STING activating adjuvant lipid, composition containing the same and use thereof

Abstract

The present invention relates to the technical field of vaccine adjuvants, and specifically to STING activating adjuvant lipids, compositions and uses thereof. The STING activating adjuvant lipid provided by the present invention includes a structure as shown in formula (I) or formula (II), which can be used as a vaccine adjuvant and can significantly enhance the therapeutic effect of mRNA vaccines or drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine adjuvants, and in particular to STING activating adjuvant lipids, compositions containing the same, and uses thereof. Background Art

[0002] In recent years, tumor immunotherapy based on messenger RNA (mRNA) has attracted widespread attention due to its significant clinical potential. Compared with DNA vaccines, mRNA can cause transient expression of tumor antigens while avoiding the possibility of insertion mutations. In addition, mRNA can improve the therapeutic effect 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. Inspired by the advantages of mRNA cancer vaccines, more than twenty mRNA-based immunotherapies have shown anti-tumor potential in preclinical and clinical studies.

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

[0004] Stimulator of interferon genes (STING) is a cytoplasmic pattern recognition receptor that is essential for spontaneously inducing anti-tumor T cell immunity. STING activators can assist mRNA nanovaccines in inducing the secretion of type I interferon (IFN-I), thereby promoting the cross-presentation of tumor antigens, the proliferation and activation of T lymphocytes, and the direct killing of tumors. However, the targeting of existing STING-activating vaccine adjuvant lipids still needs to be improved, especially the targeting of target organs and antigen-presenting cells in target organs is still insufficient, which makes it difficult to meet the needs of effective drug delivery and therapeutic efficiency. Therefore, the development of a new generation of STING-activating vaccine adjuvant lipids is of great significance for improving mRNA tumor therapy. Summary of the invention

[0005] In order to overcome the above problems existing in the prior art, the present invention provides a STING activating adjuvant lipid, a composition comprising the same and its use. The STING activating vaccine adjuvant lipid provided by the present invention has the advantages of simple preparation method, strong targeting, good activation effect on T cells, etc., and it has been verified that when it is used in mRNA anti-tumor vaccines, it can effectively improve the survival rate of mice.

[0006] In order to achieve the above object, the first aspect of the present invention provides a compound or its N-oxide compound, solvate, pharmaceutically acceptable salt or stereoisomer, wherein the compound comprises a structure represented by formula (I) or formula (II):

[0007] , ,

[0008] In formula (I) and formula (II):

[0009] L is unsubstituted C 2-5 Straight chain alkylene;

[0010] M1 is -OC(O)-, -C(O)OCH2-, -C(O)NHCH2- or -OCH2-;

[0011] M2 and M3 are -OC(O)- or -O-, respectively, and M2 and M3 are the same or different;

[0012] R1 is a substituted or unsubstituted N-containing heterocyclic ring;

[0013] R2 and R3 are unsubstituted C 16-24 In the straight chain alkenyl group, R2 and R3 are the same or different.

[0014] In the compounds of formula (I) or (II) provided by the present invention, L may be an unsubstituted C2 straight chain alkylene group (i.e., ethylene), a C3 straight chain alkylene group (i.e., propylene), a C4 straight chain alkylene group (i.e., butylene) or a C5 straight chain alkylene group (i.e., pentylene).

[0015] In the above-mentioned compounds of formula (I) or formula (II) provided by the present invention, R2 may be C 16 Straight chain alkenyl, C 17 Straight chain alkenyl, C 18 Straight chain alkenyl, C 19 Straight chain alkenyl, C 20 Straight chain alkenyl, C 21 Straight chain alkenyl, C 22 Straight chain alkenyl, C 23 Straight chain alkenyl or C 24 Straight chain alkenyl.

[0016] In the above-mentioned compounds of formula (I) or formula (II) provided by the present invention, R3 can be C 16 Straight chain alkenyl, C 17 Straight chain alkenyl, C 18 Straight chain alkenyl, C 19 Straight chain alkenyl, C 20 Straight chain alkenyl, C 21 Straight chain alkenyl, C 22 Straight chain alkenyl, C 23 Straight chain alkenyl or C24 Straight chain alkenyl.

[0017] In the present invention, there is no particular limitation on the number and position of carbon-carbon double bonds in R2 and R3, as long as the number of carbon atoms meets the above requirements. For example, the number of carbon-carbon double bonds in R2 and R3 can be 1, 2 or 3.

[0018] The unsubstituted N-containing heterocycle refers to a cyclic group containing N atoms, for example, a (hetero)cyclic group formed by replacing at least one C with N on the basis of a cycloalkyl group. The N-containing heterocycle may contain 1-3 N atoms, preferably the number of C atoms in the N-containing heterocycle is not less than 3, preferably 4-8. In some embodiments, the N-containing heterocycle may also contain other elements besides C and N, such as S, O, etc. Preferably, the N in the N-containing heterocycle is connected to L in the compound of formula (I) or formula (II).

[0019] According to a preferred embodiment of the present invention, L is -(CH2)2- or -(CH2)3-.

[0020] According to a preferred embodiment of the present invention, M1 is -OC(O)-, -C(O)OCH2-, -C(O)NHCH2- or -OCH2-.

[0021] According to a preferred embodiment of the present invention, M2 is -OC(O)- or -O-.

[0022] According to a preferred embodiment of the present invention, M3 is -OC(O)- or -O-.

[0023] Preferably, M2 and M3 are the same or different.

[0024] According to a preferred embodiment of the present invention, wherein R1 is or .

[0025] According to a preferred embodiment of the present invention, wherein R2 is an unsubstituted C 16-20 Straight chain alkenyl.

[0026] According to a preferred embodiment of the present invention, wherein R3 is an unsubstituted C 16-20 Straight chain alkenyl.

[0027] According to some preferred embodiments of the present invention, the compound has any one of the following structures of YK-1301 to YK-1309:

[0028] ,

[0029] ,

[0030] ,

[0031] ,

[0032] ,

[0033] ,

[0034] ,

[0035] ,

[0036] .

[0037] According to some particularly preferred embodiments of the present invention, the compound has any one of the following structures of YK-1301 to YK-1304:

[0038] , ,

[0039] , .

[0040] The second aspect of the present invention provides a lipid composition comprising an adjuvant lipid, wherein the adjuvant lipid comprises the compound described in the first aspect, or its N-oxide compound, solvate, pharmaceutically acceptable salt or stereoisomer.

[0041] In the lipid composition provided by the present invention, the adjuvant lipid can be provided only by the compound of the first aspect, or its N-oxide compound, solvate, pharmaceutically acceptable salt or stereoisomer, or the adjuvant lipid can also include other compounds commonly used as adjuvant lipids in the art.

[0042] According to a preferred embodiment of the present invention, the molar percentage of the adjuvant lipid in the lipid composition is 2.5-20%. That is, based on the total molar amount of various lipids contained in the lipid composition, the content of the adjuvant lipid accounts for 2.5-20%.

[0043] For example, the molar percentage of adjuvant lipid in the lipid composition can be 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, or a range consisting of any two of the above values, or any intermediate value in the range.

[0044] Preferably, the molar percentage of the adjuvant lipid in the lipid composition is 5-15%.

[0045] According to a preferred embodiment of the present invention, the molar percentage of the adjuvant lipid in the lipid composition is 10%.

[0046] According to a preferred embodiment of the present invention, the lipid composition further comprises a first lipid composition of permanent anionic lipids, permanent cationic lipids and neutral lipids.

[0047] In the lipid composition provided by the present invention, the first lipid composition may contain only permanent anionic lipids, permanent cationic lipids and neutral lipids, or may further contain other types of lipids (but not adjuvant lipids or lipid compounds and derivatives thereof that can act as adjuvants).

[0048] In the lipid composition provided by the present invention, the first lipid composition (mainly) has an organ / tissue / cell targeting function, and preferably the first lipid composition can also have a certain function of inducing an immune response against an antigen.

[0049] In some preferred embodiments of the present invention, the first lipid composition can be provided by the "lipid composition targeting antigen presenting cells" provided in CN202410420862.3, and the entire content of this application is incorporated herein by reference.

[0050] In the lipid composition provided by the present invention, the adjuvant lipid and the first lipid composition cooperate with each other, so that the lipid composition has good organ / tissue / cell targeting and also has a strong immune cell activation effect.

[0051] In order to ensure that the lipid composition has good targeting and good immune cell activation function, according to a preferred embodiment of the present invention, the molar percentage ratio of the adjuvant lipid, permanent anionic lipid, permanent cationic lipid and neutral lipid in the lipid composition is 2.5-20:10-33:40-60:20-40.

[0052] According to a preferred embodiment of the present invention, the permanent anionic lipid is selected from any one of the group consisting of 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylthioureido)ethyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylureido)ethyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-propylureido)ethyl) phosphate and (R)-2,3-bis(oleoyloxy)propyl-(2-(3-butylureido)ethyl) phosphate and their salts, or a combination of at least two of them.

[0053] According to a preferred embodiment of the present invention, the permanent cationic lipid is selected from any one or a combination of at least two of the group consisting of 1,2-dioctadecenyloxy-3-methylammonium propane, (2,3-dioleyloxypropyl)trimethylammonium and salts thereof.

[0054] According to a preferred embodiment of the present invention, 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.

[0055] It should be understood that when preparing drugs, the compound provided in the first aspect of the present invention, or its N-oxide compound, solvate, pharmaceutically acceptable salt or stereoisomer can be used alone as an adjuvant lipid, or the lipid composition provided in the second aspect of the present invention can be used as an adjuvant lipid.

[0056] The third aspect of the present invention provides a pharmaceutical composition, which comprises the lipid composition described in the second aspect and an active ingredient;

[0057] The active ingredient is selected from any one of the group consisting of nucleic acid molecules, small molecule compounds, polypeptides or proteins, or a combination of at least two of them.

[0058] According to a preferred embodiment of the present invention, the dosage of the active component and the lipid composition is such that the charge ratio of positive charge to negative charge in the pharmaceutical composition is 1:2 to 1:5.

[0059] According to a preferred embodiment of the present invention, the active ingredient is a nucleic acid molecule capable of encoding one or more antigens.

[0060] According to a preferred embodiment of the present invention, the antigen is a disease-associated antigen, or the nucleic acid molecule or antigen can induce an immune response against the disease-associated antigen or a cell expressing the disease-associated antigen.

[0061] According to a preferred embodiment of the present invention, the nucleic acid molecule is RNA encoding one or more antigens.

[0062] According to a preferred embodiment of the present invention, the pharmaceutical composition further comprises at least one auxiliary component. The auxiliary component can be any auxiliary agent or excipient that can be used for drug preparation, especially for biopharmaceutical preparation, in the art.

[0063] According to a preferred embodiment of the present invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

[0064] According to a preferred embodiment of the present invention, the pharmaceutical composition further comprises one or more hydrophobic small molecules, permeability enhancing molecules, carbohydrates, polymers, surface altering agents, functionalized lipids or cytokines.

[0065] The fourth aspect of the present invention provides the compound described in the first aspect, or its N-oxide compound, solvate, pharmaceutically acceptable salt or stereoisomer; the lipid composition described in the second aspect; or the use of the pharmaceutical composition described in the third aspect in delivering active ingredients to antigen presenting cells in target organs or tissues;

[0066] The active ingredient is selected from any one of the group consisting of nucleic acid molecules, small molecule compounds, polypeptides or proteins, or a combination of at least two of them.

[0067] According to a preferred embodiment of the present invention, 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.

[0068] Preferably, the target organ or tissue is the spleen.

[0069] According to a preferred embodiment of the present invention, the antigen presenting cells are selected from any one or a combination of at least two of the group consisting of B cells, NK cells, cDC cells, pDC cells and macrophages.

[0070] It should be understood that the above-mentioned uses provided by the present invention may include both therapeutic and diagnostic uses as well as non-therapeutic and non-diagnostic uses. For example, therapeutic uses may include using the compounds provided by the present invention, or their N-oxygen compounds, solvates, pharmaceutically acceptable salts or stereoisomers, or using the lipid compositions provided by the present invention to package (drug) active ingredients and deliver them to target organs / tissues / cells, or using the pharmaceutical compositions of the present invention to deliver the active ingredients contained therein to target organs / tissues / cells, thereby achieving the effects of treating diseases, improving symptoms, regulating physiological activities in the body, etc.; diagnostic uses may include packaging active ingredients for disease diagnosis in the compounds provided by the present invention, or their N-oxygen compounds, solvates, pharmaceutically acceptable salts or stereoisomers, or lipid compositions, so that the active ingredients are delivered to target organs / tissues / cells, thereby achieving the purpose of disease diagnosis; non-therapeutic / non-diagnostic purposes may include using the compounds provided by the present invention, or their N-oxygen compounds, solvates, pharmaceutically acceptable salts or stereoisomers, or using the lipid compositions provided by the present invention to encapsulate active ingredients and deliver them to target organs / tissues / cells for non-therapeutic, non-diagnostic purposes such as scientific research and detection (such as disease mechanism research, drug action mechanism research, new drug development, drug screening, etc.).

[0071] The fifth aspect of the present invention provides the compound described in the first aspect, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer; the lipid composition described in the second aspect; or the use of the pharmaceutical composition described in the third aspect in the preparation of a drug.

[0072] According to a preferred embodiment of the present invention, the medicament is used for treating and / or preventing a disease or disorder in a subject in need thereof.

[0073] According to a preferred embodiment of the present invention, the drug is selected from any one of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide drugs and protein drugs, or a combination of at least two of them.

[0074] According to a preferred embodiment of the present invention, the disease or disorder is characterized by malfunction of an organ, protein or gene, or abnormal activity of a protein or polypeptide.

[0075] According to a preferred embodiment of the present invention, the disease or condition is selected from any one or a combination of at least two of the group consisting of infectious diseases, tumors and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases and metabolic diseases.

[0076] According to a preferred embodiment of the present invention, the infectious disease is selected from any one or a combination of at least two of the group consisting of diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies and herpes.

[0077] According to a preferred embodiment of the present invention, the tumor is selected from any one or a combination of at least two of the group consisting of breast cancer, ovarian cancer, lung cancer, pancreatic cancer, kidney cancer, gastric cancer, lymphoma, colon cancer, liver cancer, melanoma, bladder cancer, cervical cancer and prostate cancer.

[0078] According to a preferred embodiment of the present invention, the subject is a mammal.

[0079] According to a preferred embodiment of the present invention, the mammal is any one selected from the group consisting of humans, non-human primates, companion animals, exotic species, livestock animals and edible animals.

[0080] According to a preferred embodiment of the present invention, the drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

[0081] According to a preferred embodiment of the present invention, the drug is administered intravenously or intramuscularly.

