Cholesterol-modified cationic liposome tumor vaccine, preparation method therefor, and use thereof

Abstract

The present invention belongs to the technical field of cancer immunotherapy, and particularly relates to a cholesterol-modified cationic liposome tumor vaccine, a preparation method therefor, and use thereof. In order to solve the problems of poor targeting and strong side effects of TLR agonists in anti-tumor treatment, the present invention provides a cationic liposome prepared from a cholesterol-modified 1V209 molecule, a cationic lipid component, cholesterol, and DSPE-PEG2000, and then the cationic liposome and ovalbumin 5 form the tumor vaccine by electrostatic adsorption. Animal experiments show that the vaccine can induce antigen-specific CD8+ T cells, activate lymphocytes, and generate stronger antigen cross-presentation, more memory T cells, antibodies, and cytokines. Prophylactic inoculation with the vaccine can significantly delay the progression of mouse melanoma and lymphoma and prolong the survival of mice. The combination use of the vaccine and a PD-1 checkpoint inhibitor can further enhance the anti-tumor effect. Therefore, the vaccine is a promising cancer vaccine.

Description

Cholesterolated cationic liposome tumor vaccine and its preparation method and application Technical Field

[0001] The present invention belongs to the technical field of cancer immunotherapy, and in particular relates to a cholesterolated cationic liposome tumor vaccine and a preparation method and application thereof. Background Art

[0002] Vaccines are a valuable and cost-effective immunotherapy for preventing infectious diseases and cancer. Tumor vaccines have attracted widespread attention due to their specific immunity and long-term immune memory. Although protein or peptide subunit vaccines have been rapidly developed due to their ease of manufacture, higher safety, and enhanced quality control, they are rapidly cleared from the body, resulting in poor immunogenicity and transient anti-tumor immune responses. In order to obtain a strong and long-lasting adaptive anti-tumor immune response in tumor vaccines, it is very important to select a suitable adjuvant that can both deliver antigens and enhance immune function. The most commonly used aluminum-based adjuvants cannot produce the expected anti-tumor immune response due to their weak stimulation of cell-mediated immunity. Therefore, in the process of clinical vaccination, it is particularly urgent to explore an effective, safe adjuvant that can simultaneously enhance both humoral and cellular immune responses.

[0003] Dendritic cells (DCs) are the most effective and important antigen presenting cells (APCs), which have the functions of taking up and presenting antigens, activating immune responses, and regulating humoral and cellular immunity. Adjuvants activate APCs and release the natural killing function of cytotoxic T lymphocytes (CTLs), thereby killing tumors or pathogens. Various adjuvants, including Toll-like receptor (TLR) agonists, can activate the innate immune receptors on the surface of APCs, causing APCs to present antigens, release cytokines, and then release them to CD8 + T cells provide co-stimulatory signals. Ideal cancer vaccines should not only form a "depot effect" at the injection site to maintain antigen exposure to DCs, but also deliver the vaccine to draining lymph nodes (LNs) to activate T and B cells, which is crucial for inducing effective and long-term anti-tumor immunity. To achieve this goal, a promising strategy is to apply nanoparticle vaccines, which can protect antigens from degradation, activate dendritic cells, and co-deliver antigens and adjuvants to draining lymph nodes to prime the immune system.

[0004] TLRs have long been considered adjuvants of adaptive immune responses. As key receptors of innate immunity, TLRs activate a variety of immune cells, especially DCs, through the adaptor protein (MyD88, TRAM, TRIF, MAL) pathway. At the same time, the use of TLR agonists as immune adjuvants through nanoparticle delivery systems has been shown to significantly enhance immune responses and significantly improve antigen cross-presentation. However, they have been reported to have considerable toxicity in some cases, which may be due to their widespread dissemination in the body and activation of myeloid cells, leading to cytokine storms. Although nanodelivery systems can reduce the toxicity of TLR agonists to a certain extent, the complexity and low biocompatibility of many nanodelivery systems also limit the clinical application of TLR agonists. Therefore, there is an urgent need for a nanodrug delivery system that can reduce the toxicity of TLR agonists and achieve clinical translation.

[0005] Summary of the Invention

[0006] In order to solve the technical problems of poor targeting and large side effects of Toll-like receptor (TLR) agonists in anti-tumor treatment, the present application provides a cationic liposome, a cationic liposome tumor vaccine and its application in anti-tumor. The cationic liposome tumor vaccine is named 1V209-Cho-Lip + + OVA, which is a cholesterol (Chol)-modified 1V209 molecule (1V209-Cho) with the positive lipid components, cholesterol and 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG 2000 ) prepared into cationic liposomes (1V209-Cho-Lip + ), and then the liposomes were electrostatically adsorbed with ovalbumin (OVA) to prepare 1V209-Cho-Lip + +OVA vaccine.

[0007] To achieve the above application objectives, the technical solutions adopted in this application are as follows:

[0008] In the first aspect, a cholesterol-modified 1V209 cationic liposome (1V209-Cho-Lip + ), which includes a cholesterol (Chol)-modified 1V209 molecule (1V209-Cho), a positive lipid component, cholesterol, and 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG 2000 ); wherein the structural formula of 1V209 is as shown in Formula I,

[0009] The structural formula of 1V209-Cho is shown in Formula II:

[0010] The positive lipid component is selected from at least one of trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA) or 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol).

[0011] Furthermore, the positive lipid component is selected from trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP) or trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA).

[0012] Preferably, the positive lipid component is trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP).

[0013] Among them, the 1V209-Cho-Lip + Positive lipid components, cholesterol, 1V209-Cho and DSPE-PEG 2000 The molar ratio is 60~70:27~37:1~8:1~8.

[0014] Preferably, the positive lipid components, cholesterol, 1V209-Cho and DSPE-PEG 2000 The molar ratio is 61~65:30~35:2~4:2~4.

[0015] More preferably, the positive lipid components, cholesterol, 1V209-Cho and DSPE-PEG 2000 The molar ratio is 62:32:3:3.

[0016] Among them, the 1V209-Cho-Lip + It is composed of 1V209-Cho, positive lipid components, cholesterol and DSPE-PEG 2000 Prepared by rotary evaporation or ethanol injection method.

[0017] Furthermore, the rotary evaporation method comprises the following steps: 2000 Dissolved in chloroform and methanol, formed into a lipid film under rotary evaporation at 37°C, hydrated, and sonicated to form cationic liposomes 1V209-Cho-Lip + .

[0018] The hydration is carried out in PBS at 37° C. for 1 hour.

[0019] Specifically, the ultrasonic treatment was performed in a water bath for 30 minutes, followed by ultrasonic treatment using an 80W probe ultrasonic instrument for 2 minutes, with the ultrasonic frequency being on for 5 seconds and off for 5 seconds.

[0020] Furthermore, the ethanol injection method comprises the following steps: 2000 Dissolve in ethanol and dimethyl sulfoxide, then add dropwise to the aqueous solution at 45-65°C, remove the organic solvent, and the cationic liposome 1V209-Cho-Lip is obtained. + .

[0021] The condition for the dropwise addition of ethanol by injection method is that the temperature is 45 to 55°C.

