Adjuvant lipids containing a benzothiophene structure for activating the sting pathway and compositions thereof

Abstract

The application relates to the technical field of vaccine adjuvants, in particular to an adjuvant lipid containing a benzothiophene structure for activating a STING pathway and a composition thereof. The STING-activated adjuvant lipid provided by the application can be used as a vaccine adjuvant and can significantly enhance the therapeutic effect of an mRNA vaccine or a drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of vaccine adjuvants, in particular to an adjuvant lipid containing a benzothiophene structure for stimulating the STING pathway and a composition thereof. BACKGROUND

[0002] In recent years, messenger ribonucleic acid (mRNA)-based tumor immunotherapy has attracted widespread attention. Compared with DNA vaccines, mRNA vaccines can cause transient expression of tumor antigens and avoid the possibility of mutations at the genomic level. In addition, mRNA can improve therapeutic efficacy by inducing stronger humoral and cellular responses. Compared with traditional protein-based vaccines, mRNA vaccines have many advantages, such as high biocompatibility, non-toxicity, and simple and scalable production processes.

[0003] In the prior art, in order to reduce the high innate immunogenicity of in vitro transcribed mRNA molecules and improve their biological tolerance and translation efficiency, nucleotide modifications, such as 1-methyl pseudouracil (m1ψ), are usually introduced during transcription. However, this modification, while reducing immunogenicity, can also weaken the recognition of mRNA by pattern recognition receptors, thereby reducing the strength of the innate immune response triggered thereby. This response is crucial for effectively activating dendritic cells (DCs, which are the main cells of antigen-presenting cells) and initiating subsequent immune responses.

[0004] The stimulator of interferon genes (STING) is a cytoplasmic pattern recognition receptor that is essential for the spontaneous induction of anti-tumor T cell immunity. STING activators can assist mRNA nanovaccines in inducing type I interferon (IFN-I) secretion, thereby promoting the cross-presentation of tumor antigens, the proliferation and activation of T lymphocytes, and the direct killing of tumors. STING-activated vaccine adjuvant lipids are a class of vaccine adjuvants that combine STING activators with lipid carriers (such as liposomes, lipid nanoparticles, etc.) to form a vaccine adjuvant, which can simultaneously achieve "STING pathway activation" and "efficient delivery" in vaccination, thereby significantly enhancing the immune response of the body to vaccine antigens. However, the targeting of existing STING-activated vaccine adjuvant lipids still needs to be improved, especially the targeting of target organs and antigen-presenting cells in target organs, which is still insufficient, making it difficult to meet the needs of effective drug delivery and therapeutic efficiency. Therefore, the development of a new generation of STING-activated vaccine adjuvant lipids is of great significance for improving mRNA tumor therapy. SUMMARY

[0005] The application provides a STING activating adjuvant lipid containing a benzothiophene structure, which has the advantages of simple preparation method, strong targeting, etc.

[0006] The application provides a compound of formula (I) or a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof,

[0007] ;

[0008] L1 is C 2-3 a straight-chain alkylene group;

[0009] L2 is C 1-12 a straight-chain alkylene group or is absent;

[0010] L3 is C 1-6 a straight-chain alkylene group;

[0011] M1 is -C(O)O-, -OC(O)-, -NHC(O)- or -C(O)NH-;

[0012] M2 and M3 are -OC(O)- or -C(O)O- respectively;

[0013] X is N or CH;

[0014] R1 is C 5-25 a straight-chain alkenyl group or C 5-25 a straight-chain alkyl group;

[0015] R2 is C 5-30 a straight-chain or branched alkenyl group or C 5-30 a straight-chain or branched alkyl group.

[0016] In some embodiments, L1 is -(CH2)2-, -(CH2)3- or , and the a end is connected to X.

[0017] In some embodiments, L2 is C 2-6 a straight-chain alkylene group or is absent.

[0018] In some embodiments, L2 is -(CH2)3-, -(CH2)4-, -(CH2)5- or is absent.

[0019] In some embodiments, L3 is C 2-6 a straight-chain alkylene group.

[0020] In some embodiments, L3 is -(CH2)-, -(CH2)4-, -(CH2)5- or -(CH2)6-.

[0021] In some embodiments, M1 is -C(O)O- b or -C(O)NH- b , preferably -C(O)O- b , b end is connected with L1.

[0022] In some embodiments, M2 is -C(O)O- c , c end is connected with R1.

[0023] In some embodiments, M3 is -C(O)O- d , d end is connected with R2.

[0024] In some embodiments, R1 is C 9-17 straight chain alkenyl or C 9-17 straight chain alkyl, preferably C 17 straight chain alkenyl or C 10-11 straight chain alkyl.

[0025] In some embodiments, R1 is , or .

[0026] In some embodiments, R2 is C 9-20 straight chain or branched chain alkenyl, or C 9-20 straight chain or branched chain alkyl, preferably C 17 straight chain alkenyl or C 15-18 branched chain alkyl.

[0027] In some embodiments, R2 is , , , or .

[0028] In some embodiments, the compound of formula (I) is a compound shown in formula (Ia),

[0029] ;

[0030] wherein R 2a and R 2b are independently C 5-10 straight chain alkyl; L1, L2, L3 and R1 are defined as above.

[0031] In some embodiments, the compound of formula (I) is any one of the following compounds:

[0032] ,

[0033] 、

[0034] 、

[0035] 、

[0036] 、

[0037] 、

[0038] 、

[0039] 、

[0040] or

[0041] .

[0042] The present application provides a lipid composition comprising an adjuvant lipid, wherein the adjuvant lipid is a compound of formula (I) as described above, or a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof.

[0043] In some embodiments, the adjuvant lipid is present in the lipid composition in a molar content of 2.5-20%, for example 5%, 10% or 15%.

[0044] In some embodiments, the lipid composition further comprises a permanently anionic lipid, a neutral lipid and a further permanently cationic lipid.

[0045] In some embodiments, the molar percentages of the adjuvant lipid, the permanently anionic lipid, the permanently cationic lipid and the neutral lipid are (2.5-20) : (10-33) : (20-60) : (20-40), preferably (2.5-20) : 25 : (30-47.5) : 25, more preferably 10 : 25 : 40 : 25.

[0046] In some embodiments, the permanently anionic lipid is selected from any one or a combination of at least two of the group consisting of 2-acetamidoethyl((R)-2,3- bis(oleoyloxy)propyl)phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylthioureido)ethyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylureido)ethyl) phosphate, (R)-2,3- bis(oleoyloxy)propyl-(2-(3-propylureido)ethyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl- (2-(3-butylureido)ethyl) phosphate, and salts thereof, preferably 2-acetamidoethyl((R)-2,3- bis(oleoyloxy)propyl)phosphate sodium salt.

[0047] In some embodiments, the permanently cationic lipid is selected from any one or a combination of at least two of the group consisting of 1,2-dioleoxy-3-methylammonium propane, (2,3-dioleoxypropyl)trimethylammonium, and salts thereof, preferably 1,2-dioleoxy-3- methylammonium propane chloride salt.

[0048] In some embodiments, the neutral lipid is selected from any one or a combination of at least two of the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, distearoylphosphatidylcholine, and salts thereof, preferably 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine.

[0049] The present application provides a pharmaceutical composition comprising the lipid composition as described above and an active ingredient;

[0050] The active ingredient is selected from any one or a combination of at least two of the group consisting of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.

[0051] In some embodiments, the active ingredient and the lipid composition are adjusted in amounts such that the charge ratio of positive charge to negative charge in the pharmaceutical composition is 1:2 to 1:5.

[0052] In some embodiments, the active ingredient is a nucleic acid molecule capable of encoding one or more antigens. The antigen can be a disease-associated antigen, or the nucleic acid molecule or antigen can elicit an immune response against a disease-associated antigen or a cell expressing a disease-associated antigen.

[0053] In some embodiments, the nucleic acid molecule is an RNA encoding one or more antigens.

[0054] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0055] In some embodiments, the pharmaceutical composition further comprises one or more hydrophobic small molecules, permeation enhancing molecules, carbohydrates, polymers, surface altering agents, functionalized lipids, or cytokines.

[0056] The present application provides use of a compound of Formula (I) as described above, or a solvate, pharmaceutically acceptable salt, or stereoisomer thereof, a lipid composition as described above, or a pharmaceutical composition as described above, in the manufacture of a medicament for delivering an active ingredient into an antigen presenting cell in a target organ or tissue;

[0057] The active ingredient is selected from any one or a combination of at least two of the group consisting of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.

[0058] In some embodiments, the target organ or tissue is selected from any one or a combination of at least two of the group consisting of spleen, liver, lymph, muscle, and lung.

[0059] In some embodiments, the antigen presenting cell is selected from any one or a combination of at least two of the group consisting of a B cell, an NK cell, a cDC cell, a pDC cell, and a macrophage.

[0060] The present application provides use of a compound of Formula (I) as described above, or a solvate, pharmaceutically acceptable salt, or stereoisomer thereof, a lipid composition as described above, or a pharmaceutical composition as described above, in the manufacture of a medicament;

[0061] In some embodiments, the medicament is selected from any one or a combination of at least two of the group consisting of a nucleic acid drug, a genetic vaccine, a small molecule drug, a polypeptide drug, and a protein drug.

[0062] In some embodiments, the medicament is for treating a disease or disorder characterized by malfunction of an organ, protein, or gene function, or abnormal activity of a protein or polypeptide, or deficiency or hypofunction of a cytokine.

[0063] In some embodiments, the disease or disorder is selected from any one or a combination of at least two of the group consisting of an infectious disease, a neoplastic and proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, a metabolic disease, a congenital immunodeficiency disease, an acquired immunodeficiency syndrome, a congenital granulomatous disease, and an immunocompromised state after chemotherapy / radiation therapy.

[0064] In some embodiments, the infectious disease is selected from any one or a combination of at least two of a disease caused by a coronavirus, an influenza virus, or an HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, herpes, X-linked severe combined immunodeficiency, interferon gamma receptor deficiency disease, IL-12 / IL-23 receptor deficiency disease, congenital granulomatous disease, and selective IgA deficiency disease.

[0065] In some embodiments, the tumor is selected from any one or a combination of at least two of breast cancer, ovarian cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer, lymphoma, colon cancer, liver cancer, melanoma, bladder cancer, cervical cancer, and prostate cancer.

[0066] In some embodiments, the subject of administration of the drug is a mammal.

[0067] In some embodiments, the subject of administration of the drug is any one selected from the group consisting of a human, a non-human primate, a companion animal, an exotic species, a livestock animal, and a food-producing animal.

[0068] In some embodiments, the route of administration of the drug is intravenous, intramuscular, intradermal, subcutaneous, intranasal, or inhalation.

