Vaccine adjuvant lipid compound based on Toll-like receptor stimulant and application thereof

Abstract

The invention discloses a Toll-like receptor stimulant-based vaccine adjuvant lipid compound and application thereof, and particularly discloses a compound as shown in a formula (I), a stereoisomer thereof, an N-oxygen compound thereof, a solvate thereof or a pharmaceutically acceptable salt thereof. The mRNA-TLP composition prepared from the adjuvant lipid compound provided by the invention has significantly improved targeting to mouse spleen antigen presenting cells, and can significantly inhibit tumor growth. # imgabs0 #

Description

Technical Field

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

[0002] In recent years, tumor immunotherapy based on messenger RNA (mRNA) has attracted widespread attention due to its significant clinical potential. Compared with DNA vaccines, mRNA can cause transient expression of tumor antigens while avoiding the possibility of insertion mutations. In addition, mRNA can improve therapeutic efficacy by inducing stronger humoral and cellular responses. Compared with traditional protein-based vaccines, mRNA vaccines have many advantages, such as biocompatibility, non-toxicity, and simple and scalable production processes. 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-methylpseudouracil (m1ψ) have been incorporated into in vitro transcribed mRNA sequences. However, the problem with this modification is that it may impair innate immunogenicity, which is crucial for activating dendritic cells (DCs, the main antigen-presenting cells).

[0004] Toll-like receptors (TLRs), as a family of transmembrane proteins, are innate immune receptors that are directly or indirectly responsible for detecting pathogen-associated molecular patterns (PAMPs). They can trigger a series of biosynthetic responses 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 improve the ability of antigen presentation. These molecular mechanisms are essential for activating innate and adaptive immune responses and can effectively stimulate the transition from innate immunity to adaptive immunity. The incorporation of lipid-modified TLRs into mRNA delivery systems to enhance the amplitude and duration of adaptive immune responses has been increasingly studied. Therefore, the development of a new generation of vaccine adjuvant lipids based on Toll-like receptor 7 and 8 agonists is of great significance for improving mRNA tumor therapy. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the fact that the existing vaccine adjuvant lipid compound based on Toll-like receptor 7 and 8 agonists has a single structure and the prepared mRNA composition has poor targeting to antigen presenting cells, and to provide a vaccine adjuvant lipid compound based on Toll-like receptor agonists and its application. The mRNA-TLP composition prepared by the adjuvant lipid compound in the present invention has significantly improved 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 stereoisomer, its N-oxygen compound, its solvate, or its pharmaceutically acceptable salt:

[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 Alkoxy substituted C 1-5 alkyl;

[0012] R2 is or ,

[0013] Where 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, R9 and R 10 Independently for C 10-20 Alkyl or C 10-20 Alkenyl.

[0016] In some embodiments, 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;

[0017] 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.

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

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

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

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

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

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

[0024] In some embodiments, R9 and R 10 Independently for C 16-20 Alkenyl.

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

[0026] In some embodiments, M1 is -NH- or -OC(O)HN-, and the nitrogen terminus is linked 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 , end a is connected to R2.

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

[0030] 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.

[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 of the following compounds:

[0034] , , , , , , or .

[0035] The present invention provides a lipid composition, which comprises a substance Z, wherein the substance Z is a compound of formula (I) as described above, a stereoisomer thereof, an N-oxy compound thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof.

[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, permanent anionic lipids, permanent cationic lipids and neutral lipids. The molar percentage ratio of the substance Z, permanent anionic lipids, permanent cationic lipids and neutral lipids 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(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.

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

[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-dioctadecenyloxy-3-methylammonium propane, (2,3-dioleyloxypropyl)trimethylammonium and salts thereof.

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

[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, distearoylphosphatidylcholine, and salts thereof.

[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 the substance Z, 2-acetylaminoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate, 1,2-dioctadecenyloxy-3-methylammonium propane (chloride salt) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; the molar percentages of the substance Z, 2-acetylaminoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate, 1,2-dioctadecenyloxy-3-methylammonium propane (chloride salt) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine are (2.5-20):25:(30-47.5):25; preferably (5-15):25:(35-45):25, and more preferably 10:25:40:25.

[0050] In some embodiments, the lipid composition comprises the substance Z, a cationic lipid, a neutral lipid, a structural lipid and a polymer conjugated lipid. The molar percentage ratio of the substance Z, the cationic lipid, the neutral lipid, the structural lipid and the 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 following groups consisting of compounds (1) to (7):

[0052] (1) A compound represented by formula (II), a stereoisomer thereof, an N-oxide thereof, a solvate thereof 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;

[0053] (II)

[0054] (2) A compound represented by formula (III), a stereoisomer thereof, an N-oxide thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, wherein G1 is C 2~8 Alkylene; G2 is C 2~8Alkylene; 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-;

[0055] (III)

[0056] (3) A compound represented by formula (IV), 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; 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-;

[0057] (IV)

[0058] (4) A compound represented by formula (V), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, 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 ;

[0059] (V)

[0060] (5) A compound represented by formula (VI), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G 1 and G 2 Each independently is C6~10 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;

[0061] (VI)

[0062] (6) A compound represented by formula (VII), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, 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;

[0063] (VII)

[0064] (7) A compound represented by formula (VIII), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof,

[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 derivatives thereof.

[0072] In some embodiments, the neutral lipid is selected from 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-diundecanoyl-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 hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-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), dipalmitoylphosphatidylglycerol (DPPG), palmitoyl Any one of the group consisting of oleylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine (LPE), or a combination of at least two thereof.

[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 structured lipid is selected from any one or a combination of at least two of the group consisting of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and corticosteroids.

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

[0077] In some embodiments, the molar percentage of the structured 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 distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) and methoxypolyethylene glycol ditetradecanoyl acetamide (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 includes 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; preferably 10: 40: 10: 38.5: 1.5.

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

[0083] The present invention provides a pharmaceutical composition, which comprises: (A) a therapeutic agent and / or a preventive agent, which comprises 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;

[0084] (B) Lipid composition as described above.

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

[0086] In some embodiments, the mass ratio of the lipid composition to the therapeutic agent or preventive 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 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.

[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-associated antigen, or the nucleic acid molecule or antigen is capable of eliciting an immune response against a disease-associated antigen or a cell expressing a disease-associated 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 may be a pharmaceutically acceptable 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 altering agents, functionalized lipids, or cytokines.

[0096] The present invention also provides a use of the 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 a use of the substance Z, lipid composition or pharmaceutical composition described above in the preparation of a medicament for treating a disease or condition. The disease or condition is preferably characterized by malfunctioning or abnormal protein or polypeptide activity.

[0098] In some embodiments, the disease or condition is selected from any one or a combination of at least two of 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 condition in a mammal in need thereof. The mammal may be selected from any one of the group consisting of humans, non-human primates, companion animals, exotic species, livestock animals, and food animals, or a combination of at least two thereof.

[0102] In some embodiments, the drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

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

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

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

[0106] Definition of terms

[0107] All publications and patents mentioned in the present invention are hereby incorporated into the present invention by reference in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in the present invention, then the purposes and terms of the present invention shall prevail.

[0108] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

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

[0110] Except in the examples or otherwise indicated, all numbers stating quantitative properties such as dosage in the specification and claims should be understood to be modified in all cases by the term "about". It should also be understood that any numerical range recited in the present invention is intended to include all subranges within the range and any combination of the various endpoints of the range or subrange.

