Preparation and application of solidified self-microemulsifying vaccine

Abstract

The application provides a nanocarrier, which comprises a nanosphere core and a nanosphere shell, wherein the nanosphere core comprises squalene; the nanosphere shell comprises a surfactant; and the nanosphere core and the nanosphere shell are combined through hydrophobic interaction. The nanocarrier prepared by the application can continuously regulate the surface properties, and the vaccine prepared by the nanocarrier has the advantages of high efficiency, low toxicity, convenience in preparation and storage, and the like. The nanocarrier overcomes the defects of low antigen-specific T cell induction efficiency of a traditional antigen carrier.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and in particular, relates to the preparation and application of lipid nanogel for inhalation, more particularly, to a nanocarrier, a vaccine, a method for preparing the nanocarrier, a method for preparing the vaccine, a pharmaceutical composition, and use of the pharmaceutical composition in the preparation of a medicament. BACKGROUND

[0002] Vaccines were originally mainly used to prevent infectious diseases. With the in-depth study of vaccines, vaccines are used to prevent or treat more diseases, such as tumors. Tumor vaccines can be divided into four categories: cell vaccines, polypeptide vaccines, viral vaccines, and nucleic acid vaccines. Tumor vaccines can activate the cellular immunity and humoral immunity of patients by loading different forms of tumor antigens into the body to ultimately achieve the purpose of eliminating tumors. Among them, the polypeptide vaccine is composed of tumor antigen epitopes and adjuvants. The immune dominance of antigen epitopes enables the polypeptide vaccine to activate more focused immune responses. However, most of the current research and development of polypeptide vaccines are still in the preclinical and clinical trial stages. The main challenges faced by polypeptide vaccines are summarized as follows: (1) the vaccine has less backflow in the draining lymph nodes; (2) the antigen-presenting cells have weak uptake of the vaccine; (3) the intracellular antigen cross-presentation is poor, and the selection of antigen epitopes and adjuvants. The above reasons limit the development and application of tumor polypeptide vaccines.

[0003] Most of the tumor polypeptide vaccines in clinical trials at the present stage use Montanide as an adjuvant. It is an antigen carrier with adjuvant function, which can enhance immune responses through prolonging the release time of antigens, recruiting antigen-presenting cells, and lymphocytes, etc. However, according to the clinical data statistics, the use of Montanide is accompanied by a high frequency of side effects, including muscle pain, fever, headache, gastrointestinal disorders, etc. At the same time, the characteristic of Montanide staying in the injection site for a long time induces anergy T cells, thereby weakening the therapeutic effect of the polypeptide tumor vaccine.

[0004] Preclinical studies are also constantly optimizing delivery vehicles that can be used for polypeptide tumor vaccines. Including nanodisks and albumin-binding carriers. Nanodisks are a kind of nanomaterial with a particle size of about 10 nm that can load tumor antigens and adjuvants, and can induce 20-30% antigen-specific T cells in mice. While using Montanide to load the same tumor antigens and adjuvants, only about 2% of antigen-specific T cells are induced. Albumin-binding carriers are a kind of feature that can target lymph nodes based on albumin, and use lipophilic structures to modify antigens and adjuvants to bind to albumin in vivo to enhance the purpose of vaccine lymphatic backflow. By optimizing the lipophilic structure used to modify the antigen and adjuvant, eventually 20-30% of antigen-specific T cells can be induced in mice. However, whether it is nanodisk or albumin, its inherent composition-structure relationship determines that it is difficult to continuously regulate the surface properties by changing the components, which to some extent restricts the delivery efficiency of the polypeptide vaccine and the strength of the immune response that can be activated.

[0005] Therefore, there is an urgent need in the art to design a new polypeptide vaccine delivery system to enhance the anti-tumor effect of the vaccine, thereby improving the possibility of polypeptide vaccine market application. SUMMARY

[0006] The present application is based on the inventors' discovery and understanding of the following facts and problems:

[0007] In order to solve the problems of high side effects and low activation efficiency of antigen-specific T cells of the current delivery vehicle, the present application develops a new polypeptide vaccine delivery system to prepare a highly efficient, low-toxic, easy-to-prepare and store tumor polypeptide vaccine. Based on the solid self-emulsifying drug delivery system, the inventors provide a kind of nanometer particle loaded with tumor antigen and immune adjuvant with continuously adjustable surface properties. In vitro experiments have proved that the vaccine preparation technology can form a stable and uniform delivery vehicle with a particle size of about 20 nm. A highly efficient, easy-to-store tumor polypeptide vaccine carrier is achieved by a simple method.

[0008] In a first aspect of the present application, a nanocarrier is provided. According to embodiments of the present application, the nanocarrier comprises a nanosphere core comprising squalene; and a nanosphere shell comprising a surfactant; the nanosphere core and the nanosphere shell are combined by hydrophobic interaction. According to embodiments of the present application, the nanocarrier delivery system can provide protection for the premature degradation of adjuvants and has good stability. When wrapping or coating antigens, the nanocarrier can also protect the immunogen from being prematurely hydrolyzed by proteases.

[0009] According to embodiments of the present application, the above-mentioned nanocarrier can further comprise at least one of the following additional technical features:

[0010] According to an embodiment of the present application, the surfactant is selected from at least one of polyethylene glycol succinate, Tween 80, propylene glycol monolaurate, polyethylene glycol 15-hydroxystearate, propylene glycol caprylate and other non-ionic surfactants.

[0011] According to an embodiment of the present application, the surfactant is selected from a mixture of polyethylene glycol succinate and Tween 80. In an embodiment of the present application, the selection of polyethylene glycol succinate and Tween 80 as surfactants can enhance tissue penetration and phagocytosis effects.

[0012] According to an embodiment of the present application, the mass ratio of the mixture of polyethylene glycol succinate and Tween 80 is 1:6 to 6:1.

[0013] According to an embodiment of the present application, the mass ratio of squalene to surfactant is 1:3.5-1:10, preferably 1:7. The inventors have found that the ratio of squalene to surfactant has a significant impact on tissue distribution in vivo and immune response. Within the preferred ratio range, the delivery of the carrier under the mass ratio condition significantly improves the accumulation of the vaccine in the draining lymph nodes and induces the generation of about 80% of antigen-specific T cells, cures tumors and prevents the recurrence of tumors.

[0014] In a second aspect of the present application, the present application provides a vaccine. According to an embodiment of the present application, the vaccine comprises the nano-carrier of the first aspect of the present application, an antigen to be loaded and an adjuvant.

[0015] According to an embodiment of the present application, the vaccine can enhance anti-tumor T cell immune response and thus improve the therapeutic effect.

[0016] According to an embodiment of the present application, the above vaccine can further comprise at least one of the following additional technical features:

[0017] According to an embodiment of the present application, the antigen to be loaded is pre-modified by a phospholipid. The inventors have found that the antigen modified by a phospholipid can avoid premature degradation. The antigen modified by a phospholipid has amphiphilic properties and can be inserted into the surface of the nanoparticle to form a stable nanostructure.

[0018] According to an embodiment of the present application, the mass ratio of the antigen to be loaded to the squalene is not higher than 4 μg: 1 mg. According to an embodiment of the present application, the use of the above mass ratio can ensure that the particle size of the nano-carrier is within the range of 20 nm.

