WO2021077566A1 - 二价锰在制备免疫增强药物或抗肿瘤药物中的应用 - Google Patents

二价锰在制备免疫增强药物或抗肿瘤药物中的应用 Download PDF

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WO2021077566A1
WO2021077566A1 PCT/CN2019/124098 CN2019124098W WO2021077566A1 WO 2021077566 A1 WO2021077566 A1 WO 2021077566A1 CN 2019124098 W CN2019124098 W CN 2019124098W WO 2021077566 A1 WO2021077566 A1 WO 2021077566A1
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manganese
tumor
divalent
mice
cells
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French (fr)
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蒋争凡
吕梦泽
王晨光
张睿
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北京大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/32—Manganese; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants

Definitions

  • the invention belongs to the field of medicine, and specifically relates to the application of divalent manganese in the preparation of immune enhancing drugs or antitumor drugs.
  • the treatment methods for tumors currently mainly include surgery, radiation therapy, chemotherapy, cell adoptive transfer therapy and immune checkpoint inhibitor therapy.
  • adoptive transfer therapy of cells represented by Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T), as well as programmed cell death protein-1 (PD-1) Antibody (anti-PD-1) and cytotoxic T lymphocyte antigen-4 (Cytotoxic T lymphocyte antigen-4, CTLA-4) antibody (anti-CTLA-4) is an immunotherapy represented by immune checkpoint inhibitor therapy It is the main tumor treatment method.
  • PD-1 can prevent patients with many other types of tumors.
  • the effective rate of patients with broken antibodies is only about 20%. Therefore, how to effectively enhance the body's own anti-tumor immune response to enhance the effect of tumor treatment has also become a research hotspot in recent years.
  • the body's immune system can be divided into innate immunity and adaptive immunity.
  • the innate immune system can also be called innate immunity and non-specific immunity. It is widely found in animals, plants, fungi and insects.
  • the innate immune system reacts quickly and has a wide range of functions. It is the body's first line of defense against pathogen invasion and is also activated.
  • adaptive immunity also known as acquired immunity or specific immunity.
  • Immune cells mainly involved in the natural immune response include monocytes, macrophages, natural killer cells, dendritic cells, neutrophils, and neutrophils. Basic granulocytes (basophils), eosinophils (eosinophils) and mast cells (mast cells), etc.
  • the killing of tumor cells in the body mainly relies on CD8 + T cells (which belong to the adaptive immune system), and this group of cells cannot function without the assistance of antigen-presenting cells (APC) such as dendritic cells.
  • APC antigen-presenting cells
  • the innate immune system can recognize different pathogen-associated molecular patterns (PAMPS) and damage-associated molecular patterns (DAMPS) through various pattern recognition receptors (PRR). ), and then induce the production of a series of cytokines including type I interferon to resist the invasion of pathogens.
  • PAMPS pathogen-associated molecular patterns
  • DAMPS damage-associated molecular patterns
  • PRR pattern recognition receptors
  • cGAS cyclic GMP-AMP synthase
  • cGAMP cyclic dinucleotide 2', 3'-cyclic GMP-AMP
  • Dead tumor cells will be engulfed by dendritic cells, and then release the DNA in them, activate the cGAS-STING pathway of dendritic cells, induce the production of type I interferon, promote the maturation and activation of dendritic cells, and promote their antigen crossover Cross-presentation, thereby enhancing the priming effect of dendritic cells on CD8 + T cells, thereby enhancing the specific killing of these CD8 + T cells on tumors.
  • this group of CD8 + T also It is called cytotoxic T lymphocytes.
  • the present invention provides the use of divalent manganese to prepare dendritic cells, macrophages or T cell activators and/or sensitizers.
  • the invention also provides the application of divalent manganese for preparing anti-tumor drugs.
  • the present invention provides that divalent manganese is used in the preparation of dendritic cell activator/sensitizer, macrophage activator/sensitizer, or T cell (eg CD8 + T cell) activator/sensitizer Applications.
  • the divalent manganese stimulates dendritic cells or macrophages to produce type I interferon.
  • the divalent manganese stimulates dendritic cells to up-regulate the expression of CD80 and/or CD86.
  • the dendritic cells are dendritic cells derived from structures or tissues such as bone marrow, lungs, lymph nodes, and peripheral blood.
  • the dendritic cells may be derived from mammals.
  • the macrophages are macrophages derived from structures or tissues such as bone marrow, abdominal cavity, lungs, lymph nodes, and peripheral blood.
  • the macrophages may be derived from mammals.
  • the divalent manganese promotes the proliferation of CD8-positive T cells (CD8 + T cells), or promotes the proliferation of CD8-positive T cells that specifically recognize antigens.
  • the antigen is a tumor antigen; a
  • the antigen may be ovalbumin (OVA).
  • OVA ovalbumin
  • the divalent manganese promotes the infiltration of CD8 positive T cells in the tumor.
  • the present invention provides an activator/sensitizer for dendritic cells, macrophages or CD8-positive T cells, which includes divalent manganese.
  • the activator/sensitizer can be administered to a subject in vivo or in vitro, and the subject can be a mammal.
  • the present invention provides an application of an activator/sensitizer of dendritic cells, macrophages or CD8-positive T cells including divalent manganese in cellular immunotherapy drugs.
  • the present invention provides the application of divalent manganese in the preparation of drugs for treating or preventing tumors.
  • the divalent manganese is used as a sensitizer of an anti-tumor agent, which can significantly improve the therapeutic effect of the anti-tumor agent on tumors, or significantly reduce the dosage of the anti-tumor agent.
  • the anti-tumor drug is administered to a subject who has been diagnosed with a tumor (in accordance with the criteria of a phase I clinical trial).
  • the anti-tumor agent is an immune checkpoint inhibitor and/or a chemotherapeutic drug, such as PD-1 antibody, PD-L1 antibody, cyclophosphamide.
  • a chemotherapeutic drug such as PD-1 antibody, PD-L1 antibody, cyclophosphamide.
  • the dosage of the anti-tumor drug is 0.5 times or less of the effective dose of the anti-tumor drug when administered alone, preferably 0.01-0.5 times, more preferably 0.1-0.5 Times, most preferably, 0.3-0.5 times.
  • the present invention provides an anti-tumor pharmaceutical composition, including an anti-tumor agent and divalent manganese.
  • the anti-tumor pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the present invention provides the application of divalent manganese in preparing vaccine compositions for preventing tumors.
  • the tumor-preventing vaccine can be administered to subjects who do not have tumors in advance to activate and enhance the specific killing effect on tumor cells in the body, thereby playing a tumor-preventing effect.
  • the present invention provides a vaccine composition for preventing tumors, the vaccine composition comprising the divalent manganese.
  • the vaccine further includes an antigen, and the antigen may be a tumor antigen, such as an OVA antigen; further, the OVA antigen contains a SIINFEKL peptide fragment.
  • the vaccine further includes a pharmaceutically acceptable carrier.
  • the divalent manganese can be in the form of free manganese ions, divalent manganese salts, or other divalent manganese sources that can be converted into the form of divalent manganese, for example, the divalent manganese salt is pharmaceutically acceptable Salt, selected from manganese chloride, manganese bromide, manganese iodide, manganese sulfate, manganese nitrate, manganese perchlorate, manganese acetate, manganese carbonate, manganese borate, manganese phosphate, manganese hydrobromide, manganese tartrate, fumaric acid Manganese, manganese maleate, manganese lactate, manganese benzenesulfonate, manganese pantothenate, manganese ascorbate and any combination thereof.
  • the divalent manganese salt is pharmaceutically acceptable Salt, selected from manganese chloride, manganese bromide, manganese iodide, manganese sulf
  • the divalent manganese is Mn 2 OHPO 4 colloid.
  • the Mn 2 OHPO 4 colloid is made of phosphate, MnCl 2 and physiological saline, and the phosphate is selected from Na 3 PO 4 , K 3 PO 4 or other types; in one embodiment, the manganese ion colloid is The concentrations of phosphate ion and divalent manganese ion are not less than 25mM and 20mM, respectively, and the molar concentration ratio of phosphate ion to divalent manganese ion is 1.25:1.
  • the Mn 2 OHPO 4 colloid is 100 ⁇ L 0.2M MnCl 2 solution, 50 ⁇ L 0.5 M Na 3 PO 4 solution is added, and 850 ⁇ L normal saline is added overnight to prepare Mn 2 OHPO 4 colloid.
  • the tumor is malignant melanoma, lung cancer, colon cancer, lymphoma, ovarian cancer, gastric cancer, breast cancer, pancreatic cancer, cholangiocarcinoma, kidney cancer, liver cancer, sarcoma, bladder cancer, tumor One or any combination of cervical cancer, leukemia, head and neck cancer, other subcutaneous solid tumors, etc.
  • the tumor is primary or metastatic.
  • the tumor is a tumor that is not sensitive to immune checkpoint inhibitors (anti-PD-1/anti-PD-L1) or chemotherapeutic drugs, such as skin melanoma and colon cancer.
  • the tumor is ovarian cancer.
  • the divalent manganese or anti-tumor pharmaceutical composition can be an intravenous injection preparation, a nasal drop preparation (including aerosolized form and mucosal administration), an oral preparation, an intradermal injection preparation, a subcutaneous injection preparation or an intramuscular injection preparation .
  • a nasal drop preparation including aerosolized form and mucosal administration
  • an oral preparation an intradermal injection preparation, a subcutaneous injection preparation or an intramuscular injection preparation .
  • it is preferably a subcutaneous injection preparation or an intramuscular injection preparation.
  • the amount of the divalent manganese or antitumor agent is a therapeutically effective amount.
  • divalent manganese ions can enhance the subject's ability to resist multiple types of tumors through different ways, and further discovered that divalent manganese ions can activate dendritic cells and macrophages from various sources And T cells, promote dendritic cells to produce type I interferon, promote the proliferation of CD8 + T cells or enhance their specific binding to antigens, thereby enhancing immune response and inhibiting tumor cell proliferation.
  • divalent manganese ions have a good sensitizing effect on other anti-tumor agents, such as combined use with the immune checkpoint inhibitor anti-PD-1 or the chemotherapeutic drug cyclophosphamide (cyclophosphamide monohydrate).
  • CTX chemotherapeutic drug cyclophosphamide
  • CTX combined use can significantly enhance the therapeutic effect of anti-PD-1 or CTX on tumors, and thus can significantly reduce the dosage of anti-tumor agents such as anti-PD-1 and CTX without affecting the therapeutic effect of tumors.
  • divalent manganese ions can be administered in a flexible manner when used in combination, and can be administered together with other anti-tumor agents or administered separately.
  • divalent manganese ions such as nasal drops including Nebulized inhalation or mucosal absorption
  • the combined use of divalent manganese ions can reduce the dosage of chemotherapy drugs, which will also hopefully reduce the side effects of chemotherapy on patients.
  • the combination of divalent manganese ions can reduce the dosage of anti-PD-1, which is expected to reduce the economic burden of patients and benefit more patients.
  • Figure 1 shows the construction of a mouse tumor model.
  • 1A indicates the method of tumor inoculation, the time of inoculation and the final treatment method;
  • 1B indicates the successful construction of the mouse subcutaneous tumor model;
  • 1C indicates the successful construction of the mouse lung tumor metastasis model.
  • Figure 2 shows the inhibition of melanoma in the blank control Con without addition of Mn 2+ and the mice with Mn 2+ administered in Example 2, showing that nasal instillation of Mn 2+ can enhance the mice’s resistance to subcutaneous melanoma and melanoma lungs
  • 2A shows that Mn 2+ can significantly delay the subcutaneous growth rate of mouse melanoma B16F10
  • 2B is a mouse subcutaneous tumor photographed with a live imager at different time points
  • 2C and D show that the subcutaneous tumor was killed 2 weeks after inoculation Rats, statistical analysis of the complete tumor tissues and the corresponding tumor tissue weights obtained by dissection.
  • 2E-G shows the lung tumor growth two weeks after tail vein inoculation of B16F10 melanoma, as well as the statistical analysis of the number of tumor metastases and the weight of the intact lung.
  • Figure 3 shows that Mn 2+ can enhance the ability of mice to resist multiple types of tumors through different administration routes, including the blank control Con and Mn 2+ treatment groups.
  • Figures 3A and B show that nasal instillation of Mn 2+ can significantly delay the growth rate of mouse Lewis lung cancer LLC inoculated subcutaneously, and significantly prolong the survival time of mice.
  • 3C and D show that Mn 2+ instilled in the nasal cavity can significantly delay the growth rate of mouse T lymphoma E.G7 subcutaneously inoculated, and significantly inhibit the size of the tumor.
  • 3E-F showed that injection of Mn 2+ through the tail vein can significantly delay the growth rate of mouse colon cancer MC38 inoculated subcutaneously, and significantly inhibit the size of the tumor.
  • 3G shows that intratumor injection of Mn 2+ can significantly delay the growth rate of subcutaneously inoculated mouse melanoma B16-OVA, and significantly increase the survival rate of mice for 40 days.
  • 32 mice in the control group were all 40 days before tumor inoculation
  • 13 mice were still alive at 40 days after tumor inoculation.
  • Figure 4 shows that Mn 2+ enhances the anti-tumor ability of mice not through direct killing of tumor cells.
  • 4A indicates that B16F10, LLC and L929 cells were directly treated with 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400, 800 ⁇ M MnCl 2 in vitro, and the survival rate of the cells was measured by the MTT method.
  • 4B means that mice were inoculated subcutaneously with melanoma B16F10 on both sides, and then intratumoral injection with saline or Mn 2+ was performed on one side, and then the tumor size on the other side was measured and recorded, and the survival rate of the mice was recorded at the same time.
  • 4C shows the difference in survival rate of mice in 4B.
  • Figure 5 shows that Mn 2+ treatment does not cause significant side effects in mice.
  • 5A, B indicate that mice were given Mn 2+ by nasal instillation seven times, once every 2 days, and the weight of the mice was recorded for a total of 14 days.
  • Mn 2+ treatment will not affect the body weight of the mice.
  • 5C and D indicate a total of three groups of mice, namely the non-treatment group NC, the control group Con and the Mn 2+ treatment group. Mn 2+ was given by nasal drip, once every 2 days, a total of seven times, after each The groups were no longer processed, and the weight of the mice and the survival rate of the mice in the corresponding group were recorded.
  • 5E-J represent the corresponding tissues obtained by dissection of the mouse in Figure C and D, and record the weight.
  • 5K shows the results of eosin-hematoxylin stained sections of some tissues and organs in Figure EJ.
  • Figure 6 shows that Mn 2+ enhances the anti-tumor ability of mice is mainly mediated by CD8 + T cells, and can enhance the infiltration of T cells in tumors.
  • 6A-C showed that in Rag1 knockout mice, Mn 2+ treatment could not delay the growth rate and tumor size of melanoma B16F10 subcutaneously inoculated in mice.
  • 6D-F showed that in ⁇ 2M knockout mice, Mn 2+ treatment could not delay the growth rate and tumor size of melanoma B16F10 subcutaneously inoculated in mice.
  • 6G-I shows the degree of T cell infiltration in melanoma B16F10 tumors inoculated subcutaneously in mice after Mn 2+ treatment by flow cytometry.
  • 6J shows CD8 positive cells in melanoma B16F10, colon cancer MC38 and Lewis lung cancer LLC tumors inoculated subcutaneously in mice by immunofluorescence section after Mn 2+ treatment.
  • Figure 7 shows that Mn 2+ can enhance the activity of tumor infiltrating CD8 + T cells and its ability to specifically kill.
  • 7A-D show that after Mn 2+ treatment, the infiltration of IFN ⁇ + CD8 + T cells and TNF ⁇ + CD8 + T cells in the mouse subcutaneous melanoma B16F10 increased significantly.
  • 7E-H show the Mn 2+ treatment, IFN ⁇ subcutaneously in mouse T lymphoma E.G7 + CD8 + CD8 + T cells and infiltration of tumor antigen specific recognition E.G7 SIINFEKL T cells were significantly increased.
  • mice 7I-K represents Mn 2+ after treatment, the mice were inoculated subcutaneously B16F10 melanoma infiltration of CD8 + T cells to an RNA sequence, CD8 + T and other related transcription Tbx21 significantly upregulated on tumor and CD8 + T
  • the killing-related granzymes Granzymes and perforin Perforin were significantly up-regulated, and some cytokines also changed.
  • Figure 8 shows that Mn 2+ can promote the activation of dendritic cells (DC) in vitro and in vivo.
  • 8A, B show that Mn 2+ can activate mouse bone marrow-derived DC (BMDC) and macrophages (BMDM) to produce type I interferon.
  • 8C and D show that Mn 2+ can significantly promote the expression of CD80 and CD86 in mouse bone marrow-derived DCs in vitro.
  • 8E-H shows that Mn 2+ can significantly promote the expression of CD80 and CD80 in mouse lungs and inguinal lymph nodes in vivo.
  • CD86. 8I, J showed that Mn 2+ can promote the expression of CD86 by DCs in PBMC of peripheral blood mononuclear cells of tumor patients in vitro.
  • Figure 9 shows that Mn 2+ promotes the killing of tumor cells by DC and CD8 + T cells in vitro.
  • 9A, B show that Mn 2+ can promote the killing of B16F10-OVA tumor cells by OT-I CD8 + T cells that specifically recognize OVA antigen in vitro.
  • Figure 10 shows that Mn 2+ can act as an adjuvant to help the body produce more antigen-specific CD8 + T cells.
  • 10A, B indicate that immunizing mice with OVA antigen and Mn 2+ in advance can significantly enhance the resistance of mice to B16-OVA tumors inoculated subcutaneously, and significantly extend the survival time of mice.
  • 10C, D indicate that immunizing mice with OVA and Mn 2+ in advance can help mice to significantly increase the production of OVA peptide SIINFEKL-specific CD8 + T cells.
  • 10E, F show that stimulating mice with OVA and Mn 2+ can enhance the specific killing of OVA cells in vivo.
  • 10G, H showed that stimulating mice with OVA and Mn 2+ can significantly enhance the proliferation of CD8 + T cells that specifically recognize OVA antigen in mice.
  • Figure 11 shows that the cGAS-STING pathway plays an important role in the anti-tumor immune response of Mn 2+.
  • 11-A shows that in cGAS (CGas) knockout or STING (Tmem173) knockout mice, Mn 2+ stimulation can no longer cause mice to produce type I interferon.
  • 11-B, C show that at in vitro levels, Mn 2+ cannot activate STING (Tmem173) knockout mouse bone marrow-derived DC (BMDC) and macrophages (BMDM) to produce type I interferon.
  • 11-DF showed that under the background of STING (Tmem173) knockout mice, the therapeutic effect of Mn 2+ on melanoma B16F10 subcutaneously inoculated was greatly reduced.
  • 11G, H indicate that under the background of STING (Tmem173) knockout mice, after immunizing mice with OVA antigen and Mn 2+ in advance, Mn 2+ can no longer enhance the resistance of mice to subcutaneously inoculated B16-OVA tumors, nor can it Extend the survival time of mice.