[0082] According to a preferred embodiment of the present invention, the dosage of the drug is 0.001-10 mg / kg.

[0083] The present invention further provides a method for treating and / or preventing a disease, which comprises administering a drug in a therapeutically effective amount to a subject in need thereof, wherein the drug comprises the compound described in the first aspect, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer; the lipid composition described in the second aspect; or the pharmaceutical composition described in the third aspect.

[0084] In some preferred embodiments of the present invention, the drug can be a drug encapsulated by a carrier, wherein the carrier includes the compound described in the first aspect, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer; or the carrier includes the lipid composition described in the second aspect.

[0085] The beneficial effects of this application include at least:

[0086] The adjuvant lipid compound and lipid composition provided by the present invention can be used for encapsulating active ingredients of biological drugs such as nucleic acids (such as mRNA, etc.) to form corresponding biological drugs.

[0087] Taking a nucleic acid drug with mRNA as an active ingredient as an example, the mRNA composition prepared by adding the adjuvant lipid provided by the present invention to the lipid composition has at least the following advantages:

[0088] 1. Good particle size and uniform particle distribution;

[0089] 2. Can significantly increase the protein expression of antigens in the body, muscle or spleen of subjects (such as mice);

[0090] 3. The percentage of antigen-presenting cells expressing antigens in the spleen was significantly increased;

[0091] 4. Significantly increase the maturity of antigen-presenting cells in the spleen;

[0092] 5. Significantly increase the activation state of T cells in the spleen;

[0093] 6. The volume of tumors was significantly reduced and the survival rate of mice was significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings of the present invention will be briefly introduced below. It should be understood that the drawings in the following description only relate to some specific embodiments of the present invention, rather than limiting the present invention.

[0095] Figure 1 The total fluorescence intensity of the living mice and their organs 6 hours after intravenous injection of Fluc-mRNA pharmaceutical compositions prepared with YK-1301 to YK-1309, compound 120, compound 45, E-Ac7-Dog, compound 4, and compound 6.2.c, or Fluc-mRNA-TLP compositions without adjuvant lipids into mice.

[0096] Figure 2 This is the blank control group and the mouse spleen cell flow cytometry experiment diagram (eGFP) 24 hours after intravenous injection of the eGFP-mRNA drug composition added with YK-1301 to YK-1304 respectively into the mouse body.

[0097] Figure 3 This is the flow cytometry experiment diagram (CD86) of mouse spleen cells in the blank control group and 24 hours after intravenous injection of the eGFP-mRNA pharmaceutical composition containing YK-1302 or compound 120 into mice.

[0098] Figure 4 The blank control group and the mouse spleen cells flow cytometry experiment (CD69) 24 hours after intravenous injection of the mRNA drug composition containing YK-1302, YK-1305 or E-Ac7-DOg into the mouse.

[0099] Figure 5 The stimulation of cytokines IFN-α, IL-12 and TNF-α in the serum of B16F10-OVA mice vaccinated with OVA mRNA pharmaceutical compositions supplemented with YK-1301 to YK-1304, Compound 120, or OVA mRNA-TLP compositions without adjuvant lipids.

[0100] Figure 6 Figure 2 shows the in vivo tumor growth of B16F10-OVA mice inoculated with OVA mRNA pharmaceutical compositions supplemented with YK-1301 to YK-1304 or OVA mRNA-TLP compositions without adjuvant lipids.

[0101] Figure 7 The survival rate of B16F10-OVA mice vaccinated with the OVA mRNA pharmaceutical composition supplemented with YK-1301 to YK-1304 or the OVA mRNA-TLP composition without adjuvant lipid. DETAILED DESCRIPTION

[0102] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described more clearly and completely in conjunction with the accompanying drawings of the present invention. Obviously, the specific implementation described is only a part of the implementation of the present invention, not all of the implementations. Based on the described implementation of the present invention, all other implementations obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0103] The present invention may be implemented in other specific forms without departing from the basic attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features of any other embodiment or multiple other embodiments to obtain other embodiments. The present invention includes other embodiments obtained by such combination.

[0104] All publications and patents mentioned in the present invention are hereby incorporated into the present invention by reference in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in the present invention, then the purposes and terms of the present invention shall prevail.

[0105] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0106] Unless otherwise specified, all technical and scientific terms used in the present invention have the common meanings in the field to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition in the present invention shall prevail.

[0107] Except in the examples or otherwise indicated, all numbers stating quantitative properties such as dosage in the specification and claims should be understood to be modified in all cases by the term "about". It should also be understood that any numerical range recited in the present invention is intended to include all subranges within the range and any combination of the various endpoints of the range or subrange.

[0108] In addition, when referring to a number or a numerical range, the term "about" means that the number or numerical range mentioned is within the typical tolerance range of the art, within the experimental variability or in the approximate value of the statistical experimental error, and therefore the number or numerical range can be, for example, between 1% and 15% of the number or numerical range. For example, "about" can be understood as about 2 standard deviations of the mean value, and when "about" is present before a series of numbers or ranges, it should be understood that "about" can modify each number in the numbers in the series or range.

[0109] The words "include", "contain" or "comprises" and the like used in the present invention mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unrecorded elements. The terms "contain" or "includes" used in the present invention may be open, semi-closed or closed. In other words, the term also includes "essentially consisting of" or "consisting of".

[0110] The term "pharmaceutically acceptable" in the present invention means that the compound or composition is chemically and / or toxicologically compatible with other ingredients constituting the formulation and / or with humans or mammals for preventing or treating diseases or disorders.

[0111] The term "adjuvant lipid" in the present invention refers to lipids (compounds or compositions) that can be used as adjuvants for drugs or vaccines. Adjuvant lipids play a conventional adjuvant role in drugs or vaccines, such as non-specifically changing or enhancing the body's specific immune response to antigens, playing an auxiliary role, etc. For example, adjuvant lipids can also play a role in reducing the amount of immune substances, improving the body's protective ability, and inducing a long-term, efficient specific immune response in the body.

[0112] The term "subject" or "patient" in the present invention may include mammalian subjects. For example, the mammalian subject may be selected from one or more of humans, non-human primates, companion animals, exotic species, livestock animals, and food animals.

[0113] The term "treatment" as used in the present invention refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of the disease to cure, alleviate, mitigate, improve or affect the disease or the symptoms of the disease. In the context of the present invention, unless otherwise specifically stated, the term "treatment" may also include prevention.

[0114] In the present invention, the term "antigen" includes any molecule, preferably a peptide or protein, containing at least one epitope capable of eliciting an immune response and / or an epitope to which an immune response is directed. Preferably, an antigen in the context of the present invention is a molecule that, optionally after processing, induces an immune response that is preferably specific for the antigen or a cell expressing the antigen. In particular, "antigen" relates to a molecule that, optionally after processing, is presented by an MHC molecule and specifically reacts with a T lymphocyte (T cell).

[0115] Therefore, the antigen or its fragment should be able to be recognized by the T cell receptor. Preferably, if recognized by the T cell receptor, the antigen or fragment can induce clonal expansion of T cells carrying T cell receptors that specifically recognize the antigen or fragment in the presence of a suitable co-stimulatory signal. In the context of an embodiment of the present invention, the antigen or fragment is preferably presented by a cell, preferably by an antigen presenting cell and / or a diseased cell, in the context of an MHC molecule, which results in an immune response against the antigen or a cell expressing the antigen.

[0116] According to the present invention, any suitable antigen is contemplated as a candidate for an immune response, wherein the immune response is preferably a cellular immune response.

[0117] The antigen is preferably a product corresponding to or derived from a naturally occurring antigen. The naturally occurring antigen may include or may be derived from an allergen, virus, bacteria, fungus, parasite and other infectious agents and pathogens, or the antigen may also be a tumor antigen. According to the present invention, the antigen may correspond to a naturally occurring product, for example, a viral protein or a portion thereof.

[0118] The term "pathogen" refers to pathogenic microorganisms and includes viruses, bacteria, fungi, unicellular organisms and parasites. Examples of pathogenic viruses include, but are not limited to, human immunodeficiency virus (HIV), cytomegalovirus (CMV), herpes simplex virus (HSV), hepatitis A virus (HAV), HBV, HCV, papillomavirus and human T-lymphotrophic virus (HTLV). Unicellular organisms include, but are not limited to, malarial parasites, trypanosomes, amoebas, etc.

[0119] The term "disease associated antigen" refers to all antigens with pathogenic significance, and includes "tumor antigens". According to the present invention, it is desirable to induce an immune response against a disease associated antigen or a cell expressing a disease associated antigen and presenting the disease associated antigen preferably in the context of an MHC molecule. Preferably, the disease associated antigen is a naturally occurring antigen. In one embodiment, the disease associated antigen is expressed in a diseased cell and is preferably presented by the cell's MHC molecules.

[0120] The antigen encoded by the RNA (i.e., the therapeutic and / or preventive agent) contained in the (lipid composition) nanoparticle of the present invention should induce an immune response to the disease-associated antigen to be targeted or a cell expressing the disease-associated antigen to be targeted. Therefore, the antigen encoded by the RNA contained in the nanoparticle of the present invention may correspond to or may include a disease-associated antigen or one or more immunogenic fragments thereof, such as one or more MHC binding peptides of a disease-associated antigen. Therefore, the antigen encoded by the RNA contained in the nanoparticle of the present invention may be a recombinant antigen.

[0121] Active ingredient (eg, therapeutic and / or prophylactic agent)

[0122] The lipid composition of the present invention can be used to deliver active pharmaceutical ingredients, and any substance with therapeutic / preventive activity can be used as an active pharmaceutical ingredient, such as a therapeutic agent and / or a preventive agent. Based on this, the present invention further provides a (pharmaceutical) composition, which comprises a lipid composition provided by the present invention, for delivering active pharmaceutical ingredients. The composition of the present invention can include one or more therapeutic agents and / or preventive agents (as active pharmaceutical ingredients). Active pharmaceutical ingredients can be encapsulated in the lipid composition provided by the present invention or combined with the lipid composition.

[0123] According to some preferred embodiments of the present invention, the active ingredient includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides and proteins, preferably nucleic acid molecules.

[0124] For example, the active ingredient is a vaccine or compound capable of inducing an immune response. Therefore, in some preferred embodiments, the active ingredient may be a nucleic acid molecule (such as DNA or RNA) capable of encoding one or more antigens.

[0125] The lipid composition of the present invention can (as a carrier) deliver the active ingredient to a target organ in a subject (such as a mammal), and in particular can deliver it to a target cell in the target organ. Therefore, the present invention also provides a method for treating a disease or condition in a subject in need thereof, which method comprises administering to the subject a composition comprising the above-mentioned active ingredient and / or contacting the subject's cells with the composition.

[0126] Therapeutic and / or prophylactic agents include biologically active substances and may be alternatively referred to as "active agents," "active ingredients," and the like. Therapeutic and / or prophylactic agents may be substances that, upon delivery to a cell or organ, cause a desired change in the cell or organ, or in other body tissues or systems. Such species may be used to treat one or more diseases, disorders, or conditions. In some embodiments, the therapeutic and / or prophylactic agent is a small molecule drug that can be used to treat a specific disease, disorder, or condition.Examples of drugs that can be used in the composition include, but are not limited to, anti-neoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), anti-tumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, arabinoside), anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blocking agents (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines

[0013] In some embodiments, the present invention includes but is not limited to: antibiotics (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, sedatives, and imaging agents.

[0127] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that causes an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracenedione, The invention relates to a radioactive ion, for example, anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and analogs or homologues thereof. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. Vaccines include compounds and preparations that can provide immunity to one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis and tuberculosis and may include nucleic acid molecules (e.g., mRNA) encoding infectious disease-derived antigens and / or epitopes. Vaccines may also include compounds and preparations that direct an immune response against cancer cells and may include nucleic acid molecules (e.g., mRNA) encoding tumor cell-derived antigens, epitopes and / or new epitopes. Compounds that induce an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species.Other therapeutic and / or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, razithromycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan (busulfan), and oxazolidinone (oxazolidinone). sulfan), dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP, cisplatin), anthracyclines such as daunorubicin (formerly daunomycin) and doxorubicin, antibiotics such as dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), gentamicin and amikacin, and antimitotic agents such as vincristine, vinblastine, taxol and maytansine.

[0128] In other embodiments, the therapeutic and / or preventive agent is a protein. The therapeutic protein in the nanoparticles that can be used in the present invention includes, but is 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, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0129] In some embodiments, the therapeutic and / or preventive agent can be a polynucleotide or a nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The broadest meaning of the term "polynucleotide" includes any compound and / or substance that is an oligonucleotide chain or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used according to the present invention include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger mRNA (mRNA), its hybrid; RNAi inducing factor; RNAi factor; siRNA; shRNA; miRNA; antisense RNA; ribozyme; catalytic DNA; RNA that induces triple helix formation; aptamer, etc. In some preferred embodiments, the therapeutic and / or preventive agent is RNA. The RNA that can be used in the compositions and methods described in the present invention can be selected from, but is not limited to, the group consisting of: shortmer, antagomir, antisense RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA) and mixtures thereof. In certain embodiments, the RNA is mRNA.

[0130] In some embodiments, the therapeutic and / or preventive agent is mRNA. mRNA can encode any polypeptide of interest, including any natural or non-natural polypeptide or polypeptide modified in other ways. The polypeptide encoded by mRNA can have any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by mRNA can have a therapeutic effect when expressed in a cell.

[0131] In other embodiments, the therapeutic agent and / or preventive agent is siRNA. siRNA can selectively reduce the expression of a gene of interest or down-regulate the expression of the gene. For example, the selection of siRNA can make the gene silencing relevant to a specific disease, disease or condition after the composition comprising the siRNA is administered to a subject in need. siRNA can include a sequence complementary to the mRNA sequence of a gene or protein of interest to encode. In some embodiments, siRNA can be an immunomodulatory siRNA.

[0132] In certain embodiments, the therapeutic and / or preventive agent is sgRNA and / or cas9 mRNA. sgRNA and / or cas9 mRNA can be used as a gene editing tool. For example, the sgRNA-cas9 complex can affect the mRNA translation of a cellular gene.

[0133] In some embodiments, the therapeutic agent and / or preventive agent is shRNA or its encoding vector or plasmid. shRNA can be produced inside the target cell after the appropriate construct is delivered to the nucleus. The construct and mechanism associated with shRNA are well-known in the relevant field.

[0134] Disease or condition

[0135] The composition / carrier of the present invention can deliver therapeutic agents and / or preventive agents to subjects or patients, thereby achieving the treatment and / or prevention of diseases or conditions. The therapeutic agents and / or preventive agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides or proteins. Therefore, the composition of the present invention can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs. Due to the wide variety of the above-mentioned therapeutic agents and / or preventive agents, the composition of the present invention can be used to treat or prevent a variety of diseases or conditions.

