Vaccine adjuvant lipid compound based on RLRs receptor agonist, composition containing vaccine adjuvant lipid compound and application

Abstract

The invention relates to an adjuvant lipid compound based on an RLRs small molecule agonist type, a composition containing the adjuvant lipid compound and application of the adjuvant lipid compound, and particularly discloses a compound as shown in a formula (I), and a stereoisomer, pharmaceutically acceptable salt or solvate of the compound. The compound provided by the invention is used as a constituent part of an mRNA or drug molecule delivery carrier, enters cytoplasm of immune cells of a body, and can directly participate in RIG-I activated IRF3 and NF-kB dependent innate immune response.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and in particular to a vaccine adjuvant lipid compound based on a RIG-I like receptors (RLRs) agonist, a composition containing the same, and uses thereof. Background Art

[0002] Adjuvant lipids are substances that can improve the immunogenicity of mRNA vaccines. They can enhance the ability of vaccines to stimulate the body to produce antibodies, thereby improving the protective effect of vaccines. Compared with traditional aluminum adjuvants, oil emulsion adjuvants, and natural polysaccharide adjuvants, which are used in large quantities, adjuvant lipids are a new type of adjuvant that participates in the composition of mRNA vaccine delivery vector LNP. By combining with mRNA to form a stable complex, it can play a good adjuvant role at low doses.

[0003] RLRs-type adjuvant lipids combine small molecule agonists that can activate RLRs signaling pathways, such as amantadine compounds (Amantadine-assembled nanostimulator enhances dimeric RBDantigenelicited cross-neutralization against SARS-CoV-2 strains. Nano Today43 (2022) 101393), with the design concept of cationic lipid compounds. As an immunomodulatory molecule, amantadine regulates its immune function through reasonable structural modification and transformation, and enhances its effect as an adjuvant to optimize the immune response of the vaccine. Amantadine-type adjuvant lipids, as structural components of LNPs, enhance the delivery of mRNA in the cytoplasm, and further dissociate the resulting RLRs small molecule agonists that can participate in RIG-I activation of IRF3 and NF-kB-dependent innate immune responses. It can not only sense viral RNA, but also activate the antiviral state of cells by inducing the production of IFNs and inflammatory factors, thereby limiting the replication and spread of the virus.

[0004] mRNA vaccine technology plays a huge role in protecting human health. Currently, many mRNA vaccines still face problems such as short protective efficacy and rapid decline in antibody levels. Therefore, it is urgent to develop adjuvant lipids that can enhance the immunogenicity of mRNA vaccines to improve the protective effect of vaccines. Summary of the invention

[0005] To solve the above technical problems, the present invention provides an adjuvant lipid compound based on the RLRs small molecule agonist type, a composition containing the same and its use. The compound provided by the present invention is used as a component of an mRNA or drug molecule delivery carrier, enters the cytoplasm of the body's immune cells, and can directly participate in RIG-I activation of IRF3 and NF-kB-dependent innate immune responses, and exerts a highly efficient adjuvant effect at a low dose.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a compound, a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof, characterized in that the structure of the compound is as shown in formula (I),

[0008] Formula (I);

[0009] G1 is selected from , or ;

[0010] L1 and L2 are the same or different and are independently selected from C 1~10 (For example, it can be C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 ) alkylene or a single bond;

[0011] X and Y are the same or different and are independently selected from NH, O or a single bond;

[0012] R1 and R2 are the same or different and are independently selected from or unsubstituted C 6~25 (For example, it can be C6, C8, C 10 , C 12 , C 14 , C 16 , C 18 , C 20 , C 22 , C 24 , C 25 etc.) a straight or branched hydrocarbon group, the C 6~25 The straight chain or branched hydrocarbon group has 0 to 3 (e.g., 0, 1, 2, or 3) C=C double bonds, wherein the double bonds are of E or Z type; when R1 is When X is NH; when R2 is When Y is NH;

[0013] When G1 is selected from or When at least one of R1 and R2 is .

[0014] In some embodiments, G1 is selected from or .

[0015] In some embodiments, L1 is selected from C 3~7 Alkylene or a single bond.

[0016] In some embodiments, L2 is selected from C 3~7 Alkylene or a single bond.

[0017] In some embodiments, X and Y are the same or different and are each independently selected from NH or O.

[0018] In some embodiments, R1 is selected from unsubstituted C having 0 to 2 Z-type C=C double bonds. 10~18 (For example, it can be C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 ) Straight chain hydrocarbon, unsubstituted C 14~18 (For example, it can be C 14 , C 15 , C 16 , C 17 , C 18 ) branched alkyl or .

[0019] In some embodiments, R1 is selected from , , or .

[0020] In some embodiments, R1 is selected from , , or .

[0021] In some embodiments, R2 is selected from C having 0 to 2 Z-type C=C double bonds. 10~19 (For example, it can be C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 ) Straight chain hydrocarbon, unsubstituted C 14~18(For example, it can be C 14 , C 15 , C 16 , C 17 , C 18 ) branched alkyl or .

[0022] In some embodiments, R2 is selected from , , or .

[0023] In some embodiments, R2 is selected from , , or .

[0024] In some embodiments, G1 is ; L1, L2 are the same or different, each independently selected from C 3~7 Alkylene or single bond; X and Y are both O; R1 is unsubstituted C 10~18 Straight chain alkyl, R2 is selected from or .

[0025] In some embodiments, G1 is ; L1, L2 are the same or different, each independently selected from C 3~7 Alkylene or single bond; X is O, R1 is unsubstituted C 14~18 Branched alkyl; Y is NH, R2 is .

[0026] In some embodiments, the compound has one of the following structures:

[0027] ,

[0028] ,

[0029] ,

[0030] ,

[0031] ,

[0032] ,

[0033] ,

[0034] ,

[0035] or

[0036] .

[0037] In some embodiments, the compound is compound YK-1705 having the following structure:

[0038] .

[0039] In some embodiments, the compound is compound YK-1707 having the following structure:

[0040] .

[0041] In some embodiments, the compound is compound YK-1709 having the following structure:

[0042] .

[0043] In a second aspect, the present invention provides a composition comprising a lipid composition, wherein the lipid composition comprises an adjuvant lipid; the adjuvant lipid comprises the compound described in the first aspect, its stereoisomer, pharmaceutically acceptable salt or solvate.

[0044] In some embodiments, the molar percentage of the adjuvant lipid in the lipid composition is 0.1% to 50% (eg, 0.1%, 5%, 10%, 20%, 30%, 40%, 50%, etc.).

[0045] In some embodiments, the lipid composition further comprises a cationic lipid and a neutral lipid.

[0046] In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:1 (for example, it can be 1:1, 2:1, 4:1, 6:1, 10:1, 15:1, etc.).

[0047] In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is 4:1 to 6:1.

[0048] In some embodiments, the cationic lipid is selected from any one or a combination of at least two of the following compounds (1) to (7):

[0049] (1) A compound represented by formula (II), a stereoisomer, a pharmaceutically acceptable salt or a solvate 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 12～25Branched chain alkyl;

[0050] Formula (II);

[0051] (2) A compound represented by formula (III), a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof, wherein G1 is C 2~8 Alkylene; G2 is C 2~8 Alkylene; L1 is selected from -C(O)O- or -OC(O)-; L2 is selected from -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 selected from HO(CH2)2- or HO(CH2)3-; G4 is selected from HO(CH2)2- or HO(CH2)3-; L is selected from -(CH2)2-, -(CH2)3- or -(CH2)4-;

[0052] Formula (III);

[0053] (3) A compound represented by formula (IV), its stereoisomer, pharmaceutically acceptable salt or solvate, 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 selected from HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N (CH3)(CH2)2- or CH3CH2NH(CH2)2-;

[0054] Formula (IV);

[0055] (4) A compound represented by formula (V), a stereoisomer, a pharmaceutically acceptable salt or a solvate 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 selected from -CH3, -CH2CH3 or -CH2CH2OH ;

[0056] Formula (V);

[0057] (5) A compound represented by formula (VI), a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof, wherein G 1 and G 2 Each independently is C6-C 10 Alkylene; G 3 C1-C 12 Alkylene; R 1 and R 2 Each independently selected from C6-C 24 Alkyl or C6-C 24 Alkenyl; R 3 Selected from OR 5 、N、-C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 C1-C 12 Hydrocarbon; R 5 Selected from H or C1-C6 hydrocarbon group;

[0058] Formula (VI);

[0059] (6) A compound represented by formula (VII), a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof, wherein R4 is selected from -(CH2) n Q or -(CH2) n CHQR; Q is selected from -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R), -N(H)C(S)N(H)(R), -N(R)S(O)2R or heterocycle; n is selected from 1, 2 or 3; R is C 1-8 Alkyl; X is selected from H or C 1-8 alkyl;

[0060] Formula (VII);

[0061] (7) The compound DLIN-MC3-DMA, its stereoisomers, pharmaceutically acceptable salts or solvates shown below,

[0062] DLIN-MC3-DMA.

