Method for preparing novel dendritic cell vaccine and use thereof

Abstract

Provided in the present invention are a method for preparing a novel dendritic cell vaccine and use thereof. Specifically, disclosed in the present invention is a method for preparing modified dendritic cells, comprising: culturing dendritic cells in the presence of vitamin K2, thereby obtaining dendritic cells capable of inhibiting cell death caused by tumor antigen sensitization. The modified dendritic cells can enhance T-cell activation and tumor cell killing ability. Also disclosed in the present invention is a method for tumor immunotherapy using the dendritic cells in combination with T-cell vaccines, existing tumor vaccines, immune checkpoint inhibitors, or radiotherapy and chemotherapy.

Description

A novel method for preparing a dendritic cell vaccine and its application. Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method for preparing a novel dendritic cell vaccine and its application. Background Technology

[0002] Immunotherapy is a novel approach to cancer treatment that activates or enhances a patient's own immune system to recognize and attack cancer cells. While existing cancer immunotherapies offer new hope for cancer treatment, they still have some limitations.

[0003] The efficacy of immunotherapy varies significantly among patients. Some patients may experience a significant response to immunotherapy, while others may not see any noticeable effect. This is primarily attributed to the spatiotemporal heterogeneity of tumors and individual patient differences. For some patients with advanced cancer, the efficacy of immunotherapy may not be apparent. This may be because the immune system of these patients is severely damaged, making it difficult for them to effectively combat the attack of cancer cells.

[0004] Immunotherapy, as a novel treatment method, is relatively expensive. This is mainly due to research and development costs, production costs, and patent protection. For ordinary families, immunotherapy can impose a significant financial burden. While immunotherapy has potential advantages, it also carries serious risks of side effects. Common adverse reactions include fatigue, fever, rash, and immunosuppression. These side effects can reduce the patient's quality of life and may affect the effectiveness of treatment. In severe cases, it may even be necessary to discontinue immunotherapy.

[0005] Immunotherapy is typically a slow process, requiring a longer period to observe the response. This can impact a patient's life and work. Furthermore, prolonged treatment may increase the patient's financial burden and psychological stress.

[0006] Currently, some immunotherapies are only applicable to specific types of cancer. Their application in other cancer types is still in the research and exploration stage. This means that not all cancer patients are suitable for immunotherapy. Furthermore, immunotherapy may also face problems such as inconsistent treatment responses and drug resistance. Identifying the most effective cancer-associated antigens and developing efficient and targeted delivery systems are among the technical challenges facing immunotherapy. Moreover, tumor cells may exhibit immune tolerance, making it difficult for the immune system to recognize and attack them. Simultaneously, tumor cells may also evade the immune system's attack through escape mechanisms.

[0007] Dendritic cell (DC) vaccines are a type of tumor immunotherapy. Dendritic cells are multifunctional antigen-presenting cells that play a crucial role in initiating the body's immune response and maintaining immune tolerance. DC vaccines activate immune cells in the body by loading tumor-associated antigens, thereby attacking and eliminating tumor cells. These vaccines have shown some efficacy in clinical trials for various cancers.

[0008] However, when using existing methods to prepare anti-tumor DC vaccines, DC cells are often over-activated by tumor antigens, resulting in a large number of them dying during the preparation process, leading to low vaccine preparation efficiency.

[0009] Based on the current state of research on tumor immunotherapy drugs, there is still a need in this field for more effective, safe, and economical cancer treatment drugs. Summary of the Invention

[0010] The purpose of this invention is to provide a more effective, safe, and economical dendritic cell vaccine formulation, its preparation method, and its application.

[0011] In a first aspect of the present invention, a method for preparing modified dendritic cells is provided, comprising the following steps:

[0012] (S1) provides dendritic cells;

[0013] (S2) In the presence of vitamin K2, dendritic cells with stronger antigen presentation ability are obtained by loading tumor antigens and culturing them. These are the modified dendritic cells.

[0014] In another preferred embodiment, the concentration of vitamin K2 is 50 nM-10 mM, more preferably 5 μM-1 mM, and even more preferably 50-100 μM.

[0015] In another preferred embodiment, the culture time is 1-100h, more preferably 10-80h, and even more preferably 15-40h.

[0016] In another preferred embodiment, the method is an in vitro method.

[0017] In another preferred embodiment, the method is a non-therapeutic method.

[0018] In another preferred embodiment, the dendritic cells are selected from the patient's own dendritic cells or dendritic cells derived from non-human mammals.

[0019] In another preferred embodiment, the non-human mammal is a rodent or a non-human primate, preferably including mice, rats, rabbits, and monkeys.

[0020] In another preferred embodiment, step (S1) further includes:

[0021] (S1a) Provide bone marrow cells of non-human mammalian origin, which are cultured in vitro into immature dendritic cells;

[0022] (S1b) The immature dendritic cells are induced in vitro to obtain the mature dendritic cells.

[0023] In another preferred embodiment, in step (S1b), the mature dendritic cells are obtained from bone marrow cells of non-human mammalian origin, induced in vitro by GM-CSF, TNFα and LPS.

[0024] In another preferred embodiment, the patient's own dendritic cells are prepared by the following steps:

[0025] (S1c) Provides mononuclear cells derived from the patient's own peripheral blood, which are then induced in vitro to obtain the mature dendritic cells.

[0026] In another preferred embodiment, in step (S1c), the mature dendritic cells are obtained from mononuclear cells derived from the patient's own peripheral blood, induced in vitro by IL-4 and GM-CSF.

[0027] In another preferred embodiment, the modified dendritic cells have antigen-presenting capabilities, thereby significantly increasing T cell activation.

[0028] In a second aspect of the invention, an improved dendritic cell is provided, said dendritic cell being prepared by the method described in the first aspect of the invention.

[0029] In another preferred embodiment, under conditions of exposure to tumor antigen, the survival rate of the modified dendritic cells is significantly improved compared to the survival rate of dendritic cells not treated with vitamin K2.

[0030] In another preferred embodiment, the significant improvement refers to the ratio F1 / F0 of the improved dendritic cell survival rate F1 to the untreated dendritic cell survival rate F0 under the condition of exposure to tumor antigen being 1 to 3; more preferably 1.5 to 2.5; and even more preferably 1.8 to 2.2.

[0031] In another preferred embodiment, during the contact process, the dendritic cells come into contact with the tumor antigen.

[0032] In another preferred embodiment, the tumor antigen is a tumor cell lysate.

[0033] In another preferred embodiment, the tumor antigen lysis buffer is obtained by lysing tumor cells in the same number as dendritic cells.

[0034] In another preferred embodiment, the tumor cells are selected from cancer cells of the group consisting of: lung cancer, gastrointestinal cancer, glioma, sarcoma, epithelial carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumors, breast cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, or combinations thereof.

[0035] In another preferred embodiment, the modified dendritic cells refer to dendritic cells that can inhibit tumor antigen sensitization leading to cell death.

