Manganese adjuvanted whole cell tumor vaccine and methods of making and using the same

Abstract

The present application provides a whole-cell tumor vaccine loaded with a manganese adjuvant, and a preparation method and application thereof. Specifically, the present application provides a whole-cell tumor vaccine loaded with a manganese adjuvant, which comprises: (a) tumor cells; (b) anionic lipid nanoparticles covalently coupled to the tumor cells to form a "tumor cell-anionic lipid nanoparticle" binary complex; and (c) a manganese adjuvant loaded on the "tumor cell-anionic lipid nanoparticle" binary complex to form a "tumor cell-anionic lipid nanoparticle-manganese adjuvant" ternary complex. The whole-cell tumor vaccine loaded with the manganese adjuvant of the present application not only retains the morphology and all possible antigens of the tumor cells, but also in situ loads a very high loading amount of the manganese adjuvant on the tumor cells, generates a strong immune response level, and thus synergistically and efficiently stimulates an immune reaction, thereby significantly inhibiting tumor growth.

Description

Technical Field

[0001] This invention belongs to the fields of medicine and tumor immunotherapy, specifically relating to whole-cell tumor vaccines loaded with manganese adjuvants, their preparation methods, and applications. Background Technology

[0002] Tumor vaccines activate T cells in patients by introducing tumor antigens (including tumor cells, proteins or peptides, and genes carrying these antigens), thereby inducing an immune response to kill tumor cells. This is a promising approach to tumor immunotherapy.

[0003] Whole-Tumor-Cell Vaccines are derived from the patient's own or allogeneic tumor cells as the immune source. These cells are inactivated through treatments such as irradiation, heating, or freezing, and then injected into the body for tumor immunotherapy. The advantage of whole-cell tumor vaccines lies in their inclusion of all possible tumor cell antigens, thus providing a broad spectrum of coverage and avoiding antigen omissions that can occur when using only a subset of antigens. However, vaccines prepared by directly inactivating tumor cells have relatively weak immunogenicity and are insufficient to induce a strong tumor immune response. Therefore, adjuvants are often used in combination with whole-cell tumor vaccines to enhance their immunogenicity. However, the immunotherapeutic efficacy of some existing whole-cell tumor vaccines combined with adjuvants remains unsatisfactory.

[0004] Therefore, there is an urgent need in this field to develop tumor vaccines with advantages such as stronger and more efficient stimulation of immune responses. Summary of the Invention

[0005] The purpose of this invention is to provide a tumor vaccine with advantages such as stronger and more efficient immune response, as well as its preparation method and application.

[0006] In a first aspect of the invention, a whole-cell tumor vaccine loaded with manganese adjuvant is provided, the whole-cell tumor vaccine loaded with manganese adjuvant comprising:

[0007] (a) Tumor cells;

[0008] (b) Anionic lipid nanoparticles, said anionic lipid nanoparticles covalently coupled to said tumor cells, forming a "tumor cell-anionic lipid nanoparticle" binary complex; and

[0009] (c) Manganese adjuvant, wherein the manganese adjuvant is loaded onto the binary complex of “tumor cell-anionic lipid nanoparticle” to form a ternary complex of “tumor cell-anionic lipid nanoparticle-manganese adjuvant”.

[0010] In another preferred embodiment, the tumor cells are tumor cells whose surface is modified with functional groups.

[0011] In another preferred embodiment, the tumor cells are tumor cells whose surface has been modified with thiolation.

[0012] In another preferred embodiment, the anionic lipid nanoparticles are coupled to the tumor cells via disulfide bonds.

[0013] In another preferred embodiment, the "tumor cell-anionic lipid nanoparticle-manganese adjuvant" ternary complex is loaded with Mn. 2+ ion.

[0014] In another preferred embodiment, the tumor cells are inactivated tumor cells.

[0015] In another preferred embodiment, the tumor cells are inactivated by a treatment selected from the group consisting of irradiation, liquid nitrogen treatment, or a combination thereof.

[0016] In another preferred embodiment, the tumor cells are selected from the group consisting of tumor cells of solid tumors, tumor cells of hematologic malignancies, or combinations thereof.

[0017] In another preferred embodiment, the tumor cells include cells selected from the group consisting of: lung cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, cervical cancer, esophageal cancer, thyroid cancer, pancreatic cancer, bladder cancer, kidney cancer, ovarian cancer, endometrial cancer, glioma, or combinations thereof.

[0018] In another preferred embodiment, the prostate cancer cells include: (a) castration-resistant prostate cancer cells or (b) androgen-sensitive prostate cancer cells.

[0019] In another preferred embodiment, the anionic lipid nanoparticles themselves have a Zeta potential of -40mV to -30mV.

[0020] In another preferred embodiment, after loading with manganese adjuvant, the zeta potential of the anionic lipid nanoparticles is -20mV to -10mV.

[0021] In another preferred embodiment, the anionic lipid nanoparticles have a particle size of 20–500 nm; more preferably 50–300 nm; and even more preferably 100–200 nm.

[0022] In another preferred embodiment, the polydispersity index of the anionic lipid nanoparticles is 0.2 to 0.8; preferably 0.2 to 0.6; and more preferably 0.2 to 0.4.

[0023] In another preferred embodiment, the anionic lipid nanoparticles contain: anionic phospholipids, auxiliary lipids (such as cholesterol), PEGylated lipids, and optional other functional components.

[0024] In another preferred embodiment, the anionic phospholipid is selected from the group consisting of: DSPA, DPPA, DMPA, DLPA, DOPA, or combinations thereof, preferably DSPA.

[0025] In another preferred embodiment, the PEGylated lipid is selected from the group consisting of DMG-PEG, DSG-PEG, DSPE-PEG, DSPE-MPEG, DPPE-MPEG, or combinations thereof.

[0026] In another preferred embodiment, the PEGylated lipid further comprises a maleimide group.

[0027] In another preferred embodiment, the composition of the anionic lipid nanoparticles is selected from the group consisting of: DSPA / DSPE-PEG 2000-MAL / Chol, DPPG / DPPC / Chol, PS / DOPC / Chol, DMPG / HSPC / Chol / DSPE-PE G2000, or combinations thereof; preferably DSPA / DSPE-PEG2000-MAL / Chol;

[0028] Chol refers to cholesterol.

[0029] In another preferred embodiment, the molar ratio of anionic phospholipids:PEGylated lipids:cholesterol compounds in the anionic lipid nanoparticles satisfies 5:2:3.

[0030] In another preferred embodiment, the manganese adjuvant is a compound containing manganese ions, preferably a manganese ion salt.

[0031] In another preferred embodiment, the manganese ion is selected from the group consisting of divalent manganese ions, tetravalent manganese ions, or combinations thereof.

[0032] In another preferred embodiment, the manganese ion salt is selected from the group consisting of manganese chloride, manganese sulfate, manganese carbonate, manganese phosphate, manganese pyrophosphate, manganese polyphosphate, potassium permanganate, or combinations thereof, preferably manganese chloride.

[0033] In another preferred embodiment, the concentration of the manganese ion salt in the prepared vaccine composition is 1–10 mM, more preferably 1–5 mM, and even more preferably 3–5 mM.

[0034] In another preferred embodiment, in the whole-cell tumor vaccine loaded with manganese adjuvant (i.e., the ternary complex of "tumor cells-anionic lipid nanoparticles-manganese adjuvant"), the loading of manganese adjuvant is 1-20 μg of manganese ions per million tumor cells, more preferably 2-15 μg of manganese ions per million tumor cells, and even more preferably 3-10 μg of manganese ions per million tumor cells, such as about 5-6 μg of manganese ions / 10 6 One tumor cell.

