WO2025199993A1 - Cholesterol-modified cationic liposome tumor vaccine, preparation method therefor, and use thereof - Google Patents

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

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

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.

Dendritic cells (DCs) are the most potent and important antigen-presenting cells (APCs), capable of uptake and presentation of antigens, activating immune responses, and regulating humoral and cellular immunity. Adjuvants activate APCs, potentially unleashing the natural killer function of cytotoxic T lymphocytes (CTLs), thereby killing tumors or pathogens. Various adjuvants, including Toll-like receptor (TLR) agonists, can activate innate immune receptors on the surface of APCs, enabling APCs to present antigens, release cytokines, and subsequently provide co-stimulatory signals to CD8 ⁺ T cells. Ideal cancer vaccines should not only create 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 antitumor immunity. To achieve this goal, a promising strategy is the use of nanoparticle vaccines, which can protect antigens from degradation, activate dendritic cells, and co-deliver the antigen and adjuvant to draining LNs to prime the immune system.

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 because they spread widely in the body and activate 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. Delivery system.

Summary of the Invention

In order to solve the technical problems that Toll-like receptor (TLR) agonists have poor targeting and large side effects 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 a positive lipid component, 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 are electrostatically adsorbed with ovalbumin (OVA) to prepare 1V209-Cho-Lip ⁺ +OVA vaccine.

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

In a first aspect, a cholesterol-modified 1V209 cationic liposome (1V209-Cho-Lip ⁺ ) is provided, comprising 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 1V209 structural formula is as shown in Formula I,

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

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).

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

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

The molar ratio of the positive lipid component, cholesterol, 1V209-Cho and DSPE-PEG ₂₀₀₀ in the 1V209-Cho-Lip ⁺ is 60-70:27-37:1-8:1-8.

Preferably, the molar ratio of the positive lipid component, cholesterol, 1V209-Cho and DSPE-PEG ₂₀₀₀ is 61-65:30-35:2-4:2-4.

More preferably, the molar ratio of the cationic lipid component, cholesterol, 1V209-Cho and DSPE-PEG ₂₀₀₀ is 62:32:3:3.

The 1V209-Cho-Lip ⁺ is prepared by rotary evaporation or ethanol injection of 1V209-Cho, positive lipid components, cholesterol and DSPE-PEG ₂₀₀₀ .

Furthermore, the rotary evaporation preparation includes the following steps: dissolving the positive lipid component, cholesterol, 1V209-Cho and DSPE-PEG ₂₀₀₀ in chloroform and methanol, forming a lipid film under rotary evaporation at 37°C, hydrating, and ultrasonicating to form cationic liposomes 1V209-Cho-Lip ⁺ .

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

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.

Furthermore, the ethanol injection method comprises the following steps: dissolving the positive lipid component, cholesterol, 1V209-Cho and DSPE-PEG ₂₀₀₀ in ethanol and dimethyl sulfoxide, then adding dropwise to an aqueous solution at 45-65° C., and removing the organic solvent to obtain cationic liposomes 1V209-Cho-Lip ⁺ .

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

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

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;

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.

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.

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

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.

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

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

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

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

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

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

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

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

In a second aspect, the present invention provides a method for preparing the above-mentioned 1V209-Cho-Lip ⁺ , comprising the following steps: preparing 1V209-Cho with a positive lipid component, cholesterol and DSPE-PEG2000 by rotary evaporation or ethanol injection.

Furthermore, the rotary evaporation preparation includes the following steps: dissolving the positive lipid component, cholesterol, 1V209-Cho and DSPE-PEG ₂₀₀₀ in chloroform and methanol, forming a lipid film under rotary evaporation at 37°C, hydrating, and ultrasonicating to form cationic liposomes 1V209-Cho-Lip ⁺ .

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

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.

Furthermore, the ethanol injection method comprises the following steps: dissolving the positive lipid component, cholesterol, 1V209-Cho and DSPE-PEG ₂₀₀₀ in ethanol and dimethyl sulfoxide, then adding dropwise to an aqueous solution at 45-65° C., and removing the organic solvent to obtain cationic liposomes 1V209-Cho-Lip ⁺ .

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

In a third aspect, the present invention provides a cationic liposome tumor vaccine comprising the above cationic liposome (1V209-Cho-Lip ⁺ ) and a tumor-associated antigen.

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

Furthermore, the molar ratio of the antigen to 1V209-Cho in the cationic liposome (1V209-Cho-Lip ⁺ ) is (32-37): (0.8-1.2);

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

More preferably, the present invention provides a cationic liposome tumor vaccine (1V209-Cho-Lip ⁺ +OVA), which comprises the above-mentioned cationic liposome (1V209-Cho-Lip ⁺ ) and ovalbumin (OVA).

The tumor vaccine is obtained by adding the above-mentioned 1V209-Cho-Lip ⁺ into OVA and encapsulating OVA through electrostatic adsorption.

In a fourth aspect, the present invention provides a method for preparing the above-mentioned cationic liposome tumor vaccine (1V209-Cho-Lip ⁺ +OVA), comprising the following steps: the above-prepared 1V209-Cho-Lip ⁺ and OVA are loaded with OVA by electrostatic adsorption.