[0036] In another preferred embodiment, the tumor antigen sensitization leading to cell death includes cell death caused by tumor antigen-induced overactivation of dendritic cells.

[0037] In another preferred embodiment, the vitamin K2 does not affect the activation of the dendritic cells and can inhibit cell death caused by excessive activation of dendritic cells induced by tumor antigens.

[0038] In another preferred embodiment, the modified dendritic cells have antigen-presenting capabilities, thereby significantly increasing T cell activation.

[0039] In a third aspect of the invention, a use of vitamin K2 is provided for preparing a formulation or composition that serves as a protectant for dendritic cells.

[0040] In another preferred embodiment, the protective agent is used to inhibit cell death caused by excessive activation of dendritic cells.

[0041] In another preferred embodiment, the formulation or composition is used to inhibit cell death caused by excessive activation of dendritic cells.

[0042] In another preferred embodiment, the formulation or composition is used to inhibit cell death caused by tumor antigen-induced overactivation of dendritic cells.

[0043] In another preferred embodiment, the cell death refers to the death of dendritic cells.

[0044] In another preferred embodiment, the formulation or composition is a formulation or composition for experimental or clinical use.

[0045] In another preferred embodiment, the dosage form of the preparation is selected from the group consisting of: injections and lyophilized preparations.

[0046] In a fourth aspect of the invention, a method for preparing sensitized dendritic cells is provided, wherein the dendritic cells described in the second aspect of the invention are contacted with a tumor antigen to form sensitized dendritic cells.

[0047] In another preferred embodiment, the method for loading the tumor antigen is selected from the group consisting of:

[0048] (1) Direct loading method: The tumor antigen (such as polypeptide, protein, tumor cell lysate, mRNA (liposome-encapsulated), tumor-derived exosome) is directly added to the culture medium of the dendritic cells, so that the dendritic cells can take up and process the antigen;

[0049] (2) Electroporation: The tumor antigen is introduced into the dendritic cells using electroporation technology;

[0050] (3) Genetic engineering technology: The gene encoding the tumor antigen is introduced into the dendritic cells using genetic engineering technology, causing it to be expressed and processed into an antigen within the cells; or

[0051] (4) Cell fusion technology: fusion cells with antigen presentation ability are obtained by inducing the fusion of dendritic cells and tumor cells.

[0052] In another preferred embodiment, the method for obtaining the tumor antigen is selected from the group consisting of:

[0053] (1) Extraction from tumor cells: Obtain the patient's tumor cells through surgery, biopsy or other means, and extract tumor antigens using cell culture and lysis techniques;

[0054] (2) Use of tumor-associated antigen peptides: The peptides are protein fragments specifically expressed on or inside tumor cells and obtained through chemical synthesis or genetic engineering techniques;

[0055] (3) Utilizing tumor mRNA: Tumor mRNA is an mRNA molecule that encodes tumor-associated antigens. It is obtained by extracting mRNA from tumor cells and reverse transcribing it into cDNA, which is then amplified and purified using genetic engineering techniques.

[0056] In a fifth aspect of the invention, a sensitized dendritic cell is provided, said sensitized dendritic cell being prepared by the method described in the fourth aspect of the invention.

[0057] In a sixth aspect of the present invention, a method for preparing antitumor cytotoxic T lymphocytes (CTLs) is provided, comprising the steps of: co-culturing modified dendritic cells as described in the first aspect of the present invention or sensitized dendritic cells as described in the fifth aspect of the present invention with T lymphocytes to obtain cytotoxic T lymphocytes (CTLs).

[0058] In another preferred embodiment, the T lymphocytes are the subject's own T lymphocytes.

[0059] In another preferred embodiment, during co-culture, the ratio of dendritic cells to T lymphocytes is (10-500):1, more preferably (20-300):1, and even more preferably (50-200):1.

[0060] In another preferred embodiment, the cytotoxic T lymphocytes can specifically kill tumor cells.

[0061] In a seventh aspect of the invention, the use of the dendritic cells described in the second aspect of the invention in the preparation of a medicament for treating cancer or tumors is provided.

[0062] In another preferred embodiment, the tumor is selected from the group consisting of: cancers of the central or peripheral nervous system, cancers of the endocrine or neuroendocrine system, or cancers of the hematopoietic system.

[0063] In another preferred embodiment, the tumor is selected from the group consisting of: lung cancer, gastrointestinal cancer, glioma, sarcoma, epithelial carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumor, breast cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, or combinations thereof.

[0064] In an eighth aspect of the invention, a composition for treating cancer or tumors is provided, the composition comprising dendritic cells prepared by the method described in the first aspect of the invention, dendritic cells described in the second aspect of the invention, or sensitized dendritic cells described in the fifth aspect of the invention.

[0065] In another preferred embodiment, the composition is a vaccine composition or a pharmaceutical composition.

[0066] In another preferred embodiment, the composition further comprises a pharmaceutically acceptable carrier or a vaccinologically acceptable carrier.

[0067] In another preferred embodiment, the composition further includes other tumor treatment drugs.

[0068] In another preferred embodiment, the tumor treatment drug is selected from the group consisting of: T-cell vaccines, tumor vaccines, immune checkpoint inhibitors, and radiotherapy or chemotherapy-related preparations.

[0069] In another preferred embodiment, the T-cell vaccine refers to activated T cells obtained by co-culturing the dendritic cells, and the dendritic cells combined with the T-cell vaccine are used as a combination vaccine formulation for tumor immunotherapy.

[0070] In another preferred embodiment, the tumor vaccine includes (but is not limited to): CAR T vaccine, CAR NK vaccine, CAR Macrophage vaccine, TCR-T vaccine, TIL vaccine, exosome vaccine, peptide cancer vaccine, mRNA cancer vaccine, DNA cancer vaccine, and viral vector vaccine.

[0071] In another preferred embodiment, the immune checkpoint inhibitor includes (but is not limited to): PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, and TIM-3 inhibitors.

[0072] In another preferred embodiment, the PD-1 / PD-L1 inhibitor includes (but is not limited to): pembrolizumab, nivolumab, camrelizumab, toripalimab, sintilimab, tislelizumab, atezolizumab, durvalumab, suglizumab, penaprilimab, cepalimumab, slulizumab, putelizumab, avelumab, envorimab, and trastuzumab emtansine.

[0073] In another preferred embodiment, the CTLA-4 inhibitor includes (but is not limited to): ipilimumab.

[0074] In another preferred embodiment, the bispecific inhibitors include: epalolitoprene, candunilinumab, edoxicillin, Zaijing Pharmaceuticals ZG005, LBL-024, and SHR-1701.

[0075] In another preferred embodiment, the oncology treatment drug includes (but is not limited to) temozolomide, nicolimustine, avastin, mannitol, glycerol fructose, dexamethasone, methylprednisolone, sodium valproate, carbamazepine, levetiracetam, bevacizumab, imatinib, gefitinib, auristatins, camptothecins, docarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines, or drugs containing benzodiazepines. Drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), vinca alkaloids, olstatins (e.g., olstatin E, olstatin F, MMAE, and MMAF), chlortetracycline, phenytoin, pyrethroid, pyrethroid A-chain, cobustatin, docalimidin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthraxine dione, actinomycin, diphtheria toxin Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, saccharin A chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, jatropha toxin, croton toxin, chalcogenine, Sapaonaria officinalis inhibitor, glucocorticoids, or combinations thereof.

