Liposome carrier for efficient vaccine delivery and preparation and use of liposome vaccine

Abstract

A liposome carrier is provided. A liposome vaccine is provided. A method for preparing a liposome carrier is provided. A method for preparing a liposome vaccine is provided. Use of a liposome carrier or a liposome vaccine in preparing a vaccine for treating tumors is provided.

Description

Technical Field

[0001] The present application relates to the field of drug delivery technology, and more specifically to a liposome carrier and a preparation method thereof, a liposome vaccine comprising the liposome carrier and a preparation method thereof, and uses thereof. Background Art

[0002] Tumors are one of the most lethal diseases in the world. Although traditional surgical treatment, radiotherapy and chemotherapy play a major role in tumor treatment, they are difficult to effectively inhibit tumor recurrence and metastasis. In addition to surgical treatment and radiotherapy and chemotherapy, targeted therapy and immunotherapy have become important means to improve the prognosis of tumor patients, but 80% of tumor patients do not respond to immune checkpoint monoclonal antibody blockade therapy, and the 5-year survival rate is less than 20%.

[0003] Compared with them, tumor neoantigen vaccine, as a new generation of personalized and precise immunotherapy, can effectively inhibit postoperative tumor growth, metastasis and recurrence, and prolong the survival of tumor patients. It is a cutting-edge research hotspot in the field of immunotherapy at home and abroad. It is characterized by the fact that neoantigens only exist in tumor tissue cells and are not expressed in normal cells, and have good tumor tissue cell specificity and low immune tolerance. Among them, neoantigen peptide vaccine has the advantages of easy chemical modification, stability, easy storage, multiple targets, low toxic side effects and strong anti-tumor immune response.

[0004] However, according to the analysis of clinical trial data at home and abroad, the efficiency of neoantigen vaccines in inducing immune responses in the body is still low, and the efficacy of therapeutic vaccines in inhibiting solid tumors is not ideal. Among them, antigen presenting cells (APCs) are a type of professional cells that can engulf, process and present antigen peptides to T cells. They recognize and engulf antigens through specific receptors, process them into peptide fragments and bind to MHC II molecules, and present them to the cell membrane surface, activating and stimulating the proliferation and differentiation of T lymphocytes to initiate autoimmune responses. They are the most critical lymphocytes for initiating autoimmune responses and cellular immune responses. Therefore, improving the uptake of neoantigens by APCs, the activation of APCs and the regulation of their immune functions are important ways to enhance the efficiency of antigen presentation and anti-tumor immune responses.

[0005] However, there is currently a lack of effective neoantigen vaccine delivery vectors in clinical practice, and it is impossible to effectively co-deliver neoantigens and adjuvants to the same antigen-presenting cell. The main clinical approach to neoantigen vaccines is subcutaneous injection, which is a simple injection of a mixture of neoantigen peptides and adjuvants. This method cannot effectively deliver the rare neoantigen peptides and adjuvants to the same antigen-presenting cell simultaneously. At the same time, the vaccine taken up by endocytosis is easily degraded by nucleases or proteases in lysosomes, further reducing the effective concentration of effector molecules (neoantigens and adjuvants), resulting in a weak anti-tumor immune response produced by the body and poor tumor treatment efficacy.

[0006] At present, due to the relatively stable physical and chemical properties and relatively good biosafety of liposomes, the industry generally uses lipid nanoparticles (LNPs) as the preferred encapsulation and delivery carrier for GMP commercial production of vaccines and other drugs. Liposomes are generally composed of lipid bilayers, usually containing phospholipids (such as lecithin), cholesterol and PEGylated lipids. Nowadays, liposomes have been widely used to deliver a variety of drugs and gene therapy, such as paclitaxel liposomes (Doxil) for the treatment of AIDS-related sarcomas and ovarian cancer. In addition, liposome vaccines can be used for disease treatment through intramuscular injection, subcutaneous injection, intranasal spray and other routes, such as Haemophilus influenzae vaccine, malaria vaccine, hepatitis B vaccine, etc. The existing LNPs formula is prepared by precise molar ratios of ionizable cationic lipids (MC3, C12-200), zwitterionic lipids (DSPC), cholesterol and lipid-anchored polyethylene glycol esters.

[0007] However, liposome vaccine carriers containing polyethylene glycol esters will produce PEG antibodies after vaccination, which can cause severe allergic reactions, including rash, a sudden drop in blood pressure, rapid breathing, and rapid heartbeat. In addition, the liposome carriers developed by Pfizer Pharmaceuticals for the delivery of the new coronavirus vaccine mainly stimulate B cells to produce specific antiviral antibodies for the prevention of viral infection.

[0008] A liposomal formulation that efficiently delivers neoantigen vaccines to antigen-presenting cells for personalized antitumor immunotherapy of solid tumors has not yet been developed. Summary of the invention

[0009] Provided is a liposome carrier comprising: at least one of DOTMA or DOTAP; at least one of DOPE or DSPC; and at least one of DDAB and cholesterol or its modifications; in particular, the liposome carrier comprises DOTMA, DDAB, DOPE, and cholesterol or its modifications.

[0010] Provided is a liposome vaccine, comprising a liposome carrier and a neoantigen vaccine.

[0011] Provided is a method for preparing a liposome carrier, comprising: mixing liposome carrier components in a first solvent to obtain a first solution; and mixing the first solution and a second solution, wherein the liposome carrier components include: at least one of DOTMA or DOTAP; at least one of DOPE or DSPC; and at least one of DDAB and cholesterol or its modifications; in particular, the liposome carrier components include DOTMA, DDAB, DOPE, and cholesterol or its modifications.

[0012] Provided is a method for preparing a liposome vaccine, comprising mixing a liposome carrier and a neoantigen vaccine.

[0013] Provided are the use of the liposome carrier of the present application, the liposome carrier prepared by the method for preparing the liposome carrier of the present application, the liposome vaccine of the present application, and the liposome vaccine prepared by the method for preparing the liposome vaccine of the present application in preparing drugs for treating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The synthesis route of the present invention is shown, in which DOTMA, DOPE, cholesterol, DDAB, cholesterol-CpG-ODN and neoantigen polypeptide are combined to obtain liposome vaccine, and the liposome vaccine is combined with CD205 + Schematic diagram of the binding of dendritic cells (DCs).

[0015] Figure 2 This is a cryo-electron micrograph of the liposome vaccine of the embodiment of the present application, showing the structural morphology of the liposome vaccine of the embodiment of the present application.

[0016] Figure 3A and Figure 3B The particle size and surface potential diagram of the liposome or liposome vaccine of the examples of the present application are respectively shown.

[0017] Figure 4 The graph shows the cellular uptake rate of the liposome vaccine of the examples or comparative examples of the present application by antigen presenting cells.

[0018] Figure 5 The Dil fluorescence intensity of the dendritic cells that took up the liposome vaccine of the examples or comparative examples of the present application is shown.

[0019] Figure 6 The activation rate (surface CD11c + CD80 + CD86 + expression).

[0020] Fig. 7A , 7B7C shows the fluorescence intensity of the liposome vaccine of the present application example, reflecting the ratio of the liposome vaccine of these examples to be taken up. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

[0021] Figure 8 The following table shows the serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, albumin, creatinine, urea, triglyceride, and total cholesterol of mice that took the liposome vaccine of the present example or the control group.

[0022] Fig. 9 Shown are the tumor fluorescence intensities of tumor-bearing mice injected subcutaneously, intramuscularly, and intravenously with liposome vaccines.

[0023] Fig. 10A and Fig. 10B The fluorescence intensity of tumor and the survival rate of tumor-bearing mice injected with the liposome vaccine of the comparative example or the example are shown. DETAILED DESCRIPTION

[0024] In order to further explain the technical means and effects adopted by the present application to achieve the predetermined purpose, the specific implementation methods, structures, features and effects of the present application are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0025] Terms and Definitions

[0026] In order to enable ordinary technicians in the field to understand the characteristics and effects of the present invention, the following general description and definition of the terms and expressions mentioned in the specification and claims are provided. Unless otherwise specified, all technical and scientific terms used in the text have the common meanings understood by ordinary technicians in the field for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0027] As used herein, singular terms refer to one or more than one. For example, "element" or "an element" refers to one element or more than one element. As used herein, the term "plurality" refers to at least two.

[0028] In this document, the terms "comprise", "include", "have", "contain" or any other similar terms are open conjunctions, which are intended to cover non-exclusive inclusions, indicating that the combination (such as a device, composition, method, etc.) includes the listed elements (such as each unit of the device, each component of the composition, the substantial steps of the method, etc.), but does not exclude other elements. For example, a composition or article containing a plurality of elements is not limited to these elements listed herein, but may also include other elements that are not explicitly listed but are generally inherent to the composition or article. Unless otherwise expressly stated, the term "or" refers to an inclusive "or" rather than an exclusive "or". In this document, the term "substantially consisting of..." when used to define compositions and methods means excluding other elements that have any substantial impact on the combination of the stated purpose, but does not exclude other elements that do not substantially affect the basic and novel characteristics of the invention. In this document, the term "consisting of..." and other closed conjunctions refer to combinations (units, components, substantial steps, etc.) excluding other elements, but unless otherwise stated, it is not intended to exclude trace amounts of unavoidable impurities. The embodiments defined by each of these connecting terms are within the scope of the present invention. As specific embodiments thereof, the disclosure of a technical solution including the terms "comprising", "including", "having", "containing" or any other similar terms should also be deemed to simultaneously disclose the corresponding technical solution including the terms "substantially consisting of" and "consisting of".

[0029] In this document, the terms "first", "second", "third" ... and other ordinal numbers, unless otherwise specified or further limitations are provided, are only used to distinguish objects with the same attributes in name, and are not used to describe a specific order or sequence, nor are they used to further limit these objects through these ordinal numbers themselves. It should be understood that these ordinal numbers can be interchanged under appropriate circumstances without affecting the understanding of this application. In addition, it should be understood that the disclosure of any further limited implementation methods without ordinal numbers in this application should also be regarded as the disclosure of any further limited implementation methods with these ordinal numbers. It should also be understood that the objects defined by the later ordinal numbers in this application do not mean that the same objects defined by the earlier ordinal numbers must also be included in the same implementation method.

[0030] In this article, all features or conditions defined in the form of numerical ranges or percentage ranges are only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be deemed to have covered and specifically disclosed all possible sub-ranges and individual values ​​within the range, especially integer values. For example, the range description of "1 to 8" should be deemed to have specifically disclosed all sub-ranges such as 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8 ... etc., especially the sub-ranges defined by all integer values, and should be deemed to have specifically disclosed individual values ​​such as 1, 2, 3, 4, 5, 6, 7, 8 within the range. Similarly, the range description of "between 1 to 8" should be deemed to have specifically disclosed all ranges such as 1 to 8, 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8, etc., and include its endpoint values. Unless otherwise specified, this interpretation method can be applied to all contents of the full text of the present invention, regardless of whether the scope is extensive or not.

[0031] If a quantity or other numerical value or parameter is expressed as a range, a preferred range or a series of upper and lower limits, it should be understood that all ranges consisting of any upper limit or preferred value of the range and the lower limit or preferred value of the range have been specifically disclosed herein, regardless of whether these ranges are disclosed separately. As used herein, the term "about" refers to approximate, about or near the range. When the term "about" is used for a numerical value, it modifies the numerical value by adding or subtracting the provided numerical expansion limit to form a numerical range, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the specific circumstances. In general, the term "about" is used herein to change the numerical value up and down by 10%, that is, it refers to the numerical value of the number it modifies plus 10% and the numerical value of the number it modifies minus 10%. For example, "about 50%" means within the range of 45%-55%. In addition, when a range of values ​​is mentioned herein, unless otherwise specified, the range shall include its endpoints and all integers and fractions within the range. It should also be understood that all integers and fractions are deemed to be modified by the term "about". In this article, under the premise that the purpose of the invention can be achieved, the numerical value should be understood to have the accuracy of the significant digits of the numerical value. For example, the number 40.0 should be understood to cover the range from 39.50 to 40.49.