[0136] In one embodiment, the disease or disorder is characterized by a malfunction or aberrant protein or polypeptide activity.

[0137] The reagents, compositions and methods described in the present invention can be used to treat a subject who is ill (e.g., a disease characterized by the presence of diseased cells expressing an antigen and presenting an antigenic peptide), or to prevent a subject from becoming ill. Examples of treatable and / or preventable diseases include all diseases that express one of the antigens described in the present invention. Particularly preferred diseases are infectious diseases (e.g., viral diseases) and cancer diseases. The reagents, compositions and methods described in the present invention can also be used for immunization or vaccination to prevent the diseases described in the present invention.

[0138] According to the present invention, the term "disease" refers to any pathological state, including infectious diseases and cancer diseases, in particular those forms of infectious diseases and diseases described in the present invention.

[0139] The disease to be treated according to the present invention is preferably a disease involving an antigen. According to the present invention, "disease involving an antigen" or similar expressions means that the antigen is expressed in the cells of a diseased tissue or organ. The expression in the cells of a diseased tissue or organ may be increased compared to the state of a healthy tissue or organ. In one embodiment, expression occurs only in diseased tissue, while expression is inhibited in healthy tissue. According to the present invention, diseases involving antigens include infectious diseases and cancer diseases, wherein the disease-associated antigens are preferably antigens of infectious agents and tumor antigens, respectively. Preferably, the disease involving an antigen is preferably a disease involving cells that express the antigen and present the antigen in the context of an MHC molecule (particularly class I MHC).

[0140] For example, the disease or disorder is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0141] Examples of the infectious diseases include, but are not limited to: ① viral infectious diseases, such as AIDS (HIV), hepatitis A, hepatitis B, hepatitis C, herpes zoster (varicella), rubella (Rubella, caused by rubella virus), Flaviviridae ) (e.g. yellow fever, dengue fever, epidemic encephalitis B, forest encephalitis, West Nile fever, Zika fever), diseases caused by coronavirus, diseases caused by influenza virus, rabies, hemorrhagic infectious diseases (e.g. diseases caused by Marburg virus or Ebola virus, etc.); ② bacterial infectious diseases, such as Legionnaire's disease (Legionella, Legionella ), gastric ulcer (Helicobacter pylori, Helicobacter ), cholera (Vibrio, Vibrio ), by Escherichia coli, Staphylococcus aureus ( Staphylococci ),salmonella( Salmonella ) or Streptococcus ( Streptococci ) (tetanus); ③ Infections caused by protozoan pathogens, such as malaria, sleeping sickness, leishmaniasis, toxoplasmosis, that is, infections caused by Plasmodium ( Plasmodium ), Trypanosomes ( Trypanosoma ), Leishmania ( Leishmania ) and Toxoplasma gondii; or ④ fungal infection (such as Cryptococcus neoformans ( Cryptococcus neoformans ), Histoplasma capsulatum ( Histoplasma capsulatum ), Coccidioides immitis ( Coccidioides immitis ), Blastomyces dermatitidis ( Blastomyces dermatitidis ) or Candida albicans ( Candida albicans )Infection caused by ) etc.

[0142] The carcinoma or cancer (the medical term for malignant tumor) is a class of diseases in which a group of cells exhibit uncontrolled growth (dividing beyond normal limits), invasion (invading and destroying adjacent tissues), and sometimes metastasize (spreading to other parts of the body through the lymph or blood). These three harmful properties of carcinoma distinguish it from benign tumors, which are self-limited and do not invade or metastasize. Most carcinomas form tumors, i.e., swellings or lesions formed by abnormal growth of cells (called neoplastic cells or tumor cells), but some (like leukemias) do not. According to the present invention, the term "cancer" includes, 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 and throat (ENT) cancer, breast cancer, uterine cancer, breast cancer, prostate cancer, ovarian cancer, and metastasis thereof, etc.

[0143] Malignant melanoma is a serious type of skin cancer that results from the uncontrolled growth of pigment cells called melanocytes.

[0144] According to the invention, "epithelial cancers" are malignant tumors originating from epithelial cells. This group accounts for the most common cancers, including common forms of breast, prostate, lung and colon cancer.

[0145] Lymphomas and leukemias are malignancies that arise from hematopoietic (blood-forming) cells.

[0146] Sarcomas are cancers that originate from transformed cells of one of the tissues that develop from the embryonic mesoderm. Thus, sarcomas include bone tumors, cartilage tumors, fat tumors, muscle tumors, blood vessel tumors, and hematopoietic tissue tumors.

[0147] A blastic tumor or germ cell tumor is a tumor (usually malignant) that resembles immature or embryonic tissue. Most of these tumors occur commonly in children.

[0148] Gliomas are tumors that begin in the brain or spine. They are called gliomas because they originate from glial cells. The most common site for gliomas is the brain.

[0149] Other components

[0150] The pharmaceutical compositions of the present invention may include one or more components other than those described in the preceding sections. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (eg, vitamin A or vitamin E) or sterols.

[0151] The composition may also include one or more permeability enhancing molecules, carbohydrates, polymers, surface modifiers or other components. The permeability enhancing molecules may be, for example, molecules described in U.S. Patent Application Publication No. 2005 / 0222064. The carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0152] Surface-altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, , mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornasealfa, neltenexine and erdosteine) and DNA enzymes (e.g., rhDNA enzyme). The surface-altering agent can be disposed within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent attachment or other methods).

[0153] The composition can also include one or more functionalized lipids. For example, lipids can be functionalized with alkynyl groups, which may undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized with one or more groups that effectively promote membrane penetration, cell recognition or imaging in this way. The surface of the composition can also be coupled to one or more useful antibodies. Functional groups and conjugates that can be used for targeted cell delivery, imaging and membrane penetration are well known in the art.

[0154] In addition to these components, the composition may include any substance that can be used in a pharmaceutical composition. For example, the composition may include one or more pharmaceutically acceptable (i.e., pharmaceutically acceptable) excipients or auxiliary ingredients, such as but not limited to one or more solvents, dispersion media, diluents, dispersing aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, adhesives, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc.

[0155] The term "pharmaceutically acceptable" refers to the non-toxicity of a material that does not affect the action of the active ingredients of the pharmaceutical composition. Non-pharmaceutically acceptable ingredients may be used to prepare pharmaceutically acceptable ingredients and are included in the present invention.

[0156] Suitable buffering agents for use in the compositions of the present invention include salt forms of acetic acid, salt forms of citric acid, salt forms of boric acid, and salt forms of phosphoric acid.

[0157] When used in the present invention, the term "excipient" is intended to mean all substances that may be present in the pharmaceutical composition of the present invention and which are not active ingredients, such as carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents or coloring agents. Excipients are, for example, starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0158] Suitable preservatives for use in the compositions of the present invention include benzalkonium chloride, chlorobutanol, parabens and thimerosal.

[0159] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof.

[0160] Dosage Form and Administration

[0161] The composition of the present invention can be made into a preparation in the form of solid, semisolid, liquid or gas, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The composition of the present invention can be prepared by methods well known in the pharmaceutical field. For example, a sterile injection solution can be prepared by mixing the required amount of therapeutic agent or preventive agent with the required various other ingredients mentioned above into a suitable solvent such as sterile distilled water, and then filtering and sterilizing. Surfactants can also be added to promote the formation of uniform solutions or suspensions.

[0162] For example, the composition of the invention can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In one embodiment, the composition is administered intravenously or subcutaneously.

[0163] Therapeutically effective amount

[0164] A "therapeutically effective amount" is an amount of a therapeutic agent that improves a disease or symptom when administered to a patient. A "prophylactically effective amount" is an amount of a prophylactic agent that prevents a disease or symptom when administered to a subject. The amount of a therapeutic agent that constitutes a "therapeutically effective amount" or the amount of a prophylactic agent that constitutes a "prophylactically effective amount" varies with the therapeutic and / or prophylactic agent, the disease state and its severity, the age, weight, etc. of the patient and / or subject to be treated and / or prevented. A person of ordinary skill in the art can routinely determine a therapeutically effective amount and a prophylactically effective amount based on his or her knowledge and the present invention.

[0165] The compositions of the present invention are administered in a therapeutically effective amount, which may vary not only with the specific agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be determined by the attending physician or clinician. For example, a therapeutic or preventive agent may be administered to a mammal (e.g., a human) at a dose of about 0.0001 mg / kg to about 10 mg / kg.

[0166] Antigen presenting cells

[0167] Antigen presenting cells (APCs) are cells that present (i.e., display) antigens on their surface in the context of a major histocompatibility complex (MHC). This includes situations where only one fragment or more fragments of an antigen are presented. T cells can recognize this complex with their T cell receptors (TCRs). Antigen presenting cells process antigens and present them to T cells.

[0168] Professional antigen-presenting cells are very efficient at internalizing antibodies (either by phagocytosis or by receptor-mediated endocytosis) and then displaying antigen fragments bound to class II MHC molecules on their membranes. T cells recognize and interact with the antigen-class II MHC molecule complex on the antigen-presenting cell membrane. The antigen-presenting cell then produces additional co-stimulatory signals, leading to T cell activation. The expression of co-stimulatory molecules is a typical feature of professional antigen-presenting cells.

[0169] The major types of professional antigen-presenting cells are dendritic cells (which have the broadest range of antigen presentation and are perhaps the most important antigen-presenting cells), macrophages, B cells, and certain activated epithelial cells.

[0170] Dendritic cells are a population of leukocytes, including plasmacytoid dendritic cells (pDCs) and classical dendritic cells (cDCs), which present antigens captured in peripheral tissues to T cells via both MHC class II and class I antigen presentation pathways. Dendritic cells are potent inducers of immune responses, and the activation of these cells is a key step in inducing anti-tumor immunity.

[0171] Antigen presenting cells can be loaded with peptides presented by MHC by transducing the cells with nucleic acids encoding a peptide or protein comprising the peptide to be presented, e.g., nucleic acids encoding an antigen (e.g., RNA). Transfection of dendritic cells with mRNA is a promising antigen loading technique for stimulating strong anti-tumor immunity.

[0172] The term "immunogenicity" relates to the relative efficiency of an antigen in inducing an immune response.

[0173] The terms "T cell" and "T lymphocyte" are used interchangeably in the present invention, and include helper T cells (CD4+ T cells) and cytotoxic T cells (CTL, CD8+ T cells) of cytolytic T cells.

[0174] T cells belong to a group of white blood cells called lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types (such as B cells and natural killer cells) by the presence of special receptors called T cell receptors (TCRs) on their cell surfaces. The thymus is the main organ responsible for the maturation of T cells. Several different T cell subsets have been discovered, each with different functions.

[0175] Helper T cells assist other white blood cells in the immune process, including maturation of B cells into plasma cells and activation of cytotoxic T cells and macrophages. Because they express the CD4 protein on their surface, these cells are also called CD4 +T cells. Helper T cells are activated when peptide antigens are presented to them by class II MHC molecules expressed on the surface of antigen presenting cells (APCs). After activation, they rapidly divide and secrete small proteins called cytokines that regulate or assist in active immune responses.

[0176] Cytotoxic T cells destroy diseased cells, for example, infected cells (such as virus-infected cells) and cancer cells, and are also involved in transplant rejection. Because they express the CD8 glycoprotein on their surface, these cells are also called CD8 + T cells. These cells recognize their targets by binding to antigens associated with class I MHC, which is present on the surface of nearly every cell in the body.

[0177] Most T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains, which are produced by independent T cell receptor α and β (TCRα and TCRβ) genes and are called α-TCR chain and β-TCR chain. γδ T cells represent a small subtype of T cells that have a unique T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR is composed of one γ chain and one δ chain. This group of T cells is less common than αβ T cells (2% of total T cells).

[0178] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells reside in the thymus and amplify through cell division to produce a large number of immature thymocytes. Early thymocytes express neither CD4 nor CD8 and are therefore classified as double negative (CD4-CD8-) cells. As they develop through development, they become double positive thymocytes (CD4+CD8+) and eventually mature into single positive (CD4+CD8+) cells. + CD8 - or CD4 - CD8 + ) thymocytes and are then released from the thymus into peripheral tissues.

[0179] The first signal for T cell activation is provided by the binding of the T cell receptor to a short peptide presented by the major histocompatibility complex (MHC) on another cell. This ensures that only T cells with a TCR specific for that peptide are activated. The companion cell is usually a professional antigen presenting cell (APC), usually a dendritic cell in the context of a primary response, but B cells and macrophages can also be important APCs. + T cell peptides are 8 to 10 amino acids long; they are presented by class II MHC molecules to CD4 +The peptides for T cells are longer because the binding cleft of the class II MHC molecule is open at the end.

[0180] In this article, the term "TLP" means a three-component lipid composition, specifically a composition prepared by a permanent anionic lipid, a permanent cationic lipid, and a neutral lipid at a certain molar percentage. For example, in some embodiments, TLP is a lipid composition prepared by a permanent anionic lipid ADOPE, a permanent cationic lipid DOTMA, and a neutral lipid DOPE at a certain molar percentage. TLP can encapsulate mRNA to generate a TLP-mRNA composition, as used herein.

[0181] In various embodiments of the present invention, the "TLP" group refers to a test group corresponding to a composition prepared by preparing permanent anionic lipid ADOPE, permanent cationic lipid DOTMA and neutral lipid DOPE according to a certain molar percentage without adding the adjuvant lipid of the present invention.

[0182] In some embodiments of the present invention, the permanent cationic lipid DOTMA in the TLP composition can be partially replaced by the adjuvant lipid of the present invention. For example, in some embodiments, the adjuvant lipid of the present invention partially replaces the DOTMA in the TLP composition with a certain percentage of the total lipids to prepare the corresponding composition. In the present invention, the composition obtained after encapsulating mRNA with the composition prepared in this way is called an mRNA pharmaceutical composition, as used herein.

[0183] In this article, "blank control group", "blank control" and "control" can be used interchangeably. Those skilled in the art can understand the specific technical meaning based on the context, and there will be no situation where the meaning is unclear.

[0184] The present invention provides the following solutions:

[0185] 1. A compound or its N-oxide compound, solvate, pharmaceutically acceptable salt or stereoisomer, wherein the compound comprises a structure represented by formula (I) or formula (II):

[0186] , ,

[0187] In formula (I) and formula (II):

[0188] L is unsubstituted C 2-5 Straight chain alkylene;

[0189] M1 is -OC(O)-, -C(O)OCH2-, -C(O)NHCH2- or -OCH2-;

[0190] M2 and M3 are -OC(O)- or -O-, respectively, and M2 and M3 are the same or different;

[0191] R1 is a substituted or unsubstituted N-containing heterocyclic ring;

[0192] R2 and R3 are unsubstituted C 16-24 In the straight chain alkenyl group, R2 and R3 are the same or different.