[0076] In a ninth aspect of the present invention, a method for tumor immunotherapy is provided, comprising the steps of administering to a subject in need dendritic cells prepared by the method described in the first aspect of the present invention, or sensitized dendritic cells described in the fifth aspect of the present invention, or a composition described in the eighth aspect of the present invention.

[0077] In another preferred embodiment, the method comprises treatment using sensitized dendritic cells as described in the fifth aspect of the invention or a combination of the composition described in the eighth aspect of the invention, selected from the group consisting of:

[0078] T-cell vaccines, tumor vaccine preparations, immune checkpoint inhibitors, radiotherapy or chemotherapy.

[0079] In another preferred embodiment, the T-cell vaccine refers to activated T cells obtained by co-culturing the dendritic cells, and the dendritic cell vaccine preparation combined with the T-cell vaccine is a combination vaccine preparation for tumor immunotherapy.

[0080] In another preferred embodiment, the tumor vaccine formulation includes (but is not limited to): CAR T vaccine, CARNK vaccine, CAR Macrophage vaccine, TCR-T vaccine, TIL vaccine, exosome vaccine, peptide cancer vaccine, mRNA cancer vaccine, DNA cancer vaccine, and viral vector vaccine.

[0081] In another preferred embodiment, the immune checkpoint inhibitor includes (but is not limited to): PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, and TIM-3 inhibitors.

[0082] In another preferred embodiment, the PD-1 / PD-L1 inhibitor includes (but is not limited to): pembrolizumab, camrelizumab, tislelizumab, tislelizumab, tislelizumab, atezolizumab

[0083] In another preferred embodiment, CTLA-4 inhibitors include (but are not limited to): ipilimumab.

[0084] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0085] In another preferred embodiment, the tumor is selected from the group consisting of: cancers of the central or peripheral nervous system, cancers of the endocrine or neuroendocrine system, or cancers of the hematopoietic system.

[0086] In another preferred embodiment, the tumor is selected from the group consisting of: lung cancer, gastrointestinal cancer, glioma, sarcoma, epithelial carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumor, breast cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, or combinations thereof.

[0087] In a tenth aspect of the invention, the use of dendritic cells prepared by the method described in the first aspect of the invention in the preparation of a medicament for treating viral infections is provided; the viruses include: influenza virus, enterovirus 71 (EV71), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), human T-cell phagocytic virus (HTLV), HIV, etc.

[0088] 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

[0089] Figure 1 shows that vitamin K2 significantly inhibits cell death caused by overactivation of dendritic cells. In this figure, DC represents the dendritic cell group, DC+TCL represents the DC cell group treated with tumor cell lysate (TCL), and DC+VK2+TCL represents the DC cells treated with a combination of tumor cell lysate (TCL) and vitamin K2 (VK2).

[0090] Figure 2 shows the results of dendritic cell vaccine significantly inhibiting lung cancer cell growth. In the figure, A shows the images of tumor masses in each group, B shows the changes in tumor weight, and C shows the changes in tumor volume. Placebo represents the model control group, DC represents the dendritic cell group, DC+VK2 represents the dendritic cell group treated with vitamin K2, DC+TCL represents the DC cell group treated with tumor cell lysate (TCL), and DC+TCL+VK2 represents the DC cells treated with a combination of tumor cell lysate (TCL) and vitamin K2 (VK2).

[0091] Figure 3 shows CD8 + The results of the T cell killing experiment on HBV-infected Huh7 cells, where DC represents dendritic cells, VK2 represents vitamin K2, and T represents CD8+. + T cells. Detailed Implementation

[0092] Through extensive and in-depth research, including a large-scale screening of vitamins, amino acids, and small metabolic molecules, the inventors unexpectedly discovered for the first time that vitamin K2 can significantly improve the survival rate of dendritic cells, especially inhibiting dendritic cell death induced by tumor antigen sensitization. In vivo experiments have demonstrated that vitamin K2 can enhance the anti-tumor efficiency of dendritic cells. The vaccine preparation method provided by this invention is safe, simple, and easy to operate, greatly reducing the cost and expense of producing tumor vaccines. Based on these findings, this invention was completed.

[0093] the term

[0094] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0095] As used herein, the terms “DC,” “dendritic cell,” and “dendritic cell” are used interchangeably and all refer to the dendritic cells prepared by the method described in the first aspect of this invention.

[0096] As used in this article, the terms “VK2” and “vitamin K2” are used interchangeably.

[0097] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0098] As used herein, the term "treatment" refers to the administration of an oral or topical therapeutic agent, including the tumor-targeting vaccine and compositions thereof of the present invention, to a patient who has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered to the patient in an amount that effectively relieves the symptoms of one or more diseases (therapeutic effective amount).

[0099] Vitamin K2

[0100] Vitamin K2 (VK2), CAS number 863-61-6, molecular formula C 31 H 40 O2, its structural formula is shown below:

[0101] Vitamin K2 is a fat-soluble vitamin and one of the essential vitamins for the human body. Vitamin K2 is primarily used to prevent and treat osteoporosis. It helps osteoblasts convert primary osteocalcin into active osteocalcin, thereby promoting the deposition of calcium ions from the blood into bones.

[0102] Dendritic cells (DCs)

[0103] Dendritic cells (DCs) were first discovered in 1973 by Ralph M. Steinman in the United States. They are named for the numerous dendritic or pseudopodia-like projections they produce upon maturation. DCs are currently the most potent antigen-presenting cells known, and their most important characteristic is their ability to stimulate the activation and proliferation of naive T cells. B cells and other cells can only stimulate activated T cells or memory T cells; therefore, dendritic cells (DCs) are the initiators of specific immune responses and occupy a unique position in the immune system. DCs are widely distributed throughout the body except for the brain, but their numbers are small, accounting for less than 1% of peripheral blood mononuclear cells. Based on their maturity state, DCs can be divided into DC precursors, immature DCs, migrating DCs, and mature DCs. DC precursor cells enter the epithelial sites of various solid organs and non-lymphoid tissues via the blood or lymphatic circulation, and differentiate and develop into immature DCs (iDCs) under the influence of certain cytokines. Under normal circumstances, the vast majority of DCs in the body are in an immature state; they only express low levels of MHC class II molecules, co-stimulatory molecules, and adhesion molecules, and have a weak ability to activate T cells in vitro; however, they express a large amount of FcRs and pathogen receptors, possessing a very strong ability to take up and process antigens. After taking up antigens or being stimulated by certain factors (mainly inflammatory factors such as LPS, IL1β, and TNFα), immature DCs begin to differentiate and mature. Mature DCs (mDCs) express a large number of MHC class II molecules and co-stimulatory molecules (CD80, CD40, CD86, etc.), which can effectively present processed antigens to naive T cells in the form of antigen peptide-MHC molecule complexes and activate them.

