Vaccine adjuvant lipid compounds based on RLRs receptor agonists, compositions containing the same, and uses thereof

Abstract

The present invention relates to an adjuvant lipid compound based on the small molecule agonist type of RLRs, a composition containing the same and uses thereof, and specifically discloses a compound represented by formula (I), its stereoisomers, pharmaceutically acceptable salts or solvates. The compound provided by the present invention, as a component of an mRNA or drug molecule delivery carrier, enters the cytoplasm of immune cells in the body and can directly participate in the RIG-I-activated IRF3 and NF-κB-dependent innate immune responses.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a vaccine adjuvant lipid compound based on RIG-I like receptors (RLRs) agonists, 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 adjuvants such as aluminum adjuvants, oil emulsion adjuvants, and natural polysaccharide adjuvants that require relatively large amounts, adjuvant lipids are a new type of adjuvant that participate in the composition of the mRNA vaccine delivery vector LNP. By binding to mRNA to form a stable complex, they can exert good adjuvant effects at low doses.

[0003] RLRs-type adjuvant lipids combine small molecule agonists that can activate the RLRs signaling pathway, such as amantadine compounds (Amantadine-assembled nanostimulator enhances dimeric RBD antigenelicited cross-neutralization against SARS-CoV-2 strains. Nano Today 43 (2022) 101393), with the design concept of cationic lipid compounds. As an immunomodulatory molecule, amantadine is structurally modified and transformed to regulate its immune function, enhance its adjuvant effect, and optimize the immune response of vaccines. Amantadine-type adjuvant lipids enhance the delivery of mRNA into the cytoplasm as a structural component of LNP, and the further dissociated RLRs small molecule agonists can participate in the RIG-I activation of IRF3 and NF-kB-dependent innate immune responses. They can not only sense viral RNA but also activate the antiviral state of cells by inducing the production of IFNs and inflammatory factors, restricting the replication and spread of viruses.

[0004] mRNA vaccine technology plays a huge role in protecting human health. Currently, many mRNA vaccines still face problems such as short protection periods and rapid decline in antibody levels. Therefore, there is an urgent need 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 small molecule agonist type of RLRs, a composition containing the same, and uses thereof. The compound provided by the present invention enters the cytoplasm of immune cells in the body as a component of an mRNA or drug molecule delivery carrier, and can directly participate in the RIG-I-activated IRF3 and NF-kB-dependent innate immune responses, exerting a highly efficient adjuvant effect at a low dose.

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

[0007] In a first aspect, the present invention provides a compound, its stereoisomers, pharmaceutically acceptable salts or solvates, characterized in that the structure of the compound is shown in formula (I),

[0008] Formula (I);

[0009] G1 is selected from , or ;

[0010] L1 and L2 are the same or different and are each 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 each independently selected from NH, O or a single bond;

[0012] R1 and R2 are the same or different and are each 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.) straight-chain or branched-chain hydrocarbon groups, and the C 6~25 straight-chain or branched-chain hydrocarbon group has 0 to 3 (for example, it can be 0, 1, 2, 3) C=C double bonds, where the double bond is in the E form or the Z form; when R1 is , X is NH; when R2 is , Y is NH;

[0013] When G1 is selected from or , 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 10~18 having 0 to 2 Z-form C═C double bonds (for example, it can be C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 ), a straight-chain hydrocarbon group, unsubstituted C 14~18 (for example, it can be C 14 , C 15 , C 16 , C 17 , C 18 ) a branched alkyl group 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 10~19 having 0 to 2 Z-form C═C double bonds (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 ) a straight-chain hydrocarbon group, unsubstituted C 14~18(e.g., 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 and L2 are the same or different and each independently selected from C 3~7 alkylene or a single bond; X and Y are both O; R1 is an unsubstituted C 10~18 straight-chain alkyl, and R2 is selected from or .

[0025] In some embodiments, G1 is ; L1 and L2 are the same or different and each independently selected from C 3~7 alkylene or a single bond; X is O, R1 is an unsubstituted C 14~18 branched alkyl; Y is NH, and 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, the composition comprising a lipid composition, the lipid composition comprising an adjuvant lipid; the adjuvant lipid comprising the compound described in the first aspect, its stereoisomers, pharmaceutically acceptable salts or solvates.

[0044] In some embodiments, the molar percentage of the adjuvant lipid in the lipid composition is 0.1% to 50% (for example, it can be 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) The compound represented by formula (II), its stereoisomers, pharmaceutically acceptable salts or solvates, 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 alkyl group;

[0050] Formula (II);

[0051] (2) A compound represented by formula (III), its stereoisomers, pharmaceutically acceptable salts or solvates, 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-chain or branched alkyl; R2 is C 6~25 straight-chain 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 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-chain 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), its stereoisomers, pharmaceutically acceptable salts or solvates, wherein G1 is C 1~8 alkylene; G2 is C 2~8 alkylene; R1 is C 6~25 straight-chain or branched alkyl; R2 is C 12~25 straight-chain or branched alkyl; G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is selected from -CH3, -CH2CH3 or -CH2CH2OH ;

[0056] Formula (V);

[0057] (5) A compound represented by formula (VI), its stereoisomers, pharmaceutically acceptable salts or solvates, wherein G 1 and G 2 are each independently a C6-C 10 alkylene; G 3 is a C1-C 12 alkylene; R 1 and R 2 are each independently selected from C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is selected from OR 5 、N、-C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 is a C1-C 12 hydrocarbyl; R 5 is selected from H or C1-C6 hydrocarbyl;

[0058] Formula (VI);

[0059] (6)A compound represented by formula (VII), its stereoisomers, pharmaceutically acceptable salts or solvates, 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)2, -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 shown below, its stereoisomers, pharmaceutically acceptable salts or solvates,

[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 its derivatives.

[0064] In some embodiments, 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;

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

[0066] , ,

[0067] , , , ;

[0068] The neutral lipids are selected from any one or a combination of at least two of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-doundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine or lysophosphatidylethanolamine (LPE).

[0069] The structural lipids are selected from any one or a combination of at least two of cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol or corticosteroid;

[0070] The polymer-conjugated lipid is selected from any one or a combination of at least two of distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), or methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

[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, it can be 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%, for example, 5%, 10%, 15%, 20%, 25%, etc.

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

[0076] In some embodiments, the molar percentage of the structural lipid in the lipid composition is 15-65%, for example, 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%, for example, 0.5%, 1.5%, 3%, 5%, 8%, 10%, etc.

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

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

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

[0082] In some embodiments, the molar dosage 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).

[0083] In some embodiments, the composition is a nanoparticle preparation, and the average particle size of the nanoparticle preparation is 10 nm - 300 nm (for example, it can be 10 nm, 50 nm, 100 nm, 150 nm, 200 nm, 300 nm, etc.); the polydispersity index of the nanoparticle preparation ≤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 - 200 nm; the polydispersity index of the nanoparticle preparation ≤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 agent and / or a prophylactic agent.

[0087] In some embodiments, the mass ratio of the lipid composition to the therapeutic agent and / or prophylactic agent is 10:1 - 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 prophylactic agent is 12.5:1 - 25:1.

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

[0090] In some embodiments, the dosages of the therapeutic agent and / or prophylactic agent and the lipid composition are such that the charge ratio of positive charges to negative charges in the composition is 1:(2 - 5) (for example, it can be 1:2, 1:3, 1:4, 1:5, etc.).

[0091] In some embodiments, the therapeutic agent and / or prophylactic agent comprises any one or a combination of at least two of nucleic acid molecules, small molecule compounds, polypeptides or proteins.

[0092] In some embodiments, the composition is used to deliver the therapeutic agent 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 agent 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-related antigen, or the nucleic acid molecule or antigen is capable of eliciting an immune response against the disease-related antigen or cells expressing the disease-related antigen.