[0069] In some embodiments, the dose of administration of the drug is 0.001-10 mg / kg.

[0070] It should be understood that the above uses provided by the present application can include therapeutic, diagnostic, non-therapeutic, and non-diagnostic uses. For example, the therapeutic use can include using the compound provided by the present application, or a solvate, a pharmaceutically acceptable salt, or a stereoisomer thereof, or using the lipid composition provided by the present application to package and deliver an active ingredient to a target organ / tissue / cell, or using the pharmaceutical composition of the present application to deliver the active ingredient contained therein to a target organ / tissue / cell, so as to achieve the effects of treating diseases, improving conditions, regulating physiological activities in the body, etc.; the diagnostic use can include packaging an active ingredient for disease diagnosis in the compound provided by the present application, or a solvate, a pharmaceutically acceptable salt, or a stereoisomer thereof, or a lipid composition, so as to deliver the active ingredient to a target organ / tissue / cell, thereby achieving the purpose of disease diagnosis; the non-therapeutic / non-diagnostic purpose can include using the compound provided by the present application, or a solvate, a pharmaceutically acceptable salt, or a stereoisomer thereof, or using the lipid composition provided by the present application to encapsulate an active ingredient, so as to deliver the active ingredient to a target organ / tissue / cell, for the purpose of scientific research, detection, etc. (such as disease mechanism research, drug mechanism research, new drug development, drug screening, etc.).

[0071] All publications and patents mentioned in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict between the definitions in the specification and that of a particular publication or patent incorporated by reference, the definition in the specification prevails.

[0072] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0073] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the same field as that of the disclosure. If there is an inconsistency between the definitions of terms used in the specification and that of a particular document incorporated by reference, the definition in the specification prevails.

[0074] Except in the Examples, or where otherwise explicitly indicated, all numerical quantities in the description and claims specified in terms of quantity, such as doses, should be understood to be modified in all instances by the term "about" as the term is used in the art. Also, it should be understood that any numerical range recited in this application is intended to include all sub-ranges subsumed therein.

[0075] Further, when referring to a number or a range of numbers, the term "about" means that the number or range of numbers referred to is an approximation within typical tolerances in the art, within experimental variability, or within statistical experimental error, and thus the number or range of numbers can vary, for example, between 1% and 15% of the number or range of numbers recited. For example, "about" can be understood as about 2 standard deviations from the mean, and where "about" precedes a series of numbers or a range, it should be understood that "about" can modify each of the numbers in the series or range.

[0076] In the present application, "in the structural fragment " means that the structural fragment is attached to the rest of the molecule through the bond. For example, " means that it is attached to the rest of the molecule through the "bond. ".

[0077] In the present application, the term "one or more" means 1, 2, 3, 4, 5, or 6, for example 1, 2, or 3.

[0078] In the present application, the term "alkyl" means a group having the designated number of carbon atoms (e.g., Ci, C2, C3, C4, C5, C6, C9, C 10 , C 11 , C 12 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C21 , C 23 , C 24 , C 25 , C 26 , C 27 , C 28 , C 29 , C 30 ) of straight-chained or branched, saturated, monovalent hydrocarbon radicals. Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl, , , , , or .

[0079] In the present application, the term "alkylene" refers to a saturated, divalent hydrocarbon radical resulting from the removal of two hydrogen atoms from a saturated straight-chained or branched hydrocarbon. Alkylene groups include, but are not limited to, methylene (-CH2-), ethylene {including -CH2CH2- or -CH(CH3)-}, isopropylene {including -CH(CH3)CH2- or -C(CH3)2-}, butylene {including -CH2CH2CH2- or -CH(CH2CH3)-}, and the like. , .

[0080] The term "alkenyl" refers to straight-chained or branched hydrocarbon radicals having at least one double bond, consisting solely of carbon and hydrogen atoms, having, for example, from 5 to 30 (and also, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) carbon atoms, and being attached to the rest of the molecule by a single bond. Alkenyl groups include, but are not limited to, ethenyl (vinyl, -CH=CH2), propenyl {including -CH=CHCH3 and -CH2CH=CH2), isopropenyl {including -C(CH3)=CH2}, butenyl {including -CH=CHCH2CH3 and -CH2CH2CH=CH2}, and the like. , , , , and the like.

[0081] The use of the terms "including", "containing", or "comprising" and the like in the present application means "comprising but not limited to", unless explicitly indicated to the contrary. The terms "containing" or "comprising" can be open, semi-closed or closed. In other words, the terms also include "consisting essentially of" or "consisting of".

[0082] The term "pharmaceutically acceptable" means, in the present application, that the compound or composition is compatible chemically and / or toxicologically, with the other ingredients of a formulation, and / or with the human or mammal to be treated therewith, with respect to prevention or treatment of a disease or condition.

[0083] The term "adjuvant lipid" refers to a lipid (compound or composition) that can be used as an adjuvant for a medicament or vaccine. The adjuvant lipid functions in the medicament or vaccine as a conventional adjuvant, e.g., non-specifically alters or enhances the specific immune response of the body to an antigen, functions as a helper, etc., and further, e.g., the adjuvant lipid can also function to reduce the amount of immunizing substance, increase the protective ability of the body, induce long-term, high-efficiency specific immune response of the body, etc.

[0084] The term "permanent anionic lipid" refers to a lipid having one or more hydrophobic components and a permanent anionic group (i.e., the permanent anionic lipids provided by the present application). One anionic group that can be used in the permanent anionic lipids is a phosphate group. The phosphate group can be a compound that is deprotonated and has a negative charge at a pH less than 8, 9, 10, 11, 12, 13, or 14. The hydrophobic component can be one or more C6-C 24 alkyl or alkenyl groups. The compound can have one hydrophobic group, two hydrophobic groups, or three hydrophobic groups. The permanent anionic lipid can be selected from any one or more of 2-acetamidoethyl ((R)-2, 3-bis(oleoyloxy)propyl) phosphate, (Z)-(R)-3-(phosphonooxy)propane-1, 2-diyl dioleate, and 1, 2-dioleoyl-SN-glycero-3-phospho-RAC-glycerol, and salts thereof (e.g., sodium salts, chloride salts, etc.).

[0085] The term "permanent cationic lipid" refers to a lipid having one or more hydrophobic components and a permanent cationic group. The permanent cationic lipids selected by the present application can contain a group that has a positive charge (regardless of pH). The permanent cationic lipid can be selected from any one or more of 1, 2-dioctadecenyloxy-3-methylammonium propane, (2, 3-dioleoxypropyl)trimethylammonium, and salts thereof (e.g., sodium salts, chloride salts, etc.).

[0086] The term "neutral lipid" refers to a helper lipid that is not charged or exists as a zwitterion at a selected pH. The neutral lipid can modulate nanoparticle fluidity into a lipid bilayer structure and increase efficiency by facilitating lipid phase transition, and can also affect specificity for a target organ / cell. Any neutral lipid having the above characteristics in the art, and in particular, a neutral lipid that can be used to prepare a drug carrier, can be suitable for use in the present application. The neutral lipid can include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0087] Any neutral lipid having the above-mentioned features in the art, particularly a neutral lipid that can be used to prepare a pharmaceutical carrier, can be suitable for use in the present application. For example, the neutral lipid can include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0088] The carrier component of the composition comprising the cationic lipid can include one or more neutral lipids-phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids can assemble into one or more lipid bilayers. Generally, the phospholipids can include a phospholipid moiety and one or more fatty acid moieties.

[0089] The neutral lipid moiety can be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lyso-phosphatidylcholine, and sphingomyelin. The fatty acid moiety can be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, a-linolenic acid, erucic acid, phytanic acid, eicosanoic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, the phospholipids can be functionalized with or crosslinked to one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced by a triple bond). Alkynyl groups can undergo copper-catalyzed cycloaddition reactions upon exposure to azides under appropriate reaction conditions. These reactions can be used to functionalize the lipid bilayer of the composition to facilitate membrane penetration or cell recognition, or to couple the composition to useful components such as targeting or imaging moieties (e.g., dyes).

[0090] Neutral lipids useful in these compositions can be selected from the non-limiting group consisting of 1, 2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-didodecanoyl-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-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1, 2-dilinolenoyl-sn-glycero-3-phosphocholine, 1, 2-diarachidonoyl-sn-glycero-3-phosphocholine, 1, 2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-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), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0091] The term "subject" or "patient" can include a mammalian subject in the present application. For example, the mammalian subject can be selected from one or more of a human, a non-human primate, a companion animal, an exotic species, a livestock animal, and an animal raised for food.

[0092] The term "treatment" as used herein refers to application of one or more pharmaceutical substances to a patient or subject having a disease or symptoms of a disease in order to cure, alleviate, relieve, lessen, improve, or affect the disease or symptoms of the disease. In the context of the present application, the term "treatment" can also include prophylaxis, unless explicitly indicated to the contrary.

[0093] In the present application, the term "antigen" includes any molecule, preferably a peptide or a protein, comprising at least one epitope capable of eliciting an immune response and / or an epitope against which an immune response is directed. Preferably, an antigen in the context of the present application is a molecule which, optionally after processing, induces an immune response, preferably specifically directed against the antigen or a cell expressing the antigen. In particular, an "antigen" relates to a molecule which, optionally after processing, is presented by an MHC molecule and specifically reacts with a T lymphocyte (T cell).

[0094] Accordingly, an antigen or a fragment thereof should be recognizable by a T cell receptor. Preferably, if recognized by a T cell receptor, the antigen or fragment is capable of inducing clonal expansion of a T cell carrying a T cell receptor specifically recognizing the antigen or fragment in the presence of an appropriate co-stimulatory signal. In the context of embodiments of the present application, the antigen or fragment is preferably presented by a cell, preferably by an antigen presenting cell and / or a diseased cell, in the context of an MHC molecule, which leads to an immune response against the antigen or a cell expressing the antigen.

[0095] An antigen as referred to in the present application can be any suitable antigen capable of eliciting an immune response, wherein the immune response is preferably a cellular immune response.

[0096] An antigen is preferably a product corresponding to or derived from a naturally occurring antigen. The naturally occurring antigen can include or can be derived from an allergen, a virus, a bacterium, a fungus, a parasite, and other infectious and pathogenic agents, or the antigen can also be a tumor antigen. According to the present application, an antigen can correspond to a naturally occurring product, e.g., a viral protein or a portion thereof.

[0097] The term "pathogen" relates to pathogenic microorganisms and includes viruses, bacteria, fungi, unicellular organisms and parasites. Examples of pathogenic viruses include, but are not limited to, human immunodeficiency virus (HIV), cytomegalovirus (CMV), herpes virus (HSV), hepatitis A virus (HAV), HBV, HCV, papilloma virus and human T-lymphotrophic virus (HTLV). Unicellular organisms include, but are not limited to, Plasmodium, Trypanosoma, Amoeba and the like.

