Anionic lipid compound and lipid composition and application thereof

Abstract

The present invention relates to the field of molecular biology technology, and provides anionic lipid compounds and lipid compositions and applications thereof. The anionic lipid compounds and lipid compositions provided by the present invention can be used for delivery of therapeutic agents and / or preventive agents such as nucleic acids.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to an anionic lipid compound and a lipid composition and application thereof. Background Art

[0002] Expressing tumor antigens in antigen presenting cells (APCs) by encoding mRNA to induce T cell responses against tumors has great potential in tumor immunotherapy. Since negatively charged mRNA molecules cannot directly enter antigen presenting cells, most existing delivery technologies usually inevitably deliver mRNA in large quantities to the liver and lungs. For immunotherapy, the immune response of mRNA in the lungs or liver is usually harmful. Therefore, the key issue that determines the success of mRNA immunotherapy is to deliver mRNA encoding specific antigens to antigen presenting cells (APCs) with high selectivity.

[0003] Due to the current problem of low targeting of antigen-presenting cells in existing technologies, as well as large-scale cancer genome sequencing efforts and technological advances in predicting immunogenic tumor mutations, the continuous identification of tumor-associated new antigens provides unprecedented opportunities for the development of new and improved tumor therapeutic vaccines.

[0004] Lipoplex (LPX) is a complex delivery vector developed by BioNTech that is generated by cationic lipids and neutral auxiliary lipids, and the mRNA molecule is embedded between the bilayer lipids. For example, CN109331176A discloses that it can be formed by permanent cationic (positively charged) liposomes and anionic (negatively charged) nucleic acids, and neutral lipids can also be added as auxiliary lipids. By optimizing the ratio of lipids and RNA in RNA-lipoplexes (RNA-LPX), the net charge of the formulation composition is adjusted to neutral or negative charge, and after intravenous injection, dendritic cells (DC cells) can be accurately targeted without the need for targeted modification of nanoparticles.

[0005] In the simplest case, lipoplexes are spontaneously formed by mixing nucleic acid with liposomes via a certain mixing scheme, but a variety of other schemes are also applicable. Electrostatic interactions between positively charged liposomes and negatively charged nucleic acids are the driving force for the formation of lipoplexes. In addition to the lipid components, the charge ratio between the cationic and anionic parts also plays an important role in efficient condensation and transfection. In general, an excess of positive charge in the lipoplex is considered to be necessary for efficient transfection (Templeton, N.S. et al., (1997) Nature Biotechnology 15(7): 647-652; Zhdanov, R.I. et al., (2002) Bioelectrochemistry 58(1): 53-64; Templeton, N.S. (2003) Current Medicinal Chemistry 10(14): 1279-1287). Most natural membranes are negatively charged, and the electrostatic attraction between positively charged liposome complexes and negatively charged biological membranes may play a role in cell binding and liposome complex uptake; conversely, if there is a low excess positive charge, the transfection efficiency is greatly reduced or almost zero. However, based on the reported high cytotoxicity of positively charged liposomes and liposome complexes, their application as pharmaceuticals is also problematic.

[0006] Therefore, there is an urgent need to develop improved permanently anionic lipid compounds that facilitate highly selective delivery of therapeutic and / or prophylactic agents such as nucleic acids to antigen presenting cells (APCs), and compositions comprising the same. Summary of the invention

[0007] In one aspect, the present invention provides a novel permanent anionic lipid compound, a solvate thereof, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein the structure of the permanent anionic lipid compound is shown in formula (I):

[0008] Formula (I)

[0009] in,

[0010] X1 is substituted or unsubstituted C 1-10 Straight or branched alkyl, -NH-, -C(O)O- or -O-;

[0011] X2 is O or S;

[0012] R1 is substituted or unsubstituted C 1-15 Straight or branched chain alkyl;

[0013] R2 is substituted or unsubstituted C 8-25 Alkyl or C 8-25 alkenyl;

[0014] R3 is substituted or unsubstituted C 8-25 Alkyl or C 8-25 Alkenyl.

[0015] For example, X1 can be substituted or unsubstituted C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 A straight chain alkyl group, or a substituted or unsubstituted C3, C4, C5, C6, C7, C8, C9, C 10 The branched alkyl group may also be -NH-, -C(O)O- or -O-.

[0016] For example, R1 can be substituted or unsubstituted C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 A straight chain alkyl group, or a substituted or unsubstituted C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 Branched chain alkyl.

[0017] For example, R2 can be substituted or unsubstituted C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 A straight chain or branched chain alkyl group, or a substituted or unsubstituted C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C22 , C 23 , C 24 , C 25 Straight-chain or branched alkenyl.

[0018] For example, R3 can be substituted or unsubstituted C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 A straight chain or branched chain alkyl group, or a substituted or unsubstituted C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 Straight-chain or branched alkenyl.

[0019] In the permanent anion compound provided by the present invention, X1, R1, R2 and R3 may be the same or different.

[0020] According to a preferred embodiment of the present invention, wherein X1 is substituted C 2-8 Straight chain alkyl, -NH- or -C(O)O-.

[0021] "Replaced C 2-8 "Linear alkyl" refers to 2-8 A group formed by replacing one or more H with a substituent on the basis of a straight-chain alkyl group. For example, the substituent may be selected from halogen, hydroxyl, C 1-10 At least one of the linear alkyl groups.

[0022] Preferably, X1 is a substituted C4 or C7 straight chain alkyl group, preferably having the following structure:

[0023] or , wherein "----" represents a substituent (in some preferred embodiments, the substituent is provided by R1).

[0024] Preferably, X1 is -NH-.

[0025] According to some preferred embodiments of the present invention, X2 is O.

[0026] According to some preferred embodiments of the present invention, X2 is S.

[0027] According to a preferred embodiment of the present invention, wherein R1 is an unsubstituted C 2-12 Straight chain alkyl.

[0028] Preferably, R1 is an unsubstituted C3 or C8 straight chain alkyl group.

[0029] According to a preferred embodiment of the present invention, wherein R2 is an unsubstituted C 10-20 (straight or branched) alkyl or unsubstituted C 10-20 (straight-chain or branched) alkenyl.

[0030] Preferably, R2 is unsubstituted C 17 (Straight chain) alkyl or unsubstituted C 17 (Straight chain) alkenyl.

[0031] According to a preferred embodiment of the present invention, wherein R3 is an unsubstituted C 10-20 (straight chain or branched chain) alkyl or unsubstituted (straight chain or branched chain) C 10-20 Alkenyl.

[0032] Preferably, R3 is an unsubstituted (straight chain) C 17 Alkyl or unsubstituted (straight chain) C 17 Alkenyl.

[0033] According to some preferred embodiments of the present invention, the permanent anionic lipid compound is at least one of compounds YK-1101 to YK-1113 having the following structures:

[0034] ,

[0035] ,

[0036] ,

[0037] ,

[0038] ,

[0039] ,

[0040] ,

[0041] ,

[0042] ,

[0043] ,

[0044] ,

[0045] ,or

[0046] .

[0047] Preferably, the permanent anionic lipid compound is at least one of compounds YK-1105, YK-1106, YK-1107 and YK-1108 having the above structure.

[0048] The second aspect of the present invention provides a lipid composition comprising a permanent anionic lipid, wherein the permanent anionic lipid comprises the permanent anionic lipid compound described in the first aspect, a solvate thereof, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0049] According to a preferred embodiment of the present invention, the molar percentage of the permanent anionic lipid in the lipid composition is 10-33%.

[0050] Preferably, the molar percentage of the permanent anionic lipid in the lipid composition is 20-30%.

[0051] More preferably, the molar percentage of the permanent anionic lipid in the lipid composition is 25%.

[0052] According to a preferred embodiment of the present invention, the lipid composition further comprises a permanent cationic lipid.

[0053] Preferably, the molar percentage of the permanent cationic lipid in the lipid composition is 40-60%.

[0054] Preferably, the permanent cationic lipid is any one or a combination of at least two selected from the group consisting of 1,2-dioctadecenyloxy-3-methylammonium propane, (2,3-dioleyloxypropyl)trimethylammonium and salts thereof.

[0055] According to a preferred embodiment of the present invention, the lipid composition further comprises neutral lipids.

[0056] Preferably, the molar percentage of the neutral lipid in the lipid composition is 22-40%.

[0057] Preferably, 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.

[0058] According to some preferred embodiments of the present invention, the lipid composition comprises:

[0059] (1) Permanent anionic lipids;

[0060] (2) permanent cationic lipids; and

[0061] (3) Neutral lipids;

[0062] Wherein, the molar percentage ratio of permanent anionic lipids, permanent cationic lipids and neutral lipids in the lipid composition is 10-33:40-60:22-40 (that is, in the lipid composition, the molar percentage of permanent anionic lipids is 10-30%, the molar percentage of permanent cationic lipids is 40-60%, and the molar percentage of neutral lipids is 22-40%, wherein the sum of the molar percentages of permanent anionic lipids, permanent cationic lipids and neutral lipids is 100%).

[0063] The characteristics of the permanent anionic lipids, permanent cationic lipids and neutral lipids selected in the above-mentioned lipid composition are as described above and will not be repeated here.

[0064] According to some particularly preferred embodiments of the present invention, the lipid composition comprises:

[0065] (1) Permanent anionic lipid: 10 mol%;

[0066] (2) Permanent cationic lipid: 60 mol%;

[0067] (3) Neutral lipids: 30 mol%;

[0068] or

[0069] (1) Permanent anionic lipids: 14 mol%;

[0070] (2) Permanent cationic lipids: 57 mol%;

[0071] (3) Neutral lipids: 29 mol%;

[0072] or

[0073] (1) Permanent anionic lipids: 20 mol%;

[0074] (2) Permanent cationic lipid: 40 mol%;

[0075] (3) Neutral lipids: 40 mol%;

[0076] or

[0077] (1) Permanent anionic lipid: 25 mol%;

[0078] (2) Permanent cationic lipid: 50 mol%;

[0079] (3) Neutral lipids: 25 mol%;

[0080] or

[0081] (1) Permanent anionic lipids: 33 mol%;

[0082] (2) Permanent cationic lipid: 45 mol%;

[0083] (3) Neutral lipids: 22 mol%.

[0084] The permanent anionic lipid compound and lipid composition provided by the present invention can be used for drug preparation, especially for encapsulation of active pharmaceutical ingredients (such as nucleic acids, proteins, polypeptides, small molecule compounds, etc.), so as to form drugs with certain organ / cell targeting, thereby achieving targeted treatment of diseases. In addition, the inventors have also found that after encapsulating active pharmaceutical ingredients using the permanent anionic lipid compound and / or lipid composition provided by the present invention, the obtained drug not only has improved targeting, but also can stimulate the activation of antigen presenting cells in the target organ, prompting the antigen presenting cells to express antigens, thereby improving the effect of disease prevention / treatment.

[0085] Based on the above findings, the third aspect of the present invention provides the permanent anionic lipid compound, its solvate, its pharmaceutically acceptable salt or its stereoisomer described in the first aspect, or the lipid composition described in the second aspect for use in improving the targeting of drugs to target organs and / or target cells.

[0086] For example, the permanent anionic lipid compound, its solvate, its pharmaceutically acceptable salt or its stereoisomer provided by the present invention, or the lipid composition provided by the present invention can be used to prepare drugs with improved targeting to target organs and / or target cells.

[0087] For another example, the permanent anionic lipid compound, its solvate, its pharmaceutically acceptable salt or its stereoisomer provided by the present invention, or the lipid composition provided by the present invention can be used to encapsulate the active ingredients of the drug in research activities or in the process of disease treatment / prevention, so that the targeting of the obtained drug to the target organ and / or target cell is improved. Therefore, the above-mentioned uses provided by the present invention include uses for therapeutic purposes and uses for non-therapeutic purposes.

[0088] According to some preferred embodiments of the present invention, the above-mentioned use provided by the present invention is for non-therapeutic purposes.

[0089] According to a preferred embodiment of the present invention, the molar percentage ratio of permanent anionic lipids, permanent cationic lipids and neutral lipids in the lipid composition is 10-33:40-60:22-40 (that is, in the lipid composition, the molar percentage of permanent anionic lipids is 10-30%, the molar percentage of permanent cationic lipids is 40-60%, and the molar percentage of neutral lipids is 22-40%, wherein the sum of the molar percentages of permanent anionic lipids, permanent cationic lipids and neutral lipids is 100%).

[0090] According to a preferred embodiment of the present invention, the target organ is selected from any one or a combination of at least two of the group consisting of spleen, liver and lung.

[0091] Preferably, the target organ is the spleen.

[0092] According to a preferred embodiment of the present invention, the target cell is an antigen presenting cell.

[0093] Preferably, 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.

[0094] The fourth aspect of the present invention provides the permanent anionic lipid compound described in the first aspect, its solvate, its pharmaceutically acceptable salt or its stereoisomer, or the lipid composition described in the second aspect for use in improving the activation state of antigen presenting cells in target organs and / or improving the expression of antigens by antigen presenting cells in target organs.

[0095] For example, the permanent anionic lipid compound, its solvate, its pharmaceutically acceptable salt or its stereoisomer provided by the present invention, or the lipid composition provided by the present invention can be used to prepare a drug that can increase the activation state of antigen-presenting cells in the target organ and / or increase the expression of antigens by antigen-presenting cells in the target organ.

[0096] For another example, the permanent anionic lipid compound, its solvate, its pharmaceutically acceptable salt or its stereoisomer provided by the present invention, or the lipid composition provided by the present invention can be used to encapsulate the active ingredients of the drug in research activities or in the process of disease treatment / prevention, so that after the administration of the obtained drug, the activation state of the antigen presenting cells in the target organ and / or the expression of the antigen by the antigen presenting cells in the target organ are improved. Therefore, the above-mentioned uses provided by the present invention include uses for therapeutic purposes and uses for non-therapeutic purposes.

[0097] Preferably, the above-mentioned use provided by the present invention is a use for non-therapeutic purposes.

