WO2021233237A1 - 一种肿瘤疫苗及其制备方法和应用 - Google Patents

一种肿瘤疫苗及其制备方法和应用 Download PDF

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WO2021233237A1
WO2021233237A1 PCT/CN2021/094037 CN2021094037W WO2021233237A1 WO 2021233237 A1 WO2021233237 A1 WO 2021233237A1 CN 2021094037 W CN2021094037 W CN 2021094037W WO 2021233237 A1 WO2021233237 A1 WO 2021233237A1
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membrane
protein
tumor
cells
membrane protein
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PCT/CN2021/094037
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English (en)
French (fr)
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聂广军
赵瑞芳
陈龙
覃好
赵宇亮
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国家纳米科学中心
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Priority to CN202180037006.4A priority Critical patent/CN115697390A/zh
Priority to EP21809500.8A priority patent/EP4154905A4/en
Priority to JP2022570294A priority patent/JP2023525598A/ja
Priority to US17/999,335 priority patent/US20230181705A1/en
Publication of WO2021233237A1 publication Critical patent/WO2021233237A1/zh

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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Definitions

  • This application relates to the field of biomedicine, in particular to a tumor vaccine and its preparation method and application.
  • cellular immune suppression can last for several days, and this period of time may become a window period for tumors to invade the immune system. Immunosuppression during this period often manifests as the lack of supervision of tumor cells, the decrease of NK cells, cytotoxic T lymphocytes, dendritic cells and helper T lymphocytes in peripheral blood.
  • CN104619833A describes a peptide vaccine against cancer, specifically an epitope peptide derived from UBE2T that triggers CTL, and further provides an epitope peptide derived from UBE2T or a polynucleotide encoding the polypeptide as an active ingredient Pharmaceutical composition.
  • the invention provides methods for treatment and/or prevention, and/or prevention of postoperative recurrence, and methods for inducing CTL, and methods for inducing anti-tumor immunity, which use the invention derived from UBE2T
  • CN109481418A discloses an anti-tumor nano particle and a preparation method and application thereof.
  • the provided anti-tumor nano particle includes a core, a wrapper covering the core, and an immune layer covering the wrapper.
  • the anti-tumor nanoparticles obtained by encapsulating the nanoparticles with immune adjuvants can achieve the combined therapeutic effect of photodynamic therapy and immunotherapy at the same time.
  • the current types of vaccines for preventing postoperative tumor recurrence are limited, and there is a need for vaccines with better effects, higher safety, convenient preparation and a wide range of applications in this field. Therefore, it is of great significance to develop a new type of tumor vaccine with significant effect to prevent cancer recurrence after surgery and its preparation method.
  • the present application provides a pharmaceutical combination comprising a first membrane component, the first membrane component comprising a membrane derived from the inner membrane of a bacteria, and the pharmaceutical combination further comprising other membranes derived from the bacteria.
  • a pharmaceutical combination comprising a first membrane component, the first membrane component comprising a membrane derived from the inner membrane of a bacteria, and the pharmaceutical combination further comprising other membranes derived from the bacteria.
  • the other organisms comprise cells.
  • the other organisms comprise mammalian cells.
  • the other organisms comprise tumor cells.
  • the other organisms comprise solid tumor cells.
  • the other organism is selected from the group consisting of breast cancer cells, colon cancer cells, liver cancer cells, gastric cancer cells, kidney cancer cells, pancreatic cancer cells, ovarian cancer cells, lymphoma cells, osteosarcoma cells , Glioma cells, prostate cancer cells and melanoma cells, and any combination of the above.
  • the component of the other organism comprises an immunogenic component.
  • the component of the other organism is capable of triggering an immune response to the organism.
  • the component of the other organism comprises a component derived from the cell membrane of the organism.
  • the component of the other organism comprises a tumor antigen or a functionally active fragment thereof.
  • the components of the other organisms further comprise a protein or functionally active fragments thereof selected from the following group: antigen peptide transporter 1, H-2 class II histocompatibility antigen ⁇ chain, phenol Protein kinase SYK, high-affinity immunoglobulin epsilon receptor subunit ⁇ , Ras-related C3 botulinum toxin substrate 2, tyrosine protein kinase BTK, receptor type tyrosine protein phosphatase C, Na + /K + -ATPase, ATP5A (IM CVa), ubiquinone cytochrome C reductase core protein I (IM Core I), VDAC1/porin (OM Porin), matrix cyclophilin D (Matrix CypD), interstitial cytochrome C (IMS Cytc), basement membrane specific heparan sulfate proteoglycan core protein (HSPG2), plasma membrane calcium transport ATPase 1 (ATP2b1), endo
  • the components of the other organisms further comprise a protein or functionally active fragments thereof selected from the following group: antigen peptide transporter 1, H-2 class II histocompatibility antigen ⁇ chain, phenol Protein kinase SYK, high-affinity immunoglobulin epsilon receptor subunit ⁇ , Ras-related C3 botulinum toxin substrate 2, tyrosine protein kinase BTK, receptor type tyrosine protein phosphatase C, Na + /K + -ATPase, ATP5A (IM CVa), ubiquinone cytochrome C reductase core protein I (IM Core I), VDAC1/porin (OM Porin), matrix cyclophilin D (Matrix CypD) and interstitial cytochrome C (IMS Cytc), and any combination of the above.
  • a protein or functionally active fragments thereof selected from the following group: antigen peptide transporter 1, H-2 class II histocompatibility antigen
  • it includes a second membrane component that includes a membrane derived from the cell membrane of the other organism.
  • the bacteria comprise Gram-negative bacteria and/or Gram-positive bacteria.
  • the bacteria are selected from the genera of the following group: Escherichia coli, Staphylococcus, Bacillus, Lactobacillus, Klebsiella, Brucella, Proteus, Acinetobacter And Pseudomonas, and any combination of the above.
  • the inner membrane of the bacterium comprises a protein selected from the following group or functionally active fragments thereof: cell division protein FtsZ, inner membrane protein YhcB, inner membrane protein translocation enzyme YidC, cell division protein NlpI , ABC transporter MsbA, inner membrane transporter TatA, intermembrane phospholipid transport system lipoprotein MlaA, inner membrane protein TolQ, intermembrane transport lipoprotein PqiC, outer membrane protein TolC, outer membrane introduced protein FimD, outer membrane porin OmpC, Intermembrane transport protein PqiB, major outer membrane lipoprotein Lpp, membrane-bound hemolytic wall protein transglycosylase MltB, UPF0194 membrane protein YbhG, putative membrane protein IgaA homolog, transmembrane phospholipid transport system lipoprotein MlaA, internal Membrane protein YejM, membrane-bound hemolytic wall protein transglycosylase MltA, transmembrane phospholipid transport system
  • the inner membrane of the bacterium comprises a protein or functionally active fragments thereof selected from the group consisting of FtsZ, YhcB, YidC, NlpI, MsbA, TatA, MlaA, TolQ and YebE, and the above random combination.
  • the drug combination further comprises an inner core.
  • the inner core comprises a biocompatible material.
  • the inner core contains artificial synthetic materials.
  • the inner core comprises a material selected from the group consisting of polylactic acid-glycolic acid copolymer (PLGA), metal-organic framework material (MOF), polycaprolactone (PCL), polyamide-amine (PAMAM), carbon nanotubes, graphene, gold nanoparticles, mesoporous silica nanoparticles, iron oxide nanoparticles, silver nanoparticles, tungsten nanoparticles, manganese nanoparticles, platinum nanoparticles, quantum dots, alumina nanoparticles , Hydroxyapatite nanoparticles, lipid nanoparticles (LNP), DNA nanostructures, nanohydrogels, rare earth fluoride nanocrystals and NaYF 4 nanoparticles, and any combination of the above.
  • PLGA polylactic acid-glycolic acid copolymer
  • MOF metal-organic framework material
  • PCL polycaprolactone
  • PAMAM polyamide-amine
  • carbon nanotubes graphene
  • the inner core contains substances selected from the group consisting of immune adjuvants, immune checkpoint inhibitors, nucleic acid molecules and chemotherapeutic agents, and any combination of the above.
  • the inner core comprises monophosphoryl lipid A, an immune adjuvant.
  • the inner core comprises doxorubicin, paclitaxel, docetaxel, gemcitabine, capecitabine, cyclophosphamide, fluorouracil, pemetrexed, raltitrexate, bleomycin, Daunorubicin, doxorubicin, vincristine and etoposide, and any combination of the above.
  • the inner core comprises an indoleamine 2,3-dioxygenase (IDO) inhibitor.
  • IDO indoleamine 2,3-dioxygenase
  • the inner core contains small interfering RNA (siRNA).
  • siRNA small interfering RNA
  • the diameter of the inner core is about 60 to about 100 nanometers.
  • the diameter of the inner core is about 86 nanometers.
  • the pharmaceutical combination includes a housing that includes the first membrane component and the second membrane component.
  • the outer shell includes a film in which the first film component and the second film component are fused.
  • the thickness of the shell is about 10 to about 20 nanometers.
  • the thickness of the shell is about 14 nanometers.
  • the diameter of the housing is about 100 nanometers.
  • the surface potential (Zeta potential) of the housing is about +50 mV to about -50 mV.
  • the surface potential (Zeta potential) of the housing is about -21 mV.
  • the mass ratio of the first membrane component to the second membrane component in the drug combination is 1:100 to 100:1.
  • the mass ratio of the first membrane component to the second membrane component in the drug combination is 1:100, 1:75, 1:50, 1:25, 1: 10, 1:5, 1:3, 1:1, 1:0, 0:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1 or 100:1.
  • the presence and/or ratio of the first membrane component and the second membrane component in the drug combination is confirmed by Western blotting and/or immunogold staining.
  • the pharmaceutical combination comprises particles, the particles comprising the inner core and the outer shell.
  • the mass ratio of the shell to the core material is about 1:1 to about 1:10.
  • the mass ratio of the shell to the core material is about 1:4 to about 1:6.
  • the lipopolysaccharide (LPS) content in the particles is not significantly different from the lipopolysaccharide content of mammalian cells.
  • the diameter of the particles is about 70 to about 120 nanometers.
  • the diameter of the particles is about 100 nanometers.
  • the diameter is measured by transmission electron microscopy (TEM).
  • TEM transmission electron microscopy
  • the hydrated particle size of the particles is about 150 nanometers to about 250 nanometers.
  • the hydrated particle size of the particles is about 180 nanometers.
  • the surface potential (Zeta potential) of the particles is about +50 mV to about -50 mV.
  • the surface potential (Zeta potential) of the particles is about -21mV.
  • the hydrated particle size and/or surface potential of the particles are measured by a dynamic light scattering (DLS) instrument.
  • DLS dynamic light scattering
  • the particles can maintain stability in solution.
  • the stability includes that the hydrated particle size and/or surface potential of the particles after storage for a period of time are not significantly different from those before the period of time.
  • the stability includes that the hydrated particle size and/or surface potential of the particles after being stored in phosphate buffered saline (PBS) at 4 degrees Celsius for a period of time is compared with that of the particles before the period of time. Significant differences.
  • PBS phosphate buffered saline
  • the period of time is not less than about 21 days.
  • it further comprises a substance selected from the group of immune adjuvants, immune checkpoint inhibitors, nucleic acid molecules, chemotherapeutic agents and photosensitizers, and any combination of the above.
  • it also contains the immune adjuvant monophosphoryl lipid A (MPLA).
  • MPLA immune adjuvant monophosphoryl lipid A
  • it further comprises an indoleamine 2,3-dioxygenase (IDO) inhibitor.
  • IDO indoleamine 2,3-dioxygenase
  • it also contains small interfering RNA (siRNA).
  • siRNA small interfering RNA
  • the present application also provides a vaccine, including the drug combination of the present application.
  • this application also provides a kit comprising the drug combination of the application and/or the vaccine of the application.
  • the present application also provides a method for enhancing the uptake of a target antigen by immune cells, comprising providing a drug combination, the drug combination comprising a first membrane component, the first membrane component comprising bacteria derived
  • the inner membrane of the drug combination further comprises the target antigen.
  • the drug combination includes the drug combination of the present application.
  • the immune cells comprise immune presenting cells.
  • the immune cells are selected from the group consisting of dendritic cells (DC), T lymphocytes, macrophages, and natural killer cells (NK), and any combination of the above.
  • DC dendritic cells
  • T lymphocytes T lymphocytes
  • macrophages macrophages
  • NK natural killer cells
  • the immune cells comprise bone marrow-derived dendritic cells (BMDC).
  • BMDC bone marrow-derived dendritic cells
  • the immune cells comprise CD8 positive cells and/or CD4 positive cells.
  • the method of the application can be in vitro or ex vivo. In one embodiment, the method of the application can be for non-preventive and non-therapeutic purposes.
  • the application also provides a method for activating immune cells, the method comprising administering the drug combination of the application, the vaccine of the application and/or the kit of the application.
  • the immune cells comprise immune presenting cells.
  • the immune cells are selected from the group consisting of dendritic cells (DC), T lymphocytes, macrophages, and natural killer cells (NK), and any combination of the above.
  • DC dendritic cells
  • T lymphocytes T lymphocytes
  • macrophages macrophages
  • NK natural killer cells
  • the immune cells comprise bone marrow-derived dendritic cells (BMDC).
  • BMDC bone marrow-derived dendritic cells
  • the immune cells comprise CD8 positive cells and/or CD4 positive cells.
  • the immune cells comprise the immune cells in lymph nodes and/or spleen.
  • the effect of the activation of the immune cells administered with the drug combination comprises a group selected from the group consisting of increasing antigen recognition of the immune cells compared to immune cells not administered with the drug combination
  • the expression level of the receptor, the increased expression level of the nuclear factor kappa B (NF- ⁇ B) protein of the immune cell, the increased expression and/or secretion level of the cytokine of the immune cell, and the increased mature immune cell and any combination of the above.
  • the antigen recognition receptor comprises a pattern recognition receptor (PRR).
  • PRR pattern recognition receptor
  • the antigen recognition receptor comprises a Toll-like receptor (TLR).
  • TLR Toll-like receptor
  • the antigen recognition receptor comprises TLR1, TLR2 and/or TLR6.
  • the expression of TLR4 of the immune cells administered the drug combination is substantially unchanged compared to the immune cells not administered the drug combination.
  • the cytokine comprises a pro-inflammatory cytokine.
  • the cytokine comprises interleukin (IL)-6, tumor necrosis factor (TNF)- ⁇ , IL-1 ⁇ , and/or interferon (IFN)- ⁇ .
  • IL interleukin
  • TNF tumor necrosis factor
  • IFN interferon
  • the mature immune cells comprise CD80 positive cells, CD86 positive cells and/or effector memory cells.
  • the ratio of mature immune cells includes the ratio of CD80-positive and/or CD86-positive immune cells to CD11c-positive immune cells.
  • the ratio of mature immune cells includes the ratio of immune cells with high CD44 expression and low CD62L expression to CD8-positive immune cells.
  • the method of the application can be in vitro or ex vivo. In one embodiment, the method of the application can be for non-preventive and non-therapeutic purposes.
  • the application also provides the application of the drug combination of the application, the vaccine of the application and/or the kit of the application in the preparation of medicines, which are used to enhance innate immunity and/or specific immune response.
  • the administration of the drug combination promotes the maturation of dendritic cells and/or increases the secretion of cytokines by lymphocytes compared to the non-administration of the drug combination.
  • the application does not substantially induce a systemic inflammatory response.
  • the application does not substantially induce a systemic inflammatory response, which comprises, compared with not administering the drug combination, administering the drug combination does not substantially increase the IFN- ⁇ in the serum of the subject.