[0098] The term "disease-associated antigen" refers to all antigens of pathogenic significance and includes "tumor antigens". According to the present application, it is desired to induce an immune response against a disease-associated antigen or a cell expressing a disease-associated antigen and preferably presenting a disease-associated antigen in the context of an MHC molecule. Preferably, the disease-associated antigen is a naturally occurring antigen. In one embodiment, the disease-associated antigen is expressed in a diseased cell and is preferably presented by an MHC molecule of the cell.

[0099] The antigen encoded by the RNA (i.e. the therapeutic and / or prophylactic agent) comprised in the nanoparticle according to the present application should induce an immune response against a disease-associated antigen to be targeted or a cell expressing a disease-associated antigen to be targeted. Thus, the antigen encoded by the RNA comprised in the nanoparticle according to the present application can correspond to or can comprise a disease-associated antigen or one or more immunogenic fragments thereof, e.g. one or more MHC-binding peptides of a disease-associated antigen. Thus, the antigen encoded by the RNA comprised in the nanoparticle according to the present application can be a recombinant antigen.

[0100] Active ingredient (e.g. therapeutic and / or prophylactic agent)

[0101] The lipid composition according to the present application can be used for the delivery of a pharmaceutically active ingredient, any substance having a therapeutic / prophylactic activity can be used as a pharmaceutically active ingredient, e.g. a therapeutic and / or prophylactic agent. Based thereon, the present application further provides a (pharmaceutical) composition comprising the lipid composition according to the present application for the delivery of a pharmaceutically active ingredient. The composition according to the present application can comprise one or more therapeutic and / or prophylactic agents (as pharmaceutically active ingredients). The pharmaceutically active ingredient can be encapsulated within or associated with the lipid composition according to the present application.

[0102] According to some preferred embodiments of the present application, the active ingredient comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide and a protein. Preferably, the active ingredient is a nucleic acid molecule.

[0103] For example, the active ingredient is a vaccine or a compound capable of eliciting an immune response. Thus, in some preferred embodiments, the active ingredient can be a nucleic acid molecule (such as DNA or RNA) capable of encoding one or more antigens.

[0104] The lipid compositions of the application can deliver active ingredients to target organs (as carriers) in a subject (such as a mammal), and in particular to target cells in a target organ, and thus the application also provides methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject a composition comprising an active ingredient as described above and / or contacting a cell of the subject with the composition.

[0105] Therapeutic and / or prophylactic agents include biologically active substances and can alternatively be referred to as "active agents," "active ingredients," and the like. Therapeutic and / or prophylactic agents can be substances that, upon delivery to a cell or organ, cause a desired change in that cell or organ or other body tissue or system. Such species can be used to treat one or more diseases, disorders, or conditions. In some embodiments, a therapeutic and / or prophylactic agent is a small molecule drug that can be used to treat a particular disease, disorder, or condition.Examples of drugs that can be used in the composition include, but are not limited to, anti-neoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), anti-tumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blocking agents (e.g., propranolol, timolol, and labetalol), anti-hypertensive agents (e.g., clonidine and hydralazine), anti-depressants (e.g., imipramine, amitriptyline, and doxepin), anti-convulsants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / anti-bacterial agents (e.g., gentamycin, ciprofloxacin, and cefoxitin), anti-fungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), anti-parasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, vitamins, sedatives, and imaging agents.

[0106] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, an immune response eliciting compound, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is detrimental 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, a maytansinoid 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, phosphates, cobalt, yttrium 90, samarium 153, and praseodymium. Vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis and can include nucleic acid molecules (e.g., mRNA) encoding infectious disease-derived antigens and / or epitopes. Vaccines can also include compounds and formulations that direct an immune response against cancer cells and can include nucleic acid molecules (e.g., mRNA) encoding tumor cell-derived antigens, epitopes, and / or neoepitopes. Immune response eliciting compounds can 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, lachrymatory factor (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 daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), gentamycin, and amikacin), and antimitotic agents (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0107] In other embodiments, the therapeutic and / or prophylactic agent is a protein. Therapeutic proteins that can be used in the nanoparticles of the present application include, but are not limited to, 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.

[0108] In some embodiments, the therapeutic and / or prophylactic agent can be a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term "polynucleotide" in its broadest sense encompasses any compound and / or substance that is in the form of an oligonucleotide chain or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present application include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger mRNA, hybrids thereof; RNAi inducing factors; RNAi factors; siRNA; shRNA; miRNA; antisense RNA; ribozymes; catalytic DNA; RNA that induces triple helix formation; aptamers, and the like. In some preferred embodiments, the therapeutic and / or prophylactic agent is RNA. RNA that can be used in the compositions and methods described herein can be selected from, but not limited to, the group consisting of shortmers, antagomirs, antisense RNA, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In certain embodiments, the RNA is mRNA.

[0109] In certain embodiments, the therapeutic and / or prophylactic agent is mRNA. The mRNA can encode any polypeptide of interest, including any naturally 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.

[0110] In other embodiments, the therapeutic and / or prophylactic agent is siRNA. The siRNA can be capable of selectively reducing expression of a gene of interest or down-regulating expression of the gene. For example, the siRNA can be selected such that, upon administration of a composition comprising the siRNA to a subject in need thereof, a gene associated with a particular disease, disorder, or condition is silenced. The siRNA can comprise a sequence that is complementary to a sequence of mRNA encoding a gene or protein of interest. In some embodiments, the siRNA can be an immunomodulatory siRNA.

[0111] 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 a gene editing tool. For example, the sgRNA-cas9 complex can affect mRNA translation of a gene of a cell.

[0112] In some embodiments, the therapeutic and / or prophylactic agent is an shRNA or a vector or plasmid encoding the same. The shRNA can be generated inside the target cell after delivery of the appropriate construct into the nucleus. Constructs and mechanisms associated with shRNAs are well known in the relevant art.

[0113] disease or disorder

[0114] The compositions / carriers of the present application can deliver a therapeutic and / or prophylactic agent to a subject or patient, thereby effecting 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 a nucleic acid molecule, a small molecule compound, a polypeptide or a protein. Thus, the compositions of the present application can be used to prepare a nucleic acid drug, a genetic vaccine, a small molecule drug, a polypeptide or a protein drug. Due to the wide variety of the above therapeutic and / or prophylactic agents, the compositions of the present application can be used to treat or prevent a variety of diseases or disorders.

[0115] In one embodiment, the disease or disorder is characterized by a dysfunctional or abnormal protein or polypeptide activity.

[0116] The agents, compositions and methods described herein 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 herein. Particularly preferred diseases are infectious diseases (e.g., viral diseases) and cancer diseases. The agents, compositions and methods described herein can also be used for immunization or vaccination to prevent a disease described herein.

[0117] According to the present application, the term "disease" refers to any pathological state, including infectious diseases and cancer diseases, in particular those forms of infectious diseases and diseases described herein.

[0118] According to the present application, the disease to be treated is preferably a disease characterized by a dysfunction of an organ, a protein or a gene, or an abnormality of a protein or polypeptide activity, or a deficiency or hypofunction of a cytokine.

[0119] For example, the disease or disorder is selected from the group consisting of infectious diseases, neoplastic and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, metabolic diseases, congenital immunodeficiency diseases, acquired immunodeficiency syndrome, congenital granulomatous diseases and post-chemo / radiotherapy immunodepression.

[0120] The infectious disease is selected from the group consisting of a disease caused by a coronavirus, an influenza virus, or an HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, herpes, X-linked severe combined immunodeficiency, interferon gamma receptor deficiency disease, IL-12 / IL-23 receptor deficiency disease, congenital granulomatous disease, and selective IgA deficiency disease.

[0121] Examples of the infectious disease include, but are not limited to, ① viral infectious diseases such as AIDS (HIV), hepatitis A, hepatitis B, hepatitis C, shingles (chickenpox), rubella (Rubella, caused by Rubella virus), diseases caused by Flaviviridae (e.g., yellow fever, dengue fever, epidemic encephalitis B, forest encephalitis, West Nile fever, Zika fever), diseases caused by coronaviruses, diseases caused by influenza viruses, rabies, hemorrhagic infectious diseases (e.g., diseases caused by Marburg virus or Ebola virus, etc.), and the like; 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), and the like; ③ infections caused by protozoan pathogens such as malaria, sleeping sickness, leishmaniasis, toxoplasmosis, i.e., infections caused by Plasmodium, Trypanosoma, Leishmania, and Toxoplasma, and the like; or ④ fungal infections (e.g., infections caused by Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, or Candida albicans), and the like.

[0122] The cancer or cancer (medical term for malignant tumor) is a class of diseases in which a group of cells display uncontrolled growth (division in excess of the normal limits), invasion (intrusion into and destruction of adjacent tissues), and sometimes metastasis (spread to other parts of the body through lymph or blood). These three malignant properties of cancers differentiate them from benign tumors, which are self-limited, and do not invade or metastasize. Most cancers form a tumor, i.e., a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells), but some (like leukemias) do not. According to the present application, the term "cancer" includes, but is not limited to, leukemia, seminoma, melanoma, teratoma, lymphoma, sarcoma, blastoma, neuroblastoma, glioma, glioblastoma, kidney cancer, adrenal cancer, renal cell carcinoma, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, lung cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, multiple myeloma, lymph node cancer, esophageal cancer, colon cancer, rectal cancer, bladder cancer, prostate cancer, endometrial cancer, pancreatic cancer, ear-nose-throat (ENT) cancer, breast cancer, uterine cancer, breast cancer, prostate cancer, ovarian cancer, and metastasis thereof, etc.

[0123] Malignant melanoma is a serious type of skin cancer. It is caused by uncontrolled growth of pigment cells called melanocytes.

[0124] According to the present application, "carcinoma" is a malignant tumor derived from epithelial cells. This group accounts for the most common cancers, including the common forms of breast cancer, prostate cancer, lung cancer, and colon cancer.

[0125] Lymphoma and leukemia are malignant tumors derived from hematopoietic (blood-forming) cells.

[0126] Sarcoma is a cancer of transformed cells that originate from one of several tissues that develop from the mesoderm in an embryo. Thus, sarcomas include bone tumors, cartilage tumors, fat tumors, muscle tumors, vascular tumors, and hematopoietic tissue tumors, etc.

[0127] Blastic tumor or blastoma is a tumor (usually malignant) that resembles immature or embryonic tissue. Most of these tumors are commonly found in children.

[0128] Glioma is a type of tumor that originates in the brain or spinal cord. Because it originates in glial cells, it is called a glioma. The most common site of glioma is the brain.

[0129] Other components

[0130] The pharmaceutical compositions of the present application can include one or more components in addition to those described in the foregoing sections. For example, the compositions can include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or a sterol.

