Preparation and use of liposome carrier for efficient vaccine delivery and liposome vaccine

Abstract

A liposome carrier, a liposome vaccine, preparation methods therefor, and use thereof. 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); 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-phosphorylcholine (DSPC); and a third component, comprising at least one of didodecyldimethylammonium bromide (DDAB) and cholesterol or a modifier thereof. The liposome vaccine comprises the liposome carrier and active ingredients of a vaccine and can stimulate antigen presenting cells to mature, thereby improving the immunotherapeutic effect of the liposome vaccine on tumors.

Description

Preparation and use of liposome carriers for efficient vaccine delivery and liposome vaccines 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 same and a preparation method thereof, as well as uses thereof. Background Art

[0002] Cancer is one of the most lethal diseases worldwide. While traditional surgical treatment, radiotherapy, and chemotherapy play a key role in cancer treatment, they are ineffective in preventing recurrence and metastasis. In addition to surgical treatment, radiotherapy, and chemotherapy, targeted therapy and immunotherapy have become important approaches to improve prognosis for cancer patients. However, 80% of cancer patients do not respond to immune checkpoint monoclonal antibody blockade, and the five-year survival rate is less than 20%.

[0003] In contrast, neoantigen vaccines, as a new generation of personalized, precision immunotherapy, are a cutting-edge research topic in both international and domestic immunotherapy. They effectively inhibit postoperative tumor growth, metastasis, and recurrence, and prolong survival in cancer patients. These vaccines are characterized by their unique presence in tumor tissue cells, not expressed in normal cells, and possess excellent tumor tissue cell specificity and low immune tolerance. Neoantigen peptide vaccines offer advantages such as ease of chemical modification, stability, ease of storage, multi-targeting, low toxicity and side effects, and a robust anti-tumor immune response.

[0004] However, analysis of clinical trial data from both domestic and international trials indicates that neoantigen vaccines remain inefficient in inducing immune responses, and their efficacy as therapeutic vaccines in inhibiting solid tumors remains suboptimal. Antigen presenting cells (APCs) are specialized cells that phagocytose, process, and present antigenic peptides to T cells. They recognize and ingest antigens through specific receptors, process them into peptide fragments that bind to MHC II molecules, and present them on the cell membrane, activating and stimulating the proliferation and differentiation of T lymphocytes, thereby initiating autoimmune responses. They are the most critical lymphocytes in initiating both autoimmune and cellular immune responses. Therefore, improving APC uptake of neoantigens, APC activation, and the regulation of their immune function are important approaches to enhancing antigen presentation efficiency and anti-tumor immune responses.

[0005] However, there is currently a lack of effective neoantigen vaccine delivery vectors in clinical practice, making it impossible to effectively co-deliver neoantigens and adjuvants to the same antigen-presenting cell. Subcutaneous injection of neoantigen vaccines is the main method used in clinical practice, which simply involves mixing neoantigen peptides with adjuvants. This method is unable to effectively deliver the scarce neoantigen peptides and adjuvants simultaneously to the same antigen-presenting cell. Furthermore, vaccines taken up through endocytosis are 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 and poor tumor treatment efficacy.

[0006] Currently, due to their relatively stable physicochemical properties and relatively good biosafety, lipid nanoparticles (LNPs) are widely used in industry as the encapsulation and delivery vehicles of choice for the GMP commercial production of vaccines and other drugs. Liposomes are generally composed of a lipid bilayer, typically containing phospholipids (such as lecithin), cholesterol, and PEGylated lipids. Liposomes are now widely used to deliver a variety of drugs and gene therapies, such as paclitaxel liposomes (Doxil) for the treatment of AIDS-related sarcomas and ovarian cancer. In addition, liposomal vaccines can be used to treat diseases such as Haemophilus influenzae, malaria, and hepatitis B vaccines through intramuscular injection, subcutaneous injection, and intranasal spray. Existing LNP formulations are prepared using 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, sudden drop in blood pressure, rapid breathing and rapid heartbeat. In addition, the liposome carriers developed by Pfizer Pharmaceuticals for the delivery of new coronavirus vaccines mainly stimulate B cells to produce specific antiviral antibodies to prevent viral infection.

[0008] A liposomal formulation for the efficient delivery of 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 a modified substance thereof; in particular, the liposome carrier comprises DOTMA, DDAB, DOPE, and cholesterol or a modified substance thereof.

[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 with 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 a modified product thereof; in particular, the liposome carrier components include DOTMA, DDAB, DOPE, and cholesterol or a modified product thereof.

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

[0013] Provided are 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, and their uses in preparing drugs for treating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG1 shows the synthesis route of the embodiment of the present application, the combination of DOTMA, DOPE, cholesterol, DDAB, cholesterol-CpG-ODN and neoantigen peptide to obtain liposome vaccine, and the liposome vaccine and CD205 + Schematic diagram of dendritic cell (DCs) binding.

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

[0016] FIG3A and FIG3B respectively show the particle size and surface potential diagram of the liposome or liposome vaccine according to the examples of the present application.