[0022] The synthesis method of 1V209-Cho comprises the following steps:

[0023] a. Cholesterol, EDCI, and DMAP were dissolved in DCM, and then boc-aminobutyric acid was added. After reacting at room temperature for 24 hours, DCM was added, and the organic layer was extracted, washed, dried, and concentrated to obtain a crude product, which was purified to obtain product 1;

[0024] b. Product 1 was dissolved in DCM, and 10% TFA was added. The mixture was stirred at room temperature, and DCM was added. The organic layer was extracted, washed, dried, and concentrated to obtain product 2.

[0025] c. 1V209, HATU and TEA were dissolved in DMF, and then the product 2 dissolved in DMF was added. After reacting at room temperature for 48 hours, the solvent DMF was removed to obtain a crude product, which was purified to obtain 1V209-Cho.

[0026] Specifically, the product 1 is Cho-boc, and its structural formula is shown in Formula III, and the product 2 is Cho-NH2, and its structural formula is shown in Formula IV;

[0027] Specifically, in step a, the dosage ratio of cholesterol, EDCI and DMAP is 1eq:2eq:0.1eq, the dosage of DCM is 10mL, and the dosage of boc-aminobutyric acid is 1.2eq.

[0028] Wherein, in steps a and b, the cleaning is performed by using saline solution.

[0029] Furthermore, in steps a and b, the drying is performed using anhydrous sodium sulfate.

[0030] Furthermore, in steps a and b, the concentration is vacuum concentration.

[0031] Specifically, in step a, the purification is to purify the crude product by silica gel chromatography using petroleum ether / ethyl acetate.

[0032] Wherein, in step b, the stirring time is 1 hour.

[0033] In step c, the usage ratio of 1V209, HATU and TEA is 1eq:2eq:2eq, and the usage of product 2 is 1.2eq.

[0034] Wherein, in step c, the method for removing the solvent DMF is rotary evaporation.

[0035] Furthermore, in step c, the purification is to purify the product by silica gel column chromatography using dichloromethane / methanol.

[0036] In a second aspect, the present invention provides the above-mentioned 1V209-Cho-Lip + The preparation method comprises the following steps: 1V209-Cho is prepared with a positive lipid component, cholesterol and DSPE-PEG2000 by rotary evaporation or ethanol injection.

[0037] Furthermore, the rotary evaporation method comprises the following steps: 2000 Dissolved in chloroform and methanol, formed into a lipid film under rotary evaporation at 37°C, hydrated, and sonicated to form cationic liposomes 1V209-Cho-Lip + .

[0038] The hydration is carried out in PBS at 37° C. for 1 hour.

[0039] Specifically, the ultrasonic treatment was performed in a water bath for 30 minutes, followed by ultrasonic treatment using an 80W probe ultrasonic instrument for 2 minutes, with the ultrasonic frequency being on for 5 seconds and off for 5 seconds.

[0040] Furthermore, the ethanol injection method comprises the following steps: 2000 Dissolve in ethanol and dimethyl sulfoxide, then add dropwise to the aqueous solution at 45-65°C, remove the organic solvent, and the cationic liposome 1V209-Cho-Lip is obtained. + .

[0041] The condition for the dropwise addition of ethanol by injection method is that the temperature is 45 to 55°C.

[0042] In a third aspect, the present invention provides a cationic liposome tumor vaccine, which comprises the above cationic liposome (1V209-Cho-Lip + ) and tumor-associated antigens.

[0043] Wherein, the tumor-associated antigen is the model antigen ovalbumin (OVA).

[0044] Furthermore, the antigen is combined with cationic liposomes (1V209-Cho-Lip + ) in which the molar ratio of 1V209-Cho is (32-37):(0.8-1.2);

[0045] Preferably, the molar ratio between the antigen and 1V209-Cho in the cationic liposome 1V209-Cho-Lip+ is 34:1.

[0046] More preferably, the present invention provides a cationic liposome tumor vaccine (1V209-Cho-Lip + + OVA), which includes the above cationic liposomes (1V209-Cho-Lip + ) and ovalbumin (OVA).

[0047] The tumor vaccine is the 1V209-Cho-Lip + It is obtained by adding OVA and encapsulating OVA through electrostatic adsorption.

[0048] In a fourth aspect, the present invention provides the cationic liposome tumor vaccine (1V209-Cho-Lip + +OVA) preparation method, comprising the following steps: + and OVA were obtained by electrostatic adsorption loading of OVA.

[0049] Furthermore, 1V209-Cho-Lip + Co-incubate with OVA at 25-37°C to obtain 1V209-Cho-Lip + +OVA.

[0050] In a fifth aspect, the present invention provides uses of the above-mentioned cationic liposomes and cationic liposome tumor vaccines in the preparation of drugs for preventing and / or treating tumors.

[0051] Furthermore, the anti-tumor drug is a lymph node targeting drug.

[0052] The tumor is at least one of melanoma, lymphoma, colorectal cancer, breast cancer, pancreatic ductal carcinoma, liver cancer, gastric cancer, uterine cancer, ovarian cancer, testicular cancer, basal cell carcinoma or lung cancer.

[0053] The anti-tumor drug is in the form of an injection. Preferably, the injection is administered by at least one of intramuscular injection, intravenous injection, intraperitoneal injection, or subcutaneous injection.

[0054] In a sixth aspect, the present invention provides the cationic liposome 1V209-Cho-Lip + Or a combined drug of cationic liposome tumor vaccine and other anti-tumor drugs, which is to administer the above cationic liposomes or cationic liposome tumor vaccine and other anti-tumor drugs separately or simultaneously.

[0055] Wherein, the other anti-tumor drug is an immune checkpoint inhibitor. Preferably, the other anti-tumor drug is at least one of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0056] More preferably, the other anti-tumor drug is a PD-1 inhibitor.

[0057] The tumor is at least one of melanoma, lymphoma, colorectal cancer, breast cancer, pancreatic ductal carcinoma, liver cancer, gastric cancer, uterine cancer, ovarian cancer, testicular cancer, basal cell carcinoma or lung cancer.

[0058] The combination drug is in the form of an injection. Preferably, the injection is administered by at least one of intramuscular injection, intravenous injection, intraperitoneal injection, or subcutaneous injection.

[0059] In a seventh aspect, the present invention further provides an anti-tumor drug, which is prepared using 1V209-Cho in the cationic liposome as the main active ingredient.

[0060] Furthermore, the anti-tumor drug is also added with pharmaceutically acceptable auxiliary ingredients.

[0061] Preferably, the auxiliary component is a filler, a disintegrant, a wetting agent, an antioxidant, a chelating agent, a surfactant, a flavoring agent, a chelating agent, a pH regulator or a pigment.

[0062] Furthermore, the anti-tumor drug is a lymph node targeting drug.

[0063] Specifically, the tumor is melanoma, lymphoma, colorectal cancer, breast cancer, pancreatic ductal carcinoma, liver cancer, gastric cancer, uterine cancer, ovarian cancer, testicular cancer, basal cell carcinoma or lung cancer.

[0064] Specifically, the dosage form of the anti-tumor drug is an injection. Preferably, the injection is administered by at least one of intramuscular injection, intravenous injection, intraperitoneal injection, or subcutaneous injection.