[0098] According to a preferred embodiment of the present invention, in the lipid composition, the molar percentage ratio of permanent anionic lipids, permanent cationic lipids and neutral lipids in the lipid composition is 10-33:40-60:22-40 (that is, in the lipid composition, the molar percentage of permanent anionic lipids is 10-30%, the molar percentage of permanent cationic lipids is 40-60%, and the molar percentage of neutral lipids is 22-40%, wherein the sum of the molar percentages of permanent anionic lipids, permanent cationic lipids and neutral lipids is 100%).

[0099] According to a preferred embodiment of the present invention, the target organ is selected from any one or a combination of at least two of the group consisting of spleen, liver and lung.

[0100] Preferably, the target organ is the spleen.

[0101] According to a preferred embodiment of the present invention, the target cell is an antigen presenting cell.

[0102] Preferably, 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.

[0103] According to a preferred embodiment of the present invention, the antigen is a disease-associated antigen, or the antigen can induce an immune response against the disease-associated antigen or a cell expressing the disease-associated antigen.

[0104] A fifth aspect of the present invention provides a composition comprising:

[0105] (A) a therapeutic agent and / or a preventive agent comprising any one of the group consisting of a nucleic acid molecule, a small molecule compound, a polypeptide and a protein, or a combination of at least two thereof;

[0106] (B) lipid composition;

[0107] The lipid composition includes the lipid composition described in the second aspect;

[0108] The composition is used to deliver the therapeutic agent and / or preventive agent to antigen-presenting cells in a target organ.

[0109] According to a preferred embodiment of the present invention, the therapeutic agent and / or preventive agent is a nucleic acid molecule capable of encoding one or more antigens.

[0110] 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.

[0111] According to a preferred embodiment of the present invention, the nucleic acid molecule is RNA encoding one or more antigens.

[0112] According to a preferred embodiment of the present invention, the target organ is selected from any one or a combination of at least two of the group consisting of spleen, liver and lung.

[0113] Preferably, the target organ is the spleen.

[0114] 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.

[0115] According to a preferred embodiment of the present invention, the amount of the therapeutic agent and / or preventive agent (component (A)) and the lipid composition (component (B)) is such that the charge ratio of positive charge to negative charge in the composition is 1:2 to 1:5. For example, it can be 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, or it can be a range consisting of any two of the above ratios, or any intermediate value in the range.

[0116] According to some preferred embodiments of the present invention, the composition further comprises at least one auxiliary ingredient or adjuvant.

[0117] According to some preferred embodiments of the present invention, the composition further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

[0118] According to some preferred embodiments of the present invention, the composition further comprises one or more hydrophobic small molecules, permeability enhancing molecules, carbohydrates, polymers, surface modifiers, functionalized lipids or cytokines.

[0119] A sixth aspect of the present invention provides a method for preparing a composition for delivering a therapeutic agent and / or a prophylactic agent to an antigen presenting cell in a target organ, the method comprising:

[0120] (a) dissolving a lipid in an organic solvent to form a lipid solution, wherein the lipid comprises the permanent anionic lipid, a solvate thereof, a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to the first aspect;

[0121] (b) mixing the lipid solution obtained in step (a) with water to obtain a lipid mixture;

[0122] (c) mixing the lipid mixture obtained in step (b) with a therapeutic agent and / or a preventive agent to form the composition, wherein the therapeutic agent and / or the preventive agent comprises a nucleic acid buffer solution obtained by dissolving nucleic acid molecules in a buffer solution having a pH value of 6.8-7.6.

[0123] According to a preferred embodiment of the present invention, in step (a), the lipid further comprises a permanent cationic lipid.

[0124] Preferably, in step (a), 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.

[0125] According to a preferred embodiment of the present invention, in step (a), the lipid further comprises a neutral lipid.

[0126] Preferably, in step (a), 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.

[0127] More preferably, the molar percentage ratio of permanent anionic lipids, permanent cationic lipids and neutral lipids is 10-33:40-60:22-40 (the sum of the molar percentages of permanent anionic lipids, permanent cationic lipids and neutral lipids is 100).

[0128] According to a preferred embodiment of the present invention, in step (a), the organic solvent is an alcohol solvent.

[0129] Preferably, the organic solvent comprises an alcohol having 1 to 4 carbon atoms.

[0130] According to a preferred embodiment of the present invention, in step (b), the lipid mixture can pass through a polycarbonate membrane with a pore size of 100-400 nm.

[0131] According to a preferred embodiment of the present invention, in step (c), the buffer comprises aqueous HEPES buffer.

[0132] Preferably, the buffer comprises aqueous HEPES buffer and EDTA.

[0133] According to a preferred embodiment of the present invention, in step (c), the nucleic acid molecule is a nucleic acid molecule capable of encoding one or more antigens.

[0134] Preferably, in step (c), the antigen is a disease-associated antigen, or the nucleic acid molecule or antigen is capable of inducing an immune response against a disease-associated antigen or a cell expressing a disease-associated antigen.

[0135] According to a preferred embodiment of the present invention, in step (c), the amount of the lipid mixture and the therapeutic agent and / or preventive agent is such that the charge ratio of the positive charge to the negative charge in the obtained composition is 1:2 to 1:5. For example, it can be 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, or it can be a range consisting of any two of the above ratios, or any intermediate value in the range.

[0136] The seventh aspect of the present invention provides the use of the permanent anionic lipid compound according to the first aspect, its solvate, its pharmaceutically acceptable salt or its stereoisomer, the lipid composition according to the second aspect, the composition according to the fifth aspect, or the composition prepared according to the method described in the sixth aspect in the preparation of a drug capable of inducing a cellular immune response.

[0137] The eighth aspect of the present invention provides the use of the permanent anionic lipid compound according to the first aspect, its solvate, its pharmaceutically acceptable salt or its stereoisomer, the lipid composition according to the second aspect, the composition according to the fifth aspect, or the composition prepared according to the method described in the sixth aspect in the preparation of a drug for preventing, diagnosing, treating or improving a disease, condition, abnormal condition or dysfunction in a subject.

[0138] According to a preferred embodiment of the present invention, the disease, disorder, abnormal condition or dysfunction is selected from any one of the group consisting of infectious diseases, cancer, proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases, or a combination of at least two of them.

[0139] According to a preferred embodiment of the present invention, the subject is a mammalian subject.

[0140] Preferably, the subject is selected from any one or a combination of at least two of the group consisting of humans, non-human primates, companion animals, exotic species, livestock animals, and food animals.

[0141] The permanent anionic lipid compound and lipid composition provided by the present invention can be used for nucleic acid (such as mRNA, etc.) encapsulation to form corresponding nucleic acid drugs. The mRNA composition prepared by using the permanent anionic lipid compound provided by the present invention has at least the following advantages: 1. Good particle size and uniform particle distribution; 2. Can significantly increase the protein expression of antigen in the subject (such as mouse) or in the spleen; 3. The percentage of antigen-presenting cells expressing antigen in the spleen is significantly increased; 4. The mature state of antigen-presenting cells in the spleen is significantly increased; 5. The activation state of T cells in the spleen is significantly increased.

[0142] Specifically, the permanent anionic lipid compound provided by the present invention has the following advantages:

[0143] 1. The permanent anionic lipid compound provided by the present invention has a unique chemical structure, which is different from the permanent anionic lipids disclosed in the prior art. Experimental verification found that compared with nucleic acid drugs prepared using existing permanent anionic lipids, the nucleic acid drugs prepared using the permanent anionic lipid compound provided by the present invention have significantly improved spleen targeting and spleen antigen-presenting cell targeting, and the expression of nucleic acid-carrying in the spleen and the proportion of antigen-presenting cells expressing the antigen corresponding to the nucleic acid are also significantly increased, and the maturity state of antigen-presenting cells in the spleen and the activation state of T cells are also significantly increased. For example, the differences between the permanent anionic lipid compound provided by the present invention and the prior art may include:

[0144] 1) The compounds YK-1101 to YK-1109 provided by the present invention have different groups connected to the head phosphate of 18PA (US06345442) and DOPG (see EP0172007A2);

[0145] 2) Compounds YK-1110 to YK-1113 in this application and anionic lipid compounds N-valeryl-DPPE, N-butyryl-DPPE (Enhancement of the in vivocirculation lifetime of L -a-distearoylphosphatidylcholine liposomes: importance of liposomal aggregationversus complement opsonization [J]. Biochimica et Biophysica Acta (BBA) -Biomembranes, 1997, 1329(2):370-382) and NAPE (Dietary fatty acids control thespecies of N-acyl-phosphatidylethanolamines synthesized by therapeuticallymodified bacteria in the intestinal tract. [J]. Current Forestry Reports, 2018, 4(1).), the head linking group is different and the tail chain length is different.

[0146] 2. The permanent anionic lipid compounds YK-1101 to YK-1109 provided by the present invention can prepare compositions with good particle size (particle size controlled at 220-500 nm) and uniform particle distribution (PDI < 0.5).

[0147] 1) The permanent anionic lipids YK-1101 to YK-1109 have good solubility in ethanol, and the prepared compositions have good particle size and PDI. The best one is YK-1108, which has a particle size of 235.2nm, a PDI as low as 0.1806, and good particle uniformity.

[0148] 2) The permanent anionic lipids YK-1110 to YK-1113, N-valeryl-DPPE, N-butyryl-DPPE, and NAPE have low solubility in ethanol and solid precipitation occurs during the preparation process, making them unsuitable as mRNA delivery vectors.

[0149] 3. The nucleic acid drug composition prepared from the permanent anionic lipid compound provided by the present invention can significantly increase the protein expression of the antigen in mice (corresponding to the total radiation intensity) and has obvious spleen targeting.

[0150] 1) The nucleic acid (such as mRNA) pharmaceutical composition prepared by the permanent anionic lipid of the present invention can significantly increase the protein expression of the antigen in the spleen, and the composition has good targeting to the spleen. For example, the mRNA composition prepared by YK-1108 has a total radiation intensity of 9.89×10 7 p / s, were 70.6 and 89.9 times that in the liver and lung, respectively.

[0151] 2) The mRNA compositions prepared by different permanent anionic lipids provided by the present invention have significant differences in protein expression in mice. For example, the total radiation intensity of the mRNA composition prepared by YK-1108 in the living body or spleen of mice is 5.9 times and 6.1 times that of the mRNA composition prepared by YK-1101, respectively.

[0152] 3) Compared with the existing permanent anionic lipids, the mRNA composition prepared by the permanent anionic lipids of the present invention significantly improves the protein expression in mice. For example, the total radiation intensity of the mRNA composition prepared by YK-1108 in the living body or spleen of mice is 2.6 times and 2.2 times the corresponding total radiation intensity of DOPG, respectively.

[0153] 4) The protein expression levels of mRNA compositions prepared by different permanent anionic lipids with similar structures in mice vary greatly. For example, the total radiation intensity of the mRNA composition prepared by YK-1108 in the living body or spleen of mice is 2.4 times and 2.6 times that of the corresponding total radiation intensity of YK-1109, respectively, which is a significant difference.

[0154] 5) Compared with the composition without adding anionic lipids, the composition containing permanent anionic lipids of the present invention significantly increases the protein expression in the living body or spleen of mice. For example, the total radiation intensity of the mRNA composition prepared by YK-1108 in the living body or spleen of mice is 3.9 times and 4.4 times the corresponding total radiation intensity of the large-particle LPX-RNA without adding anionic lipids.

[0155] 4. The mRNA composition prepared from the permanent anionic lipid compound provided by the present invention significantly increases the percentage of antigen presenting cells (eg, B cells, pDC cells, cDC cells, macrophages, etc.) expressing antigens in the spleen.

[0156] 1) The mRNA composition prepared by the permanent anionic lipid of the present invention can significantly increase the percentage of eGFP-positive cells in the spleen of antigen-presenting cells. For example, compared with the blank control, the mRNA composition prepared by YK-1108 increases the percentage of eGFP-positive cells in the spleen of antigen-presenting cells B cells, cDC cells, pDC cells, and macrophages by 34.0 times, 22.7 times, 16.1 times, and 202.5 times, respectively.

[0157] 2) The mRNA compositions prepared by different permanent anionic lipids of the present invention have significant differences in targeting to mouse spleen antigen presenting cells. For example, the percentage of eGFP-positive cells of the mRNA composition prepared by YK-1108 in B cells, cDC cells, pDC cells, and macrophages is 3.4 times, 4.1 times, 5.2 times, and 6.1 times that of YK-1101, respectively.

[0158] 3) Compared with the permanent anionic lipids of the prior art, the mRNA composition prepared by the permanent anionic lipids of the present invention has significantly improved targeting in mouse spleen antigen presenting cells. For example, the percentage of eGFP-positive cells of the mRNA composition prepared by YK-1108 in B cells, cDC cells, pDC cells, and macrophages in mouse spleen is 3.1 times, 2.7 times, 3.0 times, and 4.3 times the corresponding percentage of 18PA, respectively.

[0159] 4) Compared with the composition without adding anionic lipids, the mRNA composition prepared by the permanent anionic lipids of the present invention significantly improves the targeting of mouse spleen antigen presenting cells. For example, the percentage of eGFP-positive cells in B cells, cDC cells, pDC cells, and macrophages in the mouse spleen of the mRNA composition prepared by YK-1108 is 4.0 times, 4.4 times, 3.9 times, and 6.8 times the corresponding percentage of large-particle LPX-RNA without adding anionic lipids.

[0160] 5. The mRNA composition prepared from the permanent anionic lipid compound of the present invention significantly increases the maturation state of antigen presenting cells in the spleen.

[0161] 1) The mRNA composition prepared by the permanent anionic lipid provided by the present invention can significantly increase the percentage of CD86-positive antigen presenting cells in the spleen of mice. For example, compared with the blank control, the mRNA composition prepared by YK-1108 increases the percentage of CD86-positive antigen presenting cells B cells, cDC cells, pDC cells, and macrophages in the spleen by 3.0 times, 6.2 times, 5.7 times, and 5.9 times, respectively.