[0017] FIG4 shows the cellular uptake rate of the liposome vaccine of the Examples or Comparative Examples of the present application by antigen-presenting cells.

[0018] FIG5 shows the Dil fluorescence intensity of dendritic cells that have taken up the liposome vaccines of the Examples or Comparative Examples of the present application.

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

[0020] Figures 7A, 7B, and 7C show the fluorescence intensity of the liposome vaccines of the examples of the present application, reflecting the uptake rate of the liposome vaccines of these examples. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

[0021] FIG8 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 embodiment or the control group.

[0022] FIG9 shows the fluorescence intensity of tumors in tumor-bearing mice injected subcutaneously, intramuscularly, and intravenously with liposome vaccines.

[0023] FIG10A and FIG10B show the tumor fluorescence intensity and survival rate of tumor-bearing mice injected with the liposome vaccine of the comparative example or the example. DETAILED DESCRIPTION

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

[0025] Terms and Definitions

[0026] To facilitate understanding of the features and benefits of the present invention by those skilled in the art, the following provides a general description and definition of terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions 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] As used herein, the terms "comprise," "include," "have," "contain," or any similar terms are open-ended conjunctions intended to encompass non-exclusive inclusion, indicating that a combination (e.g., a device, composition, method, etc.) includes the listed elements (e.g., units of a device, components of a composition, substantial steps of a method, etc.), but does not exclude other elements. For example, a composition or article containing a plurality of elements is not limited to only those elements listed herein, but may also include other elements that are not explicitly listed but are generally inherent to the composition or article. Unless expressly stated to the contrary, the term "or" refers to an inclusive "or" rather than an exclusive "or." As used herein, the term "consisting essentially of" when used to define compositions and methods means excluding other elements that have any substantial effect on the combination for the purpose being described, but does not exclude other elements that do not substantially affect the basic and novel characteristics of the invention. As used herein, closed conjunctions such as "consisting of" refer to combinations (units, components, substantial steps, etc.) that exclude other elements, but do not mean to exclude trace amounts of unavoidable impurities unless otherwise specified. Embodiments defined by each of these transitional terms are within the scope of the present invention. Disclosure of a technical solution including the terms "comprising," "including," "having," "containing," or any other similar terms as specific embodiments thereof shall also be deemed to simultaneously disclose corresponding technical solutions 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 embodiment without ordinal numbers in this application should also be regarded as the disclosure of any further limited embodiment with these ordinal numbers. It should also be understood that the objects defined by the subsequent ordinal numbers in this application do not mean that the same objects defined by the prior ordinal numbers must also be included in the same embodiment.

[0030] Herein, all features or conditions defined in the form of numerical ranges or percentage ranges are intended for simplicity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered to have encompassed and specifically disclosed all possible subranges and individual values ​​within the range, particularly integer values. For example, a range description of "1 to 8" should be considered to have specifically disclosed all subranges such as 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8 ... and so on, particularly subranges defined by all integer values, and should be considered to have specifically disclosed individual values ​​within the range such as 1, 2, 3, 4, 5, 6, 7, 8. Similarly, a range description of "between 1 and 8" should be considered 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, and so on, including their endpoints. Unless otherwise indicated, this interpretation method applies to all contents of the present invention, regardless of whether the scope is extensive or not.

[0031] If a quantity or other 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 or preferred value for that range and any lower or preferred value for that range are specifically disclosed herein, regardless of whether such ranges are disclosed separately. As used herein, the term "about" means approximately, in the vicinity of, or in the vicinity of. When the term "about" is used in conjunction with a numerical value, it modifies the numerical value by adding or subtracting the provided numerical value 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. Generally, the term "about" is used herein to modify a numerical value by 10%, i.e., to include 10% of the numerical value plus or minus 10% of the numerical value of the modified number. 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 considered to be modified by the term "about". In this document, numerical values ​​should be understood to have the accuracy of the number of significant digits of the numerical value, provided that the purpose of the invention can be achieved. For example, the number 40.0 should be understood to cover the range from 39.50 to 40.49.

[0032] As used herein, "parts by weight" refers to parts by weight, which may be any weight unit, such as, but not limited to, kilograms, grams, pounds, etc. As used herein, "parts by mole" refers to parts by mole, which may be a 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 a patient's physiological function.

[0036] As used herein, the term "neo-antigen" refers to an antigen derived from a tumor-specific genomic mutation. For example, a neoantigen 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 neoantigen is a molecule or part of a molecule associated with a therapeutic agent (such as 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 to neoantigens that inhibit function, shorten half-life, or trigger degradation) or cellular (e.g., T cell receptor recognition of peptides derived from neoantigens), 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. Phosphothioate, phosphorodithioate, methylphosphonate, and methylphosphorothioate are stable internucleotide bonds, while phosphodiester is a naturally occurring internucleotide bond. Oligonucleotide phosphorothioate is generally synthesized as a random racemic mixture of phosphorothioate bonds with R and S configurations for the phosphorus atom.