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

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

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

[0100] In some embodiments, the therapeutic agent 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, microRNA, 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 component, and the auxiliary component can be a pharmaceutical carrier, diluent or excipient.

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

[0106] In a third aspect, the present invention provides the use of a 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 the use of a 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 a drug for treating a disease or disorder in a mammal in need thereof.

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

[0109] In some embodiments, the disease or disorder is selected from any one or a combination of at least two of infectious diseases, cancers 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 diseases caused by coronavirus, influenza virus or HIV virus, Rift Valley fever, yellow fever, rabies or various herpes.

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

[0112] In some embodiments, the administration route of the drug is intravenous, intramuscular, intradermal, subcutaneous, intranasal or inhalation.

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

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

[0115] Term Definitions

[0116] All publications and patents mentioned in the present invention are hereby incorporated into the present invention in their entirety by reference. If there is a conflict between the uses or terms used in any publications and patents incorporated by reference and the uses or terms used in the present invention, then the uses and terms of the present invention shall prevail.

[0117] The chapter headings used in the present invention are for the purpose of organizing the article only and should not be construed as limiting the subject matter described.

[0118] Unless otherwise specified, all technical and scientific terms used in the present invention have the ordinary meaning in the field to which the claimed subject matter pertains. If there are multiple definitions for a term, the definition in the present invention shall prevail.

[0119] Except as otherwise indicated in the examples or elsewhere, all numbers expressing quantitative properties such as dosages stated in the specification and claims should be understood to be modified in all instances by the term "about". It should also be understood that any numerical range recited in the present invention is intended to include all sub-ranges within that range and any combination of the endpoints of that range or sub-ranges.

[0120] In the present invention, " " in a structural fragment means that the structural fragment is connected to the rest of the molecule through this bond. For example, means that it is connected to the rest of the molecule through " ".

[0121] In the present invention, the term "alkyl" refers to a straight-chain or branched-chain, saturated monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C1, C2, C3, C4, C5, C6, C9, C 10 、C 11 、C 12 、C 16 、C 17 、C 18 、C 19 、C 20 ). Alkyl includes but is 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-chain hydrocarbon group. Alkylene includes but is not limited to: methylene (-CH2-), ethylene group {including -CH2CH2- or -CH(CH3)-, isopropylidene group {including -CH(CH3)CH2- or -C(CH3)2-, 、 . The term "alkenyl" refers to a straight-chain or branched-chain hydrocarbon group having at least one double bond, consisting only of carbon and hydrogen atoms, having, for example, 10 to 20 (such as 16, 17, 18, 19) carbon atoms, and being connected to the rest of the molecule by a single bond. Alkenyl includes but is not limited to vinyl, 、 、 , , etc.

[0123] In addition, when referring to a number or numerical range, the term "about" means that the number or numerical range mentioned is an approximation within the typical tolerances in the art, within experimental variability, or within statistical experimental error, and thus the number or numerical range can vary, for example, between 1% and 15% of the stated number or numerical range. For example, "about" can be understood as being about 2 standard deviations from the mean. When "about" is present before a series of numbers or ranges, it should be understood that "about" can modify each of the numbers in the series or range.

[0124] In addition, in the present invention, when a numerical range is used in a compound general formula and / or structural formula, it means that the number of corresponding groups within the numerical range can be any natural number within that numerical range. For example, "C A-B " means any integer from the starting point to the ending point of the number of carbon atoms, where both A and B are integers; for another example, C 1-5 represents that the number of carbon atoms is 1, 2, 3, 4, or 5; that is, when combined with other groups in the compound general formula and / or structural formula to form various possible compounds, C A-B can be used in combination with any group containing carbon atoms to define the number of carbon atoms. For example, C 1-5 alkyl / alkylene represents various possibilities of alkyl / alkylene having 1 C, 2 C, 3 C, 4 C, and / or 5 C.

[0125] The terms "comprising", "containing", or "including" and the like used in the present invention mean that the elements appearing before the word cover the elements listed after the word and their equivalents, without excluding the unrecited elements. The term "containing" or "including (comprising)" used in the present invention can be open-ended, semi-closed, and closed-ended. In other words, the term also includes "consisting essentially of...", or "consisting of...".

[0126] The term "pharmaceutically acceptable" in the present invention means that a compound or composition is chemically and / or toxicologically compatible with other components of the formulation and / or with a human or mammal for which it is used to prevent or treat a disease or disorder.

[0127] The term "subject" or "patient" in the present invention can include mammalian subjects. For example, the mammalian subject can be selected from any one or at least a combination of two of the group consisting of humans, non-human primates, companion animals, exotic species, livestock animals, and food animals.

[0128] As used herein, the term "treatment" refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from or having the symptoms of a disease, for the purpose of curing, alleviating, reducing, ameliorating or affecting the disease or the symptoms of the disease. In the context of the present invention, unless specifically stated to the contrary, the term "treatment" may also include prevention.

[0129] In the present invention, the term "antigen" includes any molecule containing at least one epitope capable of eliciting an immune response and / or an epitope against which an immune response is directed, preferably a peptide or a protein. Preferably, the antigen in the context of the present invention is a molecule which, optionally after processing, induces an immune response preferably specific for the antigen or for the cells expressing the antigen. In particular, an "antigen" refers to a molecule which, optionally after processing, is presented by MHC molecules and reacts specifically with T lymphocytes (T cells).

[0130] Thus, an antigen or a fragment thereof should be capable of being recognized by a T cell receptor. Preferably, if recognized by a T cell receptor, the antigen or fragment is capable of inducing clonal expansion of T cells carrying a T cell receptor specifically recognizing the antigen or fragment in the presence of a suitable co-stimulatory signal. In the context of an embodiment of the present invention, the antigen or fragment is preferably presented by a cell in the context of an MHC molecule, preferably by an antigen-presenting cell and / or a diseased cell, which results in an immune response against the antigen or the cells expressing the antigen.

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

[0133] The term "pathogen" refers to pathogenic microorganisms and includes viruses, bacteria, fungi, single-celled organisms and parasites. Examples of pathogenic viruses include, but are not limited to, human immunodeficiency virus (HIV), cytomegalovirus (CMV), herpes simplex virus (HSV), hepatitis A virus (HAV), HBV, HCV, papillomavirus and human T-lymphotrophic virus (HTLV). Single-celled organisms include, but are not limited to, Plasmodium, Trypanosoma, Amoeba, etc.

[0134] The term "disease-related antigen" refers to all antigens of pathogenic significance and includes "tumor antigens". According to the present invention, it is desired to induce an immune response against a disease-related antigen or a cell that expresses a disease-related antigen and preferably presents the disease-related antigen in the context of MHC molecules. Preferably, the disease-related antigen is a naturally occurring antigen. In one embodiment, the disease-related antigen is expressed in diseased cells and is preferably presented by the MHC molecules of the cells.

[0135] The antigen encoded by the RNA (i.e., the therapeutic and / or prophylactic agent) contained in the nanoparticles of the present invention (lipid composition) should induce an immune response against the disease-related antigen to be targeted or a cell that expresses the disease-related antigen to be targeted. Thus, the antigen encoded by the RNA contained in the nanoparticles of the present invention may correspond to or may comprise a disease-related antigen or one or more immunogenic fragments thereof, such as one or more MHC-binding peptides of the disease-related antigen. Thus, the antigen encoded by the RNA contained in the nanoparticles of the present invention may be a recombinant antigen.

[0136] Therapeutic and / or prophylactic agent

[0137] The lipid composition of the present invention can be used to deliver a pharmaceutically active ingredient, such as a therapeutic and / or prophylactic agent. Based on this, the present invention further provides a (pharmaceutical) composition comprising the lipid composition provided by the present invention for delivering a pharmaceutically active ingredient. The composition of the present invention may include one or more therapeutic and / or prophylactic agents (as pharmaceutically active ingredients). The pharmaceutically active ingredient may be encapsulated within the lipid composition or bound to the lipid composition.