  • 11-I, J showed that under the background of STING (Tmem173) knockout mice, OVA and Mn 2+ stimulated mice no longer in vivo to enhance the specific killing of OVA cells.
  • Figure 12 shows that Mn 2+ synergistically enhances the therapeutic effect of immune checkpoint inhibitor anti-PD-1 on tumors.
  • 12A-E indicates that the combined use of Mn 2+ and anti-PD-1 can significantly enhance the therapeutic effect of anti-PD-1 on mice subcutaneously inoculated with melanoma B16F10, and can significantly enhance the infiltration of CD8 + T cells in the tumor.
  • 12F indicates that the combined use of Mn 2+ and anti-PD-1 can significantly enhance the therapeutic effect of anti-PD-1 on mouse lung metastatic melanoma B16F10.
  • 12G indicates that for mice subcutaneously inoculated with melanoma B16F10, the combined use of Mn 2+ and anti-PD-1 can reduce the dosage of anti-PD-1 by half.
  • 12H-J shows that the combined use of Mn 2+ and anti-PD-1 can significantly enhance the therapeutic effect of anti-PD-1 on mice subcutaneously inoculated with colon cancer MC38.
  • 12K indicates that the combined use of Mn 2+ and anti-PD-1 can enhance the infiltration of CD8 + positive cells in MC38 tumors.
  • Figure 13 shows that Mn 2+ synergistically enhances the therapeutic effect of the chemotherapy drug CTX on tumors.
  • 13A-C indicate that for the treatment of mice subcutaneously inoculated with melanoma B16F10 , the combined use of Mn 2+ and the chemotherapy drug cyclophosphamide (cyclophosphamide monohydrate, CTX) can reduce the amount of CTX by half.
  • cyclophosphamide cyclophosphamide monohydrate, CTX
  • activation means activation, activation or activation
  • activator is used interchangeably with “sensitizer” and “activator”, meaning that for a given type of immune cell, according to The established mechanism produces type I interferon to activate the immune response, including promoting the proliferation of immune cells or up-regulating the expression of immunosuppressive cytokines.
  • the activator or activator of the present invention can prevent or treat diseases that reduce symptoms by regulating the activation of T cells. In one embodiment of the present invention, it includes administering an activator to promote the proliferation of CD8-positive T cells, promote antigen-specific recognition of CD8-positive T cells, or promote an immune response through other mechanisms.
  • sensitizer refers to administering a therapeutically effective amount of a molecule to a subject to increase the sensitivity of cells to anti-tumor drugs, such as increasing the sensitivity to biological drugs, chemotherapeutic drugs, and/or promoting chemical The therapeutic effect of the therapy.
  • the sensitizer can be administered together with a therapeutically effective amount of one or more other compounds or prepared as a composite formulation.
  • Other compounds include but are not limited to: compounds that promote the incorporation of chemosensitizers into target cells; control therapeutic agents, nutrition Compounds that deliver substances and/or oxygen to target cells; chemotherapeutics that act on tumors or other therapeutically effective compounds for the treatment of cancer or other diseases.
  • adding an effective amount of divalent manganese can enhance the effect of immune checkpoint inhibitors or chemotherapeutic drugs, for example, administering divalent manganese, immune checkpoint inhibitors or chemotherapeutics at a dose of 5 mg/kg per cycle When administered at 0.3-0.5 times the effective dose of the prescribed drug, the effect of the effective dose of the drug when the immune checkpoint inhibitor or chemotherapeutic drug is administered alone can be achieved.
  • Dendritic cells are the most powerful antigen-presenting cells, named for their many dendritic or pseudopod-like protrusions when they mature.
  • Type I interferon can be used as a sensitizing cytokine for dendritic cells, which is embodied in the up-regulated expression of costimulatory molecules CD80/CD86.
  • dendritic cells themselves can also produce type I interferon and other cytokines when they are stimulated. .
  • Macrophages are white blood cells located in tissues, derived from monocytes, which in turn are derived from precursor cells in the bone marrow, and participate in natural immunity and adaptive immunity in vertebrates. Macrophages can produce type I interferon and other cytokines when they are stimulated to enhance the natural immune response and adaptive immune response.
  • T lymphocytes are lymphatic stem cells derived from bone marrow. After differentiation and maturation in the thymus, they are distributed to immune organs and tissues throughout the body through lymph and blood circulation to exert immune functions. T cells can receive antigens presented by dendritic cells and other antigen-presenting cells, and then produce a series of lymphokines such as TNF ⁇ and IFN ⁇ to help kill or directly kill target cells containing corresponding antigens.
  • CD8-positive T cells belong to a class of T cells, which can develop into cytotoxic T cells after recognizing antigens presented by MHC-I molecules (such as tumor antigens), and then directly kill tumor cells.
  • tumor associated antigen refers to proteins that are specifically expressed in a limited amount of tissues and/or organs under normal conditions or at specific developmental stages.
  • tumor antigens can be found in gastric tissues (such as gastric mucosa) and reproductive organs under normal conditions. It is specifically expressed in cells (e.g., in the testis), in trophoblast tissues (e.g., in the placenta), or in germline cells; and expressed or abnormally expressed in one or more tumors or cancer tissues.
  • tumor antigens are preferably associated with the cell surface of cancer cells, and are preferably not expressed or only rarely expressed in normal tissues.
  • cancer cells can be identified by the abnormal expression of tumor antigens or tumor antigens.
  • the tumor antigen expressed by cancer cells in a subject such as a patient suffering from a cancer disease may be a self protein or a non-self protein.
  • the tumor antigens in the context of the present invention are in normal conditions, particularly in cancerous tissues or in non-essential tissues or organs (ie tissues or organs that do not cause the death of the subject when damaged by the immune system) Or it is expressed in body organs or structures that are inaccessible or difficult to reach by the immune system or protected by tolerance mechanisms (for example, by the presence of high concentrations of Treg cells).
  • the amino acid sequence of the tumor antigen may be the same between the tumor antigen expressed in normal tissue and the tumor antigen expressed in cancer tissue, or the amino acid sequence may be different, for example, only at one amino acid or at more than one amino acid It is preferably different at more than 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • tumor-associated antigen refers to an antigen molecule that exists in tumor cells, but the antigen molecule may also exist on normal cells, and its expression is usually far less than that of tumor cells; for example, tyrosinase, MART found in melanoma -1/MelanA, gp100/Pmel 17 and other antigens, such as restin in Hodgkin disease, LDLR/FUT, retrovirus H encoded env protein and HOM-RCC-1, 14 found in kidney cancer, etc., as well as galectin -9, HER2/neu, HCA519, CD20, EGFR/HER1, CA 19-9, etc.
  • tyrosinase MART found in melanoma -1/MelanA
  • gp100/Pmel 17 gp100/Pmel 17
  • other antigens such as restin in Hodgkin disease, LDLR/FUT, retrovirus H encoded env protein and HOM-RCC-1, 14 found in
  • pharmaceutically acceptable carrier used herein is not particularly limited, and pharmaceutical carriers known in the art can be selected according to different dosage forms and modes of administration, for example, they can be selected from: water, buffered aqueous solutions, isotonic salt solutions such as PBS ( Phosphate buffer), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, glycerin, hyaluronic acid, ethanol or polyalkylene glycol such as polypropylene glycol, triglyceride Ester etc.
  • PBS Phosphate buffer
  • glucose mannitol
  • dextrose dextrose
  • lactose lactose
  • starch magnesium stearate
  • cellulose magnesium carbonate
  • glycerin hyaluronic acid
  • polyalkylene glycol such as polypropylene glycol, triglyceride Ester etc.
  • the type of pharmaceutically acceptable carrier used depends inter
  • administering refers to providing a substance to a subject in a pharmacologically usable manner.
  • the pharmaceutical composition according to the present invention can be administered by any suitable route, such as oral, nasal (mucosal), intradermal, subcutaneous, intramuscular, or intravenous administration; specifically, oral, nasal drops (aerosolization) can be used. Inhalation or mucosal absorption), intratumoral injection, intramuscular injection, intravenous injection, intradermal injection or subcutaneous injection.
  • pharmaceutical effective amount As used herein, “pharmaceutical effective amount”, “therapeutically effective amount”, and “effective dose” are used interchangeably, and all refer to a dose sufficient to show its benefit to the subject to which it is administered.
  • the actual amount administered, as well as the rate and time course of administration, will depend on the condition and severity of the individual being treated.
  • the prescription of treatment (such as the decision on dosage, etc.) is ultimately the responsibility of the general practitioner and other doctors and rely on them to make decisions, usually taking into account the disease to be treated, the individual patient’s condition, the delivery site, the method of application, and what is already known to the doctor. Know other factors.
  • divalent manganese can be administered at a dose of 0.02-5 mg/kg, for example, at a dose of 0.05 mg/kg, 0.5 mg/kg, 3 mg/kg, 5 mg/kg.
  • subject means animals, including warm-blooded mammals, such as humans and non-human primates; birds; domesticated domestic or farm animals, such as cats, dogs, sheep, goats, cows, and horses. And pigs; laboratory animals such as mice, rats and guinea pigs; fish; reptiles; zoo animals and wild animals, etc.
  • Anti-tumor refers to the preventive and therapeutic effects of tumors, including but not limited to changing the microenvironment or metabolic pathways of tumor cells, killing tumor cells or inhibiting tumor cell proliferation.
  • Antitumor agents are substances that can be used to prevent and treat tumors.
  • Antitumor agents that can be used in the present invention include but are not limited to chemotherapeutics.
  • chemotherapeutic drugs refer to anti-tumor drugs used to treat cancer or treatments that combine more than one of these drugs into a cytotoxic standardized treatment regimen.
  • chemotherapeutic agent includes any anti-tumor agent, which includes small organic molecules, peptides, oligonucleotides, etc., used in any type of cancer and related processes (such as angiogenesis or metastasis), exemplary Chemotherapeutics are (but not limited to) alkylating agents such as nitrogen mustards/oxazaphosphorines (e.g., cyclophosphamide, ifosfamide), nitrosoureas (e.g., carmustine) ), triazenes (for example, temozolomide) and alkyl sulfonates (for example, busulfan), anthracycline antibiotics such as doxorubicin and n-diamycin, taxanes such as TaxolTM and docetaxel, Vinca alkaloids such as neovinblastine and vinblastine, 5-fluorouracil (5-FU), leucovorin (leucovorin),
  • alkylating agents such
  • the anti-tumor agent further includes monoclonal antibodies, interferons, biological response modifiers, and other anti-tumor drugs.
  • the anti-tumor agent is an immune checkpoint inhibitor, such as a PD-1 antibody or a PD-L1 antibody.
  • the anti-tumor agents are all known, and can be purchased commercially or can be prepared by a person skilled in the art in a known, well-known, or conventional method.
  • Immuno checkpoint inhibitors refer to those that bind to immune checkpoint proteins and block their activity and/or inhibit immune regulatory cells (for example, Treg cells, tumor-associated macrophages, etc.) from expressing the function of the immune checkpoint proteins to which they bind Molecule, compound or composition.
  • immune regulatory cells for example, Treg cells, tumor-associated macrophages, etc.
  • Immune checkpoint proteins may include but are not limited to CTLA4 (cytotoxic T lymphocyte associated protein 4, CD152), PD1 (also known as PD-1; programmed death 1 receptor), PD-L1, PD-L2, LAG- 3 (lymphocyte activation gene-3), OX40, A2AR (adenosine A2A receptor), B7-H3 (CD276), B7-H4 (VTCN1), BTLA (B and T lymphocyte attenuator, CD272), IDO ( Indoleamine 2,3-dioxygenase), KIR (killer cell immunoglobulin-like receptor), TIM 3 (T cell immunoglobulin domain and mucin domain 3), VISTA (T cell activated V Domain Ig inhibitor) and IL-2R (interleukin-2 receptor).
  • CTLA4 cytotoxic T lymphocyte associated protein 4, CD152
  • PD1 also known as PD-1; programmed death 1 receptor
  • PD-L1, PD-L2, LAG- 3 lymphocyte activation gene-3
  • PD-1 antibody or "PD-L1 antibody” refers to a drug that binds to human PD1/PDL1 (programmed death receptor), which can regulate and/or inhibit the activity of PD1 and inhibit its ligands (PDL1, PDL2, etc.) One of the) binds to the PD1 receptor, blocks the PD1/PDL1 pathway and can be an anti-PD1/L1 antibody.
  • PD1/PDL1 programmed death receptor
  • PD-1 antibody or "PD-L1 antibody” refers to a K D 1.0 ⁇ 10 -8 mol/L or lower (in one embodiment, 1.0 ⁇ 10 -8 mol/L- 1.0 ⁇ 10 -13 mol/L), in one embodiment, with a KD of 1.0 ⁇ 10 -9 mol/L or lower (in one embodiment, 1.0 ⁇ 10 -9 mol/L-1.0 ⁇ 10 -13
  • the binding affinity of mol/L is an antibody that specifically binds to human PD1 antigen or human PDL1 antigen. Binding affinity is determined using standard binding assays, such as surface plasmon resonance technology (GE-Healthcare, Uppsala, Sweden).
  • the PD-1 antibody or PD-L1 antibody is well known in the art and is commercially or clinically available, including but not limited to BMS-936559/MDX-1105 (Bristol-Myers Squibb) , MPDL3280A (Genentech), MED14736 (MedImmune), MSB0010718C (EMD Sereno), Nivolumab Nivolumab (Opdivo, Bristol Myers Squibb), Pembrolizumab (Keytruda, Merck Sharp Dohme), Atezolizumab Atezolizumab (Tecentriq, Genentech), Durvalumab (Imfinzi, Astrazeneca UK LTD), Avelumab (Bavencio, EMD Sereno), Cemiplimab (Libtayo, Regeneron), Toreplimumab (Tuoyi, Junshi Bio) and Sintilimab (Daboshu, Cinda Bio).
  • cancer and “cancer disease” and the terms “tumor” and “tumor disease” are used interchangeably herein.
  • cancer and “cancer disease” refer to at least one group of cells that show uncontrolled growth (division beyond the normal range), invasion (invasion and destruction of adjacent tissues) and sometimes metastasis (spread to the body through lymph or blood) position).
  • metastasis spread to the body through lymph or blood position.
  • the malignant characteristics of these three cancers distinguish them from benign tumors that are self-limiting and do not invade or metastasize. Most cancers form tumors, but some cancers (such as leukemia) do not.
  • tumor refers to the abnormal growth of cells (called neoplastic cells, tumorigenic cells or tumor cells), preferably the formation of swelling or lesions.
  • Tumor cell means an abnormal cell that grows through rapid uncontrolled cell proliferation and continues to grow after the stimulus that initiates new growth ceases. Tumors show partial or complete loss of structural organization and functional coordination with normal tissues, and often form unique tissue masses.
  • tumor in the present invention has the same meaning as “cancer” or “cancer disease”, and refers to a malignant disease with the above-mentioned uncontrolled growth, invasion and sometimes metastatic properties.
  • Treatment insensitivity means that the subject cannot be successfully treated or at least not successfully treated with a significantly increased probability compared to the probability of successful treatment, for example, the prescribed dose of drug still cannot be successfully suppressed The proliferation, invasion or metastasis of tumor cells.
  • Anti-GAPDH antibody (sc-25778) was purchased from Santa Cruz.
  • cGAS antibody (31659S) and STING antibody (13647S) were purchased from Cell Signaling Technology.
  • Anti-Viperin antibodies are prepared and used by published methods (13). In short, the cDNA of the antigen fragment is inserted into the pET-21b vector (Novagen) and expressed in E. coli BL21(DE3), and the recombinant protein is purified by Ni-NTA affinity column, and then injected into mice or rabbits , To obtain antiserum that can recognize the corresponding antigen. All flow cytometry antibodies were purchased from Biolegend, unless otherwise stated.
  • PE-anti-SIINFEKL-Tetramer antibody was purchased from MBL, catalog number TS-5001-1C.
  • Anti-PD-1 (29F.1A12) was purchased from BioXCell, item number #BE0273, and Rat IgG2a isotype (2A3) was purchased from BioXCell, item number #BE0089.
  • L929-ISRE (Jiang Zhengfan’s laboratory self-made cell line, stably transfected pGL3ISRE-Luciferase plasmid into L929 cells, CCL-1), BHK21( CCL-10), B16-OVA( CRL-6322) and B16F10( CRL-6475), MC38 (gifted by Zhang Yonghui, Tsinghua University School of Medicine), LLC ( CRL-1642) was cultured in DMEM (Gibco) medium supplemented with 10% FBS (Gibco), 5 ⁇ g/mL penicillin and 10 ⁇ g/mL streptomycin, E.G7-OVA ( CRL-2113) was cultured in RPMI-1640 (Gibco) medium supplemented with 10% FBS (Gibco), 5 ⁇ g/mL penicillin, and 10 ⁇ g/mL streptomycin.
  • BMDM bone marrow-derived macrophages
  • BMDCs Bone marrow-derived dendritic cells
  • RPMI-1640 (Gibco) medium containing 10ng/mL GM-CSF, 10ng/mL IL-4, and 10% FBS (Gibco)
  • the liquid was changed in half on the 3rd day, and the experiment was carried out on the 7th day.
  • Peritoneal macrophages were harvested from mice 5 days after induction of thioglycolate (BD, Sparks, MD) injection and cultured in DMEM medium supplemented with 5% FBS.
  • Sendai virus was donated by Professor Zheng Congyi of Wuhan University
  • vaccinia virus Vaccinia virus, VACV
  • Herpes simplex virus HSV-1-GFP
  • PBS peripheral blood
  • PBS diluted peripheral blood
  • Centrifuge 800g for 20 minutes at room temperature, accelerate by 1, and decelerate by 0.
  • Remove the upper layer of plasma absorb the monocytes located in the middle albuginea layer, and try to avoid the intake of platelets.
  • the wild-type mouse is C57BL/6, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd..
  • the experimental mice are generally 6-8 weeks old.
  • the experimental mice are all raised and bred in specific pathogen-free (SPF) rat houses of the Peking University Laboratory Animal Center with AAALAC certification. All operations are approved by the Peking University Laboratory Animal Center and in accordance with the United States The National Institutes of Health Laboratory Animal Care and Use Guidelines are conducted.
  • SPF pathogen-free
  • CGas-/- mice and Tmem173-/- mice were prepared by CRISPR/Cas9 method, and guide RNA (guide RNA, gRNA) was prepared with HiScribeTM T7 High Yield RNA Synthesis Kit, and Cas9 RNA was prepared with mMESSAGE mMACHINE T7 Ultra Kit.
  • guide RNA guide RNA, gRNA
  • Cas9 RNA was prepared with mMESSAGE mMACHINE T7 Ultra Kit.
  • mice fertilized eggs of super-ovulated C57BL/6 female mice were taken, and the in vitro microinjection technology Cas9 and gRNA were mixed at a concentration ratio of 2:1 (100ng/50ng/ ⁇ L) and then injected intracellularly.
  • the fertilized eggs that can develop into the two-cell stage are selected and transferred to the fallopian tubes of pseudo-pregnant ICR mice, waiting for the mice to be born.
  • the tails were cut for genotype identification, and positive mice were selected for mating, and finally homozygous knockout mice were obtained.