[0038] Implementation Method

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

[0040] In some embodiments, the liposome carrier comprises: a first component comprising or consisting of at least one of 2,3-dioleoyloxypropyl-1-trimethylammonium bromide (DOTMA) or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); a second component comprising or consisting of at least one of 1,2-bis(9Z-oleoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and a third component comprising or consisting of at least one of didodecyldimethylammonium bromide (DDAB) and cholesterol or a modification 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 a modification thereof. 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 a modification thereof. 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, 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 molar parts, particularly 0.25-10 molar parts, more particularly 0.5-10 molar parts, more particularly 1-8 molar parts, and more particularly 1-4 molar parts. In some embodiments, at least one of DOTMA or DOTAP is 0.1-10 molar parts, particularly 0.25-10 molar parts, more particularly 0.5-10 molar parts, more particularly 1-8 molar parts, and more particularly 1-4 molar parts. In some embodiments, the total amount of DOTMA or DOTAP is 0.1-10 molar parts, particularly 0.25-10 molar parts, more particularly 0.5-10 molar parts, more particularly 1-8 molar parts, and more particularly 1-4 molar parts. In some embodiments, DOTMA is 0.1-10 molar parts, particularly 0.25-10 molar parts, more particularly 0.5-10 molar parts, more particularly 1-8 molar parts, and more particularly 1-4 molar parts. In some embodiments, DOTAP is 0.1-10 molar parts, particularly 0.25-10 molar parts, more particularly 0.5-10 molar parts, more particularly 1-8 molar parts, and more particularly 1-4 molar parts. In some embodiments, DOTMA, DOTAP, or the total amount of DOTMA and DOTAP 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, 0.6, 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, 600 , 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.

[0044] In some embodiments, DDAB is 0.1-10 mole parts, particularly 0.1-5 mole parts, particularly 0.1-2 mole parts, particularly 0.25-1.5 mole parts, and particularly 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, 7.5, 8, 8.5, 9, 9.5, or 10 mole parts.

[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 parts by mole, in particular 0.1-5 parts by mole, in particular 0.25-5 parts by mole, in particular 0.25-2 parts by mole, in particular 0.5-1 parts by mole. In some embodiments, at least one of DOPE or DSPC is 0.1-10 parts by mole, in particular 0.1-5 parts by mole, in particular 0.25-5 parts by mole, in particular 0.25-2 parts by mole, in particular 0.5-1 parts by mole. In some embodiments, the total amount of DOPE or DSPC is 0.1-10 parts by mole, in particular 0.1-5 parts by mole, in particular 0.25-5 parts by mole, in particular 0.25-2 parts by mole, in particular 0.5-1 parts by mole. In some embodiments, DOPE is 0.1-10 parts by mole, in particular 0.1-5 parts by mole, in particular 0.25-5 parts by mole, in particular 0.25-2 parts by mole, in particular 0.5-1 parts by mole. In some embodiments, DSPC is 0.1-10 molar parts, particularly 0.1-5 molar parts, particularly 0.25-5 molar parts, particularly 0.25-2 molar parts, and particularly 0.5-1 molar parts. In some embodiments, DOPE, DSPC, or DOPE and DSPC combined 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, 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, particularly 0.1-5 molar parts, particularly 0.1-2 molar parts, particularly 0.1-1 molar parts, and particularly 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 , 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.

[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 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 at least one of DOTMA or DOTAP, at least one of DDAB, DOPE or DSPC, and cholesterol or modifications thereof is (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, the molar ratio of at least one of DOTMA, DDAB, DOPE or DSPC, and cholesterol or a modification thereof is (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 DOTAP, DDAB, DOPE or DSPC, and cholesterol or a modification thereof is (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, DDAB, DOPE, and cholesterol or modifications thereof is (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, 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).

[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 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, 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, 10 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 comprise, consist of, or are composed of cholesterol or cholesterol-CpG oligodeoxynucleotide (CpG-ODN). In some embodiments, the cholesterol or its modifications comprise, consist of, or are composed of cholesterol. In some embodiments, the cholesterol or its modifications comprise, consist of, or are composed of cholesterol-CpG oligodeoxynucleotide. In some embodiments, the cholesterol or its modifications comprise, consist of, or are composed of cholesterol and cholesterol-CpG oligodeoxynucleotide.