[0162] 2) The mRNA composition prepared by the permanent anionic lipid of the present invention significantly differs the maturation state of mouse spleen antigen presenting cells. The percentage of CD86 positive cells of the mRNA composition prepared by YK-1108 in B cells, cDC cells, pDC cells, and macrophages is 1.9 times, 2.4 times, 1.8 times, and 2.8 times the corresponding percentage of YK-1101, respectively.

[0163] 3) Compared with the permanent anionic lipids of the prior art, the mRNA composition prepared by the permanent anionic lipids of the present invention significantly improves the maturation state 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 composition prepared by YK-1108 is 3.1 times, 1.9 times, 1.4 times, and 2.4 times the corresponding percentage of 18PA, respectively.

[0164] 4) Compared with the composition without adding anionic lipids, the mRNA composition prepared by the permanent anionic lipids of the present invention significantly improves the maturation state of mouse 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 composition prepared by YK-1108 is 2.1 times, 2.6 times, 2.1 times, and 2.9 times the corresponding percentage of large-particle LPX-RNA without adding anionic lipids.

[0165] 6. The mRNA composition prepared from the permanent anionic lipid compound of the present invention significantly increases the activation state of T cells in the spleen.

[0166] 1) The mRNA composition prepared by the permanent anionic lipid of the present invention can significantly increase the percentage of CD69 positive cells of T cells in the spleen. For example, compared with the blank control, the mRNA composition prepared by YK-1108 increases the percentage of CD69 positive cells of T cells in the spleen by 10.8 times.

[0167] 2) The mRNA composition prepared by the permanent anionic lipid of the present invention significantly differed the activation state of mouse spleen T cells. The percentage of T cell CD69 positive cells in the mRNA composition prepared by YK-1108 was the highest, which was 3.0 times the corresponding percentage of YK-1101.

[0168] 3) Compared with the permanent anionic lipids of the prior art, the mRNA composition prepared by the permanent anionic lipids of the present invention significantly improves the activation state of mouse spleen T cells. For example, the percentage of CD69 positive cells in mouse spleen T cells of the mRNA composition prepared by YK-1108 is 1.8 times the corresponding percentage of 18PA.

[0169] 4) Compared with the composition without adding anionic lipids, the composition prepared by the present invention containing permanent anionic lipids can significantly improve the activation state of mouse spleen T cells. For example, the percentage of CD69 positive cells in mouse spleen T cells of the mRNA composition prepared by YK-1108 is 2.7 times the corresponding percentage of large-size LPX-RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0170] 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.

[0171] Figure 1 Fluorescence images of mice and mouse organs (liver, spleen, and lung) treated with the composition containing Fluc-mRNA and large-particle LPX prepared by YK-1108 and 18PA, respectively.

[0172] Figure 2 Flow cytometry analysis of mouse spleen cells of the compositions containing eGFP-mRNA prepared for the control, YK-1105, YK-1106, YK-1107 and YK-1108, respectively (eGFP).

[0173] Figure 3 Flow cytometry analysis of mouse spleen cells (CD86) of the compositions containing eGFP-mRNA prepared for blank, YK-1108, and 18PA, respectively, and large-particle LPX.

[0174] Figure 4 Flow cytometry of mouse spleens containing eGFP-mRNA and large-particle LPX prepared for YK-1101, YK-1107, and YK-1108 (CD69). DETAILED DESCRIPTION

[0175] 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.

[0176] 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.

[0177] 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.

[0178] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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".

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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).

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] The present invention provides a composition with an average size of 220-500 nm and a polydispersity coefficient of ≤0.5.

[0194] Permanent anionic lipids (compounds)

[0195] In some aspects, the present invention provides lipids having one or more hydrophobic components and permanent anionic groups (i.e., permanent anionic lipids provided by the present invention). One anionic group that can be used in the permanent anionic lipid is a phosphate group. The phosphate group can be a deprotonated compound having a negative charge at a pH below 8, 9, 10, 11, 12, 13, or 14. The hydrophobic component can be one or more C6-C 24 The compound may have one hydrophobic group, two hydrophobic groups, or three hydrophobic groups.

[0196] In some embodiments, in the lipid composition provided by the present invention, the permanent anionic lipid is present in an amount of about 10-33 mol% of the total amount of the lipid composition (that is, based on the total amount of the lipid composition, the content of the permanent anionic lipid can be 14-33 mol%). The composition can contain about 10 mol%, 15 mol%, 20 mol%, 25 mol% or 33 mol%, or any range thereof.

[0197] According to some preferred embodiments of the present invention, the permanent anionic lipid is selected from any one or a combination of at least two of the group consisting of the compound of formula (I) as described above, its solvate, its pharmaceutically acceptable salt and its stereoisomer; preferably selected from any one or a combination of at least two of the group consisting of YK-1101 to YK-1113, its solvate, its pharmaceutically acceptable salt (such as sodium salt, potassium salt, etc.) and its stereoisomer.

[0198] The inventors found that the permanent anionic lipid of the present invention has better solubility in ethanol when the tail chain contains an olefinic group, and the lipid composition (prepared with such a permanent anionic lipid has better particle size and PDI, and is more suitable as a nucleic acid delivery carrier.

[0199] Therefore, in some preferred embodiments of the present invention, the permanent anionic lipid is selected from at least one of YK-1101 to YK-1109 and their salts (such as sodium salts, potassium salts, etc.), preferably selected from any one or a combination of at least two of the group consisting of YK-1105, YK-1106, YK-1107 and YK-1108 and their salts (such as sodium salts, potassium salts, etc.).

[0200] The structural features of the above compounds YK-1101 to YK-1113 are as described above and will not be repeated here.

[0201] Permanent cationic lipids

[0202] In some aspects, the lipid compositions provided by the invention include lipids having one or more hydrophobic components and permanent cationic groups (i.e. permanent cationic lipids). The permanent cationic lipids selected for use by the present invention may contain groups having a positive charge (regardless of pH). Preferably, a permanent cationic group in the permanent cationic lipid is a quaternary ammonium group.

[0203] In some embodiments, in the lipid composition provided by the present invention, the permanent cationic lipid is present in an amount of about 40-60 mol% of the total amount of the lipid composition (that is, based on the total amount of the lipid composition, the content of the permanent cationic lipid can be 40-57 mol%). The composition can contain about 40 mol%, 45 mol%, 50 mol%, 55 mol% or 60 mol%, or any range thereof that can be derived.

[0204] According to some preferred embodiments 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 their salts (such as sodium salt, chloride salt, etc.).

[0205] Neutral lipids

[0206] Neutral lipids in the present invention refer to lipids that are uncharged or exist in the form of zwitterions at a selected pH value and play an auxiliary role. Neutral lipids may regulate the fluidity of nanoparticles into lipid bilayer structures and improve efficiency by promoting lipid phase transition, and may also affect the specificity of target organs / target cells.

[0207] Any neutral lipid having the above characteristics in the art, especially a neutral lipid that can be used to prepare a drug carrier, can be applied to the present invention. For example, the neutral lipid can include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and its derivatives.

[0208] The carrier component of the composition comprising cationic lipids can include one or more neutral lipids-phospholipids, such as one or more (many) unsaturated lipids.Phospholipids can be assembled into one or more lipid bilayers.In general, phospholipids can include a phospholipid moiety and one or more fatty acid moieties.

[0209] The neutral lipid part can be selected from the non-limiting group of the following composition: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. The fatty acid part can be selected from the non-limiting group of the following composition: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid. Also encompassed are non-natural species including natural species with modification and replacement, the modification and replacement include branching, oxidation, cyclization and alkynes. For example, phospholipids can be functionalized with one or more alkynes (e.g., one or more double bonds are replaced by triple bond alkenyl) or cross-linked with the one or more alkynes. Under appropriate reaction conditions, alkynyl may undergo copper-catalyzed cycloaddition reactions when exposed to azide. These reactions can be used to functionalize the lipid bilayer of the composition to facilitate membrane permeation or cellular recognition, or to couple the composition to useful components such as targeting or imaging moieties (eg, dyes).

[0210] The neutral lipids useful in these compositions can be selected from the non-limiting group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (DPPG), acylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.

[0211] In some embodiments, the neutral lipid comprises DSPC. In some embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises (both) DSPC and DOPE.

[0212] In some embodiments, in the lipid composition provided by the present invention, the neutral lipid is present in an amount of about 22-40 mol% of the total amount of the lipid composition (that is, based on the total amount of the lipid composition, the content of the neutral lipid can be 22-40 mol%). The composition can contain 22 mol%, 25 mol%, 29 mol%, 35 mol% or 40 mol%, or any range of neutral lipids derived therefrom.

[0213] Therapeutic and / or preventive agents

[0214] The lipid composition of the present invention can be used for delivering active pharmaceutical ingredients, such as therapeutic agents and / or preventive agents. Based on this, the present invention further provides a (pharmaceutical) composition, comprising a lipid composition provided by the present invention, for delivering active pharmaceutical ingredients. The composition of the present invention can include one or more therapeutic agents and / or preventive agents (as active pharmaceutical ingredients). Active pharmaceutical ingredients can be encapsulated in the lipid composition or combined with the lipid composition.

[0215] In some embodiments, the charge ratio of the positive charge in the lipid composition to the negative charge in the active ingredient (such as a therapeutic agent and / or a preventive agent) is 1:2 to 1:5 (that is, the charge ratio of the positive charge to the negative charge in the composition provided by the present invention is 1:2 to 1:5). In some embodiments, the charge ratio is preferably 1:2, 1:2.5, 1:3 or 1:5.

[0216] The therapeutic agent and / or preventive agent includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides and proteins, preferably nucleic acid molecules.

[0217] For example, the therapeutic and / or preventive agent is a vaccine or compound capable of causing an immune response.Therefore, in some preferred embodiments, the therapeutic and / or preventive agent can be a nucleic acid molecule capable of encoding one or more antigens.

[0218] The lipid composition of the present invention can deliver therapeutic agents and / or prophylactic agents to target cells and / or target organs in a subject (such as a mammal) (as a carrier), and thus the present invention also provides methods for treating a disease or disorder in a subject in need thereof, which methods comprise administering a composition comprising a therapeutic agent and / or prophylactic agent to the subject and / or contacting the subject's cells with the composition.

[0219] 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.

[0220] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that causes an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracenedione, The invention relates to a radioactive ion, for example, anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and analogs or homologues thereof. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. Vaccines include compounds and preparations that can provide immunity to one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis and tuberculosis and may include nucleic acid molecules (e.g., mRNA) encoding infectious disease-derived antigens and / or epitopes. Vaccines may also include compounds and preparations that direct an immune response against cancer cells and may include nucleic acid molecules (e.g., mRNA) encoding tumor cell-derived antigens, epitopes and / or new epitopes. Compounds that induce an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species.Other therapeutic and / or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, razithromycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, cyclosporine, cyclophosphamide ... The invention relates to antibiotics such as dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC), and antimitotic agents such as vincristine, vinblastine, taxol and maytansine.

[0221] In other embodiments, the therapeutic and / or prophylactic agent is a protein. The therapeutic proteins that can be used in the nanoparticles of the present invention include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), factor VIR, luteinizing hormone releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] Disease or condition

[0228] 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.

[0229] In one embodiment, the disease or disorder is characterized by a malfunction or aberrant protein or polypeptide activity.

[0230] 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.

[0231] 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.

[0232] 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).

[0233] 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.

[0234] Examples of the infectious diseases include: ① viral infectious diseases, such as AIDS (HIV), hepatitis A, hepatitis B or hepatitis C, herpes zoster (varicella), German measles (rubella virus), yellow fever, dengue fever, etc., flavivirus, coronavirus, influenza virus, rabies virus, hemorrhagic infectious diseases (Marburg virus or Ebola virus); ② bacterial infectious diseases, such as Legionnaire's disease (Legionella), gastric ulcer (Helicobacter), cholera (Vibrio), infections caused by Escherichia coli, Staphylococci, Salmonella or Streptococci (tetanus); ③ infections caused by protozoan pathogens, such as malaria, sleeping sickness, leishmaniasis, toxoplasmosis, i.e., infections caused by Plasmodium (Plasmod 4. fungal infections, such as Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis or Candida albicans.

[0235] The carcinoma or cancer (the medical term for malignant tumor) is a class of diseases in which a group of cells exhibit uncontrolled growth (dividing beyond normal limits), invasion (invading and destroying adjacent tissues), and sometimes metastasize (spreading to other parts of the body through the lymph or blood). These three harmful properties of carcinoma distinguish it from benign tumors, which are self-limited and do not invade or metastasize. Most carcinomas form tumors, i.e., swellings or lesions formed by abnormal growth of cells (called neoplastic cells or tumor cells), but some (like leukemias) do not. According to the present invention, the term "cancer" includes leukemia, seminoma, melanoma, teratoma, lymphoma, sarcoma, blastoma, neuroblastoma, glioma, glioblastoma, kidney cancer, adrenal cancer, renal cell carcinoma, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, lung cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, multiple myeloma, lymph node cancer, esophageal cancer, colon cancer, rectal cancer, bladder cancer, prostate cancer, endometrial cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, uterine cancer, breast cancer, prostate cancer, ovarian cancer, and metastases thereof.

[0236] Malignant melanoma is a serious type of skin cancer that results from the uncontrolled growth of pigment cells called melanocytes.

[0237] 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.

[0238] Lymphomas and leukemias are malignancies that arise from hematopoietic (blood-forming) cells.

[0239] Sarcomas are cancers that arise from transformed cells of one of the tissues that develop from the embryonic mesoderm. Thus, sarcomas include tumors of bone, cartilage, fat, muscle, blood vessels, and hematopoietic tissue.

[0240] 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.

[0241] 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.