  • TNF- ⁇ macrophage chemoattractant protein-1 (MCP-1), IL-12p70, IL-10, IL-23, IL-27, IL-17A, IFN- ⁇ , granulocyte-macrophage colony stimulation
  • MCP-1 macrophage chemoattractant protein-1
  • IL-12p70 IL-12p70
  • IL-10 IL-10
  • IL-23 IL-27
  • IL-17A granulocyte-macrophage colony stimulation
  • IFN- ⁇ granulocyte-macrophage colony stimulation
  • GM-CSF concentration of factor
  • the application does not substantially induce hemolytic reaction, heart damage, liver damage, spleen damage, lung damage, and/or kidney damage.
  • the application also provides the drug combination of the application, the vaccine of the application and/or the kit of the application, which are used to enhance innate immunity and/or specific immune response.
  • the administration of the drug combination promotes the maturation of dendritic cells and/or increases the secretion of cytokines by lymphocytes compared to the non-administration of the drug combination.
  • the application does not substantially induce a systemic inflammatory response.
  • the application does not substantially induce a systemic inflammatory response, which comprises, compared with not administering the drug combination, administering the drug combination does not substantially increase the IFN- ⁇ in the serum of the subject.
  • TNF- ⁇ macrophage chemoattractant protein-1 (MCP-1), IL-12p70, IL-10, IL-23, IL-27, IL-17A, IFN- ⁇ , granulocyte-macrophage colony stimulation
  • MCP-1 macrophage chemoattractant protein-1
  • IL-12p70 IL-12p70
  • IL-10 IL-10
  • IL-23 IL-27
  • IL-17A granulocyte-macrophage colony stimulation
  • IFN- ⁇ granulocyte-macrophage colony stimulation
  • GM-CSF concentration of factor
  • the application does not substantially induce hemolytic reaction, heart damage, liver damage, spleen damage, lung damage, and/or kidney damage.
  • this application also provides a method for enhancing innate immunity and/or specific immune response, which comprises administering the drug combination of the application, the vaccine of the application and/or the kit of the application to subjects in need.
  • the administration of the drug combination promotes the maturation of dendritic cells and/or increases the secretion of cytokines by lymphocytes compared to the non-administration of the drug combination.
  • the method does not substantially induce a systemic inflammatory response.
  • the method does not substantially induce a systemic inflammatory response, comprising, compared with not administering the drug combination, administering the drug combination does not substantially increase IFN- ⁇ in the serum of the subject.
  • TNF- ⁇ macrophage chemoattractant protein-1 (MCP-1), IL-12p70, IL-10, IL-23, IL-27, IL-17A, IFN- ⁇ , granulocyte-macrophage colony stimulation
  • MCP-1 macrophage chemoattractant protein-1
  • IL-12p70 IL-12p70
  • IL-10 IL-10
  • IL-23 IL-27
  • IL-17A granulocyte-macrophage colony stimulation
  • IFN- ⁇ granulocyte-macrophage colony stimulation
  • GM-CSF concentration of factor
  • the method does not substantially induce hemolytic reaction, heart damage, liver damage, spleen damage, lung damage, and/or kidney damage.
  • the application also provides the application of the drug combination of the application, the vaccine of the application and/or the kit of the application in the preparation of medicines for the prevention and/or treatment of tumors.
  • the prevention and/or treatment of a tumor comprises slowing down the rate of increase in tumor volume and/or reducing the volume of the tumor.
  • the tumor comprises the tumor that is not completely resected after tumor resection.
  • suppressing a tumor includes a tumor that regenerates after the tumor is cleared.
  • the tumor comprises a solid tumor.
  • the tumor is selected from the following group: breast tumor, colon tumor, liver tumor, stomach tumor, kidney tumor, pancreatic tumor, ovarian tumor, lymphoma, osteosarcoma, glioma, prostate cancer and melanoma Tumor and any combination of the above.
  • the application also provides the drug combination of the application, the vaccine of the application and/or the kit of the application, which are used to prevent and/or treat tumors.
  • the prevention and/or treatment of a tumor comprises slowing down the rate of increase in tumor volume and/or reducing the volume of the tumor.
  • the tumor comprises the tumor that is not completely resected after tumor resection.
  • suppressing a tumor includes a tumor that regenerates after the tumor is cleared.
  • the tumor comprises a solid tumor.
  • the tumor is selected from the following group: breast tumor, colon tumor, liver tumor, stomach tumor, kidney tumor, pancreatic tumor, ovarian tumor, lymphoma, osteosarcoma, glioma, prostate cancer and melanoma Tumor and any combination of the above.
  • the present application also provides a method for preventing and/or treating tumors, which comprises administering the drug combination of the present application, the vaccine of the present application and/or the kit of the present application to a subject in need.
  • the prevention and/or treatment of a tumor comprises slowing down the rate of increase in tumor volume and/or reducing the volume of the tumor.
  • the tumor comprises the tumor that is not completely resected after tumor resection.
  • suppressing a tumor includes a tumor that regenerates after the tumor is cleared.
  • the tumor comprises a solid tumor.
  • the tumor is selected from the following group: breast tumor, colon tumor, liver tumor, stomach tumor, kidney tumor, pancreatic tumor, ovarian tumor, lymphoma, osteosarcoma, glioma, prostate cancer and melanoma Tumor and any combination of the above.
  • the present application also provides a method for preparing the drug combination of the present application, the vaccine of the present application, and/or the kit of the present application, which comprises providing the bacterial-derived inner membrane membrane.
  • it further comprises providing the component derived from an organism other than the bacteria.
  • it further comprises mixing the membrane derived from the inner membrane of the bacteria and the components derived from other organisms other than the bacteria to provide a shell.
  • it further includes providing a core.
  • the present invention provides a tumor vaccine for preventing postoperative recurrence of cancer.
  • the tumor vaccine includes a hybrid cell membrane shell and an inner core material, and the hybrid cell membrane shell includes a gram-negative bacterial inner membrane and an entity derived from surgical resection. Tumor cell membrane.
  • the tumor vaccine involved in the present invention is a kind of nano-particles in which the core material is wrapped in a hybrid membrane.
  • the inner membrane of gram-negative bacteria is used as an immune adjuvant, and the two can be jointly delivered to the same dendritic cell. It can take advantage of the uptake and presentation of tumor antigens, which can synergistically enhance the body’s innate immunity and specific immunity, and has A certain lymph node enrichment ability has a significant effect of preventing recurrence after tumor resection, prolonging the survival period of patients, and providing a long-term protection mechanism.
  • the core material wrapped in the tumor vaccine hybrid membrane of the present invention can help maintain the rigid structure and stability of the hybrid membrane, thereby maintaining the immune enhancement of the hybrid membrane, lymphatic enrichment, and the efficacy of preventing recurrence after tumor resection surgery; At the same time, it can further contain other biologically active ingredients such as siRNA or chemotherapeutics to achieve more functions.
  • the tumor vaccine involved in the present invention has good biological safety, easy preparation of raw materials, and low preparation cost; and has a wide application range.
  • Various chemical groups can be modified on the surface of the vaccine according to actual needs, and it has broad application prospects.
  • the ratio of the molar amount of protein in the bacterial inner membrane to the solid tumor cell membrane derived from surgical resection is 1:100-100:1, such as 1:100, 1:75, 1:50, 1: 25, 1:10, 1:5, 1:3, 1:1, 1:0, 0:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1 or 100:1, etc., for example, it can be (2-3):1, and the specific point values in the above numerical range can be selected, so I won't repeat them here.
  • the ratio of the molar amount of protein in the bacterial inner membrane to the solid tumor cell membrane derived from surgical resection is specifically selected to be a numerical range of 1:100-100:1, where (2-3):1 is the numerical range with the best effect.
  • the mass ratio of the hybrid cell membrane shell to the core material is 1:(1-10), such as 1:1, 1:2, 1:3, 1:4, 1:5, 1 :6, 1:7, 1:8, 1:9, or 1:10, etc., for example, it can be 1:(4-6), the specific point values in the above numerical range can be selected, so I won’t be one by one here. Go into details.
  • the mass ratio of the hybrid cell membrane shell to the core material is specifically selected as a value range of 1:(1-10), because within this range, the surface charge of the final product is closer to the surface charge of the hybrid cell membrane shell.
  • the gram-negative bacteria include Escherichia coli, Staphylococcus, Bacillus, Lactobacillus, Klebsiella, Brucella, Proteus, Acinetobacter, or Pseudomonas Any one or a combination of at least two of the genus Monas.
  • the gram-negative bacteria involved in the present invention include but are not limited to the above types, and the inner membrane of other types of gram-negative bacteria can obtain the technical effects involved in the present invention.
  • the cell wall of gram-negative bacteria is a multi-layer structure, from the outside to the inside: the outer membrane layer (OM) is the phospholipid layer and the lipopolysaccharide layer; the middle is the thin peptidoglycan layer (PGN); the inner membrane IM( The cytoplasmic membrane) is the lipoprotein layer and the phospholipid layer.
  • the inner membrane of gram-negative bacteria is basically free of endotoxin (lipopolysaccharide LPS) and has good biological safety.
  • the tumor includes any one of breast cancer, colon cancer, liver cancer, stomach cancer, kidney cancer, pancreatic cancer, ovarian cancer, lymphoma, osteosarcoma, glioma, prostate cancer, or melanoma Or a combination of at least two.
  • the tumors involved in the present invention include but are not limited to the above-mentioned types, and other types of tumors can be applied to the technical solutions involved in the present invention.
  • the core material includes any one or at least one of PLGA, MOF, PCL, PAMAM, carbon nanotubes, graphene, gold nanoparticles, mesoporous silica nanoparticles or iron oxide nanoparticles A combination of the two.
  • the polymers involved in the present invention include but are not limited to the above types, and other biocompatible polymers can achieve the technical effects involved in the present invention.
  • the shell structure of the hybrid cell membrane is a phospholipid bilayer, which is easy to wrap on the surface of various core materials, and the method is simple, and different combinations can be made according to actual needs.
  • the particle size of the tumor vaccine for preventing cancer recurrence after surgery is 100-300nm, such as 100nm, 150nm, 200nm, 250nm or 300nm, etc., and specific points within the above numerical range can be selected. This will not repeat them one by one.
  • the core material of the tumor vaccine also contains any one or a combination of at least two of immune checkpoint inhibitors, IDO inhibitors, siRNA or chemotherapeutics; Combinations such as the combination of immune checkpoint inhibitors and IDO inhibitors, siRNA and chemotherapeutic drugs, etc., any other combination methods can be selected, and will not be repeated here.
  • the present invention provides a method for preparing a tumor vaccine for preventing postoperative recurrence of cancer as described above, characterized in that the preparation method includes the following steps:
  • step (2) Mix the gram-negative bacterial inner membrane extracted in step (1) with the solid tumor cell membrane derived from surgical resection, and extrude it with a liposome extruder to obtain hybrid membrane nanoparticles;
  • step (3) The hybrid membrane particles obtained in step (2) are mixed with the nanoparticle core and extruded with a liposome extruder to obtain the tumor vaccine for preventing cancer recurrence after surgery.
  • the mixing temperature in step (2) is 20-45°C, such as 20°C, 25°C, 30°C, 35°C, 40°C or 45°C, etc.
  • the time is 10-20 minutes, such as 10 minutes. , 12min, 15min, 18min or 20min, etc., the specific point values within the above numerical range can be selected, so I won’t repeat them here.
  • the pore size of the filter membrane of the liposome extruder described in step (2) is 300-500nm, such as 300nm, 350nm, 400nm, 450nm, or 500nm, etc., any specific value within the above numerical range The choices will not be repeated here.
  • the extrusion in step (2) refers to a cumulative back and forth extrusion 10-15 times (one round and one round counts as 1 time).
  • the mixing temperature in step (3) is 20-45°C, such as 20°C, 25°C, 30°C, 35°C, 40°C or 45°C, etc.
  • the time is 10-20 minutes, such as 10 minutes. , 12min, 15min, 18min or 20min, etc., the specific point values within the above numerical range can be selected, so I won’t repeat them here.
  • the pore size of the filter membrane of the liposome extruder described in step (3) is 100-300nm, such as 100nm, 150nm, 200nm, 250nm, or 300nm, etc., any specific value within the above numerical range The choices will not be repeated here.
  • the extrusion in step (3) refers to a cumulative back and forth extrusion 10-15 times (one round and one round counts as 1 time).
  • the nanoparticle core in step (3) is prepared by a double-emulsion method. Specifically, it can be exemplarily:
  • the organic solvent includes any one or a combination of at least two of dichloromethane, chloroform, acetone, or ethanol.
  • the volume ratio of the organic solvent to sterile distilled water is (3-7):1, for example, 3:1, 4:1, 5:1, 6:1, or 7:1, etc. Any specific point value within the numerical range can be selected, so I won’t repeat them here.
  • the temperature of the ultrasound in step (2) is 0-4°C, such as 0°C, 1°C, 2°C, 3°C or 4°C, etc.
  • the time is 2-5 minutes, such as 2min, 3min, 4min or 5min, etc.
  • the power is 20-30W, such as 20W, 22W, 25W, 28W, or 30W, etc. Any specific point value within the above numerical range can be selected, and will not be repeated here.
  • the mass fraction of the sodium cholate solution in step (3) is 1-3%, such as 1%, 2%, or 3%.
  • the volume ratio of the sodium cholate solution to the organic solvent in step (3) is (1-3):1, such as 1:1, 2:1, or 3:1.
  • the temperature of the ultrasound in step (3) is 0-4°C, such as 0°C, 1°C, 2°C, 3°C or 4°C, etc.
  • the time is 4-6min, such as 4min, 5min or 6min, etc.
  • the power is 25-35W, such as 25W, 28W, 30W, 32W, or 35W, etc. Any specific point value within the above numerical range can be selected, and will not be repeated here.
  • the mass fraction of the sodium cholate solution in step (4) is 0.3-0.7%, such as 0.3%, 0.5% or 0.7%.
  • the volume ratio of the sodium cholate solution to the organic solvent in step (4) is (8-12):1, such as 8:1, 10:1, or 12:1.
  • the stirring temperature in step (4) is 20-30°C, such as 20°C, 25°C, or 30°C, and the time is greater than 10 minutes.
  • the present invention provides an application of the tumor vaccine for preventing postoperative recurrence of cancer as described above in the preparation of drugs for enhancing the body's innate immunity and specific immune response.
  • the present invention provides an application of the above-mentioned tumor vaccine for preventing postoperative recurrence of cancer in the preparation of a medicine that promotes the secretion of pro-inflammatory cytokines by dendritic cells.
  • the present invention provides an application of the tumor vaccine for preventing postoperative recurrence of cancer as described above in the preparation of drugs for promoting the maturation of dendritic cells.
  • the present invention provides an application of the above-mentioned tumor vaccine for preventing postoperative recurrence of cancer in preparing a medicine for promoting the secretion of IFN- ⁇ from splenic lymphocytes.
  • the present invention provides the application of the above-mentioned tumor vaccine for preventing postoperative recurrence of cancer in the preparation of drugs that stimulate the immune system to secrete inflammatory factors and chemokines.
  • the present invention has the following beneficial effects:
  • the tumor vaccine involved in the present invention is a hybrid membrane-encapsulated polymer nanoparticle.
  • the present invention uses the cell membrane in the solid tumor tissue derived from surgical resection in the hybrid membrane as the tumor antigen, and creatively uses the hybrid membrane as the tumor antigen.
  • the inner membrane of gram-negative bacteria in the synapse is used as an immune adjuvant, and the two can be jointly delivered to the same dendritic cell. It can use the uptake and presentation of tumor antigens to enhance the body's innate immunity and specific immunity. And it has a certain ability of lymph node enrichment, has a significant effect of preventing recurrence after tumor resection, prolonging the survival period of patients, and providing a long-term protection mechanism.
  • the core material wrapped in the tumor vaccine hybrid membrane of the present invention can help maintain the rigid structure and stability of the hybrid membrane, thereby maintaining the immune enhancement and lymphatic enrichment of the hybrid membrane, and preventing recurrence after tumor resection surgery At the same time, it can further contain other biologically active ingredients such as siRNA or chemotherapeutics to achieve more functions.