[0193] 2. The compound according to Embodiment 1, or its N-oxide compound, solvate, pharmaceutically acceptable salt or stereoisomer, wherein L is -(CH2)2- or -(CH2)3-;

[0194] and / or, M1 is -OC(O)-, -C(O)OCH2-, -C(O)NHCH2- or -OCH2-;

[0195] and / or, M2 is -OC(O)- or -O-;

[0196] and / or, M3 is -OC(O)- or -O-;

[0197] and / or, R1 is or ;

[0198] and / or, R2 is unsubstituted C 16-20 Straight chain alkenyl;

[0199] and / or, R3 is unsubstituted C 16-20 Straight chain alkenyl.

[0200] 3. The compound according to Embodiment 1 or 2, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein the compound has any of the following structures:

[0201] ,

[0202] ,

[0203] ,

[0204] ,

[0205] ,

[0206] ,

[0207] ,

[0208] ,

[0209] .

[0210] 4. A compound according to any one of embodiments 1-3, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound has any one of the following structures:

[0211] ,

[0212] ,

[0213] ,

[0214] .

[0215] 5. A lipid composition comprising an adjuvant lipid, wherein the adjuvant lipid includes a compound described in any one of Embodiments 1-4, or an N-oxide compound, solvate, pharmaceutically acceptable salt or stereoisomer thereof.

[0216] 6. The lipid composition according to Embodiment 5, wherein the molar percentage of the adjuvant lipid in the lipid composition is 2.5-20%.

[0217] 7. The lipid composition according to embodiment 5 or 6, wherein the molar percentage of the adjuvant lipid in the lipid composition is 5-15%.

[0218] 8. A lipid composition according to any one of embodiments 5-7, wherein the molar percentage of the adjuvant lipid in the lipid composition is 10%.

[0219] 9. A lipid composition according to any one of embodiments 5-8, wherein the lipid composition also comprises a first lipid composition of permanent anionic lipids, permanent cationic lipids and neutral lipids.

[0220] 10. A lipid composition according to any one of embodiments 5-9, wherein the molar percentage ratio of the adjuvant lipid, permanent anionic lipid, permanent cationic lipid and neutral lipid in the lipid composition is 2.5-20:10-33:40-60:20-40.

[0221] 11. A lipid composition according to embodiment 9 or 10, wherein the permanent anionic lipid is selected from any one of the group consisting of 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylthioureido)ethyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylureido)ethyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-propylureido)ethyl) phosphate and (R)-2,3-bis(oleoyloxy)propyl-(2-(3-butylureido)ethyl) phosphate and salts thereof, or a combination of at least two thereof;

[0222] And / or, the permanent cationic lipid is selected from any one or a combination of at least two of the group consisting of 1,2-dioctadecenyloxy-3-methylammonium propane, (2,3-dioleyloxypropyl)trimethylammonium and salts thereof;

[0223] And / or, the neutral lipid is any one selected from the group consisting of 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine, distearoylphosphatidylcholine and salts thereof, or a combination of at least two thereof.

[0224] 12. A pharmaceutical composition, wherein the pharmaceutical composition comprises the lipid composition according to any one of embodiments 5-11 and an active ingredient;

[0225] The active ingredient is selected from any one of the group consisting of nucleic acid molecules, small molecule compounds, polypeptides or proteins, or a combination of at least two of them.

[0226] 13. A pharmaceutical composition according to embodiment 12, wherein the amount of the active ingredient and the lipid composition is such that the charge ratio of positive charge to negative charge in the pharmaceutical composition is 1:2 to 1:5.

[0227] 14. A pharmaceutical composition according to embodiment 12 or 13, wherein the active ingredient is a nucleic acid molecule capable of encoding one or more antigens.

[0228] 15. A pharmaceutical composition according to any one of embodiments 12-14, wherein the antigen is a disease-associated antigen, or the nucleic acid molecule or antigen is capable of triggering an immune response against the disease-associated antigen or cells expressing the disease-associated antigen.

[0229] 16. A pharmaceutical composition according to any one of embodiments 12-15, wherein the nucleic acid molecule is RNA encoding one or more antigens.

[0230] 17. The pharmaceutical composition according to any one of embodiments 12-16, wherein the pharmaceutical composition further comprises at least one auxiliary ingredient;

[0231] and / or, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents or excipients;

[0232] And / or, the pharmaceutical composition further comprises one or more hydrophobic small molecules, permeability enhancing molecules, carbohydrates, polymers, surface altering agents, functionalized lipids or cytokines.

[0233] 18. The compound of any one of embodiments 1-4, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer; the lipid composition of any one of embodiments 5-11; or the use of the pharmaceutical composition of any one of embodiments 12-17 for delivering an active ingredient to an antigen presenting cell in a target organ or tissue;

[0234] The active ingredient is selected from any one of the group consisting of nucleic acid molecules, small molecule compounds, polypeptides or proteins, or a combination of at least two of them.

[0235] 19. The use according to embodiment 18, wherein 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;

[0236] And / or, the antigen presenting cells are selected from any one or a combination of at least two of the group consisting of B cells, NK cells, cDC cells, pDC cells and macrophages.

[0237] 20. The use according to embodiment 18 or 19, wherein the target organ or tissue is the spleen.

[0238] 21. The compound described in any one of embodiments 1-4, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer; the lipid composition described in any one of embodiments 5-11; or the use of the pharmaceutical composition described in any one of embodiments 12-17 in the preparation of a drug.

[0239] 22. The use according to embodiment 21, wherein the medicament is used to treat and / or prevent a disease or condition in a subject in need thereof; and / or

[0240] The drug is selected from any one of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide drugs and protein drugs, or a combination of at least two of them.

[0241] 23. The use according to embodiment 22, wherein the disease or condition is characterized by organ, protein or gene dysfunction, or abnormal protein or polypeptide activity.

[0242] 24. The use according to embodiment 22 or 23, wherein the disease or condition is selected from any one or a combination of at least two of the group consisting of infectious diseases, tumors and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases and metabolic diseases.

[0243] 25. The use according to embodiment 24, wherein the infectious disease is selected from any one or a combination of at least two of the group consisting of diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies and herpes; and / or

[0244] The tumor is selected from any one or a combination of at least two of the group consisting of breast cancer, ovarian cancer, lung cancer, pancreatic cancer, kidney cancer, gastric cancer, lymphoma, colon cancer, liver cancer, melanoma, bladder cancer, cervical cancer and prostate cancer.

[0245] 26. The use according to embodiment 22, wherein the subject is a mammal.

[0246] 27. The use according to embodiment 26, wherein the mammal is selected from any one of the group consisting of humans, non-human primates, companion animals, exotic species, livestock animals and food animals.

[0247] 28. The use according to any one of embodiments 21-27, wherein the drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

[0248] 29. The use according to embodiment 28, wherein the drug is administered intravenously or intramuscularly.

[0249] 30. The use according to any one of embodiments 21-29, wherein the administration dose of the drug is 0.001-10 mg / kg.

[0250] Example

[0251] The present invention is further described below in conjunction with examples, but the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. In the specific examples of the present invention, the raw materials used can be obtained commercially. Unless otherwise specified, the percentages in the context are weight percentages, and all temperatures are given in degrees Celsius. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0252] In the following embodiments, the abbreviations represent the following meanings:

[0253] ADOPE: 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate sodium salt;

[0254] DOTMA: 1,2-dioctadeceneoxy-3-methylammonium propane (chloride salt);

[0255] DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine;

[0256] NaBH4: sodium borohydride;

[0257] Boc2O: di-tert-butyl dicarbonate;

[0258] DMAP: 4-dimethylaminopyridine;

[0259] EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0260] HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0261] DIEA: N,N-diisopropylethylamine;

[0262] DCM: dichloromethane;

[0263] DMF: N,N-dimethylformamide;

[0264] THF: tetrahydrofuran;

[0265] MeOH: methanol;

[0266] TLP: three-component lipid composition.

[0267] Example 1: Synthesis of adjuvant lipids

[0268] 1. Synthesis of YK-1301

[0269] The synthetic route of YK-1301 is as follows:

[0270]

[0271] Step 1: Synthesis of YK-1301-PM1

[0272] Dissolve YK-1301-SM (350 mg, 2.53 mmol) in DMF (5 mL). Add bromolinolene (1.8 g, 5.47 mmol) and K2CO3 (1.1 g, 7.96 mmol) in an ice-water bath. Heat to 80 degrees and stir for 3 hours. Stop the reaction, dilute with water, extract with ethyl acetate, combine the organic phases, and wash with water once. Dry the separated organic phase with anhydrous sodium sulfate, filter, and spin dry. Purify the product YK-1301-PM1 (1.3 g, 2.05 mmol, 80.8%) by silica gel chromatography (0% - 15% petroleum ether / ethyl acetate).

[0273] Step 2: Synthesis of YK-1301-PM2

[0274] YK-1301-PM1 (1.3 g, 2.05 mmol) was dissolved in THF (3 mL) and MeOH (2 mL), and sodium borohydride (387 mg, 10.23 mmol) was added under ice-water bath conditions, and then the ice-water bath was removed and stirred at room temperature for 2 hours. After the reaction was completed, 5 mL of water was added to dilute, and saturated ammonium chloride aqueous solution was slowly added dropwise to quench the reaction. Ethyl acetate was added to the reaction solution, and the liquid was separated. The aqueous phase was back-extracted twice with ethyl acetate, and the organic phases were combined and the solvent was spin-dried to obtain a crude product, which was purified by silica gel chromatography (0% - 15% petroleum ether / ethyl acetate) to obtain the product YK-1301-PM2 (870 mg, 1.37 mmol, 66.7%).

[0275] Step 3: Synthesis of YK-1301-PM3

[0276] YK-1301-PM2 (270 mg, 0.42 mmol) was dissolved in DCM (3 mL), and 4-bromobutyric acid (85 mg, 0.51 mmol), EDCI (87 mg, 0.45 mmol) and DMAP (10 mg, 0.08 mmol) were weighed in sequence, and the mixture was stirred at room temperature for 16 hours. After the reaction was completed, the mixture was filtered, wet loaded, and purified by silica gel chromatography (0% - 10% petroleum ether / ethyl acetate) to obtain the product YK-1301-PM3 (269 mg, 0.34 mmol, 80.7%).

[0277] Step 4: Synthesis of YK-1301

[0278] Dissolve YK-1301-PM3 (269 mg, 0.34 mmol) in acetonitrile (3 mL), weigh potassium carbonate (137 mg, 0.99 mmol), potassium iodide (6 mg, 0.04 mmol) and 2-ethylpiperidine (45 mg, 0.40 mmol) in turn, and stir at room temperature for 16 hours. After the reaction is complete, filter, wet sample, and purify by silica gel chromatography (0% - 10% petroleum ether / ethyl acetate) to obtain the product YK-1301 (160 mg, 0.20 mmol, 57.5%). 54 H 91 NO4, MS (ES): m / z (M + H + ) =818.9.

[0279] YK-1301: 1 H NMR (400 MHz, CDCl3) δ 7.84 - 7.62 (m, 1H), 7.40 (dd, J =15.1, 6.3 Hz, 1H), 6.98 - 6.81 (m, 1H), 5.53 - 5.28 (m, 8H), 5.06 (s, 2H), 4.02 (td, J = 6.6, 3.1 Hz, 4H), 2.87 - 2.74 (m, 4H), 2.51 (s, 2H), 2.17 - 1.93(m, 12H), 1.92 - 1.76 (m, 6H), 1.76 - 1.55 (m, 5H), 1.48 (t, J = 13.7 Hz, 5H), 1.45- 1.17 (m, 32H), 1.15 - 0.86 (m, 9H).

[0280] 2. Synthesis of YK-1302

[0281] The synthetic route of YK-1302 is as follows:

[0282]

[0283] Step 1: Synthesis of YK-1302-PM1

[0284] Dissolve YK-1302-SM1 (2.0 g, 12.98 mmol) in methanol, slowly add thionyl chloride (3.1 g, 26.06 mmol), stir at room temperature for 2 hours, and the reaction is complete by TLC. Stop the reaction, dry the solvent under reduced pressure to obtain a crude product, and purify it by silica gel chromatography (0% - 15% petroleum ether / ethyl acetate) to obtain the product YK-1302-PM1 (2.1 g, 12.49 mmol, 96.2%).

[0285] Step 2: Synthesis of YK-1302-PM2

[0286] YK-1302-PM1 (500 mg, 2.98 mmol) was dissolved in DMF, potassium carbonate (1.64 g, 11.87 mmol) and 1-bromo-cis-9-octadecene (2.27 g, 6.85 mmol) were added in sequence, and the temperature was raised to 80 o C and stirred for 5 hours. The reaction was completed after monitoring by TLC. The reaction was stopped, water was added to dilute the reaction system, ethyl acetate was added for extraction, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% - 20% petroleum ether / ethyl acetate) to obtain the product YK-1302-PM2 (1.77 g, 2.65 mmol, 89.0%).

[0287] Step 3: Synthesis of YK-1302-PM3

[0288] YK-1302-PM2 (1.77 g, 2.65 mmol) was dissolved in tetrahydrofuran and water, potassium hydroxide (743 mg, 13.24 mmol) was added, and the temperature was raised to 80 o C and stirred for 5 hours. The reaction was completed after monitoring by TLC. The reaction was stopped, water was added to dilute the reaction system, ethyl acetate was added for extraction, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain the product, YK-1302-PM3 (1.26 g, 1.92 mmol, 72.7%).

[0289] Step 4: Synthesis of YK-1302-PM4

[0290] YK-1302-PM3 (500 mg, 0.76 mmol) was dissolved in dichloromethane, and EDCI (220 mg, 1.15 mmol), DMAP (18 mg, 0.15 mmol) and 3-bromo-1-propanol (106 mg, 0.86 mmol) were weighed and added in sequence. The mixture was stirred at room temperature for 5 hours, and the reaction was completed after TLC monitoring. The reaction was stopped, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% -10% petroleum ether / ethyl acetate) to obtain the product YK-1302-PM4 (380 mg, 0.49 mmol, 64.5%).

[0291] Step 5: Synthesis of YK-1302

[0292] YK-1302-PM4 (200 mg, 0.26 mmol) was dissolved in acetonitrile, potassium carbonate (109 mg, 0.79 mmol), potassium iodide (8.7 mg, 0.05 mmol) and cycloheximide (29 mg, 0.29 mmol) were weighed in sequence, and the temperature was raised to 70 o C and stirred for 5 hours. The reaction was monitored by TLC. The reaction was stopped, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% -10% dichloromethane / methanol) to obtain the product YK-1302 (150 mg, 0.19 mmol, 73.2%). C 52 H 91 NO4, MS (ES): m / z ( M +1) =794.6.