[0063] In some embodiments, the neutral lipid is selected from any one or a combination of at least two of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide or sterol and derivatives thereof.

[0064] In some embodiments, the lipid composition further comprises any one or a combination of at least two of a cationic lipid, a neutral lipid, a structured lipid, or a polymer-conjugated lipid;

[0065] The cationic lipid is selected from any one of YK-009, YK-401, YK-305, ALC0315, SM102 or DLIN-MC3-DMA, or a combination of at least two thereof;

[0066] , ,

[0067] , , , ;

[0068] 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 Diether PC), 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), palmitoyloleoylphosphatidylethanol any one or a combination of at least two of phosphatidyl amine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine or lysophosphatidylethanolamine (LPE).

[0069] The structured lipid is selected from any one or a combination of at least two of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol or corticosteroids;

[0070] The polymer conjugated lipid is selected from any one of distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) or methoxy polyethylene glycol ditetradecanoyl acetamide (ALC-0159) or a combination of at least two thereof.

[0071] In some embodiments, the cationic lipid is YK-009.

[0072] In some embodiments, the molar percentage of the cationic lipid in the lipid composition is 30-47.5%, for example, 30%, 35%, 40%, 45%, etc.

[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 5-25%, such as 5%, 10%, 15%, 20%, 25%, etc.

[0075] In some embodiments, the structured lipid is cholesterol.

[0076] In some embodiments, the molar percentage of the structured lipid in the lipid composition is 15-65%, such as 15%, 25%, 35%, 45%, 55%, 65%, etc.

[0077] In some embodiments, the polymer-conjugated lipid is DMG-PEG2000.

[0078] In some embodiments, the molar percentage of the polymer-conjugated lipid in the lipid composition is 0.5-10%, such as 0.5%, 1.5%, 3%, 5%, 8%, 10%, etc.

[0079] In some embodiments, the lipid composition includes adjuvant lipids, cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids.

[0080] In some embodiments, the molar ratio of the adjuvant lipids, cationic lipids, neutral lipids, structural lipids and polymer conjugated lipids is (1-50):(10-75):(5-25):(15-65):(0.5-10).

[0081] The above values ​​in (1~50) may be, for example, 1, 10, 20, 30, 40, 50, etc.; the values ​​in (10~75) may be, for example, 10, 25, 45, 65, 75, etc.; the values ​​in (5~25) may be, for example, 5, 10, 15, 20, 25, etc.; the values ​​in (15~65) may be, for example, 15, 35, 55, 65, etc.; the values ​​in (0.5~10) may be, for example, 0.5, 1, 4, 8, 10, etc.

[0082] In some embodiments, the molar ratio of the adjuvant lipids, cationic lipids, neutral lipids, structural lipids and polymer conjugated lipids is (5~40):(10~50):(5~20):(15~65):(0.5~5).

[0083] In some embodiments, the composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 10nm~300nm (for example, it can be 10nm, 50nm, 100nm, 150nm, 200nm, 300nm, etc.); the polydispersity coefficient of the nanoparticle preparation is ≤50% (for example, it can be 1%, 5%, 15%, 20%, 40%, 50%, etc.).

[0084] In some embodiments, the average particle size of the nanoparticle preparation is 50 nm to 200 nm; and the polydispersity coefficient of the nanoparticle preparation is ≤20%.

[0085] In some embodiments, the cationic lipid further comprises one or more other ionizable lipid compounds.

[0086] In some embodiments, the composition further comprises a therapeutic and / or prophylactic agent.

[0087] In some embodiments, the mass ratio of the lipid composition to the therapeutic agent and / or preventive agent is 10:1 to 30:1 (for example, it can be 10:1, 15:1, 20:1, 25:1, 30:1, etc.).

[0088] In some embodiments, the mass ratio of the lipid composition to the therapeutic agent and / or preventive agent is 12.5:1 to 25:1.

[0089] In some embodiments, the mass ratio of the lipid composition to the therapeutic agent and / or preventive agent is 14:1 to 20:1.

[0090] 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 composition is 1:(2-5) (e.g., 1:2, 1:3, 1:4, 1:5, etc.).

[0091] In some embodiments, the therapeutic agent and / or preventive agent comprises any one or a combination of at least two of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein.

[0092] In some embodiments, the composition is used to deliver the therapeutic and / or prophylactic agent to antigen presenting cells in a target organ or tissue.

[0093] In some embodiments, the target organ or tissue is selected from any one or a combination of at least two of spleen, liver, lymph, muscle or lung.

[0094] In some embodiments, the antigen presenting cells are selected from any one or a combination of at least two of B cells, NK cells, cDC cells, pDC cells or macrophages.

[0095] In some embodiments, the therapeutic and / or prophylactic agent is a nucleic acid molecule capable of encoding one or more antigens.

[0096] 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.

[0097] In some embodiments, the therapeutic and / or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0098] In some embodiments, the therapeutic and / or prophylactic agent is a nucleic acid.

[0099] In some embodiments, the therapeutic and / or prophylactic agent is RNA.

[0100] In some embodiments, the therapeutic and / or prophylactic agent is DNA.

[0101] In some embodiments, the RNA is selected from any one or a combination of at least two of small interfering RNA, asymmetric interfering RNA, micro RNA, Dicer-substrate RNA, small hairpin RNA or messenger RNA.

[0102] In some embodiments, the RNA is messenger RNA.

[0103] In some embodiments, the composition further comprises one or more pharmaceutically acceptable excipients.

[0104] In some embodiments, the composition further comprises at least one auxiliary ingredient, which may be a pharmaceutically acceptable carrier, diluent or excipient.

[0105] In some embodiments, the composition further comprises one or more hydrophobic small molecules, permeability enhancing molecules, carbohydrates, polymers, surface altering agents, functionalized lipids, or cytokines.

[0106] In a third aspect, the present invention provides a use of the compound as described in the first aspect, its stereoisomers, pharmaceutically acceptable salts or solvates, or the composition as described in the second aspect in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

[0107] In a fourth aspect, the present invention provides a use of the compound as described in the first aspect, its stereoisomer, pharmaceutically acceptable salt or solvate, or the composition as described in the second aspect in the preparation of a medicament for treating a disease or condition in a mammal in need thereof.

[0108] In some embodiments, the disease or disorder is characterized by a malfunction or aberrant protein or polypeptide activity.

[0109] In some embodiments, the disease or condition is selected from any one or a combination of at least two of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, or metabolic diseases.

[0110] In some embodiments, the infectious disease is selected from any one or a combination of at least two of coronavirus, influenza virus or HIV virus-induced diseases, Rift Valley fever, yellow fever, rabies or multiple herpes.

[0111] In some embodiments, the subject to which the medicament is administered is a human.

[0112] In some embodiments, the drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0113] In some embodiments, the route of administration of the drug is subcutaneous.

[0114] In some embodiments, the administration dose of the drug is 0.001-10 mg / kg (for example, 0.001 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, etc.).

[0115] Definition of terms

[0116] 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.

[0117] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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, , .

[0122] 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-, , 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.

[0123] 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.

[0124] 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.

[0125] 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".

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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).

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] Therapeutic and / or preventive agents

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] Disease or condition

[0150] 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.

[0151] In one embodiment, the disease or disorder is characterized by a malfunction or aberrant protein or polypeptide activity.

[0152] 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.

[0153] 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.

[0154] 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).

[0155] 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.

[0156] 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.

[0157] 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.