[0131] The compositions can also include one or more penetration enhancing molecules, carbohydrates, polymers, surface altering agents, or other components. The penetration enhancing molecules can be, for example, the molecules described in U.S. Patent Application Publication No. 2005 / 0222064. The carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).

[0132] The surface altering agents can include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytics (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin beta 4, dornase alfa, neltenexine, and erdosteine), and DNases (e.g., rhDNase). The surface altering agents can be disposed within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent attachment, or other methods).

[0133] The compositions can also include one or more functionalized lipids. For example, the lipids can be functionalized with an alkyne group that can undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. In particular, the lipid bilayer can be functionalized in this way with one or more groups that can be effective in promoting membrane penetration, cell recognition, or imaging. The surface of the composition can also be coupled to one or more useful antibodies. Functional groups and conjugates useful for targeted cellular delivery, imaging, and membrane penetration are well known in the art.

[0134] In addition to these components, the compositions can include any material suitable for use in a pharmaceutical composition. For example, the composition can include one or more pharmaceutically acceptable (i.e., pharmaceutically acceptable) excipients or auxiliary ingredients 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, surface active agents, isotonic agents, thickening agents or emulsifying agents, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, and the like.

[0135] The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the active ingredients of the pharmaceutical composition. The non-pharmaceutically acceptable ingredients can be used to prepare pharmaceutically acceptable ingredients and are included in the present application.

[0136] Suitable buffers for use in the compositions of the present application include salt forms of acetic acid, salt forms of citric acid, salt forms of boric acid, and salt forms of phosphoric acid.

[0137] The term "excipient" as used in the present application is intended to mean all substances that can be present in the pharmaceutical compositions of the present application and which are not active ingredients, such as carriers, binders, lubricants, thickening agents, surface active agents, preservatives, emulsifiers, buffers, flavoring agents or coloring agents. Excipients are, for example, starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21stEdition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0138] Suitable preservatives for use in the compositions of the present application include benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0139] Examples of diluents can include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof.

[0140] Dosage forms and administration

[0141] The compositions of the present application can be prepared in a formulation to be administered in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injectables, aerosols. The compositions of the present application can be prepared by methods well known in the art of pharmacy. For example, sterile injectable solutions can be prepared by incorporating the therapeutic or prophylactic agent in the required amount in the appropriate solvent with other components as desired, as above, followed by filtered sterilization. Surfactants can be added to aid in the formation of a homogeneous solution or suspension.

[0142] For example, the compositions of the present application can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In one embodiment, the composition is administered intravenously or subcutaneously.

[0143] Therapeutically effective amount

[0144] A "therapeutically effective amount" is the amount of a therapeutic agent that, when administered to a patient, is effective to improve a disease or condition. A "prophylactically effective amount" is the amount of a prophylactic agent that, when administered to a subject, is effective to prevent a disease or condition. The amount of a therapeutic agent that constitutes a "therapeutically effective amount" or the amount of a prophylactic agent that constitutes a "prophylactically effective amount" varies according to the therapeutic agent 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. One of ordinary skill in the art can determine a therapeutically effective amount and a prophylactically effective amount in view of his knowledge and this application.

[0145] The compositions of the present application are administered in therapeutically effective amounts, which can vary according to the particular agent selected, the route of administration, and the nature of the disease treated, as well as the age, weight, and condition of the patient, and are ultimately 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 (e.g., a human).

[0146] Antigen presenting cell

[0147] An antigen presenting cell (APC) is a cell that presents (i.e., displays) an antigen in the context of a major histocompatibility complex (MHC) on its surface. This includes instances in which only one fragment or more fragments of the antigen are presented. T cells can recognize this complex with their T cell receptors (TCRs). Antigen presenting cells process antigens and present them to T cells.

[0148] Professional antigen-presenting cells are very efficient in internalizing antibodies (by phagocytosis or by receptor-mediated endocytosis) and then displaying the antigen fragment bound to a class II MHC molecule on their membrane. 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 produces an additional co-stimulatory signal, leading to T cell activation. The expression of co-stimulatory molecules is a typical feature of professional antigen-presenting cells.

[0149] 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.

[0150] Dendritic cells are a group of white blood cells, including plasmacytoid dendritic cells (pDC cells) and classical dendritic cells (cDC cells), that present antigens captured in peripheral tissues to T cells via both class II and class I MHC antigen presentation pathways. Dendritic cells are powerful inducers of the immune response, and the activation of these cells is a key step in inducing antitumor immunity.

[0151] Antigen-presenting cells can be loaded with MHC-presented peptides by transducing them with a nucleic acid encoding a peptide or protein comprising a peptide to be presented, e.g., an antigen-encoding nucleic acid (e.g., RNA). Transfection of dendritic cells with mRNA is a promising antigen loading technique to stimulate strong antitumor immunity.

[0152] The term "immunogenicity" relates to the relative efficiency of an antigen to induce an immune response.

[0153] The terms "T cell" and "T lymphocyte" are used interchangeably in the present application and include helper T cells (CD4+ T cells) and cytolytic T cells, cytotoxic T cells (CTL, CD8+ T cells).

[0154] 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 on their cell surface called the T cell receptor (TCR). The thymus is the main organ responsible for T cell maturation. Several different T cell subsets have been discovered, each with a different function.

[0155] Helper T cells assist other white blood cells in 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 known as CD4 +T cells. Helper T cells are activated when class II MHC molecules expressed on the surface of antigen presenting cells (APCs) present a peptide antigen to the helper T cell. Following activation, they rapidly divide and secrete small proteins called cytokines that modulate or assist the adaptive immune response.

[0156] Cytotoxic T cells destroy diseased cells, such as infected cells (e.g., cells infected with a virus) and cancer cells, and also participate in transplant rejection. Because they express the CD8 glycoprotein on their surface, these cells are also known as CD8 + T cells. These cells recognize their targets by binding to an antigen in association with class I MHC, which is present on the surface of almost every cell in the body.

[0157] 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 from separate T cell receptor alpha and beta (TCRa and TCRP) genes, and are referred to as the alpha-TCR chain and the beta-TCR chain. Gamma delta T cells represent a small subset 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).

[0158] All T cells are derived from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells, which are derived from hematopoietic stem cells, exist in the thymus and expand through cell division to produce a large number of immature thymocytes. Early thymocytes do not express either CD4 or CD8 and are thus classified as double negative (CD4-CD8-) cells. As they progress through development, they become double positive thymocytes (CD4+CD8+) and eventually mature into single positive (CD4 + CD8 - or CD4 - CD8 + ) thymocytes, which are then released from the thymus to the peripheral tissues.

[0159] The first signal for T cell activation is provided by the binding of the T cell receptor to a short peptide presented by a 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), often a dendritic cell in the case of a primary response, but B cells and macrophages can also be important APCs. Peptides presented by class I MHC molecules to CD8 + T cells are 8 to 10 amino acids long; peptides presented by class II MHC molecules to CD4 +Peptides for T cells are longer because the end of the binding cleft of class II MHC molecules is open.

[0160] In the present text, the term "TLP" means a three-component lipid composition, in particular a composition of a permanently anionic lipid, a permanently cationic lipid and a neutral lipid prepared in certain molar percentages. For example, in some embodiments, the TLP is a lipid composition of the permanently anionic lipid ADOPE, the permanently cationic lipid DOTMA and the neutral lipid DOPE prepared in certain molar percentages. The TLP can encapsulate mRNA, thereby generating a TLP-mRNA composition, as used in the present text.

[0161] In the present application, the "TLP" group refers to a composition prepared from the permanently anionic lipid ADOPE, the permanently cationic lipid DOTMA and the neutral lipid DOPE in certain molar percentages, without the addition of the adjuvant lipid of the present application.

[0162] In some embodiments of the present application, the permanently cationic lipid DOTMA in the TLP composition can be partially replaced by the adjuvant lipid of the present application, for example, in some embodiments, the adjuvant lipid of the present application partially replaces the DOTMA in the TLP composition in a certain percentage of the total lipid, thereby preparing a corresponding composition, which, after encapsulating mRNA, is referred to as an mRNA pharmaceutical composition, as used in the present text.

[0163] In the present text, "blank control group", "blank control" and "control" are used interchangeably, and those skilled in the art can understand the specific technical meaning according to the context, without causing ambiguity in expression.

[0164] Without deviating from the common sense in the art, the above-mentioned preferred conditions can be combined arbitrarily, thereby obtaining each preferred example of the present application.

[0165] The reagents and raw materials used in the present application are commercially available.

[0166] The positive progress effect of the present application is that the adjuvant lipid compound and the lipid composition provided by the present application can be used for the encapsulation of biological pharmaceutical active ingredients such as nucleic acids (such as mRNA, etc.), thereby forming a corresponding biological pharmaceutical.

[0167] Taking the nucleic acid pharmaceutical with mRNA as the active ingredient as an example, the mRNA pharmaceutical composition prepared by adding the adjuvant lipid provided by the present application to the lipid composition has at least the following advantages:

[0168] 1. Good particle size and uniform particle distribution;

[0169] 2. It can significantly improve the protein expression of the antigen in the body of the subject (such as a mouse) and the spleen, and has good spleen targeting;

[0170] 3. It can significantly increase the expression of immune-related cytokines;

[0171] 4. It can significantly reduce the volume of tumors and significantly increase the survival rate of mice. BRIEF DESCRIPTION OF DRAWINGS

[0172] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings of the present application will be briefly introduced below. It should be understood that the drawings described below only relate to some specific embodiments of the present application, and are not a limitation on the present application.

[0173] Figure 1 Total fluorescence intensity of the live mice and the organs of the mice after intravenous injection of the Fluc-mRNA pharmaceutical composition containing YK-2201, YK-2202, YK-2203, YK-2204, YK-2205, YK-2206, YK-2207, YK-2208, YK-2209, YK-2210, control 1 and SAL-8 or the Fluc-mRNA-TLP pharmaceutical composition without adjuvant lipids into the mice for 6 h.

[0174] Figure 2 Cytokine IFN-α, IL-12 and TNF-α stimulation in the serum of B16F10-OVA mice inoculated with OVA-mRNA pharmaceutical compositions containing YK-2201, YK-2202, YK-2203, YK-2204, YK-2205, YK-2206, YK-2207, YK-2208, YK-2209, YK-2210, control 1 and SAL-8 or OVA-mRNA-TLP pharmaceutical compositions without adjuvant lipids.

[0175] Figure 3 In vivo tumor growth of B16F10-OVA mice inoculated with OVA mRNA pharmaceutical compositions containing YK-2202, YK-2204, YK-2206 and YK-2208 or OVA mRNA-TLP pharmaceutical compositions without adjuvant lipids.