[0138] The therapeutic and / or prophylactic agent includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, and proteins. Preferably, it is a nucleic acid molecule.

[0139] For example, the therapeutic and / or prophylactic agent is a vaccine or a compound capable of eliciting an immune response. Thus, in some preferred embodiments, the therapeutic and / or prophylactic agent may be a nucleic acid molecule capable of encoding one or more antigens.

[0140] The lipid composition of the present invention can (as a carrier) deliver a therapeutic and / or prophylactic agent to target cells and / or target organs in a subject (such as a mammal). Thus, the present invention also provides methods for treating a disease or disorder in a subject in need thereof, which methods include administering to the subject a composition comprising a therapeutic and / or prophylactic agent and / or contacting the subject's cells with the composition.

[0141] Therapeutic and / or prophylactic agents include bioactive substances and are alternatively referred to as "active agents", "active ingredients", etc. A therapeutic and / or prophylactic agent can be a substance that causes a desired change in a cell or organ after being delivered to the cell or organ or other body tissues or systems. Such agents can be used to treat one or more diseases, disorders, or conditions. In some embodiments, the therapeutic and / or prophylactic agent is a small molecule drug that can be used to treat a specific disease, disorder, or condition.Examples of drugs useful in the compositions include, but are not limited to, antineoplastics (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate and streptozotocin), anti-tumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracycline, 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), β-adrenergic blockers (e.g., propranolol, timolol and labetalol), anti-hypertensives (e.g., clonidine and hydralazine), anti-depressants (e.g., imipramine, amitriptyline and doxepin), anti-spasmodics (e.g., phenytoin), anti-histamines (e.g., diphenhydramine, chlorpheniramine and promethazine), antibiotics / anti-bacterials (e.g., gentamycin, ciprofloxacin and cefoxitin), anti-fungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine and amphotericin B), anti-parasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma 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 elicits an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include but are not limited to taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and analogs or homologs thereof. Radioactive ions include but are not limited to iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, whooping cough, tetanus, plague, hepatitis, and tuberculosis and may include nucleic acid molecules (e.g., mRNA) encoding antigens and / or epitopes derived from infectious diseases. Vaccines may also include compounds and formulations that direct an immune response against cancer cells and may include nucleic acid molecules (e.g., mRNA) encoding tumor cell-derived antigens, epitopes, and / or neoepitopes. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species.Other therapeutic and / or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, CC-1065, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0143] In other embodiments, the therapeutic and / or prophylactic agent is a protein. Therapeutic proteins that can be used in the nanoparticles of the present invention include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferons, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0144] In some embodiments, the therapeutic and / or prophylactic agent can be a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The broadest meaning of the term "polynucleotide" includes any compound and / or substance in the form of an oligonucleotide chain or that can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used 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 hybrids; RNAi-inducing factors; RNAi factors; siRNA; shRNA; miRNA; antisense RNA; ribozymes; catalytic DNA; RNA that induces triple helix formation; aptamers, etc. In some preferred embodiments, the therapeutic and / or prophylactic agent is RNA. The RNA that can be used in the compositions and methods described in the present invention can be selected from, but 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), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In certain embodiments, the RNA is mRNA.

[0145] In certain embodiments, the therapeutic and / or prophylactic agent is mRNA. The mRNA can encode any polypeptide of interest, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0146] In other embodiments, the therapeutic and / or prophylactic agent is siRNA. The siRNA is capable of selectively reducing or downregulating the expression of a gene of interest. For example, the siRNA can be selected such that after administering a composition comprising the siRNA to a subject in need, the gene associated with a particular disease, disorder, or condition is silenced. The siRNA can comprise a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA can be an immunomodulatory siRNA.

[0147] In certain embodiments, the therapeutic and / or prophylactic agent is sgRNA and / or cas9 mRNA. The sgRNA and / or cas9 mRNA can be used as gene editing tools. For example, the sgRNA-cas9 complex can affect the mRNA translation of cellular genes.

[0148] In some embodiments, the therapeutic and / or prophylactic agent is an shRNA or its encoding vector or plasmid. The shRNA can be generated inside the target cell after delivery of the appropriate construct into the nucleus. The constructs and mechanisms associated with shRNA are well known in the relevant art.

[0149] disease or disorder

[0150] The compositions / carriers of the present invention can deliver a therapeutic and / or prophylactic agent to a subject or patient, thereby achieving the treatment and / or prevention of a disease or disorder. The therapeutic and / or prophylactic agent includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides or proteins. Accordingly, the compositions of the present invention can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide or protein drugs. Due to the wide variety of the above-mentioned therapeutic and / or prophylactic agents, the compositions of the present invention can be used to treat or prevent a variety of diseases or disorders.

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

[0152] The reagents, compositions and methods described in the present invention can be used to treat a subject suffering from a disease (e.g., a disease characterized by the presence of diseased cells expressing an antigen and presenting antigenic peptides), or to prevent a subject from suffering from a disease. Examples of diseases that can be treated and / or prevented encompass all diseases expressing 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 condition, including infectious diseases and cancer diseases, particularly those forms of the 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 a similar expression means that the antigen is expressed in the cells of a diseased tissue or organ. The expression in the cells of the diseased tissue or organ can be elevated compared to the state of a healthy tissue or organ. In one embodiment, the expression occurs only in the diseased tissue, while the expression in the healthy tissue is suppressed. According to the present invention, diseases involving an antigen include infectious diseases and cancer diseases, wherein the disease-related antigens are preferably the antigens of the infective agent and tumor antigens, respectively. Preferably, the disease involving an antigen is preferably a disease involving cells expressing an antigen and presenting the antigen in the context of MHC molecules (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, that is, infections caused by Plasmodium, Trypanosoma, Leishmania and Toxoplasma; or ④ fungal infections, which are caused by, for example, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis or Candida albicans.

[0157] The cancer or carcinoma (the medical term is malignant tumor) is a group of diseases in which a group of cells show uncontrolled growth (division beyond normal limits), invasion (invading and destroying adjacent tissues), and sometimes metastasis (spreading to other parts of the body through lymph or blood). These three harmful properties of cancer distinguish it from benign tumors that are self-limiting and do not invade or metastasize. Most cancers form tumors, that is, swellings or lesions formed by abnormal growth of cells (called neoplastic cells or tumor cells), but some (such as leukemia) are not. According to the present invention, the term "cancer" includes leukemia, seminoma, melanoma, teratoma, lymphoma, sarcoma, embryonal carcinoma, 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, gastric cancer, lung cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, multiple myeloma, lymph node cancer, esophageal cancer, colon cancer, rectal cancer, bladder cancer, prostate cancer, endometrial cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, uterine cancer, breast cancer, prostate cancer, ovarian cancer, and their metastases.

[0158] Malignant melanoma is a serious type of skin cancer. It is caused by the uncontrolled growth of pigment cells called melanocytes.

[0159] According to the present invention, "epithelial cancer" is a malignant tumor derived from epithelial cells. This group accounts for the most common cancers, including common forms of breast cancer, prostate cancer, lung cancer, and colon cancer.

[0160] Lymphoma and leukemia are malignant tumors derived from hematopoietic (blood-forming) cells.

[0161] Sarcoma is a cancer of transformed cells originating from one of the tissues developed from the embryonic mesoderm. Therefore, sarcomas include bone tumors, cartilage tumors, fat tumors, muscle tumors, vascular tumors, and hematopoietic tissue tumors.

[0162] Blastic tumor or embryonal carcinoma is a tumor (usually malignant) similar to immature or embryonic tissue. Most of these tumors are common in children.

[0163] Glioma is a type of tumor that begins in the brain or spinal cord. It is called glioma because it originates from glial cells. The most common site of glioma is the brain.