[0104] DC vaccines work by loading tumor-associated antigens (TAAs) to activate immune cells in the body, thereby attacking and eliminating tumor cells. These vaccines have shown some efficacy in clinical trials for various cancers.

[0105] Method for preparing dendritic cell vaccine formulation in this invention

[0106] In order to overcome the limitations of tumor immunotherapy, this invention, through extensive screening of vitamins, amino acids and small metabolic molecules, found that vitamin K2 can significantly enhance the anti-tumor efficiency of dendritic cells. In vitro experiments also proved that vitamin K2 can significantly inhibit cell death caused by excessive activation of dendritic cells, enhance the antigen presentation ability of dendritic cells and the killing ability of T cells against tumor cells.

[0107] This invention provides a method for preparing modified dendritic cells, comprising the following steps:

[0108] (S1) Obtain dendritic cells;

[0109] The dendritic cells are selected from mammals, including autologous or allogeneic bone marrow cells and peripheral blood-derived mononuclear cells, and are obtained by in vitro induction.

[0110] (S2) After treatment with vitamin K2, dendritic cells with stronger antigen presentation ability are obtained by loading tumor antigens and culturing them. These are modified dendritic cells.

[0111] In another preferred embodiment, the concentration of vitamin K2 is 5-10 mM, more preferably 0.5-1 mM, and even more preferably 50-100 μM.

[0112] In another preferred embodiment, the culture time is 1-100 h, more preferably 10-80 mM, and even more preferably 15-40 h.

[0113] In another preferred embodiment, the method is an in vitro method.

[0114] In another preferred embodiment, the method is a non-therapeutic method.

[0115] In another preferred embodiment, the dendritic cells are selected from the patient's own dendritic cells or dendritic cells derived from non-human mammals.

[0116] In another preferred embodiment, the non-human mammal is a rodent or a non-human primate, preferably including mice, rats, rabbits, and monkeys.

[0117] In another preferred embodiment, step (S1) further includes:

[0118] (S1a) Provide bone marrow cells of non-human mammalian origin, which are cultured in vitro into immature dendritic cells;

[0119] (S1b) The immature dendritic cells are induced in vitro to obtain the mature dendritic cells.

[0120] In another preferred embodiment, in step (S1b), the mature dendritic cells are obtained from bone marrow cells of non-human mammalian origin, induced in vitro by GM-CSF, TNFα and LPS.

[0121] In another preferred embodiment, the patient's own dendritic cells are prepared by the following steps:

[0122] (S1c) Provides mononuclear cells derived from the patient's own peripheral blood, which are then induced in vitro to obtain the mature dendritic cells.

[0123] In another preferred embodiment, in step (S1c), the mature dendritic cells are obtained from mononuclear cells derived from the patient's own peripheral blood, induced in vitro by IL-4 and GM-CSF.

[0124] The tumor antigen loading method is selected from the following group:

[0125] (1) Direct loading method: Dendritic cells directly take up and process the tumor antigens (peptides, proteins, tumor cell lysates, mRNA, tumor-derived exosomes);

[0126] (2) Electroporation: The tumor antigen is introduced into the dendritic cells using electroporation technology;

[0127] (3) Genetic engineering technology: The gene encoding the tumor antigen is introduced into the dendritic cells through genetic engineering technology, so that it is expressed and processed into antigen in the cells;

[0128] (4) Cell fusion technology: obtaining fused cells with antigen-presenting capabilities by inducing the fusion of dendritic cells and tumor cells.

[0129] In another preferred embodiment, the method for obtaining the tumor antigen is selected from the group consisting of:

[0130] (1) Extraction from tumor cells: Obtain the patient's tumor cells through surgery, biopsy or other means, and extract tumor antigens using cell culture and lysis techniques;

[0131] (2) Use of tumor-associated antigen peptides: The peptides are protein fragments specifically expressed on or inside tumor cells and obtained through chemical synthesis or genetic engineering techniques;

[0132] (3) Utilizing tumor antigen mRNA: Tumor mRNA is an mRNA molecule that encodes tumor-associated antigens.

[0133] (4) Using tumor antigen cDNA: mRNA from tumor cells is extracted and reverse transcribed into cDNA, which is then amplified and purified by genetic engineering techniques.

[0134] Composition and method of application

[0135] The present invention also provides a composition comprising (or containing) a therapeutically effective amount of the dendritic cells described in the second aspect of the present invention. The compositions of the present invention include pharmaceutical compositions or vaccine compositions.

[0136] In another preferred embodiment, the composition further includes other tumor treatment drugs.

[0137] Other cancer treatment drugs mentioned in this article can refer to other drugs used in cancer immunotherapy. Cancer immunotherapy is a novel approach to cancer treatment that activates or enhances the patient's own immune system to recognize and attack cancer cells. Major cancer immunotherapies include: checkpoint inhibitor therapy, CAR-T cell therapy, cancer vaccines, and adoptive cell therapy (such as TIL therapy). In addition to the above-mentioned major immunotherapies, several other immunotherapies are under investigation, such as:

[0138] Monoclonal antibody therapy: This involves injecting artificially synthesized antibodies to attack tumor cells.

[0139] Immunotoxin therapy: This involves combining a toxin with tumor-specific antigens to attack tumor cells.

[0140] Cytokine therapy: Introducing recombinant cytokines to enhance the patient's immune system's ability to attack tumors.

[0141] Cytokine gene therapy: Introducing cytokine genes into the body to activate the patient's own immune system.

[0142] Cytokine receptor gene therapy: This involves artificially synthesizing receptors that can bind to cytokines to activate the patient's own immune system.

[0143] Checkpoint inhibitors are drugs that help the immune system recognize and attack cancer cells. They enhance the killing power of immune cells by blocking the "covering" mechanisms that cancer cells use against the immune system, such as immune checkpoint molecules like PD-1 / PD-L1. These drugs include anti-CTLA-4 antibodies, anti-PD-1 antibodies, and anti-PD-L1 antibodies, and have shown significant efficacy in the treatment of various cancers.

[0144] CAR-T cell therapy is a personalized cancer immunotherapy. It utilizes a patient's own T cells, modifying them through gene editing technology to create CAR-T cells capable of recognizing and attacking cancer cells. These modified T cells are then expanded in vitro and re-injected into the patient, thereby strengthening the immune system's ability to attack cancer. CAR-T cell therapy has achieved groundbreaking progress in the treatment of hematological malignancies and some solid tumors.

[0145] A cancer vaccine is a novel type of vaccine designed to activate or train a patient's immune system to recognize and attack cancer cells. Cancer vaccines are mainly divided into two categories: preventative vaccines and therapeutic vaccines.

[0146] Preventive vaccines include the cervical cancer vaccine (HPV vaccine) and other preventive vaccines.