[0053] In some embodiments, the internucleotide linkages in the cholesterol-CpG oligodeoxynucleotide comprise modified internucleotide linkages. In some embodiments, the internucleotide linkages in the cholesterol-CpG oligodeoxynucleotide comprise phosphorothioate internucleotide linkages. In some embodiments, the internucleotide linkages in the cholesterol-CpG oligodeoxynucleotide consist of phosphorothioate internucleotide linkages. 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, the sequence set forth in SEQ ID NO:4, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% or greater 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, comprising the liposome carrier. On the one hand, the present application provides a liposome vaccine, comprising 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 comprises or consists of a peptide of less than 10 amino acids. In some embodiments, the polypeptide vaccine comprises or consists of a peptide of 11-21 amino acids. In some embodiments, the polypeptide vaccine comprises a bacterial surface antigen peptide or a viral surface antigen peptide. In some embodiments, the nucleic acid vaccine comprises 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 a polypeptide having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% or greater identity thereto. In some embodiments, the neoantigen polypeptide vaccine comprises a polypeptide as set forth in SEQ ID NO: 3, or a polypeptide having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% or greater identity thereto. In some embodiments, the neoantigen polypeptide vaccine comprises a polypeptide as set forth in SEQ ID NO: 4, or a polypeptide having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% or greater 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 liposome vaccine has a particle size of 20-300 nm. In some embodiments, the liposome vaccine is spherical in shape. In some embodiments, the liposome vaccine has 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 at least one of DDAB, 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 DOTMA, DDAB, at least one of DOPE or DSPC, and cholesterol or modifications thereof. In some embodiments, the liposome carrier components include DOTMA, DDAB, 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 an alcohol, more particularly ethanol, and more particularly anhydrous ethanol.

[0064] In some embodiments, the second solution comprises a polar solvent, particularly water. In some embodiments, the second solution is a buffer. In some embodiments, the second solution has a pK a A buffer solution having a pH between 4.2 and 6.2, particularly between 4.7 and 5.7, more particularly between 4.7 and 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 acetate-acetate buffer solution, more particularly an acetate-sodium acetate buffer solution. In some embodiments, the concentration of the buffer solution is 100-200 mM. In some embodiments, the pH 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 comprises ultrasonic treatment. In some embodiments, the ultrasonic frequency is 40-50 kHz. In some embodiments, the ultrasonic treatment lasts for 1-10 minutes, particularly 3-5 minutes.

[0066] In some embodiments, mixing the first solution and the second solution comprises injecting the first solution into the second solution. In some embodiments, mixing the first solution and the second solution comprises mixing the first solution and the second solution using a microfluidic device. In some embodiments, mixing 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 step, wherein the mixture of the first solution and the second solution is stirred. In some embodiments, the first stirring step is performed by a stirrer, such as a magnetic stirrer. In some embodiments, the stirring rate of the first stirring step is 450-600 rpm / min. In some embodiments, the duration of the first stirring step is 15-60 min, particularly 30 min.

[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, wherein the mixture of the third solution and the fourth solution is stirred. In some embodiments, the second stirring step is performed for a duration of 30-120 minutes.

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

[0072] In some embodiments, the method for preparing a liposome vaccine further comprises filtration after the dialysis. In some embodiments, the filtration is performed through a filter membrane, such as an aqueous filter membrane. In some embodiments, the filter membrane has a pore size of 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 liposome carrier components in a first solvent to obtain a first solution; mixing the first solution and a second solution to obtain a liposome carrier; and mixing the liposome carrier with the vaccine active ingredient.

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

[0075] In one aspect, 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. In one aspect, 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. In one aspect, 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. In one aspect, 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 medicament for treating tumors.

[0076] In one aspect, 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. In one aspect, 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. In one aspect, the present application provides the use of 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, in treating a tumor. In one aspect, the present application provides the use of 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, in preparing a medicament 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 medicament 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 medicament 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 medicament for treating a tumor.

[0080] In some embodiments, the tumor is a mammalian tumor. 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, fibrosarcoma, 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 lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic 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 bile duct cancer. 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, particularly intravenous injection. In some embodiments, the tumor treatment is by injection, particularly intravenous injection. In some embodiments, the tumor treatment drug is an injection, particularly 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 activating the body's anti-tumor immune response and inhibiting tumor growth.

[0088] In some embodiments, the liposome carrier of the present application can efficiently load the neoantigen polypeptide vaccine or 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, in particular, the liposome vaccine comprising the liposome carrier of the present application, is low in toxicity and biodegradable, contains no 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, in particular, the liposome vaccine comprising 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 using conventional equipment.

[0091] Antigen-presenting cells (APCs) are specialized cells that can engulf, process, and present antigenic peptides to T cells, playing a key role in initiating autoimmune responses. In some embodiments, the liposome vaccines of the present application, particularly those comprising the liposome carriers of the present application, can significantly enhance the anti-tumor immune response of the vaccine by generating 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 should not be considered as limiting the scope of protection of the present invention. Any non-essential improvements or adjustments made by those skilled in the art based on the contents of the present invention should be considered as falling within the scope of protection 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-Bis(9Z-oleoyl)-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from Xi'an Ruixi Biotechnology Co., Ltd.;

[0099] -1,2-Distearoyl-sn-glycero-3-phosphocholine (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] -Didodecyldimethylammonium bromide (DDAB) was purchased from Beijing Bailingwei Technology Co., Ltd.;

[0102] -Cholesterol-CpG-ODN bases are 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 vaccines

[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 assay, cells can locally produce cytokines after being stimulated by antigens. These cytokines are 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 ELISpot analysis system counts the number of spots on the well plate to obtain the quantitative results of the Elispot assay.