[0035] In another preferred embodiment, in the whole-cell tumor vaccine loaded with manganese adjuvant (i.e., the ternary complex of "tumor cells-anionic lipid nanoparticles-manganese adjuvant"), the weight ratio of the anionic lipid nanoparticles to the manganese ions in the loaded manganese adjuvant is 1 mg: 5-100 μg (i.e., 1000: 5-100), more preferably 1 mg: 10-50 μg, and even more preferably 1 mg: 15-40 μg, such as about 20-25 μg manganese ions / mg anionic lipid nanoparticles.

[0036] In another preferred embodiment, in the ternary complex of "tumor cells-anionic lipid nanoparticles-manganese adjuvant", the ratio of tumor cells:anionic lipid nanoparticles:manganese adjuvant satisfies:

[0037] 10 6 One tumor cell: 0.2-0.3 mg anionic lipid nanoparticles; 5-6 μg manganese ions.

[0038] In a second aspect of the invention, a vaccine composition is provided comprising a whole-cell tumor vaccine loaded with manganese adjuvant as described in the first aspect of the invention and a pharmaceutically acceptable carrier.

[0039] In another preferred embodiment, the vaccine composition is a preventive and / or therapeutic vaccine composition.

[0040] In another preferred embodiment, the vaccine composition is a pharmaceutical composition.

[0041] In another preferred embodiment, the vaccine composition is a unit dosage form.

[0042] In another preferred embodiment, the number of the "tumor cell-anionic lipid nanoparticle-manganese adjuvant" ternary complex in each unit dosage form of the vaccine composition is 1 × 10⁻⁶. 6 -1×10 8 Individual, counted as tumor cells.

[0043] In another preferred embodiment, the content of manganese adjuvant in each unit dosage form of the vaccine composition is 5.76-576 μg, calculated as manganese ions.

[0044] In a third aspect of the invention, the use of a whole-cell tumor vaccine loaded with manganese adjuvant as described in the first aspect of the invention or a vaccine composition as described in the second aspect of the invention is provided for the preparation of a medicament or formulation for the prevention and / or treatment of tumors.

[0045] In another preferred embodiment, the drug or preparation is administered to a human or non-human mammal.

[0046] In another preferred embodiment, the drug or preparation is administered to a subject diagnosed with cancer or a subject with cancer susceptibility (or risk).

[0047] In another preferred embodiment, the drug or preparation has features selected from the group consisting of:

[0048] (i) Enhance the immunogenicity of vaccines;

[0049] (ii) Synergistic effect of co-delivering antigen and adjuvant;

[0050] (iii) Inducing a strong immune response against tumor cells; and / or

[0051] (iv) Obtain significantly enhanced levels of antigen-specific antibodies.

[0052] In another preferred embodiment, the drug or preparation further includes: other drugs for treating tumors.

[0053] In another preferred embodiment, the other drugs for treating tumors are selected from the group consisting of small molecule compounds, antibody drugs, antibody-drug conjugates (ADCs), immune checkpoint inhibitors, or combinations thereof.

[0054] In another preferred embodiment, the other drugs for treating tumors are selected from the group consisting of platinum compounds (such as cisplatin, carboplatin), paclitaxel compounds (such as docetaxel, paclitaxel), 5-fluorouracil, PD-1 / PD-L1 antibodies, or combinations thereof.

[0055] In another preferred embodiment, the drug or formulation may also be administered in combination with a therapy.

[0056] In another preferred embodiment, the therapy is selected from the group consisting of surgical treatment, drug treatment, radiotherapy, or combinations thereof.

[0057] In another preferred embodiment, the timing of administration of the drug or preparation is selected from the group consisting of: postoperative, pre-chemotherapy, or when WBC counts rise to >3000 / μl.

[0058] In another preferred embodiment, the drug or preparation may be administered for four treatment courses (each course lasting 3-4 weeks) or for a lifetime.

[0059] In another preferred embodiment, the tumor is selected from the group consisting of solid tumors, hematomas, or combinations thereof.

[0060] In another preferred embodiment, the tumor is a solid tumor.

[0061] In another preferred embodiment, the tumor is selected from the group consisting of: lung cancer, breast cancer, colorectal cancer, prostate cancer, stomach cancer, liver cancer, cervical cancer, esophageal cancer, thyroid cancer, pancreatic cancer, bladder cancer, kidney cancer, ovarian cancer, endometrial cancer, glioma, or combinations thereof.

[0062] In a fourth aspect of the invention, a method for preparing a whole-cell tumor vaccine loaded with manganese adjuvant as described in the first aspect of the invention or a vaccine composition as described in the second aspect of the invention is provided, the method comprising the following steps:

[0063] (S1) Preparation of surface-thiolized tumor cells;

[0064] (S2) Preparation of anionic lipid nanoparticles by thin-film hydration method;

[0065] (S3) The tumor cells obtained in step (S1) are mixed and incubated with the anionic lipid nanoparticles obtained in step (S2), and the tumor cells with surface-coupled liposomes are collected.

[0066] (S4) The tumor cells obtained in step (S3) are incubated in a manganese ion salt solution for inactivation treatment to obtain the whole-cell tumor vaccine loaded with manganese adjuvant.

[0067] In another preferred embodiment, the incubation reagent for thiolation of the tumor cell surface in step (S1) is a 2-iminothione hydrochloride solution (or Traut's reagent).

[0068] In another preferred embodiment, the incubation temperature is room temperature.

[0069] In another preferred embodiment, step (S2) includes the following sub-steps:

[0070] (S2a) Anionic phospholipids, PEGylated lipids, auxiliary lipids (such as cholesterol) and optional other functional components are dissolved in an organic solvent and dried by rotary evaporation under reduced pressure to form a film;

[0071] (S2b) The film obtained in step (S2a) is hydrated and ultrasonically homogenized in an aqueous phase to obtain anionic lipid nanoparticles with uniform diameter.

[0072] In another preferred embodiment, the concentration of the incubation reagent is 0.5–5 mg / mL, more preferably 0.5–3 mg / mL, and even more preferably 1–2 mg / mL.

[0073] In another preferred embodiment, the incubation time is 0.5 to 3 hours, more preferably 0.5 to 2 hours, and even more preferably 0.5 to 1 hour.

[0074] In another preferred embodiment, the temperature at which the film is hydrated in the aqueous phase is 50–80°C; more preferably 60–80°C; and even more preferably 70–80°C.

[0075] In another preferred embodiment, the incubation ratio of the tumor cells to the anionic lipid nanoparticles is 10:1. 6 One tumor cell: 100–500 μg anionic lipid nanoparticles; preferably 106 For each tumor cell: 200–400 μg of anionic lipid nanoparticles, more preferably 10 μg. 6 One tumor cell: 200-250 μg anionic lipid nanoparticles.

[0076] In another preferred embodiment, the incubation time of the surface-thiolized tumor cells and anionic lipid nanoparticles is 0.5-3 h, more preferably 0.5-2 h, and even more preferably 1-2 h.

[0077] In another preferred embodiment, the concentration of the manganese ion salt solution is 1–10 mM, more preferably 1–5 mM, and even more preferably 3–5 mM.

[0078] In another preferred embodiment, the incubation time of the manganese ion salt solution is 0-2 h, more preferably 0-1 h, and even more preferably 0.15-0.3 h.

[0079] In another preferred embodiment, step (S4) includes the following sub-steps:

[0080] (S4a) The tumor cells obtained in step (S3) are incubated in a manganese ion salt solution and then subjected to two repeated freeze-thaw cycles in liquid nitrogen to inactivate them, thereby obtaining the whole-cell tumor vaccine loaded with manganese adjuvant.

[0081] In another preferred embodiment, the freeze-thaw time is 1 to 10 minutes per freeze-thaw cycle, more preferably 3 to 10 minutes per freeze-thaw cycle, and even more preferably 5 to 10 minutes per freeze-thaw cycle.