[0019] According to an embodiment of the present application, the phospholipid is selected from at least one of DOPE, DMPE, DPPE, DSPE.

[0020] According to an embodiment of the present application, the phospholipid is DOPE.

[0021] According to an embodiment of the present application, the antigen is selected from at least one of E7, TRP1, TRP2, M27, M30.

[0022] According to an embodiment of the present application, the antigen is E7.

[0023] According to an embodiment of the present application, the adjuvant is selected from at least one of cholesterin-modified CpG or poly IC, R837, MPLA and derivatives thereof. The adjuvant described in the present application can enhance immunogenicity, so that immune cells are more likely to recognize and attack pathogens, thereby improving the immune effect of the vaccine.

[0024] According to an embodiment of the present application, the adjuvant is cholesterin-modified CpG. According to an embodiment of the present application, the use of cholesterin-modified CpG adjuvant can induce the body to produce long-term and high-efficiency specific immune response, improve the protection ability of the body, and at the same time reduce the amount of immune substances and the production cost of the vaccine.

[0025] According to an embodiment of the present application, the mass ratio of the antigen to the adjuvant is 1:1.

[0026] In a third aspect of the present application, a method for preparing a nano-carrier is provided. According to an embodiment of the present application, the method comprises: performing first mixing treatment on squalene, a surfactant and a cosolvent; performing second mixing treatment on the first mixing treatment product and mannitol; and performing third mixing treatment on the second mixing treatment product and water, so as to obtain the nano-carrier. According to an embodiment of the present application, compared with the high price and strict production process of other T cell therapies such as CAR-T, TCR-T, etc., the nano-carrier prepared by the method has a T cell production efficiency of up to 80%, and the process is simple and the cost is lower.

[0027] According to an embodiment of the present application, the cosolvent is selected from at least one of ethanol, dimethyl sulfoxide and acetone.

[0028] According to an embodiment of the present application, the cosolvent is ethanol.

[0029] According to an embodiment of the present application, the method for preparing a nano-carrier can further comprise at least one of the following additional technical features:

[0030] According to an embodiment of the present application, the first mixing treatment is performed by: performing fourth mixing treatment on the surfactant and the squalene; and performing fifth mixing treatment on the fourth mixing treatment product and ethanol.

[0031] According to an embodiment of the present application, the fourth mixing process is performed at 50°C for 5-10 minutes. The aforementioned time and temperature can ensure that the surfactant is fully mixed with the squalene.

[0032] According to an embodiment of the present application, after the second mixing process and before the third mixing process, the second mixing process product is further subjected to an evaporation process. According to an embodiment of the present application, the evaporation of ethanol can prevent interference with animal model experiments.

[0033] According to an embodiment of the present application, the evaporation process is performed at 40°C for 4-6 hours. According to an embodiment of the present application, the aforementioned evaporation temperature and time can ensure that the ethanol is completely evaporated without affecting the properties of other substances.

[0034] According to an embodiment of the present application, the mass ratio of the surfactant to the squalene is 1:3.5-1:10. The inventor has found through experiments that a stable carrier with a particle size of about 20 nm can be obtained when the mass ratio of the surfactant to the squalene is in the range of 1:3.5-1:10.

[0035] According to an embodiment of the present application, the mass ratio of the surfactant to the squalene is 7:1. According to an embodiment of the present application, the inventor has found through experiments that a stable nano-carrier with a particle size of about 20 nm can be obtained when the mass ratio of the surfactant to the squalene is 7:1. Figure 2 )。

[0036] According to an embodiment of the present application, the mass-to-volume ratio of the squalene to the ethanol is 1 mg:8 μL.

[0037] According to an embodiment of the present application, in the mixture of the ethanol and the dimethyl sulfoxide, the volume ratio of the ethanol to the dimethyl sulfoxide is not less than 4:1. The inventor has found through experiments that the lower the content of the dimethyl sulfoxide, the smaller the influence on the particle size of the nano-carrier.

[0038] According to an embodiment of the present application, the mass-to-volume ratio of the mannitol to the ethanol is not less than 1.5 mg:1 μL.

[0039] In a fourth aspect of the present application, a method for preparing a vaccine is provided. According to an embodiment of the present application, the method comprises: subjecting an antigen to be loaded, a surfactant, squalene, and a cosolvent to a first mixing process; subjecting the first mixing process product to a second mixing process with mannitol; and subjecting the second mixing process product to a third mixing process with an aqueous solution containing an adjuvant, so as to obtain the vaccine.

[0040] According to an embodiment of the present application, the method for preparing the nano-carrier and the vaccine can further comprise at least one of the following additional technical features:

[0041] According to an embodiment of the present application, the surfactant is selected from at least one of polyethylene glycol succinate, Tween 80, propylene glycol monolaurate, polyethylene glycol 15-hydroxystearate, propylene glycol caprylate and other non-ionic surfactants.

[0042] According to an embodiment of the present application, the surfactant is selected from a mixture of polyethylene glycol succinate and Tween 80.

[0043] According to an embodiment of the present application, the co-solvent is selected from at least one of ethanol, dimethyl sulfoxide, acetone.

[0044] According to an embodiment of the present application, the co-solvent is a mixture of ethanol and dimethyl sulfoxide (DMSO).

[0045] According to an embodiment of the present application, the mannitol has a particle size of 200 microns. According to an embodiment of the present application, the mannitol with a particle size of 200 microns can be well adsorbed on the above-mentioned nano-carriers.

[0046] According to an embodiment of the present application, the adjuvant is selected from at least one of cholesterin-modified CpG or poly IC, R837, MPLA and derivatives thereof. According to an embodiment of the present application, the above-mentioned adjuvant has good curative effect in anti-infection and tumor adjuvant therapy.

[0047] According to an embodiment of the present application, the adjuvant is cholesterin-modified CpG. According to an embodiment of the present application, the cholesterin-modified CpG adjuvant can induce the body to produce long-term and high-efficiency specific immune response, improve the body's protection ability, and at the same time, reduce the amount of immune substances and the production cost of vaccines.

[0048] According to an embodiment of the present application, the mass ratio of the adjuvant to the mannitol is 1 ug:3 mg-1 ug:20 mg, preferably 1 ug:3.6 mg.

[0049] According to an embodiment of the present application, the method for preparing the nano-carrier and the vaccine can further comprise at least one of the following additional technical features:

[0050] According to an embodiment of the present application, the method further comprises dialysis and concentration treatment of the third mixed product to obtain the nano-carrier or the vaccine.

[0051] According to an embodiment of the present application, the dialysis treatment comprises dialysis treatment of the uniformly treated third mixed product in a dialysis buffer.

[0052] According to an embodiment of the present application, the dialysis buffer is selected from at least one of PBS buffer or 0.9% NaCl solution or 10% sucrose solution.

[0053] According to an embodiment of the present application, the pH of the dialysis buffer is 7.2-7.4, and the PBS concentration is 0.01 mol / L.

[0054] According to an embodiment of the present application, the temperature of the dialysis treatment is 4°C. According to an embodiment of the present application, the use of 4°C dialysis can maximize the activity of biological macromolecules.

[0055] According to an embodiment of the present application, the dialysis treatment time is 3-5 hours.