[0293] YK-1302: 1 H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 8.4 Hz, 1H), 7.57 (s,1H), 7.25 - 7.15 (m, 1H), 5.51- 5.27 (m, 4H), 4.50 (s, 2H), 4.07 (q, J = 6.3Hz, 4H), 2.98 (d, J = 55.7 Hz, 6H), 2.37 (d, J = 15.6 Hz, 1H), 2.04 (t, J = 10.0Hz, 7H), 1.93 - 1.80 (m, 5H), 1.75 (s, 4H), 1.65 (s, 4H), 1.56 - 1.45 (m,5H), 1.33 (d, J= 20.6 Hz, 40H), 0.91 (t, J = 6.6 Hz, 6H).

[0294] 3. Synthesis of YK-1303

[0295] The synthetic route of YK-1303 is as follows:

[0296]

[0297] Step 1: Synthesis of YK-1303-PM1

[0298] YK-1302-PM1 (200 mg, 1.19 mmol) was dissolved in DMF, potassium carbonate (492 mg, 3.56 mmol) and bromolinolene (980 mg, 2.98 mmol) were added in sequence, and the temperature was raised to 80 o C and stirred for 5 hours. The reaction was completed after monitoring by TLC. The reaction was stopped, water was added to dilute the reaction system, ethyl acetate was added for extraction, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% - 20% petroleum ether / ethyl acetate) to obtain the product YK-1303-PM1 (700 mg, 1.05 mmol, 88.5%).

[0299] Step 2: Synthesis of YK-1303-PM2

[0300] YK-1303-PM1 (600 mg, 0.90 mmol) was dissolved in tetrahydrofuran and water, potassium hydroxide (1.01 g, 18.00 mmol) was added, and the temperature was raised to 80 o C and stirred for 5 hours. The reaction was completed after monitoring by TLC. The reaction was stopped, water was added to dilute the reaction system, ethyl acetate was added for extraction, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain YK-1303-PM2 (410 mg, 0.63 mmol, 69.8%).

[0301] Step 3: Synthesis of YK-1303-PM3

[0302] YK-1303-PM2 (410 mg, 0.63 mmol) was dissolved in dichloromethane, and EDCI (180 mg, 0.94 mmol), DMAP (41 mg, 0.34 mmol) and 3-bromo-1-propanol (213 mg, 1.53 mmol) were weighed and added in sequence. The mixture was stirred at room temperature for 5 hours, and the reaction was completed after TLC monitoring. The reaction was stopped, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% -10% petroleum ether / ethyl acetate) to obtain the product YK-1303-PM3 (410 mg, 0.53 mmol, 84.3%).

[0303] Step 4: Synthesis of YK-1303.

[0304] YK-1303-PM3 (150 mg, 0.19 mmol) was dissolved in acetonitrile, potassium carbonate (81 mg, 0.59 mmol), potassium iodide (3 mg, 0.02 mmol) and cycloheximide (48 mg, 0.48 mmol) were weighed in sequence, and the temperature was raised to 70 o C and stirred for 5 hours. The reaction was completed after TLC monitoring. The reaction was stopped, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% -10% dichloromethane / methanol) to obtain the product YK-1303 (90 mg, 0.11 mmol, 58.7%). C 52 H 87 NO4, MS(ES): m / z ( M +1) = 790.6.

[0305] YK-1303: 1 H NMR (400 MHz, CDCl3) δ 7.66 (dd, J = 8.4, 2.0 Hz, 1H), 7.57(d, J = 2.0 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 5.47 - 5.32 (m, 8H), 4.38 (t, J =6.4 Hz, 2H), 4.11 - 4.04 (m, 4H), 2.81 (t, J= 6.4 Hz, 4H), 2.74 (s, 5H), 2.13- 2.05 (m, 8H), 2.04 - 1.97 (m, 2H), 1.92 - 1.83 (m, 4H), 1.72 (s, 4H), 1.64(s, 3H), 1.56 - 1.44 (m, 5H), 1.42 - 1.23 (m, 29H), 0.92 (t, J = 6.8 Hz, 6H).

[0306] 4. Synthesis of YK-1304

[0307] The synthetic route of YK-1304 is as follows:

[0308]

[0309] Step 1: Synthesis of YK-1304-PM1

[0310] Dissolve YK-1301-SM (900 mg, 6.52 mmol) in DMF (30 mL), weigh and add oleyl methanesulfonate (6.77 g, 19.53 mmol) and K2CO3 (4.50 g, 32.56 mmol) in ice-water bath, heat to 80℃ and stir for 5 hours. Stop the reaction, add water to dilute, extract with ethyl acetate, combine the organic phases, and wash with water once. Dry the separated organic phase with anhydrous sodium sulfate, filter, and spin dry. Purify the product YK-1304-PM1 (4.0 g, 6.26 mmol, 96.06%) by silica gel chromatography (0% - 15% petroleum ether / ethyl acetate).

[0311] Step 2: Synthesis of YK-1304-PM2

[0312] YK-1304-PM1 (4.0 g, 6.26 mmol) was dissolved in MeOH (20 mL), and sodium borohydride (1.18 g, 31.19 mmol) was added in batches under ice-water bath conditions, and then the ice-water bath was removed and stirred at room temperature for 4 hours. After the reaction was completed, 30 mL of water was added to dilute, and saturated ammonium chloride aqueous solution was slowly added dropwise to quench the reaction. Ethyl acetate was added to the reaction solution, and the liquid was separated. The aqueous phase was back-extracted twice with ethyl acetate, and the organic phases were combined and the solvent was dried to obtain a crude product, which was purified by silica gel chromatography (0% - 30% petroleum ether / ethyl acetate) to obtain the product YK-1304-PM2 (3.5 g, 5.46 mmol, 87.22%).

[0313] Step 3: Synthesis of YK-1304-PM3

[0314] YK-1304-PM2 (2.8 g, 4.37 mmol) was dissolved in DCM (40 mL), and 4-bromobutyric acid (1.09 g, 6.53 mmol), EDCI (1.26 g, 6.55 mmol) and DMAP (105 mg, 0.87 mmol) were weighed in sequence, and the mixture was stirred at room temperature for 16 hours. After the reaction was completed, the mixture was filtered, wet-loaded, and purified by silica gel chromatography (0% - 20% petroleum ether / ethyl acetate) to obtain the product YK-1304-PM3 (2.84 g, 3.59 mmol, 82.15%).

[0315] Step 4: Synthesis of YK-1304

[0316] Dissolve YK-1304-PM3 (1.2 g, 1.52 mmol) in acetonitrile (15 mL), weigh potassium carbonate (630 mg, 4.56 mmol), potassium iodide (50 mg, 0.3 mmol) and 2-ethylpiperidine (205 mg, 1.81 mmol) in turn, and stir at room temperature for 16 hours. After the reaction is complete, filter, wet sample, and purify by silica gel chromatography (0% - 20% petroleum ether / ethyl acetate) to obtain the product YK-1304 (1.1 g, 1.34 mmol, 88.15%). 54 H 95 NO4, MS (ES): m / z (M + H + ) =822.7.

[0317] YK-1304: 1 H NMR (400 MHz, CDCl3) δ7.15 (m, 1H), 6.89 – 6.82 (m, 3H), 5.47 – 5.44 (m, 4H) 5.14 – 5.01 (m, 2H), 3.90 – 3.88 (m, 4H), 2.46 – 2.43 (m,3H), 2.17 – 2.01 (m, 8H), 1.83 – 1.81 (m, 2H), 1.80 – 1.74 (m, 5H), 1.72 –1.59 (m, 3H), 1.51 – 1.41 (m, 2H), 1.40 – 1.28 (m, 5H), 1.27 – 1.24 (m, 44H), 0.88 – 0.80 (m, 9H).

[0318] 5. Synthesis of YK-1305

[0319] The synthetic route of YK-1305 is as follows:

[0320]

[0321] Step 1: Synthesis of YK-1305.

[0322] YK-1302-PM4 (180 mg, 0.23 mmol) was dissolved in acetonitrile, potassium carbonate (96 mg, 0.70 mmol), potassium iodide (7.7 mg, 0.20 mmol) and 2-ethylpiperidine (29 mg, 0.26 mmol) were weighed in sequence, and the temperature was raised to 70 o C and stirred for 5 hours. The reaction was completed by TLC. The reaction was stopped, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0%-10% dichloromethane / methanol) to obtain the product YK-1305 (160 mg, 0.20 mmol, 85.2%). C 53 H 93 NO4, MS(ES): m / z ( M +1) = 808.7.

[0323] YK-1305: 1 H NMR (400 MHz, CDCl3) δ 7.65 (d, J = 8.4 Hz, 1H), 7.56 (d, J =1.5 Hz, 1H), 7.04 - 6.98 (m, 1H), 5.58- 5.31 (m, 4H), 4.36 (s, 2H), 4.07 (q, J = 6.6 Hz, 4H), 2.32 (dd, J = 28.0, 10.7 Hz, 4H), 2.04 (t, J = 10.0 Hz, 9H), 1.93– 1.80 (m, 5H), 1.64 (s, 5H), 1.56 – 1.45 (m, 6H), 1.33 (d, J = 20.8 Hz, 43H),0.92 (t, J = 6.8 Hz, 9H).

[0324] 6. Synthesis of YK-1306

[0325] The synthetic route of YK-1306 is as follows:

[0326]

[0327] Step 1: Synthesis of YK-1306.

[0328] YK-1303-PM3 (200 mg, 0.26 mmol) was dissolved in acetonitrile, potassium carbonate (108 mg, 0.78 mmol), potassium iodide (6 mg, 0.04 mmol) and 2-ethylpiperidine (64 mg, 0.57 mmol) were weighed in sequence, and the temperature was raised to 70 o C and stirred for 5 hours. The reaction was completed after TLC monitoring. The reaction was stopped, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% -10% dichloromethane / methanol) to obtain the product YK-1306 (100 mg, 0.12 mmol, 48.0%). C 53 H 89 NO4, MS(ES): m / z ( M +1) = 804.9.

[0329] YK-1306: 1 H NMR (400 MHz, CDCl3) δ 7.65 (d, J = 8.4 Hz, 1H), 7.55 (s,1H), 6.90 (d, J = 8.4 Hz, 1H), 5.47 - 5.31 (m, 8H), 4.40 - 4.32 (m, 2H), 4.07(q, J = 6.4 Hz, 4H), 2.99 (s, 2H), 2.81 (t, J = 6.8 Hz, 4H), 2.74 - 2.33 (m, 2H), 2.13 - 1.98 (m, 10H), 1.90 - 1.82 (m, 4H), 1.74 (s, 4H), 1.55 - 1.46 (m, 5H), 1.45 - 1.27 (m, 32H), 0.98 - 0.88 (m, 9H).

[0330] 7. Synthesis of YK-1307

[0331] The synthetic route of YK-1307 is as follows:

[0332]

[0333] Step 1: Synthesis of YK-1307-PM1

[0334] YK-1307-SM1 (1.0 g, 5.69 mmol) was dissolved in acetonitrile and water (10 mL, volume ratio 1:1), potassium carbonate (2.36 g, 17.08 mmol) and Boc anhydride (2.73 g, 12.50 mmol) were added in sequence, and the mixture was stirred at room temperature for 5 h. The reaction was monitored by LCMS. The reaction was stopped, and water and ethyl acetate were added to the reaction solution in sequence. The aqueous phase was back-extracted twice with ethyl acetate, the organic phases were combined, and the solvent was dried to obtain a crude product. The product YK-1307-PM1 (1.3 g, 5.43 mmol, 95.5%) was purified by silica gel chromatography (0% - 60% petroleum ether / ethyl acetate).

[0335] Step 2: Synthesis of YK-1307-PM2

[0336] YK-1307-PM1 (800 mg, 3.34 mmol) was dissolved in DCM, and EDCI (2.56 g, 13.36 mmol), DMAP (82 mg, 0.67 mmol) and linoleic acid (2.34 g, 8.36 mmol) were weighed and added in turn. The mixture was stirred at room temperature for 3 hours, and the reaction was completed by TLC. The reaction was stopped, the reaction system was diluted with water, ethyl acetate was added for extraction, and the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% - 15% petroleum ether / ethyl acetate) to obtain the product YK-1307-PM2 (800 mg, 1.05 mmol, 31.3%).

[0337] Step 3: Synthesis of YK-1307-PM3

[0338] YK-1307-PM2 (400 mg, 0.52 mmol) was dissolved in 1 mL 4N HCl / dioxane and stirred at room temperature for 2 hours. The reaction was completed by TLC. The reaction was stopped and the solvent was dried under reduced pressure to obtain YK-1307-PM3 (340 mg, 0.51 mmol, 97.8%).

[0339] Step 4: Synthesis of YK-1307-PM4

[0340] YK-1307-SM2 (4.0 g, 23.95 mmol) was dissolved in methanol at 0 oC, slowly add acetyl chloride (0.5 mL), stir at room temperature for 16 hours, and the reaction is complete by TLC. Stop the reaction, add 40 mL of ethyl acetate, wash twice with saturated sodium bicarbonate, separate the liquids, dry the organic phase with anhydrous sodium sulfate, filter, and spin dry. The product YK-1307-PM4 (4.1 g, 22.65 mmol, 94.6%) is obtained.

[0341] Step 5: Synthesis of YK-1307-PM5

[0342] YK-1307-PM4 (500 mg, 2.76 mmol) was dissolved in acetonitrile, potassium carbonate (1.14 g, 8.28 mmol), potassium iodide (46 mg, 0.28 mmol) and 2-ethylpiperidine (375 mg, 3.31 mmol) were added in sequence, and the temperature was raised to 70 o C and stirred for 5 hours. The reaction was completed after monitoring by TLC. The reaction was stopped, filtered, and the solvent was evaporated under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0%-6% dichloromethane / methanol) to obtain the product YK-1307-PM5 (450 mg, 2.11 mmol, 76.4%).

[0343] Step 6: Synthesis of YK-1307-PM6

[0344] YK-1307-PM5 (450 mg, 2.11 mmol) was dissolved in a mixed solvent of THF and MeOH, and an aqueous solution of NaOH was slowly added. The mixture was stirred at room temperature for 6 hours, and the reaction was completed by TLC monitoring. The reaction was stopped, the organic solvent was removed under reduced pressure, the pH was adjusted to 1.0 with 1N HCl, and the mixture was dried by spin drying. The solid was dissolved in a mixed solvent of DCM and MeOH and filtered. The filtrate was dried by spin drying to obtain the product YK-1307-PM6 (400 mg, 2.01 mmol, 95.1%).