[0082] In a fifth aspect of the invention, a reagent combination for preparing a whole-cell tumor vaccine loaded with manganese adjuvant as described in the first aspect of the invention is provided, the reagent combination comprising:

[0083] (a) Tumor cells;

[0084] (b) Anionic lipid nanoparticles, wherein the anionic lipid nanoparticles can be covalently coupled to the tumor cells; and

[0085] (c) Manganese adjuvant, wherein the manganese adjuvant may be loaded onto the “tumor cell-anionic lipid nanoparticle” binary complex.

[0086] In a sixth aspect of the invention, a reagent kit is provided, the reagent kit comprising the reagent combination as described in the fifth aspect of the invention.

[0087] In a seventh aspect of the invention, a method for preventing or treating tumors is provided, comprising the steps of administering to a desired subject a whole-cell tumor vaccine loaded with manganese adjuvant as described in the first aspect of the invention or a vaccine composition as described in the second aspect of the invention.

[0088] In another preferred embodiment, the administration includes intramuscular injection, tail injection, subcutaneous injection, or a combination thereof.

[0089] In another preferred embodiment, the object includes an animal model.

[0090] In another preferred embodiment, the object includes a human or a non-human mammal, preferably a human.

[0091] In another preferred embodiment, the tumor is selected from the group consisting of: lung cancer, breast cancer, colorectal cancer, prostate cancer, stomach cancer, liver cancer, cervical cancer, esophageal cancer, thyroid cancer, pancreatic cancer, bladder cancer, kidney cancer, ovarian cancer, endometrial cancer, glioma, or combinations thereof.

[0092] In another preferred embodiment, the timing of administration is selected from the group consisting of: postoperative, pre-chemotherapy, or when WBC counts rise to >3000 / μl.

[0093] In another preferred embodiment, the number of applications may be four treatment courses (each course lasting 3-4 weeks) or for a lifetime.

[0094] In another preferred embodiment, the whole-cell tumor vaccine or vaccine composition is administered to the subject, thereby imparting to the subject a significantly enhanced level of antigen-specific antibodies.

[0095] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0096] Figure 1 The left image shows the particle size (left figure) and zeta potential characterization (right figure) of anionic liposomes and manganese ion-loaded liposomes in one embodiment of the present invention; wherein LNP-Mn represents manganese ion-loaded liposomes.

[0097] Figure 2 This invention illustrates an embodiment in which DiR-containing anionic liposomes were successfully loaded onto the surface of tumor cells.

[0098] Figure 3 The whole-cell tumor vaccine loaded with manganese adjuvant of the present invention has been shown to substantially retain all the antigens of tumor cells.

[0099] Figure 4 The whole-cell tumor vaccine loaded with manganese adjuvant of the present invention significantly promotes the maturation of BMDCs.

[0100] Figure 5 The whole-cell tumor vaccine loaded with manganese adjuvant of the present invention significantly activates the cGAS-STING pathway.

[0101] Figure 6 The whole-cell tumor vaccine loaded with manganese adjuvant of the present invention significantly stimulated the proliferation of CD8+ T cells when co-incubated with spleen mixed immune cells.

[0102] Figure 7 The whole-cell tumor vaccine with manganese adjuvant of the present invention has been shown to have significant therapeutic efficacy against prostate cancer in RM-1 mice.

[0103] Figure 8 The whole-cell tumor vaccine loaded with manganese adjuvant of the present invention was shown to efficiently induce an immune response in prostate cancer in RM-1 mice.

[0104] Figure 9 The whole-cell tumor vaccine loaded with manganese adjuvant of the present invention has been shown to have significant therapeutic efficacy against prostate cancer in MYC-CaP mice. Detailed Implementation

[0105] Through extensive and in-depth research and numerous screenings, the inventors have unexpectedly developed a uniquely structured whole-cell tumor vaccine loaded with manganese adjuvant. This whole-cell tumor vaccine loaded with manganese adjuvant (hereinafter referred to as "the tumor vaccine of this invention") not only preserves the morphology of tumor cells and all possible antigens, but also loads extremely high amounts of manganese adjuvant (up to approximately 6 μg / 10⁻¹) in situ onto the tumor cells. 6 (Each tumor cell) can thus synergistically and efficiently stimulate an immune response. This invention was developed based on this principle.

[0106] The tumor vaccine of this invention can co-deliver whole-cell antigen and adjuvant, exhibiting a significant synergistic effect. Experiments show that, compared to a simple whole-cell tumor vaccine or a mixture of whole-cell tumor vaccine and manganese adjuvant, the manganese adjuvant-loaded whole-cell tumor vaccine of this invention not only significantly increases the manganese adjuvant loading capacity of tumor cells, but also more effectively promotes the maturation of dendritic cells derived from bone marrow cells, fully activates the cGAS-STING signaling pathway, and promotes CD8... + T cell proliferation and activation significantly stimulate and generate a strong immune response, thereby significantly inhibiting the growth of tumor cells.

[0107] the term

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0109] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0110] As used in this article, the terms “DCs” and “dendritic cells” are used interchangeably. They refer to the most powerful professional antigen-presenting cells in the immune system, which play a crucial role in bridging innate and adaptive immunity. Their main function is to capture, process, and present antigens, activate naive T cells, and thus initiate a specific immune response.

[0111] As used in this article, the term "BMDC" refers to dendritic cells induced from mouse bone marrow cells.

[0112] As used in this article, the term "CD8" + T cells are a key type of cytotoxic lymphocyte in the adaptive immune system. They play a central role in anti-infection, anti-tumor immunity, and immune regulation by recognizing and eliminating abnormal cells such as cells infected by pathogens and mutated tumor cells.

[0113] As used in this article, the term "room temperature" refers to 4–30°C, preferably 15–25°C.

[0114] As used in this article, the term "IFN-γ" + CD8 + T cells, cytotoxic CD8 + "T cells" can be used interchangeably. They can directly kill tumor cells by secreting perforin and granzymes, while simultaneously secreting interferon IFN-γ to synergistically enhance the anti-tumor immune response, thereby inhibiting tumor growth.

[0115] abbreviations

[0116] DSPA: Distearylphosphatidyl acid

[0117] DPPA: Dipalmitoylphosphatidyl

[0118] DMPA: 1,2-dimyristoylphosphatidic acid

[0119] DLPA: Dilauroyl phosphatidyl acid

[0120] DOPA: Dioleoylphosphatidic acid

[0121] DSPE: Disteazygosylphosphatidylethanolamine

[0122] DPPE: Dipalmitoylphosphatidylethanolamine

[0123] DMG-PEG: 1,2-Dimyristic-rac-glycerol-3-polyethylene glycol, also known as PEGylated dimyristic acid glyceride

[0124] DSG-PEG: Distearatel-rac-glycerol-polyethylene glycol

[0125] DSPE-PEG: Disteazylecithin ethanolamine-polyethylene glycol

[0126] DSPE-MPEG: Methoxy-polyethylene glycol-distearatephosphatidylethanolamine

[0127] DPPE-MPEG: Methoxy-polyethylene glycol-dipalmitoylphosphatidylethanolamine

[0128] BMDC: Mouse bone marrow-derived dendritic cells

[0129] GM-CSF: Granulocyte-Macrophage Colony-Stimulating Factor

[0130] LNP: lipid nanoparticles

[0131] LNP-Mn: Liposomes loaded with manganese ions

[0132] PEG: Polyethylene Glycol

[0133] DSPE-PEG2000-MAL: Phosphatidylethanolamine-polyethylene glycol 2000-maleimide

[0134] DiR: Deep red fluorescent probe for cell membranes

[0135] Manganese adjuvant

[0136] As used herein, the term “manganese adjuvant” refers to an adjuvant containing manganese (such as manganese ions), including salts containing manganese ions (such as MnCl2, MnSO4) or oxides of Mn (such as MnO2, Mn3O4).

[0137] As used herein, the term "manganese ion" refers to the ionic state of metallic manganese, such as divalent, tetravalent, or other valence states of manganese, preferably divalent manganese ions (Mn). 2+ ).