[0065] Beneficial effects: The present invention modifies the TLR7 agonist 1V209 with cholesterol and then prepares it into cationic liposomes 1V209-Cho-Lip +Then, the cationic liposome tumor vaccine 1V209-Cho-Lip was obtained by electrostatic adsorption loading of the model antigen ovalbumin OVA. + +OVA, used to prevent tumor development. Cationic liposome tumor vaccine 1V209-Cho-Lip + +OVA is a nanovaccine that co-delivers a model antigen (OVA) and a cholesterol-modified TLR7 agonist (1V209-Cho). This vaccine can ensure DC maturation by activating TLRs in the same cells that acquire the antigen, thereby achieving CD8 + The best presentation of T cells. Animal experiments showed that 1V209-Cho-Lip + +OVA nanovaccine promoted DC cellular uptake and maturation. Compared with the rapid metabolism of free OVA, 1V209-Cho-Lip + +OVA has a "depot effect", and 1V209-Cho-Lip + +OVA can further promote the transport of ovalbumin OVA to secondary lymphoid organs. + +OVA induced strong antigen-specific CD8 + T cells, induce lymphocyte activation, produce stronger antigen cross-presentation response and more memory T cells, antibodies and cytokines. + +OVA can significantly delay the development of mouse melanoma B16F10-OVA and lymphoma E.G7-OVA transplanted tumors and prolong the survival of mice, and establish a long-term anti-tumor immune response. + + OVA more effectively inhibited tumor progression, and the combination with programmed death receptor-1 (PD-1) checkpoint inhibitors could further enhance the anti-tumor response. + +OVA) is a promising cancer vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1. Example 1 Cationic liposome tumor vaccine 1V209-Cho-Lip + +OVA preparation and characterization results: A) Cholesteryl 1V209 structure; B) Cholesteryl 1V209 cationic lipid 1V209-Cho-Lip + Particle size and electron microscopy; C) cholesterol 1V209 cationic lipid loaded with ovalbumin OVA 1V209-Cho-Lip ++OVA particle size and electron microscopy images.

[0067] Figure 2, Example 2 DCs in vitro uptake experimental results: A) dendritic cell uptake; Example 3 BMDC in vitro activation experimental results: B) after each preparation and DCs co-incubation, the expression of DCs costimulatory factors CD40, CD80, CD86; Example 4 in vitro CD8 + Results of T cell cross-presentation assay: E) CFSE-labeled OT-I CD8 + After T cells were co-cultured with DCs activated by each preparation for 72 h, CFSE-OT-I CD8 + T cell flow cytometry and quantification; F) CFSE-labeled OT-I CD8 + After co-culture of T cells with DCs activated by each preparation for 72 h, activated CD8 + T cell flow cytometry and quantitative images; data are expressed as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

[0068] Figure 3, Example 5 1V209-Cho-Lip + +OVA vaccine in vivo distribution results: A) In vivo fluorescence imaging of mice at specific times after injection; B) Ex vivo imaging and semi-quantitative analysis of inguinal and popliteal lymph node fluorescence 24 hours after injection.

[0069] Figure 4, Example 6 1V209-Cho-Lip + Figure 3: Anti-tumor efficacy of +OVA as a preventive vaccine in the B16F10-OVA tumor model. A) Schematic diagram of the immunization procedure; B) Total IgG, IgG1, IgG2a, and IgG2b titers in mouse serum on day 21 after the first immunization; C) Total IgG, IgG1, IgG2a, and IgG2b titers in mouse serum on day 28 after the first immunization.

[0070] Figure 5, Example 6 1V209-Cho-Lip + +OVA as a preventive vaccine in the B16F10-OVA tumor model. Result graphs: AD) Tumor growth in mice after injection of each formulation in the mouse prevention model; E) Survival of mice after immunization with each formulation; F) Percentage of tumor-free mice after immunization with each formulation.

[0071] Figure 6, Example 7 1V209-Cho-Lip + + OVA immune prevention mechanism study results Figure: A) Activated CD4 + T(CD4 + CD69 + T cells); B) Quantification of effector memory CD4+ T cells (CD3 + CD4 + CD44 + CD62L - ), central memory CD4 + T cells (CD3 + CD4 + CD44 + CD62L + ) and naive CD4 + T cells (CD3 + CD4 + CD44 – CD62L + C) active CD8 + T (CD8 + CD69 + Quantification of effector memory CD8 T cells in inguinal lymph nodes; D) + T cells (CD3 + CD8 + CD44 + CD62L - ), Central Memory CD8 + T cells (CD3 + CD8 + CD44 + CD62L + ) and naive CD8 + T cells (CD3 + CD8 + CD44 - CD62L + ) quantity.

[0072] Figure 7, Example 7 1V209-Cho-Lip + +OVA immune prevention mechanism study results Figure: AD) Stem cell-like CD8 + Quantification of T cells, CD4 + T cells, activated CD4 + T cells and CD8 + The number of T cells; E) B cells in the germinal center (GC) of the inguinal lymph node (GL7 + CD95 + CD19 + ) Representative flow cytometry images and their ratios; F) CD4 + T cells in inguinal lymph nodes Tfh cells (CXCR5 + PD-1 + CD4 +) Representative flow cytometry plots and ratios.

[0073] Figure 8, Example 7 1V209-Cho-Lip + +OVA immune prevention mechanism research results Figure: A) with OVA (257-264) - Representative flow cytometry images of H2Kb tetramer-specific T cell receptors and their ratios; B-C) ​​OVA (257-264) After in vitro stimulation of immune mouse spleen cells, IFN-γ + and Granzyme b + CD8 + Percentage of T cells; D H) Amounts of cytokines IFN-γ, granzyme B, TNF-α, IL-6, and IL-10 produced by splenocytes after 72 h of in vitro stimulation. Data are expressed as mean ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001).

[0074] Figure 9, Example 7 1V209-Cho-Lip + +OVA immune prevention mechanism study results Figure: AC) Secretion of inflammatory cytokines IFN-γ, TNF-α and IL-6 in the serum of mice after immunization; D) CD8 + The specific killing ability of T cells against tumors. Data are expressed as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

[0075] Figure 10, Example 8 1V209-Cho-Lip + +OVA induces long-lasting humoral and cellular immune responses and prevents tumor formation. Result Figures: A) Immunization procedure of mice; B-E) Tumor growth in each group of mice after tumor establishment; F) Proportion of tumor-free mice in each group after tumor establishment; G) Quantification of long-lived memory B cells (BMEM) in the bone marrow; H-I) ELISPOT detection of IFN-γ in the spleen and peripheral blood + cell.

[0076] Figure 11, Example 9 1V209-Cho-Lip + Figure 1: Results of an in vivo OVA immunotherapy experiment: A) Mouse immunization schedule; BG) Tumor growth in each group after tumor establishment; H) Survival of mice in each group. Data are expressed as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

[0077] Figure 12, Example 10 1V209-Cho-Lip +Figure 3: The in vivo preventive and therapeutic effects of the +OVA vaccine in the E.G7-OVA allograft tumor model. A) Mouse immunization schedule; B) Tumor growth in each group after tumor establishment; F) Survival of mice in each group. Data are expressed as mean ± SEM (***p < 0.001).

[0078] Figure 13, Example 10 1V209-Cho-Lip + +OVA vaccine in vivo preventive and therapeutic effects in the E.G7-OVA allograft tumor model: A) Mouse immunization program; BG) Tumor growth in each group of mice after tumor establishment; H) Survival of each group of mice.

[0079] Figure 14. Cholesterolated 1V209 cationic liposome vaccine 1V209-Cho-Lip + +Schematic diagram of the mechanism of action of OVA.