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

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

[0113] In the present invention, the term "alkyl" refers to a 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 ) is a linear or branched, saturated, monovalent hydrocarbon group. 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" refers to a saturated divalent hydrocarbon group obtained by removing two hydrogen atoms from a saturated straight-chain or branched hydrocarbon group. Alkylene includes, but is not limited to, methylene (-CH2-), ethylene {including -CH2CH2- or -CH(CH3)-}, isopropylene {including -CH(CH3)CH2- or -C(CH3)2-}, , .

[0115] In the present invention, the term "alkoxy" refers to a group R Y -O-, R Y The same definition is given to the term "alkyl". Alkoxy includes, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, n-hexoxy, and the like.

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

[0117] In the present invention, the term "aryl" refers to a group having a specified number of carbon atoms (e.g., C6-C 10 ) is a cyclic, unsaturated, monovalent hydrocarbon group, which is a single ring or multiple rings (for example, 2). When it is a multiple ring, the single rings share two atoms and a bond, and each ring is aromatic. The aryl group is connected to the rest of the molecule through the aromatic ring. The aryl group includes, but is not limited to, phenyl and 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 within the typical tolerance range of the art, within the experimental variability or in the approximate value of the statistical experimental error, and therefore the number or numerical range can be, for example, between 1% and 15% of the number or numerical range. For example, "about" can be understood as about 2 standard deviations of the mean 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 numbers in the series or range.

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

[0120] The words "include", "contain" or "comprises" and the like used in the present invention mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unrecorded elements. The terms "contain" or "includes" used in the present invention may be open, semi-closed or closed. In other words, the term also includes "essentially consisting of" or "consisting of".

[0121] The term "pharmaceutically acceptable" in the present invention means that the compound or composition is chemically and / or toxicologically compatible with other ingredients constituting the formulation and / or with humans or mammals for preventing or treating diseases or disorders.

[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 a combination of at least 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 refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of the disease to cure, alleviate, 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, preferably a peptide or protein, containing at least one epitope capable of eliciting an immune response and / or an epitope to which an immune response is directed. Preferably, an antigen in the context of the present invention is a molecule that, optionally after processing, induces an immune response that is preferably specific for the antigen or a cell expressing the antigen. In particular, "antigen" relates to a molecule that, optionally after processing, is presented by an MHC molecule and specifically reacts with a T lymphocyte (T cell).

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

[0126] According to the present invention, any suitable antigen is contemplated 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 may be derived from an allergen, virus, bacteria, fungus, parasite 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, unicellular 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). Unicellular organisms include, but are not limited to, malarial parasites, trypanosomes, amoebas, etc.

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

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

[0131] Therapeutic and / or preventive agents

[0132] The lipid composition of the present invention can be used for delivering active pharmaceutical ingredients, such as therapeutic agents and / or preventive agents. Based on this, the present invention further provides a (pharmaceutical) composition, comprising a lipid composition provided by the present invention, for delivering active pharmaceutical ingredients. The composition of the present invention can include one or more therapeutic agents and / or preventive agents (as active pharmaceutical ingredients). Active pharmaceutical ingredients can be encapsulated in the lipid composition or combined with the lipid composition.

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

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

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

[0136] Therapeutic and / or prophylactic agents include biologically active substances and may be alternatively referred to as "active agents," "active ingredients," and the like. Therapeutic and / or prophylactic agents may be substances that, upon delivery to a cell or organ, cause a desired change in the cell or organ, or in other body tissues or systems. Such species may 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 (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), anti-tumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, arabinoside), anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blocking agents (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines

[0013] In some embodiments, the present invention includes but is not limited to: antibiotics (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma 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 causes 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 anthracenedione, The invention relates to a radioactive ion, for example, anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and analogs or homologues 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 preparations that can provide immunity to 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 infectious disease-derived antigens and / or epitopes. Vaccines may also include compounds and preparations 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 new epitopes. Compounds that induce 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, razithromycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, cyclosporine, cyclophosphamide ... The invention relates to antibiotics such as dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC), and antimitotic agents such as vincristine, vinblastine, taxol and maytansine.

[0138] In other embodiments, the therapeutic and / or prophylactic agent is a protein. The 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, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0139] In some embodiments, the therapeutic and / or preventive agent can be a polynucleotide or a nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The broadest meaning of the term "polynucleotide" includes any compound and / or substance that is an oligonucleotide chain or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used according to 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 hybrid; RNAi inducing factor; RNAi factor; siRNA; shRNA; miRNA; antisense RNA; ribozyme; catalytic DNA; RNA that induces triple helix formation; aptamer, etc. In some preferred embodiments, the therapeutic and / or preventive agent is RNA. The RNA that can be used in the compositions and methods described in the present invention can be selected from, but is not limited to, the group consisting of: shortmer, antagomir, antisense RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA) and mixtures thereof. In certain embodiments, the RNA is mRNA.

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

[0141] In other embodiments, the therapeutic agent and / or preventive agent is siRNA. siRNA can selectively reduce the expression of a gene of interest or down-regulate the expression of the gene. For example, the selection of siRNA can make the gene silencing relevant to a specific disease, disease or condition after the composition comprising the siRNA is administered to a subject in need. siRNA can include a sequence complementary to the mRNA sequence of a gene or protein of interest to encode. In some embodiments, siRNA can be an immunomodulatory siRNA.

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

[0143] In some embodiments, the therapeutic agent and / or preventive agent is shRNA or its encoding vector or plasmid. shRNA can be produced inside the target cell after the appropriate construct is delivered to the nucleus. The construct and mechanism associated with shRNA are well-known in the relevant field.

[0144] Disease or condition

[0145] The composition / carrier of the present invention can deliver therapeutic agents and / or preventive agents to subjects or patients, thereby achieving the treatment and / or prevention of diseases or conditions. The therapeutic agents and / or preventive agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides or proteins. Therefore, the composition of the present invention can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs. Due to the wide variety of the above-mentioned therapeutic agents and / or preventive agents, the composition of the present invention can be used to treat or prevent a variety of diseases or conditions.

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

[0147] The reagents, compositions and methods described in the present invention can be used to treat a subject who is ill (e.g., a disease characterized by the presence of diseased cells expressing an antigen and presenting an antigenic peptide), or to prevent a subject from becoming ill. Examples of treatable and / or preventable diseases include 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 state, including infectious diseases and cancer diseases, in particular 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, "disease involving an antigen" or similar expressions 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 may be increased compared to the state of a healthy tissue or organ. In one embodiment, expression occurs only in diseased tissue, while expression is inhibited in healthy tissue. According to the present invention, diseases involving antigens include infectious diseases and cancer diseases, wherein the disease-associated antigens are preferably antigens of infectious agents and tumor antigens, respectively. Preferably, the disease involving an antigen is preferably a disease involving cells that express the antigen and present the antigen in the context of an MHC molecule (particularly class I MHC).

[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, i.e., infections caused by Plasmodium (Plasmod 4. fungal infections, such as Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis or Candida albicans.

[0152] The carcinoma or cancer (the medical term for malignant tumor) is a class of diseases in which a group of cells exhibit uncontrolled growth (dividing beyond normal limits), invasion (invading and destroying adjacent tissues), and sometimes metastasize (spreading to other parts of the body through the lymph or blood). These three harmful properties of carcinoma distinguish it from benign tumors, which are self-limited and do not invade or metastasize. Most carcinomas form tumors, i.e., swellings or lesions formed by abnormal growth of cells (called neoplastic cells or tumor cells), but some (like leukemias) do not. According to the present invention, the term "cancer" includes leukemia, seminoma, melanoma, teratoma, lymphoma, sarcoma, blastoma, neuroblastoma, glioma, glioblastoma, kidney cancer, adrenal cancer, renal cell carcinoma, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach 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 metastases thereof.