[0147] Cervical cancer vaccine (HPV vaccine): This is currently the most common anti-cancer preventive vaccine on the market. Cervical cancer is mainly caused by human papillomavirus (HPV) infection, so the HPV vaccine reduces the risk of cervical cancer by preventing HPV infection. Currently, bivalent, quadrivalent, and nonavalent HPV vaccines are available on the market, and they cover different types of HPV that they can prevent.

[0148] Other preventative vaccines: In addition to the HPV vaccine, preventative vaccines against other cancers are under development, such as the lung cancer vaccine LungVax. These vaccines aim to reduce the risk of cancer by training the immune system to recognize and attack specific types of cancer cells.

[0149] Therapeutic vaccines include dendritic cell (DC) vaccines, peptide cancer vaccines, mRNA cancer vaccines, DNA cancer vaccines, viral vector vaccines, and other therapeutic vaccines such as melanoma cell vaccines and breast cancer vaccines. Most of these vaccines are still in clinical trials and have not yet been widely used in clinical practice. However, studies have shown that they have some efficacy in the treatment of certain cancers.

[0150] Peptide cancer vaccines consist of chemical or biosynthetic components that target specific tumor antigen epitopes, triggering a strong immune response against those epitopes. Compared to dendritic cell (DC) vaccines, peptide cancer vaccines are simpler to design, manufacture, and administer, and are also less expensive. They are primarily used to treat liver cancer, cervical cancer, and other cancers.

[0151] mRNA vaccines are a cutting-edge approach to cancer vaccines, requiring only entry into the cytoplasm for antigen translation and expression.

[0152] DNA cancer vaccines: DNA-based cancer vaccines deliver plasmids containing genetic material encoding tumor antigens. These plasmids are absorbed by endogenous cells, which then express the encoded antigens. DNA vaccines must enter the cell nucleus to initiate transcription.

[0153] Viral vector vaccines are designed to deliver genetic material into endogenous cells to achieve antigen expression, which can trigger a strong immune response from T cells and B cells.

[0154] Adoptive cell therapy involves infusing treated immune cells or molecules into a patient to enhance their immune system. TIL (tumor-infiltrating lymphocyte) therapy is a novel type of adoptive cell therapy. It involves isolating tumor-infiltrating lymphocytes (TILs) from surgically removed tumors, expanding and culturing them in vitro, and then reinfusing them into the patient. These TILs can continuously seek out and precisely kill cancer cells, demonstrating significant efficacy against various solid tumors.

[0155] As used herein, the term "therapeutic effective dose" refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or the amount that exhibits a detectable therapeutic or preventative effect. This effect can be detected, for example, by antigen levels. Therapeutic effects also include a reduction in physiological symptoms. The precise effective dose for a given subject depends on that subject's body size and health status, the nature and severity of the condition, and the choice of the therapeutic agent and / or combination of therapeutic agents administered. Therefore, it is not useful to pre-specify an exact effective dose. However, for a given condition, the effective dose can be determined using routine laboratory testing.

[0156] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" or "vaccinologically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent (e.g., the dendritic cell therapeutic vaccine formulation of the present invention). This term refers to pharmaceutical carriers that do not induce antibodies harmful to the individual receiving the composition and do not cause excessive toxicity after administration. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, etc. These carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0157] Pharmaceutically acceptable carriers in a composition may include liquids such as water, saline, glycerol, and ethanol. Additionally, these carriers may contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc. Typically, the composition can be formulated as an injectable preparation, such as a liquid solution or suspension; it can also be formulated as a solid form suitable for reconstitution into solutions or suspensions, or as a liquid excipient, prior to injection. Liposomes are also included in the definition of pharmaceutically acceptable carriers.

[0158] The vaccine compositions of the present invention comprise an immune antigen (e.g., mature human dendritic cells of vitamin K2 of the present invention) and are generally combined with a "pharmaceutically acceptable carrier" or "vaccinologically acceptable carrier," which includes any carrier that does not itself induce antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), etc. These carriers are well known to those skilled in the art. Additionally, these carriers can act as immunostimulants ("adjuvants").

[0159] Preferred adjuvants for enhancing the effect of the immune composition include, but are not limited to: (1) aluminum salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations, such as: (a) Montanide ISA 51VG, (b) Montanide ISA 720VG; (c) MF59; (3) saponin adjuvants; (4) Freund complete adjuvant (CFA) and Freund incomplete adjuvant (IFA); (5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (GM-CSF), etc.; (6) detoxified variants of bacterial ADP-ribosylated toxins (e.g., Escherichia coli heat unstable toxin LT); and (7) other substances that act as immunostimulants to enhance the effect of the composition.

[0160] Vaccine compositions, including immunogenic compositions (e.g., may include antigens, pharmaceutically acceptable carriers, and 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 these carriers. More specifically, vaccines, including immunogenic compositions, contain an immunologically effective amount of the immunogenic composition, along with the other required components mentioned above. An “immunologically effective amount” refers to the amount administered to an individual as a single dose or a portion of a continuous dose that is effective for treatment. This dosage can be determined based on the health and physiological condition of the individual being treated, the individual’s class (e.g., human), the individual’s immune system’s ability to synthesize antibodies, the required level of protection, the vaccine formulation, the treating physician’s assessment of the medical condition, and other relevant factors. This dosage is expected to be within a relatively wide range and can be determined through routine laboratory testing.

[0161] Typically, vaccine compositions or immunogenic compositions can be formulated as injectable preparations, such as liquid solutions or suspensions; they can also be formulated as solid forms suitable for reconstitution into solutions or suspensions or liquid excipients prior to injection. The formulation may also be emulsified or encapsulated in liposomes to enhance adjuvant effects.

[0162] Once formulated into the composition of the present invention, it can be administered directly to the subject. The subject to be treated can be a mammal, especially a human.

[0163] When used as a vaccine, the vaccine composition of the present invention can be administered directly to an individual using known methods, typically via the same route of administration as conventional vaccines. Routes of administration of the pharmaceutical or vaccine composition of the present invention include (but are not limited to): subcutaneous, intradermal, intramuscular, or other parenteral routes. Routes of administration can be combined or adjusted as needed, depending on the disease condition. The vaccine composition can be administered in single or multiple doses, and may include booster doses to induce and / or maintain immunity. The vaccine composition should be administered in an "effective amount," meaning an amount sufficient to induce an immune response and effectively improve disease symptoms via the chosen route of administration.

[0164] The amount of pharmaceutical composition used in each vaccine dose is determined based on the amount that can elicit a protective immune response without significant side effects. The optimal dosage of a specific vaccine can be determined using standard research methods that include antibody titers and other responses in the subjects. Whether a booster dose is needed can be determined by monitoring the level of immunity provided by the vaccine. After assessing the antibody titer in the serum, a booster dose may be required for immunization. Administration of the pharmaceutical composition or vaccine composition can enhance the immune response to the present invention.