[0242] Other components or adjuvants

[0243] The compositions of the present invention can be administered together with supplementary immunoenhancing substances (e.g., one or more adjuvants), and can contain one or more immunoenhancing substances to further improve their effectiveness, preferably to achieve a synergistic effect of immunostimulation. The term "adjuvant" refers to a compound that prolongs or enhances or accelerates an immune response. There can be a variety of mechanisms in this regard, depending on the type of adjuvant. For example, compounds that mature DC (e.g., lipopolysaccharides or CD40 ligands) constitute a first class of suitable adjuvants. Generally, any agent (LPS, GP96, dsRNA, etc.) or cytokine (e.g., GM-CSF) that affects the immune system of the "danger signal" type can be used as an adjuvant that can enhance and / or affect an immune response in a controlled manner. CpG oligodeoxynucleotides can also be optionally used in this environment, although as explained above, they are considered to have side effects in certain environments. Particularly preferred adjuvants are cytokines, such as monokines, lymphokines, interleukins or chemokines, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFα, INF-γ, GM-CSF, LT-α, or growth factors, such as hGH. In addition, known adjuvants include aluminum hydroxide, Freund's adjuvant or oil. For example, Montanide ® ; preferably Montanide ® ISA51, lipopeptides (eg, Pam3Cys, Pam3CSK4), glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxyribonucleotides (eg, class A or class B), poly (I:C) are also suitable for use as adjuvants in the compositions of the present invention.

[0244] The composition may include one or more components other than those described in the preceding section. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (eg, vitamin A or vitamin E) or sterols.

[0245] 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).

[0246] 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).

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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).

[0252] Suitable preservatives for use in the compositions of the present invention include benzalkonium chloride, chlorobutanol, parabens and thimerosal.

[0253] 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.

[0254] Dosage Form and Administration

[0255] 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.

[0256] 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.

[0257] Therapeutically effective amount

[0258] 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.

[0259] 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.

[0260] Antigen presenting cells

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] The term "immunogenicity" relates to the relative efficiency of an antigen in inducing an immune response.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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.

[0271] 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).

[0272] 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.

[0273] The first signal for T cell activation is provided by the binding of the T cell receptor to a short peptide presented by the major histocompatibility complex (MHC) on another cell. This ensures that only T cells with a TCR specific for that peptide are activated. The companion cell is usually a professional antigen presenting cell (APC), usually a dendritic cell in the context of a primary response, but B cells and macrophages can also be important APCs. + T cell peptides are 8 to 10 amino acids long; they are presented to CD4 by class II MHC molecules + The peptides for T cells are longer because the binding cleft of the class II MHC molecule is open at the end.

[0274] Example

[0275] The present invention is further described below in conjunction with examples. However, 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.

[0276] The following abbreviations represent the following reagents:

[0277] ADOPE: 2-acetamidoethyl ((R)-2,3-bis(oleoyloxy)propyl) phosphate sodium salt;

[0278] DOTMA: 1,2-dioctadeceneoxy-3-methylammonium propane (chloride salt);

[0279] DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine;

[0280] 18PA: (Z)-(R)-3-(phosphonooxy)propane-1,2-diyl dioleate monosodium salt;

[0281] DOPG: 1,2-dioleoyl-SN-glyceryl-3-phospho-RAC-glycerol sodium salt;

[0282] pA(2′-OMe)mpG: ((2R,3S,4S,5R)-3-(((((2R,3R,4S,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-methoxytetrahydrofuran-2-yl)methyl dihydrogen phosphate;

[0283] DSPC: distearoylphosphatidylcholine;

[0284] DOTAP: (2,3-dioleyloxypropyl)trimethylammonium chloride;

[0285] HOBt: 1-hydroxybenzotriazole;

[0286] EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0287] Example 1: Synthesis of permanent anionic lipid compounds

[0288] 1. Synthesis of YK-1101

[0289] The synthetic route of YK-1101 is as follows:

[0290]

[0291] Step 1: Synthesis of YK-1101-PM1

[0292] Dissolve YK-1101-SM1 (500 mg, 1.95 mmol) in dichloromethane (10 mL), add thionyl chloride (348 mg, 2.93 mmol) dropwise under ice-water bath, and stir at room temperature for 36 hours. Stop the reaction, and dry the solvent under reduced pressure to obtain the product YK-1201-PM1 (530 mg, 1.93 mmol, 98.9%).

[0293] Step 2: Synthesis of YK-1101

[0294] DCM (10 mL) and triethylamine (486 mg, 4.8 mmol) were added to DOPE (355 mg, 0.48 mmol) and stirred to dissolve. YK-1101-PM1 (525 mg, 1.91 mmol) was added dropwise under an ice-water bath. After the addition was completed, the reaction was stirred at room temperature for 24 h. The reaction of the raw material was completed by TLC monitoring, and the reaction was stopped. The reaction solution was added to ice water and stirred, separated, and the aqueous phase was back-extracted twice with DCM. The organic phases were combined and the solvent was dried to obtain a crude product. The crude product was purified by silica gel chromatography (0-30% dichloromethane / methanol). The fraction containing the product was dried and dissolved in ethyl acetate. A saturated NaHCO3 aqueous solution was added and stirred for 5 min. The liquid was separated, dried over anhydrous sodium sulfate, and the organic phase was dried to obtain YK-1101 (23 mg, 0.02 mmol, 5%). 57 H 108 NO9P,MS(ES): m / z(MH - ) 979.8.

[0295] YK-1101: 1 H NMR (400 MHz, CDCl3) δ5.34 (s,4H), 4.37-3.49 (m, 6H), 2.30(d, 3H), 2.01 (d, 8H), 1.53 (d, 8H), 1.27 (t, 64H), 0.88 (t, 12H).

[0296] 2. Synthesis of YK-1102

[0297] The synthetic route of YK-1102 is as follows:

[0298]

[0299] DOPE (600 mg, 0.81 mmol) and YK-1102-SM1 (162 mg, 0.97 mmol) were dissolved in DCM (20 mL), and HOBt (120 mg, 0.89 mmol) and EDCI (170 mg, 0.89 mmol) were added under ice-water bath, and the reaction was stirred at room temperature for 24 h. TLC monitored the reaction to be complete, the reaction solution was washed with water once, separated, the aqueous phase was back-extracted with DCM twice, the organic phases were combined, and the solvent was dried to obtain a crude product, which was purified by silica gel chromatography (0-30% dichloromethane / methanol). The fraction containing the product was dried and dissolved in ethyl acetate, and a saturated NaHCO3 aqueous solution was added and stirred for 5 min. The liquid was separated, dried over anhydrous sodium sulfate, and the organic phase was dried to obtain YK-1102 (198 mg, 0.22 mmol, 27.4%). 49 H 92 NO9P,MS(ES): m / z(MH - )865.7.

[0300] YK-1102: 1 H NMR (400 MHz, CDCl3) δ7.73(t,1H) ,5.41-5.21 (m, 5H), 4.37(d, 1H), 4.14-4.09 (m, 1H), 3.92 (s, 4H), 3.50 (s, 2H), 2.25-2.15 (m, 5H), 1.99 (t, 7H), 1.55 (s, 6H), 1.26 (s, 47H), 0.92-0.84 (m, 12H).

[0301] 3. Synthesis of YK-1103

[0302] The synthetic route of YK-1103 is as follows:

[0303]

[0304] Step 1: Synthesis of YK-1103-PM1

[0305] Dissolve YK-1103-SM1 (5 g, 0.04 mol) in dichloromethane (7 mL), cool to 0°C, and drop methanol (0.64 g, 0.02 mol) in DCM (7 mL). Stir for 20 min after the addition is complete, warm to room temperature and continue stirring for 24 h. Stop the reaction, and dry the solvent under reduced pressure to obtain the product YK-1103-PM1 (1.25 g, 10.2 mmol, 25.9%).

[0306] Step 2: Synthesis of YK-1103

[0307] DCM (10 mL) and triethylamine (74.9 mg, 0.74 mmol) were added to DOPE (500 mg, 0.67 mmol) and stirred to dissolve. The temperature was lowered to 0°C, and YK-1103-PM1 (90.55 mg, 0.74 mmol) was added dropwise. After the addition was completed, the reaction was stirred at room temperature for 2 h. The reaction of the raw material was completed by TLC monitoring, and the reaction was stopped. The reaction solution was added to ice water and stirred, and the liquid was separated. The aqueous phase was back-extracted with DCM twice, and the organic phases were combined. The solvent was dried to obtain a crude product, which was purified by silica gel chromatography (0-30% dichloromethane / methanol). The fraction containing the product was dried and dissolved in ethyl acetate. A saturated NaHCO3 aqueous solution was added and stirred for 5 min. The liquid was separated, dried over anhydrous sodium sulfate, and the organic phase was dried to obtain YK-1103 (372 mg, 0.44 mmol, 65.2%). 44 H 80 NO 11 P, MS (ES): m / z (MH - )828.3.

[0308] YK-1103: 1 H NMR (400 MHz, CDCl3) δ 8.56(s,1H),5.38-5.21 (m, 5H), 4.16-3.97 (m, 4H),3.87 (s, 3H), 5.58 (s, 2H), 3.12-3.06 (m, 2H), 2.31-2.26(m, 4H), 2.00 (t, 8H), 1.57 (d, 4H), 1.36-1.27 (m, 40H), 0.88 (t, 6H).

[0309] 4. Synthesis of YK-1104

[0310] The synthetic route of YK-1104 is as follows:

[0311]

[0312] Step 1: Synthesis of YK-1104-PM1

[0313] Using YK-1103-SM1 (5 g, 0.04 mol) and ethanol (0.92 g, 0.02 mol) as raw materials, according to the synthesis method of YK-1103-PM1, YK-1104-PM1 (1.6 g, 11.72 mmol, 29.3%) was obtained.

[0314] Step 2: Synthesis of YK-1104

[0315] Using DOPE (200 mg, 0.27 mmol) and YK-1104-PM1 (36 mg, 0.27 mmol) as raw materials, according to the synthesis method of YK-1104, YK-1104 (108 mg, 0.12 mmol, 46.2%) was obtained. 45 H 82 NO 11 P, MS (ES): m / z (MH - )842.2.

[0316] YK-1104: 1 H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 5.38-5.29 (m, 4H), 5.22( s, 1H), 4.39-4.29 (m, 2H), 4.16-3.97 (m, 4H), 3.86 (s, 1H), 3.57 (s,1H) , 3.12-3.05(m, 2H) , 2.31-2.26(m, 4H) , 2.01-1.98 (m, 8H) , 1.58(s, 4H) ,1.32-1.26 (m, 43H) , 0.88 (t, 6H).

[0317] 5. Synthesis of YK-1105

[0318] The synthetic route of YK-1105 is as follows:

[0319]

[0320] DCM (5 mL) and triethylamine (121 mg, 1.2 mmol) were added to DOPE (300 mg, 0.4 mmol) and stirred to dissolve. The temperature was lowered to 0°C, and ethyl isocyanate (34 mg, 0.48 mmol) was added dropwise. After the addition was completed, the reaction was stirred at room temperature for 3 h. The reaction of the raw material was completed by TLC monitoring, and the reaction was stopped. The reaction solution was added to ice water and stirred, separated, and the aqueous phase was back-extracted with DCM twice. The organic phases were combined and the solvent was dried to obtain a crude product. The crude product was purified by silica gel chromatography (0-20% dichloromethane / methanol). The fraction containing the product was dried and dissolved in ethyl acetate. A saturated NaHCO3 aqueous solution was added and stirred for 5 min. The liquid was separated, dried over anhydrous sodium sulfate, and the organic phase was dried to obtain YK-1105 (220 mg, 0.26 mmol, 65.7%). 44 H 83 N2O9P,MS(ES): m / z(MH - )813.2.

[0321] YK-1105: 1H NMR (400 MHz, CDCl3) δ 5.47 - 5.20 (m, 5H), 4.42 (d, J =10.6 Hz, 1H), 4.19 (d, J = 7.0 Hz, 1H), 3.99 (d, J = 20.5 Hz, 3H), 3.48 (d, J= 33.1 Hz, 2H), 3.22 (q, J = 7.1 Hz, 2H), 3.14 - 3.02 (m, 2H), 2.42 - 2.26(m, 4H), 2.13 - 1.97 (m, 8H), 1.43 - 1.22 (m, 42H), 1.13 (t, J = 7.2 Hz, 3H),0.92 (t, J = 6.7 Hz, 6H).

[0322] 6. Synthesis of YK-1106

[0323] The synthetic route of YK-1106 is as follows:

[0324]

[0325] Dissolve n-propylamine (40 mg, 0.68 mmol) in DMF (4 mL), add TEA (69 mg, 0.68 mmol), CDI (59 mg, 0.68 mmol) in turn, stir for 30 min under nitrogen atmosphere, weigh and add DOPE (505 mg, 0.68 mmol), stir at room temperature for 3 h. Monitor the reaction of the raw material by TLC, stop the reaction, add the reaction solution to ice water, stir, separate, extract the aqueous phase twice with EA, combine the organic phases, spin dry the solvent to obtain the crude product, purify by silica gel chromatography (0-20% dichloromethane / methanol), spin dry the fraction containing the product, dissolve in ethyl acetate, add saturated NaHCO3 aqueous solution and stir for 5 min, separate, dry over anhydrous sodium sulfate, spin dry the organic phase to obtain YK-1106 (120 mg, 0.14 mmol, 20.7%). 45 H 85 N2O9P, MS(ES): m / z(MH - )828.2.

[0326] YK-1106: 1H NMR (400 MHz, CDCl3) δ 5.37 (s, 4H), 5.21 (s, 1H), 4.33 (s,1H), 3.88 (s, 4H), 3.40 (s, 1H), 3.09 (s, 1H), 2.28 (d, J = 16.7 Hz, 3H), 2.04 (d, J = 5.4 Hz, 7H), 1.60 (s, 4H), 1.46 (s, 2H), 1.31 (d, J = 6.4 Hz, 46H), 0.90 (dd, J = 15.5, 8.5 Hz, 9H).