[0345] Step 7: Synthesis of YK-1307.

[0346] YK-1307-PM6 (102 mg, 0.51 mmol) was dissolved in DMF, and HATU (233 mg, 0.61mmol) and DIEA (198 mg, 1.53 mmol) were added in turn and stirred at room temperature for 5 min. YK-1307-PM3 (340 mg, 0.51 mmol) was added to the above solution and stirred at room temperature for 5 hours. The reaction was monitored by TLC to complete. The reaction was stopped, the reaction system was diluted with water, ethyl acetate was added for extraction, the liquid was separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% - 15% dichloromethane / methanol) to obtain the product YK-1307 (30 mg, 0.04 mmol, 6.9%). 54 H 88 N2O5, MS (ES): m / z ( M +1) = 845.6.

[0347] YK-1307: 1 H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 6.96 (d, J = 2.2 Hz,2H), 6.80 (s, 1H), 5.50 - 5.17 (m,8H), 4.43 (m, J = 9.8, 5.2 Hz, 2H), 2.87 (s,7H), 2.81 (t, J = 6.4 Hz, 4H), 2.66 - 2.47 (m, 5H), 2.14 (m, J = 13.8, 7.2 Hz,12H), 1.74 (dd, J = 14.6, 7.3 Hz, 5H), 1.47 - 1.27 (m, 32H), 0.92 (t, J = 6.8 Hz,9H).

[0348] 8. Synthesis of YK-1308

[0349] The synthetic route of YK-1308 is as follows:

[0350]

[0351] Step 1: Synthesis of YK-1308-PM1

[0352] YK-1307-PM1 (450 mg, 1.88 mmol) was dissolved in DCM, and EDCI (1.44 g, 7.51 mmol), DMAP (46 mg, 0.38 mmol) and oleic acid (1.33 g, 4.71 mmol) were added in turn and stirred at room temperature for 3 hours. The reaction was monitored by TLC. The reaction was stopped, the reaction system was diluted with water, ethyl acetate was added for extraction, the liquid was separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% - 15% petroleum ether / ethyl acetate) to obtain the product YK-1308-PM1 (600 mg, 0.78 mmol, 41.5%).

[0353] Step 2: Synthesis of YK-1308-PM2

[0354] YK-1308-PM1 (350 mg, 0.46 mmol) was dissolved in 1 mL 4N HCl / dioxane and stirred at room temperature for 2 hours. The reaction was monitored by TLC to complete. The reaction was stopped and the solvent was dried under reduced pressure to obtain YK-1308-PM2 (290 mg, 0.43 mmol, 95.3%).

[0355] Step 3: Synthesis of YK-1308.

[0356] YK-1307-PM6 (86 mg, 0.43 mmol) was dissolved in DMF, and HATU (198 mg, 0.52mmol) and DIEA (167 mg, 1.29 mmol) were added in turn and stirred at room temperature for 5 min. YK-1308-PM2 (290 mg, 0.43 mmol) was added to the above solution and stirred at room temperature for 5 hours. The reaction was monitored by TLC until completion. The reaction was stopped, the reaction system was diluted with water, ethyl acetate was added for extraction, the liquid was separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% - 10% dichloromethane / methanol) to obtain the product YK-1308 (80 mg, 0.09 mmol, 21.7%). 54 H 92 N2O5, MS (ES): m / z ( M +1) = 849.7.

[0357] YK-1308: 1 H NMR (400 MHz, CDCl3) δ 8.07 - 8.04 (m, 1H), 6.98 (d, J= 14.2Hz, 2H), 6.81 (s, 1H), 5.46 - 5.33 (m, 5H), 2.57 (dd, J = 14.2, 7.0 Hz, 5H),2.07 (t, J = 11.1 Hz, 12H), 1.75 (s, 6H), 1.53 - 1.17 (m, 52H), 0.95 (m, J =13.4, 6.7 Hz, 9H).

[0358] 9. Synthesis of YK-1309

[0359] The synthetic route of YK-1309 is as follows:

[0360]

[0361] Step 1: Synthesis of YK-1309-PM1

[0362] YK-1307-PM1 (450 mg, 1.88 mmol) was dissolved in DMF, potassium carbonate (1.04 g, 7.52 mmol) and linoleyl bromide (1.29 g, 3.95 mmol) were added in sequence, and the temperature was raised to 70 o C and stirred for 5 hours. The reaction was completed after monitoring by TLC. The reaction was stopped, water was added to dilute the reaction system, ethyl acetate was added for extraction, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% - 15% petroleum ether / ethyl acetate) to obtain the product YK-1309-PM1 (890 mg, 1.21 mmol, 62.9%).

[0363] Step 2: Synthesis of YK-1309-PM2

[0364] YK-1309-PM1 (400 mg, 0.54 mmol) was dissolved in 1 mL 4N HCl / dioxane and stirred at room temperature for 2 hours. The reaction was monitored by TLC to complete. The reaction was stopped and the solvent was dried under reduced pressure to obtain YK-1309-PM2 (340 mg, 0.53 mmol, 98.5%).

[0365] Step 3: Synthesis of YK-1309.

[0366] YK-1307-PM6 (63 mg, 0.31 mmol) was dissolved in DMF, HATU (144 mg, 0.38mmol) and DIEA (163 mg, 1.26 mmol) were added in turn and stirred at room temperature for 5 min. YK-1309-PM2 (200 mg, 0.31 mmol) was added to the above solution and stirred at room temperature for 5 hours. The reaction was completed by TLC. The reaction was stopped, the reaction system was diluted with water, ethyl acetate was added for extraction, the liquid was separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was dried under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (0% - 15% dichloromethane / methanol) to obtain the product YK-1309 (77 mg, 0.09 mmol, 30.0%). 54 H 92 N2O3, MS (ES): m / z ( M +1) = 817.7.

[0367] YK-1309: 1 H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 6.47 - 6.31 (m, 3H), 5.49 - 5.28 (m, 8H), 4.34 (dd, J = 16.0, 7.4 Hz, 2H), 3.95 (t, J = 6.4 Hz, 4H),2.83 - 2.76 (m, 4H), 2.16 - 1.98 (m, 12H), 1.89 (t, J = 24.1 Hz, 4H), 1.82 -1.57 (m, 8H), 1.52 - 1.21 (m, 36H), 0.97 (dt, J = 13.6, 6.3 Hz, 9H).

[0368] 10. Synthesis of Compound 120

[0369]

[0370] Referring to the synthetic route of Example 120 in CN105164102A, 43 mg of Compound 120 was obtained.

[0371] 11. Synthesis of Compound 45

[0372]

[0373] Referring to the synthetic route of Example 10 in US20230183168A1, 59 mg of compound 45 was obtained.

[0374] 12. Synthesis of E-Ac7-DOg

[0375]

[0376] Referring to the synthetic route of Example 1 in CN115362143A, 37 mg of E-Ac7-DOg was obtained.

[0377] 13. Synthesis of Compound 4

[0378]

[0379] Referring to the synthetic route of Example 4 in WO2015095340A1, 66 mg of compound 4 was obtained.

[0380] 14. Synthesis of Compound 6.2.c

[0381]

[0382] Referring to the synthetic route of Example 4 in WO2022207938A1, 66 mg of compound 6.2.c was obtained.

[0383] Example 2: Preparation of mRNA pharmaceutical composition

[0384] A) Preparation of Fluc DNA, eGFP DNA, and OVA DNA templates

[0385] 1) Luciferase (Luciferase protein CDS), green fluorescent protein (GFP), and soft albumin (OVA) circular plasmids were constructed on the pVAX1 vector (purchased from Thermo Fisher Scientific) by EcoRV restriction enzyme digestion;

[0386] 2) Take the plasmid constructed in step 1) on the pVAX1 vector and mix it with 50 μL of E. coli competent cells Stbl2 (purchased from Thermo Fisher Scientific), incubate them on ice for 30 minutes, heat shock them at 42°C for 90 seconds, put them back on ice immediately, and incubate them on ice for 2 minutes;

[0387] 3) Add 400 μL LB medium (purchased from Thermo Fisher Scientific) and culture at 30°C with slow shaking for 45-60 minutes;

[0388] 4) Take 50-100 μL of bacterial solution and apply it on LB solid medium containing kanamycin antibiotic (100 μg / mL, purchased from Yisheng Biotechnology Co., Ltd.), and invert and culture at 37°C overnight;

[0389] 5) Sequence the obtained monoclonal colony plate to verify its correctness, pick the monoclonal colony with correct sequencing and culture it in a shaker at 30℃ with slow shaking overnight;

[0390] 6) Use an endotoxin-free plasmid extraction kit (purchased from Yisheng Biotechnology Co., Ltd.) to extract the plasmid;

[0391] 7) The extracted plasmid is digested with restriction endonucleases to form a linearized plasmid for use as a transcription template. For specific digestion steps, see Steps ① to ③.

[0392] Step ① Take 1 mg of luciferase circular plasmid and digest it with BspQ Ⅰ enzyme (purchased from Yisheng Biotechnology Co., Ltd.) at 37°C for 4 hours to form a linearized DNA transcription template (enzyme digestion system see Table 1);

[0393] Table 1 Enzyme digestion reaction system

[0394]

[0395] Step ② After the reaction is completed, add anhydrous ethanol and sodium acetate in sequence according to V 酶切反应产物 :V 无水乙醇 :V 3M醋酸钠 =1:3:1 volume ratio of anhydrous ethanol and 3M sodium acetate were added, placed at -20℃ for 1 hour, and then centrifuged at 12000 rpm to retain the precipitate;

[0396] Step ③: Wash the precipitate from step ② twice with 70% ethanol, dry the centrifuged material at 55°C for 10 minutes, and then add 1.7 mL of water for injection to dissolve it;

[0397] The concentration of the linearized plasmid in the dissolved solution was 500 ng / µL, the linearization ratio was more than 90%, and the purification recovery efficiency was 85%.

[0398] B) Preparation of Fluc mRNA, eGFP mRNA and OVA mRNA

[0399] 1) Co-transcriptional capping reaction:

[0400] Using Fluc DNA, eGFP DNA and OVA DNA prepared in A) as templates, NTP solution (NTPs) and Cap1 cap analog (Cat. No.: 10678ES80, purchased from Yisheng Biotechnology Co., Ltd.) as starting materials, mRNA was synthesized by transcription using T7 RNA polymerase. The specific reaction system is shown in Table 2. The prepared reaction system was placed in a 37°C incubator for 3 hours of shaking. The Cap1 cap analog is Cap1-GAG, which has a structure of m7G (5') ppp (5') (2'-OMeA) pG, and its molecular formula is C 32 H 43 N 15 O 24 P4.

[0401] Table 2 Co-transcription capping reaction system

[0402]

[0403] Note: All the above reagents were purchased from Yisheng Biotechnology Co., Ltd.

[0404] 2) Digestion of template DNA:

[0405] In the co-transcription capping reaction system completed in step 1) above, DNase I (purchased from Yisheng Biotechnology Co., Ltd.) was added to make the final concentration of 1 U / μg linearized plasmid, mixed well, centrifuged, and digested at 37°C for 1 hour to obtain the co-transcription capping product.

[0406] 3) Purification by lithium chloride precipitation method:

[0407] The co-transcription capping product obtained in step 2) above was purified by lithium chloride precipitation as follows:

[0408] Step ① Adding lithium chloride: Add lithium chloride solution (purchased from Thermo Fisher Scientific) to the product of step 2) above, with a final concentration of 2.8 M, and precipitate at low temperature for 2 hours;

[0409] Step ② precipitation: high-speed centrifugation at 12000 rpm for 15 minutes, retaining the precipitate;

[0410] Step ③ Washing: Wash twice with 75% ethanol and dissolve with water for injection to obtain mRNA solution. The purified mRNA solution is stored at -80°C.

[0411] Example 3: Effects of different adjuvant lipid addition amounts on mRNA pharmaceutical compositions

[0412] ADOPE, DOTMA, DOPE and YK-1301 were weighed according to the proportions in Table 3 and dissolved in ethanol to prepare an ethanol lipid complex solution (total lipid concentration was 266 mM). The ethanol lipid complex solution was quickly added to the stirring deenzyme water by ethanol injection method at a speed of 120 rmp and stirred at room temperature for 30 minutes. The resulting mixture solution was filtered through a polycarbonate membrane with a pore size of 450 nm to obtain a lipid composition solution and stored at 4°C to 8°C. The mRNA was diluted in a buffer to obtain an mRNA aqueous solution (mRNA concentration 0.5 mg / ml, the buffer was 10 mM HEPES buffer containing 0.1 mM EDTA). A sodium chloride aqueous solution (0.9% w / w) was taken with a syringe and injected into the above-prepared mRNA aqueous solution to obtain a mixed solution of mRNA and sodium chloride. The lipid complex solution was taken with a syringe and injected into the mixture of mRNA and sodium chloride, vortexed for 30 seconds, incubated at room temperature for 10 minutes, and the mRNA pharmaceutical composition with or without adjuvant lipid was obtained (the final RNA concentration was 100 μg / mL), and stored at 4°C to 8°C. The amount of mRNA and lipid complex solution was such that the charge ratio of the TLP-mRNA composition was 1: 2, and the charge ratio was calculated as follows:

[0413] The charge ratio of the mRNA pharmaceutical composition = (the molar amount of positive charge carried by the permanent cationic lipid - the molar amount of negative charge carried by the permanent anionic lipid) / (the weight of the mRNA (g) divided by the average molecular weight of the base 330 (g / mol)).

[0414] The mRNA pharmaceutical composition was diluted by mixing with an aqueous sodium chloride solution (0.9% w / w) at a volume ratio of 1:5, and the particle size and dispersion index (PDI) were measured using dynamic light scattering and a Malvern laser particle size analyzer.

[0415] Table 3 Particle size and PDI of mRNA pharmaceutical compositions with different formulations

[0416]

[0417] The results show that YK-1301 is added in an amount of 2.5 mol%, 5.0 mol%, 10.0 mol%, 15.0 mol%, and 20.0 mol% of the total lipids to partially replace the permanent cationic lipid DOTMA in the TLP composition, and the particle size and PDI of the obtained mRNA pharmaceutical composition are within the qualified range (the qualified range refers to a particle size of 220-500 nm and a PDI value <0.5). The following examples use a 10 mol% adjuvant lipid ratio to detect the characteristics of mRNA pharmaceutical compositions made with other adjuvant lipids.