[0164] Other components

[0165] The pharmaceutical composition of the present invention may include one or more components other than those described in the foregoing part. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (such as vitamin A or vitamin E) or sterols.

[0166] The composition may further comprise one or more permeation enhancing molecules, carbohydrates, polymers, surface modifiers or other components. The permeation enhancing molecules can be, for example, the molecules described in U.S. Patent Application Publication No. 2005 / 0222064. The carbohydrates can include simple sugars (such as glucose) and polysaccharides (such as glycogen and its derivatives and analogs).

[0167] The surface modifiers can include, but are not limited to, anionic proteins (such as bovine serum albumin), surfactants (such as cationic surfactants, such as dimethyldioctadecylammonium bromide), sugars or sugar derivatives (such as cyclodextrin), nucleic acids, polymers (such as heparin, polyethylene glycol and poloxamer), mucolytics (such as acetylcysteine, artemisia argyi, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexine and erdosteine) and DNAse (such as rhDNAse). The surface modifiers can be disposed within and / or on the surface of the nanoparticles of the composition (such as by coating, adsorption, covalent linkage or other methods).

[0168] The composition may further comprise one or more functionalized lipids. For example, the lipid can be functionalized with an alkynyl group that may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized in this way with one or more groups that can effectively promote membrane permeation, cell recognition or imaging. The surface of the composition can also be conjugated with one or more useful antibodies. Functional groups and conjugates useful for targeted cell delivery, imaging and membrane permeation are well known in the art.

[0169] In addition to these components, the composition can include any substance that can be used in pharmaceutical compositions. For example, the composition can include one or more pharmaceutically acceptable (e.g., medicinally acceptable) excipients or adjuvants, such as but not limited to one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc.

[0170] The term "medicinally acceptable" means that the material is non-toxic and does not affect the action of the active components of the pharmaceutical composition. Non-medicinally acceptable ingredients can be used to prepare medicinally acceptable ingredients and are included in the present invention.

[0171] Suitable buffers for the compositions of the present invention include the salt forms of acetic acid, citric acid, boric acid, and phosphoric acid.

[0172] When used in the present invention, the term "excipient" is intended to mean all substances that can be present in the pharmaceutical compositions of the present invention and that are not active ingredients, such as carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents, or coloring agents. Excipients such as 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, A.R. Gennaro; Lippincott, Williams&Wilkins, Baltimore, MD, 2006).

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

[0174] Examples of diluents can include but are not limited to calcium carbonate, sodium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, corn starch, powdered sugar, and / or combinations thereof.

[0175] Dosage Forms and Administration

[0176] The compositions of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The compositions of the present invention can be prepared by methods well-known in the pharmaceutical art. For example, a sterile injectable solution can be prepared by incorporating the required amount of the therapeutic or prophylactic agent with the various other ingredients required above into a suitable solvent such as sterile distilled water, and then filtering and sterilizing. Surfactants can also be added to facilitate the formation of a uniform solution or suspension.

[0177] For example, the compositions of the present invention can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation. In one embodiment, the composition is administered intravenously or subcutaneously.

[0178] Therapeutically effective amount

[0179] "Therapeutically effective amount" is the amount of a therapeutic agent that, when administered to a patient, can ameliorate a disease or symptom. "Prophylactically effective amount" is the amount of a prophylactic agent that, when administered to a subject, can prevent a disease or symptom. The amount of the therapeutic agent constituting the "therapeutically effective amount" or the amount of the prophylactic agent constituting the "prophylactically effective amount" varies with the therapeutic and / or prophylactic agent, the disease state and its severity, the age, weight, etc. of the patient and / or subject to be treated and / or prevented. A person of ordinary skill in the art can routinely determine the therapeutically effective amount and prophylactically effective amount based on their knowledge and the present invention.

[0180] The compositions of the present invention are administered in a therapeutically effective amount, which amount can vary not only with the particular reagent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and can ultimately be determined by the attending physician or clinician. For example, a dose of about 0.0001 mg / kg to about 10 mg / kg of the therapeutic or prophylactic agent can be administered to a mammal (such as a human).

[0181] Antigen-presenting cell

[0182] An antigen-presenting cell (APC) is a cell that presents (i.e., displays) an antigen on its surface in the context of the major histocompatibility complex (MHC). This includes cases where only one fragment or more than one fragment of the antigen is presented. T cells can recognize this complex with their T cell receptor (TCR). Antigen-presenting cells process antigens and present them to T cells.

[0183] Professional antigen-presenting cells are very efficient at internalizing antibodies (by phagocytosis or by receptor-mediated endocytosis) and then presenting antigen fragments bound to class II MHC molecules on their membranes. T cells recognize the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell and interact with it. The antigen-presenting cell then generates additional co-stimulatory signals, leading to T cell activation. Expression of co-stimulatory molecules is a characteristic feature of professional antigen-presenting cells.

[0184] The main types of professional antigen-presenting cells are dendritic cells (which have the broadest range of antigen presentation and are probably the most important antigen-presenting cells), macrophages, B cells, and certain activated epithelial cells.

[0185] Dendritic cells are a group of white blood cells that include plasmacytoid dendritic cells (pDCs) and classical dendritic cells (cDCs), which present antigens captured in peripheral tissues to T cells via two antigen presentation pathways, class II and class I MHC. Dendritic cells are powerful inducers of immune responses, and activation of these cells is a key step in inducing anti-tumor immunity.

[0186] Antigen-presenting cells can be loaded with MHC-presented peptides by transducing them with nucleic acids encoding peptides or proteins containing the peptide to be presented (e.g., nucleic acids encoding antigens (e.g., RNA)). Transfecting dendritic cells with mRNA is a promising antigen-loading technique for stimulating strong anti-tumor immunity.

[0187] The term "immunogenicity" refers 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 of the cytolytic T cell (CTL, CD8+ T cells).

[0189] T cells belong to the group of white blood cells called lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types (such as B cells and natural killer cells) by the presence of a special receptor called the T cell receptor (TCR) on their cell surface. The thymus is the main organ responsible for T cell maturation. Several different T cell subsets have been identified, each with different functions.

[0190] Helper T cells assist other white blood cells during the immune process, including functions such as maturing B cells into plasma cells and activating cytotoxic T cells and macrophages. Because they express the CD4 protein on their surface, these cells are also called CD4 +T cells. Helper T cells are activated when class II MHC molecules expressed on the surface of antigen-presenting cells (APCs) present peptide antigens to them. After activation, they divide rapidly and secrete small proteins called cytokines that regulate or assist the active immune response.

[0191] Cytotoxic T cells destroy diseased cells, such as infected cells (e.g., virus-infected cells) and cancer cells, and also participate in transplant rejection. Since they express the CD8 glycoprotein on their surface, these cells are also called CD8 + T cells. These cells recognize their targets by binding to antigens associated with class I MHC, which is present on the surface of almost every cell in the body.

[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 separate T cell receptor alpha and beta (TCRα and TCRβ) genes and are called the α-TCR chain and the β-TCR chain. Gamma-delta T cells represent a small subtype of T cells that have a unique T cell receptor (TCR) on their surface. However, in gamma-delta T cells, the TCR is composed of one gamma chain and one delta chain. This group of T cells is less common than alpha-beta 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 are present in the thymus and expand by cell division to produce a large number of immature thymocytes. Early thymocytes do not express either CD4 or CD8 and are therefore classified as double-negative (CD4-CD8-) cells. As they develop through the process of development, they become double-positive thymocytes (CD4+CD8+) and eventually mature into single-positive (CD4 + CD8 - or CD4 - CD8 + ) thymocytes, and then are released from the thymus into the 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 partner cell is usually a professional antigen-presenting cell (APC), usually a dendritic cell in the case of a primary response, but B cells and macrophages can also be important APCs. The peptides presented by class I MHC molecules to CD8 + T cells are 8 to 10 amino acids in length; the peptides presented by class II MHC molecules to CD4 +The peptides of T cells are longer because the ends of the binding clefts of class II MHC molecules are open.