[0158] Malignant melanoma is a serious type of skin cancer that results from the uncontrolled growth of pigment cells called melanocytes.

[0159] 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.

[0160] Lymphomas and leukemias are malignancies that arise from hematopoietic (blood-forming) cells.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] Other components

[0165] 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.

[0166] 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).

[0167] 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).

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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).

[0173] Suitable preservatives for use in the compositions of the present invention include benzalkonium chloride, chlorobutanol, parabens and thimerosal.

[0174] 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.

[0175] Dosage Form and Administration

[0176] 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.

[0177] 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.

[0178] Therapeutically effective amount

[0179] 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.

[0180] 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.

[0181] Antigen presenting cells

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] The term "immunogenicity" relates to the relative efficiency of an antigen in inducing an immune response.

[0188] 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.

[0189] 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.

[0190] 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 in active immune responses.

[0191] 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.

[0192] 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).

[0193] 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.

[0194] 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 to CD4 by class II MHC molecules +The peptides for T cells are longer because the binding cleft of the class II MHC molecule is open at the end.

[0195] 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.

[0196] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0197] 1. Good particle size and uniform particle distribution;

[0198] 2. As a structural component of LNP, it enhances the delivery of mRNA;

[0199] 3. The delivered mRNA showed significant protein expression in the liver and spleen of mice;

[0200] 4. It can stimulate the production of cytokines such as INF-γ and TNF-α in mouse serum and activate immune cells such as DCs cells and T cells;

[0201] 5. Improve the immunogenicity of mRNA vaccines, enhance the ability of vaccines to stimulate the body to produce antibodies, and improve the protective effect of vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0202] Figure 1 This is a graph showing the results of a cell transfection experiment of an eGFP-mRNA LNP preparation prepared with YK-009, DSPC, cholesterol, DMG-PEG2000 and adjuvant lipids in a molar ratio of 20:10:38.5:1.5:30, wherein: a is YK-1704, b is YK-1705, c is YK-1707, d is YK-1709, e is YK-009 (without adjuvant lipids), and f is 11-AM. DETAILED DESCRIPTION

[0203] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0204] 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.

[0205] The following abbreviations represent the following reagents:

[0206] YK-009: 2-octyldecyl ((decyloxy-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate; , prepared with reference to Example 1 in patent CN114044741B);

[0207] DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine;

[0208] DMG-PEG2000: 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000;

[0209] DCM: dichloromethane;

[0210] EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0211] DMAP: 4-dimethylaminopyridine;

[0212] ACN: acetonitrile;

[0213] K2CO3: potassium carbonate;

[0214] Cs2CO3: cesium carbonate;

[0215] KI: potassium iodide;

[0216] TEA: triethylamine.

[0217] Example 1: Synthesis of RLRs adjuvant lipid compounds

[0218] 1. Synthesis of YK-1701

[0219] The synthetic route is as follows:

[0220] ;

[0221] Step 1: Synthesis of YK-1701-PM1;

[0222] 6-bromohexanoic acid (10.25 g, 52.53 mmol), 3-hexyl nonyl ester (10.00 g, 43.78 mmol), EDCI (16.78 g, 87.56 mmol), DMAP (2.67 g, 21.89 mmol) were dissolved in dichloromethane (10.0 mL), and stirred at room temperature for 24 hours under nitrogen atmosphere. After the reaction was completed, water (10.0 mL) was added to the reaction solution, and then dichloromethane (50.0 mL×2) was added for extraction. The organic phases were combined and washed with saturated brine (10.0 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-1701-PM1 (16.94 g, 41.78 mmol, 95.43%). C 21 H 41 BrO2, MS(ES): m / z(M+H + )405.2.

[0223] Step 2: Synthesis of YK-1701-PM2

[0224] Using 8-bromooctanoic acid (19.23 g, 86.18 mmol), adamantane (10.00 g, 71.82 mmol), EDCI (27.54 g, 143.64 mmol), DMAP (4.39 g, 35.91 mmol) and dichloromethane (10.0 mL) as raw materials, YK-1701-PM2 (15.41 g, 43.24 mmol, 60.21%) was obtained according to the synthesis method of YK-1701-PM1. 18 H 30 BrNO, MS(ES): m / z(M+H + )356.2.

[0225] Step 3: Synthesis of YK-1701-PM3

[0226] YK-1701-PM2 (400.0 mg, 1.12 mmol), ethanolamine (342.8 mg, 5.61 mmol), and K2CO3 (465.4 mg, 3.37 mmol) were dissolved in acetonitrile (4.0 mL) and stirred at 70°C for 5 hours under nitrogen atmosphere. After the reaction was completed, the mixture was concentrated, ethyl acetate (5.0 mL) and water (5.0 mL) were added, stirred and separated, and ethyl acetate (5.0 mL×2) was added for extraction. The organic phases were combined and washed with saturated brine (5.0 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-1701-PM3 (326.4 mg, 0.97 mmol, 86.41%). 20 H 36 N2O2, MS(ES): m / z(M+H + )337.3.

[0227] Step 4: Synthesis of YK-1701

[0228] YK-1701-PM3 (326.4 mg, 0.97 mmol), YK-1701-PM1 (471.9 mg, 1.16 mmol), K2CO3 (402.2 mg, 2.91 mmol), Cs2CO3 (126.4 mg, 0.39 mmol), KI (48.3 mg, 0.29 mmol) were dissolved in acetonitrile (4.0 mL) and stirred at 70°C for 2 days. After the reaction was completed, the mixture was concentrated, ethyl acetate (5.0 mL) and water (5.0 mL) were added, stirred and separated, and ethyl acetate (5.0 mL×2) was added for extraction. The organic phases were combined and washed with saturated brine (5.0 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-1701 (97.1 mg, 0.15 mmol, 15.14%). 41 H 76 N2O4,MS(ES): m / z(M+H + )661.6.

[0229] 1H NMR (CDCl3, 400 MHz, 298 K) δ 3.93 (t, J = 8.6 Hz, 2H), 3.22 (t, J= 6.7 Hz, 2H), 2.81 (d, J = 5.8 Hz, 4H), 2.37 (t, J = 5.6 Hz, 2H), 2.12 (t, J= 5.2 Hz, 2H), 2.01 (d, J = 5.8 Hz, 2H), 1.93 (d, J = 12.3 Hz, 2H), 1.78 -1.56 (m, 13H), 1.38 - 1.32 (m, 6H), 1.16 - 1.06 (m, 33H), 0.89 (t, J = 6.7Hz, 6H).

[0230] 2. Synthesis of YK-1702

[0231] The synthetic route is as follows:

[0232] ;

[0233] Step 1: Synthesis of YK-1702

[0234] YK-1701-PM2 (400.0 mg, 1.12 mmol), ethanolamine (27.4 mg, 0.45 mmol), K2CO3 (465.4 mg, 3.37 mmol), Cs2CO3 (146.3 mg, 0.45 mmol), KI (55.9 mg, 0.34 mmol) and acetonitrile (4.0 mL) were used as raw materials to obtain YK-1702 (93.4 mg, 0.15 mmol, 34.02%) according to the method for synthesizing YK-1701. 38 H 65 N3O3, MS(ES): m / z(M+H + )612.5.

[0235] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 3.72 (t, J = 7.8 Hz, 2H), 3.31 (t, J= 6.8 Hz, 4H), 2.87 (t, J =8.2 Hz, 2H), 2.51 - 2.43 (m, 8H), 2.18 - 2.06 (s,26H), 1.83 (t, J = 7.8 Hz, 4H), 1.66 - 1.56 (m, 16H).

[0236] 3. Synthesis of YK-1703

[0237] The synthetic route is as follows:

[0238] ;

[0239] Step 1: Synthesis of YK-1703

[0240] YK-1701-PM2 (400.0 mg, 1.12 mmol), adamantane (34.0 mg, 0.22 mmol), K2CO3 (465.4 mg, 3.37 mmol), Cs2CO3 (146.3 mg, 0.45 mmol), KI (55.9 mg, 0.34 mmol) and acetonitrile (4.0 mL) were used as raw materials to obtain YK-1703 (77.6 mg, 0.11 mmol, 49.17%) according to the method for synthesizing YK-1701. 46 H 75 N3O2, MS(ES): m / z(M+H + )702.6.

