon group having 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 groups include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, , .

[0114] In the present invention, the term "alkylene" means a saturated divalent hydrocarbon group obtained by removing two hydrogen atoms from a saturated straight-chain or branched-chain hydrocarbon group. Alkylene groups include but are not limited to: methylene (-CH2-), ethylene {including -CH2CH2- or -CH(CH3)-}, isopropylidene {including -CH(CH3)CH2- or -C(CH3)2-}, , .

[0115] In the present invention, the term "alkoxy" means the group R Y -O-, where the definition of R Y is the same as that of the term "alkyl". Alkoxy groups include but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, n-hexyloxy, etc.

[0116] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group having at least one double bond, consisting only of carbon and hydrogen atoms, having, for example, 10 to 20 (such as 16, 17, 18, 19) carbon atoms, and being connected to the rest of the molecule by a single bond. Alkenyl includes, but is not limited to, vinyl, , , , , and the like.

[0117] In the present invention, the term "aryl" refers to a cyclic, unsaturated monovalent hydrocarbon group having a specified number of carbon atoms (for example, C6-C 10 ), which is monocyclic or polycyclic (for example, 2), and when polycyclic, two atoms and one bond are shared between the monocycles, and each ring has aromaticity. The aryl is connected to the rest of the molecule through an aromatic ring. Aryl includes, but is not limited to: phenyl, naphthyl.

[0118] In addition, when referring to a number or a numerical range, the term "about" means that the number or numerical range mentioned is an approximation within the typical tolerances in the art, within experimental variability, or within statistical experimental error, and thus the number or numerical range can vary, for example, between 1% and 15% of the said number or numerical range. For example, "about" can be understood as about 2 standard deviations of the average value, and when "about" is present before a series of numbers or ranges, it should be understood that "about" can modify each number in the said series or range.

[0119] Furthermore, in the present invention, when a numerical range is used in a compound general formula and / or a structural formula, it means that the number of the corresponding groups within the numerical range can be any natural number within the numerical range. For example, "C A-B " means any integer from the starting point to the ending point of the number of carbon atoms, where both A and B are integers; for another example, C 1-5 represents that the number of carbon atoms is 1, 2, 3, 4, or 5; that is, when combined with other groups in the compound general formula and / or the structural formula to form various possible compounds, C A-B can be used in combination with any group containing carbon atoms to define the number of carbon atoms. For example, C 1-5 alkyl / alkylene represents various possibilities of alkyl / alkylene having 1 C, 2 C, 3 C, 4 C, and / or 5 C.

[0120] As used in the present invention, words such as "comprising", "containing" or "including" and the like are intended to mean that the elements appearing before the word cover the elements listed after the word and their equivalents, without excluding the unrecited elements. The terms "containing" or "including (comprising)" used in the present invention can be open-ended, semi-closed and closed. In other words, the said terms also include "consisting essentially of...", or "consisting of...".

[0121] The term "pharmaceutically acceptable" in the present invention means that a compound or composition is chemically and / or toxicologically compatible with other components of the preparation and / or with humans or mammals for which it is used to prevent or treat a disease or disorder.

[0122] The term "subject" or "patient" in the present invention may include mammalian subjects. For example, the mammalian subject may be selected from any one or at least a combination of two of the group consisting of humans, non-human primates, companion animals, exotic species, livestock animals and food animals.

[0123] The term "treatment" as used in the present invention means administering one or more pharmaceutical substances to a patient or subject suffering from a disease or having the symptoms of the disease, so as to cure, relieve, mitigate, improve or affect the disease or the symptoms of the disease. In the context of the present invention, unless otherwise specifically stated, the term "treatment" may also include prevention.

[0124] In the present invention, the term "antigen" includes any molecule containing at least one epitope capable of eliciting an immune response and / or an epitope against which an immune response is directed, preferably a peptide or protein. Preferably, the antigen in the context of the present invention is such a molecule that optionally induces an immune response preferably specific for the antigen or the cells expressing the antigen after processing. In particular, "antigen" refers to such a molecule that is optionally presented by MHC molecules after processing and specifically reacts with T lymphocytes (T cells).

[0125] Therefore, an antigen or its fragment should be able to be recognized by a T cell receptor. Preferably, if recognized by a T cell receptor, the antigen or fragment is capable of inducing clonal expansion of T cells carrying a T cell receptor specifically recognizing the antigen or fragment in the presence of a suitable co-stimulatory signal. In the context of the embodiments of the present invention, the antigen or fragment is preferably presented by a cell in the context of MHC molecules, preferably by an antigen-presenting cell and / or a diseased cell, which results in an immune response against the antigen or the cells expressing the antigen.

[0126] According to the present invention, any suitable antigen is envisaged as a candidate for an immune response, wherein the immune response is preferably a cellular immune response.

[0127] The antigen is preferably a product corresponding to or derived from a naturally occurring antigen. The naturally occurring antigen may include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen may also be a tumor antigen. According to the present invention, the antigen may correspond to a naturally occurring product, for example, a viral protein or a portion thereof.

[0128] The term "pathogen" refers to pathogenic microorganisms and includes 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-lymphotrophic virus (HTLV). Single-celled organisms include, but are not limited to, Plasmodium, Trypanosoma, Amoeba, etc.

[0129] The term "disease-related antigen" refers to all antigens of pathogenic significance and includes "tumor antigens". According to the present invention, an immune response against a disease-related antigen or a cell expressing a disease-related antigen and preferably presenting the disease-related antigen in the context of MHC molecules is desired. Preferably, the disease-related antigen is a naturally occurring antigen. In one embodiment, the disease-related antigen is expressed in diseased cells and is preferably presented by the MHC molecules of the cells.

[0130] The antigen encoded by the RNA (i.e., the therapeutic and / or prophylactic agent) contained in the nanoparticles of the present invention (lipid composition) should induce an immune response against the disease-related antigen to be targeted or a cell expressing the disease-related antigen to be targeted. Thus, the antigen encoded by the RNA contained in the nanoparticles of the present invention may correspond to or may comprise a disease-related antigen or one or more immunogenic fragments thereof, such as one or more MHC-binding peptides of the disease-related antigen. Thus, the antigen encoded by the RNA contained in the nanoparticles of the present invention may be a recombinant antigen.

[0131] Therapeutic and / or prophylactic agent

[0132] The lipid composition of the present invention can be used to deliver a pharmaceutically active ingredient, such as a therapeutic and / or prophylactic agent. Based on this, the present invention further provides a (pharmaceutical) composition comprising the lipid composition provided by the present invention for delivering a pharmaceutically active ingredient. The composition of the present invention may include one or more therapeutic and / or prophylactic agents (as pharmaceutically active ingredients). The pharmaceutically active ingredient may be encapsulated within or bound to the lipid composition.

[0133] The therapeutic and / or prophylactic agent includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, and proteins. Preferably, it is a nucleic acid molecule.

[0134] For example, the therapeutic agent and / or prophylactic agent is a vaccine or a compound capable of eliciting an immune response. Thus, in some preferred embodiments, the therapeutic agent and / or prophylactic agent can be a nucleic acid molecule capable of encoding one or more antigens.

[0135] The lipid composition of the present invention can (as a carrier) deliver a therapeutic agent and / or prophylactic agent to target cells and / or target organs in a subject (such as a mammal). Thus, the present invention also provides methods for treating a disease or disorder in a subject in need thereof, which methods include administering to the subject a composition comprising a therapeutic agent and / or prophylactic agent and / or contacting the subject's cells with the composition.

[0136] Therapeutic and / or prophylactic agents include bioactive substances and are alternatively referred to as "active agents", "active ingredients", etc. Therapeutic and / or prophylactic agents can be substances that cause desired changes in a cell or organ after being delivered to the cell or organ or other body tissues or systems. Such agents can be used to treat one or more diseases, disorders, or conditions. In some embodiments, the therapeutic and / or prophylactic agent is a small molecule drug that can be used to treat a specific disease, disorder, or condition.Examples of drugs that can be used in the composition include, but are not limited to, anti-neoplastic agents (such as vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), anti-tumor agents (such as actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracycline, alkylating agents, platinum compounds, anti-metabolites, and nucleoside analogs such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (such as dibucaine and chlorpromazine), β-adrenergic blockers (such as propranolol, timolol, and labetalol), anti-hypertensive agents (such as clonidine and hydralazine), anti-depressants (such as imipramine, amitriptyline, and doxepin), anti-spasmodics (such as phenytoin), anti-histamines (such as diphenhydramine, chlorpheniramine, and promethazine), antibiotics / anti-bacterial agents (such as gentamycin, ciprofloxacin, and cefoxitin), anti-fungal agents (such as miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), anti-parasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, vitamins, sedatives, and imaging agents.

[0137] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include but are not limited to taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and analogs or homologs thereof. Radioactive ions include but are not limited to iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis and may include nucleic acid molecules (e.g., mRNA) encoding antigens and / or epitopes derived from infectious diseases. Vaccines may also include compounds and formulations that direct an immune response against cancer cells and may include nucleic acid molecules (e.g., mRNA) encoding tumor cell-derived antigens, epitopes, and / or neoepitopes. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species.Other therapeutic and / or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, CC-1065, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0138] In other embodiments, the therapeutic and / or prophylactic agent is a protein. Therapeutic proteins that can be used 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), factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferons, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0139] In some embodiments, the therapeutic and / or prophylactic agent can be a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The broadest meaning of the term "polynucleotide" includes any compound and / or substance in the form of an oligonucleotide chain or that can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used in accordance with the present invention include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger mRNA (mRNA), its hybrids; RNAi-inducing factors; RNAi factors; siRNA; shRNA; miRNA; antisense RNA; ribozymes; catalytic DNA; RNA that induces triple helix formation; aptamers, etc. In some preferred embodiments, the therapeutic and / or prophylactic agent is RNA. The RNA that can be used in the compositions and methods described in the present invention can be selected from, but 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), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In certain embodiments, the RNA is mRNA.

[0140] In certain embodiments, the therapeutic and / or prophylactic agent is mRNA. The mRNA can encode any polypeptide of interest, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0141] In other embodiments, the therapeutic and / or prophylactic agent is siRNA. The siRNA is capable of selectively reducing the expression of a gene of interest or downregulating the expression of the gene. For example, the siRNA can be selected such that after administering a composition comprising the siRNA to a subject in need, a gene associated with a particular disease, disorder, or condition is silenced. The siRNA can comprise a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA can be an immunomodulatory siRNA.

[0142] In certain embodiments, the therapeutic and / or prophylactic agent is sgRNA and / or cas9 mRNA. The sgRNA and / or cas9 mRNA can be used as gene editing tools. For example, the sgRNA-cas9 complex can affect the mRNA translation of cellular genes.

[0143] In some embodiments, the therapeutic and / or prophylactic agent is an shRNA or its encoding vector or plasmid. The shRNA can be generated inside the target cell after delivery of the appropriate construct to the nucleus. The constructs and mechanisms associated with shRNA are well known in the relevant art.

[0144] disease or disorder

[0145] The compositions / carriers of the present invention can deliver therapeutic and / or prophylactic agents to a subject or patient, thereby achieving the treatment and / or prevention of a disease or disorder. The therapeutic and / or prophylactic agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides or proteins. Accordingly, the compositions of the present invention can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide or protein drugs. Due to the wide variety of the above-mentioned therapeutic and / or prophylactic agents, the compositions of the present invention can be used to treat or prevent a variety of diseases or disorders.

[0146] In one embodiment, the disease or disorder is characterized by a malfunctioning or abnormal protein or polypeptide activity.