[0153] Malignant melanoma is a serious type of skin cancer that results from the uncontrolled growth of pigment cells called melanocytes.

[0154] According to the invention, "epithelial cancers" are malignant tumors originating from epithelial cells. This group accounts for the most common cancers, including common forms of breast, prostate, lung and colon cancer.

[0155] Lymphomas and leukemias are malignancies that arise from hematopoietic (blood-forming) cells.

[0156] Sarcomas are cancers that arise from transformed cells of one of the tissues that develop from the embryonic mesoderm. Thus, sarcomas include tumors of bone, cartilage, fat, muscle, blood vessels, and hematopoietic tissue.

[0157] A blastic tumor or germ cell tumor is a tumor (usually malignant) that resembles immature or embryonic tissue. Most of these tumors occur commonly in children.

[0158] Gliomas are tumors that begin in the brain or spine. They are called gliomas because they originate from glial cells. The most common site for gliomas is the brain.

[0159] Other components

[0160] The pharmaceutical compositions of the present invention may include one or more components other than those described in the preceding sections. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (eg, 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 may be, for example, molecules described in U.S. Patent Application Publication No. 2005 / 0222064. The carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0162] Surface-altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, , mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornasealfa, neltenexine and erdosteine) and DNA enzymes (e.g., rhDNA enzyme). The surface-altering agent can be disposed within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent attachment or other methods).

[0163] The composition can also include one or more functionalized lipids. For example, lipids can be functionalized with alkynyl groups, which may undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized with one or more groups that effectively promote membrane penetration, cell recognition or imaging in this way. The surface of the composition can also be coupled to one or more useful antibodies. Functional groups and conjugates that can be used 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 substance that can be used in a pharmaceutical composition. For example, the composition may include one or more pharmaceutically acceptable (e.g., pharmaceutically acceptable) excipients or auxiliary ingredients, such as but not limited to one or more solvents, dispersion media, diluents, dispersing aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, adhesives, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc.

[0165] The term "pharmaceutically acceptable" refers to the non-toxicity of a material that does not affect the action of the active ingredients of the pharmaceutical composition. Non-pharmaceutically acceptable ingredients may be used to prepare pharmaceutically acceptable ingredients and are included in the present invention.

[0166] Suitable buffering agents for use in the compositions of the present invention include salt forms of acetic acid, salt forms of citric acid, salt forms of boric acid, and salt forms of 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 composition of the present invention and which are not active ingredients, such as carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents or coloring agents. Excipients are, for example, starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0168] Suitable preservatives for use in 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, dicalcium 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 Form and Administration

[0171] The composition of the present invention can be made into a preparation in the form of solid, semisolid, liquid or gas, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The composition of the present invention can be prepared by methods well known in the pharmaceutical field. For example, a sterile injection solution can be prepared by mixing the required amount of therapeutic agent or preventive agent with the required various other ingredients mentioned above into a suitable solvent such as sterile distilled water, and then filtering and sterilizing. Surfactants can also be added to promote the formation of uniform solutions or suspensions.

[0172] For example, the composition of the 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 an amount of a therapeutic agent that improves a disease or symptom when administered to a patient. A "prophylactically effective amount" is an amount of a prophylactic agent that prevents a disease or symptom when administered to a subject. The amount of a therapeutic agent that constitutes a "therapeutically effective amount" or the amount of a prophylactic agent that constitutes 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 a therapeutically effective amount and a prophylactically effective amount based on his or her knowledge and the present invention.

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

[0176] Antigen presenting cells

[0177] Antigen presenting cells (APCs) are cells that present (i.e., display) antigens on their surface in the context of a major histocompatibility complex (MHC). This includes situations where only one fragment or more fragments of an antigen are 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 (either by phagocytosis or by receptor-mediated endocytosis) and then displaying antigen fragments bound to class II MHC molecules on their membranes. T cells recognize and interact with the antigen-class II MHC molecule complex on the antigen-presenting cell membrane. The antigen-presenting cell then produces additional co-stimulatory signals, leading to T cell activation. The expression of co-stimulatory molecules is a typical feature of professional antigen-presenting cells.

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

[0180] Dendritic cells are a population of leukocytes, including plasmacytoid dendritic cells (pDCs) and classical dendritic cells (cDCs), which present antigens captured in peripheral tissues to T cells via both MHC class II and class I antigen presentation pathways. Dendritic cells are potent inducers of immune responses, and the activation of these cells is a key step in inducing anti-tumor immunity.

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

[0182] The term "immunogenicity" relates 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 (CTL, CD8+ T cells) of cytolytic T cells.

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

[0185] Helper T cells assist other white blood cells in the immune process, including maturation of B cells into plasma cells and activation of 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 peptide antigens are presented to them by class II MHC molecules expressed on the surface of antigen presenting cells (APCs). After activation, they rapidly divide and secrete small proteins called cytokines that regulate or assist active immune responses.

[0186] Cytotoxic T cells destroy diseased cells, for example, infected cells (such as virus-infected cells) and cancer cells, and are also involved 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 nearly 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 independent T cell receptor α and β (TCRα and TCRβ) genes and are called α-TCR chain and 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 reside in the thymus and amplify through cell division to produce a large number of immature thymocytes. Early thymocytes express neither CD4 nor CD8 and are therefore classified as double negative (CD4-CD8-) cells. As they develop through development, they become double positive thymocytes (CD4+CD8+) and eventually mature into single positive (CD4+CD8+) cells. + CD8 - or CD4 - CD8 + ) thymocytes and are then released from the thymus into 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 companion cell is usually a professional antigen presenting cell (APC), usually a dendritic cell in the context of a primary response, but B cells and macrophages can also be important APCs. + T cell peptides are 8 to 10 amino acids long; they are presented by class II MHC molecules to CD4 +The peptides for T cells are longer because the binding cleft of the class II MHC molecule is open at the end.

[0190] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

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

[0192] The positive and progressive effect of the present invention is that, 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 of antigens in the body, muscle or spleen of subjects (such as mice);

[0195] 3. The percentage of antigen-presenting cells expressing antigens in the spleen was significantly increased;

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

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

[0198] 6. The volume of tumors was significantly reduced and the survival rate of mice was significantly increased. 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 in the following description only relate to some specific embodiments of the present invention, rather than limiting the present invention.

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

[0201] Figure 2 The flow cytometry experiment diagram of mouse spleen cells (eGFP) 24 hours after intravenous injection of blank, eGFP-mRNA-TLP composition with added YK-1202 or YK-1204, and eGFP-mRNA-TLP composition without adjuvant lipid into mice.

[0202] Figure 3Flow cytometry analysis of mouse spleen cells (CD86) 24 hours after intravenous injection of the blank, eGFP-mRNA-TLP combination supplemented with YK-1205 or TMX-201 into mice.

[0203] Figure 4 Flow cytometry analysis of mouse spleen cells (CD69) 24 hours after blank, intravenous injection of TLP compositions supplemented with YK-1204, YK-1205 or TMX-201 into mice.

[0204] Figure 5 Figure 3. Stimulation of cytokines IFN-γ and IL-12 in the serum of B16F10-OVA mice vaccinated with the OVA mRNA-TLP composition supplemented with YK-1202, YK-1204, YK-1205, or YK-1208.