[0138] In this invention, a preferred manganese adjuvant is a salt containing manganese ions. In this invention, the manganese ion salt can act as an agonist of the cGAS-STING signaling pathway, releasing Mn... 2+ In the cytoplasm, it binds to cGAS, enhancing cGAS's sensitivity to double-stranded DNA. It also enhances the affinity of cGAMP and STING on the endoplasmic reticulum surface, causing IRF3 phosphorylation, activating the NF-κB pathway, and promoting IFN-I production.

[0139] In this invention, Mn 2+It is itself a potent cGAS activator that can directly activate cGAS without relying on DNA and trigger the unique catalytic synthesis of cGAMP, thereby inducing cells to produce type I IFNs and cytokines in the absence of infection, thus exerting the effects of STING agonists.

[0140] Anionic lipid nanoparticles

[0141] As used in this article, "LNP", "liposome", "anionic lipid nanoparticle", and "anionic liposome" are interchangeable and refer to a type of lipid nanoparticle, which is a commonly used drug carrier with good biocompatibility.

[0142] This invention proposes an anionic lipid nanoparticle, comprising an outer shell formed by the hydrophilic end of a PEGylated lipid nanoparticle extending outwards, wherein the hydrophobic end of the PEGylated lipid is combined with the hydrophobic end of the anionic phospholipid, and cholesterol compounds and other auxiliary lipids help stabilize the backbone structure.

[0143] In this invention, anionic lipid nanoparticles can be used as carriers to construct vaccines by physically mixing with peptide or protein antigens. The carrier of this vaccine (i.e., the anionic lipid nanoparticles of this application) can promote the maturation of DCs and the presentation function of antigens, enhance the anti-tumor immune response, exert the effect of adjuvant, and achieve the goal of improving the therapeutic effect of the vaccine.

[0144] In this invention, the zeta potential of the anionic lipid nanoparticles is -40mV to -30mV.

[0145] In this invention, after loading with manganese adjuvant, the zeta potential of the anionic lipid nanoparticles is -20mV to -10mV.

[0146] Specifically, the anionic lipid nanoparticles have a particle size of 20–500 nm; preferably 50–300 nm; and more preferably 100–200 nm.

[0147] Specifically, the polydispersity index of the anionic lipid nanoparticles is 0.2 to 0.8; preferably 0.2 to 0.6; and more preferably 0.2 to 0.4.

[0148] In this invention, the anionic lipid nanoparticles contain: anionic phospholipids, auxiliary lipids (such as cholesterol), PEGylated lipids, and optional other functional components.

[0149] Specifically, the anionic phospholipid is selected from the group consisting of: DSPA, DPPA, DMPA, DLPA, DOPA, or combinations thereof, preferably DSPA.

[0150] Specifically, the PEGylated lipids are selected from the group consisting of DMG-PEG, DSG-PEG, DSPE-PEG, DSPE-MPEG, DPPE-MPEG, or combinations thereof.

[0151] Specifically, the PEGylated lipid further comprises maleimide groups.

[0152] Specifically, the composition of the anionic lipid nanoparticles is selected from the group consisting of: DSPA / DSPE-PEG2000-MAL / Chol, DPPG / DPPC / Chol, PS / DOPC / Chol, DMPG / HSPC / Chol / DSPE-PEG2000, or combinations thereof; preferably DSPA / DSPE-PEG2000-MAL / Chol.

[0153] Chol refers to cholesterol.

[0154] In this invention, the molar ratio of anionic phospholipids: PEGylated lipids: cholesterol compounds in the anionic lipid nanoparticles satisfies 5:2:3.

[0155] Whole-cell tumor vaccine loaded with manganese adjuvant

[0156] As used herein, the terms “whole-cell tumor vaccine loaded with manganese adjuvant of the present invention”, “tumor cell-anionic lipid nanoparticle-manganese adjuvant ternary complex of the present invention”, “whole-cell tumor vaccine loaded with manganese adjuvant”, or “ternary complex whole-cell tumor vaccine” are used interchangeably to refer to the whole-cell tumor vaccine loaded with manganese adjuvant described in the first aspect of the present invention.

[0157] This invention provides a whole-cell tumor vaccine loaded with manganese adjuvant, the whole-cell tumor vaccine loaded with manganese adjuvant comprising:

[0158] (a) Tumor cells;

[0159] (b) Anionic lipid nanoparticles, said anionic lipid nanoparticles covalently coupled to said tumor cells, forming a "tumor cell-anionic lipid nanoparticle" binary complex; and

[0160] (c) Manganese adjuvant, wherein the manganese adjuvant is loaded onto the binary complex of “tumor cell-anionic lipid nanoparticle” to form a ternary complex of “tumor cell-anionic lipid nanoparticle-manganese adjuvant”.

[0161] In another preferred embodiment, the tumor cells are tumor cells whose surface is modified with functional groups.

[0162] In another preferred embodiment, the tumor cells are tumor cells whose surface has been modified with thiolation.

[0163] In another preferred embodiment, the anionic lipid nanoparticles are coupled to the tumor cells via disulfide bonds.

[0164] In another preferred embodiment, the "tumor cell-anionic lipid nanoparticle-manganese adjuvant" ternary complex is loaded with Mn. 2+ ion.

[0165] In another preferred embodiment, the tumor cells are inactivated tumor cells.

[0166] In another preferred embodiment, the tumor cells are inactivated by a treatment selected from the group consisting of irradiation, liquid nitrogen treatment, or a combination thereof.

[0167] In another preferred embodiment, the prostate cancer cells include: (a) castration-resistant prostate cancer cells or (b) androgen-sensitive prostate cancer cells.

[0168] In another preferred embodiment, the anionic lipid nanoparticles themselves have a Zeta potential of -40mV to -30mV.

[0169] In another preferred embodiment, after loading with manganese adjuvant, the zeta potential of the anionic lipid nanoparticles is -20mV to -10mV.

[0170] In another preferred embodiment, the manganese adjuvant is a compound containing manganese ions, preferably a manganese ion salt.

[0171] In another preferred embodiment, in the whole-cell tumor vaccine loaded with manganese adjuvant (i.e., the ternary complex of "tumor cells-anionic lipid nanoparticles-manganese adjuvant"), the loading of manganese adjuvant is 1-20 μg of manganese ions per million tumor cells, more preferably 2-15 μg of manganese ions per million tumor cells, and even more preferably 3-10 μg of manganese ions per million tumor cells, such as about 5-6 μg of manganese ions / 10 6 One tumor cell.

[0172] In another preferred embodiment, in the whole-cell tumor vaccine loaded with manganese adjuvant (i.e., the ternary complex of "tumor cells-anionic lipid nanoparticles-manganese adjuvant"), the weight ratio of the anionic lipid nanoparticles to the manganese ions in the loaded manganese adjuvant is 1 mg: 5-100 μg (i.e., 1000: 5-100), more preferably 1 mg: 10-50 μg, and even more preferably 1 mg: 15-40 μg, such as about 20-25 μg manganese ions / mg anionic lipid nanoparticles.

[0173] In another preferred embodiment, in the ternary complex of "tumor cells-anionic lipid nanoparticles-manganese adjuvant", the ratio of tumor cells:anionic lipid nanoparticles:manganese adjuvant satisfies:

[0174] 106 One tumor cell: 0.2-0.3 mg anionic lipid nanoparticles; 5-6 μg manganese ions.

[0175] Preparation method of whole-cell tumor vaccine loaded with manganese adjuvant

[0176] This invention provides a method for preparing a whole-cell tumor vaccine or vaccine composition loaded with manganese adjuvant, the method comprising the following steps:

[0177] (S1) Preparation of surface-thiolized tumor cells;

[0178] (S2) Preparation of anionic lipid nanoparticles by thin-film hydration method;

[0179] (S3) The tumor cells obtained in step (S1) are mixed and incubated with the anionic lipid nanoparticles obtained in step (S2), and the tumor cells with surface-coupled liposomes are collected.