[0147] The reagents, compositions and methods described in the present invention can be used to treat a subject suffering from a disease (e.g., a disease characterized by the presence of diseased cells that express an antigen and present antigenic peptides), or to prevent a subject from suffering from a disease. Examples of diseases that can be treated and / or prevented cover all diseases that express one of the antigens described in the present invention. Particularly preferred diseases are infectious diseases (e.g., viral diseases) and cancer diseases. The reagents, compositions and methods described in the present invention can also be used for immunization or vaccination to prevent the diseases described in the present invention.

[0148] According to the present invention, the term "disease" refers to any pathological condition, including infectious diseases and cancer diseases, particularly those forms of infectious diseases and diseases described in the present invention.

[0149] The disease to be treated according to the present invention is preferably a disease involving an antigen. According to the present invention, "a disease involving an antigen" or a similar expression means that the antigen is expressed in the cells of a diseased tissue or organ. The expression in the cells of a diseased tissue or organ can be elevated compared to the state of a healthy tissue or organ. In one embodiment, the expression occurs only in the diseased tissue, while the expression in the healthy tissue is suppressed. According to the present invention, diseases involving an antigen include infectious diseases and cancer diseases, wherein the disease-related antigens are preferably the antigens of the infectious agent and tumor antigens, respectively. Preferably, the disease involving an antigen is preferably a disease involving cells that express an antigen and present the antigen in the context of MHC molecules (particularly MHC class I).

[0150] For example, the disease or disorder is selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0151] Examples of the infectious diseases include: ① viral infectious diseases, such as AIDS (HIV), hepatitis A, hepatitis B or hepatitis C, herpes zoster (varicella), German measles (rubella virus), yellow fever, dengue fever, etc., flavivirus, coronavirus, influenza virus, rabies virus, hemorrhagic infectious diseases (Marburg virus or Ebola virus); ② bacterial infectious diseases, such as Legionnaire's disease (Legionella), gastric ulcer (Helicobacter), cholera (Vibrio), infections caused by Escherichia coli, Staphylococci, Salmonella or Streptococci (tetanus); ③ infections caused by protozoan pathogens, such as malaria, sleeping sickness, leishmaniasis, toxoplasmosis, that is, infections caused by Plasmodium, Trypanosoma, Leishmania and Toxoplasma; or ④ fungal infections, which are caused by, for example, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis or Candida albicans.

[0152] The cancer or carcinoma (medical term: malignant tumor) is a group of diseases in which a group of cells shows uncontrolled growth (division beyond normal limits), invasion (invading and destroying adjacent tissues), and sometimes metastasis (spreading to other parts of the body through lymph or blood). These three harmful properties of cancer distinguish it from benign tumors that are self-limiting and do not invade or metastasize. Most cancers form tumors, that is, swellings or lesions formed by abnormal growth of cells (called neoplastic cells or tumor cells), but some (like leukemia) do not. According to the present invention, the term "cancer" includes leukemia, seminoma, melanoma, teratoma, lymphoma, sarcoma, embryonal carcinoma, neuroblastoma, glioma, glioblastoma, renal cancer, adrenal cancer, renal cell carcinoma, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, gastric cancer, lung cancer, intestinal cancer, head and neck cancer, gastrointestinal 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.

[0153] Malignant melanoma is a serious type of skin cancer. It is caused by the uncontrolled growth of pigment cells called melanocytes.

[0154] According to the present invention, "epithelial cancer" is a malignant tumor derived from epithelial cells. This group accounts for the most common cancers, including common forms of breast cancer, prostate cancer, lung cancer, and colon cancer.

[0155] Lymphoma and leukemia are malignant tumors derived from hematopoietic (blood-forming) cells.

[0156] Sarcoma is a cancer of transformed cells that originate from one of the tissues developed from the embryonic mesoderm. Therefore, sarcomas include bone tumors, cartilage tumors, fat tumors, muscle tumors, vascular tumors, and hematopoietic tissue tumors.

[0157] Blastic tumor or embryonal carcinoma is a tumor (usually malignant) similar to immature or embryonic tissue. Most of these tumors are common in children.

[0158] Glioma is a type of tumor that begins in the brain or spinal cord. It is called glioma because it originates from glial cells. The most common location of glioma is the brain.

[0159] Other components

[0160] The pharmaceutical composition of the present invention may include one or more components other than those described in the foregoing part. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0161] The composition may also include one or more permeability enhancing molecules, carbohydrates, polymers, surface modifiers or other components. The permeability enhancing molecules can be, for example, the molecules described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates can include simple sugars (such as glucose) and polysaccharides (such as glycogen and its derivatives and analogs).

[0162] Surface modifiers can include, but are not limited to, anionic proteins (such as bovine serum albumin), surfactants (such as cationic surfactants, such as dimethyldioctadecylammonium bromide), sugars or sugar derivatives (such as cyclodextrin), nucleic acids, polymers (such as heparin, polyethylene glycol and poloxamer), mucolytics (such as acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexine and erdosteine) and DNAse (such as rhDNAse). The surface modifier can be disposed within and / or on the surface of the nanoparticles of the composition (such as by coating, adsorption, covalent attachment or other methods).

[0163] The composition may also contain one or more functionalized lipids. For example, the lipid can be functionalized with an alkynyl group that may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized in this way with one or more groups that can effectively promote membrane penetration, cell recognition or imaging. The surface of the composition can also be conjugated with one or more useful antibodies. Functional groups and conjugates useful for targeted cell delivery, imaging and membrane penetration are well known in the art.

[0164] In addition to these components, the composition may include any substances that can be used in pharmaceutical compositions. For example, the composition may include one or more pharmaceutically acceptable (e.g., medicinally acceptable) excipients or auxiliary components, such as but not limited to one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc.

[0165] The term "medicinally acceptable" means that the material is non-toxic and does not affect the action of the active components of the pharmaceutical composition. Non-medicinally acceptable ingredients can be used to prepare medicinally acceptable ingredients and are included in the present invention.

[0166] Suitable buffers for the compositions of the present invention include the salt forms of acetic acid, citric acid, boric acid, and phosphoric acid.

[0167] When used in the present invention, the term "excipient" is intended to mean all substances that may be present in the pharmaceutical compositions of the present invention and that are not active ingredients, such as carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents, or coloring agents. Excipients include, for example, starch, lactose, or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, e.g., Remington’s The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro; Lippincott, Williams&Wilkins, Baltimore, MD, 2006).

[0168] Suitable preservatives for the compositions of the present invention include benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0169] Examples of diluents may include but are not limited to calcium carbonate, sodium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof.

[0170] Dosage Forms and Administration

[0171] The compositions of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The compositions of the present invention can be prepared by methods well known in the pharmaceutical art. For example, a sterile injectable solution can be prepared by incorporating the required amount of the therapeutic or prophylactic agent with the various other ingredients required above into a suitable solvent such as sterile distilled water, and then filtering and sterilizing. Surfactants can also be added to facilitate the formation of a uniform solution or suspension.

[0172] For example, the compositions of the present invention can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation. In one embodiment, the composition is administered intravenously or subcutaneously.

[0173] Therapeutically effective amount

[0174] A "therapeutically effective amount" is the amount of a therapeutic agent that, when administered to a patient, can ameliorate a disease or symptom. A "prophylactically effective amount" is the amount of a prophylactic agent that, when administered to a subject, can prevent a disease or symptom. The amount of the therapeutic agent constituting a "therapeutically effective amount" or the amount of the prophylactic agent constituting a "prophylactically effective amount" varies with the therapeutic and / or prophylactic agent, the disease state and its severity, the age, weight, etc. of the patient and / or subject to be treated and / or prevented. A person of ordinary skill in the art can routinely determine the therapeutically effective amount and the prophylactically effective amount based on their knowledge and the present invention.

[0175] The compositions of the present invention are administered in a therapeutically effective amount, which amount can vary not only with the particular reagent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and can ultimately be determined by the attending physician or clinician. For example, a dose of about 0.0001 mg / kg to about 10 mg / kg of the therapeutic or prophylactic agent can be administered to a mammal (such as a human).

[0176] Antigen-presenting cell

[0177] An antigen-presenting cell (APC) is a cell that presents (i.e., displays) an antigen on its surface in the context of the major histocompatibility complex (MHC). This includes cases where only one fragment or more than one fragment of the antigen is presented. T cells can recognize this complex with their T cell receptors (TCRs). Antigen-presenting cells process antigens and present them to T cells.

[0178] Professional antigen-presenting cells are very efficient at internalizing antibodies (by phagocytosis or by receptor-mediated endocytosis) and then presenting antigen fragments bound to class II MHC molecules on their membranes. T cells recognize the antigen-class II MHC molecule complex on the antigen-presenting cell membrane and interact with it. Then the antigen-presenting cell generates additional co-stimulatory signals, leading to T cell activation. Expression of co-stimulatory molecules is a characteristic feature of professional antigen-presenting cells.

[0179] The main types of professional antigen-presenting cells are dendritic cells (which have the broadest range of antigen presentation and are probably the most important antigen-presenting cells), macrophages, B cells, and certain activated epithelial cells.

[0180] Dendritic cells are a group of white blood cells that include plasmacytoid dendritic cells (pDC cells) and classical dendritic cells (cDC cells), which present antigens captured in peripheral tissues to T cells via two antigen presentation pathways, class II and class I MHC. Dendritic cells are powerful inducers of immune responses, and activation of these cells is a key step in inducing anti-tumor immunity.

[0181] Antigen-presenting cells can be loaded with MHC-presented peptides by transducing them with nucleic acids encoding peptides or proteins containing the peptide to be presented (e.g., nucleic acids encoding antigens (e.g., RNA)). Transfecting dendritic cells with mRNA is a promising antigen-loading technique for stimulating strong anti-tumor immunity.

[0182] The term "immunogenicity" refers to the relative efficiency of an antigen in inducing an immune response.

[0183] The terms "T cell" and "T lymphocyte" are used interchangeably in the present invention and include helper T cells (CD4+ T cells) and cytotoxic T cells of the cytolytic T cell (CTL, CD8+ T cells).

[0184] T cells belong to the group of white blood cells called lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types (such as B cells and natural killer cells) by the presence of a special receptor called the T cell receptor (TCR) on their cell surface. The thymus is the main organ responsible for T cell maturation. Several different T cell subsets have been identified, each with different functions.

[0185] Helper T cells assist other white blood cells during the immune process, including functions such as maturing B cells into plasma cells and activating cytotoxic T cells and macrophages. Because they express the CD4 protein on their surface, these cells are also called CD4 +T cells. Helper T cells are activated when class II MHC molecules expressed on the surface of antigen-presenting cells (APCs) present peptide antigens to them. After activation, they divide rapidly and secrete small proteins called cytokines that regulate or assist the active immune response.

[0186] Cytotoxic T cells destroy diseased cells, such as infected cells (e.g., virus-infected cells) and cancer cells, and also participate in transplant rejection. Because they express the CD8 glycoprotein on their surface, these cells are also called CD8 + T cells. These cells recognize their targets by binding to antigens associated with class I MHC, which is present on the surface of almost every cell in the body.

[0187] Most T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains, which are produced by separate T cell receptor α and β (TCRα and TCRβ) genes and are called the α-TCR chain and the β-TCR chain. γδ T cells represent a small subtype of T cells that have a unique T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR is composed of one γ chain and one δ chain. This group of T cells is less common than αβ T cells (2% of total T cells).

[0188] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells are present in the thymus and expand by cell division to produce a large number of immature thymocytes. Early thymocytes express neither CD4 nor CD8 and are thus classified as double-negative (CD4-CD8-) cells. As they develop through the process of development, they become double-positive thymocytes (CD4+CD8+) and eventually mature into single-positive (CD4 + CD8 - or CD4 - CD8 + ) thymocytes, which are then released from the thymus into the peripheral tissues.