[0205] Figure 6 Figure 2 shows the tumor growth in B16F10-OVA mice inoculated with the OVA mRNA-TLP composition supplemented with YK-1202, YK-1204, YK-1205, or YK-1208.

[0206] Figure 7 Survival rate of B16F10-OVA mice vaccinated with OVA mRNA-TLP composition supplemented with YK-1202, YK-1204, YK-1205, or YK-1208.

[0207] Figure 8 Figure 3. Stimulation of cytokines IFN-γ and IL-12 in the serum of B16F10-OVA mice vaccinated with the OVA mRNA-LNP composition supplemented with YK-1202, YK-1204, YK-1206, or YK-1208.

[0208] Fig. 9 Figure 2 shows tumor growth in MC38-OVA mice inoculated with OVA mRNA-LNP compositions supplemented with YK-1202, YK-1204, YK-1206, or YK-1208.

[0209] Fig.10 Survival rate of MC38-OVA mice vaccinated with OVA mRNA-LNP compositions supplemented with YK-1202, YK-1204, YK-1206, or YK-1208. DETAILED DESCRIPTION

[0210] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0211] The implementation conditions used in the embodiments can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. In the specific embodiments of the present invention, the raw materials used can 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 embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0212] The following abbreviations represent the following reagents:

[0213] ADOPE: 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate sodium salt; ( , prepared with reference to Example 1 in patent CN118001254A)

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

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

[0216] DOPG: 1,2-dioleoyl-sn-glycero-3-phospho-RAC-glycerol sodium salt;

[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: triphenylmethane.

[0228] Example 1: Synthesis of lipid-conjugated TLR adjuvants

[0229] 1. Synthesis of YK-1201

[0230] The synthetic 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), add thionyl chloride (180 mg, 1.530 mmol) dropwise in an ice-water bath, and stir at room temperature for 6 hours. Stop the reaction, dry the solvent under reduced pressure, and purify the product YK-1201-PM1 (500 mg, 0.743 mmol, 97.1%) by silica gel chromatography (0% - 15% dichloromethane / methanol).

[0234] Step 2: Synthesis of YK-1201

[0235] YK-1201-PM1 (150 mg, 0.223 mmol) and YK-1201-SM1 (84 mg, 0.270 mmol) were dissolved in DMF (3 mL), potassium carbonate (93 mg, 0.673 mmol) and potassium iodide (3 mg, 0.018 mmol) were added at room temperature, and then stirred at 70°C for 6 hours. After the reaction was completed, the temperature was lowered, ethyl acetate and water were added to the reaction solution in sequence, the liquid was separated, the aqueous phase was back-extracted twice with ethyl acetate, the organic phases were combined, the solvent was dried to obtain a crude product, and the product YK-1201 (120 mg, 0.127 mmol, 57.0%) was purified by silica gel chromatography (0% - 15% dichloromethane / methanol). 58 H 102 N6O4, MS (ES): m / z (1 / 2 M +Na + ) = 496.63.

[0236] YK-1201: 1H NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.43 - 7.35 (m, 1H), 7.26 - 7.19 (m, 1H), 6.42 (s, 2H), 4.52 - 4.44 (m, 2H), 3.99 - 3.86 (m, 6H), 3.07 - 2.96 (m, 2H), 2.94 - 2.86(m, 2H), 2.40 - 2.30 (m, 4H), 2.29 - 2.21 (m, 4H), 1.85 - 1.74 (m, 4H), 1.62- 1.40 (m, 12H), 1.29 - 1.17 (m, 45H), 1.13 - 1.05 (m, 2H), 0.96 (t, J = 7.2Hz, 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. 62 H 102 N6O4, MS (ES): m / z (1 / 2 M + Na + ) = 520.58.

[0241] YK-1202: 1H 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] YK-009 (500 mg, 0.764 mmol) and 4-bromobutyric acid (612 mg, 3.665 mmol) were dissolved in dichloromethane (5 mL), EDCI (440 mg, 2.295 mmol) and DMAP (38 mg, 0.311 mmol) were added at room temperature, and stirred at 35 ºC for 16 h. The reaction was stopped, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% - 15% dichloromethane / methanol) to obtain the product YK-1203-PM1 (480 mg, 0.598 mmol, 78.2%).

[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. 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 synthetic 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, according to 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 raw materials, according to the synthesis method of YK-1201, the product YK-1204 (60 mg, 0.0565 mmol, 31.4%) was obtained. 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 synthetic 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 raw materials, according to the synthetic method of YK-1201, the product YK-1205 (60 mg, 0.0555 mmol, 44.4%) was obtained. 66 H 108 N6O6, MS (ES): m / z (1 / 2 M + Na +) = 563.66.

[0262] YK-1205: 1 H NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.40 - 7.33 (m, 1H), 7.18 (d, J = 8.4 Hz, 2H), 7.11 -7.04 (m, 1H), 7.18 (d, J = 8.4 Hz, 2H), 5.84 (s, 2H), 4.13 - 4.08 (m, 2H), 4.01 - 3.89 (m, 8H), 2.93 - 2.86 (m, 2H), 2.38 - 2.32 (m, 4H), 2.30 - 2.24(m, 4H), 1.78 - 1.68 (m, 4H), 1.59 - 1.47 (m, 10H), 1.41 - 1.30 (m, 8H), 1.29- 1.23 (m, 42H), 0.89 - 0.80 (m, 12H).

[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 synthetic method of YK-1201, the product YK-1205 (40 mg, 0.0360 mmol, 30.0%) was obtained. 68 H 112 N6O6, MS (ES): m / z (1 / 2 M + Na + ) = 577.72.

[0267] YK-1206: 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), 4.01 - 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), return to room temperature and stir overnight, monitor the reaction by LCMS. Stop the reaction, add water and ethyl acetate to the reaction solution in sequence, separate the liquids, extract the aqueous phase with ethyl acetate twice, combine the organic phases, spin dry 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%). 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, add YK-1207-SM2 (437 mg, 2.16 mmol), return to room temperature and stir for 4 hours, monitor the reaction by TLC. Stop the reaction, purify by silica gel chromatography (0% - 50% dichloromethane / ethyl acetate) to obtain the product YK-1207-PM2 (105 mg, 0.181 mmol, 25.2%). 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, and monitor the reaction by TLC. Stop the reaction, spin dry the solvent to obtain the crude product, and purify it by silica gel chromatography (0% - 10% dichloromethane / methanol) to obtain the product YK-1207-PM3 (100 mg, 0.084 mmol, 46.9%). 64 H 106 N5O 13 P, MS (ES): m / z (M + H + ) = 1185.17.

[0277] Step 3: Synthesis of YK-1207

[0278] YK-1207-PM3 (100 mg, 0.084 mmol) was dissolved in 4M hydrogen chloride in ethyl acetate solution (1 mL), stirred at room temperature for 4 hours, and the reaction was monitored by LCMS. The reaction was stopped, the solvent was dried to obtain a crude product, and the product YK-1207 (20 mg, 0.018 mmol, 21.9%) was purified by silica gel chromatography (0% - 10% dichloromethane / methanol). 59 H 98 N5O 11 P, MS (ES): m / z (M + H + ) = 1085.13.