[0165] Furthermore, the vaccine of the present invention can be administered in combination with other immunomodulators or with other therapeutic agents.

[0166] application

[0167] The present invention also provides the use of modified dendritic cells in the preparation of medicaments for treating tumors.

[0168] In another preferred embodiment, the tumor is selected from the group consisting of: cancers of the central or peripheral nervous system, cancers of the endocrine or neuroendocrine system, or cancers of the hematopoietic system.

[0169] In another preferred embodiment, the tumor is selected from the group consisting of: lung cancer, gastrointestinal cancer, glioma, sarcoma, epithelial carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumor, breast cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, or combinations thereof.

[0170] Meanwhile, this invention provides the use of modified dendritic cells in the preparation of drugs for treating viral infections.

[0171] In another preferred embodiment, the virus is selected from the group consisting of: influenza virus, enterovirus 71 (EV71), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), human T-cell phagocytic virus (HTLV), HIV, etc.

[0172] The main innovation of this invention compared to conventional formulations is the development and provision of a stable formulation containing vitamin K2-containing human DC active ingredient, the main advantages of which include:

[0173] (1) The vaccine preparation method of the present invention uses vitamin K2 as a protective agent for dendritic cells to prevent them from being over-activated during tumor antigen stimulation, which would lead to massive cell death. The method is safe, simple and easy to operate, greatly reducing the cost and expense of producing tumor vaccines.

[0174] (2) The formulation of the present invention contains human DC of vitamin K2 (e.g., human DC of vitamin K2) as its active ingredient, which can induce the body to produce an antigen-specific immune response.

[0175] (3) The dendritic cell vaccine made using vitamin K2 in this invention can significantly inhibit tumor growth and provide new ideas for clinical tumor treatment.

[0176] 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.

[0177] Example 1: Preparation of Dendritic Cell Vaccine

[0178] In this embodiment, mouse dendritic cell vaccine and vaccine prepared by extracting mononuclear cells from patients and inducing them into dendritic cells were prepared, respectively. The specific process is as follows:

[0179] 1.1 Mouse dendritic cell vaccine:

[0180] 1) C57BL / 6 mice were euthanized by cervical dislocation and disinfected with 70% ethanol for 5 minutes.

[0181] 2) Remove the femur and tibia of the mouse under sterile conditions in a clean bench and soak them in 70% ethanol for 5 minutes.

[0182] 3) Cut both ends of the bone at the joint, draw pre-cooled RPMI-1640 complete culture medium with a syringe, insert the needle of the syringe into the bone cavity, and rinse the bone marrow into a sterile culture dish. Repeat the rinsing 3 times.

[0183] 4) Collect the cell suspension in the culture dish and filter it through a 70μm cell filter to remove debris and muscle tissue. Centrifuge the filtrate at 1000 rpm and 4℃ for 5 minutes and discard the supernatant.

[0184] 5) Wash once with PBS, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Resuspend the cells in RPMI-1640 complete medium and adjust the cell density.

[0185] 6) 2 x 10 6 The cells were seeded onto 100 mm bacterial culture dishes (bacterial culture dishes are used to inhibit the growth of macrophages and promote the maturation of suspended DCs) and cultured in 1640 RPMI medium containing 10% FBS, 2 mM L-glutamine, 1% penicillin / streptomycin, 50 μM β-mercaptoethanol, and 20 ng / ml GM-CSF (cultured in an incubator at 37°C and 5% CO2).

[0186] 7) On day 3 of culture, add RPMI-1640 complete medium containing 200 U / ml Mouse GM-CSF. On days 6 and 8 of culture, perform a half-volume medium change. After centrifuging the collected old medium to remove the supernatant, resuspend the cell pellet in fresh RPMI-1640 complete medium containing 200 U / ml Mouse GM-CSF, and then return it to the original culture dish.

[0187] 8) On day 9 of culture, add TNF-α (50 ng / ml) or LPS (1 μg / ml) and continue culturing for 1 day in an incubator at 37°C and 5% CO2 to obtain fully mature mouse dendritic cells (BMDC).

[0188] 9) Replace with RPMI-1640 complete medium and treat BMDCs with 50-100 uM vitamin K2 for 24 hours. At this time, the BMDC cell concentration is (0.8 x 10). 6 -1 x 10 6 )cells / ml.

[0189] 10) The same number of tumor cells as BMDC were sonicated in 1 ml PBS (PMSF added at a ratio of 1:100) to obtain tumor cell lysate. The tumor lysate was filtered through a 0.22 μm filter membrane and added to BMDC cell culture medium. The cells were treated for 24 hours to obtain activated mouse dendritic cells (BMDC).

[0190] 1.2 Mononuclear cells were extracted from the patient and induced to form dendritic cells for vaccine production.

[0191] 1) Collect 50ml to 200ml of blood from the patient; the specific amount varies depending on the patient's condition. Human peripheral blood mononuclear cells (PBMCs) are separated using Ficoll density gradient centrifugation (0.7 x 10). 8 -4.33 x 10 8 ).

[0192] 2) Add PBMC to the cell culture flasks, 5 x 10 g per flask. 6 -10 x 10 6 Cells were cultured in AIM-V medium (containing 1% (v / v) inactivated autologous serum, 1000 IU / ml granulocyte-macrophage colony-stimulating factor (GM-CSF), and 500 IU / ml interleukin-4 (IL-4)) and incubated at 37°C with 5% CO2 and saturated humidity. The medium was changed every three days. (PBMCs had not yet differentiated into DC cells for the first six days of culture.)

[0193] 3) On the sixth day of culture, treat the cells with 50-100 uM vitamin K2 (the cell concentration is (0.8 x -1 x 10) 6 (cells / ml) 24 hours.

[0194] 4) The same number of tumor cells as DC cells were sonicated in 1 ml PBS (PMSF added at a ratio of 1:100) to obtain tumor cell lysate. The tumor lysate was filtered through a 0.22 μm filter and added to DC cell culture medium (10% FBS (inactivated), 1-3 mM L-glutamine, 1% penicillin / streptomycin, 50 μM β-mercaptoethanol, 20-30 ng / ml GM-CSF in 1640 RPMI medium) and 10 ng / ml TNF-α was added. The cells were treated for 24 hours to obtain activated dendritic cells.

[0195] Example 2: Flow cytometry analysis of dendritic cell viability after different treatments

[0196] 2.1. Cell preparation:

[0197] Mature mouse dendritic cells (DCs) were obtained by isolating bone marrow cells from the femur and tibia of C57BL / 6 mice and treating them with 20 ng / ml GM-CSF, 50 ng / ml TNF-α, or 1 μg / ml LPS.

[0198] 2.2 The obtained DC cells were divided into three groups: a control group, a tumor cell lysate treatment group, and a tumor cell lysate and vitamin K2 combined treatment group. The specific treatment process is as described in Example 1.

[0199] Grouping and processing:

[0200] (1) DC cell group: DC cells that have not undergone any treatment.