[0189] The first signal for T cell activation is provided by the binding of the T cell receptor to a short peptide presented by the major histocompatibility complex (MHC) on another cell. This ensures that only T cells with a TCR specific for that peptide are activated. The partnering cell is usually a professional antigen-presenting cell (APC), usually a dendritic cell in the case of a primary response, but B cells and macrophages can also be important APCs. The peptides presented by class I MHC molecules to CD8 + T cells are 8 to 10 amino acids in length; the peptides presented by class II MHC molecules to CD4 +The peptides of T cells are longer because the ends of the binding clefts of class II MHC molecules are open.

[0190] Based on the common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0191] The reagents and raw materials used in the present invention are all commercially available.

[0192] The positive and progressive effects of the present invention are as follows: Compared with the prior art, the mRNA composition prepared by using the compound shown in formula (I) provided by the present invention has one or more of the following advantages:

[0193] 1. Good particle size and uniform particle distribution;

[0194] 2. Can significantly increase the protein expression level of antigens in the body (such as mice), muscles or spleens of subjects;

[0195] 3. Significantly increase the percentage of antigen-presenting cells expressing antigens in the spleen;

[0196] 4. Significantly increase the maturation state of antigen-presenting cells in the spleen;

[0197] 5. Significantly increase the activation state of T cells in the spleen;

[0198] 6. Significantly reduce the volume of tumors and significantly increase the survival rate of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0199] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings of the present invention will be briefly introduced below. It should be understood that the drawings described below only relate to some specific embodiments of the present invention and do not limit the present invention.

[0200] Figure 1 Fluorescence images of mice in vivo and mouse organs (liver, spleen) 6 hours after intravenous injection of Fluc-mRNA-TLP compositions prepared by adding YK-1202, YK-1204, YK-1205, YK-1206 or YK-1208, and Fluc-mRNA-TLP compositions without adjuvant lipids. (Left: mouse in vivo, right: mouse organs)

[0201] Figure 2 Flow cytometry experimental images of mouse spleen cells (eGFP) 24 hours after intravenous injection of blank, eGFP-mRNA-TLP compositions added with YK-1202 or YK-1204, and eGFP-mRNA-TLP compositions without adjuvant lipids into mice.

[0202] Figure 3Flow cytometry analysis of mouse spleen cells (CD86) 24 hours after intravenous injection of eGFP-mRNA-TLP compositions with or without YK-1205 or TMX-201.

[0203] Figure 4 Flow cytometry analysis of mouse spleen cells (CD69) 24 hours after intravenous injection of TLP compositions with or without YK-1204, YK-1205 or TMX-201.

[0204] Figure 5 Stimulation of cytokines IFN-γ and IL-12 in the sera of B16F10-OVA mice inoculated with OVA mRNA-TLP compositions with or without YK-1202, YK-1204, YK-1205 or YK-1208.

[0205] Figure 6 Tumor growth in B16F10-OVA mice inoculated with OVA mRNA-TLP compositions with or without YK-1202, YK-1204, YK-1205 or YK-1208.

[0206] Figure 7 Survival rate of B16F10-OVA mice inoculated with OVA mRNA-TLP compositions with or without YK-1202, YK-1204, YK-1205 or YK-1208.

[0207] Figure 8 Stimulation of cytokines IFN-γ and IL-12 in the sera of B16F10-OVA mice inoculated with OVA mRNA-LNP compositions with or without YK-1202, YK-1204, YK-1206 or YK-1208.

[0208] Figure 9 Tumor growth in MC38-OVA mice inoculated with OVA mRNA-LNP compositions with or without YK-1202, YK-1204, YK-1206 or YK-1208.

[0209] Figure 10 Survival rate of MC38-OVA mice inoculated with OVA mRNA-LNP compositions with or without YK-1202, YK-1204, YK-1206 or YK-1208. Detailed Description of the Invention

[0210] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the examples.

[0211] The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in this industry. In the specific examples of the present invention, the raw materials used can all be obtained commercially. Unless otherwise specified, the percentages in the context are weight percentages, and all temperatures are given in degrees Celsius. The technical features involved in each implementation manner of the present invention can be combined with each other as long as they do not conflict with each other.

[0212] The following abbreviated letters represent the following reagents respectively:

[0213] ADOPE: Sodium 2-acetamidoethyl ((R)-2,3-bis(oleyloxy)propyl) phosphate; ( , prepared according to Example 1 in Patent CN118001254A)

[0214] DOTMA: 1,2-Dioctadecyloxy-3-methylammonium propane (chloride);

[0215] DOPE: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine;

[0216] DOPG: Sodium 1,2-dioleoyl-sn-glycero-3-phosphate-RAC-glycerol;

[0217] DSPC: 1,2-Distearoyl-sn-glycero-3-phosphocholine;

[0218] DMG-PEG2000: 1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000;

[0219] Boc2O: Di-tert-butyl dicarbonate;

[0220] DMAP: 4-Dimethylaminopyridine;

[0221] EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0222] TsOH: p-Toluenesulfonic acid;

[0223] TFA: Trifluoroacetic acid;

[0224] DCM: Dichloromethane;

[0225] DMF: N,N-Dimethylformamide;

[0226] THF: Tetrahydrofuran;

[0227] TrCl: Triphenylmethyl chloride.

[0228] Example 1: Synthesis of TLR Adjuvant Conjugated with Lipids

[0229] Synthesis of YK-1201

[0230] The synthesis route of YK-1201 is as follows:

[0231]

[0232] Step 1: Synthesis of YK-1201-PM1

[0233] Dissolve YK-009 (500 mg, 0.765 mmol) in dichloromethane (10 mL). Under an ice-water bath, slowly add thionyl chloride (180 mg, 1.530 mmol). Stir the reaction at room temperature for 6 hours. Stop the reaction and rotary evaporate the solvent under reduced pressure. Purify the product by silica gel chromatography (0% - 15% dichloromethane / methanol) to obtain YK-1201-PM1 (500 mg, 0.743 mmol, 97.1%).

[0234] Step 2: Synthesis of YK-1201

[0235] Dissolve YK-1201-PM1 (150 mg, 0.223 mmol) and YK-1201-SM1 (84 mg, 0.270 mmol) in DMF (3 mL). Add potassium carbonate (93 mg, 0.673 mmol) and potassium iodide (3 mg, 0.018 mmol) at room temperature, and then stir the reaction at 70 °C for 6 hours. After the reaction is completed, cool down the reaction mixture. Add ethyl acetate and water to the reaction solution in sequence, separate the layers. Extract the aqueous phase with ethyl acetate twice. Combine the organic phases and rotary evaporate the solvent to obtain the crude product. Purify the product by silica gel chromatography (0% - 15% dichloromethane / methanol) to obtain YK-1201 (120 mg, 0.127 mmol, 57.0%). C 58 H 102 N6O4, MS (ES): m / z (1 / 2 M +Na + ) = 496.63.

[0236] YK-1201: 11H 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).

[0237] 2. Synthesis of YK-1202

[0238] The synthetic route of YK-1202 is as follows:

[0239]

[0240] Using YK-1201-PM1 (100 mg, 0.149 mmol) and YK-1202-SM1 (64 mg, 0.178 mmol) as raw materials, according to the synthesis method of YK-1201, the product YK-1202 (34 mg, 0.0342 mmol, 22.9%) was obtained. C 62 H 102 N6O4, MS (ES): m / z (1 / 2 M + Na + ) = 520.58.

[0241] 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).

[0242] 3. Synthesis of YK-1203

[0243] The synthetic route of YK-1203 is as follows:

[0244]

[0245] Step 1: Synthesis of YK-1203-PM1

[0246] Dissolve YK-009 (500 mg, 0.764 mmol) and 4-bromobutyric acid (612 mg, 3.665 mmol) in dichloromethane (5 mL), add EDCI (440 mg, 2.295 mmol) and DMAP (38 mg, 0.311 mmol) at room temperature, and stir at 35 ºC for 16 h. Stop the reaction, rotary evaporate the solvent under reduced pressure to obtain the crude product, and purify it by silica gel chromatography (0% - 15% dichloromethane / methanol) to obtain the product YK-1203-PM1 (480 mg, 0.598 mmol, 78.2%).

[0247] Step 2: Synthesis of YK-1203

[0248] Using YK-1203-PM1 (150 mg, 0.187 mmol) and YK-1201-SM1 (70 mg, 0.224 mmol) as raw materials, according to the synthesis method of YK-1201, the product YK-1203 (24 mg, 0.0232 mmol, 12.4%) was obtained. C 62 H 108N6O6, MS (ES): m / z (1 / 2 M + Na + ) = 539.68。

[0249] 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).

[0250] 4. Synthesis of YK-1204

[0251] The synthesis route of YK-1204 is as follows:

[0252]

[0253] Step 1: Synthesis of YK-1204-PM1

[0254] Using YK-009 (500 mg, 0.764 mmol) and 6-bromohexanoic acid (596 mg, 3.058 mmol) as raw materials, following the synthesis method of YK-1203-PM1, the product YK-1204-PM1 (520 mg, 0.626 mmol, 81.9%) was obtained.

[0255] Step 2: Synthesis of YK-1204

[0256] Using YK-1204-PM1 (150 mg, 0.180 mmol) and YK-1201-SM1 (84 mg, 0.271 mmol) as starting materials, following the synthesis method of YK-1201, the product YK-1204 (60 mg, 0.0565 mmol, 31.4%) was obtained. C 64 H 112 N6O6, MS (ES): m / z (1 / 2 M + Na + ) = 553.57.

[0257] 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).

[0258] 5. Synthesis of YK-1205

[0259] The synthesis route of YK-1205 is as follows:

[0260]

[0261] Using YK-1203-PM1 (100 mg, 0.125 mmol) and YK-1202-SM1 (66 mg, 0.184 mmol) as starting materials, following the synthesis method of YK-1201, the product YK-1205 (60 mg, 0.0555 mmol, 44.4%) was obtained. C 66 H 108 N6O6, MS (ES): m / z (1 / 2 M + Na +) = 563.66。

[0262] YK-1205: 1 1H 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).

[0263] 6. Synthesis of YK-1206

[0264] The synthetic route of YK-1206 is as follows:

[0265]

[0266] Using YK-1204-PM1 (100 mg, 0.120 mmol) and YK-1202-SM1 (52 mg, 0.144 mmol) as raw materials, according to the synthesis method of YK-1201, the product YK-1205 (40 mg, 0.0360 mmol, 30.0%) was obtained. C 68 H 112 N6O6, MS (ES): m / z (1 / 2 M + Na + ) = 577.72。

[0267] YK-1206: 11H 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).

[0268] 7. Synthesis of YK-1207

[0269] The synthetic route of YK-1207 is as follows:

[0270]

[0271] Step 1: Synthesis of YK-1207-PM1

[0272] Dissolve YK-1207-SM1 (830 mg, 2.64 mmol) in tetrahydrofuran (10 mL), cool to 0 ºC, slowly add triethylamine (1.33 g, 13.15 mmol), stir for 5 minutes, then add Boc anhydride (2.88 g, 13.20 mmol), warm to room temperature and stir overnight, monitored by LCMS. Stop the reaction, add water and ethyl acetate to the reaction mixture in turn, separate the layers, back-extract the aqueous phase with ethyl acetate twice, combine the organic phases, evaporate the solvent to obtain the crude product, and purify it by silica gel chromatography (0% - 10% dichloromethane / methanol) to obtain the product YK-1207-PM1 (830 mg, 2.00 mmol, 75.8%). C 22 H 30 N4O4, MS (ES): m / z(M + H + ) = 415.38.