[0056] According to an embodiment of the present application, the method for preparing a vaccine can further include at least one of the following additional technical features:

[0057] According to an embodiment of the present application, the mass ratio of the antigen to be loaded to the squalene is not higher than 4 μg: 1 mg. According to an embodiment of the present application, when the loaded antigen is E7, the mass ratio of the antigen to the squalene is not higher than 4 μg: 1 mg, which can prepare a tumor antigen vaccine of about 20 nm.

[0058] According to an embodiment of the present application, the antigen to be loaded is pre-modified by a phospholipid. The loaded antigen after phospholipid modification has amphiphilicity and can be inserted into the surface of the nanocarrier to form a stable nanostructure.

[0059] According to an embodiment of the present application, the phospholipid is at least one of DOPE, DMPE, DPPE, and DSPE.

[0060] According to an embodiment of the present application, the phospholipid is DOPE.

[0061] According to an embodiment of the present application, the antigen is at least one of E7, TRP1, TRP2, M27, and M30.

[0062] According to an embodiment of the present application, the antigen is E7.

[0063] In a fifth aspect of the present application, a vaccine is provided. According to an embodiment of the present application, the vaccine is obtained by the method of the fourth aspect of the present application. The inventors have found that the vaccine prepared by the method of the present application can significantly activate strong antigen-specific CD8 + T cell responses.

[0064] In a sixth aspect of the present application, a pharmaceutical composition is provided. According to an embodiment of the present application, the pharmaceutical composition includes the vaccine of the fifth aspect of the present application. The inventors have found that the pharmaceutical composition can stimulate its corresponding signaling in vivo after administration, enhance the anti-tumor T cell immune response, and thus improve the therapeutic effect, and has high safety.

[0065] According to an embodiment of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0066] In a seventh aspect of the present application, the present application provides use of the vaccine of the fifth aspect or the pharmaceutical composition of the sixth aspect in the preparation of a medicament for treating or preventing a disease.

[0067] The medicament prepared by the vaccine or the pharmaceutical composition of the present application can continuously regulate its surface properties by changing the components. By adjusting the ratio of surfactants (polyethylene glycol succinate (TPGS) and Tween 80 (TW80)), oil phase (squalene), and cosolvent (dimethyl sulfoxide and ethanol), the delivery efficiency of different types of antigens and adjuvants can be improved, and the intensity of activated immune response can be enhanced.

[0068] According to an embodiment of the present application, the disease includes cancer and infectious disease.

[0069] According to an embodiment of the present application, the cancer includes melanoma, breast cancer, and colon cancer.

[0070] According to an embodiment of the present application, the infectious disease includes tumor caused by human papilloma virus (HPV) and respiratory tract infectious disease caused by coronavirus.

[0071] Additional aspects and advantages of the present application will be given, in part, in the following description, become apparent from the following description, or be understood through practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0072] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which:

[0073] Figure 1 is a preparation flow chart of the polypeptide vaccine according to the present application; wherein, Figure A represents a homogeneous mixture and solidification process; Figure B represents a hydration process;

[0074] Figure 2 is a result chart of the influence of the excipient ratio and the antigen loading mass on the particle size according to Example 1 of the present application;

[0075] Figure 3 is a result chart of the particle size comparison of different preparations according to Example 2, Comparative Example 1 and Comparative Example 2 of the present application;

[0076] Figure 4 is a result chart of the accumulation detection of the SSE-DiR preparation in the draining lymph node according to Example 3 of the present application;

[0077] Figure 5SSE-E7 / CpG activated potent antigen-specific CD8 + T cell response detection results;

[0078] Figure 6 Squalene formulation function impact detection results according to Example 4 of the present application;

[0079] Figure 7 Delivery system adjuvant function verification results according to Example 4 of the present application;

[0080] Figure 8 Three groups of formulations activated antigen-specific CD8 + T cell comparison results according to Example 5 of the present application;

[0081] Figure 9 SSE-E7 / CpG significantly inhibited subcutaneous TC-1 tumor growth results detection graph according to Example 5 of the present application;

[0082] Figure 10 SSE-E7 / CpG treatment effect dose dependence and long-term results graph according to Example 5 of the present application. DETAILED DESCRIPTION

[0083] Embodiments of the present application are described in detail below with reference to several examples illustrated in the accompanying drawings. The embodiments described below are examples and are intended to explain the present application, and should not be understood as limiting the present application.

[0084] Definitions and Explanation

[0085] In addition, the terms "first", "second", etc. are used only for the purpose of description, and should not be understood as indicating or implying relative importance or an indicated number of technical features. Therefore, the features defined as "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise specifically limited.

[0086] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and any values should be understood to be approximate, and the ranges should be understood to include values approximately around the endpoints. For ranges of values, the endpoints of the ranges are included in the ranges, and the endpoints and individual points within the ranges are combinable to form new ranges, which are also included in the ranges.

[0087] In the present disclosure, the "solid self-emulsifying (SSE) drug delivery system" refers to a process of mixing preferred surfactants, oil phase, co-solvents, phospholipid-modified tumor antigens to form a homogeneous mixture, then dispersing the mixture onto a solid carrier, evaporating the co-solvents at high temperature, dissolving the solid mixture obtained after evaporation in water containing adjuvant CpG, and obtaining an isotonic preparation with a particle size of about 20 nm after dialysis, and finally obtaining a preparation with a suitable concentration by concentration. The polypeptide antigen vaccine prepared by the above process is referred to as SSE-tumor antigen / CpG Figure 1 ), wherein Ag (antigen) represents an antigen, DOPE-Ag represents a phospholipid-modified antigen, and Cho-CpG represents a cholesteryl-modified CpG. The polypeptide antigen vaccine prepared in the present disclosure is SSE-E7 / CpG.

[0088] In the present disclosure, the "adjuvant" refers to a non-specific immune enhancer that can enhance the immune response of the body to the antigen or change the type of immune response when injected with the antigen or pre-injected into the body. In the present disclosure, the adjuvant is selected from a cholesteryl-modified CpG.

[0089] In the present disclosure, the adjuvant "CpG" activates antigen-presenting cells (APCs) through the TLR9 signaling pathway, effectively triggers mammalian immune responses, improves the anti-tumor activity of immune checkpoint inhibitors, and can be used as a good immune adjuvant for anti-infection and tumor adjuvant therapy.

[0090] In the present disclosure, the term "peptide" or "polypeptide" refers to a polymer of amino acids regardless of the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptides. The term also does not specify or exclude post-expression modifications of the polypeptide, e.g., glycosylations, acetylations, phosphorylations, lipidations, and the like. Covalent modifications to the polypeptides are expressly included. The definition includes polypeptides having one or more amino acid analogs, including, e.g., non-naturally occurring amino acids, amino acids that are not naturally occurring in related biological systems, modified mammalian system amino acids, and the like, polypeptides having substituted linkages, and other modifications known in the art, both naturally occurring and non-naturally occurring.

[0091] Nanocarrier

[0092] In one aspect of the present disclosure, a nanocarrier is provided. According to an embodiment of the present disclosure, the nanocarrier comprises: a nanosphere core comprising squalene; and a nanosphere shell comprising: a surfactant; the nanosphere core and the nanosphere shell being combined by hydrophobic interaction.