  • the tumor vaccine involved in the present invention has good biological safety, easy preparation of raw materials, and low preparation cost.
  • this application uses the components of the bacterial inner membrane as the drug combination.
  • One of the ingredients; and a wide range of applications, various chemical groups can be modified on the surface of the vaccine according to actual needs, and it has broad application prospects.
  • Figure 1 shows the transmission electron microscope images of EM-NPs, TM-NPs and HM-NPs hybrid film nanoparticles, the scale is 100nm;
  • Figure 2 shows the results of dynamic light scattering of HM-NPs hybrid film nanoparticles
  • Figure 3A-3C shows the comparison of the levels of IL-6, TNF- ⁇ and IL-1 ⁇ in the supernatant of the control group, EM-NPs group, TM-NPs group, Mix NPs group and HM-NPs group (A is IL-1 ⁇ ).
  • -6 level B is TNF- ⁇ level
  • C is IL-1 ⁇ level);
  • Figure 4 shows the comparison of CD80 and CD86 levels in the supernatant of the control group, EM-NPs group, TM-NPs group, Mix NPs group and HM-NPs group (left is CD80 level, right is CD86 level);
  • Figure 5 shows the results of ELISPOT kit detection of IFN- ⁇ secretion in the supernatant of the control group, EM-NPs group, TM-NPs group, Mix NPs group and HM-NPs group;
  • Figure 6 shows the results of using CTL-ImmunoSpot Plate Reader software to count the number of spots in the control group, EM-NPs group, TM-NPs group, Mix NPs group and HM-NPs group;
  • Figures 7A-7M show the results of the secretion levels of inflammatory factors and chemokines in the serum of mice in the control group, EM-NPs group, TM-NPs group, Mix NPs group and HM-NPs group (A, B, C, D, E, F, G, H, I, J, K, L, M correspond to IL-6, IFN- ⁇ , TNF- ⁇ , MCP-1, IL-12p70, IL-1 ⁇ , IL-10, IL-23, IL-27, IL-17A, IFN- ⁇ , GM-CSF, IL-1 ⁇ );
  • Figure 8 shows the in vivo imaging diagram of the lymph node enrichment effect of the product prepared in Example 1 in vivo;
  • Figure 9 shows a statistical result chart of the lymph node enrichment effect of the product prepared in Example 1 in vivo
  • Figure 10 shows the results of the hemolysis test of HM-NPs hybrid film nanoparticles
  • Figure 11 shows a flow chart of the test operation of Example 6.
  • Figure 12 shows in vivo imaging images of tumor fluorescence intensity of tumor-bearing mice in the control group, EM-NPs group, TM-NPs group, Mix NPs group, and HM-NPs group;
  • Figure 13 shows a graph of the tumor volume of each tumor-bearing mouse in the control group, EM-NPs group, TM-NPs group, Mix NPs group, and HM-NPs group over time;
  • Figure 14 is a graph showing the changes in tumor volume of tumor-bearing mice in the control group, EM-NPs group, TM-NPs group, Mix NPs group, and HM-NPs group over time;
  • Figure 15 shows the survival curve of tumor-bearing mice in the control group, EM-NPs group, TM-NPs group, Mix NPs group and HM-NPs group;
  • Figure 16 shows a flow chart of the test operation of Example 7.
  • Figure 17 shows the photos of tumor changes over time in the control group, Mix NPs group and HM-NPs group tumor-bearing mice;
  • Figure 18 shows the survival curve of tumor-bearing mice in the control group, Mix NPs group and HM-NPs group;
  • Figure 19 shows the results of changes in tumors of each group of mice in the postoperative restimulation test over time
  • Figure 20A-20D shows the comparison of the expression levels of IL-6, TNF- ⁇ , IL-1 ⁇ and IFN- ⁇ in the serum of each group of mice in the postoperative restimulation test (A, B, C, and D respectively correspond to IL-6, TNF- ⁇ , IL-1 ⁇ , and IFN- ⁇ ) -6, TNF- ⁇ , IL-1 ⁇ and IFN- ⁇ );
  • Figure 21A-21C shows the comparison of the levels of IL-6, TNF- ⁇ and IL-1 ⁇ in the supernatant between products with different membrane protein concentration ratios in hybrid membrane nanoparticles (A, B, and C respectively correspond to IL-6 , TNF- ⁇ and IL-1 ⁇ ).
  • Figure 22 shows the Venn diagram showing the different protein expression profiles of (left) EM and (right) TM from four batches.
  • the overlapping parts of the circles represent co-expressed proteins.
  • E1, E2, E3 and E4 represent four batches of EM samples respectively.
  • T1, T2, T3 and T4 represent four batches of TM samples respectively.
  • Figures 23A-23C show the indicated EM to TM membrane protein mass ratio (EM:TM), 1:100, 1:75, 1:50, 1:25, 1:10, 1:5, 1: 3. 1:1, 1:0, 0:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1 or 100:1)
  • EM:TM membrane protein mass ratio
  • a to C The production of IL-6, TNF- ⁇ and IL-1 ⁇ was analyzed using the corresponding specific ELISA kit.
  • n 3 biologically independent replicate samples. The data are expressed as mean ⁇ standard deviation. *p ⁇ 0.05, **p ⁇ 0.01. ns means no significant difference.
  • Figures 24A-24C show the physical and chemical properties of three membrane vesicles before encapsulating the core of PLGA nanoparticles.
  • B Hydration size and
  • Figures 26A-26B show schematic diagrams of the core/shell structure of HM-NP.
  • A Representative TEM image of a single HM-NP.
  • the diameter of the HM-NP core is 86.83 ⁇ 7.41nm (inner wire), and the shell thickness is 14.16 ⁇ 2.07nm (outer wire).
  • Figures 27A-27B show the physical and chemical stability of HM-NP resuspended in PBS before and after lyophilization.
  • Figures 28A-28B show the results of particle size and charge of EM-NP, TM-NP and HM-NP in PBS at different time intervals at 4°C.
  • A The size of EM-NP, TM-NP and HM-NP and
  • Figures 29A-29E show a schematic diagram of the construction of a cancer vaccine HM-NP and a diagram of the characterization results of EM-NP, TM-NP and HM-NP.
  • A Preparation of HM-NP. The tumor was surgically removed from the tumor-bearing mice to obtain TM. The amplified E. coli DH5 ⁇ was treated with lysozyme to remove the cell wall, and the protoplasts were obtained by low-speed centrifugation, and then the EM was prepared using extraction buffer.
  • PLGA NP is prepared using the double emulsion method. The two membranes were mixed and repeatedly physically extruded through a liposome extruder with a 400nm filter membrane pore size.
  • HM-NP HM-NP
  • B TEM images of EM-NP, TM-NP and HM-NP. Scale bar, 100nm.
  • C Size distribution and Zeta potential of EM-NP, TM-NP and HM-NP.
  • Figure 30 shows the whole protein expression profile of EM-NP, TM-NP and HM-NP.
  • the protein profiles of EM-NPs, TM-NPs and HM-NPs were analyzed by SDS-PAGE and then stained with Coomassie brilliant blue.
  • TM rectangle representative protein on tumor cell plasma membrane (TM) of resected autologous tumor tissue;
  • EM rectangle protein band present in the prominent molecular weight range, derived from E. coli plasma membrane (EM).
  • Each lane is loaded with the same amount of protein (10 micrograms). The experiment was carried out 3 times, with 3 biological replicates in each group.
  • Figures 31A-31B show TEM images of HM-NPs with immunogold labeling of FtsZ (dark arrow, 5nm gold NPs) and Na + /K + -ATPase (light arrow, 10nm gold NPs), and then Use uranyl acetate for negative staining.
  • A Use three TEM observation areas to calculate the percentage of hybrid film nanoparticles simultaneously marked with 5 nm and 10 nm gold nanoparticles (AuNP) in the HM-NP formulation.
  • AuNP gold nanoparticles
  • Figure 32 shows the laser confocal fluorescence images of the internalization of EM-NPs-RhoB, TM-NPs-RhoB and HM-NPs-RhoB into the lysosomal compartment of BMDC.
  • Three kinds of fluorescently labeled membrane nanoparticles were incubated with BDMCs for 1 hour, and observed by confocal microscope. The experiment was carried out 3 times, with 3 biological replicates in each group.
  • Rhodamine B-loaded NP second column; lysosomal compartment: first column; bright field (BF): third column; fusion image (Merge): fourth column.
  • Scale bar 20 ⁇ m.
  • Figure 33 shows the Pearson correlation coefficient analysis result graph between the lysosomal compartment and the membrane NP.
  • Figure 34 shows the co-incubation of bacterial inner membrane (EM) with BMDCs at different concentrations (0, 0.12, 0.25, 0.50 and 1.00 mg/mL), and the designated time points (0, 3, 8, 24, 48 hours)
  • the cell supernatant of ), the -NP in BMDC the result of detecting the content of the pro-inflammatory cytokine IL-6 in the cell supernatant by a specific ELISA kit.
  • the cell supernatant of the -NP in BMDC
  • the curve represents the change in IL-6 secretion within 48 hours.
  • the production of IL-6 is positively correlated with the concentration of EM-NPs.
  • the data are expressed as mean ⁇ standard deviation. The experiment was carried out 3 times, with 3 biological replicates in each group.
  • Figures 35A-35K show the results of HM-NPs enhancing tumor antigen uptake, activating the expression of TLRs on the surface of BMDC, and co-delivering antigen and adjuvant to BMDC.
  • A Co-localization analysis of EM-NP, TM-NP and HM-NP (rhodamine B; second column) and lysosomal compartment (first column) in BMDC by confocal microscope (1 hour incubation) . The third column, bright field. Scale bar, 5 ⁇ m.
  • FIG. 1 Western blot analysis of a series of BMDCs membrane TLR protein and NF- ⁇ B protein in membrane-coated nanoparticles. Each lane contains an equal amount of protein.
  • FIG. 1 Schematic diagram of activation of TLR and its downstream signal pathways.
  • J Immunofluorescence experiment of confocal laser scanning micrograph of mouse BMDC treated with mixed NP or HM-NP for 8 hours.
  • DAPI labeled nuclei first column; Na + /K + -ATPase labeled TM: second column; FtsZ labeled EM: third column. Scale bar, 20 ⁇ m.
  • Figures 36A-36B show the verification results of enhanced accumulation of HM-NPs in draining lymph nodes (LNs).
  • B Quantification of the average fluorescence intensity in panel A. The data are expressed as mean ⁇ standard deviation. The experiment was carried out at least three times. Statistical significance is calculated by using Bonferroni multiple comparison test through two-way analysis of variance, ****p ⁇ 0.0001, ns means no significant difference.
  • Figures 37A-37G show the verification results of HM-NPs promoting the maturation of DCs in LN and the activation of splenic T cells in vivo.
  • a and B Flow cytometry analysis of CD80 + and CD86 + DC in the inguinal lymph nodes of 4T1 tumor-bearing mice after subcutaneous inoculation with membrane NP. Determine the proportion of autologous membrane antigen-specific T cells in IFN- ⁇ ELISPOT analysis and flow cytometry.
  • CD Representative results of IFN- ⁇ ELISPOT analysis and the quantitative number of spots in the corresponding 4T1 tumor-bearing mice (5 per group).
  • HM-NPs cause low-level systemic inflammation.
  • Figure AD Bonferroni's multiple comparison test was used to calculate statistical significance through one-way analysis of variance.
  • Figures 38A-38I show the verification results that HM-NPs cause low-level systemic inflammatory response.
  • An ELISA kit based on Luminex magnetic beads was used to detect the concentration of inflammatory cytokines and chemokines in the serum, and the BD Accuri C6 FACS flow cytometer and ProcartaPlex Analysis software were used for analysis. The data are expressed as mean ⁇ standard deviation. The experiment was carried out three times. Statistical significance was calculated by single-factor ANOVA using Bonferroni's multiple comparison test.
  • FIGS 39A-39E show the results of verification that vaccination with HM-NPs can induce tumor regression in the mouse 4T1-Luc mouse tumor model.
  • A shows the scheme of animal experiment design. Female BALB/c mice were inoculated subcutaneously with murine 4T1-Luc breast tumor cells. When the volume of the tumor reaches about 300 mm 3 , it is removed by surgery. Then on the 3rd, 5th, and 9th days after the operation, the mice were immunized with the designated vaccine formulation, and the tumor growth of the mice was monitored by the IVIS imaging system every week.
  • B Small tumors carrying 4T1-Luc tumors on different days Bioluminescence image of a rat's body.
  • C and D Individual and average tumor growth curves.
  • Figures 40A-40G show the results of verification that HM-NP vaccination can inhibit tumor recurrence in a variety of mouse tumor models.
  • C average tumor growth curve
  • D single tumor growth curve
  • E survival curve
  • (F) Survival curve of each group in the B16F10 melanoma model (n 10 mice per group).
  • Figures 41A-41B show the results of verification that HM-NP has higher therapeutic efficacy than three dosage forms of MPLA and tumor membrane preparation or HM vesicle preparation in the mouse CT-26 tumor model.
  • Female BALB/c mice were subcutaneously inoculated with CT-26 colon adenocarcinoma cells (2 ⁇ 10 5 cells per mouse), and then proceeded according to the above-mentioned surgical procedure and inoculation schedule.
  • the three vaccine formulations of MPLA include TM-NP+free MPLA, MPLA anchored on the tumor membrane (TM-MPLA-NPs) and PLGA nanoparticles containing MPLA with tumor membrane (TM-NPs@MPLA).
  • PLGA-free heterozygous membrane vesicles were subcutaneously inoculated into postoperative mice to compare the therapeutic potential of PLGA NP (HM NP) coated with heterozygous membrane to prevent tumor recurrence.
  • A Tumor growth curve of a single mouse in each group and
  • B average tumor growth curve and survival curve.
  • CR Complete remission. The data are expressed as mean ⁇ standard deviation. The experiment was carried out at least three times.
  • Statistical significance is calculated by two-way analysis of variance (ANOVA), Bonferroni's growth curve multiple comparison test and log-rank (Mantel-Cox) test of the log survival curve, *p ⁇ 0.05, **p ⁇ 0.01, ** **p ⁇ 0.0001, ns means no significant difference.
  • Figures 42A-42I show that HM-NP vaccination can provide long-term protective anti-tumor immunity; both innate immunity and adaptive immunity are critical to the effectiveness of the vaccine to verify the results.
  • ELISA measures the levels of pro-inflammatory cytokines in the serum of mice inoculated with CT-26 or 4T1 cells
  • C IL-6,
  • D IL-1 ⁇ ,
  • E TNF- ⁇ and
  • Spleen cells were isolated from naive mice or mice treated with HM-NPs and tumor eradicated (90 days after primary tumor inoculation).
  • mice depleting specific immune cell subpopulations NK cells, macrophages, CD8 + T cells or CD4 + T cells
  • NK cells NK cells, macrophages, CD8 + T cells or CD4 + T cells
  • the data are expressed as mean ⁇ standard deviation.
  • the significance of the statistical data was analyzed by two analysis of variance using Bonferroni multiple comparison test, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 43 shows the results of depletion of specific immune cell subpopulations during HM-NP treatment.
  • the antibody was depleted by intraperitoneal injection to knock down the corresponding immune cell subsets one day before the start of HM-NP treatment.
  • FIGS 44A-44B show that after HM-NP treatment, in order to effectively suppress tumors, both innate immunity and adaptive immunity are necessary to verify the results.
  • Figure 45 shows the results of the hemolysis test with different concentrations of HM-NP.
  • Figure 46 shows representative microscopic images of H&E stained sections of major organs (ie, heart, liver, spleen, lung, and kidney) of mice from each group at the end of the tumor regression experiment in the 4T1-Luc model. The experiment was carried out 3 times, with 3 biological replicates in each group. Scale bar, 50 ⁇ m.
  • Figures 47A-47D show the results of evaluating the liver and kidney functions of mice in all groups at the end of the tumor regression experiment of the 4T1-Luc model.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • BUN blood urea nitrogen
  • CREA creatinine.