[0180] (S4) The tumor cells obtained in step (S3) are incubated in a manganese ion salt solution for inactivation treatment to obtain the whole-cell tumor vaccine loaded with manganese adjuvant.

[0181] In another preferred embodiment, the incubation reagent for thiolation of the tumor cell surface in step (S1) is a 2-iminothione hydrochloride solution (or Traut's reagent).

[0182] In another preferred embodiment, the incubation temperature is room temperature.

[0183] In another preferred embodiment, step (S2) includes the following sub-steps:

[0184] (S2a) Anionic phospholipids, PEGylated lipids, auxiliary lipids (such as cholesterol) and optional other functional components are dissolved in an organic solvent and dried by rotary evaporation under reduced pressure to form a film;

[0185] (S2b) The film obtained in step (S2a) is hydrated and ultrasonically homogenized in an aqueous phase to obtain anionic lipid nanoparticles with uniform diameter.

[0186] In another preferred embodiment, the concentration of the incubation reagent is 0.5–5 mg / mL, more preferably 0.5–3 mg / mL, and even more preferably 1–2 mg / mL.

[0187] In another preferred embodiment, the incubation time is 0.5 to 3 hours, more preferably 0.5 to 2 hours, and even more preferably 0.5 to 1 hour.

[0188] In another preferred embodiment, the incubation ratio of the tumor cells to the anionic lipid nanoparticles is 10:1. 6One tumor cell: 100–500 μg anionic lipid nanoparticles; preferably 10 6 For each tumor cell: 200–400 μg of anionic lipid nanoparticles, more preferably 10 μg. 6 One tumor cell: 200-250 μg anionic lipid nanoparticles.

[0189] In another preferred embodiment, step (S4) includes the following sub-steps:

[0190] (S4a) The tumor cells obtained in step (S3) are incubated in a manganese ion salt solution and then subjected to two repeated freeze-thaw cycles in liquid nitrogen to inactivate them, thereby obtaining the whole-cell tumor vaccine loaded with manganese adjuvant.

[0191] In another preferred embodiment, the freeze-thaw time is 1 to 10 minutes per freeze-thaw cycle, more preferably 3 to 10 minutes per freeze-thaw cycle, and even more preferably 5 to 10 minutes per freeze-thaw cycle.

[0192] The vaccine composition of the present invention

[0193] The present invention also provides a vaccine composition. A preferred vaccine composition is a prophylactic vaccine composition. The vaccine composition of the present invention can be a monovalent or multivalent vaccine composition.

[0194] These vaccines contain high-purity, high-titer tumor antigens and are typically combined with pharmaceutically acceptable carriers, including any carrier that does not itself induce antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolizing macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid agglomerates (such as oil droplets or liposomes), and inactive viral particles. These carriers are well known to those skilled in the art. Additionally, these carriers can act as immunostimulants (or adjuvants).

[0195] The vaccine compositions of the present invention (including tumor antigens, pharmaceutically acceptable carriers and / or adjuvants) typically contain diluents such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc., may be present in the vaccine compositions.

[0196] More specifically, vaccines, including immunogenic compositions, contain an immunogenically effective dose of tumor antigen, as well as the other required components mentioned above. An "immunogenically effective dose" refers to the amount administered to an individual, either as a single or continuous dose, that is effective for treatment or prevention. This dose is determined based on the animal's (e.g., human) physiological condition, the immune system's ability to synthesize antibodies, the required level of protection, and other relevant factors.

[0197] In this invention, vaccine compositions or immunogenic compositions can be formulated into injectable formulations, such as liquid solutions or emulsions; they can also be formulated into solid forms suitable for reconstitution into solutions or suspensions, or liquid excipients, prior to injection. The formulation can also be emulsified or encapsulated in liposomes to enhance adjuvant effects under the aforementioned pharmaceutically acceptable carriers.

[0198] The conventional method is to administer the immunogenic composition via injection through a parenteral route (subcutaneous or intramuscular). Other formulations suitable for different routes of administration include oral and transdermal applications. Therapeutic doses can be administered as single-dose or multi-dose regimens. The vaccine composition of the present invention can be administered in combination with other immunomodulators.

[0199] In this invention, a vaccine composition is provided comprising a whole-cell tumor vaccine loaded with manganese adjuvant as described in the first aspect of the invention and a pharmaceutically acceptable carrier.

[0200] In another preferred embodiment, the vaccine composition is a unit dosage form.

[0201] In another preferred embodiment, the number of the "tumor cell-anionic lipid nanoparticle-manganese adjuvant" ternary complex in each unit dosage form of the vaccine composition is 1 × 10⁻⁶. 6 -1×10 8 Individual, counted as tumor cells.

[0202] In another preferred embodiment, the content of manganese adjuvant in each unit dosage form of the vaccine composition is 5.76-576 μg, calculated as manganese ions.

[0203] The main advantages of this invention include:

[0204] (a) The whole-cell tumor vaccine loaded with manganese adjuvant of the present invention not only preserves the tumor antigen very well, but also has an extremely high manganese adjuvant loading, for example, the manganese ion loading can reach about 22 μg / mg or higher on a liposome basis, and the manganese adjuvant loading can be as high as about 6 μg / 10 on a tumor cell basis. 6 One tumor cell.

[0205] (b) The tumor vaccine of the present invention, as a complete ternary complex, can co-deliver antigens and adjuvants, exhibiting a synergistic effect. The tumor vaccine of the present invention not only significantly promotes the maturation and effective activation of dendritic cells derived from bone marrow cells, releasing manganese ions into the cytoplasm to fully activate the cGAS-STING signaling pathway, but also efficiently presents tumor antigen signals to T cells, promoting CD8+. + T cell proliferation and activation significantly enhance immunogenicity, strengthen anti-tumor immune responses, and generate a strong immune response.

[0206] (c) In vivo experiments show that the whole-cell tumor vaccine loaded with manganese adjuvant of the present invention can effectively inhibit tumor growth and has important clinical significance for the prevention and treatment of tumors.

[0207] (d) The whole-cell tumor vaccine loaded with manganese adjuvant provided by the present invention uses whole tumor cells as antigens and contains all possible antigens of tumor cells, thus having a broad coverage spectrum and avoiding the problem of antigen omission caused by using only a portion of the antigens to prepare the vaccine.

[0208] (e) The whole-cell tumor vaccine loaded with manganese adjuvant of the present invention provides a metal ion delivery platform. If necessary, the manganese ion salt solution portion (e.g., about 5-25%) can be replaced with other metal salt solutions, such as Al. 3+ Ca 2+ Zn 2+ Mg 2+ and Fe 2+ / 3+ This allows for the co-delivery of different metal ions and tumor antigens, which can directly activate innate or adaptive immunity.

[0209] (f) The whole-cell tumor vaccine formulation with manganese adjuvant of the present invention is simple to formulate and easy to prepare, and is expected to be applied in the field of vaccine production.

[0210] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0211] Example 1: Preparation method of whole-cell tumor vaccine loaded with manganese adjuvant

[0212] In this embodiment, the preparation method of the whole-cell tumor vaccine loaded with manganese adjuvant is as follows:

[0213] (1) Collect tumor cells, wash with PBS solution, resuspend with Traut's reagent, incubate with shaking at room temperature for a period of time, and then wash the cells with PBS solution by centrifugation to obtain tumor cells with increased surface thiol density.

[0214] (2) Preparation of anionic liposomes containing DSPE-PEG2000-MAL by thin-film hydration method: DSPA, DSPE-PEG2000-MAL, and cholesterol were prepared in a molar ratio of 5:2:3 and dissolved in methanol. After complete dissolution, the methanol was evaporated using a vacuum rotary evaporator, forming a thin film at the bottom of the flask. The hydrated film was stirred in an 80°C water bath. Subsequently, it was homogenized by ultrasound to obtain liposomes with an average diameter of 100–200 nm.