[0032] Herein, "parts by weight" means the number of parts by weight, which may be any weight unit, such as but not limited to kilograms, grams, pounds, etc. Herein, "parts by mole" means the number of parts by mole, which may be mole or multiples thereof.

[0033] As used herein, "polypeptide" and "protein" are used interchangeably to refer to a molecule having two or more amino acid residues interconnected by peptide bonds.

[0034] As used herein, the term "tumor" refers to abnormal growth of cells or tissues, particularly including the growth of malignant tumors ("cancer"). The term "tumor" or "cancer" includes not only various tumors or cancers, but also metastases of various tumors or cancers.

[0035] As used herein, the term "active ingredient" or "active pharmaceutical ingredient" refers to an ingredient in a drug (e.g., a vaccine) that provides biological activity or pharmacological activity, or has a direct effect on the diagnosis, cure, alleviation, treatment or prevention of a disease, or has a direct effect on restoring, correcting or changing the physiological function of a patient.

[0036] As used herein, the term "neo-antigen" refers to an antigen derived from a tumor-specific genomic mutation. For example, a neo-antigen may be produced by the expression of a mutant protein in a tumor sample caused by a non-synonymous single nucleotide mutation, or by the expression of an alternative open reading frame caused by a mutation-induced frameshift. A neo-antigen is a molecule or part of a molecule associated with a therapeutic agent (e.g., a peptide, protein, or gene therapy) that is recognized by the immune system and is capable of inducing an immune response. The nature of the immune response may be humoral (e.g., neutralizing antibodies against neo-antigens that inhibit function, shorten half-life, or trigger degradation) or cellular (e.g., T cell receptor recognition of peptides derived from neo-antigens), or both.

[0037] As used herein, the term "internucleotide bond" refers to a chemical bond connecting two nucleotides by the non-basic portion of a nucleotide. Examples of internucleotide bonds include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, methylphosphorothioate, etc. Phosphorothioate, phosphorodithioate, methylphosphonate, and methylphosphorothioate are stable internucleotide bonds, while phosphodiester is a naturally occurring internucleotide bond. Oligonucleotide phosphorothioates are generally synthesized as random racemic mixtures of phosphorothioate bonds of the R and S configurations for the phosphorus atom.

[0038] Implementation

[0039] In one aspect, the present application provides a liposome carrier.

[0040] In some embodiments, the liposome carrier comprises: a first component comprising at least one of 2,3-dioleoyloxypropyl-1-trimethylammonium bromide (DOTMA) or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or consisting thereof; a second component comprising at least one of 1,2-di(9Z-oleoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or consisting thereof; and a third component comprising at least one of didodecyldimethylammonium bromide (DDAB) and cholesterol or its modifications, or consisting thereof. In some embodiments, the liposome carrier comprises at least one of DOTMA or DOTAP, at least one of DDAB, DOPE or DSPC, and optionally cholesterol or its modifications. In some embodiments, the liposome carrier comprises at least one of DOTMA or DOTAP, optionally at least one of DDAB, DOPE or DSPC, and cholesterol or its modifications. In some embodiments, the liposome carrier comprises at least one of DOTMA or DOTAP, at least one of DDAB, DOPE or DSPC, and cholesterol or a modification thereof. In some embodiments, the liposome carrier comprises at least one of DOTMA or DOTAP, DDAB, DOPE, and cholesterol or a modification thereof. In some embodiments, the liposome carrier comprises at least one of DOTMA or DOTAP, DDAB, DOPE, and cholesterol or a modification thereof. In some embodiments, the liposome carrier comprises DOTMA, DDAB, at least one of DOPE or DSPC, and cholesterol or a modification thereof. In some embodiments, the liposome carrier comprises DOTMA, DOPE, DDAB, and cholesterol or a modification thereof.

[0041] In some embodiments, the liposome carrier comprises a liposome core and a bilayer membrane, wherein the liposome core comprises at least one of DOTMA and DOTAP, the bilayer membrane comprises at least one of DOPE or DSPC, and the liposome core comprises DDAB or the bilayer membrane comprises cholesterol or a modification thereof. In some embodiments, the liposome carrier comprises DOTMA and DDAB, and the bilayer membrane comprises DOPE and cholesterol or a modification thereof.

[0042] In some embodiments, the first component comprises or consists of DOTMA. In some embodiments, the first component comprises or consists of DOTAP. In some embodiments, the first component comprises or consists of DOTMA and DOTAP. In some embodiments, the second component comprises or consists of DOPE. In some embodiments, the second component comprises or consists of DSPC. In some embodiments, the second component comprises or consists of DOPE and DSPC. In some embodiments, the third component comprises or consists of DDAB. In some embodiments, the third component comprises or consists of cholesterol or a modification thereof. In some embodiments, the third component comprises or consists of DDAB and cholesterol or a modification thereof. In some embodiments, the first component comprises or consists of DOTMA, and the third component comprises or consists of DDAB. In some embodiments, the first component comprises or consists of DOTAP, and the third component comprises or consists of DDAB. In some embodiments, the second component comprises or consists of DOPE, and the third component comprises or consists of cholesterol or a modification thereof. In some embodiments, the second component comprises or consists of DSPC, and the third component comprises or consists of cholesterol or a modification thereof. In some embodiments, the first component comprises or consists of DOTMA, the second component comprises or consists of DOPE, and the third component comprises or consists of DDAB and cholesterol or a modification thereof. In some embodiments, the first component comprises or consists of DOTMA, the second component comprises or consists of DSPC, and the third component comprises or consists of DDAB and cholesterol or a modification thereof. In some embodiments, the first component comprises or consists of DOTAP, the second component comprises or consists of DOPE, and the third component comprises or consists of DDAB and cholesterol or a modification thereof. In some embodiments, the first component comprises or consists of DOTAP, the second component comprises or consists of DSPC, and the third component comprises or consists of DDAB and cholesterol or a modification thereof.

[0043] In some embodiments, the first component is 0.1-10 mole parts, in particular 0.25-10 mole parts, more particularly 0.5-10 mole parts, more particularly 1-8 mole parts, and more particularly 1-4 mole parts. In some embodiments, at least one of DOTMA or DOTAP is 0.1-10 mole parts, in particular 0.25-10 mole parts, more particularly 0.5-10 mole parts, more particularly 1-8 mole parts, and more particularly 1-4 mole parts. In some embodiments, DOTMA or DOTAP is 0.1-10 mole parts, in particular 0.25-10 mole parts, more particularly 0.5-10 mole parts, more particularly 1-8 mole parts, and more particularly 1-4 mole parts. In some embodiments, DOTMA is 0.1-10 mole parts, in particular 0.25-10 mole parts, more particularly 0.5-10 mole parts, more particularly 1-8 mole parts, and more particularly 1-4 mole parts. In some embodiments, DOTAP is 0.1-10 mole parts, particularly 0.25-10 mole parts, more particularly 0.5-10 mole parts, more particularly 1-8 mole parts, and more particularly 1-4 mole parts. In some embodiments, DOTMA, DOTAP, or DOTMA and DOTAP combined are 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.6, 0.7, 0.8, 0.9, 10, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 280, 290, 300, 310, 320, 330, 340, 360, 380, 390, 400, 410, 420, 430, 440, 460, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610 , 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mole parts.

[0044] In some embodiments, DDAB is 0.1-10 mole parts, in particular 0.1-5 mole parts, in particular 0.1-2 mole parts, in particular 0.25-1.5 mole parts, in particular 0.5-1 mole parts. In some embodiments, DDAB is 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.55, 0.6, 0.65, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75

[0045] In some embodiments, the molar ratio of the first component to DDAB is (0.1-10):(0.1-10), in particular (0.25-10):(0.1-5), in particular (0.5-10):(0.1-2), in particular (1-8):(0.25-1.5), in particular (1-4):(0.5-1). In some embodiments, the molar ratio of at least one of DOTMA or DOTAP to DDAB is (0.1-10):(0.1-10), in particular (0.25-10):(0.1-5), in particular (0.5-10):(0.1-2), in particular (1-8):(0.25-1.5), in particular (1-4):(0.5-1). In some embodiments, the molar ratio of the sum of DOTMA or DOTAP to DDAB is (0.1-10):(0.1-10), in particular (0.25-10):(0.1-5), in particular (0.5-10):(0.1-2), in particular (1-8):(0.25-1.5), in particular (1-4):(0.5-1). In some embodiments, the molar ratio of DOTMA to DDAB is (0.1-10):(0.1-10), in particular (0.25-10):(0.1-5), in particular (0.5-10):(0.1-2), in particular (1-8):(0.25-1.5), in particular (1-4):(0.5-1). In some embodiments, the molar ratio of DOTAP to DDAB is (0.1-10):(0.1-10), particularly (0.25-10):(0.1-5), particularly (0.5-10):(0.1-2), particularly (1-8):(0.25-1.5), particularly (1-4):(0.5-1).

[0046] In some embodiments, the second component is 0.1-10 mole parts, especially 0.1-5 mole parts, especially 0.25-5 mole parts, especially 0.25-2 mole parts, especially 0.5-1 mole parts. In some embodiments, at least one of DOPE or DSPC is 0.1-10 mole parts, especially 0.1-5 mole parts, especially 0.25-5 mole parts, especially 0.25-2 mole parts, especially 0.5-1 mole parts. In some embodiments, DOPE or DSPC is 0.1-10 mole parts, especially 0.1-5 mole parts, especially 0.25-5 mole parts, especially 0.25-2 mole parts, especially 0.5-1 mole parts. In some embodiments, DOPE is 0.1-10 mole parts, especially 0.1-5 mole parts, especially 0.25-5 mole parts, especially 0.25-2 mole parts, especially 0.5-1 mole parts. In some embodiments, DSPC is 0.1-10 molar parts, in particular 0.1-5 molar parts, in particular 0.25-5 molar parts, in particular 0.25-2 molar parts, in particular 0.5-1 molar parts. In some embodiments, DOPE, DSPC, or DOPE and DSPC combined are 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.55 , 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mole parts.

[0047] In some embodiments, cholesterol or its modifications are 0.1-10 molar parts, in particular 0.1-5 molar parts, in particular 0.1-2 molar parts, in particular 0.1-1 molar parts, in particular 0.25-0.5 molar parts. In some embodiments, cholesterol or its modifications are 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.55, 0.6, 0.6 7, 8.5, 9, 9.5, or 10 mole parts.

[0048] In some embodiments, the molar ratio of the second component to cholesterol or its modifications is (0.1-10):(0.1-10), particularly (0.1-5):(0.1-5), particularly (0.25-5):(0.1-2), particularly (0.25-2):(0.1-1), particularly (0.5-1):(0.25-0.5). In some embodiments, the molar ratio of at least one of DOPE or DSPC to cholesterol or its modifications is (0.1-10):(0.1-10), particularly (0.1-5):(0.1-5), particularly (0.25-5):(0.1-2), particularly (0.25-2):(0.1-1), particularly (0.5-1):(0.25-0.5). In some embodiments, the molar ratio of the sum of DOPE or DSPC to cholesterol or its modifications is (0.1-10):(0.1-10), particularly (0.1-5):(0.1-5), particularly (0.25-5):(0.1-2), particularly (0.25-2):(0.1-1), particularly (0.5-1):(0.25-0.5). In some embodiments, the molar ratio of DOPE to cholesterol or its modifications is (0.1-10):(0.1-10), particularly (0.1-5):(0.1-5), particularly (0.25-5):(0.1-2), particularly (0.25-2):(0.1-1), particularly (0.5-1):(0.25-0.5). In some embodiments, the molar ratio of DSPC to cholesterol or its modifications is (0.1-10):(0.1-10), particularly (0.1-5):(0.1-5), particularly (0.25-5):(0.1-2), particularly (0.25-2):(0.1-1), particularly (0.5-1):(0.25-0.5).