[0201] (2) DC cell group treated with tumor cell lysate (DC+TCL): DC cells were treated with tumor cell lysate (TCL) for 24 hours. Here, TCL is LLC lung cancer cell lysate.

[0202] (3) DC cell group treated with tumor cell lysate and vitamin K2 (DC+VK2+TCL): DC cells were treated with 50-100uM vitamin K2 for 24 hours, and then treated with tumor cell lysate for 24 hours.

[0203] 2.3 After staining the three groups of cells with cell viability dyes (including LiveDead BV510-A), flow cytometry analysis was performed to obtain the cell viability status.

[0204] The results are shown in Figure 1: Compared with the dendritic cells in the DC group (survival rate 99.75%), the survival rate of DC cells in the DC+TCL group was only 15.31%, indicating that the dendritic cells treated with tumor cell lysate died significantly due to overactivation; while the survival rate of DC cells in the DC+VK2+TCL group was 32.64%, indicating that the addition of vitamin K2 to DC cells can significantly inhibit the DC cell death caused by overactivation of DC cells induced by tumor cell lysate.

[0205] Example 3: Dendritic cell vaccine enhanced with CD8 + The ability of T cells to kill tumor cells

[0206] 3.1 Establishment of a dendritic cell antigen presentation model

[0207] 1) Related preparations: Expand the culture of mouse lung cancer LLC cells, mouse intestinal cancer MC38 cells, and mouse breast cancer 4T1 cells in a cell culture incubator.

[0208] 2) Cell line detection: The cells were tested for mycoplasma contamination using a mycoplasma kit. Once the cells met the requirements, the three cell lines were seeded into 12-well plates with 0.5 x 10 cells per well. 6 .

[0209] 3) Remove the spleen and lymph nodes of the mouse through... CD8 + T-cell isolation kit (stem cell) to isolate and obtain mice CD8 + T cells.

[0210] 4) CD8 + T cells and dendritic cells (100:1) were co-cultured in 1640 medium (containing 1ug / ml CD3 antibody) for 48 hours; the dendritic cell grouping here is as shown in 3.2 Grouping and Treatment below.

[0211] 5) Activate CD8 + T cells were added to the tumor cells in step 1), and CD8+ was added to each well. + The T cell count is 1 x 10 5 Three days later, the number of tumor cells was counted.

[0212] 3.2 Grouping and Processing: The specific processing procedure is as described in Example 1.

[0213] (1) Control group (Con): No treatment was given.

[0214] (2) DC cell group treated with tumor cell lysate (DC+TCL): DC cells were treated with tumor cell lysate (TCL) for 24 hours. Here, TCL is the lysate of the corresponding tumor cells to be tested.

[0215] (3) DC cell group treated with tumor cell lysate and vitamin K2 (DC+VK2+TCL): DC cells were treated with 50-100uM vitamin K2 for 24 hours, and then treated with tumor cell lysate for 24 hours.

[0216] 3.3 Experimental Results

[0217] As shown in Table 1, in CD8 + In experiments on the killing of LLC lung cancer cells, MC38 colon cancer cells, and 4T1 breast cancer cells by T cells, compared with the DC+TCL group, the CD8+VK2 group showed significantly lower levels of CD8+ cells. + T cells have a stronger tumor-killing ability.

[0218] Table 1 Note: DC represents dendritic cells, TLC represents tumor cell lysate, and VK2 represents vitamin K2.

[0219] Example 4: Dendritic cell vaccine inhibits lung cancer cell tumorigenesis

[0220] 4.1 Preparation of a mouse model of lung cancer

[0221] 1) Preparation of relevant cell lines: Rat lung cancer LLC cells were cultured in DMEM medium in a cell culture incubator.

[0222] 2) Cell line detection: Use a mycoplasma kit to detect whether the cells are contaminated with mycoplasma. Once the cells meet the requirements, they will proceed to the next stage of mouse experiments.

[0223] 3) Subcutaneous inoculation of tumor cells into mice:

[0224] 100 μl of insulin was injected subcutaneously into the right thigh of the mouse using a syringe. 6 LLC cells. (It should be noted that all equipment was sterilized before the operation, and the mice were wiped with alcohol during the experiment); the mice were placed under a heat lamp to restore their body temperature, and after they were fully awakened, they were returned to the animal center's feeding room.

[0225] 4) Based on the principle of balanced tumor volume and body weight of tumor-bearing mice, they were divided into the following 5 groups, with 6 mice in each group.

[0226] Model control group (Placebo): 200 μL of normal saline was injected into the tail vein;

[0227] Dendritic cell group (DC): Untreated DC cells (200 μL, cell count 1 x 10⁻⁶) were injected via tail vein. 6 ).

[0228] Vitamin K2-treated DC group (DC+VK2): 50-100 μM vitamin K2-treated DC cells (200 μL, cell count 1 x 10⁻⁶) were injected via tail vein. 6 ).

[0229] DC group treated with tumor cell lysate (DC+TCL): DC cells treated with LLC lung cancer cell lysate (200 μL, cell count 1 x 10⁻⁶) were injected via tail vein. 6 ).

[0230] DC group treated with tumor cell lysate and vitamin K2 (DC+TCL+VK2): DC cells (200 μL, cell count 1 x 10⁻⁶ cells) treated with LLC lung cancer cell lysate and 50-100 μM vitamin K2 were injected via tail vein. 6 ).

[0231] On days 5 and 10 after subcutaneous tumor implantation, DC cells treated in different ways as shown in the above grouping were injected into the tail vein of mice. The drugs were administered twice in total. Tumor growth was observed. The cages were changed every three days. One week after the injection of DC cells, the size of the in situ tumor was measured. The measurement was taken every two days for the first six days, and then once a day thereafter.

[0232] 5) Observe the mice's physical condition 10 days after injecting DC cells. If any mice die, stop feeding them immediately. If no mice die, stop feeding them 15 days after injection.

[0233] 6) All mice were euthanized by spinal dislocation, and the subcutaneous tumors of the five groups of mice were removed and weighed.

[0234] The experimental results are shown in Figure 2, and the tumor weight and tumor volume are shown in Table 2.

[0235] Table 2

[0236] The results of the AC plot in Figure 2 show that, compared with the control group (Placebo), the DC group, DC+VK2 group, and DC+TCL group did not show significant effects on the changes in tumor volume and tumor weight of lung cancer. Among them, the DC group and DC+VK2 group may have no significant effect on tumor growth because the DC cells in them had not yet been activated. The DC+TCL group may have been caused by the over-activation of DC cells by tumor cell lysate, leading to DC cell death and thus failing to inhibit tumor cell growth. However, the DC+TCL+VK2 group could significantly reduce tumor volume (inhibition rate 91% = (1-111.98 / 1238.05)*100) and tumor weight (inhibition rate 92.9% = (1-0.1 / 1.4)*100). The DC vaccine treated with tumor cell lysate and vitamin K2 could significantly inhibit lung cancer cell tumorigenesis, indicating that vitamin K2 can significantly inhibit DC cell death caused by over-activation of DC cells by tumor cell lysate.