[0241] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 2.86 (t, J = 3.8 Hz, 4H), 2.61 (t, J= 6.3 Hz, 4H), 2.53 (t, J = 5.6 Hz, 4H), 2.45 (t, J = 5.2 Hz, 2H), 2.38 -2.16 (m, 39H), 2.03 (t, J = 6.7 Hz, 4H), 1.76 - 1.69 (m, 16H).

[0242] 4. Synthesis of YK-1704

[0243] The synthetic route is as follows:

[0244] ;

[0245] Step 1: Synthesis of YK-1704-PM1

[0246] Using 4-bromobutyric acid (12.66 g, 75.82 mmol), n-decanol (10.00 g, 63.18 mmol), EDCI (24.22 g, 126.36 mmol), DMAP (3.86 g, 31.59 mmol) and dichloromethane (60.0 mL) as raw materials, YK-1704-PM1 (13.41 g, 43.64 mmol, 69.08%) was obtained according to the synthesis method of YK-1701-PM1. 14 H 27 BrO2, MS(ES): m / z(M+H + )307.1.

[0247] Step 2: Synthesis of YK-1704-PM2

[0248] Using 6-bromohexanoic acid (9.13 g, 46.79 mmol), heptadecanonol (10.00 g, 38.99 mmol), EDCI (14.95 g, 77.98 mmol), DMAP (2.38 g, 19.50 mmol) and dichloromethane (20.0 mL) as raw materials, YK-1704-PM2 (14.03 g, 32.36 mmol, 83.00%) was obtained according to the method for synthesizing YK-1701-PM1. 23 H 45 BrO2,MS(ES): m / z(M+H + )433.3.

[0249] Step 3: Synthesis of YK-1704-PM3

[0250] YK-1704-PM2 (400.0 mg, 0.92 mmol), adamantane (418.7 mg, 2.77 mmol), K2CO3 (382.6 mg, 2.77 mmol) and acetonitrile (4.0 mL) were used as raw materials and YK-1701-PM3 was synthesized to obtain YK-1704-PM3 (312.8 mg, 0.62 mmol, 67.28%). 33 H 61 NO2, MS(ES): m / z(M+H + )504.5.

[0251] Step 4: Synthesis of YK-1704

[0252] YK-1704-PM3 (312.8 mg, 0.62 mmol), YK-1704-PM1 (381.5 mg, 1.24 mmol), K2CO3 (257.4 mg, 1.86 mmol), Cs2CO3 (80.9 mg, 0.25 mmol), KI (30.9 mg, 0.19 mmol) and acetonitrile (4.0 mL) were used as raw materials and YK-1704 (144.0 mg, 0.20 mmol, 31.77%) was obtained according to the method for synthesizing YK-1701. 47 H 87 NO4, MS(ES): m / z(M+H + )730.7.

[0253] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 4.57 (t, J = 6.3 Hz, 1H), 4.23 (t, J= 5.6 Hz, 2H), 2.57 - 2.56 (m, 6H), 2.42 (t, J = 3.8 Hz, 2H), 2.15 (t, J =6.3 Hz, 2H), 1.98 - 1.86 (m, 15H), 1.70 - 1.53 (m, 10H), 1.46 - 1.36 (m, 40H), 0.93 (t, J = 6.8 Hz, 9H).

[0254] 5. Synthesis of YK-1705

[0255] The synthetic route is as follows:

[0256] ;

[0257] Step 1: Synthesis of YK-1705-PM1

[0258] YK-1701-PM1 (400.0 mg, 0.99 mmol), adamantane (447.6 mg, 2.96 mmol), K2CO3 (409.0 mg, 2.96 mmol) and acetonitrile (4.0 mL) were used as raw materials and YK-1705-PM1 (325.1 mg, 0.68 mmol, 69.26%) was obtained according to the method for synthesizing YK-1701-PM3. 31 H 57 NO2, MS(ES): m / z(M+H + )476.4.

[0259] Step 2: Synthesis of YK-1705

[0260] YK-1705-PM1 (325.1 mg, 0.68 mmol), YK-1704-PM1 (629.8 mg, 2.05 mmol), K2CO3 (283.3 mg, 2.05 mmol), Cs2CO3 (89.0 mg, 0.27 mmol), KI (34.0 mg, 0.20 mmol) and acetonitrile (4.0 mL) were used as raw materials and YK-1705 (55.8 mg, 0.08 mmol, 11.63%) was obtained according to the method for synthesizing YK-1701. 45 H 83 NO4, MS(ES): m / z(M+H + )702.6.

[0261] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 4.13 (dd, J = 8.2 Hz, 4H), 2.47 -2.46 (m, 6H), 2.32 (t, J = 7.6 Hz, 2H), 2.05 (t, J = 8.6 Hz, 2H), 1.88 - 1.70(m, 15H), 1.60 - 1.36 (m, 10H), 1.36 - 1.19 (m, 35H), 0.98 (t, J = 6.7 Hz,9H).

[0262] 6. Synthesis of YK-1706

[0263] The synthetic route is as follows:

[0264] ;

[0265] Step 1: Synthesis of YK-1706

[0266] YK-1705-PM1 (270 mg, 0.57 mmol), linoleic acid (315.8 mg, 1.13 mmol), EDCI (239.9 mg, 1.25 mmol), DMAP (38.2 mg, 0.31 mmol) and dichloromethane (10.0 mL) were used as raw materials and YK-1706 (90 mg, 0.08 mmol, 21.48%) was obtained according to the method for synthesizing YK-1701-PM1. 49 H 87 NO3, MS(ES):m / z(M+H +)738.7.

[0267] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 5.43-5.29 (m, 4H), 4.13 (d, J = 19.9Hz, 2H), 3.27 - 3.21 (m, 2H), 2.82 (m, 2H), 2.32 - 2.16 (m, 10H), 1.98 - 1.76 (m, 13H), 1.58 - 1.52 (m, 8H), 1.36 - 1.26 (m, 37H), 0.88 (t, J = 6.7 Hz, 9H).

[0268] 7. Synthesis of YK-1707

[0269] The synthetic route is as follows:

[0270] ;

[0271] Step 1: Synthesis of YK-1707-PM1

[0272] Using 6-bromohexanoic acid (9.0 g, 46.13 mmol), 2-octyldecanol (12.44 g, 45.99 mmol), EDCI (14.95 g, 77.98 mmol), DMAP (2.38 g, 19.50 mmol) and dichloromethane (20.0 mL) as raw materials, YK-1707-PM1 (13.2 g, 30.45 mmol, 66.21%) was obtained according to the method for synthesizing YK-1701-PM1.

[0273] Step 2: Synthesis of YK-1707-PM2

[0274] YK-1704-PM1 (400.0 mg, 0.92 mmol), adamantane (418.7 mg, 2.77 mmol), K2CO3 (382.6 mg, 2.77 mmol) and acetonitrile (4.0 mL) were used as raw materials and YK-1707-PM2 (330.0 mg, 0.64 mmol, 69.06%) was obtained according to the method for synthesizing YK-1701-PM3. 34 H 63 NO2, MS(ES): m / z(M+H + )518.5.

[0275] Step 3: Synthesis of YK-1707

[0276] YK-1707-PM2 (330.0 mg, 0.64 mmol), YK-1704-PM1 (381.5 mg, 1.24 mmol), K2CO3 (257.4 mg, 1.86 mmol), Cs2CO3 (80.9 mg, 0.25 mmol), KI (30.9 mg, 0.19 mmol) and acetonitrile (4.0 mL) were used as raw materials and YK-1707 (120.0 mg, 0.16 mmol, 25.31%) was obtained according to the method for synthesizing YK-1701. 48 H 89 NO4, MS(ES): m / z(M+H + )744.7.

[0277] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 4.17 (t, J = 6.3 Hz, 2H), 3.86 (d, J= 5.6 Hz, 2H), 2.50 - 2.46 (m, 6H), 2.32 (t, J = 3.8 Hz, 2H), 2.05 (m, 2H), 1.89 - 1.76 (m, 17H), 1.60 - 1.56 (m, 4H), 1.36 - 1.19 (m, 44H), 0.90 (t, J =6.7 Hz, 9H).