[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 expressed Hepa 1-6 neoantigen 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; and 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 stock 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. The stock solutions were evenly mixed by ultrasonic treatment for 3–5 min 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 vigorously stirred for 30 minutes using a magnetic stirrer (450-600 rpm / min) to preliminarily obtain the liposome carrier solution of Example 1.

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

[0119] Figure 1 shows a schematic diagram of the synthesis route of Example 1 of the present application, in which DOTMA, DOPE, cholesterol, DDAB, cholesterol-CpG-ODN, and neoantigen polypeptide / mRNA are combined to obtain a liposome vaccine, as well as the binding of the liposome vaccine to CD205+ 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. The 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 stock solution, 1 part of DOPE stock solution, and 0.25 parts of cholesterol stock solution containing cholesterol-CpG-ODN were respectively pipetted into an Eppendorf tube and mixed. The stock solutions were ultrasonically treated for 3-5 minutes to mix them evenly, thereby obtaining 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. The mother solutions were ultrasonically treated for 3-5 minutes to mix them evenly, thereby obtaining 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. The stock solutions were ultrasonically treated for 3–5 minutes to mix them evenly 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 ultrasonically treated for 3-5 minutes to mix them evenly, thereby obtaining 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. The 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. The 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. The stock solutions were evenly mixed by ultrasonic treatment for 3–5 minutes 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. The 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. The 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. The 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. The 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. The 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] Example 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. The 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. The 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. The 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 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 (100 μM, the same below) were respectively pipetted into an Eppendorf tube and mixed. The stock 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] Example 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 an Eppendorf tube and mixed. The 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] Example 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. The 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] Example 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. The 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 mother solution (100 mg / ml, the same below), 0.5 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 containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into Eppendorf tubes and mixed. The mother 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] Example 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. The 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] Example 18

[0187] 2 parts of DOTMA mother solution (100 mg / ml, the same below), 0.5 parts of DDAB mother solution (50 mg / ml, the same below), 0.5 parts of DOPE mother solution (15 mg / ml, the same below), and 0.25 parts of cholesterol mother solution containing cholesterol-CpG-ODN (100 μM, the same below) were respectively pipetted into Eppendorf tubes and mixed. The mother 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 ultrasonicated for 3-5 minutes to uniformly mix the mother solutions 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 vigorously stirred for 30 minutes using a magnetic stirrer (450-600 rpm / min) to preliminarily obtain the liposome carrier solution of Comparative Example 1.

[0192] Next, DEPC-treated water containing the neoantigen polypeptide vaccine or neoantigen mRNA vaccine was added to the liposome carrier solution from Comparative Example 1 and stirred for 0.5–2 hours. The solution was dialyzed using a 10 kDa dialysis bag to remove ethanol and sodium acetate, and then filtered through a 0.22 μm aqueous filter membrane to obtain the liposome vaccine from Comparative Example 1.

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

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

[0195] 3. Characterization of Liposomal Vaccines

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

[0197] The liposomes or liposomal vaccines of Example 1 of the present application were characterized by dynamic light scattering technology, and the results are shown in Figures 3A and 3B. Figures 3A and 3B respectively show the particle size and surface potential of the liposomes or liposomal vaccines of Example 1 of the present application. Figures 3A and 3B show that the liposomes or liposomal vaccines of Example 1 of the present application are uniformly distributed, with a particle size range of 20-300 nm and a positive charge of 30-40 mV.

[0198] Comprehensive cryo-electron microscopy and dynamic light scattering results show that the present application successfully synthesized a spherical, uniformly distributed, positively charged, and stable liposome vaccine with 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 a rate of 5×10 5 The density of each well was inoculated in a 24-well plate and incubated for 12 h with the liposome vaccine of Example 1 or Comparative Example 1 or 2, respectively, and an equal volume of PBS buffer was used as the control group. The original culture medium was discarded and washed 3 times with PBS. Flow cytometry (BD) was used to quantitatively analyze 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 uptake by antigen-presenting cells). As shown in Figure 4, compared with Comparative Example 2, the liposome delivery vehicle of Example 1 efficiently delivers the neoantigen polypeptides and the adjuvant CHO-CpG-ODN to the same antigen-presenting cells, and the ratio of double-positive antigen-presenting cells is about 60 times higher than that of Comparative Example 2.

[0201] Liposome vaccines according to Example 1 and Comparative Example 1 were prepared and labeled with a Dil membrane fluorescent dye. Flow cytometry was used to quantify and analyze the Dil fluorescence intensity of dendritic cell uptake of the liposome vaccines. As shown in Figure 5 , compared to the liposome vaccine of Comparative Example 1, the liposome vaccine of Example 1 was more efficiently taken up by antigen-presenting cells, with an uptake efficiency approximately 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 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 primarily expressed on myeloid cells such as myelocytes, promyelocytes, metamyelocytes, and non-segmented and segmented neutrophils. It is also expressed on tissue macrophages, monocytes, NK cells, activated T cells, and lymphocytes. CD80 is a transmembrane protein belonging to the B7 family, primarily expressed on dendritic cells. It binds to the T cell surface receptors CD28 and CTLA-4 and participates in T cell activation. 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, and binding to these two receptors can synergistically promote or inhibit T lymphocyte activation.