[0049] In some embodiments, the molar ratio of the first component, DDAB, the second component, and cholesterol or a modification thereof is (0.1–10):(0.1–10):(0.1–10):(0.1–10), particularly (0.25–10):(0.1–5):(0.5–5):(0.1–5), particularly (0.5–10):(0.1–2):(0.25–5):(0.1–2), particularly (1–8):(0.25–1.5):(0.25–2):(0.1–1), particularly (1–4):(0.5–1):(0.5–1):(0.25–0.5). In some embodiments, the molar ratio of at least one of DOTMA or DOTAP, at least one of DDAB, DOPE or DSPC, and cholesterol or a modification thereof is (0.1-10):(0.1-10):(0.1-10):(0.1-10), particularly (0.25-10):(0.1-5):(0.5-5):(0.1-5), particularly (0.5-10):(0.1-2):(0.25-5):(0.1-2), particularly (1-8):(0.25-1.5):(0.25-2):(0.1-1), particularly (1-4):(0.5-1):(0.5-1):(0.25-0.5). In some embodiments, the molar ratio of the sum of DOTMA or DOTAP, the sum of DDAB, DOPE or DSPC, and cholesterol or its modifications is (0.1-10):(0.1-10):(0.1-10):(0.1-10), particularly (0.25-10):(0.1-5):(0.5-5):(0.1-5), particularly (0.5-10):(0.1-2):(0.25-5):(0.1-2), particularly (1-8):(0.25-1.5):(0.25-2):(0.1-1), particularly (1-4):(0.5-1):(0.5-1):(0.25-0.5). In some embodiments, at least one of DOTMA, DDAB, DOPE or DSPC, and cholesterol or a modification thereof, is in a molar ratio of (0.1–10):(0.1–10):(0.1–10):(0.1–10), particularly (0.25–10):(0.1–5):(0.5–5):(0.1–5), particularly (0.5–10):(0.1–2):(0.25–5):(0.1–2), particularly (1–8):(0.25–1.5):(0.25–2):(0.1–1), particularly (1–4):(0.5–1):(0.5–1):(0.25–0.5).In some embodiments, at least one of DOTAP, DDAB, DOPE or DSPC, and cholesterol or a modification thereof, is in a molar ratio of (0.1-10):(0.1-10):(0.1-10):(0.1-10), particularly (0.25-10):(0.1-5):(0.5-5):(0.1-5), particularly (0.5-10):(0.1-2):(0.25-5):(0.1-2), particularly (1-8):(0.25-1.5):(0.25-2):(0.1-1), particularly (1-4):(0.5-1):(0.5-1):(0.25-0.5). In some embodiments, at least one of DOTMA or DOTAP, DDAB, DOPE, and cholesterol or modifications thereof are in a molar ratio of (0.1-10):(0.1-10):(0.1-10):(0.1-10), particularly (0.25-10):(0.1-5):(0.5-5):(0.1-5), particularly (0.5-10):(0.1-2):(0.25-5):(0.1-2), particularly (1-8):(0.25-1.5):(0.25-2):(0.1-1), particularly (1-4):(0.5-1):(0.5-1):(0.25-0.5). In some embodiments, at least one of DOTMA or DOTAP, DDAB, DSPC, and cholesterol or modifications thereof are in a molar ratio of (0.1–10):(0.1–10):(0.1–10):(0.1–10), particularly (0.25–10):(0.1–5):(0.5–5):(0.1–5), particularly (0.5–10):(0.1–2):(0.25–5):(0.1–2), particularly (1–8):(0.25–1.5):(0.25–2):(0.1–1), particularly (1–4):(0.5–1):(0.5–1):(0.25–0.5).

[0050] In some embodiments, the molar ratio of DOTMA, DDAB, DOPE, and cholesterol or modifications thereof is (0.1–10):(0.1–10):(0.1–10):(0.1–10), particularly (0.25–10):(0.1–5):(0.5–5):(0.1–5), particularly (0.5–10):(0.1–2):(0.25–5):(0.1–2), particularly (1–8):(0.25–1.5):(0.25–2):(0.1–1), particularly (1–4):(0.5–1):(0.5–1):(0.25–0.5). In some embodiments, the molar ratio of DOTAP, DDAB, DOPE, and cholesterol or modifications thereof is (0.1–10):(0.1–10):(0.1–10):(0.1–10), particularly (0.25–10):(0.1–5):(0.5–5):(0.1–5), particularly (0.5–10):(0.1–2):(0.25–5):(0.1–2), particularly (1–8):(0.25–1.5):(0.25–2):(0.1–1), particularly (1–4):(0.5–1):(0.5–1):(0.25–0.5). In some embodiments, the molar ratio of DOTMA, DDAB, DSPC, and cholesterol or its modifications is (0.1–10):(0.1–10):(0.1–10):(0.1–10), particularly (0.25–10):(0.1–5):(0.5–5):(0.1–5), particularly (0.5–10):(0.1–2):(0.25–5):(0.1–2), particularly (1–8):(0.25–1.5):(0.25–2):(0.1–1), particularly (1–4):(0.5–1):(0.5–1):(0.25–0.5). In some embodiments, the molar ratio of DOTAP, DDAB, DSPC, and cholesterol or modifications thereof is (0.1–10):(0.1–10):(0.1–10):(0.1–10), particularly (0.25–10):(0.1–5):(0.5–5):(0.1–5), particularly (0.5–10):(0.1–2):(0.25–5):(0.1–2), particularly (1–8):(0.25–1.5):(0.25–2):(0.1–1), particularly (1–4):(0.5–1):(0.5–1):(0.25–0.5).

[0051] In some embodiments, at least one of DOTMA or DOTAP (or the first component), at least one of DDAB, DOPE or DSPC (or the second component), and cholesterol or its modifications are present in a molar ratio of (0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0. 9, 0.95, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10): (0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.28, 0.3, 0.32, 0.34, 0 .36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2 , 2.3, 2.4, 2.5, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10): (0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.1 6, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.55, 0.6, 0.65, 0.7 , 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10): (0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0 .55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10).

[0052] In some embodiments, the cholesterol or its modifications include cholesterol. In some embodiments, the cholesterol or its modifications include or consist of cholesterol-CpG oligodeoxynucleotide (CpG-ODN). In some embodiments, the cholesterol or its modifications include or consist of cholesterol and cholesterol-CpG oligodeoxynucleotide.

[0053] In some embodiments, the internucleotide bond in the cholesterol-CpG oligodeoxynucleotide comprises a modified internucleotide bond. In some embodiments, the internucleotide bond in the cholesterol-CpG oligodeoxynucleotide comprises a phosphorothioate internucleotide bond. In some embodiments, the internucleotide bond in the cholesterol-CpG oligodeoxynucleotide consists of a phosphorothioate internucleotide bond. In some embodiments, in the cholesterol-CpG oligodeoxynucleotide, cholesterol is attached to the 3′-end of the CpG oligodeoxynucleotide. In some embodiments, the cholesterol-CpG oligodeoxynucleotide comprises or consists of a sequence as shown in SEQ ID NO: 4 or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more identity thereto.

[0054] In some embodiments, the molar ratio of cholesterol to cholesterol-CpG oligodeoxynucleotide is (5-1,000):1, particularly (10-500):1, particularly (20-200):1, particularly (50-100):1, particularly about 100:1. In some embodiments, the molar ratio of cholesterol to cholesterol-CpG oligodeoxynucleotide is (1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000): 1.

[0055] In one aspect, the present application provides a vaccine (vaccine active ingredient). In one aspect, the present application provides a neoantigen vaccine.

[0056] On the one hand, the present application provides a liposome vaccine, including the liposome carrier. On the one hand, the present application provides a liposome vaccine, including the liposome carrier and a vaccine active ingredient, in particular the liposome carrier and a neoantigen vaccine. In some embodiments, the vaccine active ingredient is a polypeptide vaccine or a nucleic acid vaccine. In some embodiments, the vaccine active ingredient is a polypeptide vaccine. In some embodiments, the vaccine active ingredient is a nucleic acid vaccine. In some embodiments, the polypeptide vaccine includes or consists of a peptide of less than 10 amino acids. In some embodiments, the polypeptide vaccine includes or consists of a peptide of 11-21 amino acids. In some embodiments, the polypeptide vaccine includes a bacterial surface antigen peptide or a viral surface antigen peptide. In some embodiments, the nucleic acid vaccine includes siRNA, mRNA, or circular RNA. In some embodiments, the nucleic acid vaccine is mRNA.

[0057] In some embodiments, the vaccine active ingredient includes a neoantigen vaccine. In some embodiments, the neoantigen vaccine includes a neoantigen nucleic acid vaccine or a neoantigen polypeptide vaccine. In some embodiments, the neoantigen nucleic acid vaccine is a neoantigen RNA vaccine, in particular a neoantigen mRNA vaccine. In some embodiments, the neoantigen nucleic acid vaccine includes an RNA as shown in SEQ ID NO: 1 or having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more identity thereto. In some embodiments, the neoantigen polypeptide vaccine includes a polypeptide as shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 or having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more identity thereto. In some embodiments, the neoantigen polypeptide vaccine comprises a polypeptide as set forth in SEQ ID NO:2 or having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more identity thereto. In some embodiments, the neoantigen polypeptide vaccine comprises a polypeptide as set forth in SEQ ID NO:3 or having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more identity thereto. In some embodiments, the neoantigen polypeptide vaccine comprises a polypeptide as set forth in SEQ ID NO:4 or having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more identity thereto.

[0058] In some embodiments, the liposome vaccine comprises a liposome core and a bilayer membrane, wherein the liposome core comprises the vaccine and comprises at least one of DOTMA and DOTAP, the bilayer membrane comprises at least one of DOPE or DSPC, and the liposome core comprises DDAB or the bilayer membrane comprises cholesterol or a modification thereof. In some embodiments, the liposome carrier comprises DOTMA and DDAB, and the bilayer membrane comprises DOPE and cholesterol or a modification thereof.

[0059] In some embodiments, the mass ratio of the liposome carrier to the neoantigen vaccine is (1-100):1, particularly (2-50):1, particularly (5-20):1, particularly 10:1.

[0060] In some embodiments, the particle size of the liposome vaccine is 20-300 nm. In some embodiments, the shape of the liposome vaccine is spherical. In some embodiments, the liposome vaccine carries a positive charge of 30-40 mV.

[0061] In one aspect, the present application provides a method for preparing a liposome carrier, comprising mixing liposome carrier components in a first solvent to obtain a first solution; and mixing the first solution and a second solution.

[0062] In some embodiments, the liposome carrier components include at least one of DOTMA or DOTAP, at least one of DOPE or DSPC, and at least one of DDAB and cholesterol or modifications thereof. In some embodiments, the liposome carrier components include at least one of DOTMA or DOTAP, DDAB, at least one of DOPE or DSPC, and optionally cholesterol or modifications thereof. In some embodiments, the liposome carrier components include at least one of DOTMA or DOTAP, optionally DDAB, at least one of DOPE or DSPC, and cholesterol or modifications thereof. In some embodiments, the liposome carrier components include at least one of DOTMA or DOTAP, DDAB, at least one of DOPE or DSPC, and cholesterol or modifications thereof. In some embodiments, the liposome carrier components include at least one of DOTMA or DOTAP, DDAB, DOPE, and cholesterol or modifications thereof. In some embodiments, the liposome carrier components include at least one of DOTMA or DOTAP, DDAB, DOPE, and cholesterol or modifications thereof. In some embodiments, the liposome carrier components include at least one of DOTMA, DDAB, at least one of DOPE or DSPC, and cholesterol or modifications thereof. In some embodiments, the liposome carrier components include DOTMA, DDAB, at least one of DOPE or DSPC, and cholesterol or modifications thereof. In some embodiments, the liposome carrier components include DOTMA, DOPE, DDAB, and cholesterol or modifications thereof.