Furthermore, 1V209-Cho-Lip ⁺ was co-incubated with OVA at 25-37° C. to obtain 1V209-Cho-Lip ⁺ +OVA.

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.

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

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.

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.

In a sixth aspect, the present invention provides a combination drug of the above-mentioned cationic liposome 1V209-Cho-Lip ⁺ or cationic liposome tumor vaccine and other anti-tumor drugs, which is administered separately or simultaneously with the above-mentioned cationic liposome or cationic liposome tumor vaccine and other anti-tumor drugs.

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.

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

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.

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.

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.

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

Preferably, the auxiliary ingredients are fillers, disintegrants, wetting agents, antioxidants, chelating agents, surfactants, Flavorings, chelating agents, pH adjusters or colorings.

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

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.

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.

Beneficial effects: The present invention uses cholesterol to modify the TLR7 agonist 1V209, and then prepares it into cationic liposomes 1V209-Cho-Lip ⁺ , and then loads the model antigen ovalbumin OVA by electrostatic adsorption to obtain the cationic liposome tumor vaccine 1V209-Cho-Lip ⁺ +OVA, which is used to prevent the occurrence and development of tumors. As a nanovaccine, the cationic liposome tumor vaccine 1V209-Cho-Lip ⁺ +OVA can co-deliver the model antigen (OVA) and the cholesterol-containing TLR7 agonist (1V209-Cho). The vaccine can ensure DC maturation by activating the TLR in the same cell that obtains the antigen, thereby achieving optimal presentation to CD8 ⁺ T cells. The results of animal experiments showed that the 1V209-Cho-Lip ⁺ +OVA nanovaccine promoted the cellular uptake and maturation of DC. Compared to the rapid metabolism of free OVA, 1V209-Cho-Lip ⁺ +OVA exhibits a "depot effect" and can further promote the transport of ovalbumin OVA to secondary lymphoid organs. Following intramuscular immunization, 1V209-Cho-Lip ^{+ +} OVA induced robust antigen-specific CD8 ⁺ T cell activation, lymphocyte activation, enhanced antigen cross-presentation responses, and increased memory T cells, antibodies, and cytokines. Prophylactic vaccination with 1V209-Cho-Lip ⁺ +OVA significantly delayed the progression of B16F10-OVA melanoma and E.G7-OVA lymphoma xenografts in mice, prolonged survival, and established a long-lasting antitumor immune response. In tumor treatment models, 1V209-Cho-Lip ⁺ +OVA more effectively inhibited tumor progression, and combination with a programmed death receptor-1 (PD-1) checkpoint inhibitor further enhanced the anti-tumor response. Therefore, DC-targeted vaccine (1V209-Cho-Lip ⁺ +OVA) co-delivered with antigen and cholesterol-modified TLR7 agonist (1V209-Cho) is a promising cancer vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Preparation and characterization results of cationic liposome tumor vaccine 1V209-Cho-Lip ⁺ +OVA in Example 1: A) Structural formula of cholesterol-modified 1V209; B) Particle size and electron micrograph of cholesterol-modified 1V209 cationic liposome 1V209-Cho-Lip ⁺ ; C) Particle size and electron micrograph of cholesterol-modified 1V209 cationic liposome loaded with ovalbumin OVA, i.e., 1V209-Cho-Lip ⁺ +OVA.

Figure 2. Example 2: Results of in vitro DC uptake experiment: A) Dendritic cell uptake; Example 3: Results of in vitro BMDC activation experiment: B) Expression of DCs co-stimulatory factors CD40, CD80, and CD86 after co-incubation of each preparation with DCs; Example 4: Results of in vitro CD8 ⁺ T cell cross-presentation assay: E) Flow cytometry and quantitative analysis of CFSE-OT-I CD8 ⁺ T cells after co-culture of CFSE-labeled OT-I CD8 ⁺ T cells with DCs activated by each preparation for 72 hours; F) Flow cytometry and quantitative analysis of activated CD8 ⁺ T cells after co-culture of CFSE-labeled OT-I CD8 ⁺ T cells with DCs activated by each preparation for 72 hours; Data are expressed as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

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

Figure 4. Example 6. Antitumor efficacy of 1V209-Cho-Lip ⁺ +OVA as a preventive vaccine in the B16F10-OVA tumor model. Figures: 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.

Figure 5. Example 6. Antitumor efficacy of 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 preventive model; E) Survival of mice after immunization with each formulation; F) Percentage of tumor-free mice after immunization with each formulation.

Figure 6 and Example 7 Results of the study on the immune prevention mechanism of 1V209-Cho-Lip ⁺ +OVA: A) Quantification of activated CD4 ⁺ T cells (CD4 ⁺ CD69 ⁺ T cells) in inguinal draining lymph nodes; B) Numbers 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 ⁺ ) in inguinal lymph nodes; C) Quantification of activated CD8 ⁺ T cells (CD8 ⁺ CD69 ⁺ T cells) in inguinal draining lymph nodes; D) Numbers of effector memory CD8 ⁺ T cells (CD3 ⁺ CD8 ⁺ CD44 ⁺ CD62L ⁻ ), central memory CD8 ⁺ T cells (CD3 ⁺ CD8 ⁺ CD44 ⁺ CD62L ⁺ ) and naive CD8 ⁺ The number of T cells (CD3 ⁺ CD8 ⁺ CD44 ^- CD62L ⁺ ).