[0237] Example 5: Dendritic cell vaccine enhanced with CD8 + The ability of T cells to kill HBV-infected cells

[0238] 5.1 Establishment of a dendritic cell antigen presentation model

[0239] 1) Relevant cell preparation: Expand the culture of human liver cancer Huh7 cells in a cell culture incubator.

[0240] 2) Cell line detection: The cells were tested for mycoplasma contamination using a mycoplasma kit. Once the cells met the requirements, they were seeded into 12-well plates with 0.5 x 10 cells per well. 6 Meanwhile, the cells were infected with human HBV virus.

[0241] 3) Use CD8+ T cell isolation kit (stem cell) for isolating and obtaining human-derived cells from human PBMCs. CD8+ T cells.

[0242] 4) CD8+ T cells and human dendritic cells (10:1) were co-cultured in 1640 medium (containing 1ug / ml CD3 antibody) for 48 hours; the dendritic cell grouping here is as shown in 5.2 Grouping and Treatment below.

[0243] 5) Add the activated CD8+ T cells to the Huh7 cells from step 1), with the number of CD8+ T cells added to each well being 1 x 10. 5 Three days later, the number of tumor cells was counted.

[0244] 5.2 Grouping and Processing:

[0245] (1) Control group: No treatment was given.

[0246] (2) DC cell group treated with HBV antigen (DC+Antigen(HBV)): DC cells were treated with HBV antigen for 24 hours.

[0247] (3) DC cell group treated with HBV antigen and vitamin K2 (DC+VK2+Antigen(HBV)): DC cells were treated with 50-100uM vitamin K2 for 24 hours and then treated with HBV antigen for 24 hours.

[0248] 5.3 Experimental Results

[0249] As shown in Figure 3, in the experiment of CD8+T cells killing Huh7 cells infected with HBV, CD8+T cells in the DC+Antigen(HBV)+VK2 group had stronger killing ability compared with the DC+Antigen(HBV) group.

[0250] 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 method for preparing modified dendritic cells, characterized in that, Includes the following steps: (S1) provides dendritic cells; (S2) In the presence of vitamin K2, dendritic cells with stronger antigen-presenting ability are obtained by loading tumor antigens and culturing them. These are called modified dendritic cells.

2. The method as described in claim 1, characterized in that, The tumor antigen loading method is selected from the following group: (1) Direct loading method: Dendritic cells directly take up and process the tumor antigen; (2) Electroporation: The tumor antigen is introduced into the dendritic cells using electroporation technology; (3) Genetic engineering technology: The gene encoding the tumor antigen is introduced into the dendritic cells through genetic engineering technology, so that it is expressed and processed into antigen in the cells; (4) Cell fusion technology: fusion cells with antigen-presenting ability are obtained by inducing the fusion of dendritic cells and tumor cells; The methods for obtaining the tumor antigen are selected from the following group: (1) Extraction from tumor cells: Obtain the patient's tumor cells through surgery, biopsy or other means, and extract tumor antigens using cell culture and lysis techniques; (2) Use of tumor-associated antigen peptides: The peptides are protein fragments specifically expressed on or inside tumor cells and obtained through chemical synthesis or genetic engineering techniques; (3) Utilizing tumor antigen mRNA: Tumor antigen mRNA is an mRNA molecule that encodes tumor-associated antigens; (4) Using tumor antigen cDNA: mRNA from tumor cells is extracted and reverse transcribed into cDNA, which is then amplified and purified by genetic engineering technology.

3. The method as described in claim 2, characterized in that, The tumor antigen is selected from the group consisting of: polypeptides, proteins, tumor cell lysates, mRNA, tumor-derived exosomes, or combinations thereof.

4. A use of vitamin K2, characterized in that, Used to prepare a formulation or composition, said formulation or composition serving as a protectant for dendritic cells.

5. A method for preparing anti-tumor T lymphocytes, characterized in that, The steps include: co-culturing dendritic cells prepared by the method of claim 1 with T lymphocytes to obtain anti-tumor T lymphocytes, wherein the T lymphocytes are autologous or allogeneic T lymphocytes of the subject.

6. A composition for treating cancer or tumors, characterized in that, The composition comprises dendritic cells prepared by the method of claim 1, and / or a pharmaceutically acceptable or vaccinologically acceptable carrier.

7. The composition according to claim 6, characterized in that, The composition also includes other cancer treatment agents; the cancer treatment agents are selected from the group consisting of: T-cell vaccines, tumor vaccines, immune checkpoint inhibitors, and radiotherapy or chemotherapy-related preparations. The tumor treatment drugs mentioned include: temozolomide, nicolimustine, avastin, mannitol, glycerol fructose, dexamethasone, methylprednisolone, sodium valproate, carbamazepine, levetiracetam, bevacizumab, imatinib, gefitinib, auristatins, camptothecins, docarmycins, etoposides, maytansines and maytansinoids, taxanes, benzodiazepines, or drugs containing benzodiazepines. Drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), vinca alkaloids (Vinca) Alkaloids, chlortetracycline, tebuconazole, pyrethroids, pyrethroid A-chain, cobustatin, docalimicin, dolasmidine, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthraxetine dione, actinomycin, diphtheria toxin, pseudomonadoxins (PE)A, PE40, absinthecin, absinthecin A-chain, saccharin A-chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, cumin, crotonin, chachomycin, Sapaonaria officinalis inhibitors, glucocorticoids, or combinations thereof.

8. The composition according to claim 7, characterized in that, The T-cell vaccine refers to activated T cells obtained by co-culturing the modified dendritic cells, and the modified dendritic cells combined with the T-cell vaccine are used as a combination vaccine formulation for tumor immunotherapy. The tumor vaccines include: CAR T vaccines, CAR NK vaccines, CAR Macrophage vaccines, TIL vaccines, exosome vaccines, peptide cancer vaccines, mRNA cancer vaccines, DNA cancer vaccines, and viral vector vaccines; The immune checkpoint inhibitors include: PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, and bispecific inhibitors.

9. The composition according to claim 8, characterized in that, The PD-1 / PD-L1 inhibitors include: pembrolizumab, nivolumab, camrelizumab, toripalimab, sintilimab, tislelizumab, atezolizumab, durvalumab, suglizumab, penaprilimab, cepalimumab, slulizumab, putelizumab, avelumab, envorimab, and trastuzumab emtansine. The CTLA-4 inhibitors include: ipilimumab; The bispecific inhibitors include: epalolitoprene, canduniline, edoxicillin, ZG005, LBL-024, and SHR-1701.

10. The composition according to claim 6, characterized in that, The cancer or tumor is selected from the group consisting of: lung cancer, gastrointestinal cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumor, breast cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, or combinations thereof.

11. Use of dendritic cells prepared by the method of claim 1 in the preparation of a medicament for treating viral infections; characterized in that, The viruses mentioned include: influenza virus, enterovirus 71 (EV71), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), human T-phage virus (HTLV), and HIV.

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