[0063] In some embodiments, the first solvent comprises an organic solvent, particularly alcohols, more particularly ethanol, and more particularly anhydrous ethanol.

[0064] In some embodiments, the second solution comprises a polar solvent, in particular water. In some embodiments, the second solution is a buffer. In some embodiments, the second solution has a pK a A buffer solution between 4.2–6.2, particularly between 4.7–5.7, and more particularly between 4.7–5.2. In some embodiments, the second solution comprises acetate ions. In some embodiments, the second solution is a buffer solution comprising acetate ions, particularly an acetic acid-acetate buffer solution, and more particularly an acetic acid-sodium acetate buffer solution. In some embodiments, the concentration of the buffer solution is 100–200 mM. In some embodiments, the pH value of the second solution is 5.0–5.2, particularly 5.2. In some embodiments, the second solution, particularly the polar solvent, is an antisolvent of the first solvent.

[0065] In some embodiments, the mixing of the liposome carrier components in the first solvent includes ultrasonic treatment. In some embodiments, the frequency of the ultrasonic wave is 40-50 KHz. In some embodiments, the duration of the ultrasonic treatment is 1-10 min, particularly 3-5 min.

[0066] In some embodiments, the mixing of the first solution and the second solution comprises injecting the first solution into the second solution. In some embodiments, the mixing of the first solution and the second solution comprises mixing the first solution and the second solution using a microfluidic device. In some embodiments, the mixing of the first solution and the second solution comprises ethanol injection or microfluidics.

[0067] In some embodiments, the mixing of the first solution and the second solution further comprises a first stirring to stir the mixture of the first solution and the second solution. In some embodiments, the first stirring is by stirring with a stirrer, such as a magnetic stirrer. In some embodiments, the stirring rate of the first stirring is 450-600rpm / min. In some embodiments, the duration of the first stirring is 15-60min, particularly 30min.

[0068] On the one hand, the present application provides a method for preparing a liposome vaccine, comprising mixing the liposome carrier and the vaccine active ingredient.

[0069] In some embodiments, the mixing of the liposome carrier and the vaccine active ingredient comprises mixing a third solution comprising the liposome carrier and a fourth solution comprising the vaccine active ingredient. In some embodiments, the fourth solution comprises DEPC (diethyl polycarbonate) treated water or dimethyl sulfoxide (DMSO), and the vaccine active ingredient. In some embodiments, the fourth solution comprises DEPC treated water and the vaccine active ingredient. In some embodiments, the fourth solution comprises DMSO and the vaccine active ingredient. In some embodiments, the vaccine active ingredient is a nucleic acid vaccine, and the fourth solution comprises DEPC treated water and the nucleic acid vaccine. In some embodiments, the vaccine active ingredient is a polypeptide vaccine, and the fourth solution comprises DMSO and the polypeptide vaccine.

[0070] In some embodiments, the method for preparing the liposome vaccine further comprises a second stirring step, stirring the mixture of the third solution and the fourth solution. In some embodiments, the second stirring step lasts for 30-120 min.

[0071] In some embodiments, the method for preparing the liposome vaccine further comprises dialysis after the second stirring. In some embodiments, the dialysis is performed through a dialysis bag. In some embodiments, the molecular weight cutoff of the dialysis bag is 1-100 kDa, particularly 10 kDa.

[0072] In some embodiments, the method for preparing the liposome vaccine further comprises filtration after the dialysis. In some embodiments, the filtration is by filtration through a filter membrane, for example, by filtration through an aqueous filter membrane. In some embodiments, the pore size of the filter membrane is 0.1-1 μm, particularly 0.2-0.3 μm, and more particularly 0.22 μm.

[0073] In some embodiments, the method for preparing the liposome vaccine further comprises preparing the liposome carrier. In some embodiments, the method for preparing the liposome vaccine comprises: mixing the liposome carrier components in a first solvent to obtain a first solution; mixing the first solution and the second solution to obtain the liposome carrier; and mixing the liposome carrier and the vaccine active ingredient.

[0074] In some embodiments, the present application provides the RNA shown in SEQ ID NO: 1. In some embodiments, the present application provides the polypeptide shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the present application provides the polypeptide shown in SEQ ID NO: 2. In some embodiments, the present application provides the polypeptide shown in SEQ ID NO: 3. In some embodiments, the present application provides the polypeptide shown in SEQ ID NO: 4.

[0075] On the one hand, the present application provides a method for treating a tumor in a subject, comprising administering the liposome carrier of the present application, in particular a liposome vaccine comprising the liposome carrier of the present application, to the subject. On the one hand, the present application provides the liposome carrier of the present application for treating tumors, in particular a liposome vaccine comprising the liposome carrier of the present application. On the one hand, the present application provides the use of the liposome carrier of the present application, in particular a liposome vaccine comprising the liposome carrier of the present application, in treating tumors. On the one hand, the present application provides the use of the liposome carrier of the present application, in particular a liposome vaccine comprising the liposome carrier of the present application, in preparing a drug for treating tumors.

[0076] On the one hand, the present application provides a method for treating a tumor in a subject, comprising administering a liposome carrier prepared by the method of the present application, in particular a liposome vaccine comprising a liposome carrier prepared by the method of the present application, to the subject. On the one hand, the present application provides a liposome carrier prepared by the method of the present application for treating a tumor, in particular a liposome vaccine comprising a liposome carrier prepared by the method of the present application. On the one hand, the present application provides a liposome carrier prepared by the method of the present application, in particular a liposome vaccine comprising a liposome carrier prepared by the method of the present application, and the use of the liposome carrier prepared by the method of the present application, in treating a tumor. On the one hand, the present application provides a liposome carrier prepared by the method of the present application, in particular a liposome vaccine comprising a liposome carrier prepared by the method of the present application, and the use of the liposome carrier prepared by the method of the present application, in preparing a drug for treating a tumor.

[0077] In one aspect, the present application provides a method for treating a tumor in a subject, comprising administering the liposome vaccine of the present application to the subject. In one aspect, the present application provides the liposome vaccine of the present application for treating a tumor. In one aspect, the present application provides the use of the liposome vaccine of the present application in treating a tumor. In one aspect, the present application provides the use of the liposome vaccine of the present application in preparing a drug for treating a tumor.

[0078] In one aspect, the present application provides a method for treating a tumor in a subject, comprising administering a liposome vaccine prepared by the method of the present application to the subject. In one aspect, the present application provides a liposome vaccine prepared by the method of the present application for treating a tumor. In one aspect, the present application provides the use of the liposome vaccine prepared by the method of the present application in treating a tumor. In one aspect, the present application provides the use of the liposome vaccine prepared by the method of the present application in preparing a drug for treating a tumor.

[0079] In one aspect, the present application provides a method for treating a tumor in a subject, comprising administering the neoantigen vaccine of the present application to the subject. In one aspect, the present application provides the neoantigen vaccine of the present application for treating a tumor. In one aspect, the present application provides the use of the neoantigen vaccine of the present application in treating a tumor. In one aspect, the present application provides the use of the neoantigen vaccine of the present application in preparing a drug for treating a tumor.

[0080] In some embodiments, the tumor is a tumor of a mammal. In some embodiments, the tumor is a human tumor. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a mouse.

[0081] In some embodiments, the tumor comprises liver cancer (e.g., hepatocellular carcinoma or bile duct cancer), gastric cancer, ovarian cancer, endometrial cancer, cervical cancer, colorectal cancer (e.g., cecal cancer, appendix cancer, ascending colon cancer, transverse colon cancer, descending colon cancer, sigmoid colon cancer, rectal cancer, or anal cancer), lung cancer (e.g., squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer), soft tissue sarcoma, osteosarcoma, fibrosarcoma, skin cancer (e.g., malignant melanoma), testicular cancer, breast cancer, fibro Sarcoma, neuroblastoma, brain cancer (e.g., glioma, such as ependymoma, astrocytoma, oligodendroglioma, brain stem glioma, oligoastrocytoma), bladder cancer, intestinal cancer, prostate cancer, kidney cancer (e.g., renal cell carcinoma), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), pleural mesothelioma, head and neck squamous cell carcinoma, nasopharyngeal cancer, oropharyngeal cancer, or blood cancer (e.g., acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, or lymphoma).

[0082] In some embodiments, the tumor comprises liver cancer or colorectal cancer. In some embodiments, the tumor comprises liver cancer. In some embodiments, the liver cancer comprises hepatocellular carcinoma or cholangiocarcinoma. In some embodiments, the tumor comprises colorectal cancer. In some embodiments, the colorectal cancer comprises cecal cancer, appendix cancer, ascending colon cancer, transverse colon cancer, descending colon cancer, sigmoid colon cancer, rectal cancer, or anal cancer. In some embodiments, the colorectal cancer comprises colorectal adenocarcinoma or colorectal precancerous adenoma.

[0083] In some embodiments, the tumor comprises Hepa 1-6 or MC38. In some embodiments, the tumor comprises Hepa 1-6. In some embodiments, the tumor comprises MC38.

[0084] In some embodiments, the administration is injection, especially intravenous injection. In some embodiments, the treatment of tumors is by injection, especially intravenous injection, to treat tumors. In some embodiments, the drug for treating tumors is an injection, especially an intravenous injection.

[0085] In some embodiments, the liposome vaccine of the present application has uniform and stable particle size distribution, good water solubility and biosafety, and can be used for the treatment of diseases, such as anti-tumor treatment, especially anti-tumor immunotherapy.

[0086] In some embodiments, the liposome vaccine of the present application can more efficiently deliver antigens to antigen-presenting cells and stimulate the maturation of antigen-presenting cells compared to a simple mixed form of neoantigen polypeptide vaccine or neoantigen mRNA vaccine.

[0087] In some embodiments, the vaccine of the present application, especially the liposome vaccine of the present application, can efficiently deliver antigens into antigen-presenting cells or co-deliver antigens and adjuvants into antigen-presenting cells, effectively activate the body's anti-tumor immune response, and inhibit tumor growth.

[0088] In some embodiments, the liposome carrier of the present application can efficiently load the neoantigen polypeptide vaccine or the neoantigen mRNA vaccine to form positively charged liposome particles (i.e., the liposome vaccine of the present application).

[0089] In some embodiments, the liposome vaccine of the present application, especially the liposome vaccine including the liposome carrier of the present application, is low in toxicity, biodegradable, free of polyethylene glycol (PEG) components and thus does not cause potential allergic reactions such as PEG antibodies, and has good biosafety and stability. In some embodiments, the liposome carrier of the present application, especially the liposome vaccine including the liposome carrier of the present application, significantly improves the antigen delivery and antigen presenting cell uptake efficiency of the liposome vaccine through its selected specific cationic lipids, thereby improving the anti-tumor immune response of the liposome vaccine and the immunotherapy of solid tumors by the liposome vaccine.

[0090] In some embodiments, the preparation method of the liposome carrier or liposome vaccine of the present application is simple and can be prepared by conventional equipment.

[0091] Antigen presenting cells are a type of professional cells that can engulf, process and present antigenic peptides to T cells, and play a key role in initiating autoimmune responses. In some embodiments, the liposome vaccine of the present application, especially the liposome vaccine comprising the liposome carrier of the present application, can significantly improve the anti-tumor immune response of the vaccine to the body and generate more specific T lymphocytes to kill tumors through efficient antigen uptake and effective activation.

[0092] Example

[0093] The embodiments are only used to further explain the technical solutions of the present invention and cannot be regarded as limiting the protection scope of the present invention. Non-essential improvements or adjustments made by technicians in this field based on the contents of the present invention should be regarded as falling within the protection scope of the present invention.

[0094] Materials and Equipment

[0095] Unless otherwise specified, all experimental materials used were purchased from conventional biochemical reagent stores.