[0327] 7. Synthesis of YK-1107

[0328] The synthetic route of YK-1107 is as follows:

[0329]

[0330] DCM (4 mL) and triethylamine (82 mg, 0.81 mmol) were added to DOPE (200 mg, 0.27 mmol) and stirred to dissolve. The mixture was cooled to 0°C and butyl isocyanate (32 mg, 0.32 mmol) was added dropwise. After the addition was completed, the mixture was stirred at room temperature for 3 h. The reaction of the raw material was completed by TLC monitoring. The reaction was stopped and the reaction solution was added to ice water and stirred. The mixture was separated and the aqueous phase was back-extracted with DCM twice. The organic phases were combined and the solvent was dried to obtain a crude product. The crude product was purified by silica gel chromatography (0-13% dichloromethane / methanol). The fraction containing the product was dried to dryness and dissolved in ethyl acetate. A saturated aqueous solution of NaHCO3 was added and stirred for 5 min. The mixture was separated and dried over anhydrous sodium sulfate. The organic phase was dried to dryness to obtain YK-1107 (167 mg, 0.19 mmol, 71.5%). 46 H 87 N2O9P,MS(ES): m / z(MH - )841.6.

[0331] YK-1107: 1 H NMR (400 MHz, CDCl3) δ 5.44 - 5.20 (m, 5H), 4.42 (d, J =11.8 Hz, 1H), 4.17 (s, 1H), 4.02 (s, 3H), 3.44 (s, 2H), 3.23 - 3.06 (m, 3H), 2.43 - 2.23 (m, 5H), 2.04 (d, J = 5.8 Hz, 9H), 1.53 - 1.21 (m, 48H), 0.92 (q,J = 7.3 Hz, 9H).

[0332] 8. Synthesis of YK-1108

[0333] The synthetic route of YK-1108 is as follows:

[0334]

[0335] DCM (10 mL) and triethylamine (82 mg, 0.81 mmol) were added to DOPE (200 mg, 0.27 mmol) and stirred to dissolve. The temperature was lowered to 0°C, and ethyl isothiocyanate (28 mg, 0.32 mmol) was added dropwise. After the addition was completed, the reaction was stirred at room temperature for 16 h. The reaction of the raw material was completed by TLC monitoring, and the reaction was stopped. The reaction solution was added to ice water and stirred, separated, and the aqueous phase was back-extracted twice with DCM. The organic phases were combined and the solvent was dried to obtain a crude product. The crude product was purified by silica gel chromatography (0-30% dichloromethane / methanol). The fraction containing the product was dried and dissolved in ethyl acetate. A saturated aqueous solution of NaHCO3 was added and stirred for 5 min. The liquid was separated, dried over anhydrous sodium sulfate, and the organic phase was dried to obtain YK-1108 (130 mg, 0.15 mmol, 56.4%). 44 H 83 N2O8PS, MS(ES): m / z(MH-)829.5.

[0336] YK-1108: 1 H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 5.46 - 5.35 (m, 4H), 4.44 (d, J = 11.3 Hz, 1H), 4.25 - 3.93 (m, 4H), 3.84 - 3.49 (m, 3H), 3.18 -3.06 (m, 1H), 3.00 (s, 3H), 2.92 (s, 3H), 2.57 - 2.30 (m, 6H), 2.04 (d, J =5.6 Hz, 8H), 1.44 - 1.26 (m, 39H), 1.21 (t, J = 7.1 Hz, 3H), 0.98- 0.83 (m,6H).

[0337] 9. Synthesis of YK-1109

[0338] The synthetic route of YK-1109 is as follows:

[0339]

[0340] DCM (5 mL) and triethylamine (121 mg, 1.2 mmol) were added to DOPE (300 mg, 0.4 mmol) and stirred to dissolve. The temperature was lowered to 0°C, and hexyl isothiocyanate (69 mg, 0.48 mmol) was added dropwise. After the addition was completed, the reaction was stirred at room temperature for 3 h. The reaction of the raw material was completed by TLC monitoring, and the reaction was stopped. The reaction solution was added to ice water and stirred, and the liquid was separated. The aqueous phase was back-extracted with DCM twice, and the organic phases were combined. The solvent was dried to obtain a crude product, which was purified by silica gel chromatography (0-18% dichloromethane / methanol). The fraction containing the product was dried and dissolved in ethyl acetate. A saturated NaHCO3 aqueous solution was added and stirred for 5 min. The liquid was separated, dried over anhydrous sodium sulfate, and the organic phase was dried to obtain YK-1109 (130 mg, 0.14 mmol, 35.7%). 48 H 91 N2O8PS,MS(ES): m / z(MH - )885.6.

[0341] YK-1109: 1 H NMR (400 MHz, CDCl3) δ 5.45 - 5.32 (m, 4H), 5.27 (s, 1H), 4.42 (dd, J = 12.0, 3.4 Hz, 1H), 4.20 (dd, J = 11.9, 6.5 Hz, 1H), 4.03 (t, J= 6.3 Hz, 2H), 3.84 (s, 1H), 3.49 (s, 1H), 3.15 - 3.03 (m, 5H), 2.44 - 2.27(m, 4H), 2.04 (d, J = 5.9 Hz, 8H), 1.42 - 1.24 (m, 54H), 0.92 (t, J = 6.7 Hz, 9H).

[0342] 10. Synthesis of YK-1110

[0343] The synthetic route of YK-1110 is as follows:

[0344]

[0345] DMF (4 mL) and triethylamine (50 mg, 0.49 mmol) were added to DSPE (200 mg, 0.27 mmol) and stirred to dissolve. Ethyl isothiocyanate (70 mg, 0.8 mmol) was added at room temperature and stirred at 100 °C for 1 h. The reaction was completed after LCMS monitoring, and the reaction was stopped. The solvent was dried to obtain a crude product, which was purified by silica gel chromatography (0-30% dichloromethane / methanol). The fraction containing the product was dried to dryness, dissolved in ethyl acetate, and saturated NaHCO3 aqueous solution was added and stirred for 5 min. The liquids were separated and dried over anhydrous sodium sulfate. The organic phase was dried to dryness to obtain YK-1110 (100 mg, 0.12 mmol, 43.2%). 44 H 87 N2O8PS,MS(ES): m / z (MH - ) 833.7.

[0346] YK-1110: 1 H NMR (400 MHz, CDCl3) δ 4.51 - 4.10 (m, 2H), 4.07 - 3.68 (m,5H), 3.63 - 3.39 (m, 2H), 2.41 - 2.23 (m, 4H), 2.18 - 1.93 (m, 3H), 1.32 -1.22 (m, 60H), 1.18 (t, 2H), 0.88 (t, 6H).

[0347] 11. Synthesis of YK-1111

[0348] The synthetic route of YK-1111 is as follows:

[0349]

[0350] DMF (4 mL) and triethylamine (50 mg, 0.49 mmol) were added to DSPE (200 mg, 0.27 mmol) and stirred to dissolve. Ethyl isocyanate (60 mg, 0.84 mmol) was added at room temperature and stirred at 50 °C for 2 h. The reaction was completed after LCMS monitoring, and the reaction was stopped. The solvent was dried to obtain a crude product, which was purified by silica gel chromatography (0-30% dichloromethane / methanol). The fraction containing the product was dried to dryness, dissolved in ethyl acetate, and saturated NaHCO3 aqueous solution was added and stirred for 5 min. The liquids were separated, dried over anhydrous sodium sulfate, and the organic phase was dried to dryness to obtain YK-1111 (109 mg, 0.13 mmol, 48%). 44 H 87 N2O9P,MS(ES): m / z (MH - ) 817.9.

[0351] YK-1111: 1 H NMR (400 MHz, CDCl3) δ 4.57 - 3.60 (m, 6H), 3.49 - 2.96 (m,3H), 2.35 - 2.23 (m, 3H), 2.07 - 1.66 (m, 4H), 1.35 - 1.20 (m, 60H), 1.15 -1.01 (m, 3H), 0.88 (t, 6H).

[0352] 12. Synthesis of YK-1112

[0353] The synthetic route of YK-1112 is as follows:

[0354]

[0355] DMF (4 mL) and triethylamine (50 mg, 0.49 mmol) were added to DSPE (200 mg, 0.27 mmol) and stirred to dissolve. Hexyl isothiocyanate (110 mg, 0.77 mmol) was added at room temperature and stirred at 100 °C for 1 h. The reaction was completed after LCMS monitoring, and the reaction was stopped. The solvent was dried to obtain a crude product, which was purified by silica gel chromatography (0-30% dichloromethane / methanol). The fraction containing the product was dried to dryness, dissolved in ethyl acetate, and saturated NaHCO3 aqueous solution was added and stirred for 5 min. The liquids were separated, dried over anhydrous sodium sulfate, and the organic phase was dried to dryness to obtain YK-1112 (120 mg, 0.13 mmol, 48.7%). 48 H 95 N2O8PS,MS(ES): m / z (MH - ) 889.7.

[0356] YK-1112: 1 H NMR (400 MHz, CDCl3) δ 4.58 - 4.09 (m, 2H), 4.07 - 3.64 (m,5H), 3.57 - 3.18 (m, 2H), 2.42 - 2.19 (m, 5H), 1.68 - 1.47 (m, 6H), 1.37 -1.19 (m, 64H), 0.92 - 0.83 (m, 9H).

[0357] 13. Synthesis of YK-1113

[0358] The synthetic route of YK-1113 is as follows:

[0359]

[0360] DMF (4 mL) and triethylamine (50 mg, 0.49 mmol) were added to DSPE (200 mg, 0.27 mmol) and stirred to dissolve. Isocyanic acid butyl (80 mg, 0.81 mmol) was added at room temperature and stirred at 50 ° C for 2 h. The reaction was completed after LCMS monitoring, and the reaction was stopped. The solvent was dried to obtain a crude product, which was purified by silica gel chromatography (0-30% dichloromethane / methanol). The fraction containing the product was dried to dryness, dissolved in ethyl acetate, and saturated NaHCO3 aqueous solution was added and stirred for 5 min. The liquids were separated and dried over anhydrous sodium sulfate. The organic phase was dried to dryness to obtain YK-1113 (120 mg, 0.14 mmol, 51.1%). 46 H 91 N2O9P,MS(ES): m / z (MH - ) 845.7.

[0361] YK-1113: 1 H NMR (400 MHz, CDCl3) δ 4.50 - 3.79 (m, 5H), 3.52 - 3.03 (m,3H), 2.37 - 2.21 (m, 4H), 1.64 - 1.51 (m, 4H), 1.49 - 1.20 (m, 64H), 0.93 -0.84 (m, 9H).

[0362] 14. Synthesis of N-valeryl-DPPE

[0363] The synthetic route of N-valeryl-DPPE is as follows:

[0364]

[0365] Using DPPE (300 mg, 0.43 mmol) and n-valeric acid (222 mg, 2.17 mmol) as raw materials, according to the synthesis method of YK-1102, N-valeryl-DPPE (111 mg, 0.14 mmol, 32.3%) was obtained. 42 H 82 NO9P,MS(ES): m / z(MH - )774.2.

[0366] N-valeryl-DPPE: 1H NMR (400 MHz, CDCl3) δ 5.32 - 5.12 (m, 1H), 4.45 –4.33 (m, 2H), 4.22 - 3.88 (m, 2H), 3.67 - 3.12 (m, 3H), 2.44 - 2.13 (m, 4H),1.54 - 1.47 (m, 4H), 1.42 - 1.21 (m, 56H), 0.93 - 0.83 (m, 9H).

[0367] 15. Synthesis of N-butyryl-DPPE

[0368] The synthetic route of N-butyryl-DPPE is as follows:

[0369]

[0370] Using DPPE (300 mg, 0.43 mmol) and n-butyric acid (191 mg, 2.17 mmol) as raw materials, according to the synthesis method of YK-1102, N-butyryl-DPPE (124 mg, 0.16 mmol, 36.8%) was obtained. 41 H 80 NO9P,MS(ES): m / z(MH - )760.3.

[0371] N-butyryl-DPPE: 1 H NMR (400 MHz, CDCl3) δ 5.31 - 5.14 (m, 1H), 4.43 –4.31 (m, 2H), 4.21 - 3.85 (m, 2H), 3.63 - 3.15 (m, 3H), 2.43 - 2.13 (m, 4H),1.55 - 1.43 (m, 4H), 1.40 - 1.19 (m, 54H), 0.93 - 0.83 (m, 9H).

[0372] 16. Synthesis of NAPE

[0373] The synthetic route of NAPE is as follows:

[0374]

[0375] Using DPPE (300 mg, 0.43 mmol) and n-butyric acid (556 mg, 2.17 mmol) as raw materials, according to the synthesis method of YK-1102, NAPE (87 mg, 0.09 mmol, 21.2%) was obtained.53 H 104 NO9P,MS(ES): m / z(MH - )928.5.

[0376] NAPE: 1 H NMR (400 MHz, CDCl3) δ 5.27 - 5.12 (m, 1H), 4.41 - 4.34 (m,2H), 4.24 - 3.87 (m, 2H), 3.61 - 3.13 (m, 3H), 2.46 - 2.17 (m, 4H), 1.58 -1.46 (m, 4H), 1.41 - 1.17 (m, 78H), 0.91 - 0.83 (m, 9H).

[0377] Example 2: Preparation of mRNA lipid composition

[0378] A) Preparation of Fluc DNA and eGFP DNA templates

[0379] 1) Luciferase (Luciferase protein CDS) or green fluorescent protein (GFP) circular plasmid was constructed into pVAX1 vector (purchased from Thermo Fisher Scientific) by EcoRV restriction enzyme digestion;

[0380] 2) Take the plasmid constructed on the pVAX1 vector in step 1) and mix it with 50 μL of E. coli competent cells Stbl2 (purchased from Thermo Fisher Scientific), incubate them together on ice for 30 minutes, heat shock them at 42°C for 90 seconds, and immediately put them back on ice and incubate them on ice for 2 minutes;

[0381] 3) Add 400 μL LB medium (purchased from Thermo Fisher Scientific) and culture at 30°C with slow shaking for 45-60 min;

[0382] 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;

[0383] 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;

[0384] 6) Use an endotoxin-free plasmid extraction kit (purchased from Yisheng Biotechnology Co., Ltd.) to extract the plasmid;

[0385] 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.