[0093] In the present application, the nanocarrier means a particulate drug system, having a biocompatible structure (i.e. not causing toxic reactions), being easily cleared from the organism, and having a particle size generally between 10 and 100 nanometers. The nanocarrier drug system according to the present application has excellent solubility, bioavailability and targeting, and can improve the efficacy and side effects of the drug.

[0094] A vaccine

[0095] In a second aspect of the present application, a vaccine is provided. According to embodiments of the present application, the vaccine comprises the nanocarrier of the first aspect of the present application, an antigen to be loaded, and an adjuvant.

[0096] According to embodiments of the present application, the antigen to be loaded is previously modified with a phospholipid.

[0097] It is to be noted that the antigen modified with a phospholipid according to the present application is obtained by reacting the thiol group of cysteine with a phospholipid containing a thiol reactive group.

[0098] According to embodiments of the present application, the phospholipid is DOPE.

[0099] According to embodiments of the present application, the antigen is E7.

[0100] It is to be noted that in the above reaction process, the antigen is not limited to E7, and the phospholipid is not limited to DOPE.

[0101] In particular, in order to facilitate understanding, the technical solutions of the present application are explained and described in detail as follows:

[0102] Preparation process of the antigen to be loaded modified with a phospholipid: first, an additional cysteine is added to the N-terminus of HPV 16 E7 (RAHYNIVTF), then 1 mol of E7 polypeptide with N-terminus connected cysteine is added to 1 mol of DOPE-SPDP DMF solution, and the solution is stirred at room temperature for more than 6 hours for sufficient reaction to obtain a light yellow solution. Then 10 times the volume of water is added to the reaction system, and the system is placed in a -80°C refrigerator and a freeze dryer in sequence to finally obtain DOPE-CRAHYNIVTF. In the process of preparing the vaccine, DMSO is used for dissolution.

[0103] Method for preparing a nanocarrier

[0104] In yet another aspect of the present application, a method for preparing a nanocarrier is provided. According to embodiments of the present application, the method comprises: first mixing treatment of squalene, a surfactant, and a cosolvent; second mixing treatment of the first mixing treatment product and mannitol; and third mixing treatment of the second mixing treatment product and water, so as to obtain the nanocarrier.

[0105] According to an embodiment of the present application, the surfactant is selected from a mixture of polyethylene glycol succinate and Tween 80.

[0106] According to an embodiment of the present application, the co-solvent is ethanol.

[0107] According to an embodiment of the present application, the mannitol has a particle size of 200 microns (mannitol M200).

[0108] According to an embodiment of the present application, the first mixing process is performed by: performing a fourth mixing process of the surfactant and the squalene; and performing a fifth mixing process of the fourth mixing process product and the ethanol.

[0109] According to an embodiment of the present application, the fourth mixing process is performed at 50°C for 5-10 minutes.

[0110] According to an embodiment of the present application, the method further comprises, after the second mixing process and before the third mixing process, performing an evaporation process of the second mixing process product.

[0111] According to an embodiment of the present application, the evaporation process is performed at 40°C for 4-6 hours.

[0112] According to an embodiment of the present application, the mass ratio of the surfactant to the squalene is 7:1.

[0113] According to an embodiment of the present application, the mass-to-volume ratio of the squalene to the ethanol is 1 mg:8 μL.

[0114] According to an embodiment of the present application, the mass-to-volume ratio of the mannitol to the ethanol is not less than 1.5 mg:1 μL.

[0115] Vaccine preparation method

[0116] In yet another aspect of the present application, a method for preparing a vaccine is provided. According to an embodiment of the present application, the method comprises: performing a first mixing process of an antigen to be loaded, a surfactant, squalene, and a co-solvent; performing a second mixing process of a first mixing process product and mannitol; and performing a third mixing process of a second mixing process product and an aqueous solution containing an adjuvant, so as to obtain the vaccine.

[0117] According to an embodiment of the present application, the surfactant is selected from a mixture of polyethylene glycol succinate and Tween 80.

[0118] According to an embodiment of the present application, the co-solvent is a mixture of ethanol and dimethyl sulfoxide.

[0119] According to an embodiment of the present application, the mannitol has a particle size of 200 microns (mannitol M200).

[0120] According to the embodiment of the present application, the adjuvant is selected from cholesterol-modified CpG.

[0121] According to the embodiment of the present application, the method further comprises dialysis and concentration treatment of the third mixed processing product, so as to obtain the vaccine.

[0122] According to the embodiment of the present application, the dialysis treatment comprises dialysis treatment of the homogenized third mixed processing product in a dialysis buffer.

[0123] According to the embodiment of the present application, the dialysis buffer is a PBS solution with pH of 7.2-7.4, and the PBS concentration is 0.01 mol / L.

[0124] According to the embodiment of the present application, the temperature of the dialysis treatment is 4℃.

[0125] According to the embodiment of the present application, the time of the dialysis treatment is 3-5 hours.

[0126] According to the embodiment of the present application, the mass ratio of the antigen to be loaded to the squalene is not higher than 4 μg:1 mg.

[0127] According to the embodiment of the present application, the phospholipid is DOPE.

[0128] According to the embodiment of the present application, the antigen is E7.

[0129] It should be noted that the amino acid sequence of the antigen E7 is RAHYNIVTF.

[0130] Specifically, in order to facilitate understanding, the technical solutions of the present application are explained and described in detail as follows:

[0131] The specific preparation process is as follows: taking 5 mice, single administration of 10 μg E7 / 10 μg CpG, and taking the formulation adjuvant ratio (TPGS: TW80: squalene = 5:2:1) as an example. Melt TPGS at 75°C, and use a magnetic stirrer to fully stir the melted TPGS; add 62.5 mg TPGS and 25 mg TW80 to 12.5 mg squalene in sequence, and fully mix at 50°C on the magnetic stirrer for 5 min; transfer to the magnetic stirrer at room temperature, add 100 μl of ethanol and mix for 5 min; slowly add 10 μl of 5 mg / ml E7 DMSO solution, mix at room temperature for 5 min; add the above mixture to 180 mg M200, quickly stir with a weighing spoon to make it evenly dispersed, and transfer the above mixture to a 40°C oven for 4 h; take 10 μl of 5 mg / ml Cho-CpG stock solution, add to 1 ml of water and mix thoroughly; take the mixture from the oven and add the above aqueous solution, and use a vortex shaker to fully dissolve the solid particles to obtain a clear preparation; seal the preparation in a dialysis bag, and dialyze in 2 L of 0.01 mol / L, pH 7.2-7.4 PBS solution at 4°C for 3 h; transfer the preparation after dialysis to an ultrafiltration tube, concentrate at 4°C and 3000g for 20 min. Finally, an isotonic preparation with a particle size of about 20 nm is obtained.

[0132] A vaccine

[0133] The vaccine is prepared by the vaccine preparation method described above.

[0134] In the present application, the term "vaccine" refers to an agent or composition containing an active component that effectively induces a therapeutic degree of immunity against a specific pathogen or disease in a subject. In the present application, the vaccine comprises an effective amount of an adjuvant, which effectively triggers a mammalian immune response and enhances the anti-tumor activity of an immune checkpoint inhibitor.

[0135] A pharmaceutical composition

[0136] In yet another aspect of the present application, a pharmaceutical composition is provided. According to an embodiment of the present application, the pharmaceutical composition comprises the nanocarrier of the first aspect of the present application.