[0279] YK-1207: 1H NMR (400 MHz, DMSO-d6) δ 8.44 (d, J = 8.0 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 5.34- 5.24 (m, 4H),5.07 - 4.99 (m, 1H), 4.78 - 4.68 (m, 1H), 4.25 - 4.18 (m, 1H),3.99 - 3.94 (m, 1H), 3.87 - 3.76 (m, 4H), 3.56 - 3.48 (m, 6H), 2.95 - 2.93(m, 10H), 2.16 - 2.12 (m, 2H), 2.04 - 1.92 (m, 8H), 1.44 - 1.32 (m, 9H), 1.27- 1.21 (m, 48H), 0.88 - 0.81 (m, 9H).

[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 overnight at room temperature, and monitor the reaction by TLC. The reaction of the raw material was complete, the reaction was stopped, the reaction solution was poured into a saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, the organic phases were combined, the solvent was dried to obtain a crude product, and the product YK-1208-PM1 (420 mg, 0.479 mmol, 74.9%) was purified by silica gel chromatography (0% - 10% dichloromethane / methanol). 47 H 86 C1O 10 P, MS (ES): m / z (M - H + ) = 875.84.

[0285] Step 2: Synthesis of YK-1208

[0286] YK-1208-PM1 (320 mg, 0.364 mmol) and YK-1201-SM1 (114 mg, 0.366 mmol) were dissolved in DMF (3.5 mL), potassium carbonate (150 mg, 1.10 mmol) and potassium iodide (6 mg, 0.040 mmol) were added at room temperature, and then stirred at 70 ºC for 16 h. After the reaction was completed, the temperature was lowered, and the reaction solution was poured into a saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, and the organic phases were combined and the solvent was dried to obtain a crude product, which was purified by silica gel chromatography (0% - 20% dichloromethane / methanol) to obtain the product YK-1208 (105 mg, 0.091 mmol, 24.6%). 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 was based on the synthesis 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 followed the synthetic route of Compound 23 in WO2021237055A1 to obtain 30 mg of compound 23.

[0295] 11. Synthesis of 63-15

[0296]

[0297] The synthesis of 63-15 was carried out according to the synthesis route of Compound No. 63-15 in WO2019040491A1 to obtain 42 mg of 63-15.

[0298] Example 2: Preparation of mRNA lipid composition

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

[0300] 1) Luciferase (Luciferase protein CDS), green fluorescent protein (GFP), and ovalbumin (OVA) circular plasmids were constructed on the pVAX1 vector (purchased from Thermo Fisher Scientific) by EcoRV restriction enzyme digestion;

[0301] 2) Take the plasmid constructed in step 1) on the pVAX1 vector and mix it with 50 μL of E. coli competent cells Stbl2 (purchased from Thermo Fisher Scientific), then place them on ice for 30 minutes, heat shock at 42°C for 90 seconds, and immediately put them back on ice and place them on ice for 2 minutes;

[0302] 3) Add 400 μL LB medium (purchased from Thermo Fisher Scientific) and culture at 30°C with slow shaking for 45-60 minutes;

[0303] 4) Take 50-100 μL of bacterial solution and apply it on LB solid medium containing kanamycin antibiotic (100 μg / mL, purchased from Yisheng Biotechnology Co., Ltd.), and invert and culture at 37°C overnight;

[0304] 5) Sequence the obtained monoclonal colony plate to verify its correctness, pick the monoclonal colony with correct sequencing and culture it in a shaker at 30°C overnight;

[0305] 6) Use an endotoxin-free plasmid extraction kit (purchased from Yisheng Biotechnology Co., Ltd.) to extract the plasmid;

[0306] 7) The extracted plasmid is digested with restriction endonucleases to form a linearized plasmid for use as a transcription template. For specific digestion steps, see Steps ① to ③.

[0307] Step ① Take 1 mg of luciferase circular plasmid and digest it with BspQ Ⅰ enzyme (purchased from Yisheng Biotechnology Co., Ltd.) at 37°C for 4 hours to form a linearized DNA transcription template (see Table 1 for the enzyme digestion system);

[0308] Table 1 Enzyme digestion reaction system

[0309]

[0310] Step ② After the reaction is completed, add anhydrous ethanol and sodium acetate in sequence according to V 酶切反应产物 :V 无水乙醇 :V 3M醋酸钠 =1:3:1 volume ratio of anhydrous ethanol and 3M sodium acetate were added, and the mixture was allowed to settle at -20 °C for 1 hour, and then centrifuged at 12000 rpm to retain the precipitate;

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

[0312] The concentration of the linearized plasmid in the dissolved solution was 500 ng / µL, the linearization ratio was more than 90%, and the purification recovery efficiency was 85%.

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

[0314] 1) Co-transcriptional capping reaction:

[0315] Using Fluc DNA, eGFP DNA and OVA DNA prepared in A) as templates, NTP solution (NTPs) and Cap1 cap analog (Cat. No.: 10678ES80, purchased from Yisheng Biotechnology Co., Ltd.) as starting materials, mRNA was synthesized by transcription using T7 RNA polymerase. The specific reaction system is shown in Table 2. The prepared reaction system was placed in a 37°C incubator for 3 hours of shaking. The Cap1 cap analog is Cap1-GAG, which has a 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 the above reagents were purchased from Yisheng Biotechnology Co., Ltd.

[0319] 2) Digestion of template DNA:

[0320] In the co-transcription capping reaction system completed in step 1) above, DNase I (purchased from Yisheng Biotechnology Co., Ltd.) was added to make the final concentration of 1 U / μg linearized plasmid, mixed, centrifuged, and digested at 37°C for 1 hour to obtain the co-transcription capping product.

[0321] 3) Purification by lithium chloride precipitation method:

[0322] The co-transcription capping product obtained in step 2) above was purified by lithium chloride precipitation as follows:

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

[0324] Step ② Precipitation: high-speed centrifugation at 12000 rpm for 15 minutes, retain the precipitate;

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

[0326] Example 3: Effects of different adjuvant lipid addition amounts on TLP-mRNA compositions

[0327] ADOPE, DOTMA, DOPE and YK-1202 were weighed according to the proportions in Table 3 and dissolved in ethanol to prepare an ethanol lipid complex solution (total lipid concentration was 266 mM). The ethanol lipid complex solution was quickly added to the stirring sterile enzyme-free water by ethanol injection method at a speed of 120 rmp and stirred at room temperature for 30 minutes. The resulting mixture solution was filtered through a polycarbonate membrane with a pore size of 450 nm to obtain a lipid composition solution and stored at 4°C to 8°C. mRNA was diluted in a buffer to obtain an mRNA aqueous solution (mRNA concentration 0.5 mg / mL, the buffer was 10mM HEPES buffer containing 0.1 mM EDTA). Sodium chloride aqueous solution (0.9% w / w) was taken with a syringe and injected into the above-prepared mRNA aqueous solution to obtain a mixed solution of mRNA and sodium chloride. The lipid complex solution was taken with a syringe and injected into the mixture of mRNA and sodium chloride, vortexed for 30 seconds, incubated at room temperature for 10 minutes, and a TLP pharmaceutical composition with or without adjuvant lipid was obtained (the final RNA concentration was 100 μg / mL), and stored at 4°C to 8°C. The amount of mRNA and lipid complex solution was such that the charge ratio of the TLP-mRNA composition was 1: 2, and the charge ratio was calculated as follows:

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

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

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

[0331]

[0332] The results show that the particle size and PDI of the TLP pharmaceutical composition obtained by using YK-1202 to partially replace the permanent cationic lipid DOTMA component in the TLP composition at 2.5%, 5.0%, 10.0%, 15.0%, and 20.0% are within the qualified range (particle size of 220-500nm, PDI value <0.5). The following examples use an adjuvant lipid ratio of 10%.