[0278] 8. Synthesis of YK-1708

[0279] The synthetic route is as follows:

[0280] ;

[0281] Step 1: Synthesis of YK-1708

[0282] YK-1707-PM2 (290 mg, 0.56 mmol), linoleic acid (315.8 mg, 1.13 mmol), EDCI (239.9 mg, 1.25 mmol), DMAP (38.2 mg, 0.31 mmol) and dichloromethane (10.0 mL) were used as raw materials and YK-1708 (100 mg, 0.13 mmol, 22.89%) was obtained according to the method for synthesizing YK-1701-PM1. 52 H 93 NO3, MS(ES):m / z(M+H + )780.7.

[0283] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 5.43-5.29 (m, 4H), 3.90 (d, J = 19.6Hz, 2H), 3.27 - 3.21 (m, 2H), 2.82 (m, 2H), 2.32 - 2.16 (m, 10H), 1.98 - 1.76 (m, 14H), 1.58 - 1.52 (m, 6H), 1.36 - 1.26 (m, 44H), 0.92 - 0.88 (m, 9H).

[0284] 9. Synthesis of YK-1709

[0285] The synthetic route is as follows:

[0286] ;

[0287] Step 1: Synthesis of YK-1709

[0288] YK-1705-PM1 (400.0 mg, 0.84 mmol), YK-1701-PM2 (898.7 mg, 2.52 mmol), K2CO3 (348.6 mg, 2.52 mmol), Cs2CO3 (109.6 mg, 0.34 mmol), KI (41.9 mg, 0.25 mmol) and acetonitrile (4.0 mL) were used as raw materials and YK-1709 (283.4 mg, 0.38 mmol, 44.87%) was obtained according to the method for synthesizing YK-1701. 49 H 86 N2O3, MS(ES): m / z(M+H + )751.7.

[0289] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 4.43 (dd, J = 8.2 Hz, 2H), 2.76 (dd,J = 8.2 Hz, 4H), 2.62 (dd, J = 8.2 Hz, 2H), 2.51 - 2.35 (m, 6H), 2.28 - 2.06(m, 26H), 1.88 - 1.82 (m, 6H), 1.66 - 1.49 (m, 33H), 0.96 (t, J = 6.7 Hz,6H).

[0290] 10. Synthesis of YK-1710

[0291] The synthetic route is as follows:

[0292] ;

[0293] Step 1: Synthesis of YK-1710-PM1

[0294] YK-1701-PM2 (449 mg, 1.26 mmol), adamantane (381.0 mg, 2.52 mmol), K2CO3 (348.6 mg, 2.52 mmol), Cs2CO3 (109.6 mg, 0.34 mmol), KI (41.9 mg, 0.25 mmol) and acetonitrile (4.0 mL) were used as raw materials and YK-1710-PM1 (320 mg, 0.75 mmol, 59.52%) was obtained according to the method for synthesizing YK-1701. 28 H 46 N2O, MS(ES): m / z(M+H + )427.4.

[0295] Step 2: Synthesis of YK-1710

[0296] YK-1710-PM1 (320 mg, 0.75 mmol), linoleic acid (315.8 mg, 1.13 mmol), EDCI (239.9 mg, 1.25 mmol), DMAP (38.2 mg, 0.31 mmol) and dichloromethane (10.0 mL) were used as raw materials and YK-1710 (112 mg, 0.16 mmol, 21.67%) was obtained according to the method for synthesizing YK-1701-PM1. 46 H 76 N2O3, MS(ES):m / z(M+H + )689.6.

[0297] 1 H NMR (CDCl3, 400 MHz, 298 K) δ 5.43-5.29 (m, 4H), 3.27 - 3.21 (m,2H), 2.80 (m, 2H), 2.22-2.15 (m, 12H), 1.98 -1.76 (m, 26H), 1.53 - 1.50 (m,6H), 1.30 - 1.26 (m, 20H), 0.88 (t, J = 6.7 Hz, 3H).

[0298] 11. Synthesis of Compound 11-AM

[0299] ;

[0300] The synthesis of compound 11-AM referred to the synthesis route of 11-AM in WO2021021634Al to obtain 92.0 mg of 11-AM.

[0301] 12. Synthesis of Compound A-17-2Z

[0302] ;

[0303] The synthesis of A-17-2Z refers to the synthesis route on page 2 of the text, Bioeng Transl Med. 2020;5:e10161, to obtain 105 mg of A-17-2Z.

[0304] 13. Synthesis of Compound 247-9

[0305] ;

[0306] The synthesis of compound 247-9 referred to the synthesis route of compound 247-9 in WO2023133089Al to obtain 67.5 mg of compound 247-9.

[0307] 14. Synthesis of compound BN-INL-A209

[0308] ;

[0309] The synthesis of compound BN-INL-A209 refers to the synthesis route of BN-INL-A209 in WO2024052923Al to obtain 262.0 mg of compound BN-INL-A209.

[0310] Example 2: Preparation of mRNA lipid composition

[0311] A) Preparation of Fluc DNA, eGFP DNA, and OVA DNA templates

[0312] 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;

[0313] 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), incubate them on ice for 30 minutes, heat shock them at 42°C for 90 seconds, put them back on ice immediately, and incubate them on ice for 2 minutes;

[0314] 3) Add 400 μL LB medium (purchased from Thermo Fisher Scientific) and culture at 30°C with slow shaking for 45-60 minutes;

[0315] 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;

[0316] 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;

[0317] 6) Use an endotoxin-free plasmid extraction kit (purchased from Yisheng Biotechnology Co., Ltd.) to extract the plasmid;

[0318] 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.

[0319] 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);

[0320] Table 1

[0321]

[0322] 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, placed at -20℃ for 1 hour, and then centrifuged at 12000 rpm to retain the precipitate;

[0323] 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 it;

[0324] 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%.

[0325] B) Preparation of Fluc mRNA, eGFP mRNA and OVA mRNA

[0326] 1) Co-transcriptional capping reaction:

[0327] 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.

[0328] The co-transcriptional capping reaction system is shown in Table 2.

[0329] Table 2

[0330]

[0331] Note: All the above reagents were purchased from Yisheng Biotechnology Co., Ltd.

[0332] 2) Digestion of template DNA:

[0333] 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.

[0334] 3) Purification by lithium chloride precipitation method:

[0335] The co-transcription capping product obtained in step 2) above was purified by lithium chloride precipitation as follows:

[0336] 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;

[0337] Step ② Precipitation: high-speed centrifugation at 12000 rpm for 15 minutes, retain the precipitate;

[0338] 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 ℃.

[0339] Example 3: Effects of different amounts of adjuvant lipid added on LNP-mRNA compositions

[0340] This example investigates the effect of adjuvant lipids on LNP compositions.

[0341] Experimental process:

[0342] YK-009, YK-1705, DSPC, cholesterol and DMG-PEG2000 were weighed and dissolved in ethanol according to the proportions in Table 3 to prepare an ethanol lipid solution. 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 LNPs were 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 using 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 LNP pharmaceutical compositions 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 particle size, PDI and encapsulation efficiency of the compositions with different formulations are shown in Table 3.

[0343] Table 3

[0344]

[0345] The results showed that the particle size, PDI and encapsulation efficiency of the LNP pharmaceutical composition obtained by using YK-1705 to partially replace the cationic lipid YK-009 component in the LNP composition at 5%, 10%, 20%, 30% and 40% were all within the qualified range (particle size <200 nm, PDI <0.3, encapsulation efficiency >85%). When the adjuvant lipid content was 30 mol%, the particle size, PDI and encapsulation efficiency were all the best. Then, various adjuvant lipids were studied with an adjuvant lipid ratio of 30 mol%.

[0346] Example 4: Effects of different adjuvant lipids on LNP-mRNA compositions

[0347] The structures of RLRs adjuvant lipid compounds are shown in Table 4-1, Table 4-2, and Table 4-3.