[0418] Example 4: Effects of adding different adjuvant lipids on mRNA pharmaceutical compositions

[0419] Table 4 Structure and source of adjuvant lipids used in lipid compositions

[0420]

[0421] According to the preparation method of Example 3, the adjuvant lipid in Table 4 was used to partially replace DOTMA in the TLP composition at a ratio of 10 mol% of the total lipids, that is, the permanent anionic lipid ADOPE, the permanent cationic lipid DOTMA, the neutral lipid DOPE and the adjuvant lipid were in a molar ratio of 5:8:5:2 (that is, in the lipid composition, the molar percentages of the permanent anionic lipid, the permanent cationic lipid, the neutral lipid and the adjuvant lipid were 25 mol%, 40 mol%, 25 mol% and 10 mol%, respectively) to prepare an mRNA pharmaceutical composition. The results are shown in Table 5.

[0422] Table 5 Particle size and PDI test results of mRNA pharmaceutical compositions containing different adjuvant lipids

[0423]

[0424] In Table 5, the mRNA pharmaceutical composition No. 10 directly uses TLP to encapsulate mRNA without adding adjuvant lipids, and is also referred to as the "TLP" group. TLP is an mRNA pharmaceutical composition prepared according to the method of Example 3, with the molar percentages of permanent anionic lipid ADOPE, permanent cationic lipid DOTMA and neutral lipid DOPE being 25 mol%, 50 mol% and 25 mol%, respectively.

[0425] The results showed that the adjuvant lipids YK-1301 to YK-1309 in Table 4 and compounds 120, compound 45, E-Ac7-Dog, compound 4, and compound 6.2.c were all able to prepare mRNA pharmaceutical compositions with qualified particle size and PDI (particle size controlled within 220-500 nm, PDI less than 0.5) according to the preparation method of Example 2.

[0426] Example 5: Protein expression experiment of mRNA pharmaceutical composition prepared by adding adjuvant lipid in mice

[0427] The Fluc-mRNA pharmaceutical composition prepared in Example 4 with or without adjuvant lipids was injected into female BALB / c albino mice aged 4-6 weeks and weighing 17-19 g via the tail vein (the dosage was about 20 μg Fluc-mRNA / mouse), and the fluorescent imaging substrate was injected into the mice by intraperitoneal injection 6 hours after administration. The mice moved freely for 5 minutes, and then the total radiation intensity of the protein expressed in the mouse by the mRNA carried by the mRNA pharmaceutical composition was detected by IVIS Spectrum small animal in vivo imaging (corresponding to the expression intensity of the fluorescent protein, i.e., the protein expression amount). After the sampling was completed, the mice were killed by cervical dislocation and dissected to accurately separate the internal organs of the mice: liver and spleen. The total radiation intensity of the protein expressed by Fluc-mRNA in various organs of the mouse was detected by IVIS Spectrum small animal in vivo imaging (corresponding to the expression intensity of the fluorescent protein, i.e., the protein expression amount). The results of mouse in vivo imaging and protein expression detection in the liver and spleen are shown in Tables 6 and Figure 1 .

[0428] Table 6 Experimental data of mouse in vivo and organ imaging

[0429]

[0430] It can be seen that the mRNA pharmaceutical composition containing the adjuvant lipid of the present invention can efficiently deliver mRNA to the spleen, and the delivery effect is significantly enhanced compared with the mRNA pharmaceutical composition encapsulated by the TLP composition without adding adjuvant lipid (serial number 10 in Table 6).

[0431] Compared with the mRNA pharmaceutical compositions added with adjuvant lipids in the prior art (compound 120, compound 45, E-Ac6e-Dog, compound 4, compound 6.2.c), the total radiation intensity of the spleen and the total radiation intensity of the living body were significantly enhanced after administration of the mRNA pharmaceutical compositions added with YK-1301, YK-1302, YK-1303 and YK-1304 of the present invention. For example, the total radiation intensity of the living body of the composition added with YK-1302 was 2.5 times, 3.7 times, 2.4 times, 4.7 times and 3.8 times that of the composition added with compound 120, compound 45, E-Ac7-Dog, compound 4, and compound 6.2.c, respectively, and the total radiation intensity of the spleen was 2.7 times, 2.8 times, 2.3 times, 2.8 times and 3.4 times that of the composition added with compound 120, compound 45, E-Ac7-Dog, compound 4, and compound 6.2.c, respectively.

[0432] When different adjuvant lipids with similar structures of the present invention are added to the mRNA pharmaceutical composition, the total radiation intensity of the spleen and the total radiation intensity of the living body are greatly different. For example, the total radiation intensity of the spleen and the total radiation intensity of the living body of the mRNA pharmaceutical composition added with YK-1302 are 5.3 times and 3.9 times that of the mRNA pharmaceutical composition added with YK-1309, respectively.

[0433] Example 6: Study on targeting of mouse spleen cells by mRNA pharmaceutical composition with added adjuvant lipids

[0434] 1. The eGFP-mRNA pharmaceutical composition prepared in Example 4 with or without the addition of adjuvant lipid was injected into female C57BL / 6 mice aged 4-6 weeks and weighing 17-19 g via the tail vein (the amount was such that each mouse was injected with about 80 μg of GFP-mRNA), and the mice were killed by cervical dislocation 24 hours after administration and dissected to accurately separate the spleen of the mice.

[0435] 2. Preparation of single cells

[0436] 1) Grind the separated spleen tissue to make it single cell and pass it through a cell sieve;

[0437] 2) Add 10 times the volume (about 4 mL) of red blood cell lysis buffer to lyse and remove the red blood cells in the tissue;

[0438] 3) Count the cells and take 5×10 6 cells into the flow cytometry tube (to ensure the same number of cells for each sample);

[0439] 3. Detection of immune cells in spleen tissue

[0440] 1) Add 100 μL of surface antibody MIX to each single cell suspension (see Table 7 for the composition of surface antibody MIX) and incubate at room temperature in the dark for 15 minutes (one negative control);

[0441] Table 7 Reagents and sources used in mouse spleen cell flow cytometry experiments

[0442]

[0443] 2) Add 2 mL PBS, centrifuge at 500 g for 5 minutes, and discard the supernatant;

[0444] 3) Resuspend the cells in 200 μL PBS (after filtering through a 200-mesh nylon mesh), and test on a flow cytometer Cytoflex S. Analyze the percentage of GFP content in each cell. The order of testing each cell line is as follows:

[0445] T cell GFP ratio: CD45+→CD3+→GFP+

[0446] B cell GFP ratio: CD45+→CD3-CD19+→GFP+

[0447] NK cell GFP ratio: CD45+→NK1.1+→GFP+

[0448] cDC cell GFP ratio: CD45+→F4 / 80-CD11c+→GFP+

[0449] pDC cell GFP ratio: CD45+→F4 / 80-CD11cintCD317+→GFP+

[0450] Macrophage GFP ratio: CD45+→F4 / 80+→GFP+

[0451] The mice injected with an equal volume of a solution of the lipid composition without mRNA encapsulation were set as a blank control group.

[0452] Experimental results:

[0453] The percentage of eGFP-positive cells in mouse spleen cells is shown in Table 8.

[0454] Antigen presenting cells (also known as "antigen-presenting cells") refer to a type of immune cells that can absorb, process antigens, and present the processed antigens to lymphocytes. They include dendritic cells (DC cells), B lymphocytes, and macrophages. They play a vital role in the human body, mainly including immune recognition, immune response, and immune regulation.

[0455] From the data in Table 8 and Figure 2 It can be seen that the mRNA pharmaceutical composition prepared by the lipid composition containing the adjuvant lipid of the present invention can significantly increase the percentage of cells expressing antigens in antigen-presenting cells in the spleen, and can significantly increase the percentage of cells expressing antigens in antigen-presenting cells in the spleen compared with the TLP-mRNA pharmaceutical composition without adding an adjuvant (see sequence number 10 in Table 8). For example, the mRNA pharmaceutical composition containing YK-1303 increases the percentage of eGFP-positive cells of antigen-presenting cells B cells, cDC cells, pDC cells, and macrophages in the spleen by 3.3 times, 2.1 times, 1.8 times, and 2.3 times, respectively.

[0456] Compared with the adjuvant lipids of the prior art, the targeting of the mRNA pharmaceutical composition containing the adjuvant lipids of the present invention to mouse spleen antigen presenting cells is significantly improved. For example, the percentage of eGFP-positive cells of YK-1303 in mouse spleen B cells, cDC cells, pDC cells and macrophages is 3.3 times, 3.5 times, 2.7 times and 1.8 times that of the mRNA pharmaceutical composition containing compound 4 (see sequence number 14 in Table 8), respectively.

[0457] The mRNA pharmaceutical compositions prepared with different adjuvant lipids in the present invention have significant differences in targeting of mouse spleen antigen presenting cells, and the data of YK-1301, YK-1302, YK-1303 and YK-1304 are significantly higher than those of other compounds. For example, the percentage of eGFP-positive cells of the mRNA-TLP composition prepared by YK-1303 in B cells, cDC cells, pDC cells and macrophages is 3.2 times, 3.4 times, 2.4 times and 2.5 times that of the mRNA-TLP composition prepared by YK-1308, respectively.

[0458]

[0459] Example 7: Study on the effect of mRNA pharmaceutical composition prepared with adjuvant lipid on the maturation state of mouse spleen antigen presenting cells

[0460] CD86 (cluster of differentiation 86) is a molecule expressed on antigen presenting cells, which provides co-stimulatory signals required for T cell activation and survival. The upregulation of CD86 can be used to show the maturity of spleen antigen presenting cells. In this example, an experiment to detect the upregulation of CD86 was added on the basis of the flow cytometry experiment in Example 5. The surface antibody used was Brilliant Violet 510™ anti-mouse CD86. The detection order of each cell line is as follows:

[0461] B cell CD86 ratio: CD45+→CD3-CD19+→CD86+

[0462] NK cell CD86 ratio: CD45+→NK1.1+→CD86+

[0463] cDC cell CD86 ratio: CD45+→F4 / 80-CD11c+→CD86+

[0464] pDC cell CD86 ratio: CD45+→F4 / 80-CD11cintCD317+→CD86+

[0465] Macrophage CD86 ratio: CD45+→F4 / 80+→CD86+

[0466] The mice injected with an equal volume of a solution of the lipid composition without mRNA encapsulation were set as a blank control group.

[0467] Experimental results:

[0468] The percentage of CD86 positive cells in mouse spleen cells is shown in Table 9.

[0469] It can be seen that the mRNA pharmaceutical composition prepared by the adjuvant lipid of the present invention can significantly increase the percentage of CD86-positive cells of antigen-presenting cells in the spleen. Compared with the mRNA pharmaceutical composition without the addition of adjuvant lipid (see sequence number 10 in Table 9), the mRNA pharmaceutical composition prepared by the adjuvant lipids YK-1301, YK-1302, YK-1303 and YK-1304 of the present invention can significantly improve the maturity of mouse spleen antigen-presenting cells. For example, the percentage of CD86-positive cells of B cells, cDC cells, pDC cells and macrophages in the mouse spleen of the mRNA pharmaceutical composition with the addition of YK-1302 is 2.1 times, 2.5 times, 1.7 times and 1.4 times the corresponding percentages of the mRNA pharmaceutical composition without the addition of adjuvant lipid (see sequence number 10 in Table 9), respectively.

[0470] Reference Figure 3 It can be seen that compared with the adjuvant lipids of the prior art, the mRNA pharmaceutical compositions prepared by the adjuvant lipids YK-1301, YK-1302, YK-1303 and YK-1304 of the present invention significantly improve the maturation state of mouse spleen antigen presenting cells. For example, the mRNA pharmaceutical composition prepared by YK-1304 has a CD86 positive cell percentage of B cells, cDC cells, pDC cells and macrophages in the mouse spleen that is 2.1 times, 2.4 times, 1.8 times and 2.5 times the corresponding percentage of the mRNA pharmaceutical composition prepared by compound 120, respectively.

[0471] In the mRNA pharmaceutical compositions prepared by different adjuvant lipids provided by the present invention, there are significant differences in the percentage of CD86 positive cells in spleen antigen presenting cells. For example, the percentage of CD86 positive cells in B cells, cDC cells, pDC cells and macrophages in the mouse spleen of the mRNA pharmaceutical composition prepared by YK-1304 is 2.2 times, 2.3 times, 1.7 times and 2.3 times the corresponding percentages of the mRNA pharmaceutical composition prepared by YK-1309.

[0472]

[0473] Example 8: Study on T cell activation in the spleen of mice using mRNA-TLP compositions prepared with adjuvant lipids

[0474] CD69 is a marker of T cell activation and one of the earliest markers to be upregulated after T cell activation. The upregulation of CD69 can be used to show the activation of spleen T cells. In this example, an experiment to detect the upregulation of CD69 was added based on the flow cytometry experiment in Example 5. The surface antibody was APC anti-mouse CD69, and the cell line detection order was as follows:

[0475] T cell CD69 ratio:CD45 + →CD3 + →CD69 +

[0476] Experimental results:

[0477] The percentage of CD69 positive T cells in mouse spleen is shown in Table 10.

[0478] Table 10 The percentage of CD69 positive T cells in mouse spleen

[0479]

[0480] The mice injected with an equal volume of a solution of the lipid composition without mRNA encapsulation were set as a blank control group.

[0481] The mRNA pharmaceutical compositions prepared using the adjuvant lipids of the present invention can significantly increase the percentage of T cell CD69-positive cells in the spleen, and can significantly increase the percentage of T cell CD69-positive cells in the spleen compared with the mRNA-TLP compositions without the addition of adjuvant lipids (see sequence number 10 in Table 10). For example, compared with TLP without the addition of adjuvant, the mRNA pharmaceutical compositions prepared using lipid compositions with the adjuvant lipids YK-1301, YK-1302, YK-1303 and YK-1304 of the present invention make the percentage of T cell CD69-positive cells in the spleen 1.8 times, 1.8 times, 1.8 times, and 1.7 times that of the mRNA pharmaceutical compositions prepared by TLP, respectively, which is a significant improvement.

[0482] Reference Figure 4 It can be seen that compared with the adjuvant lipids of the prior art, the mRNA pharmaceutical composition prepared by the adjuvant lipid of the present invention significantly improves the activation state of mouse spleen T cells. For example, the percentage of CD69 positive cells of T cells in the mouse spleen of the mRNA pharmaceutical composition prepared by YK-1302 is 2.0 times the corresponding percentage of the mRNA pharmaceutical composition of E-Ac7-DOg.

[0483] The mRNA pharmaceutical compositions prepared by different adjuvant lipids of the present invention make the percentage of T cell CD69 positive cells in the mouse spleen significantly different. For example, the mRNA pharmaceutical compositions prepared by YK-1301, YK-1302, YK-1303 and YK-1304 make the percentage of T cell CD69 positive cells in the mouse spleen significantly higher than that of other compositions. After the administration of the mRNA pharmaceutical composition prepared by YK-1302, the percentage of T cell CD69 positive cells was the highest, which was 1.9 times that of the lowest YK-1307.