[0273] Step 2: Synthesis of YK-1207-PM2

[0274] Dissolve YK-1207-PM1 (300 mg, 0.72 mmol) in dichloromethane (3.5 mL), cool to 0 ºC, add DMAP (531 mg, 4.30 mmol), stir for 5 minutes, then add YK-1207-SM2 (437 mg, 2.16 mmol), warm to room temperature and stir for 4 hours, monitor the reaction by TLC. Stop the reaction and purify by silica gel chromatography (0% - 50% dichloromethane / ethyl acetate) to obtain the product YK-1207-PM2 (105 mg, 0.181 mmol, 25.2%). C 29 H 33 N5O8, MS (ES): m / z (M + H + ) = 580.75.

[0275] Step 3: Synthesis of YK-1207-PM3

[0276] Dissolve YK-1207-PM2 (105 mg, 0.18 mmol) in dichloromethane (3 mL), add DIEA (94 mg, 0.73 mmol) and DOPE (539 mg, 0.73 mmol) at room temperature, stir overnight at room temperature, monitor the reaction by TLC. Stop the reaction, evaporate the solvent to obtain the crude product, and purify by silica gel chromatography (0% - 10% dichloromethane / methanol) to obtain the product YK-1207-PM3 (100 mg, 0.084 mmol, 46.9%). C 64 H 106 N5O 13 P, MS (ES): m / z (M + H + ) = 1185.17.

[0277] Step 3: Synthesis of YK-1207

[0278] Dissolve YK-1207-PM3 (100 mg, 0.084 mmol) in ethyl acetate solution of 4M hydrogen chloride (1 mL), stir at room temperature for 4 hours, monitor the reaction by LCMS. Stop the reaction, evaporate the solvent to obtain the crude product, and purify by silica gel chromatography (0% - 10% dichloromethane / methanol) to obtain the product YK-1207 (20 mg, 0.018 mmol, 21.9%). C 59 H 98 N5O 11 P, MS (ES):m / z (M + H + ) = 1085.13.

[0279] YK-1207: 11H 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).

[0280] 8. Synthesis of YK-1208

[0281] The synthetic route of YK-1208 is as follows:

[0282]

[0283] Step 1: Synthesis of YK-1208-PM1

[0284] Dissolve 5-chlorovaleraldehyde (77 mg, 0.639 mmol) in dichloromethane (10 mL). Add DOPG (500 mg, 0.645 mmol) and p-toluenesulfonic acid (62 mg, 0.33 mmol) at room temperature. Stir the mixture overnight at room temperature and monitor the reaction by TLC. When the raw materials are completely reacted, stop the reaction. Pour the reaction solution into saturated sodium bicarbonate aqueous solution, extract with dichloromethane, combine the organic phases, and evaporate the solvent to obtain the crude product. Purify the crude product by silica gel chromatography (0% - 10% dichloromethane / methanol) to obtain the product YK-1208-PM1 (420 mg, 0.479 mmol, 74.9%). C 47 H 86 ClO 10 P, MS (ES): m / z (M - H + ) = 875.84.

[0285] Step 2: Synthesis of YK-1208

[0286] Dissolve YK-1208-PM1 (320 mg, 0.364 mmol) and YK-1201-SM1 (114 mg, 0.366 mmol) in DMF (3.5 mL). Add potassium carbonate (150 mg, 1.10 mmol) and potassium iodide (6 mg, 0.040 mmol) at room temperature. Then stir the reaction mixture at 70 ºC for 16 h. After the reaction is completed, cool down the temperature. Pour the reaction solution into saturated sodium bicarbonate aqueous solution, extract with ethyl acetate, combine the organic phases, and evaporate the solvent to obtain the crude product. Purify the crude product by silica gel chromatography (0% - 20% dichloromethane / methanol) to obtain the product YK-1208 (105 mg, 0.091 mmol, 24.6%). C 65 H 110 N5O 10 P, MS (ES): m / z (M + H + ) = 1153.26.

[0287] 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).

[0288] 9. Synthesis of TMX 201

[0289]

[0290] The synthesis of TMX 201 refers to the synthetic route of Compound A in WO2011134669A to obtain 25 mg of TMX201.

[0291] 10. Synthesis of Compound 23

[0292]

[0293]

[0294] The synthesis of Compound 23 refers to the synthetic route of Compound 23 in WO2021237055A1, and 30 mg of Compound 23 was obtained.

[0295] 11. Synthesis of 63 - 15

[0296]

[0297] The synthesis of 63 - 15 refers to the synthetic route of Compound No. 63 - 15 in WO2019040491A1, and 42 mg of 63 - 15 was obtained.

[0298] Example 2: Preparation of mRNA Lipid Composition

[0299] A) Preparation of Fluc DNA, eGFP DNA, and OVA DNA Templates

[0300] 1) The luciferase (Luciferase protein CDS), green fluorescent protein (GFP), and ovalbumin (OVA) circular plasmids were digested with EcoRV and ligated to the pVAX1 vector (purchased from Thermo Fisher Scientific).

[0301] 2) The plasmid constructed on the pVAX1 vector in step 1) was mixed with 50 μL of Escherichia coli competent cells Stbl2 (purchased from Thermo Fisher Scientific), and the mixture was incubated on ice for 30 minutes, heat - shocked at 42 °C for 90 seconds, immediately placed back on ice, and incubated on ice for 2 minutes.

[0302] 3) 400 μL of LB medium (purchased from Thermo Fisher Scientific) was added, and the mixture was cultured with slow shaking on a shaker at 30 °C for 45 - 60 minutes.

[0303] 4) 50 - 100 μL of the bacterial solution was spread on an LB solid medium containing kanamycin antibiotic (100 μg / mL, purchased from Yeasen Biotech Co., Ltd.), and cultured upside - down at 37 °C overnight.

[0304] 5) The resulting monoclonal colony plate was sequenced to verify its correctness, and the monoclonal colony with correct sequencing was picked and cultured with slow shaking on a shaker at 30 °C overnight.

[0305] 6) The plasmid was extracted using an endotoxin - free large - scale plasmid extraction kit (purchased from Yeasen Biotech Co., Ltd.).

[0306] 7) The plasmid obtained by extraction was digested with restriction enzymes to linearize the plasmid for use as a transcription template. The specific digestion process steps refer to steps ① - ③.

[0307] Step ①: Take 1 mg of luciferase circular plasmid and digest it at 37 °C for 4 hours with BspQ I enzyme (purchased from Yeasen Biotech Co., Ltd.) to obtain a linearized DNA transcription template (the digestion system is shown in Table 1).

[0308] Table 1 Digestion reaction system

[0309]

[0310] Step ②: After the reaction is completed, add anhydrous ethanol and sodium acetate in sequence. Add anhydrous ethanol and 3M sodium acetate according to the volume ratio of V 酶切反应产物 :V 无水乙醇 :V 3M醋酸钠 = 1:3:1, place it at -20 °C for precipitation for 1 hour, and then centrifuge at 12000 rmp to retain the precipitate.

[0311] Step ③: Wash the precipitate from Step ② twice with 70% ethanol. Place the centrifuged material in an oven at 55 °C to dry for 10 minutes, and then add 1.7 mL of injection water to dissolve it.

[0312] The concentration of the linearized plasmid in the dissolution solution is 500 ng / µL, the linearization ratio is over 90%, and the purification and recovery efficiency is 85%.

[0313] B) Preparation of Fluc mRNA, eGFP mRNA and OVA mRNA

[0314] 1) Co-transcription capping reaction:

[0315] Using the Fluc DNA, eGFP DNA and OVA DNA prepared in A) as templates, NTP solution (NTPs) and Cap1 cap analog (product number: 10678ES80, purchased from Yeasen Biotech Co., Ltd.) as starting materials, transcribe and synthesize mRNA through T7 RNA polymerase. The specific reaction system is shown in Table 2. Place the prepared reaction system in a constant temperature incubator at 37 °C and shake it for reaction for 3 h. The above Cap1 cap analog is Cap1-GAG, which has the structure of m7G (5') ppp (5') (2'-OMeA) pG, and its molecular formula is C 32 H 43 N 15 O 24 P4.

[0316] Table 2 Co-transcription capping reaction system

[0317]

[0318] Note: All of the above reagents are purchased from Yeasen Biotech Co., Ltd.

[0319] 2) Digest the template DNA:

[0320] Add DNase I (purchased from Yeasen Biotech Co., Ltd.) to the co-transcriptional capping reaction system after the completion of the reaction in step 1) above to a final concentration of 1 U / μg linearized plasmid. After mixing, centrifuge and incubate at 37 °C for 1 hour to obtain the co-transcriptional capping product.

[0321] 3) Purification by lithium chloride precipitation:

[0322] Purify the co-transcriptional capping product obtained in step 2) above by lithium chloride precipitation. The method is as follows:

[0323] Step ① Add lithium chloride: Add lithium chloride solution (purchased from Thermo Fisher Scientific) to the product of step 2) above to a final concentration of 2.8 M, and precipitate at low temperature for 2 hours;

[0324] Step ② Precipitation: Centrifuge at 12,000 rmp for 15 minutes and retain the precipitate;

[0325] Step ③ Washing: Wash twice with 75% ethanol and dissolve with water for injection to obtain the mRNA solution. The purified mRNA solution is stored at -80 °C.

[0326] Example 3: Effect of different adjuvant lipid addition amounts on the TLP-mRNA composition

[0327] Weigh ADOPE, DOTMA, DOPE, and YK-1202 according to the ratio in Table 3 and dissolve them in ethanol to prepare an ethanol lipid complex solution (total lipid concentration is 266 mM). Rapidly add the ethanol lipid complex solution to the sterile and enzyme-free water under stirring by the ethanol injection method at a rotation speed of 120 rmp, stir at room temperature for 30 minutes, filter the resulting mixture solution through a polycarbonate membrane with a pore size of 450 nm to obtain the lipid composition solution, and store it at 4 °C to 8 °C. Dilute the mRNA in the buffer to obtain an mRNA aqueous solution (mRNA concentration is 0.5 mg / mL, and the buffer is 10 mM HEPES buffer containing 0.1 mM EDTA). Use a syringe to take the sodium chloride aqueous solution (0.9% w / w) and inject it into the above-prepared mRNA aqueous solution to obtain a mixture of mRNA and sodium chloride. Use a syringe to take the lipid complex solution and inject it into the above mixture of mRNA and sodium chloride, vortex for 30 seconds, and incubate at room temperature for 10 minutes to obtain the TLP drug composition with or without adjuvant lipid (RNA final concentration is 100 μg / mL), and store it at 4 °C to 8 °C. Among them, the amounts of mRNA and lipid complex solution are such that the charge ratio of the TLP-mRNA composition is 1:2. The calculation method of the charge ratio is as follows:

[0328] Charge ratio of the mRNA-TLP composition = (molar amount of positive charges carried by the permanent cationic lipid - molar amount of negative charges carried by the permanent anionic lipid) / (weight of mRNA (g) divided by the average molecular weight of bases 330 (g / mol)).

[0329] The mRNA-TLP composition was diluted by mixing it with an aqueous sodium chloride solution (0.9% w / w) at a volume ratio of 1:5, and the particle size and polydispersity index (PDI) were measured using a Malvern laser particle size analyzer by dynamic light scattering.

[0330] Table 3 Particle size and PDI of compositions with different formulations

[0331]

[0332] The results showed that when YK-1202 was used to partially replace DOTMA, the permanent cationic lipid in the TLP composition, at 2.5%, 5.0%, 10.0%, 15.0%, and 20.0%, the particle size and PDI of the resulting TLP pharmaceutical composition were within the qualified range (particle size of 220 - 500 nm, PDI value < 0.5). In the following examples, an adjuvant lipid ratio of 10% was used.