[0206] As shown in Figures 5-6, the liposome vaccine of Example 1 has a high delivery efficiency, enhances the immune stimulation of CD11c, CD80, and CD86, and promotes the maturation of dendritic cells. It can also be seen that compared with simple mixed neoantigen vaccines or mRNA vaccines, the liposome vaccine of the present application can more effectively 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 BMDCs were seeded at a density of 100 cells / well in 24-well plates and co-treated with the Dir-labeled liposome vaccines of Comparative Examples 1-7 for 12 hours (three replicates per group). After washing with PBS buffer to remove unabsorbed liposomes, the treated BMDCs were resuspended in PBS solution, and the Dir fluorescence of the liposomes in the BMDCs was detected by flow cytometry.

[0210] The results are shown in Figure 7A. Figure 7A shows the fluorescence intensity of the liposome vaccines of the examples of this application, reflecting the proportion of liposome vaccines of these examples that were taken up. As can be seen, the liposome vaccines of Examples 1-7 were all taken up to some extent, and the liposome vaccine of Example 1 was taken up significantly more 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 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 liposome vaccines prepared based on them can effectively stimulate the maturation of antigen-presenting cells, thereby improving the anti-tumor immune response of the liposome vaccine and improving the immunotherapeutic effect of the liposome vaccine on solid tumors; among them, liposome carriers composed of DOTMA, DDAB, DOPE, and cholesterol or its modifications, and 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 immunotherapeutic 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 BMDCs were seeded at a density of 100 cells / well in 24-well plates and treated with the Dir-labeled liposome vaccines of Comparative Examples 1, 1A, 8–12, and 8A–12A for 12 hours (three replicates per group). After washing with PBS buffer to remove unabsorbed liposomes, the treated BMDCs were resuspended in PBS, and the Dir fluorescence of the liposomes within the BMDCs was detected by flow cytometry.

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

[0215] Immature mouse bone marrow-derived dendritic cells (BMDCs) were cultured at a rate of 5×10 5 BMDCs were seeded at a density of 100 cells / well in 24-well plates and co-treated with the Dir-labeled liposome vaccines of Comparative Examples 1 or 13–18 for 12 hours (three replicates per group). After washing with PBS buffer to remove unabsorbed liposomes, the treated BMDCs were resuspended in PBS solution, and the Dir fluorescence of the liposomes in the BMDCs was detected by flow cytometry.

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

[0217] The liposome vaccines of Examples 14, 16, and 18 were taken up in greater amounts.

[0218] According to the results of the above examples, it can be seen that the liposome carrier containing at least one of DOTMA or DOTAP, at least one of DDAB, DOPE or DSPC, and cholesterol or its modifications 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), 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) liposome carriers, the liposome vaccines prepared based on which are particularly capable of efficiently stimulating 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.

[0219] 7. Biosafety of Liposomal Vaccines

[0220] 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) for three injections, with an interval of 4 days.

[0221] On day 7, the mice in the control group and the group treated with the liposome vaccine of Example 1 were euthanized, and their serum was collected to detect serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, albumin, creatinine, urea, triglycerides, and total cholesterol.

[0222] The results are shown in FIG8 . 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 Example 1 has good biosafety and can be used for the treatment of diseases, such as anti-tumor treatment, especially anti-tumor immunotherapy.

[0223] 8. Antitumor effects of liposome vaccines injected in different ways on in situ liver cancer in mice

[0224] 6–8 week old male C57BL / 6 mice were selected and Hepa 1-6-luc mouse liver cancer cells (1×10 6 / mouse) were inoculated into the liver lobe of mice to establish an in situ liver cancer model, and the small animal fluorescence imaging system (PE) was used to verify the successful establishment of the mouse tumor-bearing model.

[0225] Tumor-bearing mice were divided into four groups (7 mice per group) and injected subcutaneously, intramuscularly, and intravenously with 200 μL of the liposome vaccine of Example 1. A control group was injected intravenously with an equal volume of PBS buffer. The injections were performed once every 4 days for a total of 3 times.

[0226] The changes in tumor fluorescence intensity were dynamically monitored in situ using a small animal fluorescence imaging system (PE). Figure 9 shows tumor fluorescence images on days 0 and 28.

[0227] As shown in Figure 9, 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.

[0228] 9. Antitumor Effect and Survival Rate of Intravenous Liposome Vaccine on Orthotopic Hepatocellular Carcinoma in Mice

[0229] 6–8 week old male C57BL / 6 mice were selected and Hepa 1-6-luc mouse liver cancer cells (1×10 6 / mouse) were inoculated into the liver lobe of mice to establish an in situ liver cancer model; the successful establishment of the mouse tumor-bearing model was verified using a small animal fluorescence imaging system (PE).