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

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

Figure 9. Example 7: Results of the 1V209-Cho-Lip ⁺ +OVA immunopreventive mechanism study: Figures A-C) Secretion of inflammatory cytokines IFN-γ, TNF-α, and IL-6 in mouse serum after immunization; D) Tumor-specific cytotoxicity of CD8 ⁺ T cells. Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 10, Example 8 1V209-Cho-Lip ⁺ + OVA induces long-lasting humoral and cellular immune responses to prevent tumor formation Results: A) Mouse immunization program; B-E) Tumor growth in each group of mice after tumor establishment; F) No tumor in each group of mice after tumor establishment Tumor ratio; G) Quantification of long-lived memory B cells (BMEM) in the bone marrow; H I) ELISPOT detection of IFN-γ ⁺ cells in the spleen and peripheral blood.

Figure 11. Results of the in vivo immunotherapy experiment with 1V209-Cho-Lip ⁺ +OVA in Example 9: 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).

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

Figure 13. In vivo preventive and therapeutic effects of 1V209-Cho-Lip ⁺ +OVA vaccine in Example 10 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.

Figure 14. Schematic diagram of the mechanism of action of the cholesterol-modified 1V209 cationic liposome vaccine 1V209-Cho-Lip ⁺ +OVA.

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

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.

1V209 is a synthetic small molecule TLR7 agonist with anti-tumor effects but strong systemic immunotoxicity. This invention innovatively chemically links cholesterol to 1V209 to create 1V209-Cho, aiming to improve its pharmacokinetics and enable lymph node targeting. This liposome is then fabricated into nanoscale cationic liposomes, 1V209-Cho-Lip ⁺ , which are then electrostatically adsorbed with ovalbumin (OVA) to yield 1V209-Cho-Lip ⁺ +OVA. The lymph node targeting ability of 1V209-Cho-Lip ⁺ +OVA has been verified in vivo.

In the first aspect, in one embodiment of the present invention, a cationic liposome vaccine 1V209-Cho-Lip ⁺ +OVA is first prepared, which is prepared by combining the cholesterol-modified 1V209 molecule 1V209-Cho with the positive lipid component, cholesterol and DSPE-PEG ₂₀₀₀ to form the cationic liposome 1V209-Cho-Lip ⁺ , and then adsorbing the ovalbumin OVA onto the surface of the cationic liposome 1V209-Cho-Lip ⁺ .

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

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

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

The molar ratio of the positive lipid component, cholesterol, 1V209-Cho and DSPE-PEG ₂₀₀₀ is 60-70:27-37:1-8:1-8.

Preferably, the molar ratio of the positive lipid component, cholesterol, 1V209-Cho and DSPE-PEG ₂₀₀₀ is 61-65:30-35:2-4:2-4.

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

Cationic liposomes 1V209-Cho-Lip ⁺ can be prepared using methods commonly used in the art. Rotary evaporation or ethanol infusion are common alternatives. The rotary evaporation method generally involves dissolving the cationic lipid components, cholesterol, 1V209-Cho, and DSPE-PEG ₂₀₀₀ in chloroform and methanol, then rotary evaporating at 37°C to form a lipid film. This film is then hydrated and sonicated to form the cationic liposomes 1V209-Cho-Lip ⁺ .

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

In a specific embodiment of the present invention, the cationic liposome vaccine 1V209-Cho-Lip ⁺ +OVA is prepared by electrostatic adsorption, that is, the prepared cationic 1V209-Cho-Lip ⁺ is co-incubated with ovalbumin OVA at 25-37° C. for 1 hour.

In the second aspect, in a specific embodiment of the present invention, the present invention evaluated the preventive and therapeutic effects of the cationic liposome vaccine 1V209-Cho-Lip ⁺ +OVA on mouse melanoma (B16F10-OVA) and mouse lymphoma (EG7-OVA). The experimental results showed that 1V209-Cho-Lip ⁺ +OVA exhibited 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. 1V209-Cho-Lip ⁺ +OVA immunization promoted antigen-specific humoral and cytotoxic T lymphocyte (CTL) responses, subsequently inhibiting tumor growth and prolonging mouse survival. In addition, the 1V209-Cho-Lip ⁺ +OVA cationic liposome vaccine was able to induce long-lasting antigen-specific CD8 ⁺ memory T cell immunity and prevent tumor formation 6 months after the first immunization. At the same time, in therapeutic B16F10-OVA melanoma and E.G7-OVA lymphoma models, the 1V209-Cho-Lip ⁺ +OVA cationic liposome vaccine effectively inhibited tumor progression when combined with immune checkpoint blockers (ICB). Targeted co-delivery of antigens for cancer immunotherapy by 1V209-Cho-Lip ⁺ +OVA cationic liposomes The mechanism of TLR7 agonist is shown in FIG14 .

Thirdly, in specific examples of the present invention, the efficacy of cholesterol-modified 1V209 cationic lipids (1V209-Cho-Lip ⁺ ) in treating CT26 colorectal cancer ascites tumors was evaluated. The results demonstrated that 1V209-Cho-Lip ⁺ exhibited significant anti-tumor activity in the CT26 subcutaneous tumor model.