[0333] Example 4: Effect of adding different adjuvant lipids on the TLP-mRNA composition

[0334] Table 4 Structures of adjuvant compounds in the anionic lipid composition

[0335]

[0336]

[0337] According to the preparation method of Example 3, the adjuvant lipid compounds in Table 4 were used to partially replace DOTMA in the TLP composition at a ratio of 10 mol%, that is, the permanent anionic lipid ADOPE, the permanent cationic lipid DOTMA, the neutral lipid DOPE, and the adjuvant lipid were prepared in a molar ratio of 5:8:5:2 (that is, 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) to prepare the mRNA-TLP pharmaceutical composition, and the results are shown in Table 5.

[0338] Table 5 Detection results of particle size and PDI of mRNA-TLP compositions containing different adjuvant lipids

[0339]

[0340] The results showed that the adjuvant lipids YK-1201 to YK-1208 in Table 4, as well as compounds 23, 63-15, and TMX-201, could all prepare mRNA compositions with qualified particle size and PDI (particle size controlled within 220 - 500 nm and PDI less than 0.5) according to the preparation method of Example 3.

[0341] Example 5: Protein Expression Experiment of mRNA-TLP Composition Prepared by Adding Adjuvant Lipids in Mice

[0342] The Fluc-mRNA-TLP compositions with or without adjuvant lipids prepared in Example 4 were intravenously injected into female BALB / c albino mice aged 4 - 6 weeks and weighing 17 - 19 g through the tail vein (the dosage was about 20 μg Fluc-mRNA / mouse). After 6 hours of administration, the fluorescence imaging substrate was intraperitoneally injected into the mice. The mice were allowed to move freely for 5 minutes, and then the total radiation intensity of the protein expressed by the mRNA carried by the mRNA composition in the mice was detected by an IVIS Spectrum small animal in vivo imager (corresponding to the fluorescence protein expression intensity, that is, the protein expression level). After sampling, the mice were sacrificed by cervical dislocation and dissected, and the internal organs of the mice: liver and spleen were precisely separated. The total radiation intensity of the protein expressed by Fluc-mRNA in each organ of the mice was detected by an IVIS Spectrum small animal in vivo imager (corresponding to the fluorescence protein expression intensity, that is, the protein expression level). The results of mouse in vivo imaging and protein expression detection in each internal organ are shown in Table 6 and Figure 1 .

[0343] Table 6 Experimental Data of Mouse In Vivo and Organ Imaging

[0344]

[0345] It can be seen that adding the adjuvant lipids of the present invention to the mRNA-TLP composition can efficiently deliver mRNA to the spleen of animals, and the delivery effect is significantly enhanced compared with the TLP composition without adding adjuvant lipids.

[0346] Compared with adding adjuvant lipids of the prior art (compounds 23, 63-15, and TMX-201), the compositions adding YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 of the present invention showed significant enhancement in both the total radiation intensity of the spleen and the total radiation intensity in vivo. For example, for the composition adding YK-1204, the total radiation intensity of the spleen was 4.4 times, 4.7 times, and 4.3 times that of the compositions adding compounds 23, 63-15, and TMX-201 respectively, and the in vivo radiation intensity was 3.3 times, 3.5 times, and 2.9 times that of the compositions adding compounds 23, 63-15, and TMX-201 respectively.

[0347] For compositions adding different adjuvant lipids with similar structures to those of the present invention, there are significant differences in the total radiation intensity of the spleen and the total radiation intensity in vivo. For example, for the composition adding YK-1206, the total radiation intensity of the spleen and the total radiation intensity in vivo are 2.7 times and 3.3 times respectively of those of the composition adding YK-1201.

[0348] Example 6: Targeting study on mouse spleen cells of mRNA-TLP composition adding adjuvant lipid

[0349] 1. Inject the eGFP-mRNA-TLP composition with or without adjuvant lipid prepared in Example 3 into female C57BL / 6 mice at 4-6 weeks old and weighing 17-19 g via the tail vein (the dosage is such that each mouse is injected with about 80 μg eGFP-mRNA), and sacrifice the mice by cervical dislocation 24 hours after administration and perform dissection to precisely isolate the spleens of the mice.

[0350] 2. Prepare single cells

[0351] 1) Grind the isolated spleen tissue to make the spleen tissue single-celled and pass through a cell sieve;

[0352] 2) Add 10 times the volume (about 4 mL) of red blood cell lysate to lyse and remove the red blood cells in the tissue;

[0353] 3) Count the cells and take 5×10 6 cells into a flow tube (ensure that the number of cells taken between samples is the same);

[0354] 3. Detection of immune cells in spleen tissue

[0355] 1) Add 100 μL of surface antibody MIX to each single-cell suspension (the components of surface antibody MIX are shown in Table 7 in detail), and incubate in the dark at room temperature for 15 minutes (one negative control);

[0356] Table 7 Reagents and sources in flow cytometry experiment of mouse spleen cells

[0357]

[0358] 2) Add 2 mL of PBS, centrifuge at 500 g for 5 minutes and discard the supernatant;

[0359] 3) Resuspend the cells with 200 μL of PBS, (after filtering through a 200-mesh nylon net), and detect on the flow cytometer Cytoflex S. Analyze the percentage of GFP content in each cell. The detection order of each cell line is as follows:

[0360] Proportion of GFP in T cells: CD45 + →CD3 + →GFP+

[0361] Percentage of B cells with GFP: CD45 + →CD3 - CD19 + →GFP +

[0362] Percentage of NK cells with GFP: CD45 + →NK1.1 + →GFP +

[0363] Percentage of cDC cells with GFP: CD45 + →F4 / 80 - CD11c + →GFP +

[0364] Percentage of pDC cells with GFP: CD45 + →F4 / 80 - CD11c int CD317 + →GFP +

[0365] Percentage of macrophages with GFP: CD45 + →F4 / 80 + →GFP +

[0366] Experimental results:

[0367] The percentage of eGFP-positive cells in mouse spleen cells is shown in Table 8.

[0368] Table 8 Percentage of eGFP-positive cells in mouse spleen cells

[0369]

[0370] Antigen-presenting cells (also known as "antigen-presenting cells") refer to a type of immune cells that can uptake, process antigens, and present the processed antigens to lymphocytes, including dendritic cells (DC cells), B lymphocytes, and macrophages, which play a crucial role in the human body, mainly including immune recognition, immune response, and immune regulation.

[0371] From the data in Table 8 and Figure 2It can be seen that the mRNA-TLP composition prepared from the adjuvant lipid of the present invention can significantly increase the percentage of cells expressing antigens in antigen-presenting cells in the spleen. Compared with the TLP-mRNA composition without an adjuvant, it can significantly increase the percentage of cells expressing antigens in antigen-presenting cells in the spleen. For example, the mRNA-TLP composition prepared by adding YK-1204 increased the percentage of eGFP-positive cells in antigen-presenting cells B cells, cDC cells, pDC cells, and macrophages in the spleen by 2.1-fold, 1.7-fold, 1.7-fold, and 1.7-fold, respectively.

[0372] Compared with the adjuvants of the prior art, the mRNA-TLP composition prepared from the adjuvant lipid of the present invention has significantly improved targeting to antigen-presenting cells in the mouse spleen. For example, the percentages of eGFP-positive cells in B cells, cDC cells, pDC cells, and macrophages in the mouse spleen of YK-1204 were 1.9-fold, 1.5-fold, 1.5-fold, and 1.7-fold that of TMX-201, respectively.

[0373] There are significant differences in the targeting of mRNA-TLP compositions prepared from different adjuvant lipids of the present invention to antigen-presenting cells in the mouse spleen, and the data of YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 are significantly higher than those of other compounds. For example, the percentages of eGFP-positive cells in B cells, cDC cells, pDC cells, and macrophages of the mRNA-TLP composition prepared by YK-1206 were 2.2-fold, 1.8-fold, 1.7-fold, and 1.9-fold that of the mRNA-TLP composition prepared by YK-1203, respectively.

[0374] Example 7: Study on the maturation state of antigen-presenting cells in the mouse spleen of the mRNA-TLP composition prepared from adjuvant lipid

[0375] CD86 (cluster of differentiation 86) is a molecule expressed on antigen-presenting cells that provides co-stimulatory signals required for T cell activation and survival, and the maturation state of spleen antigen-presenting cells can be shown by the upregulation of CD86. In this example, on the basis of the flow cytometry experiment in Example 5, an experiment to detect the upregulation of CD86 was added, and the surface antibody used was Brilliant Violet 510™ anti-mouse CD86. The detection sequence for each cell line is as follows:

[0376] Proportion of CD86 in B cells: CD45 + →CD3 - CD19 + →CD86 +

[0377] Proportion of CD86 in NK cells: CD45+ →NK1.1 + →CD86 +

[0378] Ratio of CD86 in cDC cells: CD45 + →F4 / 80 - CD11c + →CD86 +

[0379] Ratio of CD86 in pDC cells: CD45 + →F4 / 80 - CD11c int CD317 + →CD86 +

[0380] Ratio of CD86 in macrophages: CD45 + →F4 / 80 + →CD86 +

[0381] Experimental results:

[0382] The percentage of CD86-positive cells in mouse spleen cells is shown in Table 9.

[0383] Table 9 Percentage of CD86-positive cells in mouse spleen cells

[0384]

[0385] It can be seen that the mRNA-TLP composition prepared from the adjuvant lipid of the present invention can significantly increase the percentage of CD86-positive cells in antigen-presenting cells in the spleen. Compared with the mRNA-TLP composition without adjuvant, the mRNA-TLP compositions prepared from the adjuvant lipids YK-1202, YK-1204 to YK-1206, and YK-1208 of the present invention can significantly improve the maturation state of antigen-presenting cells in the mouse spleen. For example, in the mRNA-TLP composition added with YK-1205, the percentages of CD86-positive cells in B cells, cDC cells, pDC cells, and macrophages in the mouse spleen are 1.1 times, 1.5 times, 1.6 times, and 1.7 times the corresponding percentages of TLP without adjuvant lipid, respectively.

[0386] Compared with the adjuvants of the prior art, the mRNA-TLP compositions prepared from the adjuvant lipids YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 of the present invention significantly improve the maturation state of antigen-presenting cells in the mouse spleen. For example, in the mRNA-TLP composition prepared from YK-1205, the percentages of CD86-positive cells in B cells, cDC cells, pDC cells, and macrophages in the mouse spleen are 1.3 times, 1.9 times, 1.7 times, and 1.4 times the corresponding percentages of TMX-201, respectively. ( Figure 3 )

[0387] Among the mRNA-TLP compositions prepared from different adjuvant lipids provided by the present invention, there are significant differences in the percentages of CD86-positive cells in spleen antigen-presenting cells. For example, in the mRNA-TLP composition prepared from YK-1206, the percentage of CD86-positive cells in B cells in the mouse spleen is 1.8 times the corresponding percentage of YK-1207.

[0388] Example 8: Study on the activation of T cells in the mouse spleen of the mRNA-TLP composition prepared from adjuvant lipids

[0389] CD69 is a marker of T cell activation and belongs to one of the earliest markers that are upregulated after T cell activation. The activation of spleen T cells can be shown by the upregulation of CD69. In this example, based on the flow cytometry experiment in Example 5, an experiment to detect the upregulation of CD69 was added. The surface antibody is APC anti-mouse CD69, and the cell line detection sequence is as follows:

[0390] Proportion of T cell CD69: CD45 + →CD3 + →CD69 +

[0391] Experimental results:

[0392] The percentages of CD69-positive cells in mouse spleen T cells are shown in Table 10.