[0080] Figure 15. The therapeutic effect of cholesterol-modified 1V209 cationic liposomes on CT26 colorectal cancer ascites tumors in Example 11: A) Schematic diagram of the treatment procedure of cholesterol-modified 1V209 cationic liposomes in mice with CT26 ascites tumors; B) Statistical graph of tumors and tumor weights in each group of mice after treatment. DETAILED DESCRIPTION

[0081] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clear, the application is further described in detail below in conjunction with the embodiments. Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by ordinary technicians in this field.

[0082] 1V209 is a synthetic small molecule TLR7 agonist with anti-tumor effects but strong systemic immunotoxicity. The present invention creatively chemically links cholesterol to 1V209 to obtain 1V209-Cho, in order to improve its pharmacokinetics and enable it to target lymph nodes; it is manufactured into nano-sized cationic liposomes 1V209-Cho-Lip + Then, 1V209-Cho-Lip was obtained by electrostatic adsorption of ovalbumin OVA. + +OVA. 1V209-Cho-Lip was verified in vivo + +OVA has the ability to target lymph nodes.

[0083] In one embodiment of the present invention, the cationic liposome vaccine 1V209-Cho-Lip is first prepared. + +OVA, which is a cholesterol-modified 1V209 molecule 1V209-Cho with positive lipid components, cholesterol and DSPE-PEG 2000Prepared into cationic liposomes 1V209-Cho-Lip + Then ovalbumin OVA was adsorbed onto the cationic liposome 1V209-Cho-Lip + Surface prepared.

[0084] The positive lipid component is at least one of trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA) or 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol).

[0085] Preferably, the cationic lipid component can be selected from trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP) and trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA).

[0086] In a specific embodiment of the present invention, the cationic lipid component used is trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP).

[0087] Among them, the positive lipid components, cholesterol, 1V209-Cho and DSPE-PEG 2000 The molar ratio between them is 60~70:27~37:1~8:1~8.

[0088] Preferably, the positive lipid components, cholesterol, 1V209-Cho and DSPE-PEG 2000 The molar ratio between them is 61~65:30~35:2~4:2~4.

[0089] In a specific embodiment of the present invention, the positive lipid components, cholesterol, 1V209-Cho and DSPE-PEG 2000 The molar ratio is 62:32:3:3.

[0090] Among them, cationic liposome 1V209-Cho-Lip + , can be prepared using methods commonly used in the art. For example, rotary evaporation or ethanol injection is a common alternative method. The general operation of the rotary evaporation method is: positive lipid components, cholesterol, 1V209-Cho and DSPE-PEG 2000 Dissolved in chloroform and methanol, formed into a lipid film under rotary evaporation at 37°C, hydrated, and sonicated to form cationic liposomes 1V209-Cho-Lip + .

[0091] The general operation of the ethanol injection method is to inject positive lipid components, cholesterol, 1V209-Cho and DSPE-PEG 2000Dissolve in ethanol and dimethyl sulfoxide, then add dropwise to the aqueous solution at 45-65°C, remove the organic solvent, and the cationic liposome 1V209-Cho-Lip is obtained. + .

[0092] In a specific embodiment of the present invention, the cationic liposome vaccine 1V209-Cho-Lip + +OVA is prepared by electrostatic adsorption, and the cationic 1V209-Cho-Lip + Incubate with ovalbumin (OVA) at 25-37°C for 1 hour.

[0093] In a second aspect, in a specific embodiment of the present invention, the present invention evaluates the cationic liposome vaccine 1V209-Cho-Lip + +OVA on the prevention and treatment of mouse melanoma (B16F10-OVA) and mouse lymphoma (EG7-OVA). + +OVA showed good anti-tumor effects in both tumor models. 1V209-Cho-Lip therapy activated antigen-presenting dendritic cells (DCs) in the tumor microenvironment and sentinel lymph nodes (SLN). These activated DCs (mature DCs) can present tumor-associated antigens (TAAs) to T cells, thereby promoting the induction of tumor-specific adaptive immunity. + +OVA immunization promoted antigen-specific humoral and cytotoxic T lymphocyte (CTL) responses, subsequently inhibiting tumor growth and prolonging mouse survival. + +OVA cationic liposome vaccine can induce persistent antigen-specific CD8 + Memory T cell immunity can still prevent tumor formation 6 months after the first immunization. At the same time, in the therapeutic B16F10-OVA melanoma and E.G7-OVA lymphoma models, 1V209-Cho-Lip + +OVA cationic liposome vaccine can effectively inhibit tumor progression when combined with immune checkpoint blockers (ICB). + The mechanism of targeted co-delivery of antigens and TLR7 agonists by +OVA cationic liposomes for cancer immunotherapy is shown in FIG14 .

[0094] In a third aspect, in a specific embodiment of the present invention, the cholesterol-modified 1V209 cationic lipid 1V209-Cho-Lip was evaluated. + The experimental results showed that 1V209-Cho-Lip +It has a good anti-tumor effect in the CT26 subcutaneous tumor model.

[0095] In a fourth aspect, in one embodiment of the present invention, the cationic liposome vaccine 1V209-Cho-Lip + An anti-tumor drug is prepared using 1V209-Cho as the main active ingredient in OVA. This anti-tumor drug has lymph node targeting properties and is typically formulated as an injection. It can be used to prevent and treat melanoma, lymphoma, colorectal cancer, breast cancer, pancreatic ductal carcinoma, liver cancer, gastric cancer, uterine cancer, ovarian cancer, testicular cancer, basal cell carcinoma, or lung cancer.

[0096] Specific examples will be listed below to explain the scheme of the present invention. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0097] The main materials used in the following examples are as follows:

[0098] 1V209 was purchased from Selleck. Mouse B16F10-OVA cells and EG7-OV cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI1640 or DMEM medium (containing 10% fetal bovine serum (Gibco), 100 U mL -1 Penicillin G and 100 U mL -1 The cells were cultured with streptomycin sulfate (STS) and maintained in an incubator at 37°C and 5% CO2. Female C57BL / 6 mice (6–8 weeks old, 18–20 g) were purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China). All animal experiments were performed in accordance with the guidelines evaluated and approved by the Ethics Committee of Sichuan University.

[0099] Among them, 1V209-Cho was prepared by the following synthetic means:

[0100] Step a, cholesterol, EDCI and DMAP were dissolved in 10 mL of DCM in a ratio of 1 eq: 2 eq: 0.1 eq, and then 1.2 eq of boc-aminobutyric acid was added. After reacting at room temperature for 24 hours, 25 mL of DCM was added, and the organic layer was extracted, washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain a crude product, which was purified by silica gel chromatography using petroleum ether / ethyl acetate to obtain product 1;

[0101] Product 1:

[0102] Step b: Product 1 was dissolved in 5 mL of DCM, and 10% TFA was added. The mixture was stirred at room temperature for 1 h, and 25 mL of DCM was added. The organic layer was extracted, washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain product 2.

[0103] Product 2:

[0104] In step c, 1V209, HATU, and TEA were dissolved in 5 mL of DMF in a ratio of 1 eq: 2 eq: 2 eq, and then the product 2 dissolved in DMF was added in an amount of 1.2 eq. After reacting at room temperature for 48 hours, the solvent DMF was removed by rotary evaporation to obtain a crude product, which was purified by silica gel chromatography using dichloromethane / methanol to obtain 1V209-Cho.