Fourthly, in one embodiment of the present invention, 1V209-Cho in the cationic liposome vaccine 1V209-Cho-Lip ⁺⁺ OVA of the present invention can also be used to prepare an anti-tumor drug as the main active ingredient. This anti-tumor drug has lymph node targeting and is generally formulated as an injection. This anti-tumor drug 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.

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.

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

1V209 was purchased from Selleck. Mouse B16F10-OVA 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 streptomycin sulfate) 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.

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

Step a: Cholesterol, EDCI, and DMAP were dissolved in 10 mL of DCM at a ratio of 1 eq:2 eq:0.1 eq. 1.2 eq of boc-aminobutyric acid was then added. The mixture was reacted at room temperature for 24 hours, and then 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 a crude product, which was purified by silica gel chromatography using petroleum ether/ethyl acetate to obtain product 1.

Product 1:

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.

Product 2:

In step c, 1V209, HATU and TEA were dissolved in 5 mL of DMF at a ratio of 1 eq: 2 eq: 2 eq, and then the product 2 dissolved in DMF was added. The amount of product 2 was 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.

Example 1 Preparation and Characterization of Cationic Liposome Vaccine 1V209-Cho-Lip ⁺ +OVA

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) were dissolved in chloroform/methanol (v/v = 10:1). The organic solvent was then evaporated by rotary evaporation at 37°C to form a lipid film. After hydration with PBS at 37°C for 1 hour, the film was intermittently sonicated in water for 30 minutes, followed by sonication with an 80W probe sonicator for 120 seconds to form cholesterol-loaded 1V209 cationic liposomes (1V209-Cho-Lip ⁺ ). OVA was then added (OVA:1V209-Cho molar ratio = 34:1) and the film was shaken at room temperature for 1 hour to allow electrostatic adsorption of OVA to the cationic liposomes, resulting in 1V209-Cho-Lip ⁺ + OVA. Blank-Lip ⁺ +OVA was prepared using a similar method, but without the addition of 1V209-Cho.

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 1V209-Cho-Lip ⁺ +OVA was approximately 190 nm, larger than that of 1V209-Cho-Lip ⁺ (Figure 1B,C). Transmission electron microscopy (FE-TEM) revealed that the average particle size of 1V209-Cho-Lip ⁺ +OVA was approximately 200 nm, consistent with the DLS data. Dynamic light scattering (DLS) and FE-TEM analysis demonstrated that the cholesterol-modified 1V209 cationic liposome vaccine 1V209-Cho-Lip ⁺ +OVA had been successfully prepared.

Example 2 DCs in vitro uptake experiment

Because DCs are the most potent APCs for activating naive T cells, efficient internalization of 1V209-Cho-Lip ⁺ +OVA by DCs is crucial. Fluorescent 1V209-Cho-Lip ⁺ +OVA ^F was constructed using OVA labeled with fluorescein isothiocyanate (FITC) to investigate the cellular uptake of 1V209-Cho-Lip ⁺ +OVA. Mouse DCs were seeded in 24-well plates. OVA ^F , Blank-Lip ⁺ +OVA ^F , and 1V209-Cho-Lip ⁺ +OVA ^F (with equal amounts of OVA-FITC) were then added and incubated with DCs for 4 hours. After incubation, the culture medium was aspirated, the cells were washed three times with PBS, and the cells were suspended in 200 μL of PBS for analysis by flow cytometry. The experimental results are shown in Figure 2A. Compared with free OVA ^F , both the 1V209-Cho-Lip ⁺ +OVA ^F group and the Blank-Lip ⁺ +OVA ^F group were better taken up by DCs. In contrast, the fluorescence intensity of the 1V209-Cho-lip ⁺ +OVA ^F group was significantly higher than that of the Blank-Lip ⁺ +OVA ^F group, indicating that 1V209-Cho-Lip ⁺ +OVA ^F has an advantage in DC uptake.

Example 3 BMDC in vitro activation experiment

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 co-stimulatory molecules such as CD40, CD86, and CD80. Therefore, an in vitro activation experiment of bone marrow-derived dendritic cells (BMDCs) was performed.

Bone marrow cells were isolated from the femur and skull 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 to generate immature BMDCs. BMDCs were then plated in 24-well cell culture plates (10 ⁶ cells per well). BMDCs were treated with PBS, OVA, Blank-Lip ⁺ OVA, and 1V209-Cho-lip ⁺ OVA (10 μg mL ^-1 OVA, 5 μg mL ^-1 1V209-Cho) for 48 hours. Flow cytometry was used to assess the expression of co-stimulatory molecules (CD80, CD40, and CD86) on BMDCs to evaluate their immunostimulatory effects. The results are shown in Figures 2B, 2C, and 2D. Compared with the PBS control group, incubation with 1V209-Cho-Lip ⁺ +OVA significantly upregulated the important indicators of DC maturation, CD40 (1.77 times), CD80 (1.38 times), and CD86 (2.20 times). These data indicate that 1V209-Cho-Lip ⁺ +OVA is an effective nanovaccine that is beneficial to the maturation of DCs.