[0096] -2,3-Dioleoyloxypropyl-1-trimethylammonium bromide (DOTMA) was purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.;

[0097] -1,2-Dioleoyl-3-trimethylammonium propane (DOTAP) was purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.;

[0098] -1,2-Di(9Z-oleoyl)-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from Xi'an Ruixi Biotechnology Co., Ltd.;

[0099] -1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC) was purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.;

[0100] -Cholesterol was purchased from Shanghai Titan Technology Co., Ltd., molecular weight 386.55;

[0101] -Didodecyl dimethyl ammonium bromide (DDAB) was purchased from Beijing Bailingwei Technology Co., Ltd.;

[0102] -Cholesterol-CpG-ODN bases were fully thiolated, the sequence is shown in SEQ ID NO: 4, purchased from Shanghai Bioengineering Co., Ltd.;

[0103] -1,2-Epoxytetradecane was purchased from TCI;

[0104] -DiI was purchased from Suzhou Yuheng Biotechnology Co., Ltd.;

[0105] -CD80, CD86, and CD11c flow cytometry antibodies were purchased from eBioscience, USA;

[0106] -Nano particle size potentiometer purchased from Malvern Instruments;

[0107] - Empty plasmid PresentER-Cassette mCherry was purchased from Addgene.

[0108] 1. Construction of neoantigen vaccine

[0109] The immunogenicity of peptides or nucleic acids was tested in mice using ELISpot assays, and multiple neoantigen sequences were screened from Hepa 1–6 (mouse hepatoma cell line) or MC38 (mouse colorectal cancer cell line).

[0110] In the ELISpot test, cells can locally produce cytokines after being stimulated by antigens, and these cytokines will be captured by specific monoclonal antibodies pre-fixed on the well plate; after cell lysis, the captured cytokines bind to the biotin-labeled secondary antibody and then to the avidin-labeled alkaline phosphatase; after incubation with the substrate BCIP / NBT, purple spots appear on the PVDF well plate, indicating that the corresponding cells have produced cytokines; finally, the number of spots on the well plate is counted by the ELISpot analysis system to obtain the quantitative results of the Elispot test.

[0111] According to the screened neoantigen sequences, the corresponding neoantigen polypeptide vaccines or neoantigen nucleic acid vaccines are synthesized respectively.

[0112] The mRNA sequence screened for Hepa 1-6 cells is shown in SEQ ID NO: 1 (the expression of Hepa 1-6 neoantigens is composed of Mapk3, Lmf1, Samd9l, Traf7, Dtnb, Lbr and Ptpn2 genes); the polypeptide sequence screened for Hepa 1-6 cells is shown in SEQ ID NO: 2; the polypeptide sequence screened for MC38 cells is shown in SEQ ID NO: 3.

[0113] 2. Preparation of liposome vaccine

[0114] DOTMA, DOTAP, DDAB, DOPE, DSPC, cholesterol, and cholesterol-CpG-ODN were dissolved in anhydrous ethanol to prepare mother solutions.

[0115] Example 1 (Vaccine + DOTMA + DDAB + DOPE + Cholesterol / Cholesterol-CpG-ODN)

[0116] 1 part of DOTMA stock solution (100 mg / ml, the same below), 0.5 part of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 0.25 part of cholesterol stock solution containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 1.

[0117] The mixed solution of Example 1 was injected into a sodium acetate buffer solution (pH 5.0-5.2), and stirred vigorously for 30 min using a magnetic stirrer (450-600 rpm / min) to preliminarily obtain the liposome carrier solution of Example 1.

[0118] Then, a DMSO solution (1 mg) containing a neoantigen polypeptide vaccine or a DEPC-treated aqueous solution containing a neoantigen mRNA vaccine (10 μg) was added to the liposome carrier solution of Example 1 and stirred for 0.5–2 h. The ethanol and sodium acetate components were removed by dialysis using a dialysis bag (10 KDa), and then filtered through a 0.22 μm aqueous filter membrane to obtain the liposome vaccine of Example 1.

[0119] Figure 1 The synthesis route of Example 1 of the present application shows that the combination of DOTMA, DOPE, cholesterol, DDAB, cholesterol-CpG-ODN, and neoantigen polypeptide / mRNA obtains a liposome vaccine, and the liposome vaccine is combined with CD205 + Schematic diagram of the binding of dendritic cells (DCs).

[0120] Example 1A (Vaccine + DOTMA + DDAB + DOPE + Cholesterol)

[0121] 1 part of DOTMA stock solution (100 mg / ml, the same below), 0.5 part of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 0.25 part of cholesterol stock solution (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 min to obtain the mixed solution of Example 1A.

[0122] The steps for preparing the liposome vaccine of Example 1A from the mixed solution of Example 1A refer to the corresponding steps of Example 1.

[0123] Example 2 (Vaccine + DOTMA + DOPE + Cholesterol / Cholesterol-CpG-ODN)

[0124] 1 part of DOTMA mother solution, 1 part of DOPE mother solution, and 0.25 parts of cholesterol mother solution containing cholesterol-CpG-ODN were respectively pipetted into an Eppendorf tube and mixed, and these mother solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 2.

[0125] The steps for preparing the liposome vaccine of Example 2 from the mixed solution of Example 2 refer to the corresponding steps of Example 1.

[0126] Example 3 (Vaccine + DOTMA + DDAB + DOPE)

[0127] 1 part of DOTMA mother solution, 0.5 part of DDAB mother solution, and 1 part of DOPE mother solution were respectively pipetted into an Eppendorf tube and mixed, and the mother solutions were uniformly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 3.

[0128] The steps for preparing the liposome vaccine of Example 3 from the mixed solution of Example 3 refer to the corresponding steps of Example 1.

[0129] Example 4 (Vaccine + DOTAP + DDAB + DOPE + Cholesterol / Cholesterol-CpG-ODN)

[0130] 1 part of DOTAP stock solution (100 mg / ml, the same below), 0.5 part of DDAB stock solution, 1 part of DOPE stock solution, and 0.25 part of cholesterol stock solution containing cholesterol-CpG-ODN were respectively pipetted into an Eppendorf tube and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 4.

[0131] The steps for preparing the liposome vaccine of Example 4 from the mixed solution of Example 4 refer to the corresponding steps of Example 1.

[0132] Example 5 (Vaccine+DOTAP+DDAB+DOPE)

[0133] 1 part of DOTAP mother solution, 0.5 part of DDAB mother solution, and 1 part of DOPE mother solution were respectively pipetted into an Eppendorf tube and mixed. The mother solutions were uniformly mixed by ultrasonic treatment for 3-5 min to obtain the mixed solution of Example 5.

[0134] The steps for preparing the liposome vaccine of Example 5 from the mixed solution of Example 5 refer to the corresponding steps of Example 1.

[0135] Example 6 (Vaccine + DOTMA + DDAB + DSPC + Cholesterol / Cholesterol-CpG-ODN)

[0136] 1 part of DOTMA mother solution, 0.5 part of DDAB mother solution, 1 part of DSPC mother solution (15 mg / ml, the same below), and 0.25 part of cholesterol mother solution containing cholesterol-CpG-ODN were respectively pipetted into an Eppendorf tube and mixed, and these mother solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 6.

[0137] The steps for preparing the liposome vaccine of Example 6 from the mixed solution of Example 6 refer to the corresponding steps of Example 1.

[0138] Example 7 (Vaccine + DOTMA + DSPC + Cholesterol / Cholesterol-CpG-ODN)

[0139] 1 part of DOTMA mother solution, 1 part of DSPC mother solution, and 0.25 parts of cholesterol mother solution containing cholesterol-CpG-ODN were respectively pipetted into an Eppendorf tube and mixed, and these mother solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 7.

[0140] The steps for preparing the liposome vaccine of Example 7 from the mixed solution of Example 7 refer to the corresponding steps of Example 1.

[0141] Example 8

[0142] 1 part of DOTMA stock solution (100 mg / ml, the same below), 1 part of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 min to obtain the mixed solution of Example 8.

[0143] The steps for preparing the liposome vaccine of Example 8 from the mixed solution of Example 8 refer to the corresponding steps of Example 1.

[0144] Example 8A

[0145] 1 part of DOTMA stock solution (100 mg / ml, the same below), 1 part of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 min to obtain the mixed solution of Example 8A.

[0146] The steps for preparing the liposome vaccine of Example 8A from the mixed solution of Example 8A refer to the corresponding steps of Example 1.

[0147] Example 9

[0148] 1 part of DOTMA stock solution (100 mg / ml, the same below), 2 parts of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 9.

[0149] The steps for preparing the liposome vaccine of Example 9 from the mixed solution of Example 9 refer to the corresponding steps of Example 1.

[0150] Example 9A

[0151] 1 part of DOTMA stock solution (100 mg / ml, the same below), 2 parts of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 min to obtain the mixed solution of Example 9A.

[0152] The steps for preparing the liposome vaccine of Example 9A from the mixed solution of Example 9A refer to the corresponding steps of Example 1.

[0153] Example 10

[0154] 1 part of DOTMA stock solution (100 mg / ml, the same below), 4 parts of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 10.

[0155] The steps for preparing the liposome vaccine of Example 10 from the mixed solution of Example 10 refer to the corresponding steps of Example 1.

[0156] Example 10A

[0157] 1 part of DOTMA stock solution (100 mg / ml, the same below), 4 parts of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 min to obtain the mixed solution of Example 10A.

[0158] The steps for preparing the liposome vaccine of Example 10A from the mixed solution of Example 10A refer to the corresponding steps of Example 1.

[0159] Embodiment 11

[0160] 1 part of DOTMA stock solution (100 mg / ml, the same below), 6 parts of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 11.

[0161] The steps for preparing the liposome vaccine of Example 11 from the mixed solution of Example 11 refer to the corresponding steps of Example 1.

[0162] Example 11A

[0163] 1 part of DOTMA stock solution (100 mg / ml, the same below), 6 parts of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 min to obtain the mixed solution of Example 11A.

[0164] The steps for preparing the liposome vaccine of Example 11A from the mixed solution of Example 11A refer to the corresponding steps of Example 1.

[0165] Example 12

[0166] 1 part of DOTMA stock solution (100 mg / ml, the same below), 8 parts of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 12.

[0167] The steps for preparing the liposome vaccine of Example 12 from the mixed solution of Example 12 refer to the corresponding steps of Example 1.

[0168] Example 12A

[0169] 1 part of DOTMA mother solution (100 mg / ml, the same below), 8 parts of DDAB mother solution (50 mg / ml, the same below), 1 part of DOPE mother solution (15 mg / ml, the same below), and 0.25 parts of cholesterol mother solution (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed, and these mother solutions were evenly mixed by ultrasonic treatment for 3-5 min to obtain the mixed solution of Example 12A.

[0170] The steps for preparing the liposome vaccine of Example 12A from the mixed solution of Example 12A refer to the corresponding steps of Example 1.

[0171] Embodiment 13

[0172] 1 part of DOTMA stock solution (100 mg / ml, the same below), 0.5 part of DDAB stock solution (50 mg / ml, the same below), 4 parts of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into Eppendorf tubes and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 13.

[0173] The steps for preparing the liposome vaccine of Example 13 from the mixed solution of Example 13 refer to the corresponding steps of Example 1.

[0174] Embodiment 14

[0175] 4 parts of DOTMA stock solution (100 mg / ml, the same below), 0.5 parts of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into Eppendorf tubes and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 14.

[0176] The steps for preparing the liposome vaccine of Example 14 from the mixed solution of Example 14 refer to the corresponding steps of Example 1.

[0177] Embodiment 15

[0178] 0.1 parts of DOTMA stock solution (100 mg / ml, the same below), 1 part of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 2 parts of cholesterol stock solution containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into Eppendorf tubes and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 15.

[0179] The steps for preparing the liposome vaccine of Example 15 from the mixed solution of Example 15 refer to the corresponding steps of Example 1.