[0137] According to an embodiment of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0138] In the present context, the term "pharmaceutically acceptable" indicates that the pharmaceutical composition is capable of administration to a subject without producing an adverse, physiologically- mediated response in the subject that outweighs the medicinal benefits derived from its administration. For example, "pharmaceutically acceptable excipient" refers to an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and preferably examples of such excipients or diluents include, but are not limited to: water, saline, Ringer's solution, dextrose, mannitol, dextran, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, 5% human serum albumin, nanocarriers, and non-aqueous vehicles such as fixed oils.

[0139] In the present context, the term "pharmaceutically acceptable carrier" can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Specific examples can be one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and combinations thereof. In many cases, isotonic agents will be included, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the pharmaceutical composition. Of course, the pharmaceutically acceptable carrier can further include minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.

[0140] The effective amount of the active ingredient described in the present application can vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art (e.g., through clinical trials) according to various factors. The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, several separate dosages can be administered daily, or the dosage can be proportionally reduced, as required by the exigencies of the therapeutic situation.

[0141] The nanocarriers / vaccines of the present application can be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms. For example, liquid, aerosol, semi-solid, and solid dosage forms, etc., including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, nanocarriers, and suppositories. The typical pharmaceutical composition is in the form of an injection solution or infusion solution. The nanocarriers can be administered by aerosol inhalation, intravenous infusion, injection intramuscularly, or subcutaneous injection.

[0142] Use of the vaccine or pharmaceutical composition in the manufacture of a medicament

[0143] In still another aspect of the present application, the present application provides use of the aforementioned vaccine or pharmaceutical composition in the preparation of a medicament for treating or preventing a disease.

[0144] According to embodiments of the present application, the disease includes cancer and infectious disease. In this regard, the cancer includes melanoma, breast cancer, colon cancer, etc.; the infectious disease includes tumor caused by human papillomavirus (HPV), respiratory tract infectious disease caused by coronavirus, etc.

[0145] The nanocarriers and polypeptide tumor vaccines prepared by the method described in the present application can form nanoparticles loaded with tumor antigens and immunoadjuvants with continuously adjustable surface properties by changing the ratio of surfactants, cosolvents, and oil phase (squalene). In addition, embodiments of the present application have confirmed through in vitro experiments that the vaccine preparation process can form a stable and uniform delivery carrier with a particle size of about 20 nm, and it has been confirmed in a mouse model that the polypeptide vaccine can significantly inhibit the growth of tumors and produce a memory immune in the cured mice.

[0146] The embodiments will be described in detail below. Unless otherwise specified, the techniques or conditions in the embodiments are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the reagents or instruments used are all conventional products that can be obtained commercially.

[0147] In the present application, SSE-DiR refers to a solid self-emulsifying delivery system carrying a fluorescent dye.

[0148] Example 1: Screening of the proportion of excipients and the mass of loaded antigens

[0149] In the process of screening the proportion of excipients, the inventors prepared SSE without loading drugs by adjusting the mass ratio of surfactants and squalene. When adjusting the surfactants, the mass ratio of TPGS:TW80 was always maintained at 1:1. Taking surfactant:squalene = 2:1 as an example, the preparation steps are as follows:

[0150] 1. Prepare a homogeneous mixture. Melt TPGS at 75°C and use a magnetic stirrer to stir the melted TPGS thoroughly. Add 10 mg of TW80 and 10 mg of TPGS to 10 mg of squalene in sequence. Under the condition of 50°C, gently stir for 5 min to obtain a homogeneous mixture. Add 80 μl of ethanol to the mixture, and gently stir for 5 min at room temperature to obtain a homogeneous excipient mixture.

[0151] 2. Solidification. Add the homogeneous mixture obtained in the first step to 140 mg of mannitol M200 and stir thoroughly to make the excipients uniformly dispersed on the surface of the M200 particles. Place in a 40°C oven for 4 h to obtain a dry powder. Dissolve the powder in 1 ml of water and fully dissolve it by vortex oscillation.

[0152] The non-drug-loaded SSEs of squalene = 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 and the non-drug-loaded SSEs of TPGS:TW80:squalene = 5:2:1, 5:2:1.5, 5:2:2, 5:2:2.1, 5:2:2.5, 5:2:3 were prepared in sequence according to the above method.

[0153] The particle size and distribution were analyzed by a laser scattering particle size analyzer, and the results were as follows: Figure 2 A-B shows that the surfactant of squalene mass ratio not less than 3.5:1 can obtain a nano-carrier of about 20 nm.

[0154] The inventors found in experiments that the mass ratio of the loaded antigen and the excipient is related to the antigen, and the ratio corresponding to different antigens is different. Taking E7 as an example, preparations with different antigen / excipient ratios were prepared, and the particle size and distribution were analyzed by a laser scattering particle size analyzer. The mass ratio of the antigen and the excipient was determined according to the results. The preparation process is as follows:

[0155] First, the excipient TPGS:TW80:squalene = 5:2:1 was fixed. Taking 6.3 mg of squalene and 20 μg of E7 as an example, 75°C was used to melt TPGS, and a magnetic stirrer was used to fully stir the melted TPGS; 31.5 mg of TPGS and 12.6 mg of TW80 were sequentially added to 6.3 mg of squalene, and fully mixed on a 50°C magnetic stirrer for 5 min; the magnetic stirrer was transferred to room temperature, 50 μl of ethanol was added and mixed for 5 min; 4 μl of 5 mg / ml E7 DMSO solution was slowly added, and mixed at room temperature for 5 min; the above mixture was added to 100 mg of M200, and a weighing spoon was used to quickly stir to make it evenly dispersed, and the above mixture was transferred to a 40°C oven for 4 h; 4 μl of 5 mg / ml Cho-CpG stock solution was taken and added to 1 ml of water to fully mix; the mixture in the oven was taken out and added to the above aqueous solution, and a vortex oscillator was used to fully dissolve the solid particles to obtain the preparation.

[0156] The remaining ratio preparations were prepared according to the above method, which were 6.3 mg of squalene and 25 μg of E7; 6.3 mg of squalene and 30 μg of E7; 6.3 mg of squalene and 40 μg of E7. The detection results of the particle size analyzer were particle size 20.19 nm and PDI 0.204 (6.3 mg of squalene and 20 μg of E7); particle size 19.69 nm and PDI 0.233 (6.3 mg of squalene and 25 μg of E7); particle size 27.92 nm and PDI 0.523 (6.3 mg of squalene and 30 μg of E7); particle size 106.9 nm and PDI 0.334 (6.3 mg of squalene and 40 μg of E7), as shown inFigure 2 The results show that the mass ratio of the loaded antigen E7 to squalene is not higher than 4 μg E7: 1 mg squalene.