Example 4 Determination of CD8 ⁺ T cell cross-presentation in vitro

For antigen cross-presentation studies, CD8 ⁺ T cells from OT-1 transgenic mice were purified using a mouse CD8 ⁺ T cell isolation kit (Stemcell Technologies) and labeled with CFSE. Activated DCs (3× ¹⁰⁵ cells) were then cocultured with 3× ¹⁰⁵ CFSE-labeled CD8 ⁺ T cells in RPMI medium for 72 hours. The cells were then harvested and incubated with a Perp-Cy5.5-labeled anti-mouse CD3 antibody (Biolegene), a PE-labeled anti-mouse CD8 antibody (Biolegene), and a BV421-labeled anti-mouse CD69 antibody (Biolegene) for 30 minutes. Finally, the cells were washed twice with PBS and analyzed using a NovoCyte flow cytometer. As shown in Figures 2E and 2F, coculture with 1V209-Cho-Lip ⁺ OVA-treated DCs increased CD8 ⁺ T cell proliferation by 2.1-fold. Furthermore, after co-culture with 1V209-Cho-Lip ⁺ OVA-treated DCs, CD8 ⁺ T cell activation was 5.8-fold higher than that of PBS-treated DCs. Blank-Lip ⁺ OVA-treated DCs had little effect on CD8 ⁺ T cell proliferation. CD8 ⁺ T cell activation was also significantly lower than that of 1V209-Cho-Lip ⁺ OVA-treated DCs. In summary, 1V209-Cho-Lip ⁺ OVA significantly enhanced DCs' cross-presentation of extracellular antigens in vitro, which may play an important role in inducing antigen-specific CTL activity.

Example 5 Distribution in vivo

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 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). The mice were killed at the last time point. Inguinal and popliteal lymph nodes were harvested and fixed for imaging (Figure 3B). The fluorescence intensity of the inguinal lymph nodes in the 1V209-Cho-Lip ⁺ +OVA ^F group was stronger than that in the OVA ^F and Blank-Lip ⁺ +OVA ^F groups and remained at a high level 24 hours after injection, indicating that 1V209-Cho-Lip ⁺ +OVA ^F had strong migration and retention in the inguinal lymph nodes. At 12 and 24 hours, the fluorescence intensity at the injection site of the 1V209-Cho-Lip ⁺ +OVA ^F and Blank-Lip ⁺ +OVA ^F groups was significantly stronger than that of the OVA ^F group, consistent with previous reports that cationic liposomes have the ability to form a reservoir at the injection site. The fluorescence intensity of the inguinal lymph nodes in the Blank-Lip ⁺ +OVA ^F group was weaker. In vitro imaging showed that the fluorescence intensity of the inguinal lymph nodes in the 1V209-Cho-Lip ⁺ +OVA ^F group was significantly higher than that in the Blank-Lip ⁺ +OVA ^F group and the OVA ^F group. At the same time, the fluorescence intensity of the popliteal lymph nodes in the 1V209-Cho-Lip ⁺ +OVA ^F group was also higher than that in the other groups (Figure 3A). Semi-quantitative in vitro imaging showed that the fluorescence intensity of the inguinal lymph nodes and popliteal lymph nodes in the 1v209-Cho-Lip ⁺ +OVA ^F group was 2.06 times and 5.52 times that of the OVA ^F group, respectively (Figure 3B). In summary, 1V209-Cho-Lip ⁺ +OVA ^F not only has a reservoir effect at the injection site, but also can deliver antigens to the draining lymph nodes.

Example 6 Antitumor Efficacy of 1V209-Cho-Lip ⁺ +OVA as a Preventive Vaccine in the B16F10-OVA Tumor Model

Female C57BL/6J mice were randomly divided into phosphate buffer saline (PBS) group, OVA group, Blank-Lip ⁺ +OVA group, and 1V209-Cho-lip ⁺ +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, mice were subcutaneously inoculated with 1×10 ⁶ B16F10-OVA cells in the right flank (Figure 4A). Tumor growth and survival were observed, and tumor volume was calculated using the formula: volume (cm ³ ) = (width) ² × length/2. Serum was obtained on days 21 and 28, and serum OVA-specific IgG, IgG1, IgG2a, and IgG2b titers were determined by ELISA (Figures 4B and 4C). Compared with OVA and PBS immunization, intramuscular immunization with Blank-Lip ⁺ +OVA and 1V209-Cho-Lip ⁺ +OVA induced higher serum OVA-specific IgG levels, with the 1V209-Cho-Lip ⁺ +OVA group eliciting the highest levels. Blank-lip ⁺ +OVA immunization primarily induced Th2-related IgG1, while 1V209-Cho-Lip ⁺ +OVA induced IgG1 as well as Th1-related IgG2a and IgG2b. Compared with the Blank-Lip ⁺ +OVA, OVA, and PBS groups, the 1V209-Cho-Lip ⁺ +OVA group exhibited the best antitumor effect and significantly prolonged mouse survival, with complete tumor regression in 62.5% of mice (Figures 5A-D, 5F). Moderate tumor inhibition was observed in the Blank-Lip ⁺ +OVA group due to the adjuvant effect of cationic liposomes (Figures 5C, 5E). Taken together, these studies demonstrate that 1V209-Cho-Lip ⁺ +OVA can serve as an effective vaccine to prevent tumor development and progression after inoculation with OVA-expressing tumor cells.