[0180] Example 16

[0181] 2 parts of DOTMA stock solution (100 mg / ml, the same below), 0.5 parts of DDAB stock solution (50 mg / ml, the same below), 1 part of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into Eppendorf tubes and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 16.

[0182] The steps for preparing the liposome vaccine of Example 16 from the mixed solution of Example 16 refer to the corresponding steps of Example 1.

[0183] Embodiment 17

[0184] 1 part of DOTMA stock solution (100 mg / ml, the same below), 2 parts of DDAB stock solution (50 mg / ml, the same below), 2 parts of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 17.

[0185] The steps for preparing the liposome vaccine of Example 17 from the mixed solution of Example 17 refer to the corresponding steps of Example 1.

[0186] Embodiment 18

[0187] 2 parts of DOTMA stock solution (100 mg / ml, the same below), 0.5 parts of DDAB stock solution (50 mg / ml, the same below), 0.5 parts of DOPE stock solution (15 mg / ml, the same below), and 0.25 parts of cholesterol stock solution containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into Eppendorf tubes and mixed, and these stock solutions were evenly mixed by ultrasonic treatment for 3-5 minutes to obtain the mixed solution of Example 18.

[0188] The steps for preparing the liposome vaccine of Example 18 from the mixed solution of Example 18 refer to the corresponding steps of Example 1.

[0189] Comparative Example 1 (Vaccine+DOTMA+DOPE)

[0190] 1 part of DOTMA mother solution and 1 part of DOPE mother solution were respectively pipetted into an Eppendorf tube and mixed, and the mother solutions were uniformly mixed by ultrasonic treatment for 3-5 minutes to obtain a mixed solution of Comparative Example 1.

[0191] The mixed solution of Comparative Example 1 was injected into a sodium acetate buffer solution (pH 5.0-5.2), and stirred vigorously for 30 min using a magnetic stirrer (450-600 rpm / min) to preliminarily obtain a liposome carrier solution of Comparative Example 1.

[0192] Then, DEPC-treated water containing neoantigen polypeptide vaccine or neoantigen mRNA vaccine was added to the liposome carrier solution of Comparative Example 1 and stirred for 0.5-2h. The ethanol and sodium acetate components were removed by dialysis bag (10KDa), and then filtered through a 0.22μm aqueous filter membrane to obtain the liposome vaccine of Comparative Example 1.

[0193] Comparative Example 2 (Vaccine + Cholesterol-CpG-ODN)

[0194] The cholesterol mother solution containing cholesterol-CpG-ODN was drawn into an Eppendorf tube and ultrasonically treated for 3-5 minutes. The obtained solution was injected into a sodium acetate buffer solution (pH 5.0-5.2) and vigorously stirred for 30 minutes using a magnetic stirrer (450-600rpm / min). Then, DEPC-treated water containing a neoantigen polypeptide vaccine or a neoantigen mRNA vaccine was added thereto and stirred for 0.5-2 hours. The dialysis bag (10KDa) was dialyzed to remove the ethanol and sodium acetate components, and then filtered through a 0.22μm aqueous filter membrane to obtain the composition of Comparative Example 2, which did not form a liposome carrier structure.

[0195] 3. Characterization of Liposomal Vaccine

[0196] The structural morphology of the liposome vaccine in Example 1 of the present application was characterized using cryo-electron microscopy. The results are shown in Figure 2 . Figure 2 This is a cryo-electron micrograph of the liposome vaccine according to an embodiment of the present application. Figure 2 It is shown that according to the method of the examples of the present application, a spherical and uniform liposome vaccine is obtained.

[0197] The liposome or liposome vaccine of Example 1 of the present application was characterized by dynamic light scattering technology. The results are shown in Figure 3A , 3B . Figure 3A and Figure 3B The particle size and surface potential diagram of the liposome or liposome vaccine of the examples of the present application are respectively shown. Figure 3A , 3B It is shown that the liposomes or liposome vaccine of Example 1 of the present application are evenly distributed, have a particle size in the range of 20-300 nm, and carry a positive charge of 30-40 mV.

[0198] Based on the results of cryo-electron microscopy and dynamic light scattering, it can be seen that the present application successfully synthesized a spherical liposome vaccine with uniform particle size distribution, positive charge, and stability, and has good water solubility.

[0199] 4. Uptake of liposomal vaccines by bone marrow-derived dendritic cells

[0200] The liposome vaccines of Example 1 and Comparative Example 2 were prepared with FAM-labeled neoantigen polypeptides and Cy5-labeled CHO-CpG-ODN. Immature mouse bone marrow-derived dendritic cells (BMDCs) were cultured at 5×10 5 The cells were inoculated in a 24-well plate at a density of 1000 cells / well and incubated with the liposome vaccine of Example 1 or Comparative Example 1 or 2 for 12 h, respectively, and an equal volume of PBS buffer was used as a control group. The original culture medium was discarded and washed three times with PBS. The ratio of FAM-labeled neoantigen polypeptides and Cy5-labeled CHO-CpG-ODN double-positive antigen-presenting cells (i.e., the cellular uptake rate of liposome vaccine by antigen-presenting cells) was quantitatively analyzed by flow cytometry (BD). Figure 4 As shown, compared with Comparative Example 2, the liposome delivery vector of Example 1 efficiently co-delivers the neoantigen polypeptide and the adjuvant CHO-CpG-ODN into the same antigen presenting cell, and the proportion of double-positive antigen presenting cells is about 60 times higher than that of Comparative Example 2.

[0201] The liposome vaccines of Example 1 and Comparative Example 1 labeled with Dil membrane fluorescent dye were prepared. The Dil fluorescence intensity of the liposome vaccine taken up by dendritic cells was quantified and counted using flow cytometry. Figure 5As shown, compared with the liposome vaccine of Comparative Example 1, the liposome vaccine of Example 1 is efficiently taken up by antigen presenting cells, and its uptake efficiency is about 64 times higher than that of Comparative Example 1.

[0202] It can be seen that compared with the neoantigen vaccine or mRNA vaccine in a simple mixed form, the liposome vaccine of the present application can more efficiently deliver the antigen into antigen-presenting cells, or co-deliver the antigen and adjuvant into antigen-presenting cells.

[0203] 5. Liposomal vaccines activate bone marrow-derived dendritic cells

[0204] Immature mouse bone marrow-derived dendritic cells (BMDCs) were cultured at a rate of 5×10 5 The cells were inoculated in a 24-well plate at a density of 100 cells / well and co-treated with the liposome vaccine of Comparative Example 1, Comparative Example 2 or Example 1 for 48 hours. The cells were centrifuged (800 g, 5 min) and the treated dendritic cells were incubated with CD11c-APC, CD80-PE, and CD86-PE-Cy7 fluorescent antibodies in the dark for 30 minutes to label CD11c, CD80, and CD86 antigens. The cells were washed with PBS three times and the activation rate of the dendritic cells after incubation (surface CD11c + CD80 + CD86 + The results are shown in Figure 6 .

[0205] CD11c is an adhesion protein that is mainly expressed in myeloid cells such as myelocytes, promyelocytes, metamyelocytes, non-segmented and segmented neutrophils, and is also expressed in tissue macrophages, monocytes, NK cells, activated T cells and lymphocytes. CD80 is a transmembrane protein that belongs to the B7 family and is mainly expressed in dendritic cells. It binds to the T cell surface receptors CD28 and CTLA-4 and participates in the activation process of T cells. CD86, also known as B7-2, also belongs to the B7 family and is expressed on the surface of dendritic cells. It is a ligand for CD28 and CTLA-4. Binding to these two receptors can synergistically promote or inhibit the activation of T lymphocytes.

[0206] Depend on Figure 5 –6 It can be seen that the liposome vaccine of Example 1 has a higher delivery efficiency, enhances the immunostimulatory effects of CD11c, CD80, and CD86, and promotes the maturation of dendritic cells. It can also be seen that compared with the neoantigen vaccine or mRNA vaccine in a simple mixed form, the liposome vaccine of the present application can more efficiently stimulate the maturation of antigen presenting cells.

[0207] 6. Comparison of liposome vaccine uptake in different examples

[0208] Comparison of liposome vaccine uptake in liposome carriers with different components

[0209] Immature mouse bone marrow-derived dendritic cells (BMDCs) were cultured at a rate of 5×10 5 The cells were inoculated in a 24-well plate at a density of 100 cells / well and treated with the Dir-labeled liposome vaccines of Comparative Examples 1-7 for 12 hours (three replicate wells per group). After washing with PBS buffer to remove the unabsorbed liposomes, the treated BMDCs were resuspended in PBS solution, and the liposome Dir fluorescence in the BMDCs was detected by flow cytometry.

[0210] The results are as follows Fig. 7A . Fig. 7A The fluorescence intensity of the liposome vaccine of the examples of the present application is shown, reflecting the proportion of the liposome vaccine of these examples being taken up. It can be seen that the liposome vaccines of Examples 1-7 are all taken up to a certain extent, and the amount of the liposome vaccine of Example 1 taken up is significantly greater than that of Examples 2-7, reaching a significant difference of p < 0.0001.

[0211] According to the results of the above examples, it can be seen that the liposome carriers composed of at least one of DOTMA or DOTAP, at least one of DDAB, DOPE or DSPC, and cholesterol or its modifications, and the liposome vaccines prepared based on them can effectively stimulate the maturation of antigen-presenting cells, and can improve the anti-tumor immune response of the liposome vaccine and improve the immunotherapy effect of the liposome vaccine on solid tumors; among them, the liposome carriers composed of DOTMA, DDAB, DOPE, and cholesterol or its modifications, and the liposome vaccines prepared based on them can particularly effectively stimulate the maturation of antigen-presenting cells, thereby more effectively improving the anti-tumor immune response of the liposome vaccine and improving the immunotherapy effect of the liposome vaccine on solid tumors.

[0212] Comparison of liposome vaccine uptake in liposome carriers with different ratios

[0213] Immature mouse bone marrow-derived dendritic cells (BMDCs) were cultured at a rate of 5×10 5 The cells were inoculated in a 24-well plate at a density of 100 cells / well and treated with the Dir-labeled liposome vaccines of Comparative Examples 1, 1A, 8-12, and 8A-12A for 12 hours (three replicate wells per group). After washing with PBS buffer to remove the unabsorbed liposomes, the treated BMDCs were resuspended in PBS solution, and the liposome Dir fluorescence in the BMDCs was detected by flow cytometry.

[0214] The results are as follows Figure 7B . Figure 7BThe fluorescence intensity of the liposome vaccine of the embodiment of the present application is shown, reflecting the proportion of the liposome vaccine of these embodiments being taken in. It can be seen that the amount of liposome vaccine taken in Examples 1, 1A, 8, and 8A is significantly greater than the amount of liposome vaccine taken in Examples 9-12, 9A-12A, and the amount of cholesterol-CpG-ODN-containing embodiments (Examples 1 and 8) is also significantly higher than the corresponding embodiments not containing cholesterol-CpG-ODN (Examples 1A and 8A).

[0215] Immature mouse bone marrow-derived dendritic cells (BMDCs) were inoculated in 24-well plates at a density of 5×105 / well and co-treated with the Dir-labeled liposome vaccine of Comparative Examples 1 or 13-18 for 12 hours (three replicates per group). After washing with PBS buffer to remove the unabsorbed liposomes, the treated BMDCs were resuspended in PBS solution, and the liposome Dir fluorescence in BMDCs was detected by flow cytometry.

[0216] The results are as follows Figure 7C . Figure 7C The fluorescence intensity of the liposome vaccine of the present application is shown, reflecting the proportion of the liposome vaccine of these examples being taken in. It can be seen that the amount of the liposome vaccine of Examples 1, 14, 16, and 18 taken in is significantly greater than the amount of the liposome vaccine of Examples 13, 15, and 17 taken in, reaching a significant difference of p < 0.0001, and the amount of the liposome vaccine of Example 1 taken in is also greater than the amount of the liposome vaccine of Examples 14, 16, and 18 taken in.