[0176] Figure 4 Survival rate of B16F10-OVA mice inoculated with OVA mRNA pharmaceutical compositions containing YK-2202, YK-2204, YK-2206 and YK-2208 or OVA mRNA-TLP pharmaceutical compositions without adjuvant lipids. DETAILED DESCRIPTION

[0177] The application will be further described with reference to the following examples without however being limited thereto. The conditions employed in the examples can be further adjusted according to different requirements of specific use, and the conditions not mentioned are the conventional conditions in the industry. In the specific examples of the application, the raw materials used can be obtained through marketing. Unless otherwise specified, the percentages in the context are weight percentages, and all temperatures are given in degrees Celsius. The technical features involved in each embodiment of the application can be combined with each other as long as they do not conflict with each other.

[0178] In the following examples, the meanings of the abbreviations are as follows:

[0179] MSA-2: 5,6-dimethoxy-γ-oxo-benzo[b]thiophene-2-butanoic acid;

[0180] ADOPE: 2-acetamidoethyl((R)-2,3-bis(oleoyloxy)propyl) phosphate sodium salt;

[0181] DOTMA: 1,2-dioctadecyloxy-3-methylammonium propane chloride;

[0182] DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine;

[0183] DMAP: 4-dimethylaminopyridine;

[0184] EDCI: 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride;

[0185] DIEA: N,N-diisopropylethylamine;

[0186] DCM: dichloromethane;

[0187] THF: tetrahydrofuran;

[0188] TLP: three-component lipid composition.

[0189] Example 1: Synthesis of adjuvant lipids

[0190] 1. Synthesis of YK-2201

[0191]

[0192] MSA-2, 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (48 mg, 0.16 mmol) was dissolved in DCM (1.5 mL), YK-101 (reference patent CN116178193B paragraph

[0383] synthesis), 3-hexyl nonyl-6-((4-(dodecyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate (100 mg, 0.16 mmol), EDCI (47 mg, 0.25 mmol), DMAP (4 mg, 0.03 mmol) were weighed in sequence, and the reaction was stirred at room temperature for 18 hours. After the reaction was completed, it was dried and wet-loaded, and the product YK-2201 (130 mg, 0.15 mmol, 89.6%) was purified by silica gel chromatography (0% - 30% dichloromethane / ethyl acetate). C 51 H 85 NO9S, MS (ES): m / z (M+H) + = 888.72.

[0193] YK-2201: 1 H NMR (400 MHz, CDCl3) δ 7.91 - 7.97 (m, 1H), 7.30 - 7.36(m, 2H), 4.69 (s, 1H), 4.15 - 4.18 (m, 1H), 4.07 - 4.12 (m, 5H), 4.00 (d, J =10 Hz, 7H), 3.34 - 3.45 (m, 4H), 3.12 - 3.29 (m, 3H), 2.81 - 2.86 (m, 2H),2.70 - 2.75 (m, 1H), 2.46 - 2.53 (m, 3H), 2.29 - 2.37 (m, 3H), 2.14 - 2.26(m,2H), 1.89 - 1.94 (m, 2H), 1.62 - 1.80 (m, 9H), 1.42 - 1.48 (m, 5H), 1.31 -1.33 (m, 19H), 0.91 (t, J = 6.4 Hz, 12H).

[0194] 2. Synthesis of YK-2202

[0195]

[0196] MSA-2 (200 mg, 0.68 mmol) was dissolved in THF (10 mL), YK-009, 2-octyldecyl-6-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate (reference patent CN114044741B paragraph

[0281] synthesis) (670 mg, 1.02 mmol), EDCI (390 mg, 2.04 mmol), DMAP (83 mg, 0.68 mmol) were weighed in turn and stirred at room temperature for 16 hours. After the reaction was completed, it was filtered, wet loaded and purified by silica gel chromatography (0% - 80% petroleum ether / ethyl acetate) to obtain the product YK-2202 (362 mg, 0.39 mmol, 57.4%). C 54 H 91 NO9S, MS (ES): m / z (M + H) + = 931.38.

[0197] YK-2202: 1 H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H), 7.25 - 7.26 (m, 2H),4.00 - 4.15 (m, 4H), 3.95 - 3.98 (m, 8H), 3.30 - 3.33 (m, 2H), 2.68 - 2.80(m, 4H), 2.45 - 2.49 (m, 4H), 2.28 - 2.34 (m, 4H), 2.04 (m, 2H), 1.57 - 1.65(m, 9H), 1.24 - 1.33 (m, 42H), 0.86 - 0.89 (m, 9H).

[0198] 3. Synthesis of YK-2203

[0199]

[0200] MSA-2 (200 mg, 0.68 mmol) was dissolved in THF (10 mL), DOPE (760 mg, 1.02 mmol), EDCI (390 mg, 2.04 mmol), DMAP (83 mg, 0.68 mmol) were weighed in turn and stirred at room temperature for 16 hours. After the reaction was completed, it was filtered, wet loaded and purified by silica gel chromatography (0% - 80% petroleum ether / ethyl acetate) to obtain the product YK-2203 (185 mg, 0.18 mmol, 26.5%). C 55 H 90 NO12 PS, MS (ES): m / z (M-H) - = 1019.21; m / z (M + H) + = 1021.21.

[0201] YK-2203: 1 H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.25 - 7.26 (m, 2H),5.20 - 5.43 (m, 4H), 4.20 - 4.43 (m, 2H), 3.95 - 3.98 (m, 4H), 3.91 - 3.93(m, 4H), 3.25 - 3.28 (m, 2H), 2.82 - 2.91 (m, 3H), 2.20 - 2.34 (m, 5H), 1.95- 2.02 (m, 9H), 1.28 - 1.30 (m, 1H), 1.25 - 1.26 (m, 3H), 1.10 - 1.22 (m,45H), 0.86 - 0.89 (m, 9H).

[0202] 4. Synthesis of YK-2204

[0203]

[0204] MSA-2 (200 mg, 0.68 mmol) was dissolved in THF (5 mL), YK-002 (reference patent CN114044741B paragraph

[0203] synthesis) (454 mg, 0.68 mmol), EDCI (156 mg, 0.82 mmol), DMAP (8 mg, 0.07 mmol) were weighed in turn, and stirred at room temperature for 12 hours. After the reaction was completed, it was spin-dried, wet-loaded, and purified by silica gel chromatography (0%-30% dichloromethane / ethyl acetate) to obtain the product YK-2204 (415 mg, 0.44 mmol, 64.7%). C 55 H 93 NO9S, MS (ES): m / z (M + H) + = 944.81.

[0205] YK-2204: 1H NMR (400 MHz, CDC13) δ 8.45 (s, 1H), 8.11 (s, 1H), 7.45(s, 1H), 4.45 (t, J = 4.8 Hz, 2H), 4.22 (t, J = 4.8 Hz, 2H), 4.02 (s, 6H),3.87 - 3.92 (m, 4H), 2.96 - 3.06 (m, 6H), 2.61 (t, J = 6.4 Hz, 2H), 2.22 -2.31 (m, 4H), 1.51 - 1.72 (m, 6H), 1.29 - 1.38 (m, 49H), 0.84 - 0.89 (m, 9H).

[0206] 5. Synthesis of YK-2205

[0207]

[0208] MSA-2 (200 mg, 0.68 mmol) was dissolved in THF (5 mL), YK-006 (reference patent CN114044741B paragraph

[0250] synthesis) (435 mg, 0.68 mmol), EDCI (156 mg, 0.82 mmol), DMAP (8 mg, 0.07 mmol) were weighed in turn, and stirred at room temperature for 10 hours. After the reaction was completed, it was rotary evaporated, wet loaded, and purified by silica gel chromatography (0%-30% dichloromethane / ethyl acetate) to obtain the product YK-2205 (483 mg, 0.53 mmol, 77.6%). C 53 H 89 NO9S, MS (ES): m / z (M + H) + = 916.83.

[0209] YK-2205: 1 H NMR (400 MHz, CDC13) δ 7.88 (s, 1H), 7.54 (s, 1H), 7.17(s, 1H), 4.85 (t, J = 4.8 Hz, 2H), 4.35 (t, J = 4.8 Hz, 2H), 4.11 (s, 6H),3.63 - 3.85 (m, 10 H), 2.57 - 2.81 (m, 6H), 2.11 - 2.32 (m, 10H), 1.61 - 1.93(m, 41H), 1.14 - 1.29 (m, 9H).

[0210] 6. Synthesis of YK-2206

[0211]

[0212] MSA-2 (200 mg, 0.68 mmol) was dissolved in THF (5 mL), YK-003 (reference patent CN114044741B paragraph

[0215] synthesis) (464 mg, 0.68 mmol) was weighed in turn, EDCI (156 mg, 0.82 mmol), DMAP (8 mg, 0.07 mmol), and stirred at room temperature for 12 hours. After the reaction was completed, it was rotary evaporated, wet loaded, and purified by silica gel chromatography (0%-30% dichloromethane / ethyl acetate) to obtain the product YK-2206 (389 mg, 0.41 mmol, 59.7%). C 56 H 95 NO9S, MS (ES): m / z (M + H) + = 958.91.

[0213] YK-2206: 1 H NMR (400 MHz, CDCl3) δ 8.03 (s, 1H), 7.77 (s, 1H), 7.21(s, 1H), 4.31 (t, J = 4.8 Hz, 2H), 4.03 (t, J = 4.8 Hz, 2H), 3.97 (s, 6H),3.12 - 3.44 (m, 8H), 2.88 (t, J = 6.4 Hz, 2H), 2.45 - 2.58 (m, 6H), 1.99 -2.12 (m, 12H), 1.32 - 1.59 (m, 45H), 0.98 - 1.19 (m, 9H).

[0214] 7. Synthesis of YK-2207

[0215]

[0216] MSA-2 (200 mg, 0.68 mmol) was dissolved in THF (5 mL), YK-108 (reference patent CN116178193B paragraph

[0447] synthesis) (435 mg, 0.68 mmol) was weighed in turn and added, EDCI (156 mg, 0.82 mmol), DMAP (8 mg, 0.07 mmol), and the reaction was stirred at room temperature for 12 hours. After the reaction was completed, it was rotary evaporated, wet loaded, and purified by silica gel chromatography (0%-30% dichloromethane / ethyl acetate) to obtain the product YK-2207 (387 mg, 0.42 mmol, 62.1%). C 53 H 89 NO9S, MS (ES): m / z (M + H) + = 916.83.

[0217] YK-2207: 1 H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 7.56 (s, 1H), 7.02(s, 1H), 4.23 - 4.52 (m, 4H), 4.13 (s, 6H), 3.91 - 4.01 (m, 10H), 3.46 - 3.55(m, 10H), 3.04 - 3.12 (m, 6H), 1.55 - 1.81 (m, 41H), 0.88 - 0.94 (m, 9H).