  • the data are expressed as mean ⁇ standard deviation. Using Bonferroni's multiple comparison test, statistical significance was analyzed by one-way analysis of variance.
  • Figure 48 shows the distribution of E. coli inner membrane proteins.
  • Figures 49A-49B show the results of the preparation of HM-NPs hybrid membrane nano-vaccine from the inner membrane of bacteria of the same genus to inhibit postoperative recurrence of colon cancer model.
  • A the morphology of hybrid membrane nanoparticles prepared from different bacterial inner membranes and tumor membranes.
  • B Survival curves of mice after immunotherapy with hybrid membrane nanoparticles prepared from different bacteria.
  • Figures 50A-50C show the results of HM-NPs hybrid membrane nano-vaccine of different tumor cell membranes in inhibiting the postoperative recurrence of the corresponding tumor model.
  • A the construction of tumor model and the schematic diagram of the immune procedure.
  • B The morphology of hybrid membrane nanoparticles prepared from different tumor membrane cell membranes and bacterial inner membranes.
  • C Survival curve of mice after immunotherapy with hybrid membrane nano-vaccine HM-NPs prepared from cell membranes of liver cancer, gastric cancer, kidney cancer, pancreatic cancer, ovarian cancer, lymphoma, osteosarcoma, glioma, prostate cancer and melanoma .
  • Figure 51 shows the hybrid membrane encapsulating the PLGA polymer, extruded through a liposome extruder with a pore size of 200nm, and the surface potential of the hybrid membrane shell can be increased from +50mV to -50mV by doping with polymers of different charges. .
  • bacteria inner membrane generally refers to a layer of bacterial membrane structure.
  • bacteria can have multiple structures, from the outside to the inside, the cell wall, the bacterial outer membrane, the bacterial inner membrane, and the cytoplasm.
  • the inner membrane of the bacteria of the present application may only be an inner bacterial membrane structure close to the cytoplasm.
  • the term “pharmaceutical combination” generally refers to a product produced by the mixing or combination of one or more active ingredients.
  • drug combination can be administered to a patient separately, collectively or sequentially (without a specific time limit), wherein such administration provides a therapeutically effective level of the first membrane component of the present application and derived from other than the bacteria in the patient. Components of living organisms.
  • the term "membrane component” generally refers to a component having a component in the membrane structure.
  • the membrane components of the present application may include natural or synthetic biofilm components.
  • the membrane component of the present application may not include a phospholipid component, and the membrane component of the present application may only include membrane proteins, membrane lipids, membrane sugar molecules, membrane glycoproteins, or membrane lipoproteins of biological membranes.
  • the membrane component of the present application may include functionally active fragments of the membrane component and other arbitrary domains.
  • the term "organism” generally refers to an organism with a boundary structure.
  • the organism of the present application may be an object with life.
  • the organism of the present application may be a cellular organism with a cell structure or a virus organism without a cell structure.
  • the organisms of the present application may include bacteria, prokaryotes, eukaryotes, and the aforementioned tissues, cells, or non-cellular structures.
  • immunogenicity generally refers to the ability to cause an immune response (stimulation of the production of specific antibodies and/or the proliferation of specific T cells).
  • biocompatibility generally means that the material has compatibility with the host.
  • biocompatibility can mean that the material does not trigger abnormal blood reactions, immune reactions, and/or tissue reactions.
  • Gram-positive bacteria or "Gram-negative bacteria” usually refers to the basic characteristics of Gram staining reaction, bacteria can be divided into two main categories: G positive (G+) and G Negative (G-). After the former is stained, the bacterial cells can still retain the blue-violet color of the original crystal violet. After the latter is stained, the bacterial cells can first remove the color of the original crystal violet and bring the complex saffron or sand yellow red.
  • IL-6 generally refers to a type of cytokine.
  • IL-6 can be found in GenBank accession number P05231.
  • the IL-6 protein of the present application may also cover its functionally active fragments, and is not limited to substances containing functionally active fragments of IL-6 produced after processing and/or modification in cells.
  • IL-6 of the present application may include functionally active fragments of IL-6 and other arbitrary domains.
  • IFN- ⁇ generally refers to one of the cytokines.
  • IFN- ⁇ can be found in GenBank accession number P01579.
  • the IFN- ⁇ protein of the present application may also cover its functionally active fragments, and is not limited to substances containing IFN- ⁇ functionally active fragments produced after processing and/or modification in cells.
  • IFN- ⁇ of the present application may include functionally active fragments of IFN- ⁇ and other arbitrary domains.
  • Interleukin-1 ⁇ or "IL-1 ⁇ ” generally refers to one of the cytokines.
  • IL-1 ⁇ can be found in GenBank accession number P01584.
  • the IL-1 ⁇ protein of the present application can also cover its functionally active fragments, and is not limited to substances containing IL-1 ⁇ functionally active fragments produced after processing and/or modification in cells.
  • IL-1 ⁇ of the present application may include functionally active fragments of IL-1 ⁇ and other arbitrary domains.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • TNF- ⁇ can be found in GenBank accession number P01375.
  • the TNF- ⁇ protein of the present application may also cover its functionally active fragments, and is not limited to substances containing functionally active fragments of TNF- ⁇ produced after processing and/or modification in cells.
  • the TNF- ⁇ of the present application may include functionally active fragments of TNF- ⁇ and other arbitrary domains.
  • lipopolysaccharide usually refers to Lipopolysaccharide (English abbreviation LPS).
  • LPS can refer to the components of the outer wall of the cell wall of Gram-negative bacteria, and is a substance (glycolipid) composed of lipids and polysaccharides.
  • FtsZ generally refers to a protein in the inner membrane of bacteria.
  • FtsZ can be used to detect whether the hybrid membrane contains membrane components derived from the bacterial inner membrane.
  • FtsZ can be found in GenBank accession number P0A9A6.
  • the FtsZ of the present application may include functionally active fragments of FtsZ and other arbitrary domains.
  • the inner membrane of the bacteria may also contain lipopolysaccharide (LPS, commonly known as endotoxin) and phosphatidylethanolamine (PE), and the inner membrane of the cell may also contain minor lipid categories, such as N-acylated PE (acyl-PE) And O-acylated PG (acyl-PG), etc., and can be used to detect whether the hybrid membrane contains membrane components derived from bacterial inner membrane.
  • LPS lipopolysaccharide
  • PE phosphatidylethanolamine
  • minor lipid categories such as N-acylated PE (acyl-PE) And O-acylated PG (acyl-PG), etc.
  • the lipopolysaccharide molecule is located in the outer layer of the outer cell membrane, and can be composed of two parts, a hydrophilic polysaccharide chain and a hydrophobic lipid A.
  • the amphiphilic molecule phospholipid phthalethanolamine can be the lipid molecule with the highest content in the intracellular membrane of Gram-negative bacteria, accounting for about 77%; anionic lipid phosphatidylglycerol (PG) accounts for about 13%; cardiolipin (cardiolipin) , CL) accounted for about 10%.
  • the inner membrane of bacteria is rich in lipoproteins and lipids.
  • Phosphatidylethanolamine can be the most important phospholipid molecule of Gram-negative bacteria. It plays a very important role in maintaining the normal morphology of cells, cell growth and differentiation, and substance synthesis.
  • Bacterial membranes can have a diversity of amphiphilic lipids, including the common phosphatidylglycerol, phosphatidylethanolamine and cardiolipin, the less common phosphatidylcholine, phosphatidylinositol and many other membrane lipids, such as ornithine Acid lipids, glycolipids, sphingolipids, etc.
  • Na+/K+-ATPase generally refers to a protein of the cell membrane.
  • it may be a protein of mammalian cell membrane.
  • Na+/K+-ATPase can be used to detect whether the hybrid membrane contains membrane components from other sources besides the bacterial inner membrane.
  • Na+/K+-ATPase can be found in GenBank accession number P13637.
  • the Na+/K+-ATPase of the present application may include functionally active fragments of Na+/K+-ATPase and other arbitrary domains.
  • the present application provides a pharmaceutical combination, which may include a first membrane component, the first membrane component may include a membrane derived from the inner membrane of a bacteria, and the pharmaceutical combination may also include a membrane derived from the bacteria Component of other organisms.
  • the pharmaceutical combination of the present application may include membranes derived from the inner membrane of bacteria, as well as immunogenic substances derived from other organisms.
  • the drug combination of the present application may include a membrane derived from the inner membrane of bacteria, as well as tumor antigens.
  • the pharmaceutical combination of the present application may include a membrane derived from the inner membrane of a bacteria, and a cell membrane and/or a shell of a non-cellular membrane structure from which the antigen is derived.
  • the other organisms described in this application may comprise cells.
  • other organisms of this application may contain non-cellular structures.
  • other organisms of this application may comprise mammalian cells.
  • the other organisms of the present application may comprise non-mammalian cells, for example prokaryotic cells.
  • other organisms of the application may include tumor cells.
  • other organisms of the application may include solid tumor cells.
  • other organisms of the present application can be selected from the following group: breast cancer cells, colon cancer cells, liver cancer cells, gastric cancer cells, kidney cancer cells, pancreatic cancer cells, ovarian cancer cells, lymphoma cells, osteosarcoma cells, glial cells Tumor cells, prostate cancer cells and melanoma cells, and any combination of the above.
  • the component of the other organism may include an immunogenic component.
  • the component of the other organism may be able to elicit an immune response to the organism.
  • the component of the other organism may include a component derived from the cell membrane of the organism.
  • the component of the other organism may comprise a tumor antigen or a functionally active fragment thereof.
  • the component of the other organism may include an immunogenic protein or an immunogenic fragment thereof on the cell membrane of the other organism.
  • the component of the other organism may comprise a protein or a functionally active fragment thereof selected from the following group: antigen peptide transporter 1, H-2 class II histocompatibility antigen ⁇ chain, tyrosine protein kinase SYK , High-affinity immunoglobulin epsilon receptor subunit ⁇ , Ras-related C3 botulinum toxin substrate 2, tyrosine protein kinase BTK, receptor type tyrosine protein phosphatase C, Na + /K + -ATPase, ATP5A (IM CVa), ubiquinone cytochrome C reductase core protein I (IM Core I), VDAC1/porin (OM Porin), matrix cyclophilin D (Matrix CypD) and interstitial cytochrome C (IMS Cytc) , And any combination of the above.
  • antigen peptide transporter 1 H-2 class II histocompatibility antigen ⁇ chain
  • the pharmaceutical combination of the present application may include a second membrane component that includes a membrane derived from the cell membrane of the other organism.
  • the second membrane component of the present application may be a cell membrane containing an immunogenic protein.
  • the second membrane component of the present application can also be prepared by binding immunogenic proteins to natural and/or artificial cell membranes.
  • the bacteria may include Gram-negative bacteria and/or Gram-positive bacteria.
  • the bacteria are selected from the genera of the following group: Escherichia coli, Staphylococcus, Bacillus, Lactobacillus, Klebsiella, Brucella, Proteus, Acinetobacter And Pseudomonas, and any combination of the above.
  • the bacteria are selected from the following group: Escherichia, Staphylococcus, Bacillus, Lactobacillus and Pseudomonas.
  • the bacteria may include Escherichia coli and/or Staphylococcus aureus.
  • the inner membrane of the bacteria may comprise a protein or functionally active fragments thereof selected from the following groups: FtsZ, YhcB, YidC, NlpI, MsbA, TatA, MlaA, TolQ and YebE, and the above Any combination of.
  • the drug combination of the present application may also include an inner core.
  • the inner core of the drug combination of the present application can be used to support the membrane component of the present application.
  • the core of the drug combination of the present application may also contain biocompatible materials.
  • the core of the drug combination of the present application may also contain artificial synthetic materials.
  • a particulate and/or non-particle structured material known in the art that can be covered or partially covered by the film of the present application can be used in the core of the drug combination of the present application.
  • the core of the drug combination of the present application may also contain a substance selected from the following group: polylactic acid-glycolic acid copolymer (PLGA), metal-organic framework material (MOF), polycaprolactone (PCL), polyamide- Amine (PAMAM), carbon nanotubes, graphene, gold nanoparticles, mesoporous silica nanoparticles, iron oxide nanoparticles, silver nanoparticles, tungsten nanoparticles, manganese nanoparticles, platinum nanoparticles, quantum dots, alumina nanoparticles Particles, hydroxyapatite nanoparticles, lipid nanoparticles (LNP), DNA nanostructures, nanohydrogels, rare earth fluoride nanocrystals, and NaYF 4 nanoparticles, and any combination of the above.
  • PLGA polylactic acid-glycolic acid copolymer
  • MOF metal-organic framework material
  • PCL polycaprolactone
  • PAMAM polyamide- Amine
  • the core of the drug combination of the present application may also contain substances selected from the following group: immune adjuvants, immune checkpoint inhibitors, nucleic acid molecules and chemotherapeutic agents, and any combination of the above.
  • the core of the drug combination of the present application may also include monophosphoryl lipid A (MPLA), R848, CpG, poly(I:C), and any combination of the above.
  • MPLA monophosphoryl lipid A
  • R848 CpG
  • poly(I:C) any combination of the above.
  • the core of the drug combination of the present application may also contain doxorubicin, paclitaxel, docetaxel, gemcitabine, capecitabine, cyclophosphamide, fluorouracil, pemetrexed, raltitrexate, bleomycin , Daunorubicin, doxorubicin, vincristine and etoposide, and any combination of the above.
  • the core of the drug combination of the present application may also include an indoleamine 2,3-dioxygenase (IDO) inhibitor.
  • IDO indoleamine 2,3-dioxygenase
  • the core of the drug combination of the present application may also include small interfering RNA (siRNA).
  • siRNA small interfering RNA
  • the diameter of the inner core of the drug combination of the present application may be about 60 to about 100 nanometers.
  • the size of the inner core of the drug combination of the present application may not substantially affect the effect of the drug combination of the present application.
  • the diameter of the core of the drug combination of the present application may be about 60 to about 100 nanometers, about 70 to about 100 nanometers, about 80 to about 100 nanometers, about 90 to about 100 nanometers, about 60 to about 90 nanometers, about 70 nanometers. To about 90 nanometers, or about 80 to about 90 nanometers.
  • the diameter of the inner core of the drug combination of the present application may be about 86 nanometers.
  • the diameter of the inner core of the present application can be measured by a transmission electron microscope.
  • the pharmaceutical combination of the present application may include a housing, and the housing may include the first film component and the second film component.
  • the casing of the present application may include a film obtained by fusing the first film component and the second film component.
  • the outer shell of the present application may include natural and/or artificially synthesized biofilms, as well as proteins of bacterial inner membranes and biofilms of other organisms.
  • the outer shell of the present application may include natural or synthetic biofilms, as well as immunogenic proteins of bacterial inner membranes and immunogenic proteins of other organisms.
  • the outer shell of the present application may be composed of the following groups: a bacterial inner membrane containing immunogenic proteins and other biological membranes containing immunogenic proteins.
  • components derived from the inner membrane of bacteria can be used to enhance the immune response of the subject; for example, components derived from other organisms can be used to elicit an immune response from the subject to the other organisms.
  • using tumor cell membrane as a component derived from other organisms can be used to induce and/or enhance the subject's immune response to the tumor.
  • the thickness of the casing of the present application may be about 10 to about 20 nanometers.
  • the thickness of the shell of the present application may be about 10 to about 20 nanometers, about 11 to about 20 nanometers, about 12 to about 20 nanometers, about 13 to about 20 nanometers, about 14 to about 20 nanometers, about 15 to about 20 nanometers.
  • the thickness of the casing of the present application may be about 14 nanometers.
  • the diameter of the casing of the present application may be about 100 nanometers.
  • the thickness or diameter of the housing of the present application can be measured by a transmission electron microscope.
  • the surface potential (Zeta potential) of the housing of the present application may be about +50 mV to about -50 mV.
  • the surface potential (Zeta potential) of the housing of the present application may be about -21mV.