[0217] According to the results of the above examples, it can be seen that the liposome carrier with a molar ratio of at least one of DOTMA or DOTAP, at least one of DDAB, DOPE or DSPC, and cholesterol or its modification is (0.1-10):(0.1-10):(0.1-10), especially (0.25-10):(0.1-5):(0.5-5):(0.1-5), especially (0.5-10):(0.1-2):(0.25-5):(0.1-2), and the liposome vaccine prepared based on the liposome carrier can efficiently stimulate the maturation of antigen presenting cells and improve the anti-tumor immune response of the liposome vaccine. , improving the immunotherapeutic effect of liposome vaccines on solid tumors; wherein, in particular, at least one of DOTMA or DOTAP, at least one of DDAB, DOPE or DSPC, and cholesterol or its modifications, the molar ratio of which is (1-8):(0.25-1.5):(0.25-2):(0.1-1), more particularly (1-4):(0.5-1):(0.5-1):(0.25-0.5), the liposome carrier prepared based on the liposome vaccine can particularly effectively stimulate the maturation of antigen presenting cells, thereby more effectively improving the anti-tumor immune response of the liposome vaccine and improving the immunotherapeutic effect of the liposome vaccine on solid tumors.

[0218] 7. Biosafety of liposome vaccines

[0219] Male C57BL / 6 mice aged 6-8 weeks were selected and divided into two groups: one group was intravenously injected with an equal volume of PBS buffer as the control group, and the other group was intravenously injected with the liposome vaccine of Example 1 (200 μg, containing 50 μg of neoantigen polypeptide and 3 μg of cholesterol-CpG-ODN), three times with an interval of 4 days.

[0220] On the 7th day, the mice in the control group and the group treated with the liposome vaccine of Example 1 were euthanized, and the mouse serum was collected to detect serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, albumin, creatinine, urea, triglycerides, and total cholesterol.

[0221] Results Figure 8 Compared with the control group, the intravenous injection of the liposome vaccine of Example 1 did not affect the liver and kidney functions of mice, indicating that the liposome vaccine of the example has good biosafety and can be used for the treatment of diseases, such as anti-tumor treatment, especially anti-tumor immunotherapy.

[0222] 8. Antitumor effects of liposome vaccines injected in different ways on in situ liver cancer in mice

[0223] Male C57BL / 6 mice aged 6–8 weeks were selected, and Hepa 1-6-luc mouse liver cancer cells (1×106 / mouse) were inoculated into the liver lobes of mice to construct an in situ liver cancer model. The small animal fluorescence imaging system (PE) was used to verify the successful construction of the mouse tumor-bearing model.

[0224] Tumor-bearing mice were divided into four groups (7 mice in each group), and 200 μL of the liposome vaccine of Example 1 was injected subcutaneously, intramuscularly, and intravenously, respectively, and the control group was injected intravenously with an equal volume of PBS buffer, once every 4 days, for a total of 3 injections.

[0225] The small animal fluorescence imaging system (PE) was used to dynamically monitor the changes in tumor fluorescence intensity in situ. The tumor fluorescence imaging images on day 0 and day 28 are shown in Figure 2. Fig. 9 .

[0226] like Fig. 9 As shown in the results, compared with subcutaneous or intramuscular injection, intravenous liposome vaccine can more effectively inhibit the growth of in situ liver cancer in mice, providing suggestions for the administration method in the later clinical translation application process.

[0227] 9. Antitumor effect and survival rate of intravenous liposome vaccine on in situ hepatocellular carcinoma in mice

[0228] Male C57BL / 6 mice aged 6–8 weeks were selected, and Hepa 1-6-luc mouse liver cancer cells (1×106 / mouse) were inoculated into the liver lobes of mice to construct an in situ liver cancer model. The small animal fluorescence imaging system (PE) was used to verify the successful construction of the mouse tumor-bearing model.

[0229] The tumor-bearing mice were divided into three groups (7 mice in each group) and intravenously injected with 200 μL of the liposome vaccine of Example 1, Comparative Example 2, or an equal volume of PBS buffer solution (control group), once every 4 days, for a total of 3 injections.

[0230] The small animal fluorescence imaging system (PE) was used to dynamically monitor the changes in tumor fluorescence intensity in situ. The tumor fluorescence imaging images on day 0 and day 49 are shown in Figure 2. Fig. 10A ; Record the survival rate of mice, such as Fig. 10B .

[0231] like Fig. 10A and Fig. 10B As shown, the liposome vaccine after intravenous injection can effectively inhibit the growth of in situ liver cancer, and can effectively improve the survival rate and survival time of mice compared with comparative example 2. The liposome vaccine can be used as a therapeutic vaccine for solid tumor treatment and clinical transformation applications.

[0232] sequence

[0233]

[0234] Note: “*” indicates that the natural phosphate internucleotide bonds are replaced by thiophosphate internucleotide bonds.

[0235] The above-mentioned implementation modes are only preferred implementation modes of the present application and cannot be used to limit the scope of protection of the present application. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present application shall fall within the scope of protection required by the present application.

Claims

1. A liposome carrier, characterized in that: include: A first component comprising at least one of 2,3-dioleoyloxypropyl-1-trimethylammonium bromide (DOTMA) or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); a second component comprising at least one of 1,2-bis(9Z-oleoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); as well as a third component comprising didodecyldimethylammonium bromide (DDAB) and cholesterol or a modification thereof; in, At least one of DOTMA or DOTAP, at least one of DDAB, DOPE or DSPC, and cholesterol or a modification thereof, in a molar ratio of (1-4):(0.5-1):(0.5-1):(0.25-0.5); and The cholesterol or its modification is cholesterol or cholesterol-CpG oligodeoxynucleotide having a sequence as shown in SEQ ID NO:

4.

2. The liposome carrier according to claim 1, characterized in that The first component includes DOTMA.

3. The liposome carrier according to claim 2, characterized in that The molar ratio of DOTMA to DDAB is (0.1–10):(0.1–10).

4. The liposome carrier according to claim 2, characterized in that The molar ratio of DOTMA to DDAB is (1–8):(0.25–1.5).

5. The liposome carrier according to claim 2, characterized in that The molar ratio of DOTMA to DDAB is (1–4):(0.5–1).

6. The liposome carrier according to claim 1, characterized in that The first component includes DOTAP.

7. The liposome carrier according to claim 6, characterized in that The molar ratio of DOTAP to DDAB is (0.1–10):(0.1–10).

8. The liposome carrier according to claim 6, characterized in that The molar ratio of DOTAP to DDAB is (1–8):(0.25–1.5).

9. The liposome carrier according to claim 6, characterized in that The molar ratio of DOTAP to DDAB is (1–4):(0.5–1).

10. The liposome carrier according to claim 1, characterized in that The second component includes DOPE.

11. The liposome carrier according to claim 10, characterized in that The molar ratio of DOPE to cholesterol or its modifications is (0.1–10):(0.1–10).

12. The liposome carrier according to claim 10, characterized in that The molar ratio of DOPE to cholesterol or its modifications is (0.25–2):(0.1–1).

13. The liposome carrier according to claim 10, characterized in that The molar ratio of DOPE to cholesterol or its modifications is (0.5–1):(0.25–0.5).

14. The liposome carrier according to claim 1, characterized in that The second component includes DSPC.

15. The liposome carrier according to claim 14, characterized in that The molar ratio of DSPC to cholesterol or its modifications is (0.1–10):(0.1–10).

16. The liposome carrier according to claim 14, characterized in that The molar ratio of DSPC to cholesterol or its modifications was (0.25–2):(0.1–1).

17. The liposome carrier according to claim 14, characterized in that The molar ratio of DSPC to cholesterol or its modifications is (0.5–1):(0.25–0.5).

18. The liposome carrier according to claim 1, characterized in that The first component includes DOTAP, and the second component includes DSPC.

19. The liposome carrier according to claim 18, characterized in that The molar ratio of DOTAP, DDAB, DSPC, and cholesterol or its modifications is (1-4):(0.5-1):(0.5-1):(0.25-0.5).

20. The liposome carrier according to claim 1, characterized in that The first component includes DOTAP, and the second component includes DOPE.

21. The liposome carrier according to claim 20, characterized in that The molar ratio of DOTAP, DDAB, DOPE, and cholesterol or its modifications is (1-4):(0.5-1):(0.5-1):(0.25-0.5).

22. The liposome carrier according to claim 1, wherein The first component includes DOTMA, and the second component includes DOPE.

23. The liposome carrier according to claim 22, characterized in that The molar ratio of DOTMA, DDAB, DOPE, and cholesterol or its modifications is (1-4):(0.5-1):(0.5-1):(0.25-0.5).

24. The liposome carrier according to claim 1, wherein The first component includes DOTMA, and the second component includes DSPC.

25. The liposome carrier according to claim 24, characterized in that The molar ratio of DOTMA, DDAB, DSPC, and cholesterol or its modifications is (1-4):(0.5-1):(0.5-1):(0.25-0.5).

26. The liposome carrier according to claim 1, wherein The cholesterol or its modification includes cholesterol-CpG oligodeoxynucleotide.

27. The liposome carrier according to claim 26, wherein The cholesterol or its modified substance includes cholesterol and cholesterol-CpG oligodeoxynucleotide.

28. The liposome carrier according to claim 27, characterized in that The molar ratio of cholesterol to cholesterol-CpG oligonucleotide is (5–1,000):

1.

29. The liposome carrier according to claim 27, characterized in that The molar ratio of cholesterol to cholesterol-CpG oligodeoxynucleotide is (20–200)∶1.

30. The liposome carrier according to claim 27, wherein The molar ratio of cholesterol to cholesterol-CpG oligodeoxynucleotide is (50–100)∶1.

31. A method for preparing a liposome carrier, characterized in that: include: mixing liposome carrier components in a first solvent to obtain a first solution; as well as mixing the first solution and the second solution, Wherein, the liposome carrier components include: A first component comprising at least one of DOTMA or DOTAP; A second component comprising at least one of DOPE or DSPC; and A third component includes DDAB and cholesterol or a modification thereof; and in, At least one of DOTMA or DOTAP, at least one of DDAB, DOPE or DSPC, and cholesterol or a modification thereof, in a molar ratio of (1-4):(0.5-1):(0.5-1):(0.25-0.5); and The cholesterol or its modification is cholesterol or cholesterol-CpG oligodeoxynucleotide having a sequence as shown in SEQ ID NO:

4.

32. The method for preparing the liposome carrier according to claim 31, characterized in that: The first component includes DOTMA.

33. The method for preparing the liposome carrier according to claim 32, characterized in that: The molar ratio of DOTMA to DDAB is (0.1–10):(0.1–10).

34. The method for preparing the liposome carrier according to claim 32, characterized in that: The molar ratio of DOTMA to DDAB is (1–8):(0.25–1.5).

35. The method for preparing the liposome carrier according to claim 32, characterized in that: The molar ratio of DOTMA to DDAB is (1–4):(0.5–1).

36. The method for preparing the liposome carrier according to claim 32, characterized in that: The first component includes DOTAP.

37. The method for preparing the liposome carrier according to claim 36, characterized in that: The molar ratio of DOTAP to DDAB is (0.1–10):(0.1–10).

38. The method for preparing the liposome carrier according to claim 36, characterized in that: The molar ratio of DOTAP to DDAB is (1–8):(0.25–1.5).

39. The method for preparing the liposome carrier according to claim 36, characterized in that: The molar ratio of DOTAP to DDAB is (1–4):(0.5–1).

40. The method for preparing the liposome carrier according to claim 31, characterized in that: The second component includes DOPE.

41. The method for preparing the liposome carrier according to claim 40, characterized in that: The molar ratio of DOPE to cholesterol or its modifications is (0.1–10):(0.1–10).