[0105] Example 1 Cationic liposome vaccine 1V209-Cho-Lip + Preparation and characterization of OVA

[0106] First, DOTAP, cholesterol, 1V209-Cho and 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](ammonium salt) (DSPE-PEG 2000 ) (molar ratio = 62:32:3:3) was dissolved in chloroform / methanol (v / v = 10:1), and then the organic solvent was evaporated by rotary evaporation at 37°C to form a lipid film. After hydration with PBS at 37°C for 1 hour, it was sonicated intermittently in water for 30 minutes, and then sonicated with an 80W probe sonicator for 120 seconds to form cholesterol-modified 1V209 cationic liposomes (1V209-Cho-Lip + ), then added OVA (OVA to 1V209-Cho molar ratio of 34:1), vibrated at room temperature for 1h, and combined with cationic liposomes by electrostatic adsorption to obtain 1V209-Cho-Lip + +OVA. Blank-Lip was prepared using a similar method + +OVA, but no 1V209-Cho was added.

[0107] The particle size distribution and Zeta potential of the liposomes were characterized by DLS (Malvern Zetasizer Nano ZS), and the morphology of the liposomes was observed by TEM (H-600, Hitachi, Japan). + The particle size of +OVA is about 190nm, which is larger than that of 1V209-Cho-Lip + Meanwhile, transmission electron microscopy (FE-TEM) showed that 1V209-Cho-Lip+ The average particle size of +OVA was about 200 nm, which was consistent with the DLS data. Dynamic light scattering (DLS) and FE-TEM analysis showed that the cholesterol-modified 1V209 cationic liposome vaccine 1V209-Cho-Lip + +OVA has been successfully prepared.

[0108] Example 2 DCs in vitro uptake experiment

[0109] Since DCs are the most powerful type of APCs to activate natural T cells, 1V209-Cho-Lip + + It is crucial for OVA to be effectively internalized by DCs. OVA was labeled with fluorescein isothiocyanate (FITC) to construct a fluorescent 1V209-Cho-Lip + +OVA F , explore 1V209-Cho-Lip + + OVA cellular uptake. Mouse DCs were inoculated into 24-well plates. OVA was then added F , Blank-Lip + +OVA F and 1V209-Cho-Lip + +OVA F (equal amount of OVA-FITC) and incubated with DCs for 4 hours. After incubation, the culture medium was aspirated and washed 3 times with PBS. The cells were then suspended in 200 μL PBS and analyzed by flow cytometry. The experimental results are shown in Figure 2A. F Compared to 1V209-Cho-Lip + +OVA F Group and Blank-Lip + +OVA F In contrast, 1V209-Cho-lip + +OVA F The fluorescence intensity of the group was also significantly higher than that of the Blank-Lip + +OVA F Group. Shows 1V209-Cho-Lip + +OVA F It has advantages in DCs uptake.

[0110] Example 3 BMDC in vitro activation experiment

[0111] Dendritic cells are key APCs required for priming naive T cells and activating T cell-mediated immune responses. However, their ability to activate T cells depends primarily on their maturation state. Dendritic cell maturation is typically accompanied by upregulation of costimulatory molecules such as CD40, CD86, and CD80. Therefore, an in vitro activation experiment of bone marrow-derived dendritic cells (BMDCs) was performed.

[0112] Bone marrow cells were isolated from the femur and tibia of BALB / c mice and cultured for 6 days in complete RPMI-1640 medium supplemented with 20 ng mL -1 GM-CSF, 50 μM β-mercaptoethanol, and 10 ng mL -1 IL-4 produced immature BMDCs. BMDCs were then added to a 24-well cell culture plate (10 6 PBS, OVA, Blank-Lip + +OVA and 1V209-Cho-lip + +OVA (10 μg mL -1 OVA, 5 μg mL -1 1V209-Cho) was used to treat BMDCs for 48 h, and the expression of co-stimulatory molecules (CD80, CD40, and CD86) on BMDCs was detected by flow cytometry to evaluate its immunostimulatory effect. The results are shown in Figures 2B, 2C, and 2D. Compared with the PBS control group, 1V209-Cho-Lip + After incubation with +OVA, the important indicators of DC maturation, CD40 (1.77 times), CD80 (1.38 times) and CD86 (2.20 times) were significantly upregulated. The above data showed that 1V209-Cho-Lip + +OVA is an effective nanovaccine that facilitates the maturation of DCs.

[0113] Example 4 In vitro CD8 + Assay for T cell cross-presentation

[0114] In antigen cross-presentation studies, mouse CD8 + CD8 T cell isolation kit (Stemcell Technologies) was used to purify the CD8 T cells of OT-1 transgenic mice. + T cells were then labeled with CFSE, and the activated DCs (3×10 5 cells) and 3×10 5 CFSE-labeled CD8 +T cells were co-cultured in RPMI medium for 72 h. The cells were collected and incubated with anti-mouse CD3 antibody labeled with Perp-Cy5.5 (Biolegene), anti-mouse CD8 antibody labeled with PE (Biolegene), and anti-mouse CD69 antibody labeled with BV421 (Biolegene) for 30 min. Finally, the cells were washed twice with PBS and analyzed using a NovoCyte flow cytometer. The results are shown in Figures 2E and 2F. + After co-culture with OVA-treated DCs, CD8 + T cell proliferation increased by 2.1 times. + After co-culture with OVA-treated DCs, CD8 + T cell activation was 5.8-fold higher than that of PBS-treated DCs. + +OVA-treated DCs to CD8 + There is little effect on the proliferation of T cells. + T cell activation was also significantly lower than that of 1V209-Cho-Lip + +OVA-treated DC. In summary, 1V209-Cho-Lip + +OVA can significantly enhance the cross-presentation of extracellular antigens by DCs in vitro, which may play an important role in inducing antigen-specific CTL activity.

[0115] Example 5 Distribution in vivo

[0116] An effective vaccine delivery system not only needs to form a reservoir at the injection site, but also needs to have the ability to deliver antigens to the lymph nodes. In order to observe the in vivo distribution of different cationic liposome vaccines after administration, we encapsulated OVA-FITC instead of OVA in different cationic liposomes and injected 50 μg of OVA-FITC into the left leg muscle of C57BL / 6J mice. The distribution of labeled cationic liposomes in the inguinal draining lymph nodes was monitored in real time. In vivo fluorescence imaging was performed using the Lumina III imaging system (PerkinElmer, USA) at 4, 12, and 24 hours (Figure 3A). At the last time point, the mice were killed, and the inguinal and popliteal lymph nodes were removed and fixed for imaging (Figure 3B). 1V209-Cho-Lip + +OVA F The fluorescence intensity of the inguinal lymph nodes of the group was stronger than that of OVA F and Blank-Lip + +OVA F group, and remained at a high level 24 hours after injection, indicating that 1V209-Cho-Lip + +OVAF There was strong migration and retention in the inguinal lymph nodes. + +OVA F Group and Blank-Lip + +OVA F The fluorescence intensity at the injection site of the group was significantly stronger than that of OVA F The results of the Blank-Lip group indicate that cationic liposomes have the ability to form a reservoir at the injection site, consistent with previous reports. + +OVA F The fluorescence intensity of the inguinal lymph nodes in the group was weak. + +OVA F The fluorescence intensity of the inguinal lymph nodes in the group was significantly higher than that in the Blank-Lip group. + +OVA F Group and OVA F Group. At the same time, 1V209-Cho-Lip + +OVA F The fluorescence intensity of the popliteal lymph nodes in the group with 1v209-Cho-Lip was also higher than that in the other groups (Figure 3A). + +OVA F The fluorescence intensity of inguinal lymph nodes and popliteal lymph nodes in the two groups were OVA F In conclusion, 1V209-Cho-Lip + +OVA F Not only does it have a depot effect at the injection site, but it can also deliver antigens to draining lymph nodes.