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

[0334] Table 4 Structures of adjuvant compounds used in anionic lipid compositions

[0335]

[0336]

[0337] According to the preparation method of Example 3, the adjuvant lipid compound in Table 4 was used to partially replace the proportion of 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 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. The results are shown in Table 5.

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

[0339]

[0340] The results showed that the adjuvant lipids YK-1201~YK-1208 in Table 4 and compounds 23, 63-15, and TMX-201 were all able to prepare mRNA compositions with qualified particle size and PDI (particle size controlled within 220-500 nm, 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 lipid in mice

[0342] The Fluc-mRNA-TLP composition prepared in Example 4 with or without adjuvant lipids was injected into female BALB / c albino mice aged 4-6 weeks and weighing 17-19 g via the tail vein (the dosage was about 20 μg Fluc-mRNA / mouse), and the mice were intraperitoneally injected with fluorescent imaging substrate 6 hours after administration. The mice were allowed to move freely for 5 minutes, and then the total radiation intensity of the protein expressed in the mouse by the mRNA carried by the mRNA composition was detected by IVIS Spectrum small animal in vivo imager (corresponding to the expression intensity of fluorescent protein, i.e., the protein expression amount). After the sampling was completed, the mice were killed by cervical dislocation and dissected to accurately separate the mouse's internal organs: liver and spleen. The total radiation intensity of the protein expressed by Fluc-mRNA in various organs of the mouse was detected by IVIS Spectrum small animal in vivo imager (corresponding to the expression intensity of fluorescent protein, i.e., the protein expression amount). The results of mouse in vivo imaging and protein expression detection in various internal organs are shown in Tables 6 and Figure 1 .

[0343] Table 6 Experimental data of mouse in vivo and organ imaging

[0344]

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

[0346] Compared with the addition of prior art adjuvant lipids (compounds 23, 63-15 and TMX-201), the total radiation intensity of the spleen and the total radiation intensity of the living body were significantly enhanced by adding the compositions of YK-1202, YK-1204, YK-1205, YK-1206 and YK-1208 of the present invention. For example, the total radiation intensity of the spleen of the composition added with YK-1204 was 4.4 times, 4.7 times and 4.3 times that of the composition added with compounds 23, 63-15 and TMX-201, respectively, and the radiation intensity of the living body was 3.3 times, 3.5 times and 2.9 times that of the composition added with compounds 23, 63-15 and TMX-201, respectively.

[0347] The composition with different adjuvant lipids of similar structure added in the present invention has great difference in spleen total radiation intensity and living body total radiation intensity. For example, the composition with YK-1206 added has 2.7 times and 3.3 times of the composition with YK-1201 added.

[0348] Example 6: Targeting study of mouse spleen cells with mRNA-TLP composition containing adjuvant lipids

[0349] 1. The eGFP-mRNA-TLP composition prepared in Example 3 with or without the addition of adjuvant lipid was injected into female C57BL / 6 mice aged 4-6 weeks and weighing 17-19 g via the tail vein (the amount was such that each mouse was injected with about 80 μg of GFP-mRNA), and the mice were killed by cervical dislocation 24 hours after administration and dissected to accurately separate the spleen of the mice.

[0350] 2. Preparation of single cells

[0351] 1) Grind the separated spleen tissue to make it single cell and pass it through a cell sieve;

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

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

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

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

[0356] Table 7 Reagents and sources used in mouse spleen cell flow cytometry experiments

[0357]

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

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

[0360] T cell GFP ratio:CD45 + →CD3 + →GFP+

[0361] B cell GFP ratio:CD45 + →CD3 - CD19 + →GFP +

[0362] NK cell GFP ratio:CD45 + →NK1.1 + →GFP +

[0363] cDC cell GFP ratio: CD45 + →F4 / 80 - CD11c + →GFP +

[0364] pDC cell GFP ratio:CD45 + →F4 / 80 - CD11c int CD317 + →GFP +

[0365] Macrophage GFP ratio: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 The 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 absorb, process antigens, and present the processed antigens to lymphocytes. They include dendritic cells (DC cells), B lymphocytes, and macrophages. They play a vital 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 by the adjuvant lipid of the present invention can significantly increase the percentage of cells expressing antigens in antigen-presenting cells in the spleen, and can significantly increase the percentage of cells expressing antigens in antigen-presenting cells in the spleen compared with the TLP-mRNA composition without the addition of adjuvant. For example, the mRNA-TLP composition prepared by adding YK-1204 increases the percentage of eGFP-positive cells in antigen-presenting cells B cells, cDC cells, pDC cells, and macrophages in the spleen by 2.1 times, 1.7 times, 1.7 times, and 1.7 times, respectively.

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

[0373] The mRNA-TLP compositions prepared with different adjuvant lipids in the present invention have significant differences in targeting to mouse spleen antigen presenting cells, 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 percentage of eGFP-positive cells of the mRNA-TLP composition prepared by YK-1206 in B cells, cDC cells, pDC cells and macrophages is 2.2 times, 1.8 times, 1.7 times and 1.9 times that of the mRNA-TLP composition prepared by YK-1203, respectively.

[0374] Example 7: Study on the maturation status of mouse spleen antigen presenting cells using mRNA-TLP compositions prepared with adjuvant lipids

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

[0376] B cell CD86 ratio:CD45 + →CD3 - CD19 + →CD86 +

[0377] NK cell CD86 ratio:CD45+ →NK1.1 + →CD86 +

[0378] cDC cell CD86:CD45 ratio + →F4 / 80 - CD11c + →CD86 +

[0379] pDC cell CD86:CD45 ratio + →F4 / 80 - CD11c int CD317 + →CD86 +

[0380] Macrophage CD86:CD45 ratio + →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 by the adjuvant lipid of the present invention can significantly increase the percentage of CD86 positive cells in the spleen of antigen presenting cells. Compared with the mRNA-TLP composition without adjuvant, the mRNA-TLP composition prepared by the adjuvant lipids YK-1202, YK-1204 to YK-1206, and YK-1208 of the present invention can significantly improve the maturity of mouse spleen antigen presenting cells. For example, the percentage of CD86 positive cells of B cells, cDC cells, pDC cells and macrophages in the mouse spleen of the mRNA-TLP composition with the addition of YK-1205 is 1.1 times, 1.5 times, 1.6 times and 1.7 times the corresponding percentage of TLP without adjuvant lipid.

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

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

[0388] Example 8: Study on T cell activation in the spleen of mice using mRNA-TLP compositions prepared with adjuvant lipids

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

[0390] T cell CD69 ratio:CD45 + →CD3 + →CD69 +

[0391] Experimental results:

[0392] The percentage of CD69 positive T cells in mouse spleen is shown in Table 10.

[0393] Table 10 The percentage of CD69 positive T cells in mouse spleen

[0394]

[0395] The mRNA-TLP compositions prepared by using the adjuvant lipids of the present invention can significantly increase the percentage of T cell CD69-positive cells in the spleen, and can significantly increase the percentage of T cell CD69-positive cells in the spleen compared with the mRNA-TLP compositions without the addition of adjuvant lipids. For example, compared with TLP without the addition of adjuvant, the mRNA-TLP compositions prepared by adding the adjuvant lipids YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 of the present invention make the percentage of T cell CD69-positive cells in the spleen 1.5 times, 1.7 times, 1.7 times, 1.6 times, and 1.6 times that of TLP, respectively, which is significantly improved.