  • Cultivate the target cells such as B16F10/B16F0, or MC38, LLC, E.G7 and other cells, collect the cells with 4°C pre-cooled PBS, centrifuge at 300g for 5 minutes, repeat 2 times to fully remove the medium components, and count according to the modeling determining the required amount of cells were resuspended volume, under normal circumstances, when B16F10 modeling using a cold PBS 4 °C resuspended to 3 ⁇ 10 5 -5 ⁇ 10 5 th th / 100 ⁇ L, and MC38, LLC and made E.G7 Resuspend to 1 ⁇ 10 6 /100 ⁇ L with 4°C pre-cooled PBS during the mold.
  • the 1M MnCl 2 stock solution was diluted with normal saline to 20 ⁇ L per mouse at a dose of 5 mg/kg MnCl 2 per mouse.
  • Another group of mice of the same age was treated with nasal saline drip as the control group.
  • the 1M MnCl 2 stock solution was diluted with PBS to 300 ⁇ L per mouse at a dose of 5 mg/kg MnCl 2 per mouse, every 2 It is injected once a day, usually a total of 7 injections.
  • Another group containing the same number of mice was injected with PBS through the tail vein as a control group.
  • Grouping settings include: control group (IgG2a isotype, Clone2A3, BioXCell), Mn 2+ treatment group, anti-PD-1 monoclonal antibody treatment group (Clone 29F.1A12, BioXCell), and Mn 2+ +anti-PD-1 combination Use groups.
  • Mn 2+ adopts the operation method in 1 or 2 above.
  • Anti-PD-1 monoclonal antibody is diluted with normal saline on the 3rd, 7th, and 11th day after tumor inoculation, and each mouse is injected intraperitoneally with 200 ⁇ g/200 ⁇ L.
  • the combination therapy group is generally injected with antibody first, and then treated with Mn 2+.
  • Grouping settings include: control group, Mn 2+ treatment group, chemotherapy drug treatment group (Cyclophosphamide monohydrate, CTX, TargetMol T0707), and Mn 2+ + chemotherapy drug CTX combined use group.
  • Mn 2+ adopts the operation method in 1 or 2 above, and CTX is diluted with a solvent containing 5% DMSO+30% PEG 300+5% Tween80+ddH 2 O on the 5th, 9th, and 13th day after tumor inoculation.
  • Each mouse was injected intraperitoneally at a dose of 100 mg/kg (or reduced the dose of chemotherapy drugs according to the experimental design).
  • the combined treatment group generally received chemotherapy drugs first, and then Mn 2+ treatment.
  • the long axis and short axis are measured with a vernier caliper. The measurement is performed every 1-2 days. After recording for 2-3 weeks, CO 2 is used for mice according to the requirements of experimental animal welfare. After euthanasia, the mice were dissected and the entire tumor tissue was isolated for photographing or weighing records.
  • mice 100 ⁇ g OVA, 100 ⁇ g OVA+20 ⁇ g colloidal manganese preparation per 100 ⁇ L was prepared with normal saline, and 6-8 weeks old wild-type or gene-deficient C57BL/6 mice were taken and performed on the thighs of the hind legs. Each mouse was injected intramuscularly with 100 ⁇ L. After the initial immunization, the immunity was enhanced on the 7th day and the 14th day, and the tumor cell line stably transfected with OVA protein was inoculated on the 21st day. The unimmunized group was used as a control to observe and record the tumor growth. Finally, the mice were euthanized with CO 2.
  • mice 100 ⁇ g OVA, 100 ⁇ gOVA+20 ⁇ g colloidal manganese per 100 ⁇ L was prepared with normal saline, and 6-8 weeks old wild-type or genetically deficient C57BL/6 mice were taken in the hind thighs Each mouse was injected intramuscularly with 100 ⁇ L. After the initial immunization, the immunity was increased on the 7th day and the 14th day respectively, and the immunized mice were set as controls. This batch of mice was defined as recipient mice.
  • mice On the 21st day, take a new batch of wild-type C57BL/6 mice (donor mice), sacrifice and take out their spleen cells, prepare a single cell suspension and divide them into two groups, each with 0.5 ⁇ M Stain with 5 ⁇ M CFSE in a 37°C cell incubator for 10 minutes, and wash with PBS. Then the spleen cells stained with 5 ⁇ M CFSE were stained with OVA 257-264 peptide (10 ⁇ g/mL) in a 37°C cell incubator for 90 minutes. After washing twice with PBS, the cells were counted.
  • CD8 + T lymphocyte proliferation in mice Take 6-8 week old CD45.1 + OT-I mice, dissect their spleen tissue, prepare a single cell suspension, and separate CD8 + T with magnetic beads The cells were then stained with 1 ⁇ M CFSE at 37°C for 20 minutes. After washing twice, 2 ⁇ 10 6 CFSE-labeled CD8 + T cells were injected back into a new batch of 6-8 weeks old CD45 through tail vein injection .2 + wild-type C57BL/6 mice were injected with 100 ⁇ g OVA and 100 ⁇ g OVA+colloidal manganese (Mn 2+ ) preparations into the groin subcutaneously one day later, and set up a control group.
  • Mn 2+ colloidal manganese
  • mice were sacrificed on the 3rd day after the tail vein injection of CD8 + T cells.
  • the inguinal lymph nodes were dissected to prepare a single cell suspension, and the proportion of low-expressing CFSE (CFSElow) cells was detected by flow cytometry and flow cytometry. , And do statistical analysis.
  • the detailed experimental method can refer to the existing literature (14). Pass 2f-TGH-ISRE (for determination of human type I interferon) or L929-ISRE (for determination of mouse type I interferon) into 96-well cell plates in advance to ensure that the cell density is above 80% during the determination. Add 100 ⁇ L of the cell supernatant to be tested and the human or mouse-derived type I interferon standard serial dilution (R&D system) into the 96-well cell plate, set up parallel wells according to the experimental requirements, and place them in a 37°C incubator 4-5 hour.
  • R&D system human or mouse-derived type I interferon standard serial dilution
  • a vacuum suction pump was used to aspirate the supernatant of the stem cells, 30 ⁇ L of cell lysate (Promega) was added to each well, lysed for 30 minutes, 10 ⁇ L of cell lysate was transferred to a 96-well plate for detection, and 10 ⁇ L of firefly luciferase substrate ( Promega), after mixing, put it into the multifunctional microplate reader workstation to measure the fluorescence intensity, draw a standard curve based on the fluorescence reading of the standard, and convert the concentration of type I interferon in the sample.
  • mice adopts the Mantel-Cox test.
  • the significance analysis is marked as follows: ns, not significant, p>0.05; *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001.
  • Wild-type C57BL/6 mice of 6-8 weeks of age were inoculated with 2 ⁇ 10 5 B16F10 melanoma cells through tail vein injection, and after they were transferred, the lung metastasis model was constructed, which usually takes 2 weeks.
  • mice 5 ⁇ 10 5 B16F10 cells in PBS were subcutaneously injected into the groin, and they were divided into two groups.
  • the experimental group was instilled into the nasal cavity at a dose of 5 mg/kg 24 hours after tumor inoculation.
  • the physiological saline solution of MnCl 2 was injected, and the control group was injected with an equal volume of physiological saline solution once every 2 days. After the tumor grows to be palpable, start to measure the size of the tumor, record it every 2 days, and draw the tumor growth curve.
  • mice To be tested, intraperitoneally inject 150mg/kg of firefly luciferase substrate D-luciferin in physiological saline solution, then anesthetize the mice, 10 minutes after the injection of the substrate, put the mice into the IVIS in vivo imaging system for imaging, and Generate images.
  • mice in experiment (A) After the tumors of the mice in experiment (A) grew for 2 weeks, the mice were sacrificed by CO 2 and the mice were dissected to obtain complete tumor tissues and photographed and recorded.
  • Wild-type C57BL/6 mice aged 6-8 weeks were inoculated with 2 ⁇ 10 5 B16F10 melanoma cells in PBS suspension via tail vein injection.
  • the experimental group was instilled into the nasal cavity at a dose of 5 mg/kg 24 hours after tumor inoculation.
  • the physiological saline solution of MnCl 2 was instilled in the nasal cavity of the control group with an equal volume of physiological saline solution, once every 2 days.
  • the mice were sacrificed by CO 2 and the mice were dissected to obtain intact lung tissues, which were photographed and recorded (top). Then fix for HE staining (lower picture).
  • Example 3 Mn 2+ can have a therapeutic effect on multiple tumor types through different administration methods
  • 1 ⁇ 10 6 LLC cells were subcutaneously inoculated into the groin of wild-type C57BL/6 mice of 6-8 weeks of age.
  • the experimental group was inoculated with a 5 mg/kg dose of MnCl 2 in physiological saline solution 24 hours after tumor inoculation. In the group, an equal volume of saline solution was instilled into the nasal cavity, once every 2 days.
  • E.G7 cells were subcutaneously inoculated into the inguinal groin of wild-type C57BL/6 mice of 6-8 weeks of age. 24 hours after tumor inoculation, the experimental group was infused with MnCl 2 physiological saline solution in the nasal cavity at a dose of 5 mg/kg. In the control group, an equal volume of saline solution was instilled in the nasal cavity, once every 2 days. When the size of the tumor can be touched, start to record the size and draw the tumor growth curve.
  • mice 6-8 weeks old wild-type C57BL/6 mice were injected with 1 ⁇ 10 6 MC38 cells in the tail vein. 24 hours after tumor inoculation, the experimental group was injected with MnCl 2 PBS solution in the tail vein at a dose of 5 mg/kg. The control group An equal volume of PBS solution was injected into the tail vein, once every 2 days. When the size of the tumor can be touched, start to record the size and draw the tumor growth curve.
  • mice On the 18th day after tumor inoculation in experiment (E), the mice were sacrificed with CO 2 and the complete tumor tissue was obtained by dissection, photographed and recorded, and weighed for statistical analysis.
  • Different types of cells including B16F10, LLC and L929 cells, are seeded in the full medium in a 24-well plate in advance. After the cell density has grown to about 95%, change to serum-free medium, and add 0, 3.125, 6.25, 12.5, 25, 50, 100, 200 , 400, 800 ⁇ M MnCl 2 solution. After culturing for 24 hours, change to a new complete medium, add 1/5 volume of MTT stock solution (stock solution is 2mg/mL MTT PBS solution) of the medium, and place it in a 37°C incubator for 1 hour. Then blot the medium and allow the cells to dry at room temperature.
  • MTT stock solution stock solution is 2mg/mL MTT PBS solution
  • lysis buffer 900 mL isopropanol + 100 mL H 2 O + 5 g SDS + 1 mL 4N HCl
  • 300 ⁇ L of lysis buffer 900 mL isopropanol + 100 mL H 2 O + 5 g SDS + 1 mL 4N HCl
  • 300 ⁇ L of lysis buffer 900 mL isopropanol + 100 mL H 2 O + 5 g SDS + 1 mL 4N HCl
  • mice Observe and record the death of mice in experiment (B), and draw a mouse death curve.
  • mice The wild-type C57BL/6 mice that were not inoculated with tumors were instilled in the nasal cavity with a 5 mg/kg dose of MnCl 2 physiological saline solution, once every 2 days, for a total of 7 times.
  • Tmem173 -/- mice that were not inoculated with tumors were instilled nasally with MnCl 2 saline solution at a dose of 5 mg/kg, once every 2 days, for a total of 7 times.
  • the control group was instilled with an equal volume of saline at the same time point.
  • the wild-type C57BL/6 mice that were not inoculated with tumors were divided into three groups, a blank control group (Negative Control, NC), a control group (Con) and an experimental group (Mn 2+ ), with 6 mice in each group.
  • a blank control group Negative Control, NC
  • a control group Con
  • Mn 2+ experimental group
  • nasal instillation was performed with a physiological saline solution of MnCl 2 at a dose of 5 mg/kg, once every 2 days, 7 times in total, and then the administration was stopped;
  • the Con group was infused with an equal volume of physiological saline solution at the same time point for nasal instillation Note:
  • the NC group does not do any processing.
  • Each group of mice was weighed every 2 days until 40 days.
  • mice in the experiment Observe the behavior of the mice in the experiment (C) every day, their diet and drinking, and record their deaths, and draw a death curve.
  • mice in experiment were sacrificed with CO 2 and dissected.
  • the lung, liver, kidney, and brain tissues in the experiment were prepared for paraffin tissue sections, stained with HE, and photographed and imaged.
  • Example 6 During Mn 2+ treatment, CD8 + T cells are mainly used to exert anti-tumor efficacy
  • Experiment (A) compares the changes in the growth of subcutaneous melanoma in Rag1 -/- mice after Mn 2+ treatment
  • Experiment (D) compares the changes in the growth of subcutaneous melanoma in ⁇ 2m -/- mice after Mn 2+ treatment
  • mice Inoculate 5 ⁇ 10 5 B16F10 cells, 1 ⁇ 10 6 MC38 cells or 1 ⁇ 10 6 LLC cells subcutaneously in the groin of wild-type C57BL/6 mice, and then start using 5 mg/kg of MnCl 24 hours after tumor inoculation 2 Nasal instillation with physiological saline solution, and a control group with an equal volume of physiological saline for nasal instillation. The mice were sacrificed and dissected to obtain their complete tumors. After sectioning, the CD8 positive cells were labeled with CD8 antibody and imaged by a fluorescence microscope.
  • Example 7 Mn 2+ can enhance the activity of tumor infiltrating CD8 + T cells and its specific killing ability
  • Experiment (FH) Mn 2+ treatment can enhance the CD8 + T cells that specifically recognize tumor antigens in mouse subcutaneous E.G7 tumors Degree of infiltration
  • TIL tumor infiltrating lymphocyte, tumor infiltrating lymphocyte.
  • mice On the 14th day after tumor inoculation, the mice were sacrificed, the complete tumor tissue was obtained by dissection, single cell suspension was prepared, cell surface staining was performed, CD8 + T cells were sorted by flow cytometry, and then sent to BGI
  • the gene expression multiple calculation method is log 2 ((Mn FPKM)/(Con FPKM))
  • the absolute value of the filtering ratio is less than 1
  • the FDR false discovery rate
  • Example 8 Mn 2+ can promote the activation of murine and human dendritic cells
  • the bone marrow-derived dendritic cells BMDC of wild-type C57BL/6 mice were treated with SeV, VACV, 100ng/mL LPS, 200 ⁇ M and 400 ⁇ M MnCl 2 for 18 hours, and then the cell culture supernatant was collected. The type I was detected by the Bioassay method. Interferon activity (A).
  • the bone marrow-derived macrophages BMDM of wild-type C57BL/6 mice were treated with SeV, VACV, 200 ⁇ M and 400 ⁇ M MnCl 2 for 18 hours, and then the cell culture supernatant was collected, and the type I interferon activity was detected by Bioassay method (B) .
  • MFI mean fluorescence intensity
  • PBMCs peripheral blood of tumor patients and treated with 100 ⁇ M MnCl 2 for 18 hours. Three parallel holes were set in each group. Then the DCs were circled by flow antibody staining and flow cytometry and the CD86 MFI was analyzed for statistics. analysis.
  • Example 9 Mn 2+ promotes the killing of tumor cells by CD8 + T cells in vitro
  • A flow cytometry
  • B do statistical analysis
  • Example 10 Mn 2+ can help the body produce more antigen-specific CD8 + T cells
  • the CD8 + T cells of OT-I transgenic mice carry the V ⁇ 2/V ⁇ 5 transgenic TCR to specifically recognize the amino acids 257-264 of the OVA protein presented by MHC-I molecules, namely SIINFEKL.
  • MHC-I molecules namely SIINFEKL.
  • CD8 + T cells Take the spleens of 6-8 weeks old CD45.1 + OT-I mice, prepare a single-cell suspension, sort out CD8 + T cells with magnetic beads, and use CFSE staining to label them. 2 ⁇ 10 6 CFSE-labeled CD8 + T cells were injected back into a new batch of 6-8 week old CD45.2 + wild-type C57BL/6 mice by tail vein injection, and 100 ⁇ g OVA and 100 ⁇ g OVA+20 ⁇ g colloidal manganese preparation were injected subcutaneously in the groin one day later. And 100 ⁇ g OVA+50 ⁇ g colloidal manganese preparation, and set up a control group. Then, the mice were sacrificed on the 3rd day after the tail vein injection of CD8 + T cells.
  • mice n 4.
  • Example 11 Mn 2+ exerts anti-tumor activity as a cGAS-STING agonist
  • mice Take 6-8 week old wild-type C57BL/6, CGas -/- and Tmem173 -/- mice, and inject 5 mg/kg of MnCl 2 in PBS solution into the mice to set up a control group. After 18 hours, blood was collected from mice, serum was separated, and the activity of type I interferon was detected by Bioassay method. Subsequently, the mice were sacrificed, and the heart, thymus and spleen were obtained by dissection. After grinding, Western Blot was used to detect the expression of the type I interferon-inducing gene Viperin. The internal reference protein GAPDH was used to indicate the amount of protein loading. In CGas -/- and Tmem173 -/- mice, Mn 2+ cannot activate their type I interferon production.
  • the bone marrow-derived dendritic cells BMDC of Tmem173 -/- mice were treated with SeV, VACV, 100ng/mL LPS, 200 ⁇ M and 400 ⁇ M MnCl 2 for 18 hours, and then the cell culture supernatant was collected. The type I interference was detected by the Bioassay method.
  • Activity (B) The bone marrow-derived macrophages BMDM of Tmem173 -/- mice were treated with SeV, VACV, 200 ⁇ M and 400 ⁇ M MnCl 2 for 18 hours, and then the cell culture supernatant was collected, and the type I interferon activity was detected by Bioassay method (C).
  • mice Inoculate 5 ⁇ 10 5 B16F10 cells subcutaneously in the groin of Tmem173 -/- mice. 24 hours after inoculation, nasal instillation with MnCl 2 saline solution at a dose of 5 mg/kg was performed. The control group was instilled with an equal volume of saline solution. , The mice were sacrificed on the 14th day after tumor inoculation, the complete tumor tissue was obtained by dissection, photographed, and the weight was recorded (D). The tumor tissue obtained by the dissection of the mouse in experiment (D) was prepared into a single-cell suspension and counted.
  • mice intramuscularly inject 100 ⁇ g OVA, 100 ⁇ g OVA+20 ⁇ g colloidal manganese preparations on the 0th, 7th, and 14th days respectively, and set up a control group.
  • the mice will be injected with 100 ⁇ g OVA and 100 ⁇ g OVA+20 ⁇ g colloidal manganese. Inoculate 3 ⁇ 10 5 B16F0-OVA cells subcutaneously into the groin.
  • G tumor growth curve
  • H death curve
  • Tmem173 -/- mice of 6-8 weeks old were injected intramuscularly with 100 ⁇ g OVA, 100 ⁇ g OVA+20 ⁇ g colloidal manganese preparations on the 0th, 7th, and 14th days respectively, and a control group was set up, which was defined as the recipient mouse.