[0117] Example 6 1V209-Cho-Lip + +OVA as a preventive vaccine in the B16F10-OVA tumor model

[0118] Female C57BL / 6J mice were randomly divided into phosphate buffer saline (PBS) group, OVA group, Blank-Lip group, + +OVA group and 1V209-Cho-lip + In the +OVA group (8 mice per group), 25 μg / mouse OVA and 5 μg / mouse 1V209-Cho were injected intramuscularly on days 0, 14, and 21, respectively. On day 28, 1×10 6 B16F10-OVA cells (Figure 4A). The tumor growth and survival of mice were observed, and the tumor volume was calculated using the formula: volume (cm 3 )=(width)2 × length / 2. Serum was collected on the 21st and 28th day, and the titers of OVA-specific IgG, IgG1, IgG2a, and IgG2b in the serum were determined by ELISA (Figures 4B and 4C). + +OVA and 1V209-Cho-Lip + +OVA intramuscular immunization induced higher serum OVA-specific IgG levels, and 1V209-Cho-Lip + The +OVA group induced the highest level of serum OVA-specific IgG. + +OVA immunization mainly induced Th2-related IgG1, while 1V209-Cho-Lip + +OVA induces IgG1 and Th1-related IgG2a and IgG2b. + Compared with the +OVA group, OVA group and PBS group, 1V209-Cho-Lip + The +OVA group had the best anti-tumor effect and significantly prolonged the survival of mice, and was able to completely regress the tumors in 62.5% of the mice (Figures 5A-D, 5F). + Moderate tumor inhibition was observed in the +OVA group (Figure 5C, 5E). Taken together, these studies indicate that 1V209-Cho-Lip + +OVA can be used as an effective vaccine to prevent the occurrence and development of tumors after inoculation of tumor cells expressing OVA.

[0119] Example 7 1V209-Cho-Lip + +OVA immune prevention mechanism research

[0120] Mice were killed 7 days after the last immunization (n=5) to investigate the effect of 1V209-Cho-Lip ++OVA immune prevention mechanism. Small inguinal lymph nodes were obtained and filtered through a 70μm filter to obtain a single cell suspension, which was then stained with Percp-cy5.5-labeled anti-mouse CD3 antibody (BioLegend), FITC-labeled anti-mouse CD8 antibody (BioLegend), APC-labeled anti-mouse CD4 antibody (BioLegend), PE-labeled anti-mouse CD69 antibody (BioLegend), BV510-labeled anti-mouse CD44 antibody (BioLegend), BV421-labeled anti-mouse CD62L antibody (BioLegend), BV421-labeled anti-mouse PD-1 antibody (BioLegend), and FITC-labeled anti-mouse TCF-1 antibody (BioLegend). As shown in Figures 6A and 6C, 1V209-Cho-Lip + After OVA immunization, activated CD4 + T cells (CD4 + CD69 + T cells) and activated CD8 + T cells (CD8 + CD69 + The percentage of T cells in the three groups was significantly increased compared with the other groups, which is crucial for inducing a significant immune response and achieving immune prevention and protection. Vaccination is an effective means to prevent pathogens from infecting the human body again. Generally speaking, memory T cells produced by the initial infection can quickly trigger a series of immune responses when infected again. In our study, we used FACS to evaluate the CD4 + and CD8 + As shown in Figures 6B and 6D, compared with the other groups, 1V209-Cho-Lip + +OVA significantly increased CD4 + and CD8 + T cells EM (CD44 + CD62L - ) percentage, reduced CD4 + and CD8 + T cells (CD44 - CD62L + ) percentage. Blank-Lip + +OVA only observed CD4 + and CD8 + T cellsT CM (CD44 + CD62L + ) percentage increased, while TEM No significant changes. Tip 1V209-Cho-Lip + +OVA can induce a strong memory T cell response and has a strong protective effect during reinfection. + PD-1 + CD8 + T cells have a stem cell-like phenotype, exert sustained anti-tumor immune responses, and expand more efficiently after ICB treatment. + After OVA immunization, stem-like CD8 + The percentage of T cells increased (Figure 7A), suggesting that it may contribute to the improvement of ICB therapy.

[0121] In addition, the mouse spleen single cell suspension was further treated with APC-labeled anti-mouse CXCR5 antibody (BioLegend), PE-CF 549-labeled anti-mouse CD95 antibody (BioLegend), BV421-labeled anti-mouse CD19 antibody (BioLegend), and FITC-labeled anti-mouse GL-7 antibody (BioLegend). + +OVA immunization significantly increased the number of CD4 + , activated CD4 + T and CD8 + To further explore the proportion of T cells in 1V209-Cho-Lip + We investigated whether 1V209-Cho-Lip could significantly enhance the GC response in mice, generate mature plasma cells and memory B cells, and thus mediate long-term protective immune responses. + B cells and follicular helper T cells (T) in the inguinal lymph nodes and spleen of mice immunized with OVA FH ). 1V209-Cho-Lip + After +OVA immunization, the GC B cells in the draining inguinal lymph nodes of mice were significantly increased (Figure 7E). Corresponding GC T cells were also observed in the draining inguinal lymph nodes. FH Significantly increased (Figure 7F). + After +OVA immunization of mice, the GC response in the peritoneal draining lymph nodes was enhanced compared with that in other groups, thereby promoting the establishment of long-term immune memory.

[0122] In addition, use OVA 257-264 (5 μg mL -1) were stimulated for 3 days and then stained with Perp-Cy5.5-labeled anti-mouse CD3 antibody (BioLegend), BV510-labeled anti-mouse CD8 antibody (BioLegend), PE-labeled anti-mouse SIINFEKL-MHC I antibody (BioLegend), PE-Cy7-labeled anti-mouse IFN-γ antibody (BioLegend), and AF647-labeled anti-mouse granzyme-B antibody (BioLegend). + Mice inoculated with PBS, OVA, and Blank-Lip + Compared with the +OVA group, the 1V209-Cho-Lip + The OVA+OVA mice significantly increased OVA-specific CD8 + In addition, the frequency of T cells was detected using 1V209-Cho-Lip + +OVA immunization not only significantly increased CD8 + In addition to IFN-γ and granzyme B, 1V209-Cho-Lip + + OVA also significantly increased the secretion of TNF-α, IL-6, and IL-10 (Figure 8F-H). In addition, the serum of mice was collected and the serum INF-γ, TNF-α, and IL-6 levels were detected by ELISA. + After mice were immunized with OVA, the secretion of inflammatory factors such as IFN-γ, TNF-α, and IL-6 in the serum increased significantly ( Figure 9A-C ).