  • the surface potential (Zeta potential) of the housing may be about +50mV to about -50mV, about -15 to about -50mV, about -21 to about -50mV, about -25 to about -50mV, about +50mV to About -25mV, about +50mV to about -21mV, about -15 to about -25mV, about -17 to about -25mV, about -19 to about -25mV, about -21 to about -25mV, about -23 to about- 25mV, about -15 to about -21mV, about -17 to about -21mV, or about -19 to about -21mV.
  • the mass ratio of the first film component to the second film component of the housing of the present application may be 1:100 to 100:1.
  • the mass ratio of the first film component to the second film component of the housing of the present application may be 1:100-100:1, such as 1:100, 1:75, 1:50, 1: 25, 1:10, 1:5, 1:3, 1:1, 1:0, 0:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1 or 100:1.
  • the mass ratio of the first film component to the second film component of the housing of the present application may be 1:100, 1:90, 1:80, 1:70, 1:60, 1:50 , 1:40, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 0 :1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 40:1, 50:1 , 60:1, 70:1, 80:1, 90:1 or 100:1.
  • the presence and/or ratio of the first membrane component and the second membrane component in the drug combination of the present application can be confirmed by Western blotting and/or immunogold staining.
  • the pharmaceutical combination of the present application may include particles, and the particles may include the inner core and the outer shell.
  • the mass ratio of the shell to the core material of the drug combination of the application may be about 1:1 to about 1:10.
  • the mass ratio of the shell to the core material of the drug combination of the application may be about 1:4 to about 1:6.
  • the mass ratio of the shell to the core material of the drug combination of the application may be about 1:1 to about 1:10, about 1:2 to about 1:10, about 1:4 to about 1:10, about 1. :6 to about 1:10, about 1:8 to about 1:10, about 1:1 to about 1:6, about 1:2 to about 1:6, or about 1:4 to about 1:6.
  • the mass ratio of the shell and core material of the drug combination of the application may be about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1. :7, about 1:8, about 1:9 or about 1:10.
  • the lipopolysaccharide (LPS) content of the drug combination of the present application may not be significantly different from the lipopolysaccharide content of mammalian cells.
  • the pharmaceutical combination of the present application may not substantially contain lipopolysaccharide derived from bacteria.
  • the diameter of the particles of the drug combination of the present application may be about 70 to about 120 nanometers.
  • the diameter of the particles of the drug combination of the present application may be about 100 nanometers.
  • the diameter of the particles of the drug combination of the present application may be about 70 to about 120 nanometers, about 80 to about 120 nanometers, about 90 to about 120 nanometers, about 100 to about 120 nanometers, about 110 to about 120 nanometers, about 70 nanometers.
  • the diameter can be measured by a transmission electron microscope (TEM).
  • the hydrated particle size of the particles may be about 150 nanometers to about 250 nanometers.
  • the hydrated particle size of the particles may be about 180 nanometers.
  • the hydrated particle size of the particles can be about 150 nanometers to about 250 nanometers, 160 nanometers to about 250 nanometers, 180 nanometers to about 250 nanometers, 200 nanometers to about 250 nanometers, 220 nanometers to about 250 nanometers, 240 nanometers to about 250 nanometers.
  • the surface potential (Zeta potential) of the particles of the present application may be about +50 mV to about -50 mV.
  • the surface potential (Zeta potential) of the particles of the present application may be about -21mV.
  • the surface potential (Zeta potential) of the particles may be about +50mV to about -50mV, about -15 to about -50mV, about -21 to about -50mV, about -25 to about -50mV, about +50mV to About -25mV, about +50mV to about -21mV, about -15 to about -25mV, about -17 to about -25mV, about -19 to about -25mV, about -21 to about -25mV, about -23 to about- 25mV, about -15 to about -21mV, about -17 to about -21mV, or about -19 to about -21mV.
  • the hydrated particle size and/or surface potential of the particles can be measured by a dynamic light scattering (DLS) instrument.
  • DLS dynamic light scattering
  • the particles may be able to maintain stability in solution.
  • the stability of the particles may include that the hydrated particle size and/or surface potential of the particles after storage for a period of time are not significantly different from those before the period of time.
  • the stability of the particles may include that there is no significant difference in the hydrated particle size and/or surface potential of the particles after storage in phosphate buffered saline (PBS) at 4 degrees Celsius for a period of time compared with that before the period of time. .
  • PBS phosphate buffered saline
  • the stability of the particles may include that there is no significant difference in the hydrated particle size and/or surface potential of the particles after storage in phosphate buffered saline (PBS) at 4 degrees Celsius for a period of time compared with that before the period of time.
  • the period of time may be no less than about 21 days.
  • the period of time may be not less than about 21 days, not less than about 20 days, not less than about 15 days, not less than about 10 days, not less than about 5 days, not less than about 4 days, no Less than about 3 days, not less than about 2 days, or not less than about 1 day.
  • the pharmaceutical combination of the present application may also include substances selected from the following group: immune adjuvants, immune checkpoint inhibitors, nucleic acid molecules, chemotherapeutic agents and photosensitizers, and any combination of the above.
  • the above-mentioned components in the pharmaceutical combination of the present application may be administered simultaneously and/or separately.
  • the pharmaceutical combination of the present application may also include monophosphoryl lipid A (MPLA).
  • the pharmaceutical combination of the present application may also include an indoleamine 2,3-dioxygenase (IDO) inhibitor.
  • the drug combination of the present application may also include small interfering RNA (siRNA).
  • the present application provides a vaccine, which may include the drug combination of the present application.
  • the present application provides a kit, which may contain the drug combination of the present application and/or the vaccine of the present application.
  • the present application also provides a method for preparing the drug combination of the present application, the vaccine of the present application, and/or the kit of the present application, which may include providing the bacterial-derived inner membrane.
  • it may further include providing the components derived from other organisms other than the bacteria.
  • it may further comprise mixing the membrane derived from the inner membrane of the bacteria and the component derived from other organisms other than the bacteria to provide the outer shell.
  • it may also include providing a kernel.
  • the present application provides a method for enhancing the uptake of a target antigen by immune cells, which may include providing a drug combination, the drug combination may include a first membrane component, and the first membrane component may include a source From the inner membrane of the bacteria, the drug combination may also include the target antigen.
  • the methods of the application can be in vitro or ex vivo.
  • the methods of the present application may be for non-preventive, non-therapeutic, and/or non-diagnostic purposes.
  • the drug combination may include the drug combination of the present application.
  • the immune cells may include immune presenting cells.
  • the immune cells may include the following groups: dendritic cells (DC), T lymphocytes, macrophages, and natural killer cells (NK), and any combination of the above.
  • DC dendritic cells
  • T lymphocytes T lymphocytes
  • macrophages macrophages
  • NK natural killer cells
  • the immune cells may include bone marrow-derived dendritic cells (BMDC).
  • BMDC bone marrow-derived dendritic cells
  • the immune cells may include CD8 positive cells and/or CD4 positive cells.
  • the present application provides a method for activating immune cells, and the method may include administering the drug combination of the present application, the vaccine of the present application, and/or the kit of the present application.
  • the methods of this application can be in vitro or ex vivo.
  • the methods of the present application may be for non-preventive, non-therapeutic, and/or non-diagnostic purposes.
  • the immune cells may include immune presenting cells.
  • the immune cells may include the following groups: dendritic cells (DC), T lymphocytes, macrophages, and natural killer cells (NK), and any combination of the above.
  • DC dendritic cells
  • T lymphocytes T lymphocytes
  • macrophages macrophages
  • NK natural killer cells
  • the immune cells may include bone marrow-derived dendritic cells (BMDC).
  • BMDC bone marrow-derived dendritic cells
  • the immune cells may include CD8 positive cells and/or CD4 positive cells.
  • the immune cells may include the immune cells in lymph nodes and/or spleen.
  • the effect of the activation of the immune cells administered the drug combination may be selected from the following group: increase the antigen of the immune cells Recognizing the expression level of the receptor, the increased expression level of the nuclear factor kappa B (NF- ⁇ B) protein of the immune cell, the increased expression and/or secretion level of the cytokine of the immune cell, and the increased maturity of the immune Proportion of cells, and any combination of the above.
  • increase the antigen of the immune cells Recognizing the expression level of the receptor the increased expression level of the nuclear factor kappa B (NF- ⁇ B) protein of the immune cell
  • NF- ⁇ B nuclear factor kappa B
  • the increase includes that the immune cell administered the drug combination has an effect selected from the above-mentioned group increased by about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.
  • the increase includes an increase in the expression level of antigen recognition receptors of the immune cells administered the drug combination by about 90%, about 80%, about 70%, compared with the immune cells not administered with the drug combination. About 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.
  • the increase includes an increase in the expression level of nuclear factor kappa B (NF- ⁇ B) protein of the immune cell administered the drug combination by about 90%, about 80%, compared to the immune cell not administered the drug combination. , About 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.
  • the increase includes an increase in the cytokine expression and/or secretion level of the immune cell by about 90% compared to the immune cell not administered with the drug combination, About 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.
  • the increase includes an increase in the ratio of the increased mature immune cells of the immune cells administered the drug combination by about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.
  • the antigen recognition receptor may include a pattern recognition receptor (PRR).
  • PRR pattern recognition receptor
  • the antigen recognition receptor may include a Toll-like receptor (TLR).
  • TLR Toll-like receptor
  • the antigen recognition receptor may comprise TLR1, TLR2 and/or TLR6.
  • the expression of TLR4 of the immune cells to which the drug combination is administered may be substantially unchanged compared to the immune cells to which the drug combination is not administered.
  • the cytokine may include a pro-inflammatory cytokine.
  • the cytokine may include interleukin (IL)-6, tumor necrosis factor (TNF)- ⁇ , IL-1 ⁇ and/or interferon (IFN)- ⁇ .
  • IL interleukin
  • TNF tumor necrosis factor
  • IFN interferon
  • the mature immune cells may include CD80-positive cells, CD86-positive cells and/or effector memory cells.
  • the ratio of mature immune cells may include the ratio of CD80-positive and/or CD86-positive immune cells to CD11c-positive immune cells.
  • the ratio of mature immune cells may include the ratio of immune cells with high CD44 expression and low CD62L expression to CD8-positive immune cells.
  • the application provides a method for enhancing innate immunity and/or specific immune response, which may include administering the pharmaceutical combination of the application, the vaccine of the application, and/or the kit of the application to a subject in need.
  • the administration of the drug combination can promote the maturation of dendritic cells and/or can increase the secretion of cytokines by lymphocytes.
  • the application may not cause systemic inflammatory response substantially.
  • the application that does not substantially induce systemic inflammatory response may include that administering the drug combination does not substantially increase the IFN- ⁇ in the serum of the subject compared with not administering the drug combination.
  • administering the drug combination does not substantially increase the IFN- ⁇ in the serum of the subject compared with not administering the drug combination.
  • TNF- ⁇ TNF- ⁇
  • MCP-1 macrophage chemoattractant protein-1
  • IL-12p70 IL-10
  • IL-23 IL-27
  • IL-17A granulocyte-macrophage colony Concentration of stimulating factor (GM-CSF) and/or IL-1 ⁇ .
  • GM-CSF granulocyte-macrophage colony Concentration of stimulating factor
  • the application may substantially not cause hemolytic reaction, heart damage, liver damage, spleen damage, lung damage, and/or kidney damage.
  • the application provides the drug combination of the application, the vaccine of the application and/or the kit of the application, which can be used to enhance innate immunity and/or specific immune response.
  • the administration of the drug combination can promote the maturation of dendritic cells and/or can increase the secretion of cytokines by lymphocytes.
  • the application may not cause systemic inflammatory response substantially.
  • the application that does not substantially induce a systemic inflammatory response may include that administering the drug combination does not substantially increase the IFN- ⁇ in the serum of the subject compared with not administering the drug combination.
  • administering the drug combination does not substantially increase the IFN- ⁇ in the serum of the subject compared with not administering the drug combination.
  • TNF- ⁇ TNF- ⁇
  • MCP-1 macrophage chemoattractant protein-1
  • IL-12p70 IL-10
  • IL-23 IL-27
  • IL-17A granulocyte-macrophage colony Concentration of stimulating factor (GM-CSF) and/or IL-1 ⁇ .
  • GM-CSF granulocyte-macrophage colony Concentration of stimulating factor
  • the application may substantially not cause hemolytic reaction, heart damage, liver damage, spleen damage, lung damage, and/or kidney damage.
  • the application provides the application of the drug combination of the application, the vaccine of the application and/or the kit of the application in the preparation of drugs, which can be used to enhance innate immunity and/or specific immune response.
  • the administration of the drug combination can promote the maturation of dendritic cells and/or can increase the secretion of cytokines by lymphocytes.
  • the application may not cause systemic inflammatory response substantially.
  • the application that does not substantially induce a systemic inflammatory response may include that administering the drug combination does not substantially increase the IFN- ⁇ in the serum of the subject compared with not administering the drug combination.
  • administering the drug combination does not substantially increase the IFN- ⁇ in the serum of the subject compared with not administering the drug combination.
  • TNF- ⁇ TNF- ⁇
  • MCP-1 macrophage chemoattractant protein-1
  • IL-12p70 IL-10
  • IL-23 IL-27
  • IL-17A granulocyte-macrophage colony Concentration of stimulating factor (GM-CSF) and/or IL-1 ⁇ .
  • GM-CSF granulocyte-macrophage colony Concentration of stimulating factor
  • the application may substantially not cause hemolytic reaction, heart damage, liver damage, spleen damage, lung damage, and/or kidney damage.
  • the present application also provides a method for preventing and/or treating tumors, which may include administering the drug combination of the present application, the vaccine of the present application and/or the kit of the present application to a subject in need.
  • the prevention and/or treatment of tumors may include slowing down the rate of increase in tumor volume and/or reducing the volume of tumors.
  • the tumor may include the tumor that is not completely resected after tumor resection.
  • tumor cells and/or tumor tissues that were not completely removed can also develop into tumors again, and such tumors can also be prevented and/or treated by the method of the present application.
  • the tumor may include a tumor that regenerates after the tumor is cleared.
  • the patient may reproduce tumors.
  • the patient is prone to tumors.
  • This new tumor produced again can also be prevented and reproduced by the method of this application. / Or treatment.
  • this new tumor produced again has at least one antigen that is the same as the original tumor.
  • the tumor may include a solid tumor.
  • the tumor can be selected from the following group: breast tumor, colon tumor, liver tumor, stomach tumor, kidney tumor, pancreatic tumor, ovarian tumor, lymphoma, osteosarcoma, glioma, prostate cancer And melanoma, and any combination of the above.
  • the application also provides the drug combination of the application, the vaccine of the application and/or the kit of the application, which can be used to prevent and/or treat tumors.
  • the prevention and/or treatment of tumors may include slowing down the rate of increase in tumor volume and/or reducing the volume of tumors.
  • the tumor may include the tumor that is not completely resected after tumor resection.
  • suppressing tumors may include tumors that regenerate after the tumors are cleared.
  • the tumor may include a solid tumor.
  • the tumor may be selected from the following group: breast tumor, colon tumor, liver tumor, stomach tumor, kidney tumor, pancreatic tumor, ovarian tumor, lymphoma, osteosarcoma, glioma, prostate cancer And melanoma, and any combination of the above.
  • the application also provides the application of the drug combination of the application, the vaccine of the application and/or the kit of the application in the preparation of medicines, which can be used to prevent and/or treat tumors.
  • the prevention and/or treatment of tumors may include slowing down the rate of increase in tumor volume and/or reducing the volume of tumors.
  • the tumor may include the tumor that is not completely resected after tumor resection.
  • suppressing tumors may include tumors that regenerate after the tumors are cleared.
  • the tumor may include a solid tumor.
  • the tumor may be selected from the following group: breast tumor, colon tumor, liver tumor, stomach tumor, kidney tumor, pancreatic tumor, ovarian tumor, lymphoma, osteosarcoma, glioma, prostate cancer And melanoma, and any combination of the above.