  • a new wild-type C57BL/6 mouse spleen was taken, a single cell suspension was prepared and aliquoted, and stained with 0.5 ⁇ M CFSE and 5 ⁇ M CFSE at 37°C for 10 minutes, and then with OVA 257-264 (10 ⁇ g /mL) Stimulate at 37°C for 90 minutes.
  • Example 12 Mn 2+ synergistically enhances the therapeutic effect of the immune checkpoint inhibitor anti-PD-1 on tumors and reduces the dosage of anti-PD-1
  • Wild-type C57BL/6 mice were subcutaneously inoculated with 5 ⁇ 10 5 B16F10 cells in the groin and divided into 4 groups, Isotype group, Mn 2+ treatment group, anti-PD-1 treatment group, and combination treatment (combo) group. Twenty-four hours after tumor inoculation, the Mn 2+ group and Combo group were instilled in the nasal cavity with a 5 mg/kg MnCl 2 physiological saline solution, once every 2 days. The Isotype group was instilled with an equal volume of normal saline at the same time point.
  • mice were injected intraperitoneally with 200 ⁇ g anti-PD-1 on the 3rd, 7th, and 11th day after tumor inoculation.
  • the Isotype group was on the 3rd, 7th, and 11th day after tumor inoculation.
  • Mice were injected intraperitoneally with 200 ⁇ g IgG2a isotype antibody.
  • A the tumor growth curve
  • the tumor tissue obtained by the dissection of the mouse in experiment (A) was prepared into a single-cell suspension and counted, a suitable amount of cells was stained with flow cytometry, and then the CD8 + T cell population (D) was detected by flow cytometry, and obtained by calculation The number of CD8 + T cells per gram of tumor, and statistical analysis (E).
  • Wild-type C57BL/6 mice were injected with 2 ⁇ 10 5 B16F10 cells through the tail vein and divided into 4 groups, Isotype group, Mn 2+ treatment group, anti-PD-1 treatment group, and combination treatment (combo) group.
  • the treatment of Mn 2+ and anti-PD-1 is the same as described in the experiment (AC).
  • Two weeks after tumor inoculation the mice were sacrificed with CO 2 and the complete lung tissues of the mice were dissected, photographed and weighed for statistical analysis.
  • mice were injected intraperitoneally with 200 ⁇ g of anti-PD-1 on the 3, 7, and 11 days after tumor inoculation, and the dose of the 1/2 anti-PD-1 treatment group was halved.
  • the tumor grows to be palpable, start to measure and record and draw the tumor growth curve.
  • the mice were sacrificed, the complete tumor tissue was obtained by dissection, and the weight was recorded for statistical analysis.
  • Each group of mice n 5.
  • Wild-type C57BL/6 mice were subcutaneously inoculated with 1 ⁇ 10 6 MC38 cells in the groin and divided into 4 groups, Isotype group, Mn 2+ treatment group, anti-PD-1 treatment group, and combination treatment (combo) group. Twenty-four hours after tumor inoculation, mice in the Mn 2+ group and combo group were injected with 5 mg/kg MnCl 2 PBS solution via the tail vein, once every 2 days. In the Isotype group, an equal volume of PBS was injected into the tail vein at the same time point. The mice in the anti-PD-1 treatment group and combo group were injected intraperitoneally with 200 ⁇ g anti-PD-1 on the 3, 7, and 11 days after tumor inoculation.
  • mice were injected intraperitoneally with 200 ⁇ g IgG2a isotype antibody.
  • H tumor growth curve
  • Example 13 Mn 2+ synergistically enhances the therapeutic effect of the chemotherapy drug CTX on tumors and reduces the amount of CTX
  • mice were injected intraperitoneally with 100 mg/kg of CTX on the 5th, 9th, and 13th days after tumor inoculation, and the dose of 1/2 CTX treatment group was halved.
  • mice in the Con group were injected intraperitoneally with an equal volume of solvent on the 5th, 9th, and 13th days after tumor inoculation.
  • A tumor growth curve
  • mice were sacrificed on the 15th day after tumor inoculation, the complete tumor tissue was obtained by dissection, photographed, and the weight was recorded for statistical analysis (B, C).
  • Each group of mice n 6.

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Abstract

二价锰在制备免疫增强药物或抗肿瘤药物中的应用，具体涉及二价锰作为树突状细胞、巨噬细胞或CD8阳性T细胞激活剂及其用于树突状细胞、巨噬细胞或CD8阳性T细胞激活剂的用途。还提供了二价锰用于增强肿瘤药物效果的增敏剂，其与抗肿瘤药物联合施用时，可以显著减少抗肿瘤药物的剂量，达到良好的治疗效果。

Description

二价锰在制备免疫增强药物或抗肿瘤药物中的应用
本申请要求申请号为201911013693.7，发明名称为“二价锰在制备免疫增强药物或抗肿瘤药物中的应用”的中国发明专利申请的优先权，该申请的内容通过引用整体结合到本文中。
技术领域
本发明属于医药领域，具体涉及二价锰在制备免疫增强药物或抗肿瘤药物中的应用。
背景技术
肿瘤的治疗手段目前主要有手术治疗，放射治疗，化学药物治疗，细胞过继转输治疗和免疫检查点抑制剂治疗。目前尤其以嵌合抗原受体T细胞免疫疗法(Chimeric Antigen Receptor T-Cell Immunotherapy,CAR-T)为代表的细胞过继转输治疗，以及程序性死亡1(Programmed cell Death protein-1,PD-1)抗体(anti-PD-1)和细胞毒性T细胞相关抗原4(Cytotoxic T lymphocyte antigen-4,CTLA-4)抗体(anti-CTLA-4)为等免疫检查点抑制剂治疗为代表的免疫治疗为主要的肿瘤治疗手段。上述治疗手段虽然在某一些特定类型的肿瘤如霍奇金淋巴癌、促纤维增生性黑色素瘤和默克尔细胞癌中有效率较高，然而对于其他多种类型的肿瘤患者，PD-1阻断抗体的患者有效率只有20％左右，因此，如何有效提升机体的自身抗肿瘤免疫反应来增强肿瘤治疗效果也成为了近年来的研究热点。
机体的免疫系统可以分为天然免疫(innate immunity)和适应性免疫(adaptive immunity)。天然免疫系统的又可称为固有免疫、非特异性免疫，广泛存在于动物、植物、真菌和昆虫之中，天然免疫系统反应迅速、作用广泛，是机体抵御病原入侵的第一道防线，也是激活适应性免疫(又称获得性免疫或特异性免疫)的基础。主要参与天然免疫应答的免疫细胞包括单核细胞(monocytes)、巨噬细胞(macrophages)、自然杀伤细胞(natural killer cells)、树突状细胞(dendritic cells)、中性粒细胞(neutrophils)、嗜碱性粒细胞(basophils)、嗜酸性粒细胞(eosinophils)以及肥大细胞(mast cells)等。
机体中杀伤肿瘤细胞主要依靠的是CD8 +T细胞(属于适应性免疫系统)，而这群细胞发挥功能离不开抗原递呈细胞(antigen-presenting cells,APC)如树突状细胞的协助，由此可见，天然免疫系统中树突状细胞的激活和活化对于后续机体内对于抗肿瘤免疫的发挥起到了至关重要的作用。
天然免疫系统可以通过各种不同的模式识别受体(pattern recognition receptors,PRR)来 识别不同的病原相关分子模式(pathogen-associated molecular patterns,PAMPS)和损伤相关分子模式(damage-associated molecular patterns,DAMPS)，进而诱导产生一系列的细胞因子包括I型干扰素来抵御病原的入侵。在不同的PRR中，cGAS(cyclic GMP-AMP synthase)可以通过识别双链DNA，合成第二信使cGAMP(cyclic dinucleotide 2’,3’-cyclic GMP-AMP)，激活下游蛋白STING(stimulator of interferon genes)，进而诱导产生I型干扰素和激活天然免疫反应。死亡的肿瘤细胞会被树突状细胞吞噬，进而释放其中的DNA，激活树突状细胞的cGAS-STING通路，诱导产生I型干扰素，促进树突状细胞的成熟活化，并促进其抗原交叉递呈(cross-presentation)作用，从而增强树突状细胞对于CD8 +T细胞的致敏(priming)作用，进而增强这些CD8 +T细胞对于肿瘤的特异性杀伤，此时这群CD8 +T也称为细胞毒性T细胞(cytotoxic T lymphocytes)。
但是，目前尚未有关于二价锰通过激活全身性反应来激活抗肿瘤反应的报道。
发明内容
针对上述问题，本发明提供用二价锰制备树突状细胞、巨噬细胞或T细胞激活剂和/或致敏剂的应用。本发明还提供二价锰用于制备抗肿瘤药物的应用。
第一方面，本发明提供了二价锰在制备树突状细胞激活剂/致敏剂、巨噬细胞激活剂/致敏剂或T细胞(例如CD8 +T细胞)激活剂/致敏剂中的应用。
在一些实施方案中，所述二价锰刺激树突状细胞或巨噬细胞产生I型干扰素。
在一些实施方案中，所述二价锰刺激树突状细胞上调表达CD80和/或CD86。
根据本发明的实施方案，所述树突状细胞是来源于骨髓、肺脏、淋巴结、外周血等结构或组织的树突状细胞。所述树突状细胞可以来源于哺乳动物。
根据本发明的实施方案，所述巨噬细胞是来源于骨髓、腹腔、肺脏、淋巴结、外周血等结构或组织的巨噬细胞。所述巨噬细胞可以来源于哺乳动物。
在另一些实施方案中，所述二价锰促进CD8阳性T细胞(CD8 +T细胞)增殖，或者促进抗原特异性识别CD8阳性T细胞的增殖，较佳地，所述抗原为肿瘤抗原；一个具体的实施例中，所述抗原可以是卵清蛋白(OVA)。在一些实施方案中，所述二价锰促进肿瘤中CD8阳性T细胞的浸润。
第二方面，本发明提供一种树突状细胞、巨噬细胞或CD8阳性T细胞的激活剂/致敏剂，其包括二价锰。在一些实施方案中，所述激活剂/致敏剂可施用于对象体内或体外，所述对象可以为哺乳动物。
第三方面，本发明提供一种包括二价锰的树突状细胞、巨噬细胞或CD8阳性T细胞的激活剂/致敏剂在细胞免疫治疗药物中的应用。
第四方面，本发明提供二价锰在制备治疗或预防肿瘤的药物中的应用。在本发明的一些实施方案中，所述二价锰作为抗肿瘤剂的增敏剂，可显著提高抗肿瘤剂对肿瘤的治疗效果，或者显著降低抗肿瘤剂的用量。在一些实施方案中，所述抗肿瘤药物施用于已经确诊患有肿瘤的对象(符合I期临床试验标准)。
根据本发明的实施方案，所述抗肿瘤剂为免疫检查点抑制剂和/或化疗药物，例如PD-1抗体、PD-L1抗体、环磷酰胺。本发明中，基于二价锰的增敏效果，所述抗肿瘤药的用量为该抗肿瘤药单独施用时有效剂量的0.5倍以下，优选地，0.01-0.5倍，更优选地，0.1-0.5倍，最优选地，0.3-0.5倍。
第五方面，本发明提供一种抗肿瘤药物组合物，包括抗肿瘤剂和二价锰。在一些实施方案中，所述抗肿瘤药物组合物还包含药学上可接受的载体。
第六方面，本发明提供二价锰在制备预防肿瘤的疫苗组合物中的应用。在一些实施方案中，所述预防肿瘤的疫苗可以提前施用于未患肿瘤的对象，激活并增强体内对于肿瘤细胞的特异性杀伤作用，起到预防肿瘤的效果。
第七方面，本发明提供一种预防肿瘤的疫苗组合物，所述疫苗组合物包括所述二价锰。在一些实施方案中，所述疫苗还包括抗原，所述抗原可以是肿瘤抗原，例如OVA抗原；进一步地，所述OVA抗原含有SIINFEKL肽段。在一些实施方案中，所述疫苗进一步包括药学上可接受的载体。
本发明中，所述二价锰可以是游离的锰离子、二价锰盐形式存在或者可以转变为二价锰形式的其他二价锰源，例如所述二价锰盐是药学上可接受的盐，选自氯化锰、溴化锰、碘化锰、硫酸锰、硝酸锰、高氯酸锰、醋酸锰、碳酸锰、硼酸锰、磷酸锰、氢溴酸锰、酒石酸锰、富马酸锰、马来酸锰、乳酸锰、苯磺酸锰、泛酸锰、抗坏血酸锰及其任意组合。在本发明的另一实施方案中，所述二价锰是Mn 2OHPO 4胶体。所述Mn 2OHPO 4胶体由磷酸盐、MnCl 2和生理盐水制成，所述磷酸盐选自Na 3PO 4、K 3PO 4或其他种类；在一个实施方案中，所述锰离子胶体中磷酸根离子和二价锰离子的浓度分别不低于25mM和20mM，磷酸根离子与二价锰离子的摩尔浓度比为1.25:1。在一个具体实施方案中，所述Mn 2OHPO 4胶体为100μL 0.2M的MnCl 2溶液，加50μL 0.5M的Na 3PO 4溶液，加850μL生理盐水放置过夜后，制备得到Mn 2OHPO 4胶体。
本发明中，作为一些实施方案，所述肿瘤为恶性黑色素瘤、肺癌、结肠癌、淋巴癌、卵 巢癌、胃癌、乳腺癌、胰腺癌、胆管癌、肾癌、肝癌、肉瘤、膀胱癌、子宫颈癌、白血病、头颈癌、其他皮下实体瘤等中的一种或任意组合。在一些实施方案中，所述肿瘤为原发性的或者转移的。优选的，所述肿瘤为对免疫检查点抑制剂(anti-PD-1/anti-PD-L1)或化疗药物治疗不敏感的肿瘤，如皮肤黑色素瘤、结肠癌。进一步优选的，所述肿瘤为卵巢癌。