[0230] Tumor-bearing mice were divided into three groups (7 mice in each group) and injected intravenously 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.

[0231] The changes in tumor fluorescence intensity were dynamically monitored in situ using a small animal fluorescence imaging system (PE). The tumor fluorescence imaging images on day 0 and day 49 are shown in Figure 10A ; the survival rate of mice was recorded as shown in Figure 10B .

[0232] As shown in Figures 10A and 10B , the liposome vaccine after intravenous injection can effectively inhibit the growth of in situ liver cancer and effectively improve the survival rate and survival time of mice compared with Comparative Example 2. This liposome vaccine can be used as a therapeutic vaccine for the treatment of solid tumors and clinical translation applications.

[0233] sequence Note: “*” indicates that the natural phosphate internucleotide linkage is replaced by a phosphorothioate internucleotide linkage.

[0234] The above-mentioned embodiments are only preferred embodiments of the present application and cannot be used to limit the scope of protection of the present application. Any non-substantial changes and replacements made by technicians in this field based on the present application shall fall within the scope of protection required by the present application.

Claims

1. A liposome carrier comprising: 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 The third component includes at least one of didodecyldimethylammonium bromide (DDAB) and cholesterol or a modified substance thereof.

2. A method for preparing a liposome carrier, comprising: 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 The third component includes at least one of DDAB and cholesterol or a modification thereof.

3. The method for preparing a liposome carrier according to claim 2, wherein: The first solvent is an organic solvent, in particular an alcohol, more in particular ethanol, more in particular anhydrous ethanol; and / or The second solution comprises a polar solvent, in particular water; and / or The second solution comprises acetate ions, in particular the second solution is a buffer comprising acetate ions, in particular an acetic acid-acetate buffer, more particularly an acetic acid-sodium acetate buffer; Optionally, the concentration of the buffer is 100–200 mM; Optionally, the pH value of the second solution is 5.0-5.2, in particular 5.2; Optionally, the mixing of the liposome carrier components in the first solvent comprises ultrasonic treatment; Optionally, the frequency of the ultrasonic wave is 40–50 KHz; Optionally, the duration of the ultrasonic treatment is 1-10 min, in particular 3-5 min; Optionally, mixing the first mixed solution and the second solution comprises injecting the first mixed solution into the second solution; Optionally, the mixing of the first mixed solution and the second solution comprises mixing the first mixed solution and the second solution using a microfluidic device; Optionally, the mixing of the first mixed solution and the second solution comprises an ethanol injection method or a microfluidics method; Optionally, the mixing of the first mixed solution and the second solution further comprises a first stirring, in particular stirring by a stirrer, such as a magnetic stirrer; Optionally, the stirring rate of the first stirring is 450-600 rpm / min; Optionally, the first stirring period is 15-60 min, in particular 30 min.

4. The liposome carrier or the method for preparing a liposome carrier according to any one of claims 1 to 3, wherein: The third component includes DDAB; Optionally, the molar ratio of at least one of DOTMA or DOTAP to DDAB is (0.1-10):(0.1-10), particularly (1-8):(0.25-1.5), more particularly (1-4):(0.5-1); Optionally, the first component comprises DOTMA and the third component comprises DDAB; Optionally, the molar ratio of DOTMA to DDAB is (0.1-10):(0.1-10), particularly (1-8):(0.25-1.5), more particularly (1-4):(0.5-1); Optionally, the first component comprises DOTAP and the third component comprises DDAB; Optionally, the molar ratio of DOTAP to DDAB is (0.1-10):(0.1-10), specifically (1-8):(0.25-1.5), more specifically (1-4):(0.5-1).

5. The liposome carrier or the method for preparing a liposome carrier according to any one of claims 1 to 4, wherein: The third component includes cholesterol or a modification thereof; Optionally, the molar ratio of at least one of DOPE or DSPC to cholesterol or its modification is (0.1-10):(0.1-10), particularly (0.25-2):(0.1-1), more particularly (0.5-1):(0.25-0.5); Optionally, the second component comprises DOPE, and the third component comprises cholesterol or a modification thereof; Optionally, the molar ratio of DOPE to cholesterol or its modification is (0.1-10):(0.1-10), particularly (0.25-2):(0.1-1), more particularly (0.5-1):(0.25-0.5); Optionally, the second component comprises DSPC, and the third component comprises cholesterol or a modification thereof; Optionally, the molar ratio of DSPC to cholesterol or its modification is (0.1-10):(0.1-10), particularly (0.25-2):(0.1-1), more particularly (0.5-1):(0.25-0.5).

6. The liposome carrier or the method for preparing a liposome carrier according to any one of claims 1 to 5, wherein: The third component includes DDAB and cholesterol or a modification thereof.

7. The liposome carrier or the method for preparing the liposome carrier according to claim 6, wherein: 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 (0.1-10):(0.1-10):(0.1-10):(0.1-10), particularly (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).