[0195] Based on the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples 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 mRNA involved in delivery has 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 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 It is a graph showing the results of cell transfection experiments of LNP formulations of eGFP-mRNA prepared by mixing YK-009, DSPC, cholesterol, DMG-PEG2000 and adjuvant lipid at a molar ratio of 20:10:38.5:1.5:30, where: a is YK-1704, b is YK-1705, c is YK-1707, d is YK-1709, e is YK-009 (without adjuvant lipid), and f is 11-A-M. DETAILED DESCRIPTION OF THE INVENTION

[0203] The technical solutions of the present invention will be further described below with reference to the drawings and through specific embodiments. However, the following examples are only simple 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 adopted in the examples can be further adjusted according to different requirements of specific use. The implementation conditions not specified are conventional conditions in the industry. In the specific examples of the present invention, the raw materials used can all be obtained commercially. Unless otherwise specified, the percentages in the context are weight percentages, and all temperatures are given in degrees Celsius. The technical features involved in various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0205] The following abbreviated letters represent the following reagents respectively:

[0206] YK-009: 2-Octyldecyl ((decoxy-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate; ( , prepared according 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 synthesis route is as follows:

[0220] ;

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

[0222] 6-Bromohexanoic acid (10.25 g, 52.53 mmol), 3-hexylnonyl ester (10.00 g, 43.78 mmol), EDCI (16.78 g, 87.56 mmol), and DMAP (2.67 g, 21.89 mmol) were dissolved in dichloromethane (10.0 mL). Under a nitrogen atmosphere, the mixture was stirred at room temperature for 24 hours. 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 reduced pressure 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), adamantylamine (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 method for synthesizing YK-1701-PM1. C 18 H 30 BrNO, MS(ES): m / z(M+H + ) 356.2.

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

[0226] Dissolve YK-1701-PM2 (400.0 mg, 1.12 mmol), ethanolamine (342.8 mg, 5.61 mmol), and K2CO3 (465.4 mg, 3.37 mmol) in acetonitrile (4.0 mL). Under a nitrogen atmosphere, stir the reaction at 70 °C for 5 hours. After the reaction is complete, concentrate the mixture, add ethyl acetate (5.0 mL) and water (5.0 mL), stir, separate the layers, extract with ethyl acetate (5.0 mL × 2), combine the organic phases, wash with saturated brine (5.0 mL × 2), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to remove the solvent. Purify the residue by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-1701-PM3 (326.4 mg, 0.97 mmol, 86.41%). C 20 H 36 N2O2, MS(ES): m / z(M+H + ) 337.3.

[0227] Step 4: Synthesis of YK-1701

[0228] Dissolve 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), and KI (48.3 mg, 0.29 mmol) in acetonitrile (4.0 mL). Stir the reaction at 70 °C for 2 days. After the reaction is complete, concentrate the mixture, add ethyl acetate (5.0 mL) and water (5.0 mL), stir, separate the layers, extract with ethyl acetate (5.0 mL × 2), combine the organic phases, wash with saturated brine (5.0 mL × 2), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to remove the solvent. Purify the residue by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-1701 (97.1 mg, 0.15 mmol, 15.14%). C 41 H 76 N2O4, MS(ES): m / z(M+H + ) 661.6.

[0229] 11H 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 synthesis route is as follows:

[0232] ;

[0233] Step 1: Synthesis of YK-1702

[0234] Using 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) as raw materials, YK-1702 (93.4 mg, 0.15 mmol, 34.02%) was obtained according to the method for synthesizing YK-1701. C 38 H 65 N3O3, MS(ES): m / z(M+H + )612.5.

[0235] 1 1H 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 synthesis route is as follows:

[0238] ;

[0239] Step 1: Synthesis of YK-1703

[0240] Using YK-1701-PM2 (400.0 mg, 1.12 mmol), adamantylamine (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) as raw materials, YK-1703 (77.6 mg, 0.11 mmol, 49.17%) was obtained according to the method for synthesizing YK-1701. C 46 H 75 N3O2, MS(ES): m / z(M+H + ) 702.6.

[0241] 1 1H 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 synthesis 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 method for synthesizing YK-1701-PM1. C 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), heptadecane nonol (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. C 23 H 45 BrO2,MS(ES): m / z(M+H + )433.3。

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

[0250] Using YK-1704-PM2 (400.0 mg, 0.92 mmol), adamantylamine (418.7 mg, 2.77 mmol), K2CO3 (382.6 mg, 2.77 mmol) and acetonitrile (4.0 mL) as raw materials, according to the method for synthesizing YK-1701-PM3, YK-1704-PM3 (312.8 mg, 0.62 mmol, 67.28%) was obtained. C 33 H 61 NO2, MS(ES): m / z(M+H + )504.5。

[0251] Step 4: Synthesis of YK-1704

[0252] Using 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) as raw materials, according to the method for synthesizing YK-1701, YK-1704 (144.0 mg, 0.20 mmol, 31.77%) was obtained. C 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 synthesis route is as follows:

[0256] ;

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

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

[0259] Step 2: Synthesis of YK-1705

[0260] Using 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) as raw materials, following the method for synthesizing YK-1701, YK-1705 (55.8 mg, 0.08 mmol, 11.63%) was obtained. C 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 synthesis route is as follows:

[0264] ;

[0265] Step 1: Synthesis of YK-1706

[0266] Using 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) as raw materials, following the method for synthesizing YK-1701-PM1, YK-1706 (90 mg, 0.08 mmol, 21.48%) was obtained. C 49 H 87 NO3, MS(ES):m / z(M+H +)738.7。

[0267] 1 1H NMR (CDCl3, 400 MHz, 298 K) δ 5.43 - 5.29 (m, 4H), 4.13 (d, J = 19.9 Hz, 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] Using YK - 1704 - PM1 (400.0 mg, 0.92 mmol), adamantylamine (418.7 mg, 2.77 mmol), K2CO3 (382.6 mg, 2.77 mmol) and acetonitrile (4.0 mL) as raw materials, YK - 1707 - PM2 (330.0 mg, 0.64 mmol, 69.06%) was obtained according to the method for synthesizing YK - 1701 - PM3. C 34 H 63 NO2, MS(ES): m / z(M + H + )518.5。

[0275] Step 3: Synthesis of YK - 1707

[0276] Using 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) as raw materials, according to the method for synthesizing YK-1701, YK-1707 (120.0 mg, 0.16 mmol, 25.31%) was obtained. C 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] Using 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) as raw materials, according to the method for synthesizing YK-1701-PM1, YK-1708 (100 mg, 0.13 mmol, 22.89%) was obtained. C 52 H 93 NO3, MS(ES):m / z(M+H + )780.7。

[0283] 1 1H NMR (CDCl3, 400 MHz, 298 K) δ 5.43 - 5.29 (m, 4H), 3.90 (d, J = 19.6 Hz, 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] Using 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) as raw materials, according to the method for synthesizing YK-1701, YK-1709 (283.4 mg, 0.38 mmol, 44.87%) was obtained. C 49 H 86 N2O3, MS(ES): m / z(M+H + ) 751.7.

[0289] 1 1H 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] Using YK-1701-PM2 (449 mg, 1.26 mmol), adamantylamine (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) as raw materials, according to the method for synthesizing YK-1701, YK-1710-PM1 (320 mg, 0.75 mmol, 59.52%) was obtained. C 28 H 46 N2O, MS(ES): m / z(M+H + )427.4.

[0295] Step 2: Synthesis of YK-1710

[0296] Using 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) as raw materials, according to the method for synthesizing YK-1701-PM1, YK-1710 (112 mg, 0.16 mmol, 21.67%) was obtained. C 46 H 76 N2O3, MS(ES):m / z(M+H + )689.6.