[0215] (3) Add anionic liposomes containing DSPE-PEG2000-MAL to the surface-thiolized tumor cells and mix them evenly. 250 μg of liposomes are added per million cells. After incubation for a period of time, the cells are washed by centrifugation with PBS solution to remove liposomes that have not bound to the cells, thus obtaining tumor cells coupled with anionic liposomes on the cell surface.

[0216] (4) Add a 5mM manganese ion salt solution to the tumor cells obtained in step (3) and mix well. Incubate for a period of time, centrifuge and wash to remove unbound metal ions and obtain tumor cells loaded with manganese adjuvant.

[0217] (5) The tumor cells obtained in step (4) are frozen and thawed twice with liquid nitrogen, freezing for 5 minutes each time, to obtain a whole-cell tumor vaccine loaded with manganese adjuvant.

[0218] Example 2: Characterization of whole-cell tumor vaccine loaded with manganese adjuvant

[0219] Based on the preparation method of Example 1, the whole-cell tumor vaccine loaded with manganese adjuvant of this example was obtained and subjected to a series of characterizations.

[0220] 2.1 Characterization of liposome particle size and surface potential

[0221] 1) Experimental method:

[0222] Anionic liposomes were prepared by thin-film hydration and incubated with a manganese salt solution. Free metal ions were removed by ultrafiltration, and the liposome particle size and zeta potential were detected by a particle size analyzer (ZS90, Malvern).

[0223] 2) Experimental results:

[0224] like Figure 1 As shown, the anionic liposomes have a particle size of about 140 nm and a zeta potential of about -30 mV; while the liposomes loaded with manganese ions have a particle size of about 177 nm and a zeta potential of about -14 mV. This indicates that the increase in zeta potential proves that manganese ions were successfully loaded on the surface of the liposomes.

[0225] 2.2 Liposome loading on cell surface

[0226] 1) Experimental method:

[0227] The collected tumor cells were resuspended in 1 mg / mL Traut's reagent and incubated for 1 h to perform surface thiolation. Anionic liposomes loaded with the fluorescent dye DiR were prepared by thin-film hydration. The thiolated tumor cells were co-incubated with the liposomes, followed by washing with PBS to remove unbound liposomes. The fluorescence intensity of DiR on the surface of the tumor cells was detected by flow cytometry, with the fluorescence detection channel for DiR being APC-Cy7.

[0228] 2) Experimental results:

[0229] like Figure 2 As shown, after surface-thiolized tumor cells were mixed with liposomes containing MAL, the DiR fluorescence intensity on the cell surface increased significantly, and the loading of manganese ions had almost no effect on the coupling between liposomes and cells.

[0230] 2.3 Antigen Retention of Manganese-Loaded Whole-Cell Tumor Vaccines

[0231] 1) Experimental method:

[0232] A quantitative amount of untreated tumor cells, tumor cells inactivated by liquid nitrogen freeze-thaw, and whole-cell tumor vaccine loaded with manganese adjuvant prepared according to Example 1 were taken. The cells were lysed using RIPA lysis buffer, and the supernatant was used to run an SDS-PAGE gel. The gel was stained with Coomassie Brilliant Blue solution and then imaged.

[0233] 2) Experimental results:

[0234] like Figure 3 As shown, the whole-cell tumor vaccine loaded with manganese adjuvant retains very complete antigens from tumor cells.

[0235] 2.4 Characterization of manganese adjuvant loading in whole-cell tumor vaccines

[0236] 1) Experimental method:

[0237] The whole-cell tumor vaccine loaded with manganese adjuvant was frozen at -80°C and then freeze-dried under vacuum for 24 hours. Subsequently, the manganese ion loading in the whole-cell tumor vaccine loaded with manganese adjuvant was determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0238] 2) Experimental results:

[0239] The results showed that the manganese ion loading in the manganese-loaded liposomes was 21.84 μg per milligram of liposomes; and the manganese ion loading in each million tumor cells was 5.76 μg.

[0240] Example 3: Whole-cell tumor vaccine loaded with manganese adjuvant significantly promotes BMDC maturation

[0241] 1) Experimental method:

[0242] Bone marrow mononuclear cells were collected from 8-week-old male C57BL / 6 mice, and the cell concentration was adjusted to 102. 6 Cells were cultured at a density of 10 cells / mL, and 20 ng / mL of cytokine GM-CSF was added. The cell suspension was seeded into 6-well plates, and half of the medium was changed every 2 days, with fresh cytokine added.

[0243] Mouse bone marrow-derived dendritic cells (BMDCs) were obtained by culturing for 7 days. The BMDCs obtained by the above method were then cultured at a rate of 1×10⁻⁶. 6 Seeds were planted at a density of cells / well in 12-well plates, and positive controls were added using lipopolysaccharide (200 ng / mL), liposomes (264 μg / mL), manganese-loaded liposomes (264 μg / mL, containing 5.76 μg of manganese ions), and whole-cell tumor antigen (1 × 10⁻⁶ cells / well). 6 (cells / mL), or whole-cell tumor vaccines loaded with manganese adjuvant (based on tumor cell count, 1×10⁻⁶). 6 Stimulation was performed using a solution containing 5.76 μg of manganese ions per mL.

[0244] After 24 h of culture, cells were collected, washed with PBS, and dead cells were labeled using the Zombie Aqua™ Fixable Viability Kit. Cells were then incubated at 4 °C for 30 min with antibody dilution buffers (PerCP / Cy5.5 anti-mouse CD11C antibody, PE anti-mouse CD80 antibody, and Brilliant Violet 785™ anti-mouse CD86 antibody). Following washing, the expression levels of the co-stimulatory molecules CD80 / CD86 on the surface of BMDCs were detected by flow cytometry.

[0245] 2) Experimental results:

[0246] The results for the expression levels of the co-stimulatory molecules CD80 / CD86 on the surface of BMDCs are shown in... Figure 4 See Table 1.

[0247] Table 1. Expression levels of co-stimulatory molecules CD80 / CD86 on the surface of BMDCs

[0248]

[0249] like Figure 4As shown, compared with untreated inactivated tumor cells, the whole-cell tumor vaccine loaded with manganese adjuvant significantly increased the expression of co-stimulatory molecules CD80 / CD86 on the surface of BMDCs and significantly promoted the maturation of BMDCs.

[0250] Example 4: Whole-cell tumor vaccine loaded with manganese adjuvant significantly activated the cGAS-STING pathway

[0251] 1) Experimental method:

[0252] The BMDC obtained by the above method is used in 2×10 6 Seeds were placed in 6-well plates at a density of cells / well, and loaded with liposomes (264 μg / mL), manganese-loaded liposomes (264 μg / mL, containing 5.76 μg of manganese ions), and whole-cell tumor antigen (1 × 10⁻⁶ cells / well), respectively. 6 (cells / mL), or whole-cell tumor vaccines loaded with manganese adjuvant (based on tumor cell count, 1×10⁻⁶). 6 Protein quantification was performed using the BCA method with supernatant containing 5.76 μg of manganese ions per mL. 20 μg of protein was then run on an SDS-PAGE gel and transferred to a PVDF membrane. The expression of cGAS-STING pathway-related proteins was detected using antibodies TBK1, p-TBK1, IRF3, p-IRF3, STING, and p-STING.

[0253] 2) Experimental results:

[0254] The expression results of cGAS-STING pathway-related proteins are shown in... Figure 5 And Table 2.

[0255] Table 2. Expression of cGAS-STING pathway-related proteins

[0256]

[0257] like Figure 5 As shown, compared with other groups, the phosphorylation levels of TBK1, IRF3 and STING proteins were increased in both the liposome-manganese adjuvant group and the whole-cell tumor vaccine group loaded with manganese adjuvant. However, unexpectedly, the increase in phosphorylation levels of TBK1, IRF3 and STING proteins was more significant in the whole-cell tumor vaccine group loaded with manganese adjuvant.