[0393] Table 10 Percentages of CD69-positive cells in mouse spleen T cells

[0394]

[0395] The mRNA-TLP compositions prepared with the adjuvant lipids of the present invention can all significantly increase the percentage of CD69-positive cells in T cells in the spleen. Compared with the mRNA-TLP compositions without adjuvant lipids, they can significantly increase the percentage of CD69-positive cells in T cells in the spleen. For example, compared with the TLP without adjuvant, the mRNA-TLP compositions prepared with the adjuvant lipids YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 of the present invention make the percentage of CD69-positive cells in T cells in the spleen 1.5 times, 1.7 times, 1.7 times, 1.6 times, and 1.6 times that of TLP respectively, showing a significant increase.

[0396] Compared with the adjuvant lipids of the prior art, the mRNA-TLP compositions prepared with the adjuvant lipids of the present invention significantly improve the activation state of T cells in the spleen of mice. For example, the percentage of CD69-positive cells in T cells in the spleen of the mRNA-TLP composition prepared with YK-1205 is 2.5 times the corresponding percentage of the mRNA-TLP composition of TMX-201. ( Figure 4 )

[0397] There are significant differences in the percentage of CD69-positive cells in T cells in the spleen of the mRNA-TLP compositions prepared with different adjuvant lipids in the present invention. For example, the percentage of CD69-positive cells in T cells in the spleen of the mRNA-TLP compositions prepared with YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 is significantly higher than that of other compositions. Among them, after administering the mRNA-TLP composition prepared with YK-1205, the percentage of CD69-positive cells in T cells is the highest, which is 1.4 times that of the lowest YK-1207.

[0398] Example 9: Stimulation effect of the mRNA-TLP composition containing adjuvant lipid on cytokines IFN-γ and IL-12

[0399] Interferon-γ (IFN-γ) is an important cytokine, mainly produced by activated T cells and natural killer (NK) cells. It plays a key role in the immune response, has multiple functions such as antiviral, antitumor, immune regulation, and promotion of inflammatory responses, and can treat various diseases. Interleukin-12 (IL-12) is a multifunctional cytokine produced by dendritic cells, macrophages, B lymphocytes, and other antigen-presenting cells, and plays an important role in regulating the immune response, promoting Th1-type immunity, enhancing cytotoxicity, and in tumor immunity. Detecting the stimulation of the mRNA-TLP composition containing adjuvant lipid on cytokines IFN-γ and IL-12 can reflect the improvement effect of adjuvant lipid on the innate immunity of the mRNA-TLP composition.

[0400] Experimental procedure:

[0401] Six hours after the tail vein injection of the OVA-mRNA-TLP (40 µg) composition prepared according to Example 4 into 8-week-old female C57BL / 6J mice, the mice were euthanized by enucleation of the eyeballs and blood was taken to obtain as much serum as possible. The contents of IFN-γ and IL-12 in the serum were measured by ELISA. At the same time, the mice injected with an equal volume of blank lipid solution were set as the blank group.

[0402] ELISA assay: Mouse IFN-γ and IL-12 were detected in mouse serum using a standard ELISA according to the manufacturer's instructions.

[0403] Table 11 Comparison of the stimulating effects of adjuvant lipid-containing mRNA-TLP compositions on cytokines IFN-γ and IL-12

[0404]

[0405] Experimental results:

[0406] As shown in Table 11 and Figure 5 As shown, compared with the mRNA-TLP composition without adjuvant lipid, the IFN-γ and IL-12 cytokines in the serum of the mRNA-TLP composition containing adjuvant lipid were significantly increased 6 hours after injection. Among them, the IFN-γ and IL-12 stimulated by YK-1202-TLP were 1.8 times and 1.5 times that of the mRNA-TLP composition without adjuvant lipid, respectively; YK-1204-TLP were 2.7 times and 3.1 times, respectively; YK-1205-TLP were 2.0 times and 1.8 times, respectively; YK-1208-TLP were 2.1 times and 1.9 times, respectively, of the mRNA-TLP composition without adjuvant lipid.

[0407] The experimental results show that by detecting the IFN-γ and IL-12 cytokines stimulated by the adjuvant lipid-containing mRNA-TLP composition, it can be proved that the adjuvant lipid of the present invention can initiate an immune stimulation program and significantly improve the innate immunity of the mRNA-TLP composition.

[0408] Example 10: Therapeutic effect of adjuvant lipid-containing mRNA-TLP composition on tumor-bearing mouse model

[0409] Experimental procedure:

[0410] 1) Establishment of B16F10-OVA mouse model: Before the study, female C57BL / 6J mice between 6 and 8 weeks of age were acclimated for at least three days. The mice had free access to food and sterile water and were housed in a 12-hour light / dark cycle at 22 °C ± 2 °C and 55% ± 15% relative humidity. B16F10-OVA cells (Zhejiang Meisen Cell Technology Co., Ltd.) were cultured in a complete medium described in the B16F10-OVA (CTCC-001-0727) instruction manual at 5% CO2 and 37 °C. The cells were collected using 0.25% trypsin-EDTA and resuspended in Dulbecco's phosphate buffered saline (DPBS). Then, 2 × 10 5 cells / 100 μL / mouse (B16F10-OVA) were transplanted subcutaneously (SC) into female C57BL / 6J mice to establish a subcutaneous B16F10-OVA tumor model. Vaccination started when the tumor volume reached about 100 mm 3 .

[0411] 2) Vaccination: For the B16F10-OVA mouse model, the enrolled C57BL / 6J mice were vaccinated with the OVA mRNA YK-1202-TLP, YK-1204-TLP, YK-1205-TLP, YK-1208-TLP compositions (40 μg) containing TLR adjuvant lipids prepared in Example 3 by tail vein injection on days 3, 7, 10, 12, 14, and 17 after injecting the cells (day 0) (each mouse was injected with a vaccine containing 40 μg of the therapeutic agent mRNA-OVA each time). At the same time, mice inoculated with an equal volume of OVA mRNA-TLP without adjuvant were set as the control group, with 8 mice in each parallel group.

[0412] 3) Tumor size and mouse survival rate: Starting from the 7th day after inoculating the tumor, the tumor diameter was measured 3 times a week. The tumor volume of C57BL / 6J mice was calculated according to the following formula: V(mm 3 ) = x × y 2 / 2, with the unit of mm, where V represents the tumor volume, x represents the long diameter of the tumor, and y represents the short diameter of the tumor. At the same time, the change in the body weight of C57BL / 6J mice was recorded 3 times a week using an electronic balance, and the survival rate was statistically analyzed.

[0413] Experimental results:

[0414] As Figure 6As shown in Table 12, on the 10th day after tumor inoculation, the mice in the blank lipid control group (Control Group) and the OVA mRNA-TLP composition group without adjuvant entered the rapid tumor growth stage, 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 adjuvant lipids all showed significant tumor growth delay. Starting from the 14th day, compared with the TLP group, the tumor volumes of the mRNA-TLP pharmaceutical compositions with adjuvant lipids also showed stronger tumor growth inhibition, and the mRNA-TLP groups prepared with YK-1204 and YK-1208 showed relatively better tumor inhibition effects.

[0415] Table 12 Tumor growth in mice during the tumor treatment process of different compositions

[0416]

[0417] In terms of survival rate, as Figure 7 shown, the mice in the blank lipid control group started to die on the 17th day after tumor inoculation and all died on the 19th day; the TLP group without adjuvant started to die on the 19th day and all died on the 27th day; the mRNA-TLP pharmaceutical compositions with adjuvant lipids YK-1202, YK-1204, YK-1205, and YK-1208 started to die on the 23rd, 25th, 25th, and 30th days respectively and all died on the 31st, 43rd, 37th, and 45th days respectively. It can be seen that the addition of adjuvant lipids significantly prolonged the survival time of the mice.

[0418] Example 11: Effects of different addition amounts of adjuvant lipids on LNP-mRNA compositions

[0419] This example investigated the effects of adjuvant lipids on LNP compositions.

[0420] Experimental procedure:

[0421] Weigh YK-009, YK-1202, DSPC, cholesterol, and DMG-PEG2000 according to the ratio in Table 12 and dissolve them in ethanol to prepare an ethanol lipid solution. Dilute eGFP-mRNA in citrate buffer (pH = 4 - 5) to obtain an aqueous mRNA solution. Use a microfluidic device to mix the ethanol lipid solution with the aqueous Fluc mRNA solution prepared from different capping structures at a flow rate of 10 mL / min and a volume ratio of 1:3 to prepare LNP with a weight ratio of total lipid to mRNA of approximately 15:1. After diluting the obtained liposomes to 10 times the volume with PBS, ultrafiltrate to remove ethanol using a 300 KDa ultrafiltration tube. Then, make up the volume to a certain volume with PBS. Finally, filter the lipid nanoparticles through a 0.2 μm sterile filter to obtain the LNP pharmaceutical composition with or without adjuvant lipid. Use dynamic light scattering and a Malvern laser particle size analyzer to measure the particle size and polydispersity index (PDI). Take 10 μL of the liposome solution, dilute it to 1 mL with RNase-free deionized water, add it to the sample cell, and measure each sample 3 times. The measurement conditions are: 90° scattering angle, 25°C. According to the manufacturer's instructions, use the Quant itRibogreen RNA Quantification Assay Kit (ThermoFisher Scientific, UK) to determine the encapsulation efficiency of the lipid nanoparticles, and the results are shown in Table 13.

[0422] Table 13 Particle size, PDI, and encapsulation rate of compositions with different formulations

[0423]

[0424] The results show that when using YK-1202 to partially replace the cationic lipid YK-009 in the LNP composition at 2.5%, 5.0%, 10.0%, 15.0%, and 20.0%, the particle size, PDI, and encapsulation rate of the obtained LNP pharmaceutical composition are all within the qualified range (particle size < 200 nm, PDI < 0.3, encapsulation rate > 85%). When the adjuvant content is 10 mol%, the particle size, PDI, and encapsulation rate reach the best state. Next, the adjuvant lipid is studied at a ratio of 10% adjuvant lipid.

[0425] Example 12: Effects of different adjuvant lipids on LNP-mRNA composition

[0426] Experimental procedure:

[0427] According to the preparation method of Example 11, the adjuvant lipid compound in Table 4 was used to partially replace the proportion of YK-009 in the LNP composition at a ratio of 10 mol%, that is, the Fluc-mRNA-LNP pharmaceutical composition was prepared with YK-009, DSPC, cholesterol, DMG-PEG2000 and the adjuvant lipid in a molar ratio of 40:10:38.5:1.5:10. Evaluation method: First, the particle size, polydispersity index (PDI) and encapsulation efficiency were measured according to the method of Example 11; then the pharmaceutical composition was added to the cell culture medium in a 96-well plate, and after continuous culture 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 by the IVIS fluorescence detection system; finally, 10 μL of CCK-8 solution was added to each well of the above-mentioned plate after 24 hours of culture, and after incubating the culture plate in an incubator for 1 hour, the absorbance at 450 nm was measured by a microplate reader to detect the cell viability. The results are shown in Table 14.

[0428] Table 14 Detection results of particle size and PDI of LNP compositions containing different adjuvants

[0429]

[0430] The results show that: In this application, the adjuvant lipids YK-1201~1208 and the adjuvant lipids 63-15 and TMX-201 disclosed in the prior art can all prepare good mRNA-LNP compositions by replacing YK-009 at a ratio of 10%. The particle size of all lipid nanoparticles is between 67 and 88 nm, the PDI value is between 0.08 and 0.13, and the encapsulation efficiency is above 90%.