[0123] Isolate spleen lymphocytes and add CD8 + Specific OVA 257-264 Peptide (10 μg mL -1 ) for 3 days, and the T cell cytotoxicity was measured. Lymphocytes (effector cells) and CFSE-labeled B16F10-OVA cells (target cells) were incubated for 6 hours at different E:T ratios (100:1, 50:1, 25:1, and 12.5:1). Flow cytometry was used to determine the frequency of B16F10-OVA cells with high CFSE expression, and the percentage of specific cytotoxicity was calculated. 1V209-Cho-Lip + CD8 + T cells showed high tumor-specific cytotoxicity (Figure 9D). The above results indicate that 1V209-Cho-Lip + +OVA nanovaccine can indeed induce a strong T cell immune response in vivo.

[0124] Example 8 1V209-Cho-Lip + +OVA induces long-lasting humoral and cellular immune responses and prevents tumor formation

[0125] Female C57BL / 6J mice were randomly divided into PBS group, OVA group, Blank-Lip group + +OVA group and 1V209-Cho-Lip + +OVA group, 5 mice per group, were injected intramuscularly with 25 μg / mouse OVA and 5 μg / mouse 1V209-Cho on days 0, 14, and 21, respectively. Orbital blood was collected from mice 5 and 6 months after the first injection. The immunization schedule is shown in Figure 10A. 6 months after the first injection, mice were subcutaneously inoculated with 1×10 6 B16F10-OVA cells. Tumor size was measured every 2 days and recorded continuously for 20 days. The tumor growth of mice is shown in Figure 10B-F. + The mice in the +OVA immunization group induced long-lasting immune memory, and almost all mice showed no tumor growth 20 days after tumor establishment (Figure 10E, 10F). + Tumor formation was observed in mice immunized with OVA, but 60% of the mice failed to prevent tumor formation on day 20 (Figures 10D and 10F). In contrast, tumor formation was observed in mice in the PBS and OVA groups 4-8 days after tumor establishment (Figures 10B and 10C).

[0126] Long-lived memory B cells (BMEM) form an important part of immune memory and provide a rapid antibody response for reinfection. The spleen of the above-mentioned mice was taken and filtered through a 70μm filter to obtain a single cell suspension, which was stained with Percp-Cy5.5-labeled anti-mouse CD3 antibody (BioLegend), PE-labeled anti-mouse CD19 antibody (BioLegend), APC-labeled anti-mouse IgD antibody (BioLegend), FITC-labeled anti-mouse CD27 antibody (BioLegend), and BV421-labeled anti-mouse IgM antibody (BioLegend). BMEM in the bone marrow was evaluated by flow cytometry (Figure 10G), 1V209-Cho-Lip + The proportion of BMEM in the +OVA immunized group was significantly higher than that in the other groups.

[0127] ELISPOT detection of CD8 cells secreting IFN-γ +T cells. Briefly, in the ELISPOT assay, immunol 2HB plates were coated with anti-IFN-γ antibodies and then blocked with PBS-BSA. Splenocytes were serially diluted and plated onto pre-coated plates supplemented with SIINFEK. The plates were further incubated for 18 h at 37°C in a 5% CO2 incubator. IFN-γ was detected using biotinylated anti-IFN-γ antibodies, alkaline phosphatase streptavidin, and BCIP dissolved in low-melting-point agarose solution. + CD8 + T cell spots. The analysis results showed that immune 1V209-Cho-Lip + IFN-γ in the spleen and blood of OVA+ mice + CD8 + The number of T cells increased significantly (Figure 10H, 10I). However, no significant tumor-specific CD8 T cells were observed in the spleen or blood of the other three groups of mice. + Memory T cells (Figures 10H, 10I).

[0128] Example 9 In vivo immunotherapy experiment

[0129] On day 0, mice were subcutaneously inoculated with 3×10 5 On the third day after inoculation of B16F10-OV cells, the tumor-bearing mice were randomly divided into 6 groups: PBS group, OVA group, Blank-Lip group + +OVA group, 1V209-Cho-Lip + +OVA group, Anti-PD-1 group and 1V209-Cho-Lip + +OVA+Anti-PD-1 group, 8 mice in each group. Each preparation was injected intramuscularly on the 3rd day after tumor establishment, once every 7 days, for a total of 3 times (25ug / mouse OVA and 5ug / mouse 1V209-Cho). Anti-PD-1 (100μg / mouse) was injected intraperitoneally every 3 days, for a total of 6 times. The treatment procedure is shown in Figure 11A. The tumor growth and survival of mice were observed. The average survival days of mice in the PBS control group were 20 days, and all mice died 26 days after tumor establishment (Figures 11B and 11H). The anti-tumor effects of the OVA group and the anti-PD-1 group were similar to those of the PBS group, with average survival days of 23 days and 24 days, respectively (Figures 11C, 11F, and 11H). While Blank-Lip + +OVA had only a slight anti-tumor effect, with an average survival time of 31 days, and all mice died 41 days after tumor establishment (Figure 11D, 11H). + + OVA was able to significantly inhibit tumor growth, and the average survival time of mice was 43 days (Figure 11E, 11H). +The combined treatment of +OVA and PD-1 was able to further inhibit tumor growth, with 75% of mice surviving until 60 days after tumor establishment (Figure 11G, 11H). + +OVA had a better effect on tumor growth, survival time and survival rate than other groups. + +OVA did help improve ICB treatment.

[0130] Example 10 1V209-Cho-Lip + In vivo preventive and therapeutic effects of +OVA vaccine in the E.G7-OVA allograft tumor model

[0131] To further evaluate 1V209-Cho-Lip + To investigate the anti-tumor effect of OVA nanovaccines in cancer prevention and treatment, we constructed another mouse T lymphoma cell line expressing OVA, E.G7-OVA (Figures 12A, 13A). In the EG7-OVA prevention model, C57BL / 6J mice were randomly assigned to four different groups: PBS group, OVA group, Blank-Lip group, and PBS group. + +OVA group and 1V209-Cho-Lip + +OVA nanovaccine group. All vaccines were injected intramuscularly on days 0, 14, and 21 (Figure 12A). 7 days after the last immunization, E.G7-OVA T lymphoma cells were inoculated. + Compared with the +OVA group, OVA group and PBS group, 1V209-Cho-Lip + The +OVA nanovaccine group showed the best tumor growth inhibition effect and significantly prolonged the survival of mice (Figure 12). + When the +OVA nanovaccine was used as a therapeutic vaccine after tumor inoculation (Figure 13A), it also effectively inhibited the growth of E.G7-OVA tumors and prolonged the survival time of mice (Figure 13B-E). + Combined treatment with OVA and PD-1 further enhanced the anti-tumor effect (Figure 13G) and further prolonged the survival time of mice (Figure 13H). These results indicate that 1V209-Cho-Lip + +OVA exhibited potent antitumor effects in both E.G7-OVA preventive and therapeutic models.