[0297] 1 1H 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-A-M

[0299] ;

[0300] The synthesis of compound 11-A-M refers to the synthesis route of 11-A-M in WO2021021634A1, and 92.0 mg of 11-A-M was obtained.

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

[0302] ;

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

[0304] 13. Synthesis of compound 247-9

[0305] ;

[0306] The synthesis of compound 247-9 refers to the synthesis route of compound 247-9 in WO2023133089A1, and 67.5 mg of compound 247-9 was obtained.

[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 WO2024052923A1, and 262.0 mg of compound BN-INL-A209 was obtained.

[0310] Example 2: Preparation of mRNA lipid composition

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

[0312] 1) The luciferase (Luciferase protein CDS), green fluorescent protein (GFP), and ovalbumin (OVA) circular plasmids were digested with EcoRV and ligated to the pVAX1 vector (purchased from Thermo Fisher Scientific);

[0313] 2) The plasmid constructed on the pVAX1 vector in step 1) was mixed with 50 μL of Escherichia coli competent cells Stbl2 (purchased from Thermo Fisher Scientific), incubated on ice for 30 minutes, heat-shocked at 42 °C for 90 seconds, immediately placed back on ice, and incubated on ice for 2 minutes;

[0314] 3) Add 400 μL of LB medium (purchased from Thermo Fisher Scientific), and incubate it in a shaker at 30 °C with slow shaking for 45 - 60 minutes;

[0315] 4) Take 50 - 100 μL of the bacterial solution and spread it on an LB solid medium containing kanamycin antibiotic (100 μg / mL, purchased from Yeasen Biotechnology Co., Ltd.), and incubate it upside down at 37 °C overnight;

[0316] 5) Sequence the obtained monoclonal colony plate to verify its correctness, pick the monoclonal colony with correct sequencing, and incubate it in a shaker at 30 °C with slow shaking overnight;

[0317] 6) Extract the plasmid using an endotoxin-free large plasmid extraction kit (purchased from Yeasen Biotechnology Co., Ltd.);

[0318] 7) Use a restriction endonuclease to digest the extracted plasmid into a linearized plasmid for use as a transcription template. The specific digestion process steps refer to steps ① - ③.

[0319] Step ①: Take 1 mg of luciferase circular plasmid, digest it at 37 °C for 4 hours (with BspQ Ⅰ enzyme, purchased from Yeasen Biotechnology Co., Ltd.) into a linearized DNA transcription template (the digestion system is shown in Table 1);

[0320] Table 1

[0321]

[0322] Step ②: After the reaction is completed, add absolute ethanol and sodium acetate in sequence. Add absolute ethanol and 3M sodium acetate according to a volume ratio of V 酶切反应产物 :V 无水乙醇 :V 3M醋酸钠 = 1:3:1, place it at -20 °C for precipitation for 1 hour, and then centrifuge at 12000 rmp to retain the precipitate;

[0323] Step ③: Wash the precipitate in step ② twice with 70% ethanol, dry the centrifuged material at 55 °C for 10 minutes, and then add 1.7 mL of injection water to dissolve it;

[0324] The concentration of the linearized plasmid in the dissolution solution is 500 ng / µL, the linearization ratio is more than 90%, and the purification and recovery efficiency is 85%.

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

[0326] 1) Co-transcriptional capping reaction:

[0327] Using the prepared Fluc DNA, eGFP DNA, and OVA DNA as templates, and NTP solution (NTPs) and Cap1 cap analog (product number: 10678ES80, purchased from Yeasen Biotech Co., Ltd.) as starting materials, mRNA was transcribed and synthesized by T7 RNA polymerase. The specific reaction system is shown in Table 2. The prepared reaction system was placed in an incubator at 37 °C and shaken for reaction for 3 h. The above Cap1 cap analog is Cap1-GAG, which has the structure of m7G (5') ppp (5') (2'-OMeA) pG, and its molecular formula is C 32 H 43 N 15 O 24 P4.

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

[0329] Table 2

[0330]

[0331] Note: The above reagents are all purchased from Yeasen Biotech Co., Ltd.

[0332] 2) Digest the template DNA:

[0333] Add DNase I (purchased from Yeasen Biotech Co., Ltd.) to the co-transcriptional capping reaction system completed in the above step 1) to make the final concentration 1 U / μg of linearized plasmid. After mixing, centrifuge and place at 37 °C for digestion for 1 hour to obtain the co-transcriptional capping product.

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

[0335] Purify the co-transcriptional capping product obtained in the above step 2) by the lithium chloride precipitation method as follows:

[0336] Step ① Add lithium chloride: Add lithium chloride solution (purchased from Thermo Fisher Scientific) to the product of the above step 2) to make the final concentration 2.8 M, and precipitate at low temperature for 2 hours;

[0337] Step ② Precipitation: Centrifuge at 12000 rmp for 15 minutes and retain the precipitate;

[0338] Step ③ Washing: Wash twice with 75% ethanol and dissolve with injection water to obtain the mRNA solution. The purified mRNA solution is stored at -80 °C.

[0339] Example 3: Effect of different addition amounts of adjuvant lipid on the LNP-mRNA composition

[0340] This example investigated the effect of adjuvant lipid on the LNP composition.

[0341] Experimental procedure:

[0342] Weigh YK-009, YK-1705, DSPC, cholesterol, and DMG-PEG2000 according to the ratio in Table 3 and dissolve them in ethanol to prepare an ethanol lipid solution. Dilute eGFP-mRNA in citrate buffer (pH = 4 - 5) to obtain an mRNA aqueous solution. Use a microfluidic device to mix the ethanol lipid solution with the Fluc mRNA aqueous solution prepared from different capping structures at a flow rate of 10 mL / min and a volume ratio of 1:3 to prepare LNP with a weight ratio of total lipid to mRNA of approximately 15:1. After diluting the obtained liposomes to 10 times the volume with PBS, ultrafiltrate to remove ethanol using a 300 KDa ultrafiltration tube. Then, make up the volume to a certain volume with PBS. Finally, filter the lipid nanoparticles through a 0.2 μm sterile filter to obtain the LNP pharmaceutical composition with or without adjuvant lipid. Use dynamic light scattering and a Malvern laser particle size analyzer to measure the particle size and polydispersity index (PDI). Take 10 μL of the liposome solution, dilute it to 1 mL with RNase-free deionized water, add it to the sample cell, and repeat the measurement 3 times for each sample. The measurement conditions are: 90° scattering angle, 25°C. According to the manufacturer's instructions, use the Quant itRibogreen RNA Quantification Kit (ThermoFisher Scientific, UK) to determine the encapsulation efficiency of the lipid nanoparticles. The particle size, PDI, and encapsulation rate results of the compositions with different formulations are shown in Table 3.

[0343] Table 3

[0344]

[0345] The results show that when using YK-1705 to partially replace the cationic lipid YK-009 in the LNP composition at 5%, 10%, 20%, 30%, and 40%, the particle size, PDI, and encapsulation rate of the obtained LNP pharmaceutical composition are within the qualified range (particle size < 200 nm, PDI < 0.3, encapsulation rate > 85%). When the content of adjuvant lipid is 30 mol%, the particle size, PDI, and encapsulation rate reach the best state. Next, various adjuvant lipids are studied at a ratio of 30 mol% of adjuvant lipid.