[0157] Example 2: Preparation and morphological characterization of SSE-E7 / CpG

[0158] According to an embodiment of the present application, SSE-E7 / CpG containing 30 μg E7 and 30 μg CpG is prepared, and the specific steps are as follows:

[0159] 1. Preparation of a homogeneous mixture. Melt polyethylene glycol succinate (TPGS) at 75 °C, and use a magnetic stirrer to fully stir the melted TPGS. Add 15.2 mg of Tween 80 (TW80) and 38 mg of TPGS to 7.6 mg of squalene in sequence. Stir gently at 50 °C for 5 min to obtain a homogeneous mixture. Add 60 μl of ethanol to the mixture, and stir gently at room temperature for 5 min to obtain a homogeneous excipient mixture. Add 6 μl of a 5 mg / ml E7 DMSO solution to the obtained excipient mixture, and continue stirring at room temperature for 5 min to obtain a homogeneous mixture of the excipient and the antigen polypeptide.

[0160] 2. Solidification. Add the homogeneous mixture obtained in step 1 to 110 mg of mannitol M200, and fully stir to uniformly disperse the excipient and the antigen polypeptide on the surface of the mannitol M200 particles. Place in a 40 °C oven for 4 h to fully evaporate the ethanol and obtain a dry powder.

[0161] 3. Hydration. Pre-add 6 μl of a Cho-CpG (5 mg / ml) stock solution to 0.6 ml of water, and dissolve the powder obtained in step 2 in water, and vortex to fully dissolve the powder to obtain a preparation.

[0162] 4. Dialysis. Seal the preparation obtained in step 3 in a dialysis bag, and place in 2 L of a 0.01 mol / L, pH 7.2-7.4 PBS solution, and dialyze at 4 °C for 3 h to obtain an isotonic preparation.

[0163] 5. Concentration. Concentrate the isotonic preparation after dialysis obtained in step 4 using an ultrafiltration tube, and the concentration conditions are 4 °C, 3000 g, and 20 min.

[0164] Drop 20 μl of the SSE-E7 / CpG slowly into 1 ml of water stirred, and analyze the particle size and distribution using a laser scattering particle size analyzer. The results show that the particle size is 19.71 nm, and the PDI is 0.147 ( Figure 3 ).

[0165] Comparative Example 1: Preparation and morphological characterization of SE without loading of a drug

[0166] To test the effect of solidification on particle size, the comparative example is self-emulsifying drug delivery system (hereinafter referred to as SE) without solidification. The preparation steps of SE are as follows:

[0167] 1. Preparation of homogeneous mixture. Melt TPGS at 75°C, and use a magnetic stirrer to fully stir the melted TPGS. Add 15.2 mg of TW80 and 38 mg of TPGS to 7.6 mg of squalene in sequence. Stir gently for 5 min at 50°C to obtain a homogeneous mixture. Add 60 μl of ethanol to the mixture, and stir gently for 5 min at room temperature to obtain a homogeneous mixture of excipients.

[0168] Take 20 μl of SE and slowly drop it into 1 ml of water under stirring, and analyze the particle size and distribution by using a laser scattering particle size analyzer. The results show that the particle size is 138.9 nm, and the PDI is 0.189 ( Figure 3 ).

[0169] Comparative Example 2: Preparation and morphological characterization of drug-free SSE

[0170] To test the effect of drug loading on particle size, the comparative example is solidified self-emulsifying drug delivery system (hereinafter referred to as SSE) without drug loading. The preparation steps of SSE are as follows:

[0171] 1. Preparation of homogeneous mixture. Melt TPGS at 75°C, and use a magnetic stirrer to fully stir the melted TPGS. Add 15.2 mg of TW80 and 38 mg of TPGS to 7.6 mg of squalene in sequence. Stir gently for 5 min at 50°C to obtain a homogeneous mixture. Add 60 μl of ethanol to the mixture, and stir gently for 5 min at room temperature to obtain a homogeneous mixture of excipients.

[0172] 2. Solidification. Add the homogeneous mixture obtained in the first step to 110 mg of M200, and fully stir to uniformly disperse the excipients on the surface of the mannitol M200 particles. Place in a 40°C oven for 4 h to fully evaporate the ethanol, and obtain a dry powder. Dissolve an appropriate amount of the powder in 1 ml of water, and fully dissolve by vortexing.

[0173] Analyze the particle size and distribution by using a laser scattering particle size analyzer. The results show that the particle size is 17.25 nm, and the PDI is 0.182 ( Figure 3 ).

[0174] Example 3: Detection of lymph node backflow of SSE injected subcutaneously

[0175] To investigate the lymph node backflow efficiency of SSE, SSE-DiR is prepared in this example to track the accumulation of the preparation in the lymph nodes. The preparation steps of SSE-DiR are as follows:

[0176] Eight groups of test groups were set up to detect the effect of the preparation on lymph node backflow, which were (TPGS:TW80:squalene = 0:7:1; 1:6:1; 2:5:1; 3:4:1; 4:3:1; 5:2:1; 6:1:1; 7:0:1) respectively.

[0177] Take TPGS:TW80:squalene = 0:7:1 as an example, the preparation process is as follows:

[0178] 1. Prepare a homogeneous mixture. Melt TPGS at 75°C, and use a magnetic stirrer to stir the melted TPGS thoroughly. Add 44.1 mg of TW80 and 0 mg of TPGS to 6.3 mg of squalene in sequence. Under the condition of 50°C, gently stir for 5 min to obtain a homogeneous mixture. Add 50 μl of ethanol solution to the mixture, and gently stir at room temperature for 5 min to obtain a homogeneous mixture of excipients.

[0179] 2. Solidification. Add the homogeneous mixture obtained in the first step to 100 mg of M200 and mix thoroughly to evenly disperse the components on the surface of the M200 particles. Place in a 40°C oven for 4 h to fully evaporate the ethanol and obtain a dry powder.

[0180] 3. Hydration. Add 1 ml of water to the powder and fully dissolve the powder by vortexing.

[0181] 4. Post-application of DiR. Slowly add 100 μl of 0.8 mg / ml DiR ethanol solution to the above solution, and slowly stir at room temperature for 1 h.

[0182] 5. Dialysis. Seal the DiR-applied preparation in a dialysis bag, and place it in 2 L of 0.01 mol / L, pH 7.2-7.4 PBS solution, and dialyze at 4°C for 3 h.

[0183] The rest of the groups only have surfactants TPGS and TW80 adjusted according to the mass ratio, and the rest of the reagents are used in the same amount as the (TPGS:TW80:squalene = 0:7:1) group. The preparation of each group is as shown in Table A. Figure 4 A.

[0184] The effect of the two surfactants in lymph node backflow was verified by detecting the backflow efficiency of SSE-DiR with different surfactant ratios in the draining lymph nodes. Subcutaneous injection of 120 μl of preparation, i.e. 9.6 μg of DiR. After 24 hours, the inguinal lymph nodes, i.e. the draining lymph nodes, were dissected out for imaging. The results are shown in Table B. Figure 4As shown in B and C, the accumulation of DiR in the draining lymph nodes showed a parabolic trend with the increase of the proportion of TPGS. This result showed that the backflow efficiency of SSE-DiR in the draining lymph nodes was related to the proportion of the two surfactants.

[0185] Example 4: SSE-E7 / CpG activated strong antigen-specific CD8 + T cell response

[0186] 1. In order to detect the activation effect of SSE-E7 / CpG on CD8 + T cells, C57 / B6J mice were subcutaneously injected with SSE-E7 / CpG (10 μg of antigen and adjuvant each) every 7 days, and the proportion of antigen-specific CD8 + T cells in the peripheral blood was detected on the 14th day.