  • the Elisa kit involved in the following examples was purchased from Abcam, USA; CD80 and CD86 antibodies were purchased from BioLegend, USA; ELISPOT kit was purchased from Beijing Dakwei Biotechnology Co., Ltd.; ProcartaPlex multiplex immunoassay kit was purchased from Thermo Fisher, USA Scientific company.
  • mice involved in the following examples are BALB/c, 6-8 weeks old, 16-18g weight, female, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and raised in the National Nanoscience Center. In the pathogen animal room, all animal experiments are carried out in strict accordance with the guidelines approved by the National Institutional Animal Care and Use Committee (IACUC).
  • IACUC National Institutional Animal Care and Use Committee
  • E. coli E. coli DH5 ⁇
  • 4T1-luciferase triple-negative breast cancer tissue cell membrane or CT26 colon cancer tissue cell membrane The cell membrane is used as a research model for solid tumor cell membranes derived from surgical resection, and PLGA is used as a research model for core materials.
  • the drug combination of this application uses mixed E. coli cytoplasmic membrane (EM) and autologous tumor membrane (TM) from resected tumor tissue to generate hybrid membrane nanoparticle vaccines (HM-NPs) for antigen and adjuvant delivery to antigen Presenting cells (APC).
  • EM E. coli cytoplasmic membrane
  • TM autologous tumor membrane
  • APC antigen Presenting cells
  • This personalized tumor vaccine is tailor-made designed to safely enhance the innate immune response and maximize its anti-tumor effect while avoiding side effects.
  • this application proves that the presence of EM can significantly increase DC uptake (p ⁇ 0.0001) of tumor antigens, activate Toll-like receptors (TLR) on its plasma membrane and promote its in vitro maturation.
  • TLR Toll-like receptors
  • HM-NP lymph nodes
  • LNs lymph nodes
  • HM-NP has achieved significant anti-tumor immunity in 4T1-Luc, B16F10, EMT-6 and CT-26 tumor models.
  • CT26 colon tumor model HM-NPs can cause a strong tumor-specific immune response, which not only prolongs the survival time of animals after surgery, but also has a long-term protective effect (up to 3 months) against tumor re-stimulation.
  • HM-NPs of the present application is an effective autologous tumor vaccine, which can successfully activate innate immunity and adaptive immune response, thereby significantly inhibiting tumor recurrence, with negligible side effects.
  • Current research shows that mixed membrane vaccine technology may be transformed into an effective clinical treatment strategy for postoperative cancer treatment.
  • EM-NPs the nanoparticle system formed by encapsulating PLGA in the inner membrane of Escherichia coli
  • EM-NPs the cell membrane encapsulating PLGA formed by the cells in the 4T1-luciferase triple-negative breast cancer tissue Nanoparticles (represented by the code TM-NPs later) system
  • 4 Hybrid membrane package composed of E. coli inner membrane and tumor cell membrane A system of nanoparticles formed by PLGA (hereafter represented by the code HM-NPs).
  • the concentration equivalents of total membrane protein in the four products remain the same, and the concentration equivalents of the two membrane proteins in Mix NPs and HM-NPs remain the same.
  • HM-NPs The preparation method of HM-NPs is as follows:
  • the operation is: inoculate Escherichia coli in LB liquid medium for culture, and expand overnight to an OD600 value of 1.2; add lysozyme with a final concentration of 2mg/mL, and Incubate for 1 hour at 37°C to lyse the cell wall; centrifuge at 3000g for 5 minutes at 4°C, discard the supernatant to obtain protoplasts; add extraction buffer (extraction buffer), use density gradient centrifugation to obtain the inner membrane of E. coli; resuspend and save Standby at -80°C;
  • each BALB/c mouse is subcutaneously inoculated with 200,000 tumor cells, and the tumor volume reaches about 300mm 3 and surgically removed under aseptic conditions Tumor tissue; cut the resulting tumor tissue into small pieces with scissors, and then add collagenase IV (1.0mg ⁇ mL -1 ), deoxyribonuclease DNase (0.1mg ⁇ mL -1 ) and hyaluronidase (0.1mg ⁇ ML -1 ) GBSS solution, placed at 37°C for 15 minutes to digest the tumor tissue; place the digested tissue on a 70 ⁇ m filter for grinding, and then filter it through a 70 ⁇ m filter.
  • collagenase IV 1.0mg ⁇ mL -1
  • deoxyribonuclease DNase 0.1mg ⁇ mL -1
  • hyaluronidase 0.1mg ⁇ ML -1
  • the filtrate is centrifuged and resuspended to obtain a single Cell suspension; add extraction buffer containing mannitol, sucrose, bovine serum albumin (BSA), Tris hydrochloric acid, EGTA and phosphatase-protease inhibitor mixture, and then use cell disrupter 30W ultrasound to disrupt cells under ice bath conditions After 5 minutes, go through the centrifugal speeds of 3000g, 10000g and 100000g, and finally obtain the cell membrane derived from tumor tissue; resuspend and freeze the cell membrane at -80°C for later use;
  • BSA bovine serum albumin
  • step (4) of the preparation method of HM-NPs to prepare PLGA nanoparticles by double-emulsion method, mix 50 ⁇ L of the prepared 2mg/mL bacterial inner membrane EM with 500 ⁇ L of the prepared 1mg/mL PLGA polymer nanoparticle solution, Then pass through a liposome extruder with a filter membrane pore size of 200 nm, and squeeze back and forth 13 times in total (one back and forth counts as 1 time), and finally EM-NPs are obtained.
  • TM-NPs The preparation method of TM-NPs is as follows:
  • step (4) of the preparation method of HM-NPs to prepare PLGA nanoparticles by double-emulsion method, and mix 50 ⁇ L of the prepared 2mg/mL tumor cell membrane TM with 500 ⁇ L of the prepared 1mg/mL PLGA polymer nanoparticle solution , And then pass through a liposome extruder with a filter membrane pore size of 200 nm, and squeeze 13 times back and forth in total (one back and forth counts as 1 time), and finally TM-NPs are obtained.
  • the prepared EM-NPs, TM-NPs and HM-NPs hybrid film nanoparticles were observed with a transmission electron microscope (FEI, Tecnai G2 20S-TWIN, 200KV, USA).
  • FEI transmission electron microscope
  • Tecnai G2 20S-TWIN, 200KV, USA The results are shown in Figure 1.
  • the results show that the above three
  • the shape of the nano-particles of the various membranes is regular round, and the size is relatively uniform.
  • the scale in the figure is 100nm.
  • the prepared HM-NPs hybrid film nanoparticles were characterized by dynamic light scattering (Zetasizer NanoZS), and the results are shown in Figure 2.
  • the figure shows that the prepared HM-NPs hybrid film nanoparticles are mainly distributed in particle size It is between 100-300nm and reaches a peak at 164.2nm (accounting for 15.8%), indicating that the prepared hybrid membrane nanoparticles have a good particle size distribution, which is conducive to entering the cell to play a biological role.
  • Example 2 explores the effects of the four products prepared in Example 1 on enhancing innate immunity and specific immunity.
  • the specific content includes:
  • BMDCs dendritic cells
  • BMDC dendritic cells
  • FITC-CD11c, PE-CD80 and PE-CD86 flow cytometry antibodies to stain the cells in the dark for 20 minutes;
  • spleen lymphocytes of each group of mice and the tumor cell membranes of the same source in the HM-NPs vaccine were incubated in a 37°C 5% CO 2 incubator for 24 hours;
  • HM-NPs hybrid membrane nanoparticles have an improved potential to enhance innate and specific immunity.
  • Example 2 explores the effects of the four products prepared in Example 1 on stimulating the immune system of mice to secrete inflammatory factors and chemokines.
  • the specific steps are as follows:
  • HM-NPs hybrid membrane nanoparticles have a certain effect on the secretion of inflammatory factors and chemokines by the systemic immune system. Compared with the blank control group, only the pro-inflammatory cytokines IL-6 and IL-1 ⁇ increased significantly, and the other 11 inflammatory factors and chemokines remained basically unchanged. It shows that the HM-NPs hybrid membrane nano-vaccine has good biological safety.
  • each group of membrane nanoparticles containing the fluorescent dye IR780 The hydrophilic fluorescent dye IR780 is encapsulated in PLGA by the double emulsion method, specifically: Take 200 ⁇ L of the hydrophilic fluorescent dye IR780 at a concentration of 1mg/mL, Add 1mL of PLGA dichloromethane solution with a concentration of 10mg/mL, colostrum for 3min; add 1% surfactant sodium cholate, re-emulsion for 5min; avoid light and rotate for 10min to remove organic solvent dichloromethane; 11000g centrifugation for 15min, and use After washing three times and twice, PLGA nanoparticles carrying fluorescent dye IR780 were obtained. Then it is mixed with each group of film particles prepared in advance. The specific steps and feeding ratio are the same as in Example 1;
  • mice were injected subcutaneously into the right back of each group of fluorescently-labeled membrane nanoparticles. After 12 hours, the lymph nodes of each group were taken, and fluorescent imaging was performed using the small animal optical 3D in vivo imaging system, and the LivingImage software was used for analysis.
  • BALB/c mice take 1mL of blood, dissolve it in 2mL PBS solution, and centrifuge at 3000rpm for 10min;
  • mice The female BALB/c mice were randomly divided into 5 groups, each with 6 mice, and 200,000 4T1-luciferase breast cancer cells were subcutaneously inoculated on the right back of each mouse to construct a mouse breast cancer model (day 0 );
  • the cell suspension was ultrasonically disrupted in an ice bath with a cell disruptor (35W, 5min); the disrupted cell suspension was centrifuged (3000g, 5min, 4°C) ); Take the supernatant from the previous step and centrifuge again (10000g, 10min, 4°C); Take the centrifuged cell supernatant and add it to the ultracentrifuge tube for ultracentrifugation (100000g, 2h, 4°C); discard after ultracentrifugation Clear, resuspend in 1mL PBS to obtain the tumor cell membrane fragments TM derived from surgical resection, and store at -80°C;
  • mice were randomly divided into 5 groups with 6 mice in each group;
  • Example 2 explores the effects of the four products prepared in Example 1 on inhibiting tumor recurrence and tumor cell re-stimulation experiments in CT26 colon cancer tumor-bearing mice undergoing tumor resection.
  • the experimental operation flow chart of this example is as follows Shown in Figure 16. Specific steps are as follows:
  • the cell suspension was ultrasonically disrupted in an ice bath with a cell disruptor (35W, 5min); the disrupted cell suspension was centrifuged (3000g, 5min, 4°C) ); Take the supernatant from the previous step and centrifuge again (10000g, 10min, 4°C); Take the centrifuged cell supernatant and add it to the ultracentrifuge tube for ultracentrifugation (100000g, 2h, 4°C); discard after ultracentrifugation Clear, resuspend in 1mL PBS to obtain the tumor cell membrane fragments TM derived from surgical resection, and store at -80°C;
  • mice Re-randomize the mice into 5 groups with 15 mice in each group;
  • mice vaccinated with HM-NPs vaccine in 1 were randomly divided into 3 groups after 60 days, with 5 mice in each group;
  • mice One group of mice was inoculated with 200,000 4T1 breast cancer cells subcutaneously on the right back of the mice, another group of mice was inoculated with 200,000 CT26 colon cancer cells subcutaneously on the right back of the mice, and the remaining group of mice were not inoculated with tumor cells ( Inoculate normal saline as a control (Control, Ctrl);
  • mice The pro-inflammatory factors (IL-6, TNF- ⁇ and IL-1 ⁇ ) secreted by the mice were significantly higher than those of the other two groups, and the IFN- ⁇ secreted by them was also increased to a certain extent compared with the mice inoculated with 4T1 breast cancer cells.
  • This example explores the dosage ratio of the gram-negative bacterial intima and the solid tumor cell membrane derived from surgical resection in the HM-NPs hybrid membrane nanoparticles involved in the present invention, which specifically includes the following content:
  • Example 1 Prepare hybrid membrane nanoparticles with different membrane protein concentration ratios according to the method of Example 1, which are specifically set as: a single gram-negative bacterial intima EM, a single surgical resection-derived solid tumor cell membrane TM, and leather
  • the mass ratios of the endometrium EM of Langerhans-negative bacteria to the solid tumor cell membrane TM derived from surgical resection are 1:100, 1:75, 1:50, 1:25, 1:10, 1:5, 1:3, 1: 1. 1:0, 0:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1 or 100:1 hybrid membrane, with an equal volume of PBS solution as a control Group (Control);
  • BMDC BALB/c mouse bone marrow-derived dendritic cells
  • a membrane nanoparticle system is prepared, which is a hybrid membrane composed of the inner membrane of Klebsiella pneumoniae (CG43 strain) and the cell membrane of cells in HepG2 liver cancer tissues, which is wrapped in a zirconium (Zr)-based MOF (metal organic framework). )
  • the system of nanoparticles formed (hereinafter represented by the code HM-NPs-2).
  • the preparation method is as follows:
  • a membrane nanoparticle system is prepared, which is a nanoparticle formed by a hybrid membrane composed of the inner membrane of Brucella PBP 39 and the cell membrane of cells in SN12-PM6SN12-PM6 renal cancer tissues wrapped with MSN (Mesoporous Silica) (Hereafter represented by the code HM-NPs-3) system.
  • the preparation method is as follows:
  • Example 9 This example explores the effects of enhancing innate immunity and specific immunity of the two products prepared in Example 9 and Example 10.
  • the specific content includes:
  • HM-NPs-2 and HM-NPs-3 hybrid membrane nanoparticles promote BMDC to secrete IL-6, TNF- ⁇ , and IL-1 ⁇ at levels similar to HM-NPs, and have a significant potential to enhance the body's innate immunity .
  • HM-NPs-2 and HM-NPs-3 hybrid membrane nanoparticles promote the expression of CD80 and CD86 on the surface of BMDC cells similar to HM-NPs, and have significant potential to stimulate the maturation of DC cells.
  • HM-NPs-2 and HM-NPs-3 hybrid membrane nanoparticles have significant potential to enhance innate and specific immunity.
  • BMDC bone marrow-derived dendritic cells
  • This application studies the innate immune stimulatory response and specific immune stimulatory response of different membrane nanoparticles in vivo and in vitro, the characterization of the physical and chemical properties of different membrane nanoparticles, and the proteomics characterization.
  • animal experiments use electronic vernier calipers to measure tumor volume. When the volume of the tumor reaches about 300 mm 3 , it is removed by surgery. The mice were then immunized with different vaccine formulations on the 3rd, 5th and 9th days after the operation.
  • mice from the HM-NP vaccination group were randomly divided into three groups, and 60 days later, they were inoculated with CT-26 colon adenocarcinoma cells or 4T1 breast cancer cells.
  • BMDCs are produced from fresh bone marrow mesenchymal stem cells of C57BL/6J mice by induced differentiation. The cells were cultured in RPMI 1640 medium containing 10% FBS and penicillin-streptomycin. The cells were added to a 1.5 mL centrifuge tube (40,000 cells/tube), and then different vaccine preparations with a final concentration of 0.4 mg ⁇ mL -1 were added, and the cells were co-cultured at 37° C. for 8 hours.
  • the cells were collected by centrifugation at 500 ⁇ g for 3 minutes, and resuspended in FACS buffer (RPMI 1640 medium supplemented with 2% FBS).
  • FACS buffer RPMI 1640 medium supplemented with 2% FBS.
  • BD Accuri C6 FACS flow cytometer (BD Biosciences, San Jose, USA) was used to evaluate the fluorescence signal, and BD Accuri C6 software was used for analysis.
  • Cy5.5-labeled membranes with fluorescently labeled lysosomes were prepared to coat NP and BMDC.
  • EM-NP, TM-NP and HM-NP were co-incubated with BMDC and imaged under a confocal laser scanning microscope (Zeiss 710, Zeiss Microsystems, Germany).