Example 7 Study on the immune prevention mechanism of 1V209-Cho-Lip ⁺ +OVA

Seven days after the last immunization, mice (n=5) were sacrificed to investigate the mechanism of 1V209-Cho-Lip ⁺ +OVA immunoprophylaxis. Inguinal lymph nodes were removed and filtered through a 70 μm filter to obtain a single-cell suspension. The suspension 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), and BV421-labeled anti-mouse PD-1. Antibody (BioLegend), FITC-labeled anti-mouse TCF-1 antibody (BioLegend). As shown in Figures 6A and 6C, after 1V209-Cho-Lip ⁺ +OVA immunization, the percentages of activated CD4 ⁺ T cells (CD4 ⁺ CD69 ⁺ T cells) and activated CD8 ⁺ T cells (CD8 ⁺ CD69 ⁺ T cells) in the inguinal draining lymph nodes were significantly increased compared with other groups, which is crucial for inducing significant immune responses 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 upon reinfection. In our study, we used FACS to evaluate the memory T cells of CD4 ⁺ and CD8 ⁺ T cells in the draining inguinal lymph nodes of mice immunized three times. As shown in Figures 6B and 6D, compared with the other groups, 1V209-Cho-Lip ⁺ +OVA significantly increased the percentage of _TEM (CD44 ⁺ CD62L ^- ) of CD4 ⁺ and CD8 ⁺ T cells, and decreased the percentage of TEM (CD44 + CD62L - ) of CD4 ⁺ and CD8 ⁺ T cells. (CD44 ^- CD62L ⁺ ) percentage. Blank-Lip ⁺ +OVA only observed an increase in the percentage of CD4 ⁺ and CD8 ⁺ T cells T _CM (CD44 ⁺ CD62L ⁺ ), while T _EM showed no significant changes. This suggests that 1V209-Cho-Lip ⁺ +OVA can induce a strong memory T cell response and has a strong protective effect during reinfection. The study found that TCF1 ⁺ PD-1 ⁺ CD8 ⁺ T cells have a stem cell-like phenotype, exert a sustained anti-tumor immune response, and expand more effectively after ICB treatment. After 1V209-Cho-Lip ⁺ +OVA immunization, the percentage of stem-like CD8 ⁺ T cells in the inguinal draining lymph nodes increased (Figure 7A), suggesting that it may help improve ICB treatment.

In addition, the mouse spleen single cell suspension was further incubated 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). The results showed that 1V209-Cho-Lip ⁺ +OVA immunization significantly increased the proportion of CD4 ⁺ , activated CD4 ⁺ T, and CD8 ⁺ T cells in the spleen of mice (Figure 7B-D). In order to further explore whether 1V209-Cho-Lip ⁺ +OVA can significantly enhance the GC response of mice, produce mature plasma cells and memory B cells, and thus mediate long-term protective immune responses. Seven days after the last immunization, we studied the B cells and follicular helper T cells ( _TFH ) in the inguinal draining lymph nodes and spleen of mice immunized with 1V209-Cho-Lip ⁺ +OVA. Immunization with 1V209-Cho-Lip ⁺ +OVA significantly increased GC B cells in the draining inguinal lymph nodes of mice (Figure 7E). A corresponding significant increase in GC T _FH was also observed in the draining inguinal lymph nodes (Figure 7F). Immunization with 1V209-Cho-Lip ⁺ +OVA enhanced GC responses in the draining peritoneal lymph nodes compared to other groups, thereby promoting the establishment of long-term immune memory.

In addition, after stimulating splenocytes with OVA _257-264 (5 μg mL ^-1 ) for 3 days, the cells were 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). The frequency of OVA-specific CD8 ⁺ T cells in mice vaccinated with 1V209-Cho-Lip ⁺ +OVA was significantly increased compared with the PBS, OVA, and Blank-Lip ^{+ +} OVA groups ( Figure 8A ). Furthermore, immunization with 1V209-Cho-Lip ⁺ OVA not only significantly increased the percentage of CD8 ⁺ IFN-γ or granzyme B-producing CTLs ( Figures 8B, 8C ), but also increased the expression of IFN-γ and granzyme B in CTLs. 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 (Figures 8F-H). In addition, the serum of mice was collected and the levels of serum INF-γ, TNF-α, and IL-6 were detected by ELISA. After mice were immunized with 1V209-Cho-Lip ⁺ +OVA, the secretion of inflammatory factors such as IFN-γ, TNF-α, and IL-6 in the serum was significantly increased (Figures 9A-C).

Splenic lymphocytes were isolated and stimulated with the CD8 ⁺ -specific OVA _257-264 peptide (10 μg mL ^-1 ) for 3 days to detect the killing activity of T cells. 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 detect the frequency of B16F10-OVA cells with high CFSE expression, and the percentage of specific killing was calculated. CD8 ⁺ T cells from mice immunized with 1V209-Cho-Lip ⁺ +OVA exhibited high tumor-specific cytotoxicity (Figure 9D). The above results indicate that the 1V209-Cho-Lip ⁺ +OVA nanovaccine can indeed induce a strong T cell immune response in vivo.