[0431] However, there are significant differences in the relative fluorescence intensity (translation efficiency of mRNA) and cell viability (cytotoxicity) of the above-prepared LNP compositions. The mRNA-LNP compositions prepared from YK-1202, YK-1204, YK-1205, YK-1206 and YK-1208 have a significantly higher relative fluorescence intensity than those of the YK-1201, YK-1203 and YK-1207 groups, and are also significantly higher than the LNP group without adjuvant lipid and the mRNA-LNP groups added with 63-15 and TMX-201. Moreover, the cytotoxicity of the mRNA-LNP compositions prepared from YK-1202, YK-1204, YK-1205, YK-1206 and YK-1208 is significantly lower than that of the YK-1201, YK-1203 and YK-1207 groups, and is at the same level as the mRNA-LNP groups without adjuvant lipid and added with 63-15 and TMX-201.

[0432] Example 13: Animal Expression of LNP-mRNA Compositions with Different Adjuvant Lipids

[0433] Experimental Procedure:

[0434] The LNP formulations containing 10 μg of Fluc-mRNA with different adjuvant lipids prepared according to Example 11 were injected into the tail muscle of female BALB / C mice aged 4 - 6 weeks and weighing 17 - 19 g. At a specific time point (6 hours) after administration, the mice were intraperitoneally injected with a fluorescence imaging substrate. The mice were allowed to move freely for 5 minutes, and then the average radiant intensity (corresponding to the fluorescence expression intensity) of the protein expressed by the mRNA carried by the LNP in the mice was detected by an IVIS Spectrum small animal in vivo imager.

[0435] After sampling was completed, the mice were euthanized with carbon dioxide, dissected, and the internal organs of the mice: liver, spleen, and lung were precisely separated. The total radiant intensity (corresponding to the fluorescence expression intensity) of the protein expressed by the mRNA carried by the LNP in each organ of the mice was detected by an IVIS Spectrum small animal in vivo imager. The results of the in vivo imaging detection of the mice are shown in Table 15.

[0436] Table 15 In Vivo Imaging Experimental Data at a Specific Time Point (6 hours) after Administration to Mice

[0437]

[0438] Experimental Results:

[0439] It can be seen that for the LNP formulations prepared from YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208, the expression levels of the delivered mRNA in the injection site, abdominal cavity, liver, and spleen of the mice were significantly higher compared to the LNP without added adjuvant lipid and the LNP with the representative adjuvant lipid TMX-201 in the prior art. For example, in the mRNA-LNP group with YK-1206 added, the expression level at the injection site of the mice was 1.7 times that of the LNP without added adjuvant lipid, and the expression in the spleen of the mice was 1.8 times that of the LNP with TMX-201 added.

[0440] There are a large number of APC cells in the spleen and muscle (injection site). By increasing the expression levels of the delivered mRNA in the spleen and muscle, it is possible to rapidly induce an immune response and produce antibodies in vivo for the mRNA vaccine. Without changing the vaccine components, the preventive and therapeutic effects can be significantly improved, which has important clinical significance.

[0441] In addition, for the LNP formulation containing Fluc-mRNA with added adjuvant lipids, the expression differences in different organs of mice were very large. The LNP formulations prepared with YK-1202, YK-1204, YK-1205, YK-1206, YK-1208, TMX-201 and without added adjuvant lipids were all expressed in the liver and spleen, but not expressed in the lungs.

[0442] Compared with the structurally similar adjuvant lipid YK-1201, for the mRNA-LNP formulations prepared with YK-1202, YK-1204, YK-1205, YK-1206, YK-1208, the expression intensity of mRNA in the liver and spleen of mice was significantly increased. For example, for the LNP formulation with added YK-1206, the expression level of mRNA at the injection site was 2.8 times that of the LNP formulation with added YK-1201, and the expression level in the spleen reached 3.8 times.

[0443] Example 14: Stimulation of cytokines IFN-γ and IL-12 by mRNA-LNP compositions containing adjuvant lipids

[0444] Experimental procedure:

[0445] Eight-week-old female C57BL / 6J mice were euthanized by exsanguination by eyeball removal 6 hours after intramuscular injection of the OVA-mRNA-LNP (5 µg) composition prepared according to Example 11 to obtain as much serum as possible. The contents of IFN-γ and IL-12 in the serum were measured by ELISA. At the same time, mice injected with an equal volume of blank lipid solution were set as the blank group.

[0446] ELISA assay: Mouse IFN-γ and IL-12 were detected in mouse serum using a standard ELISA according to the manufacturer's instructions.

[0447] Table 16 Comparison of the stimulation of cytokines IFN-γ and IL-12 by mRNA-LNP compositions containing adjuvant lipids

[0448]

[0449] Experimental results:

[0450] As shown in Table 16 and Figure 8As shown, compared with the mRNA-LNP composition without adjuvant lipid, the mRNA-LNP composition containing adjuvant lipid showed an increase in both IFN-γ and IL-12 cytokines in serum 6 hours after injection. Among them, the serum levels of cytokine IL-12 stimulated by the mRNA-LNP compositions supplemented with YK-1202-LNP, YK-1204-LNP, YK-1206-LNP, and YK-1208-LNP were 1.3-fold, 1.6-fold, 2.1-fold, and 1.5-fold those of the mRNA-LNP composition without adjuvant lipid; the serum levels of cytokine IFN-γ stimulated by the mRNA-LNP compositions supplemented with YK-1202-LNP, YK-1204-LNP, YK-1206-LNP, and YK-1208-LNP were 1.5-fold, 2.9-fold, 1.8-fold, and 5.3-fold those of the mRNA-LNP composition without adjuvant lipid.

[0451] The experimental results show that by detecting the IFN-γ and IL-12 cytokines stimulated by the mRNA-LNP composition containing adjuvant lipid, it can be 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.

[0452] Example 15: Therapeutic effect of mRNA-LNP composition containing adjuvant lipid on tumor-bearing mouse model

[0453] Experimental procedure:

[0454] 1) Establishment of MC38-OVA mouse model: Before the study, female C57BL / 6J mice between 6 and 8 weeks of age were acclimated for at least three days. The mice had free access to food and sterile water and were housed in a 12-hour light / dark cycle at 22 °C ± 2 °C and 55% ± 15% relative humidity. MC38-OVA cells were cultured in a complete medium (RPMI-1640 supplemented with 10% fetal bovine serum (FBS)) at 5% CO2 and 37 °C as described in the instructions. Cells were collected using 0.25% trypsin-EDTA and resuspended in Dulbecco's phosphate-buffered saline (DPBS). Then, 1.5 × 10 6 cells / 100 μL / mouse (MC38-OVA) were transplanted subcutaneously (SC) into female C57BL / 6J mice to establish a subcutaneous MC38-OVA tumor model. Vaccination started when the tumor volume reached about 50 mm 3 or so.

[0455] 2) Vaccination: The C57BL / 6J mice included in the group were vaccinated by intramuscular injection with the OVA mRNA-YK-1202-LNP, YK-1204-LNP, YK-1206-LNP, and YK-1208-LNP compositions containing adjuvant lipids prepared in Example 11 on the 3rd, 7th, 10th, 12th, 15th, and 19th days after injecting cells (day 0) (each mouse was injected with a vaccine containing 5 μg of therapeutic agent mRNA-OVA each time). At the same time, mice inoculated with OVA mRNA-LNP without adjuvant lipids of equal volume were set as the control group, with 8 mice in each group in parallel.

[0456] 3) Tumor size and mouse survival rate: Starting from the 7th day after inoculating the tumor, the tumor diameter was measured 3 times a week. The tumor volume of C57BL / 6J mice was calculated according to the following formula: V(mm 3 ) = x × y 2 / 2, with the unit of mm, where V represents the tumor volume, x represents the long diameter of the tumor, and y represents the short diameter of the tumor. At the same time, the change in the body weight of C57BL / 6J mice was recorded with an electronic balance 3 times a week, and the survival rate was statistically analyzed.

[0457] Experimental results:

[0458] As Figure 9 shown in and Table 17, on the 12th day after inoculating the tumor, the blank lipid control group (Control Group) and the mice of the OVA mRNA-LNP composition without adjuvant lipids entered the rapid tumor growth period, but the gap between the group without adjuvant LNP and the blank control group gradually widened over time. The OVA mRNA-LNP compositions added with adjuvant lipids all showed significant tumor growth delay. Starting from the 14th day, compared with the group without adjuvant LNP, the tumor volumes of the mRNA-LNP groups added with YK-1202, YK-1204, YK-1206, and YK-1208 also showed stronger tumor growth inhibition, and the YK-1204 and YK-1208 groups showed relatively better tumor inhibition effects.

[0459] Table 17 Tumor growth in mice during the tumor treatment process of different compositions

[0460]

[0461] In terms of the survival rate, as Figure 10As shown, the blank lipid control group started to die on the 17th day after tumor inoculation and all died on the 19th day; the mRNA-LNP group without adjuvant started to die on the 21st day and all died on the 30th day; the mRNA-LNP groups of YK-1202, YK-1204, YK-1206, and YK-1208 with adjuvant started to die on the 25th, 27th, 31st, and 29th days respectively and all died on the 33rd, 40th, 41st, and 42nd days respectively. It can be seen that after adding the adjuvant lipid of the present invention, the survival time of mice was significantly prolonged.

[0462] 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. Compared with the adjuvant lipids disclosed in the prior art, it has at least one of the following advantages:

[0463] 1. The chemical structure of the adjuvant lipid designed by the present invention is different from that of the adjuvant lipids disclosed in the prior art and is a brand-new compound.

[0464] 2. Adding the adjuvant lipid of the present invention to the mRNA-TLP composition can significantly increase the protein expression level in mice and at the same time has significant spleen targeting. Compared with the adjuvants in the prior art, the mRNA-TLP composition prepared from the adjuvant lipid of the present invention can significantly increase the protein expression level in the live body and spleen of mice.

[0465] 3. The mRNA-TLP composition prepared from the adjuvant lipid of the present invention significantly increases the percentage of antigen-presenting cells (such as B cells, pDC cells, cDC cells, and macrophages) expressing antigens in the spleen. Compared with the adjuvant lipids in the prior art, the mRNA-TLP composition prepared from the adjuvant lipid of the present invention has significantly improved targeting to antigen-presenting cells in the mouse spleen.

[0466] 4. The mRNA-TLP composition prepared from the adjuvant lipid of the present invention can significantly increase the percentage of CD86-positive cells among antigen-presenting cells in the spleen. Compared with the adjuvant lipids in the prior art, the mRNA-TLP composition prepared from the adjuvant lipid of the present invention significantly improves the maturation state of antigen-presenting cells in the mouse spleen.

[0467] 5. The mRNA-TLP composition prepared from the adjuvant lipid of the present invention can significantly increase the percentage of CD69-positive cells among T cells in the spleen. Compared with the adjuvant lipids in the prior art, the mRNA-TLP composition prepared from the adjuvant lipid of the present invention significantly improves the activation state of T cells in the mouse spleen.

[0468] 6. The mRNA-TLP composition prepared by adding the adjuvant lipid of the present invention shows a significant effect of controlling tumor growth and prolonging the survival time of tumor-bearing experimental animals in animal experiments compared with the mRNA-TLP composition without adjuvant lipid.

[0469] 7. The mRNA-LNP composition prepared by adding the adjuvant lipid (2.5% - 20 mol%) of the present invention has a good particle size (<200 nm) and a uniform particle distribution PDI (<0.3). Compared with the mRNA-LNP composition without adjuvant lipid, the mRNA-LNP composition prepared by adding the adjuvant lipid of the present invention has a significantly increased mRNA translation efficiency, a significantly reduced cytotoxicity, a significantly increased protein expression level in the injection site, abdominal cavity, liver and spleen of mice, and significantly inhibits tumor growth and prolongs the survival time of tumor-bearing experimental animals in animal experiments.