[0218] 8. Synthesis of YK-2208

[0219]

[0220] MSA-2 (200 mg, 0.68 mmol) was dissolved in THF (5 mL), YK-107 (reference patent CN116178193B paragraph

[0437] synthesis) (454 mg, 0.68 mmol) was weighed in turn and added, EDCI (156 mg, 0.82 mmol), DMAP (8 mg, 0.07 mmol), and the reaction was stirred at room temperature for 12 hours. After the reaction was completed, it was rotary evaporated, wet loaded, and purified by silica gel chromatography (0%-30% dichloromethane / ethyl acetate) to obtain the product YK-2208 (403 mg, 0.43 mmol, 62.8%). C 55 H 93 NO9S, MS (ES): m / z (M + H) + = 944.77.

[0221] YK-2208: 1 H NMR (400 MHz, CDCl3) δ 8.88 (s, 1H), 8.04 (s, 1H), 7.76(s, 1H), 4.82 - 4.99 (m, 4H), 4.57 (s, 8H), 4.12 - 4.32 (m, 8H), 3.46 - 3.58(m, 8H), 3.13 (t, J = 6.4 Hz, 2H), 2.66 - 2.88 (m, 6H), 2.31 - 2.52 (m, 6H),1.89 - 2.03 (m, 39H), 1.15 - 1.29 (m, 9H).

[0222] 9. Synthesis of YK-2209

[0223]

[0224] Step 1: Synthesis of YK-2209-PM1

[0225] Dissolve 2-hexyl-decanol (1 g, 4.1 mmol) in DCM (10 mL), add 6-bromohexanoic acid (801 mg, 4.1 mmol), EDCI (950 mg, 4.95 mmol), DMAP (50 mg, 0.41 mmol) successively, stir at room temperature for 5 hours. After the reaction is completed, spin dry, wet loading, and purify by silica gel chromatography (0% - 10% petroleum ether / ethyl acetate) to obtain the product YK-2209-PM1 (1.5 g, 3.6 mmol, 86.9%).

[0226] Step 2: Synthesis of YK-2209-PM2

[0227] Dissolve YK-2209-PM1 (1.5 g, 3.6 mmol) in acetonitrile (15 mL), add YK-004-PM2 (synthesized according to the patent CN114044741B paragraph

[0227] ) (1.0 g, 3.6 mmol), potassium carbonate (1.5 g, 10.8 mmol), potassium iodide (60 mg, 0.36 mmol) successively, stir at 70°C for 10 hours. After the reaction is completed, filter, wet loading, and purify by silica gel chromatography (0% - 15% dichloromethane / methanol) to obtain the product YK-2209-PM2 (1.2 g, 1.9 mmol, 53.5%).

[0228] Step 3: Synthesis of YK-2209

[0229] MSA-2 (200 mg, 0.68 mmol) was dissolved in THF (5 mL), YK-2209-PM2 (425 mg, 0.68 mmol), EDCI (156 mg, 0.82 mmol), DMAP (8 mg, 0.07 mmol) were added sequentially, and the reaction was stirred at room temperature for 14 hours. After the reaction was completed, it was rotary evaporated, wet loaded, and purified by silica gel chromatography (0% - 30% dichloromethane / ethyl acetate) to obtain the product YK-2209 (420 mg, 0.47 mmol, 68.5%). 52 H 87 NO9S, MS (ES): m / z (M + H) + = 902.81.

[0230] YK-2209: 1 H NMR (400 MHz, CDCl3) δ 7.95 (s, 1H), 7.55 (s, 1H), 7.37(s, 1H), 4.21 - 4.35 (m, 6H), 3.99 (s, 4H), 3.54 - 3.71 (m, 6H), 2.84 - 3.01(m, 6H), 2.34 - 2.51 (m, 4H), 1.93 - 2.12 (m, 12H), 1.45 - 1.65 (m, 37H),1.04 (s, 9H).

[0231] 10. Synthesis of YK-2210

[0232]

[0233] Step 1: Synthesis of YK-2210-PM1

[0234] Compound 27-PM1 (1.0 g, 2.3 mmol) was dissolved in acetonitrile (15 mL), YK-004-PM2 (reference patent CN114044741B paragraph

[0227] synthesis) (665 mg, 2.3 mmol), potassium carbonate (959 mg, 6.9 mmol), potassium iodide (38 mg, 0.23 mmol) were added sequentially, and the reaction was stirred at 70°C for 10 hours. After the reaction was completed, it was filtered, wet loaded, and purified by silica gel chromatography (0% - 15% dichloromethane / methanol) to obtain the product YK-2210-PM1 (800 mg, 1.3 mmol, 54.1%).

[0235] Step 2: Synthesis of YK-2210

[0236] MSA-2 (200 mg, 0.68 mmol) was dissolved in THF (5 mL), YK-2210-PM1 (435 mg, 0.68 mmol), EDCI (156 mg, 0.82 mmol), DMAP (8 mg, 0.07 mmol) were added sequentially, and the reaction was stirred at room temperature for 10 hours. After the reaction was completed, it was spin-dried, wet-loaded, and purified by silica gel chromatography (0% - 30% dichloromethane / ethyl acetate) to obtain the product YK-2210 (380 mg, 0.42 mmol, 61.0%). C 53 H 89 NO9S, MS (ES): m / z (M + H) + = 916.81.

[0237] YK-2210: 1 H NMR (400 MHz, CDCl3) δ 8.75 (s, 1H), 7.99 (s, 1H), 7.39(s, 1H), 4.71 (t, J = 4.8 Hz, 2H), 4.02 (t, J = 4.8 Hz, 2H), 3.82 (s, 6H),3.45 - 3.62 (m, 4H), 3.16 - 3.28 (m, 6H), 2.81 (t, J = 4.0 Hz, 2H), 2.41 -2.53 (m, 4H), 2.02 - 2.30 (m, 6H), 1.78 - 1.93 (m, 45H), 0.99 - 1.19 (m, 9H).

[0238] 11. Synthesis of Reference 1

[0239] Reference 1 was synthesized according to the following route:

[0240]

[0241] Step 1: Synthesis of Reference-PM1

[0242] n-Undecanol (705 mg, 4.1 mmol) was dissolved in DCM (10 mL), 6-bromohexanoic acid (801 mg, 4.1 mmol), EDCI (950 mg, 4.95 mmol), DMAP (50 mg, 0.41 mmol) were added sequentially, and the reaction was stirred at room temperature for 5 hours. After the reaction was completed, it was spin-dried, wet-loaded, and purified by silica gel chromatography (0% - 10% petroleum ether / ethyl acetate) to obtain the product Reference-PM1 (1.2 g, 3.4 mmol, 82.9%).

[0243] Step 2: Synthesis of Control-PM2

[0244] Control-PM1 (1.2 g, 3.4 mmol) was dissolved in acetonitrile (12 mL), and ethanolamine (210 mg, 3.4 mmol), potassium carbonate (1.5 g, 10.8 mmol), potassium iodide (60 mg, 0.36 mmol) were added sequentially, and stirred at 70 °C for 10 hours. After the reaction was completed, it was filtered, and wet loaded, and purified by silica gel chromatography (0% - 15% dichloromethane / methanol) to give the product Control-PM2 (700 mg, 2.1 mmol, 61.8%).

[0245] Step 3: Synthesis of Control-PM3

[0246] Synthesized according to the synthesis method of YK-2209-PM1, except that 2-hexyl-decanol and 6-bromohexanoic acid were replaced by 9-heptadecanol and 8-bromooctanoic acid, to give 1.4 g of Control-PM3.

[0247] Step 4: Synthesis of Control-PM4

[0248] Synthesized according to the synthesis method of YK-2209-PM2, except that YK-2209-PM1 and YK-004-PM2 were replaced by Control-PM3 and Control-PM2, to give 800 mg of Control-PM4.

[0249] Step 5: Synthesis of Control 1

[0250] Synthesized according to the synthesis method of YK-2209, except that YK-2209-PM2 was replaced by Control-PM4, to give 360 mg of Control 1.

[0251] 12. Synthesis of Control SAL-8

[0252]

[0253] SAL-8 is a compound in patent document CN118891043A (paragraph

[0383] ), and was synthesized according to the synthesis method in paragraph

[0368] to give 230 mg of Control SAL-8.

[0254] Example 2: Preparation of mRNA

[0255] A) Preparation of Fluc DNA, OVA DNA template

[0256] 1) Construct the luciferase (Luciferase protein CDS), ovalbumin (OVA) circular plasmid on the pVAX1 vector (purchased from Thermo Fisher Scientific) by EcoRV enzyme digestion enzyme connection;

[0257] 2) Take the plasmid constructed on the pVAX1 vector in step 1) and mix with 50 μL of E. coli competent cells Stbl2 (purchased from Thermo Fisher Scientific) uniformly, then ice bath for 30 minutes, 42 ℃ heat shock for 90 seconds, immediately put back on ice, ice bath for 2 minutes;

[0258] 3) Add 400 μL of LB medium (purchased from Thermo Fisher Scientific), and cultivate at 30 ℃ on a shaker for 45-60 minutes;

[0259] 4) Take 50-100 μL of bacterial solution and spread on LB solid medium containing kanamycin antibiotic (100 μg / mL, purchased from Yixing Biotechnology Co., Ltd.), and cultivate at 37 ℃ overnight;

[0260] 5) The obtained monoclonal colonies are plated for sequencing to verify their correctness, and the monoclonal colonies with correct sequencing are selected and cultivated at 30 ℃ on a shaker overnight;

[0261] 6) Use the endotoxin-free plasmid extraction kit (Yixing Biotechnology Co., Ltd., item number 19036ES10) to extract the plasmid;

[0262] 7) The obtained plasmid is digested with restriction enzymes to form a linearized plasmid as a transcription template, and the specific digestion process steps are shown in steps ①-③.

[0263] Step 1) Take 1 mg of luciferase circular plasmid, and digest (BspQ I enzyme, purchased from Yixing Biotechnology Co., Ltd.) at 37 ℃ for 4 hours to form a linearized DNA transcription template (enzyme digestion system is shown in Table 1);

[0264] Table 1 Enzyme digestion reaction system

[0265]

[0266] Step 2) After the reaction is completed, add anhydrous ethanol and sodium acetate in the order of V 酶切反应产物 :V 无水乙醇 :V 3M醋酸钠 =1:3:1, and place at -20 ℃ for 1 hour, and then centrifuge at 12000 rpm to reserve the precipitate;

[0267] Step 3) Wash the precipitate in step 2) with 70% ethanol for 2 times, and dry the centrifuged substance at 55 ℃ for 10 minutes, and then add 1.7 mL of water for injection to dissolve;

[0268] The concentration of the linearized plasmid in the dissolution solution was 500 ng / µL, the linearization ratio was more than 90%, and the purification recovery rate was 85%.