本发明中，所述二价锰或抗肿瘤药物组合物可以为静脉注射制剂、鼻滴制剂(包括雾化形式和黏膜施用)、口服制剂、皮内注射制剂、皮下注射制剂或者肌内注射制剂。当其作为免疫预防肿瘤的药物时，优选皮下注射制剂或者肌内注射制剂。
本发明中，所述二价锰或抗肿瘤剂的用量为治疗有效量。
有益效果
本发明的发明人出乎意料地发现二价锰离子可以通过不同途径增强施用对象抵抗多种类型肿瘤的能力，并进一步发现二价锰离子可以活化各种来源的树突状细胞、巨噬细胞和T细胞，促使树突状细胞产生I型干扰素，促进CD8 +T细胞增殖或者是增强其与抗原的特异性结合，从而增强免疫反应，抑制肿瘤细胞增殖。
本发明的发明人还出乎意料地发现二价锰离子对于其他抗肿瘤剂有良好的增敏效果，例如与免疫检查点抑制剂anti-PD-1联合使用或化疗药物环磷酰胺(cyclophosphamide monohydrate,CTX)联合使用可以显著增强anti-PD-1或CTX对于肿瘤的治疗效果，进而能够在不影响肿瘤治疗效果的前提下显著降低抗肿瘤剂例如anti-PD-1和CTX的用量。同时，二价锰离子在联用时施用方式灵活，可以与其他抗肿瘤剂一起施用或者分别施用，并不需要更改其他治疗方案的施用方法，只需要额外施用二价锰离子(例如做鼻滴包括雾化吸入或黏膜吸收)即可，有效地简化了施用方案。另外，二价锰离子的联合使用能降低化疗药物的使用剂量，这也将有望减轻化疗对患者造成的副作用。不仅如此，二价锰离子的联用能够减少anti-PD-1的使用剂量，这将有望减轻患者的经济负担，使更多的患者受益。
附图说明
下面通过对本发明的详细描述以及附图来清楚地说明本发明前面叙述的方面以及其他方面。为了举例说明本发明，在附图中的实施方案是目前优选的，然而，可以理解，本发明并不限于所公开的特定实施方案。
图1显示小鼠肿瘤模型的构建。1A表示肿瘤的接种方式和接种时间及最后处理方式；1B表示构建成功的小鼠皮下肿瘤模型；1C表示构建成功的小鼠肺部肿瘤转移模型。
图2显示了实施例2不添加Mn 2+的空白对照Con和施用Mn 2+的小鼠黑色素瘤抑制情况， 显示通过鼻腔滴注Mn 2+可以增强小鼠抵抗皮下黑色素瘤和黑色素瘤肺部转移的能力。2A显示Mn 2+能显著延缓小鼠黑色素瘤B16F10在皮下的生长速度；2B为在不同时间点用活体成像仪拍下的小鼠皮下肿瘤；2C，D显示在皮下肿瘤接种2周后处死小鼠，解剖所得的完整肿瘤组织及对应的肿瘤组织重量的统计分析。2E-G显示尾静脉注射接种B16F10黑色素瘤2周后肺部的肿瘤生长情况，以及对应的肿瘤转移灶个数和完整肺部的重量的统计学分析。
图3显示Mn 2+可以通过不同给药途径增强小鼠抵抗多种类型肿瘤的能力，包括空白对照Con和Mn 2+处理组。图3A，B显示通过鼻腔滴注Mn 2+可以显著延缓皮下接种的小鼠路易斯肺癌LLC的生长速度，并显著延长小鼠的存活时间。3C，D显示通过鼻腔滴注Mn 2+可以显著延缓皮下接种的小鼠T淋巴癌E.G7的生长速度，并显著抑制肿瘤的大小。3E-F显示通过尾静脉注射Mn 2+可以显著延缓皮下接种的小鼠结肠癌MC38的生长速度，并显著抑制肿瘤的大小。3G显示通过肿瘤内注射Mn 2+可以显著延缓皮下接种的小鼠黑色素瘤B16-OVA的生长速度，并显著提高小鼠的40天存活率，对照组32只小鼠均在肿瘤接种40天之前死亡，Mn 2+治疗组23只小鼠在肿瘤接种40天时依旧有13只存活。
图4显示Mn 2+增强小鼠抗肿瘤的能力不是通过对肿瘤细胞产生直接杀伤。4A表示在体外用0、3.125、6.25、12.5、25、50、100、200、400、800μM的MnCl 2直接处理B16F10、LLC和L929细胞，用MTT法测定细胞的存活率。4B表示在小鼠双侧皮下接种黑色素瘤B16F10，然后在一侧用生理盐水或Mn 2+做瘤内注射，之后测量并记录另一侧的肿瘤大小，同时记录小鼠的生存率。4C显示4B中小鼠的生存率差异。
图5显示Mn 2+治疗不对小鼠造成明显的副作用。5A,B表示对小鼠用鼻腔滴注的方式给Mn 2+七次，每2天一次，记录小鼠体重，一共14天，在野生型小鼠背景和STING(Tmem173)敲除小鼠背景下，Mn 2+治疗都不会对小鼠的体重造成影响。5C,D表示一共三组小鼠，分别是不处理组NC，对照组Con和Mn 2+处理组，通过鼻腔滴注的方式给Mn 2+，每2天一次，一共七次，之后每一组都不再处理，记录小鼠体重和对应组内小鼠的生存率。5E-J表示图C,D中的小鼠经过解剖取得的对应组织，记录重量。5K表示图E-J中部分组织器官的伊红-苏木精染色切片结果。
图6显示Mn 2+增强小鼠抗肿瘤能力主要由CD8 +T细胞介导，并且能增强肿瘤内T细胞的浸润。6A-C显示在Rag1敲除小鼠中，Mn 2+处理不能延缓小鼠皮下接种的黑色素瘤B16F10的生长速度和肿瘤大小。6D-F显示在β2M敲除小鼠中，Mn 2+处理不能延缓小鼠皮下接种的黑色素瘤B16F10的生长速度和肿瘤大小。6G-I显示通过流式分析在Mn 2+治疗后小鼠皮下接种的黑色素瘤B16F10瘤内的T细胞的浸润程度。6J显示在Mn 2+治疗后通过免疫荧光切片 显示小鼠皮下接种的黑色素瘤B16F10、结肠癌MC38和路易斯肺癌LLC肿瘤内的CD8阳性细胞。
图7显示Mn 2+能够增强肿瘤浸润CD8 +T细胞的活性及其特异性杀伤的能力。7A-D示在Mn 2+治疗后，小鼠皮下黑色素瘤B16F10内浸润的IFNγ +CD8 +T细胞和TNFα +CD8 +T细胞的浸润程度有显著上升。7E-H显示Mn 2+治疗后，小鼠皮下T淋巴癌E.G7内的IFNγ +CD8 +T细胞以及特异性识别E.G7肿瘤抗原SIINFEKL的CD8 +T细胞的浸润程度均有显著上升。7I-K表示在Mn 2+治疗后，将小鼠皮下接种的黑色素瘤B16F10中浸润的CD8 +T细胞进行RNA测序，与CD8 +T转录相关的Tbx21等有显著上调，与CD8 +T对于肿瘤杀伤相关的颗粒酶Granzymes和穿孔素Perforin均有显著上调，另外还有一些细胞因子也发生了变化。
图8显示Mn 2+可以在体外和体内水平促进树突状细胞(Dendritic cell,DC)的活化。8A,B显示Mn 2+可以激活小鼠骨髓来源DC(BMDC)和巨噬细胞(BMDM)产生I型干扰素。8C,D显示Mn 2+可以在体外水平显著促进小鼠骨髓来源的DC表达CD80和CD86。8E-H显示Mn 2+可以在体内水平显著促进小鼠肺脏中和腹股沟淋巴结中的DC表达CD80和CD86。8I,J显示Mn 2+可以在体外水平促进肿瘤患者外周血单个核细胞PBMC中的DC表达CD86。
图9显示Mn 2+在体外促进DC和CD8 +T细胞对于肿瘤细胞的杀伤。9A,B显示Mn 2+可以在体外促进特异性识别OVA抗原的OT-I CD8 +T细胞对于B16F10-OVA肿瘤细胞的杀伤。
图10显示Mn 2+可以起到佐剂作用帮助机体产生更多抗原特异性的CD8 +T细胞。10A,B表示提前用OVA抗原和Mn 2+免疫小鼠后可以显著增强小鼠对于皮下接种的B16-OVA肿瘤的抵抗，并显著延长小鼠的存活时间。10C,D说明提前用OVA和Mn 2+免疫小鼠后可以帮助小鼠显著增加产生OVA肽段SIINFEKL特异性的CD8 +T细胞。10E,F显示用OVA和Mn 2+刺激小鼠后可以在体内水平增强小鼠对于OVA细胞的特异性杀伤。10G,H显示用OVA和Mn 2+刺激小鼠可以显著增强小鼠体内特异性识别OVA抗原的CD8 +T细胞的增殖。
图11显示cGAS-STING通路对于Mn 2+发挥抗肿瘤免疫反应起重要作用。11-A显示在cGAS(CGas)敲除或者STING(Tmem173)敲除小鼠中，Mn 2+的刺激不能再引起小鼠产生I型干扰素。11-B,C显示在体外水平，Mn 2+不能激活STING(Tmem173)敲除小鼠骨髓来源DC(BMDC)和巨噬细胞(BMDM)产生I型干扰素。11-D-F显示在STING(Tmem173)敲除小鼠背景下，Mn 2+对皮下接种黑色素瘤B16F10的治疗效果就大幅减弱。11G,H表明在STING(Tmem173)敲除小鼠背景下，提前用OVA抗原和Mn 2+免疫小鼠后,Mn 2+不能再增强小鼠对于皮下接种的B16-OVA肿瘤的抵抗，也不能延长小鼠的存活时间。11-I,J显示在STING(Tmem173)敲除小鼠背景下，OVA和Mn 2+刺激小鼠后不能再在体内水平增强小鼠对 于OVA细胞的特异性杀伤。
图12显示Mn 2+协同增强免疫检查点抑制剂anti-PD-1对于肿瘤的治疗效果。12A-E表示Mn 2+和anti-PD-1联合使用能够显著增强anti-PD-1对于小鼠皮下接种黑色素瘤B16F10的治疗作用，同时能显著增强肿瘤内CD8 +T细胞的浸润。12F表示Mn 2+和anti-PD-1联合使用能够显著增强anti-PD-1对于小鼠肺转移型黑色素瘤B16F10的治疗作用。12G表示对于小鼠皮下接种黑色素瘤B16F10的治疗，Mn 2+和anti-PD-1联合使用后可以减少一半anti-PD-1的用量。12H-J示Mn 2+和anti-PD-1联合使用能够显著增强anti-PD-1对于小鼠皮下接种结肠癌MC38的治疗作用。12K表明Mn 2+和anti-PD-1联合使用能增强MC38肿瘤内CD8 +阳性细胞的浸润。
图13显示Mn 2+协同增强化疗药物CTX对于肿瘤的治疗效果。13A-C表示对于小鼠皮下接种黑色素瘤B16F10的治疗，Mn 2+和化疗药物环磷酰胺(cyclophosphamide monohydrate,CTX)联合使用后可以减少一半CTX的用量。
具体实施方式
定义与说明
本文中“激活”、“致敏”或“活化”具有相同的含义，“激活剂”与“致敏剂”、“活化剂”可互换使用，意指对于给定类型的免疫细胞，根据已建立的机制产生I型干扰素来激活免疫应答，包括促进免疫细胞增殖，或者上调免疫抑制性细胞因子的表达量等。本发明的激活剂或活化剂可通过调节T细胞的激活来减轻症状的疾病的预防或治疗。在本发明的一个实施方案中，包括通过施用激活剂促进CD8阳性T细胞增殖，促进抗原特异性识别CD8阳性T细胞，或者通过其他机制促进免疫应答。
本文中术语“增敏”或“增敏剂”是指为给予对象治疗有效量的分子以增加细胞对抗肿瘤药物的敏感性，例如增加对生物药、化疗药物的敏感性和/或促进用化学疗法的治疗效果。增敏剂可与治疗有效量的一种或多种其他化合物一同给药或制备为复合制剂，其他化合物包括但不限于：促进化学增敏剂掺入到靶细胞的化合物；控制治疗剂、营养物和/或氧流到靶细胞的化合物；作用于肿瘤的化疗剂或用于治疗癌症或其他疾病的其他治疗有效的化合物。在一个实施方案中，添加有效量的二价锰作可以增强免疫检查点抑制剂或者化疗药物的效果，例如以每个周期5mg/kg的剂量施用二价锰，免疫检查点抑制剂或化疗药物以规定药物有效量的0.3-0.5倍施用时，可以达到免疫检查点抑制剂或化疗药物单独施用时药物有效量的效果。
“树突状细胞”是功能最强的抗原提呈细胞，因其成熟时伸出许多树突样或伪足样突起而得名。I型干扰素可以作为树突状细胞的致敏细胞因子，体现为共刺激分子CD80/CD86等的上调表达，同时树突状细胞本身也可以在受到刺激信号时产生I型干扰素等细胞因子。
“巨噬细胞”是一种位于组织内的白血球，源自单核细胞，而单核细胞又来源于骨髓中的前体细胞，在脊椎动物体内参与天然免疫和适应性免疫。巨噬细胞在受到刺激信号时可以产生I型干扰素等细胞因子，进而增强天然免疫反应和适应性免疫反应。
“T淋巴细胞(T细胞)”是由来源于骨髓的淋巴干细胞，在胸腺中分化、发育成熟后，通过淋巴和血液循环而分布到全身的免疫器官和组织中发挥免疫功能。T细胞可以接收树突状细胞等抗原递呈细胞递呈的抗原，进而产生一系列的淋巴因子如TNFα和IFNγ等，辅助杀伤或直接杀伤含有对应抗原的靶细胞。
“CD8阳性T细胞”属于T细胞的一类，在识别MHC-I类分子递呈的抗原(例如肿瘤抗原)以后可以发育成为细胞毒性T细胞，进而对肿瘤细胞产生直接杀伤。
术语“肿瘤相关抗原”、“肿瘤抗原”、“肿瘤表达抗原”、“癌抗原”和“癌症表达抗原”是等同物，并且在本文中可互换使用。是指在正常条件下在有限量的组织和/或器官中或在特定发育阶段特异性表达的蛋白质，例如肿瘤抗原可在正常条件下在胃组织中(例如在胃黏膜中)、在生殖器官中(例如在睾丸中)、在滋养层组织中(例如在胎盘中)、或在种系细胞中特异性表达；并且在一个或更多个肿瘤或癌组织中表达或异常表达。在该情况下，“有限量”优选地意指不超过3，更优选不超过2。在本发明的上下文中，肿瘤抗原优选与癌细胞的细胞表面缔合，并且优选地在正常组织中不表达或仅很少表达。优选地，可通过肿瘤抗原或肿瘤抗原的异常表达鉴定癌细胞。在本发明的上下文中，由对象例如患有癌症疾病的患者中的癌细胞表达的肿瘤抗原可以是自身蛋白或非自身蛋白。在一些优选实施方案中，在本发明上下文中的肿瘤抗原在正常条件下特别是在癌组织中或者在非必需组织或器官(即当被免疫系统损害时不导致对象死亡的组织或器官)中或者在免疫系统不可及或很难可及或通过耐受机制(例如，通过存在高浓度的Treg细胞)进行保护的身体器官或结构中表达。肿瘤抗原的氨基酸序列在正常组织中表达的肿瘤抗原和在癌组织中表达的肿瘤抗原之间可以是相同的，或者氨基酸序列可以是不同的，例如，仅在一个氨基酸处或在多于一个氨基酸处，优选在多于2、3、4、5、6、7、8、9或10个氨基酸处不同。在一个实施方案中，肿瘤相关抗原是指在肿瘤细胞存在的抗原分子，但在正常细胞上也可能存在该抗原分子，通常其表达量远不及肿瘤细胞；例如在黑色素瘤中发现的tyrosinase,MART-1/MelanA,gp100/Pmel 17等抗原，又如Hodgkin病中的 restin,LDLR/FUT，肾癌中发现的逆转录病毒H编码的env蛋白和HOM-RCC-1，14等，还有如galectin-9，HER2/neu，HCA519，CD20，EGFR/HER1，CA 19-9等等。
本文使用的术语“药学上可接受的载体”没有特别的限定，可以根据不同剂型和施用方式选用本领域已知的药物载体，例如可选自：水、缓冲水溶液、等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、甘油、透明质酸、乙醇或聚亚烷基二醇如聚丙二醇、甘油三酯等。所用可药用载体的类型尤其依赖于组合物是否配制为用于经口、鼻(黏膜)、皮内、皮下、肌内或静脉施用。
本文使用的术语“施用”是指以在药理学上可用的方式向对象提供物质。根据本发明的药物组合物可通过任何适宜的途径施用，例如可经口、鼻(黏膜)、皮内、皮下、肌内或静脉内施用；具体而言，可采用口服、鼻滴(雾化吸入或黏膜吸收)、瘤内注射、肌内注射、静脉注射、皮内注射或者皮下注射等方法施用。
本文使用的“药物有效量”、“治疗有效量”、“有效剂量”可互换使用，均是指足以显示其对于所施用对象益处的剂量。施用的实际量，以及施用的速率和时间过程会取决于所治疗者的自身情况和严重程度。治疗的处方(例如对剂量的决定等)最终是全科医生及其它医生的责任并依赖其做决定，通常考虑所治疗的疾病、患者个体的情况、递送部位、施用方法以及对于医生来说已知的其它因素。在本发明中，二价锰可以0.02-5mg/kg的剂量施用，例如以0.05mg/kg、0.5mg/kg、3mg/kg、5mg/kg的剂量施用。
本文所使用的术语“对象”意指动物，包括温血哺乳动物，例如人和非人灵长类动物；鸟类；驯养的家养或农场动物，例如猫、狗、绵羊、山羊、牛、马和猪；实验室动物，例如小鼠、大鼠和豚鼠；鱼；爬行动物；动物园动物和野生动物等。
“抗肿瘤”是对于肿瘤的预防作用和治疗作用，包括但不限于改变肿瘤细胞微环境或者代谢途径，杀死肿瘤细胞或抑制肿瘤细胞的增殖。“抗肿瘤剂”是可用于预防肿瘤和治疗肿瘤的物质，可用于本发明的抗肿瘤剂包括但不限于化疗药物。上述“化疗药物”是指用于治疗癌症的抗肿瘤药或将多于一种的这些药物组合成细胞毒性标准化治疗方案的治疗。在本发明中，术语“化疗药物”包括任意的抗肿瘤剂，其包括用于任意类型癌症及相关进程(例如血管生成或转移)的小有机分子、肽、寡核苷酸等，示例性的化疗药物为(但不限于)烷化剂类例如氮芥类/氧氮环膦类(oxazaphosphorine)(例如，环磷酰胺、异环磷酰胺)、亚硝基脲类(例如，卡莫司汀)、三氮烯类(例如，替莫唑胺)和烷基磺酸酯(例如，白消安)、蒽环类抗生素如阿霉素和正定霉素、紫杉烷类如TaxolTM和多西他赛、长春花生物碱类如新长春碱和长春碱、5-氟尿嘧啶(5-FU)、亚叶酸(leucovorin)、伊立替康、伊达比星、丝裂霉素C、奥沙利铂、雷替曲塞、 培美曲塞(pemetrexed)、他莫昔芬、顺铂、卡铂、甲氨蝶呤、放线菌素D、米托蒽醌、博来霉素(blenoxane)、光神霉素(mithramycin)、甲氨蝶呤、紫杉醇、2-甲氧雌二醇、普马司他(prinomastat)、巴马司他(batimastat)、BAY12-9566、羧胺三唑(carboxyamidotriazole)、CC-1088、右美沙芬乙酸、二甲基咕吨酮乙酸、内皮抑素、IM-862、马立马司他(marimastat)、青霉胺、PTK787/ZK222584、RPI.4610、乳酸角鲨胺、SU5416、沙利度胺、康普立停(combretastatin)、他莫昔芬、COL-3、新伐司他(neovastat)、BMS-275291、SU6668、抗VEGF抗体、Medi-522(VitaxinII)、CAI、白介素12、IM862、阿米洛利、血管抑素、血管抑素Kl-3、血管抑素Kl-5、卡托普利、DL-α-二氟甲基鸟氨酸、DL-α-二氟甲基鸟氨酸HCl、内皮抑素、烟曲霉素(fumagillin)、除莠霉素A(herbimycinA)、4-羟基苯基维甲酰胺(4-hydroxyphenylretinamide)、胡桃醌(juglone)、层黏连蛋白、层黏连蛋白六肽、层黏连蛋白五肽、熏草菌素A(lavendustinA)、甲羟孕酮、米诺环素、胎盘核糖核酸酶抑制剂、苏拉明(suramin)、血小板反应蛋白(thrombospondin)、靶向促血管发生因子的抗体(例如，阿瓦斯汀(Avastin)、爱必妥(Erbitux)、维克替比(Vectibix)、赫赛汀(Herceptin))、拓扑异构酶抑制剂、抗微管剂、促血管发生生长因子的低分子量酪氨酸激酶抑制剂(例如特罗凯(Tarceva)、多吉美(Nexavar)、索坦(Sutent)、易瑞沙(Iressa))、GTP酶抑制剂、组蛋白脱乙酰化酶抑制剂、AKT激酶或ATP酶抑制剂、Wnt信号传导抑制剂、E2F转录因子抑制剂；mTOR抑制剂(例如驮瑞塞尔(Torisel))、α、β和γ干扰素、IL-12、基质金属蛋白酶抑制剂(例如，COL3、马立马司他(Marimastat)、巴马司他(Batimastat))、ZD6474、SUl1248、vitaxin、PDGFR抑制剂(例如，格列卫(Gleevec))、NM3和2-ME2、环肽例如西仑吉肽(cilengitide)。TheMerckIndexCD-ROM第13版中详细描述了另一些合适的化疗剂。在一个实施方案中，所述抗肿瘤剂还包括单克隆抗体、干扰素、生物反应调节剂以及其他抗肿瘤药物。在一个实施方案中，所述抗肿瘤剂为免疫检查点抑制剂，例如PD-1抗体或者PD-L1抗体。在一个实施方案中，所述抗肿瘤剂均是已知的，且可经商业途径购得或可由本领域技术人员以本身所知的、或众所周知的、或常规的方法制备。