[0258] The above results indicate that the whole-cell tumor vaccine loaded with manganese adjuvant has excellent activation ability for the cGAS-STING pathway.

[0259] Example 5: Whole-cell tumor vaccine loaded with manganese adjuvant can promote CD8 + T cell proliferation

[0260] 1) Experimental method:

[0261] After obtaining single cells from the spleen of C57BL / 6 mice, they were labeled using CFSE, and then analyzed at 1×10⁻⁶ cells per cell line. 6 Cells were seeded at a density of [number] cells / mL in 24-well plates and co-cultured for 48 h with liposomes, manganese-loaded liposomes, whole-cell tumor antigen, or whole-cell tumor vaccine loaded with manganese adjuvant. Cells were then collected, centrifuged at 500g for 5 min, and dead cells were labeled using the Zombie Aqua™ Fixable Viability Kit. Cells were then diluted with antibody dilution buffer (Alexa). 700anti-mouse CD45antibody, APC / Cyanine7 anti-mouse CD3 antibody, Brilliant Violet 650 TM Anti-mouse CD4 and PE / Cyanine7 anti-mouse CD8a antibody were incubated at 4°C for 30 min; after incubation, CD8 were measured using flow cytometry. + T cell proliferation.

[0262] 2) Experimental results:

[0263] CD8 + T cell proliferation, such as Figure 6 As shown in Table 3.

[0264] Table 3.CD8 + T cell proliferation

[0265]

[0266] like Figure 6 As shown, compared with the saline group, liposome-manganese adjuvant group, and whole-cell tumor antigen group, the CD8+ of the whole-cell tumor vaccine group loaded with manganese adjuvant was significantly higher. + The proportion of T cell proliferation was significantly increased, indicating that the whole-cell tumor vaccine loaded with manganese adjuvant significantly promoted CD8 proliferation. + T cell proliferation.

[0267] Example 6: Evaluation of therapeutic effect on tumor growth in mice

[0268] 6.1 Evaluation of the efficacy of RM-1 in inhibiting tumor growth in mice

[0269] 1) Experimental method:

[0270] Following the preparation method of the manganese-adjuvant-loaded whole-cell tumor vaccine in Example 1, a manganese-adjuvant-loaded whole-cell tumor vaccine was prepared by using RM-1 mouse prostate tumor as the whole-cell tumor antigen, loading the surface with manganese adjuvant, and then freeze-thawing and inactivating. 8 × 10⁸ mcg of the vaccine was subcutaneously injected into the right hind limb of each C57BL / 6 mouse. 3 A subcutaneous RM-1 tumor model was established using RM-1 cells in the logarithmic growth phase. RM-1 tumor-bearing mice were randomly divided into four groups (n=5). On days 2, 7, and 12, they were subcutaneously injected via the tail with equal doses of physiological saline, manganese-loaded liposomes, whole-cell tumor antigen, or manganese-adjuvant-loaded whole-cell tumor vaccine. The dosage of manganese-loaded liposomes was 1.44 mg / kg, and the dosage of whole-cell tumor antigen was 2.5 × 10⁻⁶. 8 per kg.

[0271] The day the mouse prostate cancer model was constructed was designated as day 1. Mice were randomly divided into 4 groups of 5 mice each. Based on the drug administration, the groups were designated as saline group, liposome-manganese adjuvant group, whole cell tumor antigen group, and whole cell tumor vaccine group loaded with manganese adjuvant.

[0272] According to the grouping, C57BL / 6 mice were subcutaneously injected via the tail at days 2, 7, and 12 with equal doses of physiological saline, manganese-loaded liposomes, whole-cell tumor antigen, or whole-cell tumor vaccine loaded with manganese adjuvant. The tumor volume of the mice was recorded and determined according to the formula: Tumor volume = (length × width × width) / 2.

[0273] 2) Experimental results:

[0274] Changes in tumor volume in mice, as shown in... Figure 7 and Table 4

[0275] Table 4. Tumor volume in mice

[0276]

[0277] like Figure 7 As shown, compared with the saline group, the liposome-manganese adjuvant group and the whole-cell tumor antigen group, the whole-cell tumor vaccine group loaded with manganese adjuvant showed the most significant effect in inhibiting tumor growth in mice.

[0278] The above results indicate that the whole-cell tumor vaccine loaded with manganese adjuvant of the present invention effectively inhibits the growth of prostate tumor RM-1.

[0279] 6.2 Evaluation of the therapeutic effect on MYC-CaP tumor growth in mice

[0280] 1) Experimental method:

[0281] Following the preparation method of the manganese-adjuvant-loaded whole-cell tumor vaccine in Example 1, a manganese-adjuvant-loaded whole-cell tumor vaccine was prepared by using MYC-CaP mouse prostate tumor as the whole-cell tumor antigen, loading the surface with manganese adjuvant, and then freeze-thawing and inactivating. 1×10⁻⁶ mcg was subcutaneously injected into the right hind limb of each FVB / NJ mouse. 6 A subcutaneous MYC-CaP tumor model was established using MYC-CaP cells in the logarithmic growth phase.

[0282] MYC-CaP tumor-bearing mice were randomly divided into four groups (n=5). On days 8, 13, and 18 after modeling, they were subcutaneously injected via the tail with equal doses of saline, manganese-loaded liposomes, whole-cell tumor antigen, or manganese-adjuvanted whole-cell tumor vaccine. These were designated as the saline group, liposome-manganese adjuvant group, whole-cell tumor antigen group, and manganese-adjuvanted whole-cell tumor vaccine group, respectively. The dosage of manganese-loaded liposomes was 1.44 mg / kg, and the dosage of whole-cell tumor antigen was 2.5 × 10⁻⁶ mg / kg. 8 Tumor volume per kg of mice was recorded and determined using the formula: Tumor volume = (length × width × width) / 2.

[0283] 2) Experimental results:

[0284] like Figure 9 As shown, compared with the saline group, the liposome-manganese adjuvant group and the whole-cell tumor antigen group, the whole-cell tumor vaccine group loaded with manganese adjuvant showed the most significant effect in inhibiting tumor growth in mice.

[0285] The above results demonstrate that the whole-cell tumor vaccine loaded with manganese adjuvant of the present invention effectively inhibits the growth of prostate tumor MYC-CaP.

[0286] Example 7: Evaluation of the in vivo immune effect of a whole-cell tumor vaccine loaded with manganese adjuvant

[0287] 1) Experimental method:

[0288] Following the preparation method of the manganese-adjuvant-loaded whole-cell tumor vaccine in Example 1, a manganese-adjuvant-loaded whole-cell tumor vaccine was prepared by using RM-1 mouse prostate tumor as the whole-cell tumor antigen, loading the surface with manganese adjuvant, and then freeze-thawing and inactivating. 8 × 10⁸ mcg of the vaccine was subcutaneously injected into the right hind limb of each C57BL / 6 mouse. 3 A mouse prostate cancer model was constructed using RM-1 cells in the logarithmic growth phase.

[0289] The day the mouse prostate cancer model was established was designated as day 1. Mice were randomly divided into 4 groups of 5 mice each: saline group, liposome-manganese adjuvant group, whole-cell tumor antigen group, and whole-cell tumor vaccine group loaded with manganese adjuvant. According to the group, C57BL / 6 mice were injected subcutaneously into the tail with equal doses of saline, liposomes loaded with manganese ions, whole-cell tumor antigen, or whole-cell tumor vaccine loaded with manganese ions on days 2, 7, and 12, respectively.

[0290] On day 21 after subcutaneous administration, spleens, inguinal lymph nodes, and tumor tissues of mice in each group were collected. Single-cell suspensions were prepared and stained to analyze changes in the composition and function of immune cells.