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

Female C57BL/6J mice were randomly divided into PBS, OVA, Blank-Lip ⁺ +OVA, and 1V209-Cho-Lip ⁺ +OVA groups, with 5 mice in each group. They were intramuscularly injected with 25 μg/mouse OVA and 5 μg/mouse 1V209-Cho on days 0, 14, and 21, respectively. Orbital blood was collected from the mice 5 and 6 months after the first injection. The immunization schedule is shown in Figure 10A. Six months after the first injection, mice were subcutaneously inoculated with 1×10 ⁶ B16F10-OVA cells. Tumor size was measured every 2 days for 20 days. Tumor growth in mice is shown in Figures 10B-F. Mice immunized with 1V209-Cho-Lip ⁺ +OVA induced long-lasting immune memory, with no tumor growth observed in nearly all mice 20 days after tumor establishment (Figures 10E, 10F). Although tumor formation was not observed in mice immunized with Blank-Lip ⁺ +OVA until day 16 after B16F10-OVA inoculation, 60% of mice failed to prevent tumor formation by 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 ).

Long-lived memory B cells (BMEM) form an important part of immune memory and provide a rapid antibody response to reinfection. The spleens of the above mice were removed and filtered through a 70μm filter to obtain a single-cell suspension. The suspension was then 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 assessed by flow cytometry (Figure 10G). The proportion of BMEM in the 1V209-Cho-Lip ⁺ +OVA-immunized mice was significantly higher than that in the other groups.

ELISPOT detection of CD8 ⁺ T cells secreting IFN-γ. 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 inoculated into pre-coated plates supplemented with SIINFEK. They were further incubated for 18 h at 37°C in a 5% _CO2 incubator. IFN-γ ⁺ CD8 ⁺ T cell spots were detected using biotinylated anti-IFN-γ antibodies, alkaline phosphatase streptavidin, and BCIP dissolved in low-melting-point agarose solution. The analysis results showed that the number of IFN-γ ⁺ CD8 ⁺ T cells in the spleen and blood of immunized 1V209-Cho-Lip ⁺⁺ OVA mice was significantly increased. However, no significant tumor-specific CD8 ⁺ memory T cells were observed in the spleen or blood of the other three groups of mice (Figures 10H, 10I).

Example 9 In vivo immunotherapy experiment

On day 0, mice were subcutaneously inoculated with 3×10 ⁵ B16F10-OV cells on the right flank. Three days after inoculation, tumor-bearing mice were randomly divided into six groups: PBS, OVA, Blank-Lip ⁺ +OVA, 1V209-Cho-Lip ⁺ +OVA, Anti-PD-1, and 1V209-Cho-Lip ⁺ +OVA + Anti-PD-1, with eight mice in each group. Three days after tumor establishment, each formulation (25 μg/mouse OVA and 5 μg/mouse 1V209-Cho) was injected intramuscularly once every seven days for a total of three doses. Anti-PD-1 (100 μg/mouse) was injected intraperitoneally every three days for a total of six doses. The treatment schedule is shown in Figure 11A . Tumor growth and survival of mice were observed. The average survival of mice in the PBS control group was 20 days, and all mice died 26 days after tumor establishment (Figures 11B and 11H). The anti-tumor effects of the OVA and anti-PD-1 groups were similar to those of the PBS group, with average survival times of 23 and 24 days, respectively (Figures 11C, 11F, and 11H). Blank-Lip ⁺ +OVA, on the other hand, had only a slight anti-tumor effect, with an average survival time of 31 days, and all mice died 41 days after tumor establishment (Figures 11D and 11H). 1V209-Cho-Lip ⁺ +OVA significantly inhibited tumor growth, with an average survival time of 43 days (Figures 11E and 11H). Notably, combined treatment with 1V209-Cho-Lip ⁺ +OVA and PD-1 further inhibited tumor growth, with 75% of mice surviving up to 60 days after tumor establishment (Figures 11G and 11H). In tumor treatment experiments, 1V209-Cho-Lip ⁺ +OVA had a superior effect on tumor growth, survival time, and survival rate than other groups. Furthermore, 1V209-Cho-Lip ⁺ +OVA did help improve ICB therapy.

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

To further evaluate the anti-tumor effect of 1V209-Cho-Lip ⁺ +OVA nanovaccine in cancer prevention and treatment, we constructed another OVA-expressing mouse T lymphoma cell line, 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 ⁺ +OVA group, and 1V209-Cho-Lip ⁺ +OVA nanovaccine group. All vaccines were injected intramuscularly on days 0, 14, and 21 (Figure 12A). Seven days after the last immunization, E.G7-OVA T lymphoma cells were inoculated. Compared with the Blank-Lip ⁺ +OVA group, OVA group, and PBS group, the 1V209-Cho-Lip ⁺ +OVA nanovaccine group showed the best tumor growth inhibition effect and significantly prolonged mouse survival (Figure 12). When 1V209-Cho-Lip ⁺ +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 of mice (Figures 13B-E). Notably, the combination of 1V209-Cho-Lip ⁺ +OVA and PD-1 further enhanced the anti-tumor effect (Figure 13G) and further prolonged the survival of mice (Figure 13H). These results indicate that 1V209-Cho-Lip ⁺ +OVA exhibits a potent anti-tumor effect in both E.G7-OVA prevention and treatment models.