[0386] 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 h to form a linearized DNA transcription template (enzyme digestion system see Table 1);

[0387] Table 1 Enzyme digestion reaction system

[0388]

[0389] 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 1h, and then centrifuged at 12000rmp to retain the precipitate;

[0390] Step ③ Wash the precipitate from step ② twice with 70% ethanol, dry the centrifuged material at 55°C for 10 min, and then add 1.7 mL of water for injection to dissolve it;

[0391] 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%.

[0392] B) Preparation of Fluc mRNA and eGFP mRNA

[0393] 1) Co-transcriptional capping reaction:

[0394] Using Fluc DNA and eGFP 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.

[0395] Table 2 Co-transcription capping reaction system

[0396]

[0397] Note: All the above reagents were purchased from Yisheng Biotechnology Co., Ltd.

[0398] 2) Digestion of template DNA:

[0399] 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 the linearized plasmid 1U / μg, mixed, centrifuged, and digested at 37°C for 1h to obtain the co-transcription capping product.

[0400] 3) Purification by lithium chloride precipitation method:

[0401] The co-transcription capping product obtained in step 2) above was purified by lithium chloride precipitation as follows:

[0402] 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;

[0403] Step ② Precipitation: high-speed centrifugation at 12000 rpm for 15 min, retain the precipitate;

[0404] 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℃.

[0405] C) Optimization of the ratio of lipid composition to prepare mRNA composition

[0406] 1) Preparation of lipid composition solution

[0407] Permanent anionic lipids, permanent cationic lipids and neutral lipids were weighed according to the formula 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 deenzyme water by ethanol injection method, 120 rpm, stirred at room temperature for 30 minutes, and the obtained lipid composition 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.

[0408] Table 3 Lipid composition formula

[0409]

[0410] The structures of permanent anionic lipids used to prepare lipid compositions are shown in Table 4, where YK-1101 to YK-1113, as well as N-valeryl-DPPE, N-butyryl-DPPE and NAPE were synthesized using the method in Example 1; 18PA was purchased from Adamas-beta; and DOPG was purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd. Each anionic lipid in the table was prepared using the formula in Table 3 to prepare the corresponding lipid composition, and the "content" in the formula refers to the molar percentage of the component in the lipid composition.

[0411] Table 4 Anionic lipid structures used to prepare lipid compositions

[0412]

[0413] It can be seen from Table 4 that the chemical structures of YK-1101 to YK-1113 are different from those of the permanent anionic lipids disclosed in the prior art, as follows:

[0414] 1. The groups connected to the head phosphate of YK-1101 to YK-1109 are different from those of 18PA and DOPG. Specifically, 18PA has no substituent groups, DOPG is connected to the glycerol group, and YK-1101 to YK-1109 are connected to the substituted aminoethyl group.

[0415] 2. YK-1110 to YK-1113 have different head connection groups from N-valeryl-DPPE, N-butyryl-DPPE and NAPE, and different tail chain lengths. Specifically: N-valeryl-DPPE, N-butyryl-DPPE and NAPE are connected to substituted amide groups at the head, and both lipid chains are hexadecanoic acid esters; YK-1110 to YK-1113 are connected to substituted urea groups and substituted thiourea groups, and both lipid chains are octadecanoic acid esters.

[0416] 2) mRNA composition

[0417] Dilute the mRNA in a buffer to obtain an mRNA aqueous solution (mRNA concentration 0.5 mg / ml, the buffer is a 10 mM HEPES buffer containing 0.1 mM EDTA, obtained by adding EDTA to aqueous HEPES). Use a syringe to take an aqueous sodium chloride solution (0.9% w / w) and inject it into the above-configured mRNA aqueous solution to obtain a mixture of mRNA and sodium chloride. Use a syringe to take a lipid complex solution and inject it into the above-mentioned mixture of mRNA and sodium chloride, vortex for 30 seconds, incubate at room temperature for 10 minutes, obtain an mRNA composition (RNA final concentration is 100 μg / mL), and store at 4°C to 8°C. Among them, the amount of mRNA and lipid complex solution used makes the charge ratio of the mRNA composition 1:5, 1:3, 2:5, 1:2, 2:3 and 3:4, respectively, and the charge ratio is calculated as follows:

[0418] The charge ratio of the mRNA 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)).

[0419] 3) Determination of particle size and PDI

[0420] The mRNA 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 polydispersity index (PDI) were measured using dynamic light scattering and a Malvern laser particle size analyzer.

[0421] The experimental results show that when the molar percentage of permanent anionic lipids, permanent cationic lipids and neutral lipids in the lipid composition is controlled to be (10-33%): (40-60%): (22-40%), especially when the molar percentage of permanent anionic lipids is 10-33%, the particle size and PDI of the prepared composition are at a good level (particle size is controlled within 220-500nm, PDI is less than 0.5). When the amount of permanent anionic lipids in the lipid composition is too large, for example, when the molar percentage exceeds 33%, for example, when it exceeds 40%, the system is unstable during the preparation process and a large amount of particulate solids are generated.

[0422] At the same time, when the charge ratio of the mRNA composition is controlled within the range of 1:2-1:5, its particle size is good (the particle size is controlled within the range of 220-500nm) and the particle distribution is uniform (PDI < 0.5). When the charge ratio is lower or higher than the range of 1:2-1:5, such as 2:3 and 3:4, large solid particles are precipitated.

[0423] The experimental results also show that when anionic lipids are not added to the lipid composition, mRNA compositions with qualified particle size and PDI can be prepared. On this basis, the permanent anionic lipids in Table 4 were selected to prepare mRNA compositions with a molar ratio of 1:2:1 (i.e., in the lipid composition, the molar percentages of permanent anionic lipids, permanent cationic lipids and neutral lipids were 25%, 50% and 25%, respectively) and a charge ratio of 1:2 for subsequent in vivo protein expression experiments in mice and flow cytometry experiments in mouse spleen cells to further study their performance effects.

[0424] Example 3: Effect of the type of anionic lipid on the particle size and PDI of mRNA composition

[0425] Using dynamic light scattering, a Malvern laser particle size analyzer was used to detect the particle size and PDI of the mRNA composition prepared in Example 2 in which the molar ratio of permanent anionic lipid, permanent cationic lipid and neutral lipid was 1:2:1 and the charge ratio was 1:2. The specific results are shown in Table 5.

[0426] Table 5 Particle size and PDI test results of mRNA compositions containing different permanent anionic lipids

[0427]

[0428] As can be seen from Table 5, the permanent anionic lipids YK-1101 to YK-1109 with olefin substitution in the tail chain, as well as 18PA and DOPG have good solubility in ethanol, and the prepared mRNA compositions have good particle size and PDI. Among them, the best is YK-1108, with a particle size of 235.2 nm, a PDI as low as 0.1806, and good particle uniformity.

[0429] In contrast, permanent anionic lipids YK-1110 to YK-1113, which do not contain olefin substitutions in the tail chains, as well as N-valeryl-DPPE, N-butyryl-DPPE and NAPE, have low solubility in ethanol and solid precipitation during the preparation process, indicating that the lipid compositions prepared using these anionic lipids according to the preparation method of the present invention are not suitable as mRNA delivery vectors.

[0430] From the above comparison, it can be seen that by adding permanent anionic lipids containing olefin substitution in the tail chain, a composition with good particle size (particle size controlled at 220-500nm) and uniform particle distribution (PDI<0.5) can be prepared according to the preparation method of the present invention.

[0431] Example 4: Protein expression experiment in mice

[0432] The ethanol-soluble mRNA composition screened out in Example 3 was injected into female BALB / c albino mice aged 4-6 weeks and weighing 17-19g via the tail vein (the dosage was about 20μg Fluc-mRNA / mouse), and the mice were intraperitoneally injected with fluorescent imaging substrate 6 hours after administration. The mice were allowed to move freely for 5 minutes, and then the total radiation intensity of the protein expressed in the mouse by the mRNA carried by the mRNA composition was detected by IVIS Spectrum small animal in vivo imager (corresponding to the expression intensity of fluorescent protein, i.e., the protein expression amount). After the sampling was completed, the mice were killed by cervical dislocation and dissected to accurately separate the mouse's internal organs: liver, spleen, and lungs. The total radiation intensity of the protein expressed by Fluc-mRNA in various organs of the mouse was detected by IVIS Spectrum small animal in vivo imager (corresponding to the expression intensity of fluorescent protein, i.e., the protein expression amount). The results of mouse in vivo imaging and protein expression detection in various internal organs are shown in Tables 6 and Figure 1 .

[0433] The LPX-RNA in Table 6 is a large-particle LPX-RNA without the addition of anionic lipids, which is prepared by adopting the preparation method in Example 2, according to the conditions that the concentration of the total lipid ethanol solution is 330 mM, the molar ratio of DOTMA:DOPE is 2:1, and the charge ratio of DOTMA:RNA is 2:3.

[0434] Table 6 Experimental data of mouse in vivo and organ imaging

[0435]

[0436] 1) The mRNA composition prepared by the permanent anionic lipid of the present invention can significantly increase the protein expression of the antigen in the spleen, indicating that the mRNA composition has good targeting to the spleen.

[0437] As can be seen from Table 6, the mRNA composition prepared by the permanent anionic lipids of the present invention (including YK-1105, YK-1106, YK-1107 and YK-1108, etc.) can significantly increase the protein expression of the antigen in the spleen. For example, the total radiation intensity of the spleen of the mRNA composition prepared by YK-1108 is 9.89×10 7 p / s, which are 70.6 times and 89.9 times that of the liver and lung, respectively. The total radiation intensity of the spleen is significantly higher than that of the liver and lung, indicating that the protein expression of Fluc-mRNA delivered by the mRNA composition prepared by the permanent anionic lipid of the present invention in the spleen is significantly higher than that in the liver and lung, which also shows that the lipid composition prepared by the permanent anion of the present invention can improve spleen targeting.

[0438] 2) The mRNA compositions prepared with different permanent anionic lipids of the present invention have significant differences in the amount of protein expressed in the living body and spleen of mice.

[0439] For example, the total radiation intensity of the mRNA compositions prepared from YK-1105 to YK-1108 was significantly higher than that of the mRNA compositions prepared from other compounds, among which YK-1108 had the highest total radiation intensity in the mouse live body and spleen, which were 5.9 times and 6.1 times that of the lowest YK-1101, respectively.

[0440] From the data in Table 6, it can be seen that the total radiation intensity of the proteins expressed in the living body or spleen of mice by the mRNA compositions prepared by different anionic lipids in the present invention is very different. The total radiation intensity of the mRNA compositions prepared by YK-1105 to YK-1108 is significantly higher than that prepared by other compounds, among which the total radiation intensity of the mRNA composition prepared by YK-1108 is the highest, and the total radiation intensity of the living body and spleen of mice can reach 4.33×10 7 p / s and 9.89×10 7p / s, and the total radiation intensity of YK-1105, YK-1106, and YK-1107 in mice was 3.22×10 7 p / s, 3.52×10 7 p / s and 3.58×10 7 p / s, and the total radiation intensity of the spleen was 8.82×10 7 p / s, 8.17×10 7 p / s and 8.21×10 7 p / s.

[0441] The total radiation intensity of the mRNA compositions prepared by YK-1101, YK-1102, YK-1103, YK-1104 and YK-1109 in living mice was 0.73×10 7 p / s, 0.74×10 7 p / s, 0.79×10 7 p / s, 0.77×10 7 p / s and 1.79×10 7 p / s, and the total radiation intensity of the spleen was 1.61×10 7 p / s, 1.66×10 7 p / s, 1.72×10 7 p / s, 1.84×10 7 p / s and 3.81×10 7 Among them, the corresponding total radiation intensity of YK-1101 was the lowest, with the total radiation intensity of the mouse body and spleen being 0.73×10 7 p / s and 1.61×10 7 The total radiation intensity of the mRNA compositions prepared from YK-1105 to YK-1108 in mice was 4.4, 4.8, 4.9 and 5.9 times that of YK-1101, respectively; the total radiation intensity of the spleen was 5.5, 5.1, 5.1 and 6.1 times that of YK-1101, respectively. Compared with YK-1101, the spleen targeting and protein expression in the spleen of YK-1105 to YK-1108 were significantly improved.

[0442] 3) Compared with the permanent anionic lipids in the prior art, the mRNA compositions prepared from the permanent anionic lipids YK-1105 to YK-1108 of the present invention significantly increased the protein expression levels in the living body and spleen of mice.

[0443] The total radiation intensity of the mRNA composites prepared by 18PA and DOPG in living mice was 1.84×10 7 p / s and 1.66×10 7 p / s, and 5.03×107 p / s and 4.51×10 7 p / s.

[0444] The total radiation intensity of mice in vivo of the mRNA compositions prepared respectively from YK-1105 to YK-1108 of the present invention are 1.8 times, 1.9 times, 1.9 times and 2.4 times of the corresponding total radiation intensity of 18PA, respectively, and are 1.9 times, 2.1 times, 2.2 times and 2.6 times of the mRNA compositions prepared respectively from DOPG; the total radiation intensity of the spleen of the mRNA compositions prepared respectively from YK-1105 to YK-1108 of the present invention are 1.8 times, 1.6 times, 1.6 times and 2.0 times of the corresponding total radiation intensity of 18PA, respectively, and are 2.0 times, 1.8 times, 1.8 times and 2.2 times of the corresponding total radiation intensity of DOPG, respectively.