[0348] Table 4-1

[0349]

[0350] Table 4-2

[0351]

[0352] Table 4-3

[0353]

[0354] Experimental process:

[0355] According to the preparation method of Example 3, the RLRs adjuvant lipid compounds in Table 4-1, Table 4-2, and Table 4-3 were used to partially replace the cationic lipid YK-009 in the LNP composition at a ratio of 30 mol%, that is, YK-009, DSPC, cholesterol, DMG-PEG2000 and adjuvant lipid were used in a molar ratio of 20:10:38.5:1.5:30 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 in Example 3; then the drug composition was added to the cell culture medium of the 96-well plate, and the culture was continued for 24 hours. The corresponding reagents were added according to the instructions of the Gaussia Luciferase Assay Kit, and the fluorescence expression intensity of each well was detected by the IVIS fluorescence detection system; finally, 10 μL of 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. The absorbance at 450 nm was measured by a microplate reader to detect the cell viability. The particle size and PDI test results of the LNP compositions containing different adjuvants are shown in Table 5.

[0356] Table 5

[0357]

[0358] The results show that:

[0359] (1) Physical and chemical properties of mRNA-LNP containing adjuvant lipids: The adjuvant lipids YK-1701~1710 in this application and the adjuvant lipids 11-AM, A-17-2Z, 247-9 and BN-INL-A209 disclosed in the prior art can all be used to prepare good mRNA-LNP compositions by replacing YK-009 at a ratio of 30%. The particle size of all lipid nanoparticles is between 69~100nm, the PDI value is between 0.03~0.19, and the encapsulation efficiency is above 86%;

[0360] (2) In vitro transfection activity of mRNA-LNPs containing adjuvant lipids: The relative fluorescence intensity (translation efficiency of mRNA) of the LNP compositions prepared above showed significant differences. The relative fluorescence intensity of the mRNA-LNP compositions prepared from YK-1705, YK-1707 and YK-1709 was significantly higher than that of the YK-1702, YK-1703, YK-1708 and YK-1710 groups, and was also significantly higher than that of the YK-009-LNP group without adjuvant lipids and the mRNA-LNP group with 11-AM, A-17-2Z, 247-9 and BN-INL-A209 (see Figure 1 );

[0361] (3) Cellular activity of mRNA-LNP containing adjuvant lipids: The cell viability (cytotoxicity) of the LNP compositions prepared above showed significant differences. The cell viability of the mRNA-LNP composition groups prepared by YK-1705, YK-1707 and YK-1709 reached 95%, 90% and 93%, respectively. The cytotoxicity was significantly lower than that of the YK-1702, YK-1703 and YK-1710 groups, and significantly higher than that of the YK-009-LNP group without adjuvant lipids and the mRNA-LNP group with 11-AM, A-17-2Z, 247-9 and BN-INL-A209.

[0362] Example 5: Animal expression of LNP-mRNA composition with added RLRs adjuvant lipid

[0363] Experimental process:

[0364] The LNP preparations containing 10 μg of Fluc-mRNA with different adjuvant lipids prepared according to Example 4 were injected intramuscularly into female BALB / C mice aged 4 to 6 weeks and weighing 17 to 19 g, and the mice were intraperitoneally injected with fluorescent imaging substrates 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.

[0365] 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 6.

[0366] Table 6

[0367]

[0368] Experimental results:

[0369] The expression levels of the delivered mRNA in the injection site, abdominal cavity, liver and spleen of mice prepared by LNP preparations made of YK-1705, YK-1707 and YK-1709 are significantly improved compared to YK-009-LNP without adjuvant lipids and 11-AM-LNP, 247-9-LNP and BN-INL-A209-LNP with representative adjuvant lipids in the prior art. For example, the expression level of the mRNA-LNP group with added YK-1705 at the injection site of mice can reach 1.7 times that of YK-009-LNP without added adjuvant lipids, and the expression level in the spleen of mice is 2.3 times that of 11-AM-LNP, 1.8 times that of 247-9-LNP and 3.2 times that of BN-INL-A209-LNP.

[0370] 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.

[0371] In addition, the expression of Fluc-mRNA-containing LNP preparations with added adjuvant lipids was very different in different organs of mice. YK-1705, YK-1707, YK-1709, 11-AM, 247-9, BN-INL-A209 and LNP preparations prepared without added adjuvant lipids were all expressed in the liver and spleen, but not in the lungs.

[0372] Compared with the adjuvant lipid YK-1704, the mRNA-LNP preparations prepared by YK-1705 and YK-1709 significantly increased the expression intensity of mRNA in the liver and spleen of mice. For example, the expression level of mRNA in the LNP preparations with YK-1705 added was 1.9 times that of the LNP preparations with YK-1704 added at the injection site, and the expression level in the spleen was 3.2 times.

[0373] Example 6: Stimulation of serum cytokines IFN-γ and TNF-α by mRNA-LNP compositions containing RLRs adjuvant lipids

[0374] 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. Tumor necrosis factor-α (TNF-α) is a key cytokine connecting inflammation and the immune system, mainly produced by macrophages, and has multiple biological functions such as cell proliferation, metabolic activation, inflammatory response and apoptosis. Detecting the stimulation of cytokines IFN-γ and TNF-α by mRNA-LNP compositions containing adjuvant lipids can reflect the improvement of the innate immunity of the mRNA-LNP compositions by adjuvant lipids.

[0375] Experimental process:

[0376] Female BALB / C mice aged 4 to 6 weeks and weighing 17 to 19 g were intramuscularly injected with the OVA-mRNA-LNP (10 µg) composition prepared according to the method of Example 4, and then the eyes were removed and euthanized 6 hours later to obtain as much serum as possible. The levels of IFN-γ and TNF-α 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.

[0377] ELISA assay: Mouse IFN-γ and TNF-α were detected in mouse serum using a standard ELISA assay according to the manufacturer's instructions.

[0378] Table 7 shows a comparison of the stimulating effects of mRNA-LNP compositions containing adjuvant lipids on cytokines IFN-γ and TNF-α.

[0379] Table 7

[0380]

[0381] Experimental results:

[0382] As shown in Table 7, the mRNA-LNP compositions of different RLRs adjuvant lipids in the present invention had significant differences in the levels of IFN-γ and TNF-α cytokines in the serum of mice 6 hours after injection. Among them, the serum content of cytokine IFN-γ stimulated by the mRNA-LNP composition added with YK-1705-LNP, YK-1707-LNP, and YK-1709-LNP was 2.8 times, 2.3 times, and 2.7 times that of the mRNA-LNP composition added with YK-1703-LNP; the serum content of cytokine TNF-α stimulated by the mRNA-LNP composition added with YK-1705-LNP, YK-1707-LNP, and YK-1709-LNP was 2.3 times, 1.9 times, and 1.8 times that of the mRNA-LNP composition added with YK-1706-LNP.

[0383] Compared with the mRNA-LNP composition without adjuvant lipid (YK-009-LNP), the mRNA-LNP composition containing RLRs adjuvant lipid significantly increased the IFN-γ and TNF-α cytokines in the serum 6 hours after injection. Among them, the serum content of cytokine IFN-γ stimulated by the mRNA-LNP composition with added YK-1705-LNP, YK-1707-LNP, and YK-1709-LNP was 2.5 times, 2.0 times, and 2.4 times that of the mRNA-LNP composition without adjuvant lipid (YK-009-LNP); the serum content of cytokine TNF-α stimulated by the mRNA-LNP composition with added YK-1705-LNP, YK-1707-LNP, and YK-1709-LNP was 2.1 times, 1.7 times, and 1.6 times that of the mRNA-LNP composition without adjuvant lipid (YK-009-LNP).

[0384] Compared with the mRNA-LNP composition in the prior art with 11-AM, 247-9 and BN-INL-A209 as adjuvant lipids, the serum content of cytokine IFN-γ stimulated by the mRNA-LNP composition with YK-1705-LNP added is 1.9 times, 2.0 times and 2.3 times that of the mRNA-LNP composition with 11-AM, 247-9 and BN-INL-A209 as adjuvant lipids, respectively; the serum content of cytokine TNF-α stimulated by the mRNA-LNP composition with YK-1705-LNP added is 1.6 times, 1.7 times and 1.9 times that of the mRNA-LNP composition with 11-AM, 247-9 and BN-INL-A209 as adjuvant lipids, respectively.

[0385] The experimental results show that by detecting the IFN-γ and TNF-α 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.