In summary, the present invention successfully prepared a cholesterol-modified TLR7 agonist cationic liposome vaccine platform (1V209-Cho-Lip ⁺ +OVA), which can not only stimulate DCs maturation in vitro, but also significantly enhance DCs Cross-presentation of extracellular antigens. The 1V209-Cho-Lip ⁺ +OVA vaccine can efficiently co-deliver model antigens (OVA) and cholesterol-modified TLR7 agonists (1V209-Cho) to lymph nodes and present them to DCs. 1V209-Cho-Lip ⁺ +OVA can induce potent and persistent T cell responses, lasting T cell immune memory, and protective immunity. After preventive vaccination, 1V209-Cho-Lip ⁺ +OVA is superior to other formulations in delaying tumor development 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 powerful anti-tumor effect (Figure 14). In conclusion, the cholesterolylated TLR7 agonist cationic liposome 1V209-Cho-Lip ⁺ +OVA provides an effective co-encapsulation method for antigen and adjuvant and has the potential to be developed as a highly effective and long-lasting human anticancer preventive vaccine.

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

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 (19)

Cholesterolated 1V209 cationic liposomes 1V209-Cho-Lip ⁺ are characterized by comprising a cholesterol-modified 1V209 molecule 1V209-Cho, a cationic lipid component, cholesterol, and DSPE-PEG ₂₀₀₀ ; wherein the 1V209 structural formula is shown in Formula I,
The structural formula of 1V209-Cho is shown in Formula II:
The 1V209-Cho-Lip ⁺ according to claim 1 is 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. The 1V209-Cho-Lip ⁺ according to claim 1 or 2 is characterized in that the molar ratio of the positive lipid component, cholesterol, 1V209-Cho and DSPE-PEG ₂₀₀₀ in the 1V209-Cho-Lip ⁺ is 60-70:27-37:1-8:1-8; preferably, the molar ratio of the positive lipid component, cholesterol, 1V209-Cho and DSPE-PEG ₂₀₀₀ is 61-65:30-35:2-4:2-4; more preferably, the molar ratio of the positive lipid component, cholesterol, 1V209-Cho and DSPE-PEG ₂₀₀₀ is 62:32:3:3. The 1V209-Cho-Lip ⁺ according to any one of claims 1 to 3, characterized in that it is prepared from 1V209-Cho, a positive lipid component, cholesterol, and DSPE-PEG ₂₀₀₀ by rotary evaporation or ethanol injection. The method for preparing 1V209-Cho-Lip ⁺ according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: 1V209-Cho, a positive lipid component, cholesterol and DSPE-PEG2000 are prepared by a rotary evaporation method or an ethanol injection method. A cationic liposome tumor vaccine, characterized by comprising the 1V209-Cho-Lip ⁺ according to any one of claims 1 to 4 and a tumor-associated antigen. The tumor vaccine according to claim 6, wherein the tumor-associated antigen is a model antigen ovalbumin (OVA). The tumor vaccine according to claim 6 or 7, characterized in that the molar ratio of the antigen to 1V209-Cho in 1V209-Cho-Lip ^{+ is} (32-37):(0.8-1.2); preferably, the molar ratio of the antigen to 1V209-Cho in 1V209-Cho-Lip+ is 34:1. 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 obtained by adding 1V209-Cho-Lip ⁺ to OVA and encapsulating OVA through electrostatic adsorption. The method for preparing the tumor vaccine 1V209-Cho-Lip ⁺ +OVA according to claim 9 is characterized in that it comprises the following steps: 1V209-Cho-Lip ⁺ and OVA according to any one of claims 1 to 4 are loaded with OVA by electrostatic adsorption; further, 1V209-Cho-Lip ⁺ and OVA are co-incubated at 25-37°C to obtain 1V209-Cho-Lip ⁺ +OVA. Use of the 1V209-Cho-Lip ⁺ according to any one of claims 1 to 4 or the cationic liposome tumor vaccine according to any one of claims 5 to 9 in the preparation of an anti-tumor drug. The use according to claim 11 is characterized in that the anti-tumor drug is a lymph node targeted drug. A combination drug of 1V209-Cho-Lip ⁺ according to any one of claims 1 to 4 or 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 ⁺ or the cationic liposome tumor vaccine and the other anti-tumor drugs are administered separately or simultaneously. The combination 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. An anti-tumor drug, characterized in that it is prepared from 1V209-Cho in the cationic liposome according to any one of claims 1 to 4 as a main active ingredient. The anti-tumor drug according to claim 15, characterized in that: the anti-tumor drug is further added with a pharmaceutically acceptable auxiliary ingredient; preferably, the auxiliary ingredient is at least one of a filler, a disintegrant, a wetting agent, an antioxidant, a chelating agent, a surfactant, a flavoring agent, a chelating agent, a pH adjuster or a pigment. The anti-tumor drug according to claim 16, characterized in that the anti-tumor drug is a lymph node targeted drug. 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. The use according to claim 11, the combination 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 the 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.