[0445] 4) Compared with the mRNA composition without the addition of anionic lipids, the mRNA composition containing the permanent anionic lipids YK-1105 to YK-1108 of the present invention significantly increased the protein expression level in the mouse living body and spleen.

[0446] LPX-RNA without anionic lipids (preparation method is the same as in Example 2) had a total radiation intensity of 1.12×10 7 p / s and 2.24×10 7 The total radiation intensity of the mRNA compositions prepared from YK-1105 to YK-1108 in the living mice was 2.9 times, 3.1 times, 3.2 times and 3.9 times that of the corresponding total radiation intensity of LPX-RNA, respectively, and the total radiation intensity in the spleen was 3.9 times, 3.6 times, 3.7 times and 4.4 times that of the corresponding total radiation intensity of LPX-RNA, respectively, which was significantly improved.

[0447] It can be seen from animal experiments that the permanent anionic lipids YK-1105 to YK-1108 of the present invention, whether compared with structurally similar permanent anionic lipids (including YK-1101 to YK-1104 and YK-1109 of the present application, and 18PA and DOPG in the prior art) or compared with large-particle LPX-RNA without the addition of anionic lipids, the mRNA compositions prepared by them have significantly improved the protein expression levels in living mice and spleens.

[0448] In vivo experiments further demonstrated that the mRNA compositions prepared from the anionic lipids YK-1105 to YK-1108 of the present invention can effectively deliver mRNA into the body and express it efficiently in the spleen. In addition, it is not necessarily the case that the protein expression levels of Fluc mRNA compositions prepared from permanent anionic lipids with similar structures in mice or spleens are similar. On the contrary, there is a high possibility that there will be huge differences.

[0449] Example 5: Targeted study of mouse spleen cells

[0450] 1. The ethanol-soluble mRNA composition selected in Example 3 was injected into female C57BL / 6 mice aged 4-6 weeks and weighing 17-19 g via the tail vein (the dosage was such that each mouse was injected with about 80 μg of eGFP-mRNA), and the mice were killed by cervical dislocation 24 hours after administration and dissected to accurately separate the mouse spleen.

[0451] 2. Preparation of single cells

[0452] 1) Grind the separated spleen tissue to make it single cell and pass it through a cell sieve;

[0453] 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;

[0454] 3) Count the cells and take 5×10 6 cells into the flow tube (to ensure the same number of cells between samples);

[0455] 3. Detection of immune cells in spleen tissue

[0456] 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 min (one negative control);

[0457] Table 7 Reagents and sources used in mouse spleen cell flow cytometry experiments

[0458]

[0459] 2) Add 2 mL PBS, centrifuge at 500 g for 5 min, and discard the supernatant;

[0460] 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:

[0461] T cell GFP ratio: CD45+→CD3+→GFP+

[0462] B cell GFP ratio: CD45+→CD3-CD19+→GFP+

[0463] NK cell GFP ratio: CD45+→NK1.1+→GFP+

[0464] cDC cell GFP ratio: CD45+→F4 / 80-CD11c+→GFP+

[0465] pDC cell GFP ratio: CD45+→F4 / 80-CD11cintCD317+→GFP+

[0466] Macrophage GFP ratio: CD45+→F4 / 80+→GFP+

[0467] Experimental results:

[0468] The percentage of eGFP-positive cells in mouse spleen cells is shown in Tables 8 and Figure 2 .

[0469]

[0470] 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.

[0471] 1) The mRNA composition prepared by the permanent anionic lipid of the present invention can significantly increase the percentage of antigen-presenting cells expressing antigens in the spleen.

[0472] The mRNA compositions prepared by the permanent anionic lipids of the present invention can significantly increase the percentage of antigen-presenting cells expressing antigens in the spleen. For example, compared with the blank control, the mRNA composition prepared by YK-1108 increases the percentage of eGFP-positive cells of antigen-presenting cells B cells, cDC cells, pDC cells, and macrophages in the spleen by 34.0 times, 22.7 times, 16.1 times, and 202.5 times, respectively.

[0473] 2) The mRNA compositions prepared by different permanent anionic lipids in the present invention have significant differences in targeting to mouse spleen antigen presenting cells, and the data of YK-1105 to YK-1108 are significantly higher than those of other compounds. Among them, YK-1108 has the highest percentage of cells expressing antigens in antigen presenting cells, and the percentage of eGFP-positive cells of B cells, cDC cells, pDC cells and macrophages are 3.4 times, 4.1 times, 5.2 times and 6.1 times that of the lowest YK-1101, respectively.

[0474] From the data in Table 8 and Figure 2It can be seen that the targeting properties of the mRNA compositions prepared with different anionic lipids of the present invention to mouse spleen antigen presenting cells are very different. The percentages of cells expressing antigens in antigen presenting cells of YK-1105 to YK-1108 were all very high, among which YK-1108 had the highest percentage, with the percentages of eGFP-positive cells in B cells, cDC cells, pDC cells and macrophages being 1.36%, 13.63%, 15.76% and 22.27%, respectively. The percentages of eGFP-positive cells in B cells of the mRNA compositions prepared by YK-1105, YK-1106 and YK-1107 were 0.56%, 0.93% and 1.36%, respectively; the percentages of eGFP-positive cells in cDC cells were 11.86%, 13.40% and 11.37%, respectively; the percentages of eGFP-positive cells in pDC cells were 14.09%, 16.98% and 12.14%, respectively; and the percentages of eGFP-positive cells in macrophages were 14.41%, 15.45% and 20.72%, respectively.

[0475] Among the mRNA compositions prepared by the permanent anionic lipids of the present invention, the mRNA composition prepared by YK-1101 has the lowest targeting in mouse spleen antigen presenting cells, and its eGFP-positive cell percentages in B cells, cDC cells, pDC cells, and macrophages are 0.40%, 3.30%, 3.01%, and 3.66%, respectively. The percentages of eGFP-positive cells in B cells of the mRNA compositions prepared from YK-1105 to YK-1108 were 1.4 times, 2.3 times, 3.4 times and 3.4 times that of the corresponding percentages of YK-1101; the percentages of eGFP-positive cells in cDC cells were 3.6 times, 4.1 times, 3.4 times and 4.1 times that of YK-1101, respectively; the percentages of eGFP-positive cells in pDC cells were 4.7 times, 5.6 times, 4.0 times and 5.2 times that of YK-1101, respectively; and the percentages of eGFP-positive cells in macrophages were 3.9 times, 4.2 times, 5.7 times and 6.1 times that of YK-1101, respectively, all of which were significantly improved.

[0476] 3) Compared with the permanent anionic lipids in the prior art, the mRNA compositions prepared from the permanent anionic lipids YK-1105 to YK-1108 of the present invention have significantly improved targeting in mouse spleen antigen presenting cells. For example, the percentage of eGFP-positive cells of B cells, cDC cells, pDC cells and macrophages in mouse spleen of YK-1108 is 3.1 times, 2.7 times, 3.0 times and 4.3 times that of 18PA, respectively.

[0477] The percentages of eGFP-positive cells in B cells, cDC cells, pDC cells, and macrophages prepared by 18PA were 0.44%, 5.14%, 5.34%, and 5.15%, respectively. The percentages of eGFP-positive cells in B cells prepared by YK-1105 to YK-1108 were 1.3, 2.1, 3.1, and 3.1 times that of 18PA, respectively; the percentages of eGFP-positive cells in cDC cells were 2.3, 2.6, 2.2, and 2.7 times that of 18PA, respectively; the percentages of eGFP-positive cells in pDC cells were 2.6, 3.2, 2.3, and 3.0 times that of 18PA, respectively; and the percentages of eGFP-positive cells in macrophages were 2.8, 3.0, 4.0, and 4.3 times that of 18PA, respectively, which were significantly improved.

[0478] 4) Compared with the composition without adding anionic lipids, the compositions prepared by the permanent anionic lipids YK-1105 to YK-1108 of the present invention have significantly improved targeting in mouse spleen antigen presenting cells. For example, the percentage of eGFP-positive cells of YK-1108 in B cells, cDC cells, pDC cells and macrophages in mouse spleen is 4.0 times, 4.4 times, 3.9 times and 6.8 times that of large-size LPX-RNA, respectively.

[0479] The percentages of eGFP-positive cells of large-sized LPX-RNA in B cells, cDC cells, pDC cells and macrophages were 0.34%, 3.13%, 4.05% and 3.26%, respectively. The percentages of eGFP-positive cells of mRNA compositions prepared from YK-1105 to YK-1108 in B cells were 1.6, 2.7, 4.0 and 4.0 times of the corresponding percentages of large-sized LPX-RNA, respectively; the percentages of eGFP-positive cells in cDC cells were 3.8, 4.3, 3.6 and 4.4 times of the corresponding percentages of large-sized LPX-RNA, respectively; the percentages of eGFP-positive cells in pDC cells were 3.5, 4.2, 3.0 and 3.9 times of the corresponding percentages of large-sized LPX-RNA, respectively; and the percentages of eGFP-positive cells in macrophages were 4.4, 4.7, 6.4 and 6.8 times of the corresponding percentages of large-sized LPX-RNA, respectively, which were significantly improved.

[0480] It can be seen from the spleen cell flow cytometry experiment that compared with structurally similar permanent anionic lipids (including YK-1101 of the present application and 18PA of the prior art) and large-particle LPX-RNA without added anionic lipids, the permanent anionic lipids of the present invention, such as YK-1105 to YK-1108, respectively prepared mRNA compositions significantly increased the percentage of eGFP-positive cells in B cells, cDC cells, pDC cells and macrophages.

[0481] The above experiments further prove that the mRNA compositions prepared by YK-1105 to YK-1108 of the present invention can significantly increase the percentage of antigen-presenting cells (e.g., B cells, pDC cells, cDC cells, and macrophages) in the spleen that express antigens. Moreover, it is not necessarily the case that the eGFP mRNA compositions prepared by permanent anionic lipids with similar structures have similar targeting properties to antigen-presenting cells in the spleen of mice. On the contrary, there is a high possibility that there will be huge differences.

[0482] Example 6: Study on the maturation state of mouse spleen antigen presenting cells

[0483] 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:

[0484] B cell CD86 ratio: CD45+→CD3-CD19+→CD86+

[0485] NK cell CD86 ratio: CD45+→NK1.1+→CD86+

[0486] cDC cell CD86 ratio: CD45+→F4 / 80-CD11c+→CD86+

[0487] pDC cell CD86 ratio: CD45+→F4 / 80-CD11cintCD317+→CD86+

[0488] Macrophage CD86 ratio: CD45+→F4 / 80+→CD86+

[0489] Experimental results:

[0490] The percentage of CD86 positive cells in mouse spleen cells is shown in Table 9 and Figure 3 .

[0491]

[0492] 1) The mRNA composition prepared by the permanent anionic lipid of the present invention can significantly increase the percentage of antigen presenting cells CD86 positive cells in the spleen.

[0493] The mRNA compositions prepared by the permanent anionic lipids of the present invention can significantly increase the percentage of CD86-positive cells in the spleen of antigen-presenting cells. For example, compared with the blank control, the mRNA composition prepared by YK-1108 increases the percentage of CD86-positive cells of antigen-presenting cells B cells, cDC cells, pDC cells and macrophages in the spleen to 3.0 times, 6.2 times, 5.7 times and 5.9 times, respectively, which is significantly improved.

[0494] 2) The mRNA compositions prepared by different permanent anionic lipids of the present invention significantly differ in the maturation state of mouse spleen antigen presenting cells, for example, the mRNA compositions prepared by YK-1105 to YK-1108 are significantly higher than the mRNA compositions prepared by other compounds. Among them, the mRNA composition prepared by YK-1108 has the highest percentage of CD86-positive antigen presenting cells, and the percentage of CD86-positive cells of B cells, cDC cells, pDC cells and macrophages are 1.9 times, 2.4 times, 1.8 times and 2.8 times that of the lowest YK-1101, respectively.

[0495] From the data in Table 9 and Figure 3 It can be seen that the mRNA compositions prepared from different anionic lipids of the present invention cause great differences in the maturation status of mouse spleen antigen-presenting cells. The mRNA compositions prepared from YK-1105 to YK-1108 all increased the percentage of CD86-positive cells in antigen-presenting cells to a high level, among which YK-1108 had the highest percentage, with the percentage of CD86-positive cells in B cells, cDC cells, pDC cells and macrophages reaching 1.05%, 50.26%, 75.25% and 28.51%, respectively; the mRNA compositions prepared from YK-1105, YK-1106 and YK-1107 respectively had the percentage of CD86-positive cells in B cells of 0.85%, 0.87% and 0.96%, respectively; the percentage of CD86-positive cells in cDC cells of 38.58%, 38.72% and 52.09%, respectively; the percentage of CD86-positive cells in pDC cells of 62.80%, 63.01% and 72.92%, respectively; and the percentage of CD86-positive cells in macrophages of 25.64%, 28.39% and 28.51%, respectively.

[0496] The mRNA composition prepared by YK-1101 resulted in the lowest maturity state of antigen presenting cells, with the percentages of CD86-positive cells in B cells, cDC cells, pDC cells, and macrophages being 0.56%, 20.95%, 40.69%, and 10.19%, respectively. The percentages of CD86-positive cells in B cells of the mRNA compositions prepared from YK-1105 to YK-1108 were 1.5 times, 1.6 times, 1.7 times and 1.9 times of that in YK-1101, respectively; the percentages of CD86-positive cells in cDC cells were 1.8 times, 1.8 times, 2.5 times and 2.4 times of that in YK-1101, respectively; the percentages of CD86-positive cells in pDC cells were 1.5 times, 1.5 times, 1.8 times and 1.8 times of that in YK-1101, respectively; and the percentages of CD86-positive cells in macrophages were 2.4 times, 2.5 times, 2.8 times and 2.8 times of that in YK-1101, respectively, which were significantly improved.