[0291] 2) Experimental results:

[0292] like Figure 8 As shown in Tables 5-7, compared with the administration of saline alone, manganese-loaded liposomes, or whole-cell tumor antigen alone, the administration of whole-cell tumor vaccines loaded with manganese adjuvants significantly promoted the maturation of dendritic cells (DCs) in inguinal lymph nodes and significantly promoted the activation of T cells in the lymph nodes.

[0293] The above results indicate that tumor vaccines loaded with manganese adjuvant can elicit a strong immune response under the synergistic stimulation of whole-cell antigen and manganese adjuvant in vivo.

[0294] in,

[0295] Dendritic cells (CD11c) + MHCⅡ + The expression of co-stimulatory molecules CD80 and CD86 on the surface of dendritic cells (DCs) (CD80 / CD86 belong to the co-stimulatory molecule family and are markers of dendritic cell maturation and activation) was analyzed, and the results are shown in the figure. Figure 8 A and Table 5;

[0296] Table 5. Expression of co-stimulatory molecules CD80 and CD86 on the surface of dendritic cells

[0297]

[0298] like Figure 8 As shown in Figure A, compared with the liposome-manganese adjuvant group and the whole-cell tumor antigen group, the number of dendritic cells (CD11c) in the lymph nodes of the whole-cell tumor vaccine group loaded with manganese adjuvant was significantly higher. + MHCⅡ + The expression of co-stimulatory molecules CD80 and CD86 on the surface of DCs was significantly increased, and T cell activation was significantly enhanced.

[0299] Ki67 in lymph nodes + CD8 + The proportion of T cells, the results are shown in Figure 8 B and Table 6.

[0300] Table 6. Ki67 in lymph nodes + CD8 + The proportion of T cells

[0301]

[0302]

[0303] like Figure 8 As shown in Figure B, compared with the liposome-manganese adjuvant group and the whole-cell tumor antigen group, Ki67 in the lymph nodes of the whole-cell tumor vaccine group loaded with manganese adjuvant was significantly higher. + CD8 + The proportion of T cells increased significantly.

[0304] Since Ki67 is a marker protein of cell proliferation and is expressed in actively dividing cells, the above results indicate that CD8+ cells in the manganese-adjuvanted whole-cell tumor vaccine group are in a proliferating state. + The number of T cells increased significantly, and the whole-cell tumor vaccine loaded with manganese adjuvant successfully activated CD8. + Clonal expansion of T cells and stronger antigen-specific immune responses.

[0305] Cytotoxic CD8 in lymph nodes + The proportion of T cells, the results are shown in Figure 8 C and Table 7.

[0306] Table 7. Cytotoxic CD8 in lymph nodes + The proportion of T cells

[0307]

[0308] like Figure 8 As shown in Figure C, compared with the liposome-manganese adjuvant group and the whole-cell tumor antigen group, the whole-cell tumor vaccine group loaded with manganese adjuvant showed higher levels of cytotoxic CD8 in the lymph nodes. + The proportion of T cells increased significantly.

[0309] The above results indicate that the whole-cell tumor vaccine loaded with manganese adjuvant activated a more potent anti-tumor immune response.

[0310] discuss

[0311] The cGAS-STING pathway is a cytoplasmic DNA sensor that plays a key role in the innate immune response. It is considered a bridge between innate and adaptive immunity, which helps dendritic cells mature and migrate and enhances the cytotoxic effects of cytotoxic T cells and natural killer cells. These advantages have led to widespread attention for cGAS-STING adjuvants in the field of tumor immunology.

[0312] Manganese ions, as a novel STING agonist, have great potential in enhancing the body's immune response. However, the rapid metabolism, short half-life, and low cellular entry efficiency of manganese ions as a single adjuvant limit the activation effect of the cGAS-STING signaling pathway. While some tumor vaccines utilize materials such as PEG, these often involve complex chemical synthesis and may be immunogenic, making their clinical translation difficult.

[0313] The whole-cell tumor vaccine loaded with manganese adjuvant of the present invention not only preserves the morphology of tumor cells and all possible antigens, but also loads extremely high amounts of manganese adjuvant (up to about 5 μg / 10⁻¹) in situ onto the tumor cells. 6 This allows tumor cell antigens, anionic liposome nanoparticles, and a high-load manganese adjuvant to synergistically and efficiently stimulate a strong autoimmune response, resulting in excellent antitumor effects.

[0314] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A whole-cell tumor vaccine loaded with manganese adjuvant, characterized in that, The whole-cell tumor vaccine loaded with manganese adjuvant comprises: (a) Tumor cells; (b) Anionic lipid nanoparticles, said anionic lipid nanoparticles covalently coupled to said tumor cells, forming a "tumor cell-anionic lipid nanoparticle" binary complex; and (c) Manganese adjuvant, wherein the manganese adjuvant is loaded onto the binary complex of "tumor cell-anionic lipid nanoparticles" to form a ternary complex of "tumor cell-anionic lipid nanoparticle-manganese adjuvant".

2. The whole-cell tumor vaccine as described in claim 1, characterized in that, The tumor cells are tumor cells whose surface has been modified with thiol.

3. The whole-cell tumor vaccine as described in claim 1, characterized in that, In the whole-cell tumor vaccine loaded with manganese adjuvant, the manganese adjuvant loading is 1-20 μg manganese ions per million tumor cells, preferably 2-15 μg manganese ions per million tumor cells, and more preferably 3-10 μg manganese ions per million tumor cells.

4. The whole-cell tumor vaccine as described in claim 1, characterized in that, In the aforementioned ternary complex of "tumor cells-anionic lipid nanoparticles-manganese adjuvant", the ratio of tumor cells:anionic lipid nanoparticles:manganese adjuvant satisfies the following: 10 6 One tumor cell: 0.2-0.3 mg anionic lipid nanoparticles; 5-6 μg manganese ions.

5. A vaccine composition, characterized in that, The vaccine composition comprises the whole-cell tumor vaccine with manganese adjuvant as described in claim 1 and a pharmaceutically acceptable carrier.

6. The vaccine composition according to claim 5, characterized in that, In each unit dosage form of the vaccine composition, the quantity of the "tumor cell-anionic lipid nanoparticle-manganese adjuvant" ternary complex is 1×10⁻⁶. 6 -1×10 8 Individual, counted as tumor cells.

7. The vaccine composition according to claim 5, characterized in that, In each unit dosage form of the vaccine composition, the content of manganese adjuvant is 5.76-576 μg, calculated as manganese ions.

8. The use of a whole-cell tumor vaccine loaded with manganese adjuvant as described in claim 1 or a vaccine composition as described in claim 2, characterized in that, Used to prepare drugs or preparations for the prevention and / or treatment of tumors.

9. A method for preparing a whole-cell tumor vaccine loaded with manganese adjuvant as described in claim 1 or a vaccine composition as described in claim 2, characterized in that, The method includes the following steps: (S1) Preparation of surface-thiolized tumor cells; (S2) Preparation of anionic lipid nanoparticles by thin-film hydration method; (S3) The tumor cells obtained in step (S1) are mixed and incubated with the anionic lipid nanoparticles obtained in step (S2), and the tumor cells with surface-coupled liposomes are collected. (S4) The tumor cells obtained in step (S3) are incubated in a manganese ion salt solution for inactivation treatment to obtain the whole-cell tumor vaccine loaded with manganese adjuvant.

10. A reagent combination for preparing a whole-cell tumor vaccine loaded with manganese adjuvant as described in claim 1, characterized in that, The reagent combination includes: (a) Tumor cells; (b) Anionic lipid nanoparticles, wherein the anionic lipid nanoparticles can be covalently coupled to the tumor cells; and (c) Manganese adjuvant, wherein the manganese adjuvant may be loaded onto the "tumor cell-anionic lipid nanoparticle" binary complex.

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