[0187] A total of 8 groups of preparations were detected for the activation of CD8 + T cell response, respectively (TPGS:TW80:squalene=0:7:1; 1:6:1; 2:5:1; 3:4:1; 4:3:1; 5:2:1; 6:1:1; 7:0:1).

[0188] Take TPGS:TW80:squalene=0:7:1 as an example, the preparation process is as follows:

[0189] 1) Prepare a homogeneous mixture. Melt TPGS at 75°C, and use a magnetic stirrer to stir the melted TPGS thoroughly. Add 87.5 mg of TW80 and 0 mg of TPGS to 12.5 mg of squalene in sequence. Stir gently at 50°C for 5 min to obtain a homogeneous mixture. Add 100 μl of ethanol to the mixture, and stir gently at room temperature for 5 min to obtain a homogeneous excipient mixture. Add 8 μl of 5 mg / ml E7 DMSO solution to the obtained excipient mixture, and continue to stir at room temperature for 5 min.

[0190] 2) Solidification. Add the homogeneous mixture obtained in the first step to 180 mg of M200, and stir thoroughly to disperse the excipients and antigen polypeptides uniformly on the surface of the M200 particles. Place in a 40°C oven for 4 h to volatilize ethanol and obtain a dry powder.

[0191] 3) Hydration. Add 8 μl of 5 mg / ml Cho-CpG stock solution to 1 ml of water in advance, dissolve the powder in water, and vortex to obtain the preparation. Fourth step, dialysis. Seal the preparation obtained in the third step with a dialysis bag, and put it into 2 L of 0.01 mol / L, pH 7.2-7.4 PBS solution, and dialyze at 4°C for 3 h.

[0192] 4) Concentration. The isotonic preparation after dialysis was concentrated using an ultrafiltration tube. The concentration conditions were 4°C, 3000g, and 20min.

[0193] For the remaining groups, the preparation process only adjusted the amounts of the two surfactants according to the ratio, while keeping the amounts of other components and preparation conditions unchanged. The particle size and distribution were analyzed using a laser scattering particle size analyzer, and the results are as follows: Figure 5 A shows that changes in the surfactant ratio have almost no effect on the particle size of the peptide vaccine.

[0194] Animal experiments showed that SSE-E7 / CpG prepared by using TPGS and TW80 at different mass ratios could effectively activate specific CD8. + T cell response ( Figure 5 B). Furthermore, SSE-E7 / CpG (5:2:1) can induce 77.7% of antigen-specific CD8+. + T cells, while SE-E7 / CpG (5:2:1) induced only 58.1% of antigen-specific CD8 cells. + T cells. This indicates that SSE-E7 / CpG can activate potent antigen-specific CD8+ cells. + T cell response.

[0195] 2. To further demonstrate the high efficacy of SSE-E7 / CpG, the doses of tumor antigen and immune adjuvant were halved. The preparation process remained the same as described above, with adjustments made proportionally based on the dosage. Using the same administration strategy and detection methods, the effects of SSE-E7 / CpG (5 μg each of antigen and adjuvant) on CD8 were investigated. + T cell activation status.

[0196] The results showed that SSE-E7 / CpG (5 μg each of antigen and adjuvant) could still induce highly efficient antigen-specific CD8. + T cells ( Figure 5 C).

[0197] 3. Similarly, CD8 activation is detected by the detection agent. + To investigate the effect of squalene on the efficacy of the formulation, four groups of SSE-E7 / CpG (10 μg each of antigen and adjuvant) with different squalene contents were prepared: TPGS:TW80:squalene = 5:2:0, 5:2:0.5, 5:2:1, and 5:2:2. The preparation procedure was the same as that of the 5:2:1 formulation. C57 / B6J mice in each group were subcutaneously injected with SSE-E7 / CpG containing different squalene contents every 7 days. On day 14, antigen-specific CD8+ levels in peripheral blood were measured. + T cell ratio.

[0198] The results showed that squalene did not affect the CD8 specificity of the formulation against the antigen.+ The level of activation of T cells Figure 6 ).

[0199] 4. To investigate whether the formulation itself has adjuvant effect, SSE-E7 (antigen 10 μg) was prepared in this example. The preparation procedure is as follows:

[0200] 1. Preparation of homogeneous mixture. Melt TPGS at 75°C, and use a magnetic stirrer to stir the melted TPGS thoroughly. Add 15.2 mg of Tween 80 and 38 mg of TPGS to 7.6 mg of squalene successively. Stir the mixture gently at 50°C for 5 min to obtain a homogeneous mixture. Add 60 μl of ethanol to the mixture, and stir the mixture gently at room temperature for 5 min to obtain a homogeneous mixture of excipients. Add 6 μl of 5 mg / ml E7 DMSO solution to the above obtained mixture of excipients, and continue to stir the mixture at room temperature for 5 min.

[0201] 2. Solidification. Add the homogeneous mixture obtained in the first step to 110 mg of M200, and stir the mixture thoroughly to disperse the excipients and the antigen polypeptide evenly on the surface of the M200 particles. Place the mixture in a 40°C oven for 4 h to evaporate ethanol thoroughly, and obtain a dry powder.

[0202] 3. Hydration. Dissolve the powder in 0.6 ml of water, and vortex the mixture to dissolve the powder thoroughly to obtain the formulation. Fourth step, dialysis. Seal the formulation obtained in the third step in a dialysis bag, and place the dialysis bag in 2 L of 0.01 mol / L PBS solution with pH 7.2-7.4, and dialyze the formulation at 4°C for 3 h.

[0203] 4. Concentration. Concentrate the isotonic formulation after dialysis using an ultrafiltration tube, and the concentration conditions are 4°C, 3000 g, and 20 min. Inject SSE-E7 (antigen 10 μg) subcutaneously into C57 / B6J mice every 7 days, and detect the proportion of antigen-specific CD8 + T cells in the peripheral blood on day 14.

[0204] The results show that SSE-E7 (antigen 10 μg) does not induce the production of antigen-specific CD8 + T cells Figure 7 ).

[0205] Example 5: Investigation of the therapeutic effect of SSE-E7 / CpG on TC-1 subcutaneous tumor model

[0206] 1. To compare the difference of CD8+T cell response activated by SSE-E7 / CpG, E7+CpG+Montanide and free E7+CpG, C57 / B6 mice were injected with the three formulations respectively, the administration strategy was once every 7 days, and the administration was repeated twice. The proportion of antigen-specific CD8+T cells in the peripheral blood of mice was detected on day 14. The dose of each group was 10 μg of antigen and adjuvant, and the proportion of excipients in the SSE-E7 / CpG group was (TPGS: TW80: squalene = 5:2:1).

[0207] The preparation process of SSE-E7 / CpG was consistent with Example 4, and the amount of each component was adjusted in proportion to the number of experimental animals.

[0208] E7+CpG+Montanide (10 μg of antigen and adjuvant) was prepared as follows: E7 (E7 solution without phospholipid) and CpG (cholesterol solution without connection) were added to PBS according to the required dose, and the same volume of Montanide was mixed to form an emulsion that was not soluble in water by means of an antigen emulsifying machine.

[0209] Free E7+CpG (10 μg of antigen and adjuvant) was prepared as follows: E7 (E7 solution without phospholipid) and CpG (cholesterol solution without connection) were added to PBS according to the required dose.