“免疫检查点抑制剂”是指结合免疫检查点蛋白并阻断其活性和/或抑制免疫调节细胞(例如，Treg细胞，肿瘤相关巨噬细胞等)表达其结合的免疫检查点蛋白的功能的分子，化合物或组合物。免疫检查点蛋白可包括但不限于CTLA4(细胞毒T淋巴细胞相关蛋白4，CD152)，PD1(也称为PD-1；程序性死亡1受体)，PD-L1，PD-L2，LAG-3(淋巴细胞活化基因-3)，OX40，A2AR(腺苷A2A受体)，B7-H3(CD276)，B7-H4(VTCN1)，BTLA(B和T淋巴细胞衰减子，CD272)，IDO(吲哚胺2，3-双加氧酶)，KIR(杀伤细胞免疫球蛋白样受体)，TIM 3(T细胞免疫 球蛋白结构域和粘蛋白结构域3)，VISTA(T细胞活化的V结构域Ig抑制因子)和IL-2R(白细胞介素-2受体)。
“PD-1抗体”或者“PD-L1抗体”指与人PD1/PDL1(程序性死亡受体)结合的药剂，其可调节和/或抑制PD1活性，可抑制其配体(PDL1、PDL2等)之一与PD1受体结合，阻断PD1/PDL1通路并且可以是抗PD1/L1抗体。在一个实施方案中，“PD-1抗体”或“PD-L1抗体”指以K D1.0×10 -8mol/L或更低(在一个实施方案中，1.0×10 -8mol/L-1.0×10 -13mol/L)，在一个实施方案中，以KD 1.0×10 -9mol/L或更低(在一个实施方案中，1.0×10 -9mol/L-1.0×10 -13mol/L)的结合亲和力特异性结合人PD1抗原或人PDL1抗原的抗体。结合亲和力用标准结合测定法测定，诸如表面等离振子共振技术(GE-Healthcare，Uppsala，Sweden)。在一个实施方案中，所述PD-1抗体或PD-L1抗体是本领域熟知的并且是商业上或临床上可获得的，包括但不限于BMS-936559/MDX-1105(Bristol-Myers Squibb)，MPDL3280A(Genentech)，MED14736(MedImmune)，MSB0010718C(EMD Sereno)，纳武利尤单抗Nivolumab(Opdivo，Bristol Myers Squibb)，帕博利珠单抗Pembrolizumab(Keytruda，Merck Sharp Dohme)，阿特珠单抗Atezolizumab(Tecentriq，Genentech),Durvalumab(Imfinzi，Astrazeneca UK LTD)，Avelumab(Bavencio，EMD Sereno)，Cemiplimab(Libtayo，Regeneron)，特瑞普利单抗(拓益，君实生物)和信迪利单抗(达伯舒，信达生物)。
出于本发明的目的，本文中术语“癌症”和“癌症疾病”与术语“肿瘤”和“肿瘤疾病”可互换使用。术语“癌症”和“癌症疾病”是指至少一组细胞显示出不受控制的生长(分裂超出正常范围)、侵袭(侵入和破坏邻近组织)和有时转移(通过淋巴或血液扩散至身体的其他位置)。这三种癌症的恶性特征将它们与具有自限性且不会侵袭或转移的良性肿瘤区分开。大多数癌症形成肿瘤，但有些癌症(例如白血病)则不会。术语“肿瘤”或“肿瘤疾病”是指细胞(被称为赘生性细胞、肿瘤发生性细胞或肿瘤细胞)的异常生长，优选形成肿胀或病灶。“肿瘤细胞”意指通过快速的不受控制的细胞增殖生长并在引发新生长的刺激停止之后继续生长的异常细胞。肿瘤显示出结构组织和与正常组织的功能协调的部分或完全缺失，并且通常形成独特的组织团块。在一个实施方案中，本发明所述“肿瘤”与“癌症”或“癌症疾病”具有相同含义，指具有上述不受控制的生长、侵袭和有时转移性质的恶性病变。
“治疗不敏感”意指与成功治疗的机率相比，不能成功地治疗受试者或不能至少以显著增加的可能性成功地治疗受试者，例如施用规定剂量的药物后仍然不能成功地抑制肿瘤细胞的增殖、侵袭或转移。
下文将结合具体实施例对本发明的制备方法做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
下述实施例中所用的试剂、材料等，如无特殊说明，均可从商业途径得到。
材料和方法
抗体和试剂
抗体来源如下：抗GAPDH抗体(sc-25778)购自Santa Cruz。cGAS抗体(31659S)和STING抗体(13647S)购自Cell Signaling Technology。抗蝰蛇毒素(Viperin)抗体通过已公开方法制得和使用(13)。简言之，抗原片段的cDNA插入到pET-21b载体(Novagen)并表达于大肠杆菌E.coli BL21(DE3)，通过Ni-NTA亲和柱纯化重组蛋白，然后将其注射进小鼠或兔子，获得能够识别对应抗原的抗血清。流式抗体均购自Biolegend，另有说明除外。PE-anti-SIINFEKL-Tetramer抗体购自MBL，货号TS-5001-1C。Anti-PD-1(29F.1A12)购自BioXCell，货号#BE0273，Rat IgG2a isotype(2A3)购自BioXCell，货号#BE0089。
所有化学品购自Sigma-Aldrich(St.Louis,MO)，另有说明除外。脂多糖(Sigma,L4130)、卵清蛋白(InvivoGen,#vac-pova)、
Figure PCTCN2019124098-appb-000001
-1077(Sigma,10771)、VybrantTM MTT Cell Proliferation Assay Kit(Invitrogen,V13154)、mMESSAGE mMACHINE T7 Ultra(Ambion,am1345)、HiScribe TM T7 High Yield RNA Synthesis Kit(NEB,E2040S)、MEGAclear TM Kit Purification of Transcription Reactions(Ambion,am1908)、CFSE cell division tracer kit(Biolegend,423801)、Cyclophosphamide monohydrate(TargetMol,T0707)均为商业化产品。
细胞
L929-ISRE(蒋争凡实验室自制细胞系，将pGL3ISRE-Luciferase质粒稳定转染至L929细胞，
Figure PCTCN2019124098-appb-000002
CCL-1)、BHK21(
Figure PCTCN2019124098-appb-000003
CCL-10)、B16-OVA(
Figure PCTCN2019124098-appb-000004
CRL-6322)和B16F10(
Figure PCTCN2019124098-appb-000005
CRL-6475)，MC38(由清华大学医学院张永辉老师惠赠)，LLC(
Figure PCTCN2019124098-appb-000006
CRL-1642)培养于添加有10％FBS(Gibco)、5μg/mL青霉素和10μg/mL链霉素的DMEM(Gibco)培养基中,E.G7-OVA(
Figure PCTCN2019124098-appb-000007
CRL-2113)培养于添加有10％FBS(Gibco)、5μg/mL青霉素和10μg/mL链霉素的RPMI-1640(Gibco)培养基中。骨髓来源的巨噬细胞(bone marrow-derived macrophages,BMDMs)制备BMDM时，用10mL含有L929细胞上清的诱导培养基(空DMEM:FBS:L929上清＝5:2:3)重悬骨髓细胞，随后铺至10cm细胞培养皿或 者所需要的孔板当中，诱导5-7天后可分化为BMDM，处理前换成5％FBS的DMEM培养。骨髓来源的树突细胞(bone marrow-derived dendritic cell,BMDCs)在含有10ng/mL GM-CSF、10ng/mL IL-4、10％FBS(Gibco)的RPMI-1640(Gibco)培养基中诱导，第3天半量换液，第7天进行实验。腹膜巨噬细胞收获自巯基乙醇酸盐(BD,Sparks,MD)注射诱导后5天的小鼠，培养于添加有5％FBS的DMEM培养基中。
病毒感染
仙台病毒(Sendai virus,SeV)由武汉大学郑从义教授惠赠，痘苗病毒(Vaccinia virus,VACV)由华中农业大学金梅林教授惠赠，单纯疱疹病毒(Herpes simplex virus 1,HSV-1)由武汉大学舒红兵教授馈赠。将被处理细胞预先接种于6孔板或者12孔板中。用以感染细胞诱发天然免疫反应的病毒滴度为：SeV(MOI＝0.01)，VACV(MOI＝0.01)，HSV-1-GFP(MOI＝0.01)。感染2小时后，更换为新鲜的全培养基，18-24小时后收集细胞和培养上清用于western blot或者是type I-IFN Bioassay检测。
人源样品
经供者知情同意，抽取健康成人供者或肿瘤患者的外周血(EDTA抗凝)10mL，转移至50mL离心管中，PBS稀释至25mL(外周血:PBS＝1:1.5)。取15mL Ficoll(
Figure PCTCN2019124098-appb-000008
-1077)至于50mL离心管中，将PBS稀释的外周血缓慢铺在Ficoll上层(Ficoll:稀释外周血＝3:5)。在室温条件下，800g离心20分钟，加速1，减速0。去掉上层血浆，吸取位于中间白膜层的单核细胞，尽量避免吸取血小板。补加PBS至40mL，4℃,600g离心5分钟，弃上清，重复洗涤一次，使用含有5％胎牛血清的RPMI-1640重悬细胞并培养。
小鼠
野生型小鼠为C57BL/6，购买自北京维通利华实验动物技术有限公司(Beijing Vital River Laboratory Animal Technology Co.,Ltd.)。实验小鼠年龄一般均在6-8周龄。实验小鼠都在具备AAALAC认证的北京大学实验动物中心无特定病原菌(specific pathogen-free,SPF)的鼠房中饲养和繁殖，所有操作都在北京大学实验动物中心批准的情况下，并按照美国国立卫生研究院实验动物护理和使用指南进行。
CGas-/-小鼠和Tmem173-/-小鼠由CRISPR/Cas9方法制备获得，用HiScribeTM T7 High Yield RNA Synthesis Kit制备获得引导RNA(guide RNA,gRNA)，用mMESSAGE mMACHINE T7 Ultra Kit制备获得Cas9RNA。同时，提前准备好超排的C57BL/6小鼠并与雄鼠交配，以及用于代孕的ICR雌鼠和交配用的ICR结扎雄鼠。之后取超排过的C57BL/6雌鼠的受精卵，利用体外显微注射技术Cas9和gRNA按2:1的浓度比例(100ng/50ng/μL) 混合后进行胞内注射。选取能发育成二细胞期的受精卵，转移至假孕ICR鼠的输卵管，等待小鼠出生。待小鼠长到约2周龄时剪取鼠尾进行基因型鉴定，选取阳性小鼠交配，最后得到纯合敲除小鼠。
小鼠肿瘤模型构建：
(1)黑色素瘤肺转移模型构建
培养B16F10细胞，随后用4℃预冷的PBS收集细胞，300g离心5分钟，重复2遍，以充分去除培养基成分，然后进行细胞计数，再用4℃预冷的PBS重悬细胞至2×10 5个/300μL，细胞尽量置于冰上以保证细胞活力。选取6-8周龄的C57BL/6小鼠，放置入固鼠器，用酒精擦拭其尾巴，或者用烤灯预先照射小鼠使其尾静脉充分扩张，然后用胰岛针进行尾静脉注射，细胞量为2×10 5个/300μL一只小鼠，2周之后可在肺部见到明显的黑色素瘤转移灶。
(2)皮下实体瘤模型构建
培养目的细胞如B16F10/B16F0，或者MC38、LLC、E.G7等细胞，用4℃预冷的PBS收集细胞，300g离心5分钟，重复2遍，以充分去除培养基成分，计数后根据造模所需细胞量确定重悬体积，一般情况下，B16F10造模时用4℃预冷的PBS重悬至3×10 5个-5×10 5个/100μL，而MC38、LLC和E.G7造模时用4℃预冷的PBS重悬至1×10 6个/100μL。随后将100μL细胞皮下注射至C57BL/6小鼠的腹股沟部位，一般2-3周以后可以见到明显的肿瘤形成。本文中根据实验动物福利要求，当肿瘤体积大于1000mm 3时或者肿瘤发生溃烂时对动物实施安乐死，肿瘤体积计算规则为：Volume(mm 3)＝1/2×长轴(mm)×短轴(mm)×短轴(mm)。
小鼠Mn 2+治疗以及联合治疗
(1)滴鼻治疗
在6-8周龄C57BL/6小鼠接种肿瘤后24小时，按照每只小鼠给5mg/kg的MnCl 2剂量，将1M的MnCl 2储液用生理盐水稀释至20μL每只小鼠，每2天滴鼻一次，通常共滴鼻7次。另一组相同周龄的小鼠滴鼻生理盐水作为对照组。
(2)尾静脉治疗
在6-8周龄C57BL/6小鼠接种肿瘤后24小时，按照每只小鼠给5mg/kg的MnCl 2剂量，将1M的MnCl 2储液用PBS稀释至300μL每只小鼠，每2天注射一次，通常共注射7次。另一组含有相同个数的小鼠尾静脉注射PBS作为对照组。
(3)免疫检查点抑制剂anti-PD-1联合治疗
分组设置包含：对照组(IgG2a isotype,Clone2A3,BioXCell)，Mn 2+治疗组，anti-PD-1 单抗治疗组(Clone 29F.1A12,BioXCell)，以及Mn 2++anti-PD-1联合使用组。在使用上，Mn 2+采取上述1或者2中的操作方法，anti-PD-1单抗在肿瘤接种后第3,7,11天用生理盐水稀释后每只小鼠腹腔注射200μg/200μL，联合治疗组一般是先注射抗体，再给Mn 2+处理。
(4)化疗药物联合治疗
分组设置包含：对照组，Mn 2+治疗组，化疗药物治疗组(Cyclophosphamide monohydrate,CTX,TargetMol T0707)，以及Mn 2++化疗药物CTX联合使用组。在使用上，Mn 2+采取上述1或者2中的操作方法，CTX在肿瘤接种后第5,9,13天用含有5％DMSO+30％PEG 300+5％Tween80+ddH 2O的溶剂稀释，每只小鼠按100mg/kg剂量进行腹腔注射(或者根据实验设计对化疗药物进行减量)，联合治疗组一般是先注射化疗药物，再给Mn 2+处理。在接种的肿瘤生长至大小可触及后，用游标卡尺进行长轴和短轴的测量，每1-2天测量一次，记录2-3周后，根据实验动物福利要求，用CO 2对小鼠实施安乐死，随后解剖小鼠，分离完整肿瘤组织，用于拍照或者称重记录。
小鼠免疫佐剂及肿瘤预防实验：用生理盐水配制每100μL含有100μgOVA，100μgOVA+20μg胶体锰制剂，取6-8周龄野生型或基因缺陷型C57BL/6小鼠，在后腿股部进行肌肉注射每只小鼠注射100μL。在初次免疫后，分别于第7天和第14天后增强免疫，于第21天接种稳转有OVA蛋白的肿瘤细胞系。未免疫组作为对照，观察并记录肿瘤生长情况，最后用CO 2将小鼠安乐死。
小鼠体内细胞毒性T淋巴细胞功能检测实验：用生理盐水配制每100μL含有100μgOVA,100μgOVA+20μg胶体锰制剂，取6-8周龄野生型或基因缺陷型C57BL/6小鼠，在后腿股部进行肌肉注射每只小鼠注射100μL，初次免疫后，分别于第7天和第14天后增强免疫，并设置免疫的小鼠作为对照，该批小鼠定义为受体小鼠。第21天时，取新一批适量个数的野生型C57BL/6小鼠(供体小鼠)，处死并取出其脾脏细胞，制备成单细胞悬液并等分为两组，分别用0.5μM和5μM的CFSE在37℃胞培养箱中染色10分钟，用PBS洗一遍。然后将5μM CFSE染色的脾脏细胞用OVA 257-264肽段(10μg/mL)在37℃细胞培养箱中染色90分钟。之后用PBS洗涤两次，进行细胞计数。将0.5μM和5μM CFSE染色的脾脏细胞1:1均匀混合，然后通过尾静脉注射的方式，每只受体小鼠注射4×10 6个供体小鼠的脾脏细胞。24小时后，将受体小鼠处死，并解剖取得脾脏，制备成单细胞悬液，然后通过流式细胞术技术检测CFSE阳性细胞，并计算特异性杀伤比例，计算公式为：特异性杀伤比例＝(CFSE low细胞比例－CFSE high细胞比例)/CFSE low细胞比例×100。
小鼠体内CD8 +T淋巴细胞增殖检测：取6-8周龄CD45.1 +的OT-I小鼠，解剖取得其脾 脏组织，制备成单细胞悬液，用磁珠分选出CD8 +T细胞，然后用1μM的CFSE在37℃染色20分钟，洗涤2遍后，将2×10 6个CFSE标记的CD8 +T细胞通过尾静脉注射回输给新的一批6-8周龄的CD45.2 +野生型C57BL/6小鼠，1天后分别在腹股沟皮下注射100μg OVA,100μg OVA+胶体锰(Mn 2+)制剂，并设置对照组。然后于尾静脉注射CD8 +T细胞后的第3天处死小鼠，解剖取其腹股沟淋巴结，制备单细胞悬液，利用流式抗体染色和流式细胞术检测低表达CFSE(CFSElow)细胞的比例，并做统计分析。
I型干扰素生物活性检测(Type I-interferon Bioassay)