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

[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 procedure:

[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 proportion of the cationic lipid YK-009 in the LNP composition at a ratio of 30 mol%, that is, the Fluc-mRNA-LNP pharmaceutical composition was prepared with a molar ratio of YK-009, DSPC, cholesterol, DMG-PEG2000 to the adjuvant lipid of 20:10:38.5:1.5:30. Evaluation method: First, the particle size, polydispersity index (PDI), and encapsulation efficiency were measured according to the method of Example 3; then the pharmaceutical composition was added to the cell culture medium in a 96-well plate. After continuing to culture for 24 hours, the corresponding reagents were added according to the instructions of the Gaussia Luciferase Assay Kit, and the fluorescence expression intensity of each well was detected by the IVIS fluorescence detection system; finally, 10 μL of CCK-8 solution was added to each well of the above-mentioned plate after 24 hours of culture. After incubating the culture plate 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 detection results of the LNP compositions containing different adjuvants are shown in Table 5.

[0356] Table 5

[0357]

[0358] The results show that:

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

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

[0361] (3)Cell activity of mRNA-LNP containing adjuvant lipids: There were significant differences in the cell viability (cytotoxicity) of the LNP compositions prepared above. The cell viabilities of the mRNA-LNP compositions prepared from 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 was also significantly higher than the YK-009-LNP group without adjuvant lipids and the mRNA-LNP groups with 11-A-M, A-17-2Z, 247-9, and BN-INL-A209.

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

[0363] Experimental procedure:

[0364] The LNP preparations containing 10 μg Fluc-mRNA with different adjuvant lipids prepared according to Example 4 were intramuscularly injected into female BALB / C mice aged 4 - 6 weeks and weighing 17 - 19 g. At a specific time point (6 hours) after administration, the mice were intraperitoneally injected with a fluorescence imaging substrate. The mice were allowed to move freely for 5 minutes, and then the average radiant intensity (corresponding to the fluorescence expression intensity) of the protein expressed by the mRNA carried by the LNP in the mice was detected by an IVIS Spectrum small animal in vivo imager.

[0365] After sampling, the mice were euthanized with carbon dioxide, and the mice were dissected to precisely isolate the internal organs of the mice: liver, spleen, and lung. The total radiant intensity (corresponding to the fluorescence expression intensity) of the protein expressed by the mRNA carried by the LNP in each organ of the mice was detected by an IVIS Spectrum small animal in vivo imager. The results of the in vivo imaging of the mice are shown in Table 6.

[0366] Table 6

[0367]

[0368] Experimental results:

[0369] For the LNP formulations prepared from YK-1705, YK-1707, and YK-1709, the expression levels of the delivered mRNA in the injection site, abdominal cavity, liver, and spleen of mice were significantly increased compared to YK-009-LNP without adjuvant lipid and 11-A-M-LNP, 247-9-LNP, and BN-INL-A209-LNP with representative adjuvant lipids in the prior art. For example, in the mRNA-LNP group with YK-1705 added, the expression level in the injection site of mice was 1.7 times that of YK-009-LNP without adjuvant lipid, and the expression level in the spleen of mice was 2.3 times that of 11-A-M-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 muscle (injection site). By increasing the expression levels of the delivered mRNA in the spleen and muscle, rapid induction of immune responses and antibody production can be achieved in vivo with the mRNA vaccine. Without changing the vaccine components, the preventive and therapeutic effects can be significantly improved, which has important clinical significance.

[0371] In addition, for the LNP formulations containing Fluc-mRNA with adjuvant lipids added, the expression differences in different organs of mice were very large. The LNP formulations prepared from YK-1705, YK-1707, YK-1709, 11-A-M, 247-9, BN-INL-A209, and without adjuvant lipid were all expressed in the liver and spleen, but not expressed in the lungs.

[0372] Compared with the adjuvant lipid YK-1704, for the mRNA-LNP formulations prepared from YK-1705 and YK-1709, the expression intensities of mRNA in the liver and spleen of mice were both significantly increased. For example, for the LNP formulation with YK-1705 added, the expression level of mRNA in the injection site was 1.9 times that of the LNP formulation with YK-1704 added, and the expression level in the spleen reached 3.2 times.

[0373] Example 6: Excitation effect of the mRNA-LNP composition containing RLRs adjuvant lipid on cytokines IFN-γ and TNF-α in serum

[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 the immune response and has various functions such as antiviral, antitumor, immunomodulatory, and promoting inflammatory responses, and can treat a variety of diseases. Tumor necrosis factor-α (TNF-α) is a key cytokine that links 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 induction of cytokines IFN-γ and TNF-α by the adjuvant lipid-containing mRNA-LNP composition can reflect the improvement of the innate immunity of the adjuvant lipid on the mRNA-LNP composition.

[0375] Experimental procedure:

[0376] Female BALB / C mice aged 4 - 6 weeks and weighing 17 - 19 g were euthanized by exsanguination by ophthalmic enucleation 6 hours after intramuscular injection of the OVA-mRNA-LNP (10 µg) composition prepared according to the method of Example 4 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 according to the manufacturer's instructions.

[0378] The comparison of the induction effects of the adjuvant lipid-containing mRNA-LNP composition on cytokines IFN-γ and TNF-α is shown in Table 7.

[0379] Table 7

[0380]

[0381] Experimental results:

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

[0383] Compared with the mRNA-LNP composition without adjuvant lipid (YK-009-LNP), the IFN-γ and TNF-α cytokines in the serum of the mRNA-LNP composition containing RLRs adjuvant lipid increase significantly 6 hours after injection. Among them, the serum content of cytokine IFN-γ stimulated by the mRNA-LNP compositions supplemented with YK-1705-LNP, YK-1707-LNP, and YK-1709-LNP is 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 compositions supplemented with YK-1705-LNP, YK-1707-LNP, and YK-1709-LNP is 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 compositions with 11-A-M, 247-9, and BN-INL-A209 as adjuvant lipids in the prior art, the serum content of cytokine IFN-γ stimulated by the mRNA-LNP composition supplemented with YK-1705-LNP is 1.9 times, 2.0 times, and 2.3 times that of the mRNA-LNP compositions supplemented with 11-A-M, 247-9, and BN-INL-A209 adjuvant lipids respectively; the serum content of cytokine TNF-α stimulated by the mRNA-LNP composition supplemented with YK-1705-LNP is 1.6 times, 1.7 times, and 1.9 times that of the mRNA-LNP compositions supplemented with 11-A-M, 247-9, and BN-INL-A209 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 demonstrated that the adjuvant lipids of the present invention can initiate an immune stimulation program and significantly improve the innate immunity of the mRNA-LNP composition.

[0386] Example 7: Antigen-specific cytotoxic T cells produced by the spleen stimulated by the mRNA composition containing RLRs adjuvant lipids

[0387] The strength of the T cell effect generated by the tumor mRNA-liposome complex can be judged by detecting antigen-specific CD8 + cytotoxic T cells (CD8 + T cells) in the spleen, which is crucial for the anti-tumor effect of lipid complex delivering tumor mRNA.

[0388] Experimental procedure:

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

[0390] 2. Preparation of single cells

[0391] 1) Grind the isolated spleen tissue to make the spleen tissue single-celled and pass through a cell sieve;

[0392] 2) Add 10 times the volume (about 4 mL) of red blood cell lysate to lyse and remove the red blood cells in the tissue;

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

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

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

[0396] Detect antigen-specific CD8 +The T cell experimental reagents and their sources are shown in Table 8.

[0397] Table 8

[0398]

[0399] 2), Add 2 mL of 1×RBC Lysis Buffer, incubate in the dark for 10 minutes, centrifuge at 500 g for 5 min, 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 of PBS, transfer to a clean labeled EP tube, (filtered through a 200-mesh nylon mesh), and detect on the Cytoflex S flow cytometer. Adjust the compensation with single-color compensation microspheres before loading. The detection sequence 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 lipid and the RNA composition without agonist cationic lipid is shown in Table 9.