[0386] Example 7: Stimulation of antigen-specific cytotoxic T cells produced in the spleen by mRNA compositions containing RLRs adjuvant lipids

[0387] The strength of the T cell effector generated by tumor mRNA-liposome complexes can be determined by measuring antigen-specific CD8 + Cytotoxic T cells (CD8 + T cells), which is crucial for the anti-tumor effect of lipid complex delivery of tumor mRNA.

[0388] Experimental process:

[0389] 1. The OVA-mRNA-LNP (10 µg) composition prepared in Example 6 with or without adjuvant lipids was injected into female C57BL / 6 mice aged 4-6 weeks and weighing 17-19 g via the tail vein, once on days 0, 3, and 8, for a total of 3 injections. At the same time, mice with the same volume of blank lipid solution after dilution were set as the control group, with 3 mice in each group in parallel. On the 13th day, the mice were euthanized by cervical dissection and dissected to accurately separate the mouse spleen. After preparing single cells, OVA antigen-specific CD8 + T cells in total CD8 + The percentage of T cells.

[0390] 2. Preparation of single cells

[0391] 1) Grind the separated spleen tissue to make it single cell and pass it through a cell sieve;

[0392] 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;

[0393] 3) Count the cells and take 5×10 6 cells into the flow tube (to ensure the same number of cells between samples);

[0394] 3. Detection of immune cells in spleen tissue

[0395] 1) Add 100 μL of surface antibody MIX to each single cell suspension (see Table 8 for the composition of surface antibody MIX) and incubate at room temperature in the dark for 15 minutes (one negative control);

[0396] Detection of OVA antigen-specific CD8 +The reagents and sources of T cell experiments are shown in Table 8.

[0397] Table 8

[0398]

[0399] 2) Add 2 mL of 1× RBC Lysis Buffer, protect from light for 10 minutes, centrifuge at 500 g for 5 minutes, and discard the supernatant.

[0400] 3) Add 2 mL of 1× RBC Lysis Buffer, centrifuge at 500 g for 5 min, and discard the supernatant.

[0401] 4) Add 200 μL PBS, transfer to a clean and numbered EP tube (filtered through a 200-mesh nylon mesh), and test on the flow cytometer Cytoflex S. Use single-color compensation microspheres to adjust compensation before testing. The test order is as follows: CD3 + →CD8 + →APC anti-mouse H-2Kb bound to SIINFEKL

[0402] The proportion of OVA antigen-specific T cells stimulated by the mRNA composition containing RLRs adjuvant lipids and the RNA composition without agonist cationic lipids is shown in Table 9.

[0403] Table 9

[0404]

[0405] As shown in Table 9, compared with the mRNA-LNP composition without adjuvant lipid, the mRNA-LNP composition containing RLRs adjuvant lipid was inoculated into mice three times, and the CD8 + The mRNA-LNP combination with YK-1705-LNP, YK-1707-LNP, and YK-1709-LNP stimulated the production of CD8 + The proportion of T cells was 2.6 times, 1.6 times and 2.0 times that of the mRNA-LNP composition without adjuvant lipid (YK-009-LNP). Based on the average data of the three experimental groups, compared with the mRNA-LNP composition with 11-AM, 247-9 and BN-INL-A209 as adjuvant lipid in the prior art, the addition of YK-1705-LNP composition stimulated the production of CD8 + The T cell ratios were 1.7 times, 2.0 times, and 2.3 times that of the mRNA-LNP compositions to which 11-AM, 247-9, and BN-INL-A209 adjuvant lipids were added, respectively.

[0406] The experimental results show that by detecting the mRNA composition containing RLRs adjuvant lipids to stimulate the production of antigen-specific cytotoxic T cells in the mouse spleen, it can be proved that the mRNA vaccine containing RLRs adjuvant lipids can produce very strong T cell effects in the mouse spleen.

[0407] Example 8: Stimulation of spleen cytokine IFN-γ by mRNA-LNP composition containing RLRs adjuvant lipid

[0408] Experimental process:

[0409] The OVA-mRNA-LNP (10 µg) composition prepared in Example 6 with or without adjuvant lipids was injected into female C57BL / 6 mice aged 4-6 weeks and weighing 17-19 g via the tail vein, once on days 0, 3, and 8, for a total of 3 injections. At the same time, mice with the same volume of blank lipid solution after dilution were set as the control group, with 3 mice in each group in parallel. On the 13th day, the mice were euthanized by cervical dissection and dissected to accurately separate the mouse spleen. After preparing single cells, ELISPOT was used to detect the secretion of interferon IFN-γ by activated immune cells (such as T cells) in the spleen.

[0410] ELISPOT counting assay: Mouse IFN-γ was detected in mouse splenocytes using a standard ELISPOT assay according to the manufacturer's instructions.

[0411] The comparison of the stimulating effect of mRNA-LNP compositions containing adjuvant lipids on the cytokine IFN-γ is shown in Table 10.

[0412] Table 10

[0413]

[0414] Experimental results:

[0415] As shown in Table 10, compared with the mRNA-LNP composition without adjuvant lipid, the mRNA-LNP composition containing RLRs adjuvant lipid was inoculated three times in mice, and the IFN-γ cytokine level in the mouse spleen cells was significantly increased. Among them, the number of fluorescent spots of cytokine IFN-γ stimulated by the mRNA-LNP composition with added YK-1705-LNP, YK-1707-LNP, and YK-1709-LNP was 1.8 times, 1.5 times, and 1.7 times that of the mRNA-LNP composition without adjuvant lipid (YK-009-LNP). Based on the average data of the three experimental groups, compared with the mRNA-LNP compositions in the prior art with 11-AM, 247-9, and BN-INL-A209 as adjuvant lipids, the number of fluorescent spots of cytokine IFN-γ stimulated by the mRNA-LNP composition with the addition of YK-1705-LNP was 1.3 times, 1.4 times, and 1.8 times that of the mRNA-LNP composition with the addition of 11-AM, 247-9, and BN-INL-A209 as adjuvant lipids, respectively.

[0416] The experimental results show that the mRNA-LNP composition containing adjuvant lipids can effectively activate immune cells (such as T cells) in the spleen to secrete interferon IFN-γ, which can prove 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.

[0417] The present invention designs a series of novel vaccine adjuvant lipids based on RLRs receptor agonists, which have the following advantages compared with the adjuvant lipids disclosed in the prior art:

[0418] 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.

[0419] 2. Adding the adjuvant lipid of the present invention (5%~40mol%) to prepare the mRNA-LNP composition has a good particle size (<200nm) 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.

[0420] 3. The mRNA-TLP composition prepared by the adjuvant lipid of the present invention can significantly increase the content of cytokine IFN-γ in mouse serum and spleen, can initiate the immune stimulation program, and significantly improve the innate immunity of the mRNA-LNP composition.

[0421] 4. mRNA vaccines containing RLRs adjuvant lipids can produce very strong T cell effects in mice.

[0422] 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 based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A compound, a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof, characterized in that: The structure of the compound is shown in formula (I), Formula (I); G1 is selected from , or ; L1 and L2 are the same or different and are independently selected from C 1~10 Alkylene or single bond; X and Y are the same or different and are independently selected from NH, O or a single bond; R1 and R2 are the same or different and are independently selected from or unsubstituted C 6~25 A straight chain or branched hydrocarbon group, the C 6~25 The straight chain or branched hydrocarbon group has 0 to 3 C=C double bonds, wherein the double bonds are of E or Z type; when R1 is When X is NH; when R2 is When Y is NH; When G1 is selected from or When at least one of R1 and R2 is .

2. The compound according to claim 1, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: G1 is selected from or .

3. The compound according to claim 1, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: L1 is selected from C 3~7 Alkylene or a single bond.

4. The compound according to claim 1, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: L2 is selected from C 3~7 Alkylene or a single bond.

5. The compound according to claim 1, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: X and Y are the same or different and are independently selected from NH or O.

6. The compound according to claim 1, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: R1 is selected from unsubstituted C having 0 to 2 Z-type C=C double bonds 10~18 Straight chain hydrocarbon, unsubstituted C 14~18 Branched alkyl or .

7. The compound according to claim 6, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: R1 is selected from , , or .

8. The compound according to claim 6, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: R1 is selected from , , or .

9. The compound according to claim 1, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: R2 is selected from C having 0 to 2 Z-type C=C double bonds 10~19 Straight chain hydrocarbon, unsubstituted C 14~18 Branched alkyl or .