[0484] Example 9: Stimulating effect of mRNA pharmaceutical composition containing adjuvant lipid on cytokines IFN-γ and IL-12

[0485] Interferon-α (IFN-α) is an important cytokine, mainly produced by activated T cells and natural killer (NK) cells. It plays a key role in immune response, has multiple functions such as antiviral, antitumor, immunomodulatory and promoting inflammatory response, and can treat a variety of diseases. Interleukin-12 (IL-12) is a multifunctional cytokine produced by dendritic cells, macrophages, B lymphocytes and other antigen-presenting cells. It plays an important role in regulating immune response, promoting Th1 immunity, enhancing cytotoxicity and tumor immunity. TNF-α, or tumor necrosis factor α, is a ligand of the TNF superfamily. It is a multifunctional cellular molecule that plays a core role in inflammation, apoptosis and immune system development. Detecting the stimulation of cytokines IFN-α, IL-12 and TNF-α by mRNA pharmaceutical compositions containing adjuvant lipids can reflect the improvement of the innate immunity of mRNA pharmaceutical compositions by adjuvant lipids.

[0486] Experimental process:

[0487] In this example, 6-8 week old female C57BL / 6J mice were injected with the OVA-mRNA pharmaceutical composition prepared in Example 4 (i.e., serial numbers 1 to 4, 10 to 11 in Table 5 of Example 4 (mRNA injection amount 40 µg / mouse)) through the tail vein, and the eyeballs were removed and euthanized 6 hours after blood collection to obtain as much serum as possible. The levels of IFN-α, IL-12 and TNF-α in the serum were measured by ELISA. At the same time, mice injected with an equal volume of a solution of the lipid composition without mRNA encapsulation were set as a blank control group.

[0488] ELISA assays: Mouse IFN-α, IL-12, and TNF-α were detected in mouse sera using standard ELISA assays according to the manufacturer's instructions.

[0489] Table 11 The stimulating effect of mRNA pharmaceutical compositions containing adjuvant lipids on IFN-α, IL-12 and TNF-α

[0490]

[0491] Experimental results:

[0492] As shown in Table 11 and Figure 5As shown, compared with the mRNA pharmaceutical composition without adjuvant lipid, the mRNA pharmaceutical composition prepared by the adjuvant lipid of the present invention has a significant increase in IFN-α, IL-12 and TNF-α cytokines in the serum 6 hours after injection, among which the IFN-γ stimulated by the mRNA pharmaceutical composition prepared by YK-1301~1304 is 1.6 times, 1.8 times, 1.5 times and 1.3 times that of the mRNA-TLP composition without adjuvant lipid (serial number 10), respectively; the IL-12 stimulated by the mRNA pharmaceutical composition prepared by YK-1301 to YK-1304 is 3.0 times, 2.4 times, 2.9 times and 3.5 times that of the mRNA pharmaceutical composition without adjuvant lipid, respectively; the TNF-α stimulated by the mRNA pharmaceutical composition prepared by YK-1301 to YK-1304 is 2.2 times, 1.9 times, 2.1 times and 2.4 times that of the mRNA-TLP composition without adjuvant lipid (serial number 10), respectively.

[0493] Compared with the adjuvant lipids in the prior art, the mRNA pharmaceutical compositions prepared by the adjuvant lipids of the present invention have significantly increased IFN-α, IL-12 and TNF-α cytokines in the serum 6 hours after injection, among which the IFN-γ stimulated by the mRNA pharmaceutical compositions prepared by YK-1301 to YK-1304 are 1.9 times, 2.1 times, 1.8 times and 1.6 times that of the mRNA pharmaceutical composition prepared by compound 120, respectively; the IL-12 stimulated by the mRNA pharmaceutical compositions prepared by YK-1301 to YK-1304 are 1.9 times, 1.5 times, 1.8 times and 2.2 times that of the mRNA pharmaceutical composition prepared by compound 120, respectively; and the TNF-α stimulated by the mRNA pharmaceutical compositions prepared by YK-1301 to YK-1304 are 1.7 times, 1.4 times, 1.6 times and 1.8 times that of the mRNA pharmaceutical composition (serial number 11) prepared by compound 120, respectively.

[0494] The experimental results show that by detecting the IFN-α, IL-12 and TNF-α cytokine stimulation of the mRNA pharmaceutical composition containing adjuvant lipids, it can be proved that the adjuvant lipids of the present invention can significantly improve the innate immunity of the mRNA-TLP composition.

[0495] Example 10: Therapeutic effect of mRNA pharmaceutical composition containing adjuvant lipid on tumor-bearing mouse model

[0496] Experimental process:

[0497] 1) Establishment of B16F10-OVA mouse model: Female C57BL / 6J mice between 6 and 8 weeks of age were acclimated for at least three days prior to the study. Mice had free access to food and sterile water and were housed at 22°C ± 2°C and 55% ± 15% relative humidity with a 12-hour light / dark cycle. B16F10-OVA cells were cultured in complete medium as described in the instructions at 37°C with 5% CO2. Cells were harvested using 0.25% trypsin-EDTA, resuspended in Dulbecco's phosphate-buffered saline (DPBS), and then 2 × 10 5 The B16F10-OVA cells / 100 μL / mouse were transplanted into female C57BL / 6J mice by subcutaneous (sc.) injection to establish a subcutaneous B16F10-OVA tumor model. 3 Start vaccinations around.

[0498] 2) Vaccination: For the B16F10-OVA mouse model, the C57BL / 6J mice enrolled were administered with the pharmaceutical composition containing adjuvant lipids prepared according to Example 4 (serial numbers 1 to 4, respectively named YK-1301-TLP, YK-1302-TLP, YK-1303-TLP, YK-1304-TLP) and adjuvant-free lipids (serial number 10, named TLP). The vaccination method was to vaccinate via tail vein injection on days 3, 7, 10, 12, 14, and 17 after the injection of cells (day 0) (each injection contained 40 μg of the therapeutic agent mRNA-OVA), and the mice vaccinated with an equal volume of a lipid composition solution without mRNA encapsulation were set as a blank control group, with 8 mice in each group in parallel.

[0499] 3) Tumor size and mouse survival rate: Starting from the 7th day after tumor inoculation, the tumor diameter was measured 3 times a week. The tumor volume of C57BL / 6J mice was calculated according to the following formula: V (mm 3 )=x×y 2 / 2, in mm, where V represents tumor volume, x represents the long diameter of the tumor, and y represents the short diameter of the tumor. At the same time, the changes in the body weight of C57BL / 6J mice were recorded 3 times a week using an electronic balance, and the survival rate was calculated.

[0500] Experimental results:

[0501] like Figure 6As shown in Table 12, on the 10th day after tumor inoculation, the mice in the blank control group and the OVA mRNA-TLP composition group without adjuvant entered the rapid tumor growth phase, but the growth rate of the TLP group was much lower than that of the blank control group. Correspondingly, the OVA mRNA pharmaceutical composition group with adjuvant lipids showed significant tumor growth delay.

[0502] Starting from the 14th day, compared with the TLP group, the tumor volume of the mRNA drug compositions with adjuvant lipids also showed stronger tumor growth inhibition, among which the mRNA drug compositions prepared from YK-1301 to YK-1304 showed relatively better tumor inhibition effects.

[0503] In terms of survival rate, Figure 7 As shown in Table 13, the blank control group began to die on the 17th day after tumor inoculation, and all died on the 19th day; the TLP group without adjuvant began to die on the 21st day, and all died on the 31st day; the mRNA pharmaceutical compositions with adjuvant lipids YK-1301 to YK-1304 began to die on the 25th, 25th, 27th, and 25th days, respectively, and all died on the 43rd, 42nd, 44th, and 44th days, respectively. It can be seen that the addition of adjuvant lipids significantly prolonged the survival time of mice.

[0504] Table 12 Tumor growth in mice during tumor treatment with different compositions

[0505]

[0506] Table 13 Survival rate of mice during tumor treatment with different compositions

[0507]

[0508] It can be seen from the test results of the above examples that the present invention designs a series of novel STING agonist vaccine adjuvant lipids, such as YK-1301 to YK-1304, which have the following advantages compared with the adjuvant lipids disclosed in the prior art:

[0509] 1. The chemical structure of the adjuvant lipid designed in the present invention is different from that of the adjuvant lipid disclosed in the prior art and is a completely new compound.

[0510] 2. Adding the adjuvant lipid of the present invention to the mRNA-TLP composition can significantly increase the protein expression in mice, and has significant spleen targeting. Compared with the adjuvants of the prior art, the mRNA-TLP composition prepared by the adjuvant lipid of the present invention can significantly increase the protein expression in the living mice and spleen.

[0511] 3. The mRNA pharmaceutical composition prepared by the adjuvant lipid of the present invention significantly increases the percentage of antigen-presenting cells (such as B cells, pDC cells, cDC cells and macrophages) expressing antigens in the spleen. Compared with the adjuvant lipids of the prior art, the mRNA pharmaceutical composition prepared by the adjuvant lipid of the present invention has significantly improved targeting of antigen-presenting cells in the spleen of mice.

[0512] 4. The mRNA pharmaceutical composition prepared by the adjuvant lipid of the present invention can significantly increase the percentage of CD86-positive antigen-presenting cells in the spleen. Compared with the adjuvant lipid of the prior art, the mRNA pharmaceutical composition prepared by the adjuvant lipid of the present invention significantly improves the maturation state of mouse spleen antigen-presenting cells.

[0513] 5. The mRNA pharmaceutical composition prepared by the adjuvant lipid of the present invention can significantly increase the percentage of CD69-positive T cells in the spleen. Compared with the adjuvant lipid of the prior art, the mRNA pharmaceutical composition prepared by the adjuvant lipid of the present invention significantly improves the activation state of mouse spleen T cells.

[0514] 6. The mRNA pharmaceutical composition prepared by adding the adjuvant lipid of the present invention showed significant effects in controlling tumor growth and prolonging the survival time of tumor-bearing experimental animals in animal experiments compared with the mRNA-TLP composition without adjuvant lipid.

[0515] The above is a detailed description of the present invention, the purpose of which is to enable those familiar with the technology in this field to understand the content of the present invention and implement it, and it is not intended to limit the scope of protection of the present invention. All equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that: The compound comprises a structure shown in formula (I) or formula (II): ， ， In formula (I) and formula (II): L is unsubstituted C 2-5 Straight chain alkylene; M1 is -OC(O)- or -C(O)OCH2-; M2 and M3 are -O-; R1 is or ; R2 and R3 are unsubstituted C 16-24 In a straight chain alkenyl group, R2 and R3 are the same or different.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein L is -(CH2)2- or -(CH2)3-; and / or, R2 is unsubstituted C 16-20 Straight chain alkenyl; and / or, R3 is unsubstituted C 16-20 Straight chain alkenyl.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein The compound has any of the following structures: 、 、 、 。 4. A lipid composition comprising an adjuvant lipid, characterized in that The adjuvant lipid comprises the compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof.

5. The lipid composition according to claim 4, wherein The molar percentage of the adjuvant lipid in the lipid composition is 2.5-20%.

6. The lipid composition according to claim 4, wherein The lipid composition also comprises a first lipid composition of permanent anionic lipids, permanent cationic lipids, and neutral lipids.

7. The lipid composition according to claim 6, wherein The molar percentage ratio of the adjuvant lipid, the permanent anionic lipid, the permanent cationic lipid and the neutral lipid in the lipid composition is 2.5-20:10-33:40-60:20-40.

8. The lipid composition according to claim 6 or 7, wherein The permanent anionic lipid is any one or a combination of at least two selected from the group consisting of 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylthioureido)ethyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-ethylureido)ethyl) phosphate, (R)-2,3-bis(oleoyloxy)propyl-(2-(3-propylureido)ethyl) phosphate and (R)-2,3-bis(oleoyloxy)propyl-(2-(3-butylureido)ethyl) phosphate and salts thereof; And / or, the permanent cationic lipid is selected from any one or a combination of at least two of the group consisting of 1,2-dioctadecenyloxy-3-methylammonium propane, (2,3-dioleyloxypropyl)trimethylammonium and salts thereof; And / or, the neutral lipid is any one selected from the group consisting of 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine, distearoylphosphatidylcholine and salts thereof, or a combination of at least two thereof.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the lipid composition according to any one of claims 4 to 8 and an active ingredient; The active ingredient is selected from any one of the group consisting of nucleic acid molecules, small molecule compounds, polypeptides or proteins, or a combination of at least two of them.

10. The pharmaceutical composition according to claim 9, wherein The active ingredient and the lipid composition are used in amounts such that the charge ratio of positive charge to negative charge in the pharmaceutical composition is 1:2 to 1:

5.

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

12. The pharmaceutical composition according to claim 11, wherein The antigen is a disease-associated antigen, or the nucleic acid molecule or antigen can induce an immune response against the disease-associated antigen or a cell expressing the disease-associated antigen.

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

14. The pharmaceutical composition according to claim 9, wherein The pharmaceutical composition further comprises at least one auxiliary ingredient; and / or, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents or excipients; And / or, the pharmaceutical composition further comprises one or more hydrophobic small molecules, permeability enhancing molecules, carbohydrates, polymers, surface altering agents, functionalized lipids or cytokines.

15. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof; the lipid composition of any one of claims 4-8; or the use of the pharmaceutical composition of any one of claims 9-14 in delivering an active ingredient to an antigen presenting cell in a target organ or tissue; The active ingredient is selected from any one of the group consisting of nucleic acid molecules, small molecule compounds, polypeptides or proteins, or a combination of at least two thereof; The use is non-therapeutic, non-diagnostic.

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

17. Use of the compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof; the lipid composition according to any one of claims 4 to 8; or the pharmaceutical composition according to any one of claims 9 to 14 in the preparation of a drug.

18. The use according to claim 17, wherein The medicament is for treating and / or preventing a disease or condition in a subject in need thereof; and / or The drug is selected from any one of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide drugs and protein drugs, or a combination of at least two of them.

19. The use according to claim 18, wherein The disease or condition is characterized by malfunction of an organ, protein or gene, or abnormal activity of a protein or polypeptide.

20. The use according to claim 18 or 19, wherein The disease or condition is selected from any one or a combination of at least two of the group consisting of infectious diseases, tumors and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases and metabolic diseases.

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

22. The use according to claim 18, wherein The subject is a mammal.

23. The use according to claim 22, wherein The mammal is selected from any one of the group consisting of humans, non-human primates, companion animals, exotic species, livestock animals, and food animals.

24. The use according to claim 17, wherein The drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

25. The use according to claim 17, wherein The administration dose of the drug is 0.001-10 mg / kg.

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