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

[0397] The mRNA-TLP compositions prepared by different adjuvant lipids of the present invention have significant differences in the percentage of T cell CD69 positive cells in the mouse spleen. For example, the percentage of T cell CD69 positive cells in the mouse spleen in the mRNA-TLP compositions prepared by YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 is significantly higher than that of other compositions. After the mRNA-TLP composition prepared by YK-1205 is administered, the percentage of T cell CD69 positive cells is the highest, which is 1.4 times that of the lowest YK-1207.

[0398] Example 9: Stimulation of cytokines IFN-γ and IL-12 by mRNA-TLP compositions containing adjuvant lipids

[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 immune response, has multiple functions such as antiviral, anti-tumor, immunomodulatory and promoting inflammatory response, and can treat a variety of diseases. Interleukin-12 (IL-12) is a multifunctional cytokine produced by dendritic cells, macrophages, B lymphocytes and other antigen-presenting cells. It plays an important role in regulating immune response, promoting Th1 immunity, enhancing cytotoxicity and tumor immunity. Detecting the stimulation of cytokines IFN-γ and IL-12 by mRNA-TLP compositions containing adjuvant lipids can reflect the improvement of the innate immunity of mRNA-TLP compositions by adjuvant lipids.

[0400] Experimental process:

[0401] 8-week-old female C57BL / 6J mice were injected with the OVA-mRNA-TLP (40 µg) composition prepared according to Example 4 through the tail vein, and the eyes were removed for euthanasia 6 hours after the mice were euthanized to obtain as much serum as possible. The levels 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.

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

[0403] Table 11 Comparison of the stimulating effects of mRNA-TLP compositions containing adjuvant lipids 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 mRNA-TLP composition containing adjuvant lipid had a significant increase in IFN-γ and IL-12 cytokines in the serum 6 hours after injection, among which, 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 that of the mRNA-TLP composition without adjuvant lipid, respectively; YK-1205-TLP were 2.0 times and 1.8 times that of the mRNA-TLP composition without adjuvant lipid, respectively; YK-1208-TLP were 2.1 times and 1.9 times that of the mRNA-TLP composition without adjuvant lipid, respectively.

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

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

[0409] Experimental process:

[0410] 1) Establishment of B16F10-OVA mouse model: Female C57BL / 6J mice between 6 and 8 weeks of age were acclimated for at least three days prior to the study. Mice had free access to food and sterile water and were housed at 22°C ± 2°C and 55% ± 15% relative humidity with a 12-h light / dark cycle. B16F10-OVA cells (Zhejiang Meisen Cell Technology Co., Ltd.) were cultured in complete medium as described in the instructions for B16F10-OVA (CTCC-001-0727) at 5% CO2 and 37°C. Cells were harvested using 0.25% trypsin-EDTA, resuspended in Dulbecco's phosphate-buffered saline (DPBS), and then 2 × 10 5 The B16F10-OVA cells / 100 μL / mouse were transplanted into female C57BL / 6J mice by subcutaneous (SC) injection to establish a subcutaneous B16F10-OVA tumor model. 3 Start vaccinations around.

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

[0412] 3) Tumor size and mouse survival rate: Starting from the 7th day after tumor inoculation, 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, in mm, where V represents tumor volume, x represents the long diameter of the tumor, and y represents the short diameter of the tumor. At the same time, the changes in the body weight of C57BL / 6J mice were recorded 3 times a week using an electronic balance, and the survival rate was calculated.

[0413] Experimental results:

[0414] like Figure 6As shown in Table 12, on the 10th day after tumor inoculation, mice in the blank lipid control group (Control Group) and the OVA mRNA-TLP composition group without adjuvant entered the rapid tumor growth phase, 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 volume of the mRNA-TLP pharmaceutical composition with adjuvant lipids also showed stronger tumor growth inhibition, among which 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 tumor treatment with different compositions

[0416]

[0417] In terms of survival rate, Figure 7 As shown, the blank lipid control group began to die on the 17th day after tumor inoculation, and all died on the 19th day; the TLP group without adjuvant began to die on the 19th day, and all died on the 27th day; the mRNA-TLP pharmaceutical composition with adjuvant lipids YK-1202, YK-1204, YK-1205, and YK-1208 began 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 mice.

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

[0419] This example investigates the effect of adjuvant lipids on LNP compositions.

[0420] Experimental process:

[0421] According to the ratio in Table 12, YK-009, YK-1202, DSPC, cholesterol and DMG-PEG2000 were weighed and dissolved in ethanol to prepare an ethanol lipid solution. The eGFP-mRNA was diluted in a citrate buffer (pH = 4~5) to obtain an mRNA aqueous solution. The ethanol lipid solution was mixed with the Fluc mRNA aqueous solution prepared by different capping structures at a volume ratio of 1:3 using a microfluidic device at a flow rate of 10 mL / min, and LNP was prepared with a weight ratio of total lipid to mRNA of about 15:1. The obtained liposomes were diluted to 10 times the volume with PBS, and then ultrafiltered to remove ethanol with a 300 KDa ultrafiltration tube. Then the volume was fixed to a certain volume with PBS, and finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter to obtain an LNP pharmaceutical composition with or without adjuvant lipids. The particle size and polydispersity index (PDI) were measured using a Malvern laser particle size analyzer using dynamic light scattering. Take 10 μL of liposome solution, dilute to 1 mL with RNase-free deionized water, add to the sample pool, and repeat the measurement 3 times for each sample. The measurement conditions are: 90° scattering angle, 25°C. According to the manufacturer's instructions, the encapsulation efficiency of lipid nanoparticles was determined using the Quant it Ribogreen RNA quantification kit (Thermo Fisher Scientific, UK). The results are shown in Table 13.

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

[0423]

[0424] The results showed that the particle size, PDI and encapsulation efficiency of the LNP pharmaceutical composition obtained by partially replacing the cationic lipid YK-009 component in the LNP composition with YK-1202 at 2.5%, 5.0%, 10.0%, 15.0% and 20.0% were within the qualified range (particle size <200 nm, PDI <0.3, encapsulation efficiency >85%). When the adjuvant content was 10 mol%, the particle size, PDI and encapsulation efficiency were all in the best state. Next, the adjuvant lipid was studied at a ratio of 10% adjuvant lipid.

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

[0426] Experimental process:

[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, YK-009, DSPC, cholesterol, DMG-PEG2000 and adjuvant lipid were prepared at a molar ratio of 40:10:38.5:1.5:10 to prepare the Fluc-mRNA-LNP pharmaceutical composition. Evaluation method: First, the particle size, polydispersity index (PDI) and encapsulation efficiency were determined according to the method of Example 11; then the pharmaceutical composition was added to the cell culture medium of the 96-well plate, and after continuing to 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 CCK-8 solution was added to each well of the well plate after 24 hours of culture, and the culture plate was incubated in the incubator for 1 hour, and the absorbance at 450 nm was measured by an ELISA instrument to detect the cell survival rate. The results are shown in Table 14.