10. The compound according to claim 9, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: R2 is selected from , , or .

11. The compound according to claim 9, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: R2 is selected from , , or .

12. The compound according to claim 1, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: G1 is ; L1, L2 are the same or different, each independently selected from C 3~7 Alkylene or single bond; X and Y are both O; R1 is unsubstituted C 10~18 Straight chain alkyl, R2 is selected from or .

13. The compound according to claim 1, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: G1 is ; L1, L2 are the same or different, each independently selected from C 3~7 Alkylene or single bond; X is O, R1 is unsubstituted C 14~18 Branched alkyl; Y is NH, R2 is .

14. The compound according to claim 1, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: The compound has one of the following structures: 、 、 、 、 、 、 、 、 or 。 15. The compound according to claim 1, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: The compound is compound YK-1705 having the following structure: 。 16. The compound according to claim 1, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: The compound is compound YK-1707 having the following structure: 。 17. The compound according to claim 1, its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: The compound is compound YK-1709 having the following structure: 。 18. A composition, characterized in that The composition comprises a lipid composition, and the lipid composition comprises an adjuvant lipid; the adjuvant lipid comprises the compound described in any one of claims 1 to 17, its stereoisomer, pharmaceutically acceptable salt or solvate.

19. The composition according to claim 18, characterized in that The molar percentage of the adjuvant lipid in the lipid composition is 0.1% to 50%.

20. The composition according to claim 18, characterized in that The lipid composition also includes cationic lipids and neutral lipids.

21. The composition according to claim 20, characterized in that The molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:

1.

22. The composition according to claim 21, characterized in that The molar ratio of the cationic lipid to the neutral lipid is 4:1 to 6:

1.

23. The composition according to claim 20, 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, a pharmaceutically acceptable salt or a solvate 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; Formula (II); (2) A compound represented by formula (III), a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof, wherein G1 is C 2~8 Alkylene; G2 is C 2~8 Alkylene; L1 is selected from -C(O)O- or -OC(O)-; L2 is selected from -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 selected from HO(CH2)2- or HO(CH2)3-; G4 is selected from HO(CH2)2- or HO(CH2)3-; L is selected from -(CH2)2-, -(CH2)3- or -(CH2)4-; Formula (III); (3) A compound represented by formula (IV), its stereoisomers, pharmaceutically acceptable salts or solvates, 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 selected from HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N (CH3)(CH2)2- or CH3CH2NH(CH2)2-; Formula (IV); (4) A compound represented by formula (V), a stereoisomer, a pharmaceutically acceptable salt or a solvate 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 selected from -CH3, -CH2CH3 or -CH2CH2OH; Formula (V); (5) A compound represented by formula (VI), a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof, wherein G 1 and G 2 Each independently is C6-C 10 Alkylene; G 3 C1-C 12 Alkylene; R 1 and R 2 Each independently selected from C6-C 24 Alkyl or C6-C 24 Alkenyl; R 3 Selected from OR 5 、N、-C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 C1-C 12 Hydrocarbon; R 5 Selected from H or C1-C6 hydrocarbon group; Formula (VI); (6) A compound represented by formula (VII), a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof, wherein R4 is selected from -(CH2) n Q or -(CH2) n CHQR; Q is selected from -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R), -N(H)C(S)N(H)(R), -N(R)S(O)2R or heterocycle; n is selected from 1, 2 or 3; R is C 1-8 Alkyl; X is selected from H or C 1-8 alkyl; Formula (VII); (7) The compound DLIN-MC3-DMA, its stereoisomers, pharmaceutically acceptable salts or solvates shown below, DLIN-MC3-DMA。 24. The composition according to claim 20, characterized in that The neutral lipid is selected from any one or a combination of at least two of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide or sterol and derivatives thereof.

25. The composition according to claim 18, characterized in that The lipid composition further comprises any one or a combination of at least two of cationic lipids, neutral lipids, structural lipids or polymer-conjugated lipids; The cationic lipid is selected from any one of YK-009, YK-401, YK-305, ALC0315, SM102 or DLIN-MC3-DMA, or a combination of at least two thereof; 、 、 、 、 、 ； 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-di-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 acylphosphatidylglycerol, 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 or lysophosphatidylethanolamine, or a combination of at least two thereof; The structured lipid is selected from any one or a combination of at least two of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol or corticosteroids; The polymer conjugated lipid is selected from any one of distearoylphosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol 2000 or methoxy polyethylene glycol ditetradecyl acetamide or a combination of at least two thereof.

26. The composition according to claim 25, characterized in that The lipid composition includes adjuvant lipids, cationic lipids, neutral lipids, structural lipids and polymer conjugated lipids.

27. The composition according to claim 26, characterized in that The molar ratio of the adjuvant lipid, cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid is (1-50):(10-75):(5-25):(15-65):(0.5-10).

28. The composition according to claim 27, characterized in that The molar ratio of the adjuvant lipid, cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid is (5-40):(10-50):(5-20):(15-65):(0.5-5).

29. The composition according to claim 18, characterized in that The composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 10nm-300nm; the polydispersity coefficient of the nanoparticle preparation is ≤50%.

30. The composition according to claim 29, characterized in that The average particle size of the nanoparticle preparation is 50nm-200nm; the polydispersity coefficient of the nanoparticle preparation is ≤20%.

31. The composition according to claim 23, characterized in that The cationic lipid may also include one or more other ionizable lipid compounds.

32. The composition according to claim 18, characterized in that The composition also includes a therapeutic and / or prophylactic agent.

33. The composition according to claim 32, characterized in that The mass ratio of the lipid composition to the therapeutic agent and / or preventive agent is 10:1 to 30:

1.

34. The composition according to claim 33, characterized in that The mass ratio of the lipid composition to the therapeutic agent and / or preventive agent is 12.5:1 to 25:

1.

35. The composition according to claim 34, characterized in that The mass ratio of the lipid composition to the therapeutic agent and / or preventive agent is 14:1 to 20:

1.

36. The composition according to claim 32, characterized in that The therapeutic agent and / or preventive agent includes any one of nucleic acid molecules, small molecule compounds, polypeptides or proteins, or a combination of at least two of them.

37. The composition according to claim 32, characterized in that The therapeutic and / or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

38. The composition according to claim 36, characterized in that The therapeutic and / or prophylactic agent is a nucleic acid.

39. The composition according to claim 38, characterized in that The therapeutic and / or prophylactic agent is RNA.

40. The composition according to claim 38, characterized in that The therapeutic and / or prophylactic agent is DNA.

41. The composition according to claim 39, characterized in that The RNA is selected from any one or a combination of at least two of small interfering RNA, asymmetric interfering RNA, micro RNA, Dicer-substrate RNA, small hairpin RNA or messenger RNA.

42. The composition according to claim 41, characterized in that The RNA is messenger RNA.

43. The composition according to claim 18, characterized in that The composition may further comprise one or more pharmaceutically acceptable excipients.

44. Use of the compound, stereoisomer, pharmaceutically acceptable salt or solvate thereof as claimed in any one of claims 1 to 17, or the composition as claimed in any one of claims 18 to 43 in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

45. Use of a compound, a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof as claimed in any one of claims 1 to 17, or a composition as claimed in any one of claims 18 to 43 in the preparation of a medicament for treating a disease or condition in a mammal in need thereof.

46. ​​The use according to claim 45, characterized in that The disease or disorder is characterized by malfunctioning or aberrant protein or polypeptide activity.

47. The use according to claim 45, characterized in that The disease or condition is selected from any one or a combination of at least two of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases or metabolic diseases.

48. The use according to claim 47, characterized in that The infectious disease is selected from any one or a combination of at least two of coronavirus, influenza virus or HIV virus-induced diseases, Rift Valley fever, yellow fever, rabies or multiple herpes.

49. The use according to any one of claims 44 to 48, characterized in that The subject of administration of the drug is human.

50. The use according to any one of claims 44 to 48, characterized in that The drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

51. The use according to claim 50, characterized in that The drug is administered subcutaneously.

52. The use according to any one of claims 44 to 48, characterized in that The dosage of the drug is 0.001-10 mg / kg.

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