  • BMDC was treated with HM-NP or Mix NP and imaged under a confocal laser scanning microscope.
  • the EM-NP was labeled with the primary antibody FtsZ specific to the bacterial inner membrane and the goat secondary antibody against rabbit IgG (Alexa Fluor@633 goat, Abcam, Cambridge, UK).
  • TM-NP was labeled with the primary antibody Na + /K + -ATPase specific to the tumor cell membrane and the goat secondary antibody to mouse IgG (Alexa Fluor@488 goat, Abcam, Cambridge, UK).
  • BMDCs were added to a 96-well plate (40,000 cells/well) and combined with different nanoformulations with a final concentration of 0.4 mg ⁇ mL -1 at 37°C Incubate for a total of 24 hours. Collect the supernatant, and use the corresponding specific ELISA kit to analyze the concentration of IL-6, TNF- ⁇ and IL-1 ⁇ .
  • BMDCs were added to a 1.5mL centrifuge tube (40,000 cells/tube), and the final concentration of 0.4mg ⁇ mL -1 of different nano-formulations were combined at 37°C. Incubate for 24 hours. The cells were then collected by centrifugation and resuspended in FACS buffer. FITC-CD11c, PE-CD80 and PE-CD86 were used for fluorescent labeling and were analyzed using BD Accuri C6 FACS flow cytometer and BD Accuri C6 software. To assess DC maturation in vivo, female BALB/c mice were subcutaneously inoculated with 4T1 breast cancer cells.
  • HM-NP HM-NP
  • the surgical procedures and vaccination schedule are the same as the above-mentioned research protocol.
  • Spleen cells were collected 12 hours after the last vaccination and incubated with the 4T1 cell membrane (as an antigen) for 24 hours.
  • the ELISPOT kit pre-coated with IFN- ⁇ capture antibody was used to evaluate the IFN- ⁇ release of T cells induced by different membrane NPs.
  • mice were subcutaneously inoculated with 4T1 breast cancer cells. Perform surgical and vaccination procedures as described above. According to the manufacturer's instructions, the ELISA kit based on Luminex magnetic beads is used to detect inflammatory cytokines and chemokines in the serum. The usage of capture magnetic beads and detection antibodies is 1/5 of the recommended amount.
  • mice Female BALB/c or C57BL/6 mice were subcutaneously inoculated with 4T1-Luc, CT26, B16F10 or EMT-6 tumor cells (2 ⁇ 10 5 per mouse). Perform surgical and vaccination procedures as described above. IVIS (Spectrum CT, Perkin Elmer, UK) was used to monitor the bioluminescence signal produced by 4T1-Luc cells. Measure the volume of all tumors with electronic calipers every other day. Calculate the tumor volume according to the following formula:
  • V (L ⁇ W ⁇ W)/2(L, the longest dimension; W, the shortest dimension)
  • a specific ELISA kit was used to detect and challenge the secretion of cytokines in the serum of mice.
  • mice Female BALB/c mice were subcutaneously inoculated with CT-26 colon adenocarcinoma cells (2 ⁇ 10 5 per mouse). The surgical procedure and vaccination schedule are the same as above. Intraperitoneal injection of antibodies depleted a subset of immune cells. Start one day before the start of HM-NP vaccine treatment.
  • Immune cell depletion antibody types and usage are as follows: macrophages with anti-mouse CSF1R (CD115, BioXCell; 300 micrograms injected every other day), NK cells with anti-ASGM1 (anti-mouse Asialo-GM1, BioLegend; 50 ⁇ L/ Injections, twice a week), CD8 + T cells with anti-mouse CD8 (Lyt 2.1, BioXCell; 400 ⁇ g/injection, twice a week) and CD4 + T cells with anti-mouse CD4 ( Twice a week, GK1.5, BioXCell; 200 micrograms/injection, once a week). Measure the volume of all tumors with electronic calipers every other day. The cell depletion of macrophages, NK cells, CD8 + T cells and CD4 + T cells was confirmed by flow cytometry of PBMC.
  • Poly(lactide-glycolic acid copolymer)-OH (75:25, relative molecular mass [Mr] 20,000 Da) was purchased from Jinandaigang Biotechnology Company (Beijing, China).
  • PBS Phosphate buffered saline
  • DMEM DMEM
  • RPMI 1640 medium fetal bovine serum
  • FBS fetal bovine serum
  • the BCA protein assay kit was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA).
  • the pre-coated ELISA kit for LPS detection was purchased from eBioscience (San Diego, California).
  • the rabbit antibody against FtsZ was purchased from Agrisera ( Sweden).
  • IM CVa Mouse anti-Na + /K + -ATPase, ATP5A (IM CVa), Ubiquinol-cytochrome C reductase core protein I (IM Core I), VDAC1/porin (OM Porin), matrix cyclophilin D ( Matrix CypD) and IMS cytochrome C (IMS) antibody Cytc were purchased from Abcam (Cambridge, UK). Anti-rabbit IgG-gold conjugate (gold nano size 5nm) and anti-mouse IgG-gold conjugate (gold nano size 10nm) were purchased from Abcam (Cambridge, UK). Murine IL-4 and GM-CSF were purchased from Beyotime Biotechnology (Shanghai, China).
  • ELISA kits for IL-6, TNF- ⁇ , IL-1 ⁇ and IFN- ⁇ analysis were purchased from eBioscience (San Diego, California).
  • Mouse antibodies against FITC-CD11c, PE-CD80 and PE-CD86 were purchased from BioLegend (San Diego, USA).
  • the pre-coated ELISPOT kit for IFN- ⁇ was purchased from Da Kewei Biotechnology Co., Ltd. (Beijing, China).
  • Luminex beads-based ELISA kits were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Potassium D-fluorescein was purchased from bide Pharmatech Co. Ltd (Shanghai, China).
  • CD115 anti-mouse CSF1R
  • CD8 + T cells Lyt 2.1, anti-mouse CD8
  • CD4 + T cells GK1.5, anti-mouse CD4
  • NK cell depletion antibody anti-mouse Asialo-GM1 was purchased from BioLegend (San Diego, USA). Unless otherwise stated, other chemicals were purchased from Hualide Technology Co., Ltd. (Beijing, China).
  • Mouse 4T1 breast cancer cells, 4T1-luciferase breast cancer cells, CT-26 colon tumor cells, EMT-6 breast cancer cells and B16F10 melanoma cells were purchased from the American Type Culture Collection (ATCC, Manassas, USA) .
  • 4T1, 4T1-Luc, EMT-6 and B16F10 cells were cultured in RPMI 1640 medium containing 10% FBS, 2.5 g ⁇ L -1 glucose and 0.11 g ⁇ L -1 sodium pyruvate.
  • CT-26 cells were maintained in DMEM medium supplemented with 10% FBS. All cells were incubated at 37°C and 5% CO 2 .
  • mice Female BALB/c or C57BL/6 mice were purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd. (Beijing, China) and kept under pathogen-free conditions. All animal experiments were conducted under the guidance of the Institutional Animal Care and Use Committee (IACUC) of the National Center for Nano Science and Technology of the Chinese Academy of Sciences (Beijing, China).
  • IACUC Institutional Animal Care and Use Committee
  • BMDC bone marrow-derived dendritic cells
  • BMDC is produced from BMSCs of female BALB/c mice (6 to 8 weeks old) by inducing differentiation.
  • the bones were collected and placed in 70% ethanol, and then washed with PBS.
  • the two distal ends of the bone were cut open, and the bone marrow was collected by gently washing with RPMI 1640 medium.
  • the red blood cells in the collected cells were lysed with ACK buffer, and the remaining cells were collected by centrifugation at 300 ⁇ g for 5 minutes, and then cultured in RPMI 1640 medium in a 6-well plate (1 ⁇ 10 6 cells/well).
  • E. coli-derived cytoplasmic membrane EM
  • the cytoplasmic membrane was isolated from E. coli strain DH5 ⁇ .
  • cryopreserved E. coli DH5 ⁇ cells were cultured overnight in liquid LB medium at 37°C with shaking at 200 rpm.
  • the bacteria were collected by centrifugation (3000 ⁇ g, 20 min, 4° C.), and washed with PBS 3 times.
  • the cell pellet was resuspended in 10 mL buffer A (1M sucrose, 0.2M Tris-HCl, pH 8.0). Then, lysozyme was added to a final concentration of 2 mg ⁇ mL -1 , and the cells were incubated at 37° C. with shaking at 120 rpm for 1 hour.
  • the membrane was collected by centrifugation at 113,000 ⁇ g at 4°C for 1 hour, resuspended in PBS, and stored at -80°C for subsequent experiments.
  • the BCA protein assay kit was used to quantify the protein concentration of the final membrane product.
  • a proteomics analysis H ⁇ Wayen Biotechnology Co., Ltd., Shanghai, China was performed, and a total of 2,302 proteins were detected.
  • mice were subcutaneously inoculated with murine 4T1 breast cancer cells, 4T1-Luc breast cancer cells or CT-26 colon cancer cells.
  • the tumor about 300mm 3
  • the buffer containing collagenase IV 1.0 mg ⁇ mL -1
  • DNase 0.1 mg ⁇ mL -1
  • hyaluronidase 0.1 mg ⁇ mL -1
  • the precipitate was homogenized in 2mL separation buffer (225mM mannitol, 75mM sucrose, 0.5% BSA, 0.5mM EGTA, 30mM Tris and a mixture of phosphatase and protease inhibitors), and then distributed by ultrasonic treatment (30W) in ice Bathe for 3 minutes to fully destroy the cells.
  • the cell homogenate was centrifuged at 3000 ⁇ g for 5 minutes at 4°C, and the supernatant was collected. The supernatant was further centrifuged at 10,000 ⁇ g at 4°C for 10 minutes, and the obtained supernatant was ultracentrifuged at 100,000 ⁇ g at 4°C for 2 hours.
  • the cell membrane derived from tumor tissue was resuspended in 5mM Bis-Tris buffer (pH 6.0) and stored at -80°C.
  • the BCA protein assay kit was used to determine the protein concentration of the membrane extract.
  • a proteomics analysis H ⁇ Wayen Biotechnology Co., Ltd., Shanghai, China was performed, and a total of 5,353 proteins were detected.
  • HM vesicles hybrid membrane (HM) vesicles
  • the mixed EM and TM were gently shaken at 37°C for 15 minutes using a dry bath incubator before membrane fusion.
  • HM vesicles were formed by repeated physical extrusion through a 400nm cut-off extruder (Hamilton Company, Reno, Nevada, USA).
  • Poly(lactide-glycolic acid copolymer)-OH(PLGA) nanoparticles were prepared using double emulsion method.
  • PLGA was dissolved in dichloromethane at a concentration of 10 mg ⁇ mL -1. Then, 1 mL of PLGA solution was mixed with 0.2 mL of sterile water.
  • the mixture was emulsified by ultrasonic treatment (25W) in an ice bath for 3 minutes, then 2 mL of 1% sodium cholate was added, and emulsification was performed by ultrasonic treatment (30W) in an ice bath for 5 minutes.
  • the emulsion was added dropwise to 10 mL of 0.5% sodium cholate solution and stirred at room temperature for 30 minutes.
  • PLGA NP was harvested by centrifuging the emulsion at 10,000 ⁇ g for 15 minutes and washing twice in sterile water.
  • HM-NP HM-NP
  • PLGA NP and HM vesicles were again co-extruded repeatedly with a 200:1 cut-off extruder (Hamilton Company) and a 5:1 mass ratio of polymer to membrane protein.
  • Mouse anti-Na + /K + -ATPase, ATP5A (IM CVa), Ubiquinol-cytochrome C reductase core protein I (IM Core I), VDAC1/Porin (OM Porin), matrix cyclophilin D (Matrix CypD) and IMS Cytc (IMS Cytc) antibodies are also used for detection; these are tumor-derived membrane markers.
  • the second antibody used is an anti-rabbit IgG HRP conjugated antibody, while for other primary antibodies, the second antibody is an anti-mouse IgG conjugated HRP antibody.
  • immunogold staining was performed. The sample was deposited on a carbon-coated copper grid.
  • the grid was blocked with 1 wt% bovine serum albumin (BSA) in PBS.
  • BSA bovine serum albumin
  • the EM-NP grid was stained with 10 ⁇ L of anti-rabbit FtsZ (0.5 mg ⁇ mL -1 ), and the TM-NP grid was stained with 10 ⁇ L of anti-mouse Na + /K + -ATPase (0.5 mg ⁇ mL -1 ). After one hour of incubation, the samples were washed 6 times in 1% BSA.
  • the EM-NPs grid was then stained with 10 ⁇ L of anti-rabbit IgG-gold conjugate (gold nanometer size of 5 nm) and diluted 1:20 with 1% BSA.
  • the TM-NP grid was stained with 10 ⁇ L of anti-mouse IgG-gold conjugate (gold nano size is 10 nm) and diluted 1:20 with 1% BSA. After one hour of incubation in the dark, the grid was washed 8 times with PBS. The samples were then fixed in 50 ⁇ L of 1% glutaraldehyde in PBS, and washed with sterile water 8 times, each for 2 minutes. Finally, the sample grid was stained with 10 ⁇ L of 1% uranyl acetate and inspected using TEM (HT7700, HITACHI, Japan). The HM-NP grid is prepared according to the same procedure.
  • a mixture of antibodies against FtsZ and Na + /K + -ATPase was used as the primary antibody.
  • HM-NP The engineering design of HM-NP includes three steps: 1) Preparation of EM-TM hybrid membrane (HM) vesicles, 2) Synthesis of lactide-glycolic acid polymer nanoparticles (PLGA NPs) and 3) HM coating to PLGA NP up (Figure 29A).
  • EM was isolated from E. coli strain DH5 ⁇
  • TM was isolated from autologous tumor cell membrane
  • LC-MS liquid chromatography-mass spectrometry
  • the Venn diagram in Figure 22 illustrates that the membrane protein difference between the four batches of different samples of EM ( Figure 22 left; E1, E2, E3 and E4) and TM ( Figure 22 right; T1, T2, T3 and T4) is very small .
  • EM and TM 1:100, 1:75, 1:50, 1:25, 1:10, 1:5, 1:3, 1:1, 1:0, 0:1, 3: 1, 5:1, 10:1, 25:1, 50:1, 75:1 or 100:1 protein mass ratio, fusion of different ratios of membranes, repeated extrusion through liposomes with 400nm filter membrane pore size To prepare HM.
  • Proteome Discoverer 2.4 software was used to analyze the protein in EM. Accession Number (Protein Accession Number): The unique identifier assigned to the protein by the FASTA database used to generate the report is displayed by default. Abundance: Display the abundance value of the sample before scaling and normalization. The data is expressed as the mean ⁇ standard deviation of 4 biologically independent plasma membrane samples of E. coli DH5 ⁇ .
  • these bacterial inner membrane characteristic proteins may comprise proteins or functionally active fragments thereof selected from the following group: cell division protein FtsZ, inner membrane protein YhcB, inner membrane protein translocation enzyme YidC, cell division protein NlpI, ABC transporter MsbA, Inner membrane transporter TatA, inter-membrane phospholipid transport system lipoprotein MlaA, inner membrane protein TolQ, inter-membrane transport lipoprotein PqiC, outer membrane protein TolC, outer membrane introduced protein FimD, outer membrane porin OmpC, inter-membrane transport protein PqiB, Major outer membrane lipoprotein Lpp, membrane-bound hemolytic wall protein transglycosylase MltB, UPF0194 membrane protein YbhG, putative membrane protein IgaA homolog, transmembrane phospholipid transport system lipoprotein MlaA, inner
  • Table 2A LC-MS identifies the protein expression profile of tumor membranes derived from surgically resected tumors
  • Protein accession number The unique identifier assigned to the protein by the FASTA database used to generate the report is displayed by default.
  • Abundance Display the abundance value of the sample before scaling and normalization. Data are expressed as the mean ⁇ standard deviation of 4 biologically independent tumor membrane samples from surgically resected tumors.