详细实验方法可参考已有文献(14)。提前将2f-TGH-ISRE(测定人源I型干扰素)或者L929-ISRE(测定鼠源I型干扰素)传代至96孔细胞板中，保证在测定时细胞密度在80％以上。将100μL待检测细胞上清和人源或鼠源的I型干扰素标准品梯度稀释液(R&D system)加入到96孔细胞板中，根据实验需求设置平行孔，置于37℃培养箱4-5小时。随后，用真空吸引泵吸干细胞上清，每个孔中加入30μL细胞裂解液(Promega)，裂解30分钟，取10μL细胞裂解液至检测用96孔板，再加入10μL萤火虫荧光素酶底物(Promega)，混匀后放入多功能酶标仪工作站测定荧光强度，根据标准品荧光读值绘制标准曲线，并换算出样品中I型干扰素的浓度。
统计分析
本文中的数据用mean±SEM标注呈现，数据分析采用Student’s t-test。小鼠生存曲线分析比较采用Mantel-Cox test。显著性分析标注如下：ns,not significant,p>0.05；*p<0.05；**p<0.01；***p<0.001；****p<0.0001。
实施例1.小鼠肿瘤模型的构建
实验(A)小鼠肿瘤模型构建的方法
如图1A所示，实验中所用的肿瘤接种方法简要流程图。
实验(B)小鼠皮下肿瘤模型的构建
在6-8周龄野生型C57BL/6小鼠腹股沟皮下注射10 5数量级的肿瘤细胞，待其生长，构建皮下肿瘤模型，一般需要2周。
实验(C)小鼠肺转移肿瘤模型的构建
向6-8周龄野生型C57BL/6小鼠通过尾静脉注射接种2×10 5个B16F10黑色素瘤细胞，待其转移，构建肺部转移模型，一般需要2周。
实施例2.小鼠鼻腔滴注Mn 2+对于皮下黑色素瘤和肺部转移黑色素瘤均有治疗效果
实验(A)Mn 2+治疗后小鼠皮下黑色素瘤生长的变化
在6-8周龄野生型C57BL/6小鼠腹股沟皮下注射5×10 5个B16F10细胞的PBS悬液，分为两组，实验组于肿瘤接种24小时后，按照5mg/kg剂量开始鼻腔滴注MnCl 2的生理盐水溶液，对照组鼻腔滴注等体积的生理盐水溶液，每2天一次。待肿瘤生长至可触及后，开始测量肿瘤的大小，每2天记录一次，绘制肿瘤生长曲线。对照组n＝10，实验组n＝10。
实验(B)通过活体成像比较Mn 2+治疗时不同时间点肿瘤的差异
取待检测小鼠，腹腔注射150mg/kg的萤火虫荧光素酶底物D-luciferin的生理盐水溶液，然后麻醉小鼠，注射底物10分钟后将小鼠放入IVIS活体成像系统中成像，并生成图像。
实验(C)通过解剖比较Mn 2+治疗后小鼠肿瘤的大小差异
待实验(A)中的小鼠肿瘤生长2周后，CO 2处死小鼠，解剖小鼠取得完整肿瘤组织并拍照记录。
实验(D)比较Mn 2+治疗后小鼠皮下黑色素瘤的大小差异
将实验(A)中小鼠解剖获得的所有肿瘤称重记录，并做统计学分析。
实验(E)通过解剖肺部组织比较Mn 2+治疗后小鼠肺转移肿瘤的差异
向6-8周龄野生型C57BL/6小鼠通过尾静脉注射接种2×10 5个B16F10黑色素瘤细胞的PBS悬液，实验组于肿瘤接种24小时后，按照5mg/kg剂量开始鼻腔滴注MnCl 2的生理盐水溶液，对照组鼻腔滴注等体积的生理盐水溶液，每2天一次。对照组n＝5只，实验组n＝5只。2周后，CO 2处死小鼠，解剖小鼠取得完整肺脏组织，拍照记录(图上)。随后固定做HE染色(图下)。
实验(F)比较Mn 2+治疗后小鼠肺转移肿瘤转移灶的个数
记录实验(E)中肺部组织表面的黑色素肿瘤灶点数，并做统计学分析。
实验(G)比较Mn 2+治疗后荷瘤小鼠肺脏的重量差异
记录实验(E)中整个肺部组织的重量，并做统计学分析。
实施例3.Mn 2+可以通过不同给药方式对多种肿瘤类型起到治疗效果
实验(A)比较Mn 2+治疗后小鼠皮下LLC生长的变化
在6-8周龄野生型C57BL/6小鼠腹股沟皮下接种1×10 6个LLC细胞，实验组于肿瘤接种24小时后，按照5mg/kg剂量开始鼻腔滴注MnCl 2的生理盐水溶液，对照组鼻腔滴注等体积的生理盐水溶液，每2天一次。对照组n＝7，实验组n＝7。等肿瘤生长至可触及时开始记录 大小，并绘制肿瘤生长曲线。
实验(B)比较Mn 2+治疗后LLC荷瘤小鼠的存活率变化
记录实验(A)中小鼠的死亡情况，并绘制生存曲线。
实验(C)比较Mn 2+治疗后小鼠皮下E.G7生长的变化
在6-8周龄野生型C57BL/6小鼠腹股沟皮下接种1×10 6个E.G7细胞，实验组于肿瘤接种24小时后，按照5mg/kg剂量开始鼻腔滴注MnCl 2的生理盐水溶液，对照组鼻腔滴注等体积的生理盐水溶液，每2天一次。等肿瘤大小可触及时开始记录大小，并绘制肿瘤生长曲线。
实验(D)比较Mn 2+治疗后小鼠皮下E.G7的大小差异
在实验(C)中的肿瘤接种第17天后用CO 2处死小鼠，解剖取得完整肿瘤组织，称重记录，并做统计学分析，对照组n＝8只，实验组n＝9只。
实验(E)比较Mn 2+治疗后小鼠皮下MC38生长的变化
向6-8周龄野生型C57BL/6小鼠尾静脉注射1×10 6个MC38细胞，实验组于肿瘤接种24小时后，按照5mg/kg剂量开始尾静脉注射MnCl 2的PBS溶液，对照组尾静脉注射等体积的PBS溶液，每2天一次。等肿瘤大小可触及时开始记录大小，并绘制肿瘤生长曲线。
实验(F)比较Mn 2+治疗后小鼠皮下MC38肿瘤的大小差异
在实验(E)中的肿瘤接种第18天后用CO 2处死小鼠，解剖取得完整肿瘤组织，拍照记录，并称重做统计学分析。
实验(G)比较肿瘤内注射Mn 2+治疗后，黑色素瘤荷瘤小鼠的存活率差异
取6-8周龄野生型C57BL/6小鼠，在一侧腹股沟皮下注射3×10 5个B16-OVA肿瘤细胞。在肿瘤接种后第8,10,12,14天分别瘤内注射5mg/kg的MnCl 2生理盐水溶液，记录绘制肿瘤生长曲线。对照组n＝32，实验组n＝23。
实施例4.Mn 2+在体外以及体内对于肿瘤细胞直接杀伤的效应观察
实验(A)在体外用Mn 2+处理细胞后MTT染色测定细胞死活
预先在24孔板中的全培养基中接种不同类型的细胞，包括B16F10，LLC和L929细胞。然后等细胞密度长到约95％后，换成不含血清的培养基，并加入0、3.125、6.25、12.5、25、50、100、200、400、800μM的MnCl 2溶液。培养24小时后，更换成新的全培养基，并添加培养基1/5体积的MTT储液(储液为2mg/mL的MTT PBS溶液)，置于37℃培养箱反应1小时。然后吸干培养基，在室温将细胞晾干。随后每个孔加入300μL的裂解液(900mL异丙醇+100mL H 2O+5g SDS+1mL 4N HCl)，室温裂解30分钟。然后用多功能酶标仪读取590nm 处的OD值，并减去690nm处的背景OD值，将读值做分析，以不加MnCl 2处理孔的读值为分母，分别计算出其余孔的百分比用以指征细胞存活率。
实验(B)在小鼠体内用Mn 2+做瘤内注射后观察对侧肿瘤生长情况
取6-8周龄野生型C57BL/6小鼠，在双侧腹股沟皮下分别注射1.5×10 5个B16-OVA肿瘤细胞。在肿瘤接种后第8,10,12,14天分别在一侧瘤内注射5mg/kg的MnCl 2的生理盐水溶液，记录绘制另一侧的肿瘤生长曲线，对照组n＝10，实验组n＝10。
实验(C)小鼠体内用Mn 2+做瘤内注射后观察小鼠存活率差异
观察记录实验(B)中小鼠的死亡情况，绘制小鼠死亡曲线。
实施例5.Mn 2+治疗对于小鼠的毒副作用检测
实验(A)Mn 2+处理对于野生型小鼠的体重影响(14天)
对不接种肿瘤的野生型C57BL/6小鼠用5mg/kg剂量的MnCl 2生理盐水溶液进行鼻腔滴注，每2天一次，一共7次，对照组在同样时间点鼻腔滴注等体积的生理盐水，在每次鼻腔滴注前对小鼠称重，并记录绘制体重曲线。每组小鼠n＝7。
实验(B)Mn 2+处理对于STING敲除小鼠的体重影响(14天)
对不接种肿瘤Tmem173 -/-小鼠用5mg/kg剂量的MnCl 2生理盐水溶液进行鼻腔滴注，每2天一次，一共7次，对照组在同样时间点鼻腔滴注等体积的生理盐水，在每次鼻腔滴注前对小鼠称重，并记录绘制体重曲线。每组小鼠n＝7。
实验(C)Mn 2+处理对于野生型小鼠的体重影响(40天)
将不接种肿瘤的野生型C57BL/6小鼠分为三组，空白对照组(Negative Control,NC)，对照组(Con)和实验组(Mn 2+)，每组6只小鼠。Mn 2+组用5mg/kg剂量的MnCl 2生理盐水溶液进行鼻腔滴注，每2天一次，一共7次，然后停止给药；Con组在相同时间点用等体积的生理盐水溶液进行鼻腔滴注；NC组不做任何处理。每组小鼠每2天称量一次体重，一直至40天。
实验(D)Mn 2+处理对于野生型小鼠的存活率影响(40天)
每天观察实验(C)中小鼠的行为，饮食饮水情况，并记录死亡情况，绘制死亡曲线。
实验(E-J)Mn 2+处理对于野生型小鼠的各器官质量的影响(40天)
在第40天时，用CO 2将实验(C)中的小鼠全部处死，并解剖。取得小鼠完整的心脏(E)，肝脏(F)，脾脏(G)，肺脏(H)，肾脏(I)和完整脑组织(J)，拍照记录，并分别称量记录每一只小鼠的每一种器官的重量，做统计学分析。
实验(K)Mn 2+处理对于野生型小鼠部分器官的HE染色检查(40天)
取实验(E-J)中的肺脏、肝脏、肾脏和脑组织用以石蜡组织切片制备，并用HE染色，拍照成像。
实施例6.Mn 2+治疗时主要通过CD8 +T细胞发挥抗肿瘤疗效
实验(A)比较Rag1 -/-小鼠中Mn 2+治疗后小鼠皮下黑色素瘤生长的变化
在Rag1 -/-小鼠中，通过腹股沟皮下注射的方式接种5×10 5个B16F10细胞，接种后24小时，开始用5mg/kg的MnCl 2生理盐水溶液进行鼻腔滴注，对照组在相同时间点用等体积生理盐水做鼻腔滴注。等到肿瘤大小长到可以触及时，开始测量并记录大小，用以绘制肿瘤生长曲线。对照组n＝11，实验组n＝11。
实验(B,C)比较Rag1 -/-小鼠中Mn 2+治疗后小鼠皮下黑色素瘤的大小差异
在实验(A)中小鼠的肿瘤接种后第14天，用CO 2处死小鼠，取得其完整肿瘤组织，拍照记录(B)，并称重做统计学分析(C)。
实验(D)比较β2m -/-小鼠中Mn 2+治疗后小鼠皮下黑色素瘤生长的变化
在β2m -/-小鼠中，通过腹股沟皮下注射的方式接种5×10 5个B16F10细胞，接种后24小时，开始用5mg/kg的MnCl 2生理盐水溶液进行鼻腔滴注，对照组在相同时间点用等体积生理盐水做鼻腔滴注。等到肿瘤大小长到可以触及时，开始测量并记录大小，用以绘制肿瘤生长曲线。对照组n＝6，实验组n＝6。
实验(E,F)比较β2m -/-小鼠中Mn 2+治疗后小鼠皮下黑色素瘤的大小差异
在实验(D)中小鼠的肿瘤接种后第14天，用CO 2处死小鼠，取得其完整肿瘤组织，拍照记录(E)，并称重做统计学分析(F)。
实验(G-I)Mn 2+治疗后小鼠皮下黑色素瘤内的CD4 +T，CD8 +T的浸润程度升高
在野生型C57BL/6小鼠腹股沟皮下接种5×10 5个B16F10细胞，在接种24小时后，用5mg/kg的MnCl 2生理盐水溶液进行鼻腔滴注，设置对照组用等体积生理盐水滴注，在肿瘤接种后第14天处死小鼠，解剖取得其完整肿瘤组织，然后制备成单细胞悬液并计数，取适量细胞用流式抗体染色，然后利用流式细胞术分别检测CD4阳性和CD8阳性细胞群(G)，通过计算得到每克肿瘤中CD8阳性细胞(H)和CD4阳性细胞(I)的数量，并做统计学分析。对照组n＝8，实验组n＝8。
实验(J)Mn 2+治疗后小鼠不同类型皮下肿瘤内的CD8阳性细胞浸润程度升高
在野生型C57BL/6小鼠腹股沟皮下接种5×10 5个B16F10细胞，1×10 6个MC38细胞或 者是1×10 6个LLC细胞，然后在肿瘤接种后24小时开始用5mg/kg的MnCl 2生理盐水溶液进行鼻腔滴注，设置对照组用等体积生理盐水做鼻腔滴注。处死小鼠后解剖获得其完整肿瘤，切片后利用CD8抗体标记其中CD8阳性细胞，并通过荧光显微镜成像。
实施例7.Mn 2+能够增强肿瘤浸润CD8 +T细胞的活性及其特异性杀伤的能力
实验(A,B)Mn 2+治疗后小鼠皮下黑色素瘤内IFNγ +CD8 +T细胞的浸润程度升高
在野生型C57BL/6小鼠腹股沟皮下接种5×10 5个B16F10细胞，在接种24小时后，用5mg/kg的MnCl 2生理盐水溶液进行鼻腔滴注，对照组用等体积生理盐水做鼻腔滴注，在肿瘤接种后第14天处死小鼠，解剖取得其完整肿瘤组织，制备单细胞悬液，进行细胞表面染色和胞内染色，然后利用流式细胞术检测IFNγ +CD8 +双阳性T细胞的比例(A)，计算并统计分析IFNγ +CD8 +双阳性T细胞在肿瘤中的浸润程度(B)。对照组n＝13，实验组n＝13。
实验(C,D)Mn 2+治疗后小鼠皮下黑色素瘤内TNFα +CD8 +T细胞的浸润程度升高
在野生型C57BL/6小鼠腹股沟皮下接种5×10 5个B16F10细胞，在接种24小时后，用5mg/kg的MnCl 2生理盐水溶液进行鼻腔滴注，对照组用等体积生理盐水做鼻腔滴注，在肿瘤接种后第14天处死小鼠，解剖取得其完整肿瘤组织，制备单细胞悬液，进行细胞表面染色和胞内染色，然后利用流式细胞术检测TNFα +CD8 +双阳性T细胞的比例(C)，计算并统计分析TNFα +CD8 +双阳性T细胞在肿瘤中的浸润程度(D)。对照组n＝5，实验组n＝5。
实验(E)比较Mn 2+治疗后小鼠皮下E.G7的大小差异
在野生型C57BL/6小鼠腹股沟皮下接种1×10 6个E.G7细胞，在接种24小时后，用5mg/kg的MnCl 2生理盐水溶液进行鼻腔滴注，设置对照组用等体积生理盐水滴注，在肿瘤接种后第17天处死小鼠，解剖取得其完整肿瘤组织，拍照记录。对照组n＝4，实验组n＝4。
实验(F-H)Mn 2+治疗后能增强小鼠皮下E.G7瘤内特异性识别肿瘤抗原的CD8 +T细胞 浸润程度
将实验(E)中获得的肿瘤组织制备单细胞悬液，进行细胞表面染色和胞内染色，然后利用流式细胞术检测IFNγ +CD8 +双阳性T细胞的比例(b)，计算并统计分析IFNγ +CD8 +双阳性T细胞在肿瘤中的浸润程度(c)，另外通过细胞表面染色和流式细胞术统计分析SIINFEKL +CD8 +T细胞在肿瘤中的浸润程度(d)。TIL:tumor infiltrating lymphocyte，肿瘤浸润淋巴细胞。
实验(I-K)Mn 2+治疗后能增强小鼠皮下黑色素瘤内CD8 +T细胞的肿瘤杀伤活性
在野生型C57BL/6小鼠腹股沟皮下接种5×10 5个B16F10细胞，在接种24小时后，用 5mg/kg的MnCl 2生理盐水溶液进行鼻腔滴注，设置对照组用等体积生理盐水滴注，在肿瘤接种后第14天处死小鼠，解剖取得其完整肿瘤组织，制备单细胞悬液，进行细胞表面染色，然后利用流式细胞术分选出CD8 +T细胞，然后送由华大基因做微量RNA提取和建库，做高通量测序，基因表达倍数计算方式为log 2((Mn FPKM)/(Con FPKM))，滤除比值绝对值小于1和FDR(false discovery rate)大于0.001的数据，然后将对照组的基因表达量标准化到0后显示实验组基因表达情况，绘制热图(Heatmap)。
实施例8.Mn 2+能够促进鼠源和人源树突状细胞的活化
实验(A,B)Mn 2+刺激小鼠骨髓来源的树突状细胞BMDC和小鼠骨髓来源的巨噬细胞 BMDM产生I型干扰素
将野生型C57BL/6小鼠的骨髓来源树突状细胞BMDC分别用SeV,VACV,100ng/mL LPS,200μM和400μM的MnCl 2处理18小时，然后收取细胞培养上清，利用Bioassay法检测I型干扰素活性(A)。将野生型C57BL/6小鼠的骨髓来源巨噬细胞BMDM分别用SeV,VACV,200μM和400μM的MnCl 2处理18小时，然后收取细胞培养上清，利用Bioassay法检测I型干扰素活性(B)。
实验(C,D)Mn 2+在体外促进BMDC的活化
将野生型C57BL/6小鼠的骨髓来源树突状细胞BMDC用10ng/mL LPS,200μM和400μM的MnCl 2处理18小时，每组设置3个平行孔，收取细胞后利用流式染色和流式细胞术检测分析CD80的平均荧光强度(mean fluorescence intensity,MFI)。FMO,Flow minus one，单独不染CD80，其余抗体染色与其他组一致。每组小鼠n＝3(C)。将野生型C57BL/6小鼠的骨髓来源树突状细胞BMDC用200μM的MnCl 2处理18小时，每组设置3个平行孔，收取细胞后利用流式染色和流式细胞术检测分析CD86的MFI。每组小鼠n＝3(D)。
实验(E,F)Mn 2+在促进小鼠肺脏中DC的活化
向野生型C57BL/6小鼠鼻腔滴注5mg/kg的MnCl 2生理盐水溶液，对照组鼻腔滴注等体积的生理盐水，18小时后，处死小鼠并取得其完整肺脏组织，制备单细胞悬液，然后利用流式抗体染色和流式细胞术圈出DC并分析其CD80的MFI(E)和CD86的MFI(F)。FMO,Flow minus one，即其余抗体与其他组一致，不染CD80或者CD86。对照组n＝5，实验组n＝5。
实验(G,H)Mn 2+在促进小鼠腹股沟淋巴结中DC的活化
向野生型C57BL/6小鼠的双侧腹股沟皮下总共注射5mg/kg的MnCl 2胶体锰制剂，对照组腹股沟皮下注射等体积的溶剂，18小时后，处死小鼠并取得其完整的腹股沟淋巴结，制备 单细胞悬液，然后利用流式抗体染色和流式细胞术圈出DC并分析其CD80的MFI(G)和CD86的MFI(H)。对照组n＝5，实验组n＝5。
实验(I,J)Mn 2+在体外促进人源DC的活化
取肿瘤患者的外周血分离得到PBMC，用100μM MnCl 2处理18小时，每组设置3个平行孔，然后利用流式抗体染色和流式细胞术圈出DC并分析其CD86的MFI，做统计学分析。
实施例9.Mn 2+在体外促进CD8 +T细胞对于肿瘤细胞的杀伤
实验(A,B)Mn 2+在体外促进CD8 +T细胞对于肿瘤细胞的杀伤
取6-8周龄OT-I小鼠的脾脏，制备单细胞悬液，利用磁珠阴选的方式分选得到CD8 +T细胞，然后与BMDC和预先铺板的B16F10-OVA-GFP细胞共孵育(CD8 +T细胞:BMDC:肿瘤细胞＝2:1:2)，按如图所示分组，并加入所示浓度的MnCl 2，每组设置3个平行孔，24小时后收取肿瘤细胞，利用流式细胞术分析GFP的强度(A)，并做统计学分析(B)。