[0210] The results showed that SSE-E7 / CpG (10 μg of antigen and adjuvant) could induce 83.23 ± 4.66% of antigen-specific CD8+T cells, while E7+CpG+Montanide and free E7+CpG could only induce 3.52 ± 1.96% and 5.40 ± 0.77% of antigen-specific CD8+T cells respectively. Figure 8 ).

[0211] 2. To investigate the anti-tumor effect of SSE-E7 / CpG, the therapeutic effect of SSE-E7 / CpG (10 μg of antigen and adjuvant) was evaluated using a TC-1 subcutaneous tumor mouse model. C57 / B6 mice were subcutaneously inoculated with 2 x 10 5 TC-1, and subcutaneous administration was performed on the 10th day after inoculation, once every 7 days, and the administration was repeated twice. The size of the tumor was tracked every 2-3 days during the period to evaluate the therapeutic effect.

[0212] The preparation process of SSE-E7 / CpG (10 μg of antigen and adjuvant) was consistent with Example 4, and the amount of each component was adjusted in proportion to the number of experimental animals.

[0213] The results show that SSE-E7 / CpG not only significantly inhibits tumor growth, but also causes the subcutaneous tumors of all mice in the experimental group to disappear on day 26 after tumor inoculation. The therapeutic effect of the E7+CpG+Montanide (10 μg of each of the antigen and adjuvant) and free E7+CpG (10 μg of each of the antigen and adjuvant) groups is significantly weaker than that of SSE-E7 / CpG (10 μg of each of the antigen and adjuvant). Figure 9

[0214] 3. In order to investigate the anti-tumor effect of different doses of SSE-E7 / CpG, the therapeutic effect of SSE-E7 / CpG at 10 μg, 5 μg and 2.5 μg was evaluated using a mouse model of TC-1 subcutaneous tumors. C57 / B6 mice were subcutaneously inoculated with 2 x 10 5 TC-1, and subcutaneous administration was performed on day 10 after inoculation, once every 7 days, for a total of 2 times. The size of the tumor was tracked every 2-3 days during this period, and the therapeutic effect was evaluated based on the size.

[0215] The preparation of SSE-E7 / CpG was prepared at a benchmark of 10 μg, and the preparations for the 5 μg and 2.5 μg groups were obtained by diluting the 10 μg preparation. The preparation process for the 10 μg preparation was consistent with that of Example 4, and the amounts of the components were adjusted in proportion to the number of experimental animals.

[0216] The results show that SSE-E7 / CpG (10 μg of each of the antigen and adjuvant) has the strongest inhibitory effect on tumors, and the subcutaneous tumors of all mice in the 10 μg group disappeared on day 24 after tumor inoculation. The subcutaneous tumors of all mice in the 5 μg group disappeared on day 28 after tumor inoculation. Only one mouse in the 2.5 μg group reached a cure on day 24, and the tumors of the other two mice began to recur on day 24 Figure 10

[0217] 4. In order to investigate the memory immune response, the cured mice in the three groups of different doses received a second tumor inoculation on day 42. The inoculation site was the opposite dorsal side of the first inoculation site, and the amount of TC-1 inoculated was 2 x 10 5 .

[0218] The results show that the cured mice did not exhibit tumor growth after receiving a second TC1 inoculation, indicating that the cured mice produced a memory immune response Figure 10

[0219] ​​​In summary, through the detection results of the tumor polypeptide vaccine process in the preparation form characterization, in vivo lymph node backflow, inhibition of tumor cell growth and other aspects, the results show that the solid self-emulsifying drug delivery system significantly reduces the particle size of the preparation, and the insertion of tumor antigen polypeptide and immune adjuvant has negligible effect on the particle size of the preparation. Further experimental verification results show that the lymph node backflow effect of subcutaneous injection of SSE-tumor antigen / CpG is significantly improved, indicating that SSE-tumor antigen / CpG has excellent tumor inhibition effect.

[0220] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0221] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A vaccine, characterized in that, This includes nanocarriers, antigens to be loaded, and adjuvants; The nanocarrier comprises: a nanosphere core comprising squalene; and a nanosphere shell comprising a surfactant selected from a mixture of polyethylene glycol succinate and Tween 80; the nanosphere core and the nanosphere shell are bonded together by hydrophobic interaction. The antigen to be loaded is pre-modified with phospholipids; the phospholipid is DOPE; the antigen is E7; the adjuvant is cholesterol-modified CpG; the mass ratio of squalene to surfactant is 1:3.5-1:10; the mass ratio of antigen to squalene is no higher than 4 μg: 1 mg; the mass ratio of antigen to adjuvant is 1:1; the mass ratio of the mixture of polyethylene glycol succinate and Tween 80 is 1:6 to 6:

1. The vaccine preparation method includes: subjecting the antigen to be loaded, surfactant, squalene, and cosolvent to a first mixing treatment; subjecting the product of the first mixing treatment to a second mixing treatment with mannitol; and subjecting the product of the second mixing treatment to a third mixing treatment with an aqueous solution containing adjuvant to obtain a solidified self-microemulsion vaccine; wherein the cosolvent is a mixture of ethanol and dimethyl sulfoxide; and the mass ratio of the adjuvant to the mannitol is 1 µg: 3 mg to 1 µg: 20 mg.

2. The vaccine according to claim 1, characterized in that, The mass ratio of squalene to surfactant is 1:

7.

3. The vaccine according to claim 1, characterized in that, In the vaccine preparation method, the co-solvent is a mixture of ethanol and dimethyl sulfoxide, wherein the volume ratio of ethanol to dimethyl sulfoxide is not less than 4:

1.

4. The vaccine according to claim 1, characterized in that, The mass-to-volume ratio of squalene to ethanol is 1 mg: 8 µL.

5. The vaccine according to claim 1, characterized in that, In the vaccine preparation method, the mannitol has a particle size of 200 micrometers.

6. The vaccine according to any one of claims 1-5, characterized in that, The vaccine preparation method further includes: dialysis and concentration of the third mixed product to obtain the vaccine.

7. The vaccine according to claim 6, characterized in that, The dialysis process includes dialysis of a homogenized third mixture in a dialysis buffer.

8. The vaccine according to claim 7, characterized in that, The dialysis buffer is selected from at least one of PBS buffer, 0.9% NaCl solution, or 10% sucrose solution.

9. The vaccine according to claim 8, characterized in that, The dialysis buffer is a PBS solution with a pH of 7.2-7.4; the concentration of the PBS is 0.01 mol / L.

10. The vaccine according to claim 7, characterized in that, The dialysis treatment was performed at a temperature of 4°C.

11. The vaccine according to claim 7, characterized in that, The dialysis treatment lasts for 3 to 5 hours.

12. The vaccine according to claim 1, characterized in that, The mass ratio of the adjuvant to the mannitol is 1 µg: 3.6 mg.

13. A pharmaceutical composition, characterized in that, include: The vaccine according to any one of claims 1-12.

14. The pharmaceutical composition according to claim 13, characterized in that, Further includes: Pharmaceutically acceptable carriers or excipients.

15. Use of the pharmaceutical composition of claim 13 or 14 in the preparation of a medicament for the treatment or prevention of tumors caused by human papillomavirus (HPV).

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