[0403] Table 9

[0404]

[0405] As shown in Table 9, compared with the mRNA-LNP composition without adjuvant lipid, after three inoculations in mice, the content of CD8 + T cells in mouse spleen cells was significantly increased by the mRNA-LNP composition containing RLRs adjuvant lipid. Among them, the mRNA-LNP compositions supplemented with YK-1705-LNP, YK-1707-LNP, and YK-1709-LNP stimulated the production of CD8 + T cell ratios were 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 compositions with 11-A-M, 247-9, and BN-INL-A209 as adjuvant lipids in the prior art, the mRNA-LNP composition supplemented with YK-1705-LNP stimulated the production of CD8 + T cell ratios were 1.7 times, 2.0 times, and 2.3 times that of the mRNA-LNP compositions supplemented with 11-A-M, 247-9, and BN-INL-A209 adjuvant lipids, respectively.

[0406] The experimental results show that by detecting the generation of antigen-specific cytotoxic T cells in the spleens of mice stimulated by the mRNA composition containing RLRs adjuvant lipids, it can be demonstrated that the mRNA vaccine containing RLRs adjuvant lipids can produce a very strong T cell effect in the spleens of mice.

[0407] Example 8: Stimulation of Splenic Cytokine IFN-γ by mRNA-LNP Composition Containing RLRs Adjuvant Lipids

[0408] Experimental procedure:

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

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

[0411] The comparison of the stimulation of cytokine IFN-γ by the mRNA-LNP composition containing adjuvant lipids 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 significantly increased the level of IFN-γ cytokine in mouse splenocytes after three inoculations in mice. Among them, the number of fluorescent spots of cytokine IFN-γ stimulated by the mRNA-LNP compositions added with 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 with 11-A-M, 247-9, and BN-INL-A209 as adjuvant lipids in the prior art, the number of fluorescent spots of cytokine IFN-γ stimulated by the mRNA-LNP composition added with YK-1705-LNP was 1.3 times, 1.4 times, and 1.8 times that of the mRNA-LNP compositions added with 11-A-M, 247-9, and BN-INL-A209 adjuvant lipids, respectively.

[0416] The experimental results show that by detecting the mRNA-LNP composition containing adjuvant lipid, it can well activate immune cells (such as T cells) in the spleen to secrete interferon IFN-γ, which can prove that the adjuvant lipid of the present invention can initiate an immune stimulation program and significantly improve the innate immunity of the mRNA-LNP composition.

[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 by the present invention is different from that of the adjuvant lipids disclosed in the prior art and is a brand-new compound.

[0419] 2. By adding the adjuvant lipid of the present invention (5% - 40 mol%) to prepare the mRNA-LNP composition, the particle size is good (<200 nm), and the particle distribution is uniform PDI (<0.3). Compared with the mRNA-LNP composition without adjuvant lipid, the mRNA-LNP composition prepared by adding the adjuvant lipid of the present invention has a significantly increased mRNA translation efficiency, a significantly reduced cytotoxicity, and a significantly increased protein expression level in the mouse injection site, abdominal cavity, liver, and spleen.

[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 an immune stimulation program, and significantly improve the innate immunity of the mRNA-LNP composition.

[0421] 4. The mRNA vaccine containing RLRs adjuvant lipids can generate very strong T cell effects in mice.

[0422] The above has described the present invention in detail. The purpose is to enable those skilled in this field of technology to understand the content of the present invention and implement it. However, this should not limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the compound is shown in formula (I), Formula (I); 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 ; or, 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 .

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: When R1 is unsubstituted C 10~18 In the case of a straight chain alkyl group, R1 is selected from or .

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: When R1 is unsubstituted C 14~18 When the alkyl group is branched, R1 is selected from , or .

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound has one of the following structures: 、 、 。 5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound is compound YK-1705 having the following structure: 。 6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound is compound YK-1707 having the following structure: 。 7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound is compound YK-1709 having the following structure: 。 8. 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 according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof.

9. The composition according to claim 8, characterized in that The molar percentage of the adjuvant lipid in the lipid composition is 0.1% to 50%.

10. The composition according to claim 8, characterized in that The lipid composition also includes cationic lipids and neutral lipids.

11. The composition according to claim 10, characterized in that The molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:

1.

12. The composition according to claim 11, characterized in that The molar ratio of the cationic lipid to the neutral lipid is 4:1 to 6:

1.

13. The composition according to claim 10, characterized in that The cationic lipid is selected from any one or a combination of at least two of the following compounds (1) to (7): (1) A compound represented by formula (II), or a pharmaceutically acceptable salt thereof, wherein G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; G3 is C 1～3 Alkylene; L1 is C 6～15 Straight chain alkyl; L2 is C 12～25 Branched chain alkyl; Formula (II); (2) A compound represented by formula (III), or a pharmaceutically acceptable salt thereof, wherein G1 is C 2~8 Alkylene; G2 is C 2~8 Alkylene; L1 is 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), or a pharmaceutically acceptable salt thereof, wherein: G1 is C 1~6 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~20 Straight or branched alkyl; R2 is C 12~25 Branched alkyl; G3 is 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), or a pharmaceutically acceptable salt thereof, wherein G1 is C 1~8 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~25 Straight or branched alkyl; R2 is C 12~25 Straight or branched alkyl; G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is selected from -CH3, -CH2CH3 or -CH2CH2OH; Formula (V); (5) A compound represented by formula (VI) or a pharmaceutically acceptable salt 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) or a pharmaceutically acceptable salt 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 shown below, or a pharmaceutically acceptable salt thereof, DLIN-MC3-DMA。 14. The composition according to claim 10, 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.

15. The composition according to claim 8, 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-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, 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.

16. The composition according to claim 15, characterized in that The lipid composition includes adjuvant lipids, cationic lipids, neutral lipids, structural lipids and polymer conjugated lipids.

17. The composition according to claim 16, 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).

18. The composition according to claim 17, 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).

19. The composition according to claim 8, 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%.

20. The composition according to claim 19, characterized in that The average particle size of the nanoparticle preparation is 50nm-200nm; the polydispersity coefficient of the nanoparticle preparation is ≤20%.

21. The composition according to claim 13, characterized in that The cationic lipid may also include one or more other ionizable lipid compounds.

22. The composition according to claim 8, characterized in that The composition also includes a therapeutic and / or prophylactic agent.

23. The composition according to claim 22, characterized in that The mass ratio of the lipid composition to the therapeutic agent and / or preventive agent is 10:1 to 30:

1.

24. The composition according to claim 23, 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.

25. The composition according to claim 24, characterized in that The mass ratio of the lipid composition to the therapeutic agent and / or preventive agent is 14:1 to 20:

1.

26. The composition according to claim 22, 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.

27. The composition according to claim 22, characterized in that The therapeutic and / or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

28. The composition according to claim 26, characterized in that The therapeutic and / or prophylactic agent is a nucleic acid.

29. The composition according to claim 28, characterized in that The therapeutic and / or prophylactic agent is RNA.

30. The composition according to claim 28, characterized in that The therapeutic and / or prophylactic agent is DNA.

31. The composition according to claim 29, 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.

32. The composition according to claim 31, characterized in that The RNA is messenger RNA.

33. The composition according to claim 8, characterized in that The composition may further comprise one or more pharmaceutically acceptable excipients.

34. Use of the compound according to any one of claims 1 to 7, a pharmaceutically acceptable salt thereof, or the composition according to any one of claims 8 to 33 in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

35. Use of a compound as described in any one of claims 1 to 7, a pharmaceutically acceptable salt thereof, or a composition as described in any one of claims 8 to 33 in the preparation of a medicament for treating a disease or condition based on an RLRs receptor agonist in a mammal in need thereof.

36. The use according to claim 34 or 35, characterized in that The subject of administration of the drug is human.

37. The use according to claim 34 or 35, characterized in that The drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

38. The use according to claim 37, characterized in that The drug is administered subcutaneously.

39. The use according to claim 34 or 35, characterized in that The dosage of the drug is 0.001-10 mg / kg.

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