[0470] The above has described the present invention in detail. The purpose is to enable those skilled in this field to understand the content of the present invention and implement it, but it cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered by the protection scope of the present invention.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof: ； L1 is C 1-10 Alkylene or C 1-4 Alkylene-C 6-10 Aryl-C 1-4 Alkylene; M1 is -NH- or -OC(O)HN-; L2 is C 1-8 Alkylene, -R 3a C(O)OR 4a -、 or , where R 3a , R 3c , R 3d , R 4a , R 4c , R 4d and R5 are independently C 1-8 Alkylene; R1 is C 1-5 Alkyl or C 1-6 Alkoxy substituted C 1-5 alkyl; R2 is or , in, L3 and L4 are independently C 1-7 Alkylene; R6 and R7 are independently C 7-28 alkyl; R8 is C 1-7 Alkylene, R9 and R 10 Independently for C 10-20 Alkyl or C 10-20 Alkenyl.

2. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound represented by formula (I) satisfies any one or a combination of at least two selected from the group consisting of the following conditions: (1) L1 is C 1-6 Straight chain alkylene, L2 is C 1-6 Straight chain alkylene C(O)OC 1-4 Straight chain alkylene- a or , a end and R 2 Connect; R 3d , R 4d and R5 are independently C 1-4 Straight chain alkylene; Or, L1 is C 1-4 Straight chain alkylene-C 6-10 Aryl-C 1-4 Straight chain alkylene, L2 is C 1-4 Straight chain alkylene or C 1-6 Straight chain alkylene C(O)OC 1-4 Straight chain alkylene a , a end and R 2 connect; (2) R1 is C 1-5 Straight chain alkyl; (3) L3 is C 1-4 Straight chain alkylene; (4) L4 is C 4-6 Straight chain alkylene; (5) R6 is C 8-12 Straight chain alkyl; (6) R7 is C 16-20 Branched chain alkyl; (7) R8 is C 1-4 Straight chain alkylene; (8) R9 and R 10 Independently for C 16-20 Alkenyl.

3. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound represented by formula (I) satisfies any one or a combination of at least two selected from the group consisting of the following conditions: (1) L1 is -(CH2)4-, -CH2C((CH3)2)- b or , end b is connected to M1; (2) M1 is -NH-; (3) L2 is -(CH2)2-, -(CH2)3C(O)O(CH2)2- a 、-(CH2)5C(O)O(CH2)2- a , or , end a is connected to R2; (4) R1 is -(CH2)3CH3 or -CH2OCH2CH3; (5) R2 is or 。 4. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: L1 is -(CH2)4-, L2 is -(CH2)5C(O)O(CH2)2- a or , end a is connected to R2; Or, L1 is , L2 is -(CH2)2-, -(CH2)3C(O)O(CH2)2- a or -(CH2)5C(O)O(CH2)2- a , end a is connected to R2.

5. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound represented by formula (I) is any of the following compounds: 、 、 、 、 、 、 or 。 6. A lipid composition, characterized in that The lipid composition comprises a substance Z, and the substance Z is a compound represented by formula (I) as described in any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

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

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

9. The lipid composition according to claim 6, characterized in that The lipid composition comprises the substance Z, permanent anionic lipids, permanent cationic lipids and neutral lipids.

10. The lipid composition according to claim 9, characterized in that The lipid composition satisfies any one or a combination of at least two selected from the group consisting of the following conditions: (1) the permanent anionic lipid is any one selected from 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, or a combination of at least two thereof; (2) the permanent cationic lipid is selected from any one of the group consisting of 1,2-dioctadecenyloxy-3-methylammonium propane, (2,3-dioleyloxypropyl)trimethylammonium and their salts, or a combination of at least two thereof; (3) the neutral lipid is any one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, distearoylphosphatidylcholine and salts thereof, or a combination of at least two thereof; (4) The molar percentage ratio of the substance Z, permanent anionic lipid, permanent cationic lipid and neutral lipid is (2.5-20): (10-33): (20-60): (20-40).

11. The lipid composition according to claim 9, characterized in that The lipid composition satisfies any one or a combination of at least two selected from the group consisting of the following conditions: (1) The permanent anionic lipid is 2-acetylaminoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate sodium salt; (2) the molar percentage of the permanent anionic lipid in the lipid composition is 25%; (3) The permanent cationic lipid is 1,2-dioctadeceneoxy-3-methylammonium propane chloride; (4) 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 molar percentage of the neutral lipid in the lipid composition is 25%.

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

13. The lipid composition according to claim 12, characterized in that The molar percentage ratio of the 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 lipid composition according to claim 12, characterized in that The cationic lipid is selected from any one or a combination of at least two of the following compounds (1) to (7): (1) A compound represented by formula (II) or a pharmaceutically acceptable salt thereof, wherein G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; G3 is C 1～3 Alkylene; L1 is C 6～15 Straight chain alkyl; L2 is C 12～25 Branched chain alkyl; (II); (2) A compound represented by formula (III) or a pharmaceutically acceptable salt thereof, wherein G1 is C 2~8 Alkylene; G2 is C 2~8 Alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6~25 Straight or branched alkyl; R2 is C 6~25 Straight or branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2-, -(CH2)3- or -(CH2)4-; (III); (3) A compound represented by formula (IV) or a pharmaceutically acceptable salt thereof, wherein: G1 is C 1~6 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~20 Straight or branched alkyl; R2 is C 12~25 Branched alkyl; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(C H2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-; (IV); (4) A compound represented by formula (V) or a pharmaceutically acceptable salt thereof, wherein G1 is C 1~8 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~25 Straight or branched alkyl; R2 is C 12~25 Straight or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is -CH3, -CH2CH3 or -CH2CH2OH; (V)； (5) A compound represented by formula (VI) or a pharmaceutically acceptable salt thereof, wherein G 1 and G 2 Each independently is C 6~10 Alkylene; G 3 C 1~12 Alkylene; R 1 and R 2 Each independently is C 6~24 Alkyl or C 6~24 Alkenyl; R 3 OR 5 、N、-C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 C 1~12 Hydrocarbon; R 5 H or C 1~6 Hydrocarbon; (WE); (6) A compound represented by formula (VII) or a pharmaceutically acceptable salt thereof, wherein R4 is -(CH2) n Q and -(CH2) n CHQR; Q is -OR, -OH, -O(CH2) n wherein n is 1, 2 or 3; R is C(S)(O)R or heterocyclic; wherein n is 1, 2 or 3; and R is C(O)R or heterocyclic; wherein n is 1, 2 or 3; and R is C(O)N(R)2, -N(H)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), -N(H)C(S)N(R), -N(H)C(S)N(H)(R), -N(R)S(O)2R or heterocyclic; n is 1, 2 or 3; and R is C(O) 1-8 Alkyl; X is H or C 1-8 alkyl; (VII); (7) a compound represented by formula (VIII) or a pharmaceutically acceptable salt thereof, (VIII)。 15. The lipid composition according to claim 12, characterized in that The neutral lipid is selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide and sterol.

16. The lipid composition according to claim 12, characterized in that The lipid composition satisfies any one or a combination of at least two selected from the group consisting of the following conditions: (1) The cationic lipid is selected from any one of the group consisting of YK-009, YK-401, YK-305, ALC0315, SM102 and DLIN-MC3-DMA, or a combination of at least two of them: 、 、 、 、 、 ； (2) The neutral lipid is selected from 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-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, -O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 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, 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, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoyl any one of the group consisting of phosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine, or a combination of at least two thereof; (3) the structured lipid is selected from any one of the group consisting of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and corticosteroids, or a combination of at least two thereof; (4) The polymer conjugated lipid is selected from any one of the group consisting of distearoylphosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristoyl-sn-glycero-3-methoxy polyethylene glycol 2000 and methoxy polyethylene glycol ditetradecanoyl acetamide, or a combination of at least two thereof.

17. The lipid composition according to claim 16, characterized in that The lipid composition satisfies any one or a combination of at least two selected from the group consisting of the following conditions: (1) The cationic lipid is YK-009; (2) 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 and / or 1,2-distearoyl-sn-glycero-3-phosphocholine; (4) the molar percentage of the neutral lipid in the lipid composition is 10%; (5) The structural lipid is cholesterol; (6) the molar percentage of the structured lipid in the lipid composition is 38.5%; (7) The polymer conjugated lipid is 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000; (8) The molar percentage of the polymer conjugated lipid in the lipid composition is 1.5%.

18. The lipid composition according to any one of claims 6 to 17, characterized in that The lipid composition also includes one or more cell penetrating peptides.

19. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (A) a therapeutic agent and / or a preventive agent comprising any one of the group consisting of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein, or a combination of at least two thereof; (B) The lipid composition of any one of claims 9 to 17.

20. The pharmaceutical composition according to claim 19, characterized in that The pharmaceutical composition satisfies any one or a combination of at least two selected from the group consisting of the following conditions: (1) The therapeutic agent and / or preventive agent and the lipid composition according to any one of claims 9 to 11 are used in such an amount that the charge ratio of positive charge to negative charge in the pharmaceutical composition is 1:(2-5); (2) The mass ratio of the lipid composition according to any one of claims 12 to 17 to the therapeutic agent or preventive agent is (12.5-25): 1; (3) The pharmaceutical composition is used to deliver the therapeutic agent and / or preventive agent to antigen-presenting cells in a target organ or tissue.

21. The pharmaceutical composition according to claim 20, characterized in that The target organ or tissue is selected from any one or a combination of at least two of the group consisting of spleen, liver, lymph, muscle and lung; And / or, the antigen presenting cells are selected from any one or a combination of at least two of the group consisting of B cells, NK cells, cDC cells, pDC cells and macrophages.

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

23. The pharmaceutical composition according to claim 22, characterized in that The nucleic acid molecule is RNA encoding one or more antigens.

24. The pharmaceutical composition according to claim 22, characterized in that The antigen is a disease-associated antigen, or the nucleic acid molecule or antigen can induce an immune response against the disease-associated antigen or a cell expressing the disease-associated antigen.

25. The pharmaceutical composition according to claim 19, characterized in that The pharmaceutical composition further comprises at least one auxiliary ingredient; And / or, the pharmaceutical composition further comprises one or more hydrophobic small molecules, permeability enhancing molecules, carbohydrates, polymers, surface altering agents, functionalized lipids or cytokines.

26. The pharmaceutical composition according to claim 25, characterized in that The auxiliary component is a pharmaceutically acceptable carrier, diluent or excipient.

27. Use of a compound of formula (I) as described in any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, a lipid composition as described in any one of claims 6 to 18, or a pharmaceutical composition as described in any one of claims 19 to 26 in the preparation of a nucleic acid drug, a gene vaccine, a small molecule drug, a polypeptide or a protein drug.

28. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 5, a lipid composition as described in any one of claims 6 to 18, or a pharmaceutical composition as described in any one of claims 19 to 26 in the preparation of a medicament for treating a disease or condition; characterized in that: The disease or disorder is characterized by malfunctioning or aberrant protein or polypeptide activity.

29. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 5, a lipid composition as described in any one of claims 6 to 18, or a pharmaceutical composition as described in any one of claims 19 to 26 in the preparation of a medicament for treating a disease or condition; characterized in that: The disease or disorder is selected from any one or a combination of at least two of the group consisting of infectious diseases, tumor and proliferative diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases.

30. The use according to claim 29, characterized in that The infectious disease is a disease caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies or herpes; And / or, the tumor is breast cancer, ovarian cancer, lung cancer, pancreatic cancer, kidney cancer, gastric cancer, lymphoma, colon cancer, liver cancer, melanoma, bladder cancer, cervical cancer or prostate cancer.

31. The use according to any one of claims 27 to 29, characterized in that The drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation; And / or, the administration dosage of the drug is 0.001~10 mg / kg.

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