[0269] B) Preparation of Fluc mRNA and OVA mRNA

[0270] 1) Co-transcription capping reaction:

[0271] Fluc DNA and OVA DNA prepared in A) were used as templates, NTP solution (NTPs) and Cap1 cap analog (product number: 10678ES80, purchased from Yixing Biotechnology Co., Ltd.) were used as starting materials, and mRNA was synthesized by T7 RNA polymerase transcription. The specific reaction system is shown in Table 2. The prepared reaction system was placed in a 37 ℃ constant temperature incubator and shaken 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.

[0272] Table 2 Co-transcription capping reaction system

[0273]

[0274] Note: The above reagents are purchased from Yixing Biotechnology Co., Ltd.

[0275] 2) Digestion of template DNA:

[0276] DNase I (purchased from Yixing Biotechnology Co., Ltd.) was added to the co-transcription capping reaction system of the above step 1) to make the final concentration 1 U / µg of linearized plasmid, mixed, centrifuged, and placed at 37℃ for 1 hour to obtain the co-transcription capping product.

[0277] 3) Lithium chloride precipitation method for purification:

[0278] The co-transcription capping product obtained in the above step 2) was purified by lithium chloride precipitation method as follows:

[0279] 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 2.8M, and precipitate at low temperature for 2 hours;

[0280] Step ② Precipitation: High-speed centrifugation at 12000 rmp for 15 minutes, and keep the precipitate;

[0281] Step 3: Washing: Wash twice with 75% ethanol, and dissolve with RNase-free water to obtain mRNA solution. The purified mRNA solution is stored at -80°C.

[0282] Example 3: Effect of the ratio of adjuvant lipids on the particle size and PDI of TLP-mRNA pharmaceutical composition

[0283] The ADOPE, DOTMA, DOPE and YK-2201 are weighed according to the proportions in Table 3, dissolved in ethanol, and an ethanol-lipid complex solution (total lipid concentration of 266 mM) is prepared. The ethanol-lipid complex solution is quickly added to RNase-free water under stirring at a speed of 120 rpm, and the mixture is stirred at room temperature for 30 minutes. The resulting mixture is filtered through a polycarbonate membrane with a pore size of 450 nm to obtain a lipid composition solution, which is stored at 4-8°C.

[0284] The mRNA obtained in Example 2 is diluted in 10 mM HEPES buffer containing 0.1 mM EDTA to obtain an aqueous mRNA solution with a concentration of 0.5 mg / mL. An aqueous sodium chloride solution (0.9% w / w) is taken with a syringe and injected into the above prepared aqueous mRNA solution to obtain an aqueous sodium chloride solution of mRNA.

[0285] The ethanol-lipid complex solution is injected into the above aqueous sodium chloride solution of mRNA with a syringe, vortexed for 30 seconds, and incubated at room temperature for 10 minutes to obtain a TLP-mRNA pharmaceutical composition (final RNA concentration of 100 μg / mL), which is stored at 4-8°C. The amounts of mRNA and lipid complex solution are used to obtain a TLP-mRNA composition with a charge ratio of 1:2, and the calculation method is as follows:

[0286] The charge ratio of the TLP-mRNA pharmaceutical composition = (the positive charge molar amount of the permanent cationic lipid - the negative charge molar amount of the permanent anionic lipid) / (the weight of mRNA (g) divided by the average molecular weight of the base 330 (g / mol)).

[0287] The TLP-mRNA pharmaceutical composition is diluted with an aqueous sodium chloride solution of 0.9% w / w at a volume ratio of 1:5, and the particle size and polydispersity index (PDI) are measured by dynamic light scattering using a Malvern laser particle size analyzer.

[0288] Table 3 Particle size and PDI of TLP-mRNA pharmaceutical composition with different formulations

[0289]

[0290] The results show that, by adding YK-2201 in an amount of 2.5 mol%, 5.0 mol%, 10.0 mol%, 15.0 mol%, 20.0 mol% of total lipids, to partially replace the permanent cationic lipid DOTMA in the TLP composition, the particle size and PDI of the obtained TLP-mRNA drug composition are both within the qualified range (the qualified range refers to a particle size of 220-500 nm and a PDI value <0.5).

[0291] The following examples detect the properties of TLP-mRNA drug compositions prepared with other adjuvant lipids or TLP-mRNA drug compositions without adjuvant lipids at an adjuvant lipid proportion of 10 mol%.

[0292] Example 4: Effect of different adjuvant lipids on the particle size and PDI of TLP-mRNA drug compositions

[0293] According to the preparation method of Example 3, adjuvant lipids YK-2201 ~ YK-2210, control 1 and SAL-8 are used to partially replace DOTMA in TLP, that is, the permanent anionic lipid ADOPE, the permanent cationic lipid DOTMA, the neutral lipid DOPE and the adjuvant lipid are prepared into TLP-mRNA drug compositions at a molar ratio of 5:8:5:2 (that is, the molar percentages of the permanent anionic lipid, the permanent cationic lipid, the neutral lipid and the adjuvant lipid in the lipid composition are 25 mol%, 40 mol%, 25 mol% and 10 mol%, respectively) at a proportion of 10 mol% of total lipids, and the results are shown in Table 4.

[0294] Table 4 Particle size and PDI of TLP-mRNA drug compositions containing different adjuvant lipids

[0295]

[0296] In Table 4, No. 11 does not add the adjuvant lipids of the present application, also known as the “TLP” group. TLP is an mRNA drug composition prepared according to a method similar to Example 3, but without adding adjuvant lipids, with the molar percentages of the permanent anionic lipid ADOPE, the permanent cationic lipid DOTMA and the neutral lipid DOPE being 25 mol%, 50 mol% and 25 mol%, respectively.

[0297] The results show that the adjuvant lipids YK-2201 ~ YK-2210 and control 1 can all prepare mRNA drug compositions with qualified particle size and PDI (the particle size is controlled within 220-500 nm, and the PDI is less than 0.5) according to the preparation method of Example 2.

[0298] Example 5: Protein expression experiment in mice

[0299] The TLP-mRNA pharmaceutical composition prepared in Example 4 was injected into 4-6 week old female BALB / c albino mice weighing 17-19 g via tail vein (about 20 μg Fluc-mRNA per mouse), and 6 hours after administration, the fluorescent imaging substrate was injected into the mice by intraperitoneal injection, the mice were allowed to move freely for 5 minutes, and then the total radiation intensity of the protein expressed in the mice by the mRNA (corresponding to the expression intensity of the fluorescent protein, i.e. the amount of protein expression) was detected by IVIS Spectrum small animal live imaging instrument. After the sampling was completed, the mice were sacrificed by cervical dislocation and dissected, and the internal organs of the mice: liver, spleen were accurately separated. Since the mRNA molecule carries luciferase CDS, the total radiation intensity of the protein expressed in the mouse organs by Fluc-mRNA (corresponding to the expression intensity of the fluorescent protein, i.e. the amount of protein expression) can be detected by IVIS Spectrum small animal live imaging instrument. The results of live imaging of mice and detection of protein expression in liver and spleen are shown in Table 5 and Figure 1 .

[0300] Table 5 Data of mouse live and organ imaging experiments

[0301]

[0302] It can be seen that the TLP-mRNA pharmaceutical composition containing the adjuvant lipid of the present application can efficiently deliver mRNA to the spleen, and the delivery effect is significantly enhanced compared with the mRNA pharmaceutical composition packaged by the TLP composition without the addition of adjuvant lipid (No. 11). For example, the composition containing YK-2202, the total radiation intensity of the live and the total radiation intensity of the spleen are 1.9 times and 1.8 times that of the TLP composition without the addition of adjuvant, respectively.

[0303] Compared with the TLP-mRNA pharmaceutical composition containing the adjuvant lipid of the prior art (Control 1, Control SAL-8), the administration of the TLP-mRNA pharmaceutical composition containing YK-2201~YK-2210 of the present application significantly enhances the total radiation intensity of the spleen and the total radiation intensity of the live, for example, the total radiation intensity of the live of the composition containing YK-2202 is 3.1 times and 3.3 times that of the composition containing Control 1 and Control SAL-8, respectively, and the total radiation intensity of the spleen is 2.7 times and 3.2 times that of the composition containing Control 1 and Control SAL-8, respectively.

[0304] Example 6: Adjuvant lipid-containing TLP-mRNA pharmaceutical composition enhances expression of cytokines

[0305] Interferon-alpha (IFN-a) is an important cytokine, mainly produced by activated T cells and natural killer (NK) cells. It plays a key role in immune response, with anti-viral, anti-tumor, immune regulation and promotion of inflammatory response, etc. functions, and can treat a variety of diseases. Interleukin-12 (IL-12) is a multifunctional cytokine produced by dendritic cells, macrophages, B lymphocytes and other antigen-presenting cells, which plays an important role in regulating immune response, promoting Th1 type immunity, enhancing cytotoxicity and in tumor immunity. TNF-a, tumor necrosis factor alpha, is a ligand of the TNF superfamily. It is a pleiotropic cell molecule that plays a central role in inflammation, apoptosis and immune system development. By comparing the expression levels of cytokines IFN-a, IL-12 and TNF-a in different experimental groups of TLP-mRNA drug compositions, the effect of adjuvant lipids on the innate immunity triggered by the corresponding TLP-mRNA drug compositions can be reflected.

[0306] Experimental procedure:

[0307] In this example, 6-8 week old female C57BL / 6J mice were tail vein injected with OVA-mRNA drug composition prepared according to Example 4 (mRNA injection amount 40 µg per mouse) 6 hours later, and then euthanized by enucleation to obtain as much serum as possible. The content of IFN-a, IL-12 and TNF-a in the serum was measured by ELISA, and mice injected with the same volume of solution of lipid composition without encapsulated mRNA were set as blank control group.

[0308] ELISA assay: The levels of IFN-a, IL-12 and TNF-a in mouse serum were determined by standard ELISA according to the manufacturer's instructions.

[0309] Table 6 Expression levels of IFN-a, IL-12 and TNF-a in different experimental groups of TLP-mRNA drug compositions

[0310]

[0311] Experimental results:

[0312] As shown in Table 6 and Figure 2Compared with group 11, the mRNA drug compositions containing the adjuvant lipids of the present application in groups 1-10 had significant increases in IFN-a, IL-12 and TNF-a cytokines in serum 6 hours after injection, wherein the IFN-a levels of groups 1-10 were 1.5 times, 2.0 times, 1.6 times, 2.0 times, 1.9 times, 2.1 times, 1.6 times, 2.0 times, 1.8 times and 1.7 times those of group 11, respectively; the IL-12 levels of groups 1-10 were 2.4 times, 3.5 times, 2.6 times, 3.2 times, 3.0 times, 3.4 times, 2.5 times, 3.1 times, 3.0 times and 2.9 times those of group 11, respectively; and the TNF-a levels of groups 1-10 were 1.8 times, 2.3 times, 1.7 times, 2.5 times, 2.2 times, 2.1 times, 1.9 times, 2.1 times, 2.3 times and 2.2 times those of group 11, respectively.