[0428] Table 14 Particle size and PDI test results of LNP compositions containing different adjuvants

[0429]

[0430] The results show that the adjuvant lipids YK-1201-1208 in the present application and the adjuvant lipids 63-15 and TMX-201 disclosed in the prior art can all be used to 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, the relative fluorescence intensity (mRNA translation efficiency) and cell viability (cytotoxicity) of the LNP compositions prepared above were significantly different. The relative fluorescence intensity of the mRNA-LNP compositions prepared by YK-1202, YK-1204, YK-1205, YK-1206 and YK-1208 was significantly higher than that of the YK-1201, YK-1203 and YK-1207 groups, and was also significantly higher than that of the LNP groups without adjuvant lipids and the LNP groups with 63 The cytotoxicity of the mRNA-LNP groups prepared by YK-1202, YK-1204, YK-1205, YK-1206 and YK-1208 was significantly lower than that of the YK-1201, YK-1203 and YK-1207 groups, and was at the same level as the mRNA-LNP groups without adjuvant lipids and the mRNA-LNP groups with 63-15 and TMX-201.

[0432] Example 13: Animal expression of LNP-mRNA compositions with different adjuvant lipids

[0433] Experimental process:

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

[0435] After sampling, the mice were euthanized with carbon dioxide, dissected, and their internal organs were precisely separated: liver, spleen, and lungs. The total radiation intensity (corresponding to the fluorescence expression intensity) of the proteins expressed by the mRNA carried by LNP in various organs of the mice was detected by IVIS Spectrum small animal in vivo imaging instrument. The results of the in vivo imaging test of 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 the expression of mRNA delivered by LNP preparations prepared by YK-1202, YK-1204, YK-1205, YK-1206, and YK-1208 at the injection site, abdominal cavity, liver, and spleen of mice is significantly improved compared to LNPs without adjuvant lipids and LNPs with TMX-201, a representative adjuvant lipid in the prior art. For example, the expression of mRNA-LNP group with YK-1206 at the injection site of mice can reach 1.7 times that of LNP without adjuvant lipids, and the expression in the spleen of mice can reach 1.8 times that of LNP with TMX-201.

[0440] There are a large number of APC cells in the spleen and muscles (injection sites). By increasing the expression of delivered mRNA in the spleen and muscles, mRNA vaccines can quickly induce immune responses and produce antibodies in vivo. Without changing the vaccine components, the prevention and treatment effects can be significantly improved, which has important clinical significance.

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

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

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

[0444] Experimental process:

[0445] 8-week-old female C57BL / 6J mice were intramuscularly injected with the OVA-mRNA-LNP (5 µg) composition prepared according to Example 11, and then their eyes were removed and euthanized 6 hours later to obtain as much serum as possible. The levels 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 assay according to the manufacturer's instructions.

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

[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 increased the IFN-γ and IL-12 cytokines in the serum 6 hours after injection. Among them, the serum content of cytokine IL-12 stimulated by the mRNA-LNP composition added with YK-1202-LNP, YK-1204-LNP, YK-1206-LNP, and YK-1208-LNP was 1.3 times, 1.6 times, 2.1 times, and 1.5 times that of the mRNA-LNP composition without adjuvant lipid; the serum content of cytokine IFN-γ stimulated by the mRNA-LNP composition added with YK-1202-LNP, YK-1204-LNP, YK-1206-LNP, and YK-1208-LNP was 1.5 times, 2.9 times, 1.8 times, and 5.3 times that 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 lipids, it can be proved that the adjuvant lipids of the present invention can initiate the 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 process:

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

[0455] 2) Vaccination: The C57BL / 6J mice enrolled in the group were vaccinated by intramuscular injection with OVAmRNA-YK-1202-LNP, YK-1204-LNP, YK-1206-LNP, and YK-1208-LNP compositions containing adjuvant lipids prepared according to Example 11 on days 3, 7, 10, 12, 15, and 19 after the injection of cells (day 0) (each injection contained 5 μg of the therapeutic agent mRNA-OVA vaccine). At the same time, mice vaccinated with an equal volume of OVA mRNA-LNP without adjuvant lipids 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 tumor inoculation, 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, in mm, where V represents tumor volume, x represents the long diameter of the tumor, and y represents the short diameter of the tumor. At the same time, the changes in the body weight of C57BL / 6J mice were recorded 3 times a week using an electronic balance, and the survival rate was calculated.

[0457] Experimental results:

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

[0459] Table 17 Tumor growth in mice during tumor treatment with different compositions

[0460]

[0461] In terms of survival rate, Fig.10As shown, the blank lipid control group began to die on the 17th day after tumor inoculation, and all died on the 19th day; the mRNA-LNP group without adjuvant began 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 adjuvants began to die on the 25th day, the 27th day, the 31st day, and the 29th day, and all died on the 33rd, 40th, 41st, and 42nd day, respectively. It can be seen that the addition of the adjuvant lipid of the present invention significantly prolonged the survival time of mice.

[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, which have at least one of the following advantages compared with the adjuvant lipids disclosed in the prior art:

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

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

[0465] 3. The mRNA-TLP composition prepared by 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 of the prior art, the mRNA-TLP composition prepared by the adjuvant lipid of the present invention has significantly improved targeting of antigen-presenting cells in the spleen of mice.

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

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

[0468] 6. The mRNA-TLP composition prepared by adding the adjuvant lipid of the present invention showed significant effects 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. Adding the adjuvant lipid of the present invention (2.5%~20mol%) to prepare the mRNA-LNP composition 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 significantly increased the translation efficiency of mRNA, significantly reduced cytotoxicity, and significantly increased the protein expression in the injection site, abdominal cavity, liver and spleen of mice. In animal experiments, tumor growth is significantly inhibited and the survival time of tumor-bearing experimental animals is prolonged.

[0470] The above is a detailed description of the present invention, the purpose of which is to enable those familiar with the technology in this field to understand the content of the present invention and implement it, and it is not intended to limit the scope of protection of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A compound represented by formula (I), its stereoisomer, its N-oxide compound, its solvate, or its pharmaceutically acceptable salt: ； L1 is C 1-10 Alkylene or C 1-4 Alkylene-C 6-10 Aryl-C 1-4 Alkylene; M1 is -NH-, -OC(O)HN-, -C(O)O- or -O-; L2 is C 1-8 Alkylene, -R 3a C(O)OR 4a -、-R 3b OR 4b -、 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; 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) according to claim 1, its stereoisomer, its N-oxide compound, its solvate, or its pharmaceutically acceptable salt, 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) according to claim 1, its stereoisomer, its N-oxide compound, its solvate, or its pharmaceutically acceptable salt, 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) according to claim 1, its stereoisomer, its N-oxide compound, its solvate, or its pharmaceutically acceptable salt, 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) according to claim 1, its stereoisomer, its N-oxide compound, its solvate, or its pharmaceutically acceptable salt, 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 substance Z, and the substance Z is a compound represented by formula (I) as described in any one of claims 1 to 5, a stereoisomer thereof, an N-oxide compound thereof, a solvate thereof, 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), a stereoisomer thereof, an N-oxide thereof, a solvate thereof 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), a stereoisomer thereof, an N-oxide thereof, a solvate thereof 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), 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; 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), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, 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), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, 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), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, 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), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, 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, sterol and derivatives thereof.

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 (DOPE) and / or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (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 (DMG-PEG2000); (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 according to 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, its stereoisomer, its N-oxide compound, its solvate, 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 nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

28. Use of a compound of formula (I) as described in any one of claims 1 to 5, a stereoisomer thereof, an N-oxy compound thereof, a solvate thereof, 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 medicament for treating a disease or condition.

29. The use according to claim 28, characterized in that The disease or disorder is characterized by malfunction or aberrant protein or polypeptide activity.

30. The use according to claim 28, characterized in that The disease or condition is selected from any one or a combination of at least two of the group consisting of infectious diseases, tumors and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases.

31. The use according to claim 30, 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.

32. The use according to claim 27 or 28, 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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