[0132] In summary, the present invention successfully prepared a cholesterol-modified TLR7 agonist cationic liposome vaccine platform (1V209-Cho-Lip ++ OVA), this vaccine can not only stimulate DCs maturation in vitro, but also significantly enhance DCs cross-presentation of extracellular antigens. + +OVA vaccine can efficiently co-deliver model antigen (OVA) and cholesterol-modified TLR7 agonist (1V209-Cho) to lymph nodes and present them to DCs. + +OVA can induce potent and long-lasting T cell responses, long-lasting T cell immune memory, and protective immunity. + +OVA was superior to other formulations in delaying tumor progression and improving survival. Therapeutic tumor challenge showed that 1V209-Cho-Lip + +OVA significantly inhibited tumor progression. More strikingly, 1V209-Cho-Lip + + OVA combined with PD-1 treatment can effectively inhibit the occurrence and development of tumors and exert a strong anti-tumor effect (Figure 14). In short, the cholesterol-modified TLR7 agonist cationic liposome 1V209-Cho-Lip + +OVA provides an effective method for co-encapsulation of antigen and adjuvant and has the potential to be developed into a highly effective and long-lasting human anticancer preventive vaccine.

[0133] Example 11 Therapeutic Effect of Cholesterolized 1V209 Cationic Liposomes on CT26 Colorectal Cancer Ascites

[0134] To further investigate the anti-tumor effects of cholesteryl-1V209 cationic liposomes, we investigated their tumor-suppressing effects in the CT26 colorectal cancer ascites tumor model. Mice were intraperitoneally injected with CT26 tumor cells. Five days later, the tumor-bearing mice were randomly divided into three groups: a control group, a blank cationic liposome group, and a cholesteryl-1V209 cationic liposome group. Treatment was administered 5, 10, and 15 days after tumor establishment. Mice were sacrificed 20 days after tumor establishment, and the peritoneal tumors were removed, photographed, and weighed. The results, as shown in Figure 15, show that cholesteryl-1V209 cationic liposomes significantly inhibited the growth of CT26 tumor cells in the mouse peritoneal cavity. This demonstrates that cholesteryl-1V209 cationic liposomes exhibit a potent anti-tumor effect in the CT26 ascites tumor model.

Claims

1. Cholesterolized 1V209 cationic liposomes 1V209-Cho-Lip + , characterized in that: It includes a cholesterol-modified 1V209 molecule 1V209-Cho, a positive lipid component, cholesterol, and DSPE-PEG 2000 ; Wherein, the structural formula of 1V209 is as shown in Formula I, The structural formula of 1V209-Cho is shown in Formula II:

2. 1V209-Cho-Lip according to claim 1 + , characterized in that: The positive lipid component is selected from at least one of trimethyl-2,3-dioleoyloxypropylammonium bromide, trimethyl-2,3-dioleyloxypropylammonium chloride or 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol; preferably, the positive lipid component is selected from trimethyl-2,3-dioleoyloxypropylammonium bromide or trimethyl-2,3-dioleyloxypropylammonium chloride; more preferably, the positive lipid component is trimethyl-2,3-dioleoyloxypropylammonium bromide.

3. 1V209-Cho-Lip according to claim 1 or 2 + , characterized in that: The 1V209-Cho-Lip + Positive lipid components, cholesterol, 1V209-Cho and DSPE-PEG 2000 The molar ratio is 60-70:27-37:1-8:1-8; preferably, the positive lipid component, cholesterol, 1V209-Cho and DSPE-PEG 2000 The molar ratio is 61-65:30-35:2-4:2-4; More preferably, the positive lipid component, cholesterol, 1V209-Cho and DSPE-PEG 2000 The molar ratio is 62:32:3:

3.

4. 1V209-Cho-Lip according to any one of claims 1 to 3 + , characterized in that: It is composed of 1V209-Cho and positive lipid components, cholesterol and DSPE-PEG 2000 Prepared by rotary evaporation or ethanol injection method.

5. 1V209-Cho-Lip according to any one of claims 1 to 4 + The preparation method is characterized in that: The following steps are involved: 1V209-Cho was prepared by rotary evaporation or ethanol injection with positive lipid components, cholesterol and DSPE-PEG2000.

6. Cationic liposome tumor vaccine, characterized in that: Containing the 1V209-Cho-Lip according to any one of claims 1 to 4 + and tumor-associated antigens.

7. The tumor vaccine according to claim 6, characterized in that: The tumor-associated antigen is the model antigen ovalbumin OVA.

8. The tumor vaccine according to claim 6 or 7, characterized in that: The antigen and 1V209-Cho-Lip + The molar ratio of 1V209-Cho in 1V209-Cho is (32-37): (0.8-1.2); preferably, the molar ratio between the antigen and 1V209-Cho in 1V209-Cho-Lip+ is 34:

1.

9. The tumor vaccine according to any one of claims 6 to 8, characterized in that: The cationic liposome tumor vaccine is 1V209-Cho-Lip + +OVA, which includes 1V209-Cho-Lip + and ovalbumin OVA; preferably, the tumor vaccine is 1V209-Cho-Lip + It is obtained by adding OVA and encapsulating OVA through electrostatic adsorption.

10. The tumor vaccine 1V209-Cho-Lip according to claim 9 + +OVA preparation method, characterized by: The method comprises the following steps: + and OVA were loaded with OVA by electrostatic adsorption; further, 1V209-Cho-Lip + Co-incubate with OVA at 25-37°C to obtain 1V209-Cho-Lip + +OVA.

11. 1V209-Cho-Lip according to any one of claims 1 to 4 + Or use of the cationic liposome tumor vaccine according to any one of claims 5 to 9 in the preparation of anti-tumor drugs.

12. The use according to claim 11, characterized in that: The anti-tumor drug is a lymph node targeting drug.

13. 1V209-Cho-Lip according to any one of claims 1 to 4 + Or the combined drug of the cationic liposome tumor vaccine according to any one of claims 5 to 9 and other anti-tumor drugs, characterized in that: 1V209-Cho-Lip is administered separately or simultaneously + or cationic liposome tumor vaccines and other anti-tumor drugs.

14. The combined drug according to claim 13, characterized in that: The other anti-tumor drug is an immune checkpoint inhibitor; preferably, the other anti-tumor drug is at least one of a PD-1 inhibitor, a PD-L1 inhibitor or a CTLA-4 inhibitor; more preferably, the other anti-tumor drug is a PD-1 inhibitor.

15. An anti-tumor drug, characterized in that: The invention is prepared by using 1V209-Cho as the main active ingredient in the cationic liposomes according to any one of claims 1 to 4.

16. The anti-tumor drug according to claim 15, characterized in that: The anti-tumor drug is further added with pharmaceutically acceptable auxiliary ingredients; preferably, the auxiliary ingredients are at least one of fillers, disintegrants, wetting agents, antioxidants, chelating agents, surfactants, flavoring agents, chelating agents, pH regulators or pigments.

17. The anti-tumor drug according to claim 16, characterized in that: The anti-tumor drug is a lymph node targeting drug.

18. The use according to claim 11, the combined drug according to claim 13 or 14, or the anti-tumor drug according to any one of claims 15 to 17, characterized in that: The tumor is at least one of melanoma, lymphoma, colorectal cancer, breast cancer, pancreatic ductal carcinoma, liver cancer, gastric cancer, uterine cancer, ovarian cancer, testicular cancer, basal cell carcinoma or lung cancer.

19. The use according to claim 11, the combined drug according to claim 13 or 14, or the anti-tumor drug according to any one of claims 15 to 17, characterized in that: The dosage form of the anti-tumor drug or combination drug is an injection; preferably, the administration route of the injection is at least one of intramuscular injection, intravenous injection, intraperitoneal injection or subcutaneous injection.

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