[0313] Compared with the experimental groups (groups 12 and 13) of the adjuvant lipids of the prior art, groups 1-10 had significant increases in IFN-a, IL-12 and TNF-a cytokines in serum 6 hours after injection, wherein the IFN-a levels of groups 1-10 were 1.1 times, 1.5 times, 1.2 times, 1.4 times, 1.4 times, 1.5 times, 1.2 times, 1.4 times, 1.3 times and 1.3 times those of group 12, respectively; the IL-12 levels of groups 1-10 were 1.4 times, 2.0 times, 1.5 times, 1.8 times, 1.8 times, 2.0 times, 1.4 times, 1.8 times, 1.8 times and 1.7 times those of group 12, respectively; and the TNF-a levels of groups 1-10 were 1.1 times, 1.5 times, 1.1 times, 1.6 times, 1.4 times, 1.3 times, 1.2 times, 1.3 times, 1.4 times and 1.4 times those of group 12, respectively.

[0314] The experimental results show that by detecting the stimulation of IFN-a, IL-12 and TNF-a cytokines by the mRNA drug compositions containing the adjuvant lipids, it can be proved that the adjuvant lipids of the present application can significantly enhance the expression of cytokines and improve the corresponding innate immune improvement effect of the mRNA-TLP composition.

[0315] Example 7: Therapeutic effect of TLP-mRNA drug compositions containing adjuvant lipids on tumor-bearing mouse models

[0316] Experimental process:

[0317] 1) Establishment of B16F10-OVA mouse model: 6 to 8 weeks old female C57BL / 6J mice were acclimated for at least three days prior to the start of the study. The mice had free access to food and sterile water and were housed in a 12-hour light / dark cycle at 22 °C ± 2 °C and 55% ± 15% relative humidity. B16F10-OVA cells were cultured in complete medium as described in the instructions at 5% CO2, 37 °C. Cells were collected using 0.25% trypsin-EDTA, resuspended in Dulbecco's Phosphate Buffered Saline (DPBS), and then about 2 × 10 5 cells / 100 μL / mouse (B16F10-OVA) were transplanted into female C57BL / 6J mice by subcutaneous (sc.) injection to establish a subcutaneous B16F10-OVA tumor model, and the mice were inoculated with vaccines when the tumor volume reached 30-50 mm 3 around.

[0318] 2) Inoculation: For the B16F10-OVA mouse model, C57BL / 6J mice enrolled were administered with the pharmaceutical composition containing adjuvant lipids prepared in Example 4 (the pharmaceutical compositions containing adjuvant lipids YK-2202, YK-2204, YK-2206, YK-220 were named YK-2202-TLP group, YK-2204-TLP group, YK-2206-TLP group, YK-2208-TLP group, respectively) and the pharmaceutical composition without adjuvant lipids (named TLP group). The inoculation method was to inoculate the mice by tail vein injection (each mouse was injected with 40 μg of therapeutic mRNA-OVA-containing vaccine each time) at 3, 7, 10, 12, 14, 17 days after cell injection (day 0), and mice inoculated with the same volume of solution of the lipid composition without encapsulated mRNA were set as the blank control group, with 8 mice in each group in parallel.

[0319] 3) Tumor size and mouse survival rate: The tumor diameter was measured 3 times a week starting from the 7th day after tumor inoculation. The tumor volume of C57BL / 6J mice was calculated according to the following formula: V (mm 3 )=x×y 2 / 2, unit: mm, where V represents the tumor volume, x represents the long diameter of the tumor, and y represents the short diameter of the tumor. The body weight change of C57BL / 6J mice was recorded with an electronic balance 3 times a week, and the survival rate was calculated.

[0320] Experimental results:

[0321] As Figure 3As shown in Table 7, on day 10 after tumor inoculation, mice in both the blank control group and the group containing the adjuvant-free OVA mRNA-TLP composition entered the rapid tumor growth phase, but the growth rate of the TLP group was much lower than that of the blank control group. In contrast, the OVA mRNA drug composition groups containing adjuvant lipids all showed significant tumor growth delay.

[0322] Starting on day 14, compared with the TLP group, the mRNA drug compositions containing adjuvant lipids also showed stronger tumor growth inhibition, and the mRNA drug compositions containing YK-2202, YK-2204, YK-2206 and YK-2208 all showed relatively better tumor inhibition effects.

[0323] In terms of survival rate, such as Figure 4 As shown in Table 8, mice in the blank control group began to die on day 17 after tumor inoculation, and all died by day 20; mice in the TLP group began to die on day 25, and all died by day 33; mice in the TLP-mRNA drug combination group containing adjuvant lipids YK-2202, YK-2204, YK-2206, and YK-2208 began to die on days 33, 30, 31, and 30, respectively, and all died on days 44, 40, 43, and 41, respectively. It can be seen that compared with the experimental group without adjuvant lipids, the mice in the experimental group with adjuvant lipids had significantly longer survival time.

[0324] Table 7. Tumor growth in mice after drug administration

[0325]

[0326] Table 8. Survival rate of mice after drug administration

[0327]

[0328]

[0329] The above detailed description of this application is intended to enable those skilled in the art to understand and implement its contents, but it should not be construed as limiting the scope of protection of this application. All equivalent changes or modifications made in accordance with the spirit and essence of this application should be included within the scope of protection of this application.

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, wherein, The compound is any one of the following compounds: 、 、 、 、 or 。 2. A compound, or a pharmaceutically acceptable salt thereof, wherein, The compound is .

3. A compound, or a pharmaceutically acceptable salt thereof, wherein, The compound is .

4. A lipid composition comprising an adjuvant lipid, wherein the adjuvant lipid is the compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof.

5. The lipid composition of claim 4, wherein, The adjuvant lipid is present in the lipid composition at a molar content of 2.5-20%.

6. The lipid composition of claim 4, wherein, The lipid composition further comprises a permanently anionic lipid, a neutral lipid, and a permanently cationic lipid.

7. The lipid composition of claim 4, wherein, The molar percentage of the adjuvant lipid, the permanently anionic lipid, the permanently cationic lipid, and the neutral lipid is (2.5-20) : (10-33) : (20-60) : (20-40).

8. The lipid composition according to claim 6 or 7, wherein, The lipid composition satisfies any one or a combination of at least two of the following conditions (1) to (4): (1) the permanently anionic lipid is any one or a combination of at least two of the group consisting of 2-acetylaminoethyl((R)-2,3-bis(oleoyloxy)propyl)phosphonate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylthioureido)ethyl)phosphonate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylureido)ethyl)phosphonate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-propylureido)ethyl)phosphonate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-butylureido)ethyl)phosphonate, and salts thereof; (2) the permanently cationic lipid is any one or a combination of at least two of the group consisting of 1,2-dioctadecenyloxy-3-methylammonium propane, (2,3-dioleoxypropyl)trimethylammonium, and salts thereof; (3) the neutral lipid is any one or a combination of at least two of the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, distearoylphosphatidylcholine, and salts thereof; (4) the molar percentage of the adjuvant lipid, the permanently anionic lipid, the permanently cationic lipid, and the neutral lipid is 10 : 25 : 40 :

25.

9. A pharmaceutical composition comprising the lipid composition of any one of claims 4-8 and an active ingredient; The active ingredient is any one or a combination of at least two of the group consisting of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.

10. The pharmaceutical composition of claim 9, wherein, The active ingredient is present in the pharmaceutical composition at a weight ratio to the lipid composition of 1:2 to 1:

5.

11. The pharmaceutical composition of claim 9, wherein, The active ingredient is a nucleic acid molecule capable of encoding one or more antigens.

12. The pharmaceutical composition of claim 11, wherein, The antigen is a disease-associated antigen, or the nucleic acid molecule or antigen is capable of eliciting an immune response against a disease-associated antigen or a cell expressing a disease-associated antigen.

13. The pharmaceutical composition according to claim 11 or 12, wherein, The nucleic acid molecule is an RNA encoding one or more antigens.

14. The pharmaceutical composition of claim 9, wherein, The pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients; or, the pharmaceutical composition further comprises one or more hydrophobic small molecules, penetration-enhancing molecules, carbohydrates, polymers, surface-altering agents, functionalized lipids, or cytokines.

15. Use of a compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, a lipid composition of any one of claims 4-8, or a pharmaceutical composition of any one of claims 9-14 in the manufacture of a medicament for delivering an active ingredient into antigen presenting cells in a target organ or tissue; the active ingredient is any one or a combination of at least two selected from the group consisting of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein.

16. The use according to claim 15, wherein, the target organ or tissue is any one or a combination of at least two selected from the group consisting of spleen, liver, lymph, muscle and lung; or, the antigen presenting cells are any one or a combination of at least two selected from the group consisting of B cells, NK cells, cDC cells, pDC cells and macrophages.

17. Use of a compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, a lipid composition of any one of claims 4-8, or a pharmaceutical composition of any one of claims 9-14 in the manufacture of a medicament.

18. The use according to claim 17, wherein, the medicament is any one or a combination of at least two selected from the group consisting of a nucleic acid drug, a genetic vaccine, a small molecule drug, a polypeptide drug and a protein drug.

19. The use of claim 17, wherein, the medicament is for treating a disease or disorder characterized by malfunction of an organ, a protein or a gene function, or abnormal activity of a protein or a polypeptide, or deficiency or hypofunction of a cytokine.

20. The use according to claim 19, wherein, the disease or disorder is any one or a combination of at least two selected from the group consisting of an infectious disease, a tumor and a proliferative disease, a genetic disease, an autoimmune disease, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, a metabolic disease, acquired immunodeficiency syndrome and immunosuppression after chemotherapy / radiotherapy.

21. The use of claim 19, wherein, the disease or disorder is diabetes or a congenital immunodeficiency disease.

22. The use of claim 19, wherein, the disease or disorder is congenital granulomatous disease.

23. The use of claim 20, wherein, the infectious disease is any one or a combination of at least two selected from the group consisting of a disease caused by a coronavirus, an influenza virus or an HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies and herpes; or, the tumor is any one or a combination of at least two selected from the group consisting of breast cancer, ovarian cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer, lymphoma, colon cancer, liver cancer, melanoma, bladder cancer, cervical cancer and prostate cancer.

24. The use of claim 17, wherein, the administration route of the medicament is intravenous, intramuscular, intradermal, subcutaneous, intranasal or inhalation; or, the administration dose of the medicament is 0.001-10 mg / kg.

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