实施例10.Mn 2+可帮助机体产生更多抗原特异性的CD8 +T细胞
实验(A,B)胶体锰Mn 2OHPO 4作为免疫增强剂增强机体的抗肿瘤反应
取6-8周龄野生型C57BL/6小鼠，在第0,7,14天分别肌肉注射100μg OVA,100μg OVA+20μg胶体锰制剂，并设置对照组，于首次免疫后第21天在小鼠腹股沟皮下接种3×10 5个B16-F0-OVA细胞，等肿瘤长到可触及时开始测量并记录大小，绘制肿瘤生长曲线(A)。同时每天观察小鼠，记录小鼠的死亡曲线(B)。每组小鼠n＝8。
实验(C,D)胶体锰Mn 2OHPO 4可以促进抗原特异性识别CD8 +T细胞的产生
取6-8周龄野生型C57BL/6小鼠，在第0,7,14天分别肌肉注射100μg OVA,100μg OVA+20μg胶体锰制剂，并设置对照组。于首次免疫后第21天处死小鼠，并解剖取得其脾脏组织，制备单细胞悬液，利用流式抗体染色和流式细胞术检测SIINFEKL +CD8 +T细胞的比例(C)，并做统计分析(D)。每组小鼠n＝3。
实验(E,F)胶体锰Mn 2OHPO 4可以促进OT-I CD8 +T细胞对于含有OVA抗原细胞的 特异性杀伤
OT-I转基因小鼠的CD8 +T细胞携带有Vα2/Vβ5的转基因TCR用以特异性识别由MHC-I类分子递呈的OVA蛋白的第257-264位氨基酸即SIINFEKL。我们取6-8周龄野生型C57BL/6小鼠，在第0,7,14天分别肌肉注射100μg OVA,100μg OVA+20μg胶体锰制剂，并设置对照组，定义为受体小鼠。在第21天时取新一批相同周龄的野生型C57BL/6小鼠的 脾脏，制备单细胞悬液并等分，分别用0.5μM CFSE和5μM CFSE染色，再用OVA 257-264(10μg/mL)刺激。然后将两部分染色的脾脏细胞1:1混合后通过尾静脉回输给免疫过的小鼠，24小时后，将受体小鼠的脾脏取出，制备单细胞悬液，然后通过流式细胞术检测CFSE标记的细胞(E)，并计算特异性杀伤比例(F)。每组小鼠n＝5。
实验(G,H)胶体锰Mn 2OHPO 4可以促进OT-I CD8 +T细胞的增殖
取6-8周龄CD45.1 +OT-I小鼠的脾脏，制备成单细胞悬液后用磁珠分选出CD8 +T细胞，用CFSE染色标记，将2×10 6个CFSE标记的CD8 +T细胞通过尾静脉注射回输给新的一批6-8周龄的CD45.2 +野生型C57BL/6小鼠，1天后分别在腹股沟皮下注射100μg OVA,100μg OVA+20μg胶体锰制剂和100μg OVA+50μg胶体锰制剂，并设置对照组。然后于尾静脉注射CD8 +T细胞后的第3天处死小鼠，解剖取其腹股沟淋巴结，制备单细胞悬液，利用流式抗体染色和流式细胞术检测低表达CFSE(CFSElow)细胞的比例(G)，并做统计分析(H)。每组小鼠n＝4。
实施例11.Mn 2+作为cGAS-STING激动剂发挥抗肿瘤活性
实验(A)静脉给Mn 2+能激活小鼠全身性I型干扰素反应并且是cGAS-STING通路依赖 的。
取6-8周龄的野生型C57BL/6、CGas -/-和Tmem173 -/-小鼠，将5mg/kg剂量的MnCl 2的PBS溶液注射给小鼠，设置对照组。18小时后，采集小鼠血液，分离血清，利用Bioassay法检测其中的I型干扰素活性。随后处死小鼠，解剖取得其心脏、胸腺和脾脏，研磨后利用免疫印迹法(Western Blot)检测其中的I型干扰素诱导基因Viperin的表达，内参蛋白GAPDH用于指示蛋白上样量。在CGas -/-和Tmem173 -/-小鼠中，Mn 2+不能激活其I型干扰素产生。
实验(B,C)Tmem173 -/-BMDC和Tmem173 -/-BMDM中Mn 2+处理不能激活I型干扰素 产生
将Tmem173 -/-小鼠的骨髓来源树突状细胞BMDC分别用SeV,VACV,100ng/mL LPS,200μM和400μM的MnCl 2处理18小时，然后收取细胞培养上清，利用Bioassay法检测I型干扰素活性(B)。将Tmem173 -/-小鼠的骨髓来源巨噬细胞BMDM分别用SeV,VACV,200μM和400μM的MnCl 2处理18小时，然后收取细胞培养上清，利用Bioassay法检测I型干扰素活性(C)。
实验(D-F)在Tmem173 -/-小鼠背景下，Mn 2+的肿瘤治疗效果就大幅减弱
在Tmem173 -/-小鼠腹股沟皮下接种5×10 5个B16F10细胞，在接种24小时后，用5mg/kg剂量的MnCl 2生理盐水溶液进行鼻腔滴注，设置对照组用等体积生理盐水滴注，在肿瘤接种后第14天处死小鼠，解剖取得其完整肿瘤组织，拍照，并记录重量(D)。将实验(D)中小鼠解剖取得的肿瘤组织制备成单细胞悬液并计数，取适量细胞用流式抗体染色，然后利用流式细胞术检测CD4和CD8阳性细胞，并通过计算得到每克肿瘤中CD4阳性细胞(E)和CD8阳性的细胞(F)的数量，并做统计学分析。对照组n＝4，实验组n＝4。
实验(G，H)在Tmem173 -/-小鼠背景下，胶体锰Mn 2OHPO 4的肿瘤免疫预防效果就大 幅减弱
取6-8周龄Tmem173 -/-小鼠，在第0,7,14天分别肌肉注射100μg OVA,100μg OVA+20μg胶体锰制剂，并设置对照组，于首次免疫后第21天在小鼠腹股沟皮下接种3×10 5个B16F0-OVA细胞，等肿瘤长到可触及时开始测量并记录大小，绘制肿瘤生长曲线(G)。同时每天观察小鼠，记录小鼠的死亡曲线(H)。每组小鼠n＝8。
实验(I,J)在Tmem173 -/-小鼠背景下，胶体锰Mn 2OHPO 4不能有效促进OT-I CD8 +T 细胞对于含有OVA抗原细胞的特异性杀伤
取6-8周龄Tmem173 -/-小鼠，在第0,7,14天分别肌肉注射100μg OVA,100μg OVA+20μg胶体锰制剂，并设置对照组，定义为受体小鼠。在第21天时取新的野生型C57BL/6小鼠的脾脏，制备单细胞悬液并等分，分别用0.5μM CFSE和5μM CFSE在37℃染色10分钟，之后再用OVA 257-264(10μg/mL)在37℃刺激90分钟。然后将两部分染色的脾脏细胞1:1混合后通过尾静脉回输给免疫过的受体小鼠，24小时后，将受体小鼠的脾脏取出，制备成单细胞悬液，然后通过流式细胞术检测CFSE标记的细胞(I)，并计算特异性杀伤比例(J)。每组小鼠n＝6。
实施例12.Mn 2+协同增强免疫检查点抑制剂anti-PD-1对于肿瘤的治疗效果并减少anti-PD-1的用量
实验(A-C)Mn 2+和anti-PD-1联合使用能显著抑制小鼠皮下黑色素瘤生长
在野生型C57BL/6小鼠腹股沟皮下接种5×10 5个B16F10细胞，并分为4组，Isotype组，Mn 2+治疗组，anti-PD-1治疗组，联合治疗(combo)组。在肿瘤接种24小时后，Mn 2+组和Combo组用5mg/kg的MnCl 2生理盐水溶液进行鼻腔滴注，每2天滴注一次。Isotype组在相同时间点用等体积生理盐水滴注。anti-PD-1治疗组和combo组在肿瘤接种后第3,7,11天用200μg anti-PD-1对小鼠进行腹腔注射，同时，Isotype组在肿瘤接种后第3,7,11天用200μg  IgG2a isotype抗体腹腔注射小鼠。待肿瘤生长至可触及时开始测量记录并绘制肿瘤生长曲线(A)。在肿瘤接种后第15天处死小鼠，解剖取得其完整肿瘤组织，拍照，并记录重量做统计学分析(B,C)。每组小鼠n＝6。
实验(D,E)Mn 2+和anti-PD-1联合使用能显著增强小鼠皮下黑色素瘤中CD8 +T细胞的 浸润
将实验(A)中小鼠解剖取得的肿瘤组织制备成单细胞悬液并计数，取适量细胞用流式抗体染色，然后利用流式细胞术检测CD8 +T细胞群(D)，并通过计算得到每克肿瘤中CD8 +T细胞的数量，并做统计学分析(E)。
实验(F)Mn 2+和anti-PD-1联合使用能显著抑制小鼠肺部黑色素瘤的转移
通过尾静脉向野生型C57BL/6小鼠注射2×10 5个B16F10细胞，并分为4组，Isotype组，Mn 2+治疗组，anti-PD-1治疗组，联合治疗(combo)组。Mn 2+和anti-PD-1的治疗与实验(A-C)中描述的一致。在肿瘤接种2周后用CO 2处死小鼠，解剖取得小鼠完整的肺脏组织，拍照并称重，做统计学分析。
实验(G)对于小鼠皮下黑色素瘤的治疗，Mn 2+和anti-PD-1联合使用能有效减少 anti-PD-1的用量
在野生型小鼠腹股沟皮下接种5×10 5个B16F10细胞，分为Isotype组，anti-PD-1治疗组，Mn 2+治疗组，1/2剂量anti-PD-1治疗组，1/2剂量anti-PD-1联合Mn 2+治疗组。在肿瘤接种24小时后，Mn 2+治疗组和1/2anti-PD-1+Mn 2+组用5mg/kg MnCl 2生理盐水溶液进行鼻腔滴注，每2天一次。anti-PD-1治疗组在肿瘤接种后第3,7,11天用200μg剂量的anti-PD-1对小鼠进行腹腔注射，1/2anti-PD-1治疗组剂量减半。待肿瘤生长至可触及时开始测量记录并绘制肿瘤生长曲线。并在第13天处死小鼠，解剖取得其完整肿瘤组织，并记录重量做统计学分析。每组小鼠n＝5。
实验(H-J)Mn 2+和anti-PD-1联合使用能显著抑制小鼠皮下MC38肿瘤的生长
在野生型C57BL/6小鼠腹股沟皮下接种1×10 6个MC38细胞，并分为4组，Isotype组，Mn 2+治疗组，anti-PD-1治疗组，联合治疗(combo)组。在肿瘤接种24小时后，Mn 2+组和combo组小鼠用5mg/kg的MnCl 2PBS溶液进行尾静脉注射，每2天一次。Isotype组在相同时间点用等体积PBS尾静脉注射。在肿瘤接种后第3,7,11天用200μg anti-PD-1对anti-PD-1治疗组和combo组小鼠进行腹腔注射，同时，Isotype组在肿瘤接种后第3,7,11天用200μg IgG2a isotype抗体腹腔注射小鼠。待肿瘤大小生长至可触及时开始测量记录并绘制肿瘤生长曲线(H)。在肿瘤接种后第15天处死小鼠，解剖取得其完整肿瘤组织，拍照(I)，并记录重 量做统计学分析(J)。每组小鼠n＝6。
实验(K)实验(H-J)Mn 2+和anti-PD-1联合使用能提高小鼠皮下MC38肿瘤内CD8 阳性细胞的浸润程度
取实验(G)中的肿瘤做组织切片，利用特异性抗体标记其中CD8阳性细胞，通过荧光显微镜成像。
实施例13.Mn 2+协同增强化疗药物CTX对于肿瘤的治疗效并减少CTX的用量
实验(A-C)Mn 2+协同增强化疗药物CTX对于肿瘤的治疗效并减少CTX的用量
在野生型小鼠腹股沟皮下接种5×10 5个B16F10-OVA细胞，分为Con组，CTX治疗组，Mn 2+治疗组，1/2剂量CTX治疗组，1/2剂量CTX联合Mn 2+治疗组。在肿瘤接种24小时后，Mn 2+治疗组和1/2CTX+Mn 2+组用5mg/kg MnCl 2生理盐水溶液进行鼻腔滴注，每2天一次。Con组在相同时间点用等体积生理盐水滴注。CTX治疗组在肿瘤接种后第5,9,13天用100mg/kg剂量的CTX对小鼠进行腹腔注射，1/2CTX治疗组剂量减半。同时，Con组在肿瘤接种后第5,9,13天用等体积的溶剂腹腔注射小鼠。待肿瘤生长至可触及时开始测量记录并绘制肿瘤生长曲线(A)。在肿瘤接种后第15天处死小鼠，解剖取得其完整肿瘤组织，拍照，并记录重量做统计学分析(B,C)。每组小鼠n＝6。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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Claims (18)

  1. 二价锰在制备树突状细胞、巨噬细胞或T细胞激活剂或致敏剂中的应用。
  2. 根据权利要求1所述的应用，所述二价锰刺激树突状细胞或巨噬细胞产生I型干扰素，或者刺激树突状细胞上调表达CD80和/或CD86。
  3. 根据权利要求1所述的应用，所述二价锰促进CD8阳性T细胞增殖，优选地，促进抗原特异性识别CD8阳性T细胞的增殖，或者促进肿瘤中CD8阳性T细胞的浸润。
  4. 根据权利要求1-3任一项所述的应用，所述二价锰是游离的锰离子、二价锰盐或者可以转变为二价锰形式的其他二价锰源，优选地，所述二价锰盐选自氯化锰、溴化锰、碘化锰、硫酸锰、硝酸锰、高氯酸锰、醋酸锰、碳酸锰、硼酸锰、磷酸锰、氢溴酸锰、酒石酸锰、富马酸锰、马来酸锰、乳酸锰、苯磺酸锰、泛酸锰、抗坏血酸锰、磷酸锰盐、碳酸锰盐和氢氧化锰中的任一种及其任意组合，更优选地，所述二价锰是MnCl 2或者Mn 2OHPO 4胶体。
  5. 一种树突状细胞、巨噬细胞或T细胞激活剂或致敏剂，其包括二价锰，所述二价锰是游离的锰离子、二价锰盐或者可以转变为二价锰形式的其他二价锰源，优选地，所述二价锰盐选自氯化锰、溴化锰、碘化锰、硫酸锰、硝酸锰、高氯酸锰、醋酸锰、碳酸锰、硼酸锰、磷酸锰、氢溴酸锰、酒石酸锰、富马酸锰、马来酸锰、乳酸锰、苯磺酸锰、泛酸锰、抗坏血酸锰、磷酸锰盐、碳酸锰盐和氢氧化锰中的任一种及其任意组合，更优选地，所述二价锰是MnCl 2或者Mn 2OHPO 4胶体。
  6. 根据权利要求5所述的树突状细胞、巨噬细胞或T细胞激活剂或致敏剂在细胞免疫治疗药物中的应用。
  7. 二价锰在制备治疗或预防肿瘤的药物中的应用，所述二价锰是游离的锰离子、二价锰盐或者可以转变为二价锰形式的其他二价锰源，优选地，所述二价锰盐选自氯化锰、溴化锰、碘化锰、硫酸锰、硝酸锰、高氯酸锰、醋酸锰、碳酸锰、硼酸锰、磷酸锰、氢溴酸锰、酒石酸锰、富马酸锰、马来酸锰、乳酸锰、苯磺酸锰、泛酸锰、抗坏血酸锰、磷酸锰盐、碳酸锰盐和氢氧化锰中的任一种及其任意组合，更优选地，所述二价锰是MnCl 2或者Mn 2OHPO 4胶体。
  8. 根据权利要求7所述的应用，所述肿瘤包括恶性黑色素瘤、肺癌、结肠癌、淋巴癌、卵巢癌、胃癌、乳腺癌、胰腺癌、胆管癌、肾癌、肝癌、肉瘤、膀胱癌、子宫颈癌、白血病、头颈癌或者其他皮下实体瘤等中的至少一种。
  9. 根据权利要求7或8所述的应用，所述肿瘤是对免疫检查点抑制剂和/或化疗药物治疗不 敏感的肿瘤，优选地，所述肿瘤是对PD-1抗体、PD-L1抗体，环磷酰胺治疗不敏感的肿瘤。
  10. 根据权利要求7或8所述的二价锰在制备治疗或预防肿瘤的药物中的应用，其特征在于，所述二价锰用于制备抗肿瘤剂的增敏剂，所述抗肿瘤剂为免疫检查点抑制剂和/或化疗药物，优选地，所述抗肿瘤剂为PD-1抗体、PD-L1抗体、环磷酰胺中的至少一种。
  11. 一种抗肿瘤药物组合物，包括
    1)抗肿瘤剂，
    2)二价锰。
    优选地，所述抗肿瘤药物组合物包括：3)药学上可接受的载体；
    优选地，所述抗肿瘤剂为免疫检查点抑制剂和/或化疗药物，更优选地，所述抗肿瘤剂为PD-1抗体、PD-L1抗体、环磷酰胺中的至少一种；
    优选地，所述二价锰是游离的锰离子、二价锰盐或者可以转变为二价锰形式的其他二价锰源，优选地，所述二价锰盐选自氯化锰、溴化锰、碘化锰、硫酸锰、硝酸锰、高氯酸锰、醋酸锰、碳酸锰、硼酸锰、磷酸锰、氢溴酸锰、酒石酸锰、富马酸锰、马来酸锰、乳酸锰、苯磺酸锰、泛酸锰、抗坏血酸锰、磷酸锰盐、碳酸锰盐和氢氧化锰中的任一种及其任意组合，更优选地，所述二价锰是MnCl 2或者Mn 2OHPO 4胶体。
  12. 根据权利要求11所述的抗肿瘤药物组合物，所述抗肿瘤药物组合物为静脉注射制剂、鼻滴制剂(包括雾化剂和黏膜用剂型)、口服制剂、皮内注射制剂、皮下注射制剂或者肌内注射制剂。
  13. 一种疫苗组合物，包括
    1)二价锰，以及
    2)一种或多种抗原，
    优选地，所述二价锰是游离的锰离子、二价锰盐或者可以转变为二价锰形式的其他二价锰源，优选地，所述二价锰盐选自氯化锰、溴化锰、碘化锰、硫酸锰、硝酸锰、高氯酸锰、醋酸锰、碳酸锰、硼酸锰、磷酸锰、氢溴酸锰、酒石酸锰、富马酸锰、马来酸锰、乳酸锰、苯磺酸锰、泛酸锰、抗坏血酸锰、磷酸锰盐、碳酸锰盐和氢氧化锰中的任一种及其任意组合，更优选地，所述二价锰是MnCl 2或者Mn 2OHPO 4胶体；
    优选地，所述抗原为肿瘤抗原或者病毒抗原。
  14. 根据权利要求13所述的疫苗组合物用于制备抗肿瘤或者抗感染药物中的应用。
  15. 一种锰离子胶体，其特征在于，由磷酸盐、锰盐和水溶液组成；优选地，所述磷酸盐选自Na 3PO 4、K 3PO 4或其他磷酸盐，所述锰盐为MnCl 2，所述水溶液为生理盐水；同样优选 地，所述水溶液还包含助溶剂、稳定剂和/或缓冲剂中的一种或多种。
  16. 根据权利要求15所述的锰离子胶体，其特征在于，所述锰离子胶体中磷酸根离子和二价锰离子的浓度分别不低于25mM和20mM，磷酸根离子与二价锰离子的摩尔浓度比为1.25：1。
  17. 根据权利要求15或16所述的锰离子胶体在制备抗肿瘤药物或者制备疫苗组合物中的应用。
  18. 根据权利要求15或16所述的锰离子胶体在制备树突状细胞、巨噬细胞或T细胞激活剂或致敏剂中的应用。
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CN114028559A (zh) * 2021-12-28 2022-02-11 广东粤港澳大湾区国家纳米科技创新研究院 一种铝锰复合纳米晶及其制备方法和应用
CN115715805A (zh) * 2022-11-14 2023-02-28 山东大学 一种基于钒的仿生纳米材料及其制备方法与应用
CN115715805B (zh) * 2022-11-14 2024-01-19 山东大学 一种基于钒的仿生纳米材料及其制备方法与应用
CN117105271A (zh) * 2023-08-29 2023-11-24 首都医科大学附属北京儿童医院 碳酸锰纳米sting激动剂及其制备方法与应用
CN117105271B (zh) * 2023-08-29 2024-05-31 首都医科大学附属北京儿童医院 碳酸锰纳米sting激动剂及其制备方法与应用

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