8. The liposome carrier or the method for preparing a liposome carrier according to any one of claims 1 to 7, wherein: The first component includes DOTMA, the second component includes DOPE, and the third component includes DDAB and cholesterol or a modification thereof.

9. The liposome carrier or the method for preparing the liposome carrier according to claim 8, wherein: DOTMA, DDAB, DOPE, and cholesterol or their modifications 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), more particularly (1–4):(0.5–1):(0.5–1):(0.25–0.5).

10. The liposome carrier or the method for preparing a liposome carrier according to any one of claims 1 to 9, wherein: The cholesterol or its modification includes cholesterol-CpG oligodeoxynucleotide, especially the cholesterol or its modification is cholesterol and cholesterol-CpG oligodeoxynucleotide; Optionally, the cholesterol-CpG oligodeoxynucleotide has a sequence as shown in SEQ ID NO: 4; Optionally, the molar ratio of cholesterol to cholesterol-CpG oligodeoxynucleotide is (5-1,000):1, particularly (20-200):1, and more particularly (50-100):

1.

11. A liposome vaccine comprising: A liposome carrier as claimed in any one of claims 1 or 4 to 10, or prepared by the method of any one of claims 2 to 10; and Active ingredients of vaccines.

12. A method for preparing a liposome vaccine, comprising: mix A liposome carrier as claimed in any one of claims 1 or 4 to 10, or prepared by the method of any one of claims 2 to 10; and Active ingredients of vaccines.

13. The method for preparing a liposome vaccine according to claim 12, 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; Optionally, the fourth solution includes DEPC-treated water or dimethyl sulfoxide, and the vaccine active ingredient; Optionally, the vaccine active ingredient is a nucleic acid vaccine, and the fourth solution includes DEPC-treated water and the nucleic acid vaccine; Optionally, the vaccine active ingredient is a polypeptide vaccine, and the fourth solution includes dimethyl sulfoxide and the polypeptide vaccine; Optionally, the method for preparing the liposome vaccine further comprises a second stirring step of stirring a mixture of the third solution and the fourth solution; Optionally, the second stirring time is 30–120 min; Optionally, the method for preparing the liposome vaccine further comprises dialysis after the second stirring, in particular dialysis through a dialysis bag; Optionally, the molecular weight cut-off of the dialysis bag is 1-100 kDa, in particular 10 kDa; Optionally, the method for preparing the liposome vaccine further comprises filtering, in particular filtering through a filter membrane, in particular an aqueous filter membrane; Optionally, the pore size of the filter membrane is 0.1-1 μm, in particular 0.2-0.3 μm, more in particular 0.22 μm.

14. The liposome vaccine or the method for preparing a liposome vaccine according to any one of claims 11 to 13, wherein: The active ingredient of the vaccine is a polypeptide vaccine or a nucleic acid vaccine; Optionally, the vaccine active ingredient is a polypeptide vaccine, in particular, the polypeptide vaccine comprises a bacterial surface antigen peptide or a viral surface antigen peptide, and / or comprises a peptide of 11-21 amino acids, Optionally, the vaccine active ingredient is a nucleic acid vaccine, particularly siRNA, mRNA, or circular RNA, more particularly mRNA; Optionally, the vaccine active ingredient includes a neoantigen vaccine, in particular a neoantigen nucleic acid vaccine or a neoantigen polypeptide vaccine; Optionally, the neoantigen nucleic acid vaccine comprises the RNA shown in SEQ ID NO: 1; Optionally, the neoantigen polypeptide vaccine comprises a polypeptide as shown in SEQ ID NO: 2 or SEQ ID NO: 3; Optionally, the particle size of the liposome vaccine is 20–300 nm; Optionally, the liposome vaccine is spherical in shape; Optionally, the liposome vaccine has a positive charge of 30-40 mV.

15. The liposome carrier according to any one of claims 1 or 4 to 10, the liposome carrier prepared by the method according to any one of claims 2 to 10, the liposome vaccine according to claim 11 or 14, or the liposome vaccine prepared by the method according to any one of claims 12 to 14, for treating tumors; Optionally, the tumor comprises liver cancer, gastric cancer, ovarian cancer, endometrial cancer, cervical cancer, colorectal cancer, lung cancer, soft tissue sarcoma, osteosarcoma, fibrosarcoma, skin cancer, testicular cancer, breast cancer, fibrosarcoma, neuroblastoma, brain cancer, bladder cancer, intestinal cancer, prostate cancer, kidney cancer, pancreatic cancer, pleural mesothelioma, head and neck squamous cell carcinoma, nasopharyngeal cancer, oropharyngeal cancer, or blood cancer; Optionally, the tumor comprises: Liver cancer, such as hepatocellular carcinoma or bile duct cancer; or Colorectal cancer, such as cancer of the cecum, appendix, ascending colon, transverse colon, descending colon, sigmoid colon, rectum, or anus, in particular colorectal adenocarcinoma or colorectal precancerous adenoma.

Images