42. The method for preparing the liposome carrier according to claim 40, wherein: The molar ratio of DOPE to cholesterol or its modifications is (0.25–2):(0.1–1).

43. The method for preparing the liposome carrier according to claim 40, characterized in that: The molar ratio of DOPE to cholesterol or its modifications is (0.5–1):(0.25–0.5).

44. The method for preparing the liposome carrier according to claim 31, wherein: The second component includes DSPC.

45. The method for preparing a liposome carrier according to claim 44, wherein: The molar ratio of DSPC to cholesterol or its modifications is (0.1–10):(0.1–10).

46. ​​The method for preparing a liposome carrier according to claim 44, wherein: The molar ratio of DSPC to cholesterol or its modifications was (0.25–2):(0.1–1).

47. The method for preparing a liposome carrier according to claim 44, wherein: The molar ratio of DSPC to cholesterol or its modifications is (0.5–1):(0.25–0.5).

48. The method for preparing the liposome carrier according to claim 31, characterized in that: The first component includes DOTAP, and the second component includes DSPC.

49. The method for preparing a liposome carrier according to claim 48, wherein: The molar ratio of DOTAP, DDAB, DSPC, and cholesterol or its modifications is (1-4):(0.5-1):(0.5-1):(0.25-0.5).

50. The method for preparing the liposome carrier according to claim 31, characterized in that: The first component includes DOTAP, and the second component includes DOPE.

51. The method for preparing the liposome carrier according to claim 50, characterized in that: The molar ratio of DOTAP, DDAB, DOPE, and cholesterol or its modifications is (1-4):(0.5-1):(0.5-1):(0.25-0.5).

52. The method for preparing the liposome carrier according to claim 31, wherein: The first component includes DOTMA, and the second component includes DOPE.

53. The method for preparing the liposome carrier according to claim 52, characterized in that: The molar ratio of DOTMA, DDAB, DOPE, and cholesterol or its modifications is (1-4):(0.5-1):(0.5-1):(0.25-0.5).

54. The method for preparing the liposome carrier according to claim 31, wherein: The first component includes DOTMA, and the second component includes DSPC.

55. The method for preparing the liposome carrier according to claim 54, characterized in that: The molar ratio of DOTMA, DDAB, DSPC, and cholesterol or its modifications is (1-4):(0.5-1):(0.5-1):(0.25-0.5).

56. The method for preparing the liposome carrier according to claim 31, wherein: The cholesterol or its modification includes cholesterol-CpG oligodeoxynucleotide.

57. The method for preparing the liposome carrier according to claim 56, characterized in that: The cholesterol or its modified substance includes cholesterol and cholesterol-CpG oligodeoxynucleotide.

58. The method for preparing the liposome carrier according to claim 57, wherein: The molar ratio of cholesterol to cholesterol-CpG oligonucleotide is (5–1,000):

1.

59. The method for preparing the liposome carrier according to claim 57, wherein: The molar ratio of cholesterol to cholesterol-CpG oligodeoxynucleotide is (20–200)∶1.

60. The method for preparing the liposome carrier according to claim 57, wherein: The molar ratio of cholesterol to cholesterol-CpG oligodeoxynucleotide is (50–100)∶1.

61. The method for preparing the liposome carrier according to claim 31, wherein: The first solvent includes an organic solvent.

62. The method for preparing the liposome carrier according to claim 31, wherein: The first solvent includes ethanol.

63. The method for preparing the liposome carrier according to claim 31, characterized in that: The second solution includes a polar solvent.

64. The method for preparing a liposome carrier as claimed in claim 31, characterized in that: The second solution includes water.

65. The method for preparing the liposome carrier according to claim 31, wherein: The second solution is a buffer solution.

66. The method for preparing a liposome carrier according to claim 65, wherein: The concentration of the buffer was 100-200 mM.

67. The method for preparing the liposome carrier according to claim 31, wherein: The second solution has a pK a Buffer between 4.2–6.

2.

68. The method for preparing the liposome carrier according to claim 31, wherein: The second solution is a buffer solution including acetate ions.

69. The method for preparing the liposome carrier according to claim 31, wherein: The second solution is acetic acid-acetate buffer.

70. The method for preparing the liposome carrier according to claim 31, characterized in that: The pH value of the second solution is 5.0-5.

2.

71. The method for preparing the liposome carrier according to claim 31, wherein: The mixing of the liposome carrier components in the first solvent includes ultrasonic treatment.

72. The method for preparing a liposome carrier as claimed in claim 71, characterized in that: The frequency of the ultrasonic wave is 40-50 KHz.

73. The method for preparing the liposome carrier according to claim 71, characterized in that: The duration of the ultrasonic treatment is 1-10 min.

74. The method for preparing the liposome carrier according to claim 31, wherein: The mixing of the first solution and the second solution includes injecting the first solution into the second solution.

75. The method for preparing the liposome carrier according to claim 31, wherein: The mixing of the first solution and the second solution comprises mixing the first solution and the second solution using a microfluidic device.

76. The method for preparing the liposome carrier according to claim 31, wherein: The mixing of the first solution and the second solution comprises an ethanol injection method or a microfluidics method.

77. The method for preparing a liposome carrier as claimed in claim 31, characterized in that: The mixing of the first solution and the second solution further comprises a first stirring step of stirring the mixture of the first solution and the second solution.

78. The method for preparing the liposome carrier according to claim 77, characterized in that: The first stirring is stirring by a magnetic stirrer.

79. The method for preparing a liposome carrier as claimed in claim 77, characterized in that: The stirring rate of the first stirring is 450-600 rpm / min.

80. The method for preparing the liposome carrier according to claim 77, wherein: The duration of the first stirring is 15-60 min.

81. A liposome vaccine, characterized in that include: The liposome carrier according to any one of claims 1 to 30, or the liposome carrier prepared by the method according to any one of claims 31 to 80; and Vaccine active ingredients; Wherein, the vaccine active ingredients include neoantigen vaccine.

82. The liposome vaccine of claim 81, wherein The active ingredient of the vaccine is a polypeptide vaccine or a nucleic acid vaccine.

83. The liposome vaccine of claim 82, wherein The active ingredient of the vaccine is a polypeptide vaccine.

84. The liposome vaccine of claim 83, wherein The polypeptide vaccine comprises peptides of 11-21 amino acids.

85. The liposome vaccine of claim 83, wherein The polypeptide vaccine comprises bacterial surface antigen peptides or viral surface antigen peptides.

86. The liposome vaccine of claim 82, wherein The active ingredient of the vaccine is a nucleic acid vaccine.

87. The liposome vaccine of claim 86, wherein The nucleic acid vaccine includes siRNA, mRNA, or circular RNA.

88. The liposome vaccine of claim 86, wherein The nucleic acid vaccine is mRNA.

89. The liposome vaccine of claim 81, wherein The neoantigen vaccine includes a neoantigen nucleic acid vaccine.

90. The liposome vaccine of claim 81, wherein The neoantigen vaccine includes a neoantigen polypeptide vaccine.

91. The liposome vaccine of claim 89, wherein The neoantigen nucleic acid vaccine includes the RNA shown in SEQ ID NO:

1.

92. The liposome vaccine of claim 90, wherein The neoantigen polypeptide vaccine comprises a polypeptide as shown in SEQ ID NO:2 or SEQ ID NO:

3.

93. The liposome vaccine of claim 81, wherein The particle size of the liposome vaccine is 20-300 nm.

94. The liposome vaccine of claim 81, wherein The liposome vaccine is spherical in shape.

95. The liposome vaccine of claim 81, wherein The liposome vaccine has a positive charge of 30–40 mV.

96. A method for preparing a liposome vaccine, characterized in that: include: mix The liposome carrier according to any one of claims 1 to 30, or the liposome carrier prepared by the method according to any one of claims 31 to 80; and Vaccine active ingredients; Wherein, the vaccine active ingredients include neoantigen vaccine.

97. The method for preparing a liposome vaccine according to claim 96, wherein: The active ingredient of the vaccine is a polypeptide vaccine or a nucleic acid vaccine.

98. The method for preparing a liposome vaccine according to claim 97, wherein: The active ingredient of the vaccine is a polypeptide vaccine.

99. The method for preparing a liposome vaccine according to claim 98, wherein: The polypeptide vaccine comprises peptides of 11-21 amino acids.

100. The method for preparing a liposome vaccine according to claim 98, wherein: The polypeptide vaccine comprises bacterial surface antigen peptides or viral surface antigen peptides.

101. The method for preparing a liposome vaccine according to claim 97, wherein: The active ingredient of the vaccine is a nucleic acid vaccine.

102. The method for preparing the liposome vaccine according to claim 101, wherein: The nucleic acid vaccine includes siRNA, mRNA, or circular RNA.

103. The method for preparing the liposome vaccine according to claim 101, characterized in that: The nucleic acid vaccine is mRNA.

104. The method for preparing the liposome vaccine according to claim 96, wherein: The neoantigen vaccine includes a neoantigen nucleic acid vaccine.

105. The method for preparing the liposome vaccine according to claim 96, wherein: The neoantigen vaccine includes a neoantigen polypeptide vaccine.

106. The method for preparing the liposome vaccine according to claim 104, wherein: The neoantigen nucleic acid vaccine includes the RNA shown in SEQ ID NO:

1.

107. The method for preparing the liposome vaccine according to claim 104, characterized in that: The neoantigen polypeptide vaccine comprises a polypeptide as shown in SEQ ID NO:2 or SEQ ID NO:

3.

108. The method for preparing the liposome vaccine according to claim 96, wherein: The particle size of the liposome vaccine is 20-300 nm.

109. The method for preparing the liposome vaccine according to claim 96, wherein: The liposome vaccine is spherical in shape.

110. The method for preparing the liposome vaccine according to claim 96, wherein: The liposome vaccine has a positive charge of 30–40 mV.

111. The method for preparing a liposome vaccine according to claim 96, wherein: The mixing of the liposome carrier and the vaccine active ingredient comprises mixing a third solution comprising the liposome carrier and a fourth solution comprising the vaccine active ingredient.

112. The method for preparing the liposome vaccine according to claim 111, characterized in that: The fourth solution includes DEPC-treated water or dimethyl sulfoxide, and the vaccine active ingredient.

113. The method for preparing the liposome vaccine according to claim 111, characterized in that: The vaccine active ingredient is a nucleic acid vaccine, and the fourth solution includes DEPC-treated water and the nucleic acid vaccine.

114. The method for preparing the liposome vaccine according to claim 111, characterized in that: The vaccine active ingredient is a polypeptide vaccine, and the fourth solution includes dimethyl sulfoxide and the polypeptide vaccine.

115. The method for preparing the liposome vaccine according to claim 111, characterized in that: The method for preparing the liposome vaccine further comprises a second stirring step of stirring the mixed solution of the third solution and the fourth solution.

116. The method for preparing the liposome vaccine according to claim 115, characterized in that: The duration of the second stirring is 30-120 min.

117. The method for preparing the liposome vaccine according to claim 115, wherein: The method for preparing the liposome vaccine further comprises dialysis after the second stirring.

118. The method for preparing the liposome vaccine according to claim 117, characterized in that: The dialysis is performed through a dialysis bag.

119. The method for preparing a liposome vaccine according to claim 118, wherein: The molecular weight cut-off of the dialysis bag is 1-100 kDa.

120. The method for preparing the liposome vaccine according to claim 117, wherein: The method for preparing the liposome vaccine further comprises filtration after the dialysis.

121. The method for preparing the liposome vaccine according to claim 120, wherein: The filtration is by filtration through a membrane filter.

122. The method for preparing the liposome vaccine according to claim 121, characterized in that: The pore size of the filter membrane is 0.1–1 μm.

123. Use of the liposome vaccine as claimed in any one of claims 81 to 95, or the liposome vaccine prepared by the method as claimed in any one of claims 96 to 122, in the preparation of a medicament for treating liver cancer.

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