[0497] 3) Compared with the permanent anionic lipids in the prior art, the mRNA compositions prepared from the permanent anionic lipids YK-1105 to YK-1108 of the present invention significantly improved the maturation state of mouse 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 composition prepared by YK-1108 was 3.1 times, 1.9 times, 1.4 times and 2.4 times the corresponding percentage of 18PA, respectively.

[0498] The percentages of CD86 positive cells in B cells, cDC cells, pDC cells and macrophages prepared by 18PA were 0.34%, 26.76%, 53.68% and 11.89%, respectively. The percentages of CD86 positive cells in B cells prepared by YK-1105 to YK-1108 were 2.5 times, 2.6 times, 2.8 times and 3.1 times of the corresponding percentages of 18PA, respectively; the percentages of CD86 positive cells in cDC cells were 1.4 times, 1.4 times, 1.9 times and 1.9 times of the corresponding percentages of 18PA, respectively; the percentages of CD86 positive cells in pDC cells were 1.2 times, 1.2 times, 1.4 times and 1.4 times of the corresponding percentages of 18PA, respectively; and the percentages of CD86 positive cells in macrophages were 2.0 times, 2.2 times, 2.4 times and 2.4 times of the corresponding percentages of 18PA, respectively, which were significantly improved.

[0499] 4) Compared with the composition without adding anionic lipids, the compositions prepared by the permanent anionic lipids YK-1105 to YK-1108 of the present invention significantly improved the maturation state 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 spleen of mice with YK-1108 was 2.1 times, 2.6 times, 2.1 times and 2.9 times the corresponding percentage of large-size LPX-RNA, respectively.

[0500] The percentages of CD86-positive cells of the mRNA composition prepared from large-particle LPX-RNA were 0.49%, 19.50%, 36.44% and 9.98% in B cells, cDC cells, pDC cells and macrophages, respectively. The percentages of CD86-positive cells in B cells of the mRNA compositions prepared from YK-1105 to YK-1108 were 1.7 times, 1.8 times, 2.0 times and 2.1 times of the corresponding percentages of large-sized LPX-RNA, respectively; the percentages of CD86-positive cells in cDC cells were 2.0 times, 2.0 times, 2.7 times and 2.6 times of the corresponding percentages of large-sized LPX-RNA, respectively; the percentages of CD86-positive cells in pDC cells were 1.7 times, 1.7 times, 2.0 times and 2.1 times of the corresponding percentages of large-sized LPX-RNA, respectively; and the percentages of CD86-positive cells in macrophages were 2.4 times, 2.6 times, 2.8 times and 2.9 times of the corresponding percentages of large-sized LPX-RNA, respectively, which were significantly improved.

[0501] It can be seen from the spleen cell flow cytometry experiment that when the mRNA composition contains the permanent anionic lipids of the present invention (such as YK-1105 to YK-1108), the percentage of CD86-positive cells of B cells, cDC cells, pDC cells, and macrophages is significantly increased compared with the mRNA composition containing structurally similar permanent anionic lipids (such as YK-1101 of the present invention and 18PA of the prior art) or compared with the mRNA composition of large-particle LPX-RNA without adding anionic lipids.

[0502] This experiment further proves that the mRNA compositions prepared by YK-1105 to YK-1108 of the present invention can significantly increase the maturity of antigen presenting cells (e.g., B cells, pDC cells, cDC cells, macrophages) in the spleen. In addition, the percentage of CD86 positive cells in antigen presenting cells in the spleen of mice is not necessarily close for eGFP mRNA compositions prepared by different permanent anionic lipids with similar structures. On the contrary, they may be very different.

[0503] Example 7: Study on T cell activation in mouse spleen

[0504] 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:

[0505] T cell CD69 ratio:CD45 + →CD3 + →CD69 +

[0506] Experimental results:

[0507] The percentage of CD69 positive T cells in mouse spleen is shown in Table 10 and Figure 4 .

[0508] Table 10 The percentage of CD69 positive T cells in mouse spleen

[0509] 1) The mRNA composition prepared by the permanent anionic lipid of the present invention can significantly increase the percentage of CD69-positive T cells in the spleen.

[0510] The mRNA compositions prepared using the permanent anionic lipids of the present invention can significantly increase the percentage of T cell CD69 positive cells in the spleen. For example, compared with the blank control, the mRNA composition prepared by YK-1108 increases the percentage of T cell CD69 positive cells in the spleen by 10.8 times, which is a significant improvement.

[0511] 2) The mRNA compositions prepared from different permanent anionic lipids of the present invention significantly differed in the activation state of mouse spleen T cells. For example, the mRNA compositions prepared from YK-1105 to YK-1108 were significantly higher than those prepared from other compounds. After administration of the mRNA composition containing YK-1108, the percentage of T cell CD69 positive cells was the highest, which was 3.0 times that of the lowest YK-1101.

[0512] From the data in Table 10 and Figure 4It can be seen that the mRNA compositions prepared by different anionic lipids of the present invention have very different activation conditions of mouse spleen T cells. Among them, the mRNA compositions prepared by YK-1105 to YK-1108 have a high percentage of T cell CD69 positive cells, among which YK-1108 has the highest percentage, which can reach 56.60%; after applying the mRNA compositions prepared by YK-1105, YK-1106 and YK-1107, the percentage of T cell CD69 positive cells was 49.19%, 48.51% and 49.52% respectively.

[0513] The activation of spleen T cells was the lowest after administration of the mRNA composition prepared by YK-1101, with a CD69 positive cell percentage of 18.79%. After administration of the mRNA compositions prepared by YK-1105 to YK-1108, the CD69 positive cell percentages of T cells were 2.6 times, 2.6 times, 2.6 times, and 3.0 times that of YK-1101, respectively.

[0514] 3) Compared with the permanent anionic lipids of the prior art, the mRNA compositions prepared from the permanent anionic lipids YK-1105 to YK-1108 of the present invention significantly enhance the activation state of mouse spleen T cells. For example, the percentage of CD69 positive cells in mouse spleen T cells of the mRNA composition prepared from YK-1108 is 1.8 times the corresponding percentage of the mRNA composition prepared from 18PA.

[0515] The percentage of CD69 positive cells in T cells of the mRNA composition prepared by 18PA was 31.2%. The percentage of CD69 positive cells in T cells of the mRNA compositions prepared by YK-1105 to YK-1108 were 1.6 times, 1.6 times, 1.6 times and 1.8 times that of 18PA, respectively, which was significantly improved.

[0516] 4) Compared with the composition without adding anionic lipids, the mRNA composition prepared by the permanent anionic lipids YK-1105 to YK-1108 of the present invention significantly improved the activation state of mouse spleen T cells. For example, the percentage of CD69 positive cells in mouse spleen T cells of the mRNA composition prepared by YK-1108 was 2.7 times the corresponding percentage of large-particle LPX-RNA.

[0517] The percentage of CD69 positive cells in T cells of large-sized LPX-RNA was 21.14%. The percentage of CD69 positive cells in T cells of YK-1105 to YK-1108 were 2.3 times, 2.3 times, 2.3 times and 2.7 times of that of large-sized LPX-RNA, respectively, which was significantly improved.

[0518] It can be seen from the spleen cell flow cytometry experiment that the permanent anionic lipids YK-1105 to YK-1108 designed in the present invention, whether compared with structurally similar permanent anionic lipids (including YK-1101 in the present application and 18PA in the prior art) or compared with large-particle LPX-RNA without the addition of anionic lipids, the mRNA composition significantly improves the percentage of CD69 positive cells in T cells.

[0519] This experiment further proves that the mRNA compositions prepared by YK-1105 to YK-1108 of the present invention can significantly increase the activation state of T cells in the spleen. In addition, different eGFP mRNA compositions prepared by different permanent anionic lipids with similar structures are not necessarily similar in the percentage of CD69 positive cells of T cells in the mouse spleen. On the contrary, they may be very different.

[0520] 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 permanent anionic lipid compound, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that: The permanent anionic lipid compound is at least one of compounds YK-1105 to YK-1108 having the following structures: 、 、 ,or 。 2. A lipid composition comprising a permanent anionic lipid, characterized in that The permanent anionic lipid comprises the permanent anionic lipid compound according to claim 1, a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

3. The lipid composition according to claim 2, wherein The molar percentage of the permanent anionic lipid in the lipid composition is 10-33%.

4. The lipid composition according to claim 3, wherein The molar percentage of the permanent anionic lipid in the lipid composition is 20-30%.

5. The lipid composition according to claim 3 or 4, wherein The molar percentage of the permanent anionic lipid in the lipid composition is 25%.

6. The lipid composition according to claim 2, wherein The lipid composition further comprises a permanent cationic lipid; And / or, the lipid composition further comprises a neutral lipid.

7. The lipid composition according to claim 6, wherein The molar percentage of the permanent cationic lipid in the lipid composition is 40-60%; And / or, the molar percentage of the neutral lipid in the lipid composition is 22-40%.

8. The lipid composition according to claim 6 or 7, wherein 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. Use of the permanent anionic lipid compound according to claim 1, its pharmaceutically acceptable salt or its stereoisomer, or the lipid composition according to any one of claims 2 to 8 in improving the targeting of drugs to target organs and / or target cells, wherein the use is for non-therapeutic purposes, the target organ is the spleen, and the target cell is an antigen presenting cell.

10. Use of the permanent anionic lipid compound according to claim 1, its pharmaceutically acceptable salt or its stereoisomer, or the lipid composition according to any one of claims 2 to 8 in improving the activation state of antigen-presenting cells in target organs and / or improving the expression of antigens by antigen-presenting cells in target organs, wherein the use is for non-therapeutic purposes and the target organ is the spleen.

11. The use according to claim 9 or 10, wherein The molar percentage ratio of the permanent anionic lipid, the permanent cationic lipid and the neutral lipid in the lipid composition is 10-33:40-60:22-40.

12. The use according to claim 9 or 10, wherein 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.

13. A composition comprising: (A) a therapeutic agent and / or a preventive agent comprising any one of the group consisting of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein, or a combination of at least two thereof; (B) lipid composition; It is characterized in that The lipid composition comprises the lipid composition of any one of claims 2-8; The composition is used to deliver the therapeutic agent and / or preventive agent to antigen-presenting cells in a target organ, which is the spleen.

14. The composition according to claim 13, wherein The therapeutic and / or prophylactic agent is a nucleic acid molecule capable of encoding one or more antigens.

15. The composition according to claim 13 or 14, 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.

16. The composition according to claim 15, wherein The nucleic acid molecule is RNA encoding one or more antigens.

17. The composition according to claim 13, wherein 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.

18. The composition according to claim 13, wherein The therapeutic and / or preventive agent and the lipid composition are used in such an amount that the charge ratio of positive charges to negative charges in the composition is 1:2 to 1:

5.

19. The composition according to claim 13, wherein The composition further comprises at least one auxiliary ingredient or adjuvant; and / or, the composition further comprises one or more pharmaceutically acceptable carriers, diluents or excipients; And / or, the composition further comprises one or more hydrophobic small molecules, permeability enhancing molecules, carbohydrates, polymers, surface altering agents, functionalized lipids or cytokines.

20. A method for preparing a composition for delivering a therapeutic agent and / or a prophylactic agent to an antigen presenting cell in a target organ, characterized in that: The method includes: (a) dissolving a lipid in an organic solvent to form a lipid solution, wherein the lipid comprises the permanent anionic lipid according to claim 1, a pharmaceutically acceptable salt thereof or a stereoisomer thereof; (b) mixing the lipid solution obtained in step (a) with water to obtain a lipid mixture; (c) mixing the lipid mixture obtained in step (b) with a therapeutic agent and / or a preventive agent to form the composition, wherein the therapeutic agent and / or the preventive agent comprises a nucleic acid buffer solution obtained by dissolving nucleic acid molecules in a buffer solution having a pH value of 6.8-7.

6.

21. The method according to claim 20, wherein: In step (a), the lipid further comprises a permanent cationic lipid; And / or, in step (a), the lipid further comprises a neutral lipid.

22. The method according to claim 21, wherein: In step (a), 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, in step (a), 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.

23. The method according to any one of claims 20 to 22, wherein: The molar percentage ratio of the permanent anionic lipid, the permanent cationic lipid and the neutral lipid is 10-33:40-60:22-40.

24. The method according to claim 20, wherein: In step (a), the organic solvent is an alcohol solvent; and / or, in step (b), the lipid mixture can pass through a polycarbonate membrane with a pore size of 100-400 nm; and / or, in step (c), the buffer comprises aqueous HEPES buffer; And / or, in step (c), the nucleic acid molecule is a nucleic acid molecule capable of encoding one or more antigens.

25. The method according to claim 20 or 24, wherein: In step (a), the organic solvent comprises an alcohol having 1 to 4 carbon atoms; and / or, in step (c), the buffer comprises aqueous HEPES buffer and EDTA; And / or, in step (c), 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.

26. The method of claim 20, wherein: In step (c), the lipid mixture and the therapeutic agent and / or preventive agent are used in such amounts that the charge ratio of positive charge to negative charge in the obtained composition is 1:2 to 1:

5.

27. Use of the permanent anionic lipid compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer thereof, the lipid composition according to claims 2-8, the composition according to any one of claims 13-19, or the composition prepared by the method according to any one of claims 20-26 in the preparation of a drug capable of inducing a cellular immune response.

28. Use of the permanent anionic lipid compound of claim 1, its pharmaceutically acceptable salt or stereoisomer thereof, the lipid composition of claims 2-8, the composition of any one of claims 13-19, or the composition prepared by the method of any one of claims 20-26 in the preparation of a drug for preventing, diagnosing, treating or ameliorating a disease, condition, abnormal condition or dysfunction in a subject.

29. The use according to claim 28, wherein The disease, disorder, abnormal condition or dysfunction is selected from any one or a combination of at least two of the group consisting of infectious diseases, cancer, proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases; And / or, the subject is a mammalian subject.

30. The use according to claim 28 or 29, wherein The subject is selected from any one or a combination of at least two of the group consisting of humans, non-human primates, companion animals, exotic species, livestock animals, and food animals.

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