  • the characteristic proteins of these other organisms may include proteins or functionally active fragments thereof selected from the following group: antigen peptide transporter 1, H-2 class II histocompatibility antigen ⁇ chain, tyrosine protein kinase SYK, high affinity Immunoglobulin epsilon receptor subunit ⁇ , Ras-related C3 botulinum toxin substrate 2, tyrosine protein kinase BTK, receptor type tyrosine protein phosphatase C, Na + /K + -ATPase, ATP5A (IM CVa), ubiquinone cytochrome C reductase core protein I (IM Core I), VDAC1/porin (OM Porin), matrix cyclophilin D (Matrix CypD), interstitial cytochrome C (IMS Cytc), basement membrane Specific heparan
  • TEM images show that all three membrane vesicle preparations (EM, TM and HM) have similar sizes, with a diameter of about 100 nm. Compared with TM and HM vesicles, the Zeta potential of EM vesicles is more negative ( Figures 24A-24C).
  • PLGA NP was prepared using the double emulsion method.
  • HM-NP was obtained by repeatedly co-extruding the PLGA core and HM vesicles through a liposome extruder with a 200 nm filter membrane pore size. Similarly, by repeatedly co-extruding the PLGA core and EM vesicles or TM vesicles through a liposome extruder with a 200nm filter membrane pore size, EM coated PLGA NP (EM-NP) and TM coatings can be produced PLGA NP (TM-NP).
  • TEM images show that HM-NPs have a uniform spherical nanostructure with a core and a shell ( Figure 29B and Figure 26A-26B).
  • the diameters of the PLGA core and membrane shell of HM-NP are 86.83 ⁇ 7.41nm and 14.16 ⁇ 2.07nm, respectively (FIGS. 26A-26B).
  • the surface charge of HM-NPs is -21.3mV (Figure 29C), which is basically unchanged compared with the charge of HM vesicles ( Figure 24C), indicating that At this ratio, the HM surface is complete and has reached saturation.
  • Dynamic light scattering (DLS) analysis showed that the hydrated particle size of HM-NP was about 180 nm ( Figure 29C), which was similar to EM-NP and TM-NP.
  • HM-NP membrane-coated NPs
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • TM-NP TM-NP
  • FtsZ is a bacterial cell division protein, which exists on both EM-NP and HM-NP ( Figure 29D top), and a series of mammalian cell proteins, including Na + /K + -ATPase, ATP5A (IM CVa), Ubiquinol-cytochrome C reductase core protein I (IM Core I), VDAC1/Porin (OM Porin), matrix cyclophilin D (Matrix CypD) and IMS Cytc (IMS Cytc) are all clearly present in TM- NP and HM-NP ( Figure 29D bottom).
  • HM-NPs enhance the uptake of tumor antigens, activate the expression of TLRs on the surface of BMDC, and simultaneously deliver the antigen and adjuvant to the detection of BMDC
  • TLRs can recognize PAMPs on the bacterial surface and initiate an innate immune response.
  • EM-NPs membrane nanoparticles
  • TM-NPs membrane nanoparticles
  • Mix NPs and HM-NPs membrane nanoparticles
  • this application first examines the ability and changes of EM-NPs alone to stimulate the secretion of pro-inflammatory cytokines after co-incubation with BMDCs for a certain period of time.
  • concentration gradients (0.12mg/mL, 0.25mg/mL, 0.50mg/mL, 1.00mg/mL) of EM-NPs and BMDCs were incubated together, and selected different time points (0h, 3h, 8h, 24h, 48h) Collect the cell supernatant of each group, and use the kit to detect the secretion level of the pro-inflammatory cytokine IL-6 in the supernatant.
  • HM-NPs This striking cytokine release profile in HM-NPs is attributable to the adjuvant properties of bacterial cell plasma membrane components, because TM-NPs lack the ability to stimulate BMDC.
  • TM-NPs lack the ability to stimulate BMDC.
  • HM-NPs are more effective than Mix NPs in stimulating the secretion of IL-6 and IL-1 ⁇ (p ⁇ 0.0001).
  • tumor autoantigens and immune adjuvants have a stronger potential to improve the intensity of immune stimulation on the same nanoparticle.
  • this application uses immunofluorescence experiments to further study the co-localization efficiency of EM and TM components in BMDC treated with HM-NP and Mix NP.
  • the red fluorescently labeled FtsZ antibody and the green fluorescently labeled Na + /K + -ATPase antibody were used to identify EM and TM, respectively (Figure 35J).
  • Immunofluorescence images showed that when BMDC was treated with HM-NPs, the co-localization was significantly greater than that in the Mix NP group (p ⁇ 0.0001) (mean 81.53% vs 52.39%; Figure 35K).
  • HM-NP has an enhanced ability to deliver EM and TM to the same DC compared to mixed samples.
  • Co-delivery of antigen and adjuvant to DC can enhance the immunogenicity of tumor antigens and the efficacy of cancer vaccines in immunotherapy.
  • the results of this application verify that HM-NP can facilitate such co-presentation.
  • HM-NPs promote the maturation of DCs in LNs and activate the detection of tumor membrane antigen-specific T cells
  • mice were subcutaneously inoculated with murine 4T1 breast cancer cells. When the volume of the tumor reaches about 300 mm 3 , it is removed by surgery. Most of the tumor tissue of each mouse was excised to prepare vaccine preparations, and the remaining approximately 1% was used to simulate the presence of residual micro-tumors in the operating bed of the clinic. On the second day after surgery, the mice were imaged, tumors were measured and randomly divided into five groups. The mice were immunized with different vaccine formulations on the 3rd, 5th and 9th days after the operation. Twelve hours after the last vaccination, this application assessed the presence of stimulatory molecules on DC in the inguinal lymph nodes.
  • TM-NPs Compared with in vitro (Figure 35H-35I), the efficacy of TM-NP in vivo is enhanced (Figure 37A-37B), which may be because TM-NPs enter mice as damage-associated molecular patterns (Damage-associated molecular patterns, DAMPs) are recognized by sensitized immune cells (such as DCs) in the immune system, which stimulates the maturation of DCs to a certain extent.
  • DAMPs damage-associated molecular patterns
  • the HM-NP treatment group showed the highest expression of costimulatory markers, which is consistent with the in vitro maturation results (Figure 35H-35I).
  • LN lymph nodes
  • this application subcutaneously injects membrane NP labeled with the fluorescent dye IR-780 into BALB/c mice. After 12 hours, the inguinal LN was excised and imaged using an in vivo imaging system (IVIS; Figure 36A). Fluorescence signals were observed in all groups, and the fluorescence intensity of the HM-NP group was 3.3 times that of the EM-NP group and 12.4 times that of the TM-NP group ( Figure 36B), indicating that HM-NPs migrated to LNs most effectively Perform tumor antigen presentation.
  • IVIS in vivo imaging system
  • Enhancing the stimulation of the patient's immune system during cancer immunotherapy and the integration of bacterial membrane components in preparations can lead to life-threatening side effects, such as cytokine storms.
  • the application inoculated female BALB/c mice with murine 4T1 breast cancer cells subcutaneously. Perform surgical and vaccination procedures as described above.
  • the concentration of inflammatory cytokines and chemokines in the serum is detected by an ELISA kit based on Luminex magnetic beads.
  • HM-NPs can enhance both innate immunity and adaptive immunity, and stimulate systemic inflammation with low response, indicating that the preparation has the potential to safely trigger the efficacy of anti-tumor immunotherapy.
  • HM-NP vaccination can induce tumor regression in murine 4T1-Luc tumor model
  • this application sets out to evaluate the ability of HM-NP vaccine to inhibit tumor recurrence (Figure 39A).
  • the present application according to the surgical operation and vaccination procedures, subcutaneously injected murine 4T1-Luc breast cancer cells into female BALB/c mice, and IVIS was used to monitor the subcutaneous tumors of the mice before and after the operation ( Figure 39B). IVIS images showed that the tumor volume gradually increased in all non-HM-NP vaccination groups. As shown in Figure 39C-39E, tumor growth was not affected by EM-NP or TM-NP vaccination.
  • mice treated with the HM-NP vaccine showed stronger tumor suppression ability, with a survival rate of about 92%, further confirming the benefits of using the hybrid membrane nanoplatform to co-deliver tumor antigens and adjuvants.
  • no tumor recurrence was observed in the mice that survived to the end of the experiment (day 60), indicating that the survival rate of the animals was significantly higher than that of other groups.
  • HM-NP vaccine can inhibit the detection of tumor regression in a variety of murine tumor models
  • mice vaccinated with HM-NP did not have tumor recurrence within 60 days, while the median survival time of the Mix NP group was about 37 days ( Figure 40E). It is worth noting that all mice immunized with HM-NPs in this experiment achieved effective tumor recurrence inhibition, with an inhibition rate as high as 100%.
  • CT26 and 4T1-luc models have higher immunogenicity and a stronger response to immunotherapy.
  • this application tested the preparation in two tumor models with lower immunogenicity, B16F10 melanoma and EMT-6 breast tumor models. Similar to the results of this application in the CT26 and 4T1-Luc models, more than 90% of the mice vaccinated with HM-NP did not see tumor recurrence within 60 days, while it was observed in Mix NP and the control group 40%-100% of tumors recur. B16F10 melanoma ( Figure 40F) and EMT-6 breast tumor ( Figure 40G) models. Similarly, the results of this application show that the vaccine based on the hybrid membrane strategy can be applied to a variety of solid tumor models and has significant therapeutic efficacy.
  • This application also compares the effects of the bacterial inner membrane used as a vaccine adjuvant with the commercially available adjuvant monophosphoryl lipid A (MPLA) in a mouse CT-26 tumor model ( Figures 41A-41B).
  • MPLA monophosphoryl lipid A
  • this application also studies the anti-tumor efficacy of hybrid membrane vesicles without PLGA core.
  • EM-based cancer vaccine formulations consistently showed higher treatment than other tumor membrane antigen-containing formulations including HM vesicles and three MPLA-containing groups (TM-NP+free MPLA, TM-MPLA-NP and TM-NP) Efficacy, Figure 41A-41B.
  • HM-NP vaccination can protect the detection of tumor readjustment in CT26 tumor model
  • mice in the HM-NPs vaccination group from the CT-26 tumor model were randomly divided into three groups, and they were respectively inoculated with saline, CT-26 colon adenocarcinoma cells or 4T1 breast cancer cells.
  • mice inoculated with CT-26 tumor cells showed complete tumor elimination and 100% tumor inhibition.
  • the mice in the normal saline group did not have tumor recurrence within 90 days.
  • mice inoculated with 4T1 cells reached 1000 cm 3 after 3 weeks, indicating that the protective effect provided by HM-NP is a CT-26 specific immune response.
  • this application found that compared with the 4T1 vaccinated group, the serum concentration of pro-inflammatory cytokines (including IL-6, IL-1 ⁇ and TNF- ⁇ ) in mice inoculated with CT-26 cells increased.
  • CT26 tumor re-excitation (challenge) model Figure 42C-42E
  • the secretion of the specific immune cytokine IFN- ⁇ also increased in the CT-26 vaccinated group ( Figure 42F).
  • HM-NPs also increased effector memory T cells compared to naive T cells ( Figure 42G-42H). Due to the presence of the same tumor antigen, these results are consistent with a stronger immune response. Therefore, the HM-NP vaccine not only provides immunity against tumor recurrence, but also provides specific long-term protection.
  • Tumor suppression after HM-NP vaccination requires analysis of innate and adaptive immunity
  • NK cells and macrophages play an important role in innate immunity, and CD4 + T cells and CD8 + T cells are essential for adaptive immune responses.
  • CT-26 murine colon adenocarcinoma cells were subcutaneously inoculated to female BALB/c mice. When the volume of the tumor reaches about 300 mm 3 , it is removed by surgery. Then, this application randomly divides the mice into six different groups. Depletion antibodies against immune cell surface markers were injected intraperitoneally (Table 3).
  • mice were inoculated with HM-NP or saline (as a control) 3 times on the 3rd, 5th and 9th days after the operation. As shown in Figure 42I and Figure 44A, when the immune system of the mice vaccinated with HM-NP remains intact, the tumor remission rate reaches 100%.
  • the peripheral blood of the mouse was analyzed by flow cytometry to confirm the depletion of macrophages (CSF1R antibody), NK cells (ASGM1 antibody), CD8 + T cells (CD8 antibody) or CD4 + T cells (CD4 antibody) (Figure 43).
  • CSF1R antibody macrophages
  • ASGM1 antibody NK cells
  • CD8 + T cells CD8 antibody
  • CD4 + T cells CD4 antibody
  • HM-NP liver enzymes
  • BUN and CREA serum concentrations of renal function markers
  • This experiment uses the basic process of proteome identification based on mass spectrometry, that is, the MS/MS mass spectrometry data is subjected to a series of optimization processes and then compared with the database for similarity scores for protein identification. Upload each original data file and the corresponding database to Maxquant 1.5.8.3 software. After the search is completed, remove the Reverse and Potential Contaminant proteins, and perform statistics on the search results: The total protein (non- -redundant) 218, total peptide (include redundant) 441, specific peptide (razor+unique) 430, total spectrum (MS/MS Identified) 1230. This application calculates the location of the cell where the protein is located, and finds that the protein located in the cytosol accounts for the largest proportion, as high as 34.63%. The specific results are shown in Tables 4 and 5:
  • Cytoplasm 34.63 Large cytoplasmic ribosomal subunit 10.51 Cytoplasm 10.12 Outer membrane boundary periplasmic space 7.39 Cytoplasmic small ribosomal subunit 7.00 membrane 6.23 Plasma membrane 5.06 Periplasmic space 3.89 Outer cell membrane 3.89 Components of the membrane 3.50 Components of plasma membrane 1.56 Components of the outer cell membrane 1.17 Pore complex 1.17 GroEL-GroES complex 0.78 Exo-deoxyribonuclease VII complex 0.78 Outer membrane 0.78 Nucleosides 0.78 ATP binding cassette (ABC) transporter complex 0.78
  • OmpA Outer membrane protein A, which is commonly found on the surface of gram-negative bacteria and has a ⁇ -barrel structure; functionally, they have the function of providing permeability for the outer membrane and maintaining the stability of the outer membrane structure; in bacterial life activities It plays a vital role in the innate immune system: it can act as a bacterial adhesin and invasin, participate in the formation of biofilms, and also serve as a receptor for a variety of bacteriophages, and is an important target for the innate immune system.
  • OmpC Outer membrane protein C, as a pore protein in the outer membrane of Gram-negative bacteria, allowing small molecules to diffuse passively on the outer membrane.
  • OmpX Outer membrane protein X, an important bacterial outer membrane protein, belongs to the Ail protein family. This protein participates in the adhesion and invasion of bacteria to cells in other bacteria, resists the host's immune defense, etc., and is closely related to the virulence of bacteria. Therefore, it is considered to be a candidate target for vaccine development.
  • OsmE Osmotic induced lipoprotein E, which is related to osmotic pressure response
  • LamB Maltoporin, involved in the transport of maltose and maltodextrin, which is essential for the transport of dextrin containing more than three glucosyl moieties.
  • the hydrophobic pathway of aromatic residues ("greasy slip") is used to guide and select sugars for transport through channels. It can also be used as a receptor for several bacteriophages (including lambda);
  • MipA MltA interacting protein, which can be used as a scaffold protein required to form a complex with MrcB/PonB and MltA.
  • the complex may play a role in the expansion and separation of wall protein vesicles;
  • Lpp The outer membrane lipoprotein Lpp, which interacts covalently and non-covalently with peptidoglycan. This interaction helps maintain the structural and functional integrity of the cell envelope;
  • SlyB the outer membrane lipoprotein SlyB, directly regulated by the PhoP gene, which is mainly involved in regulating the expression of bacterial virulence genes;
  • Outer membrane protein W as the outer membrane pore protein of Gram-negative bacteria, is the receptor for colistin S4, and can be used as a target for drug screening;
  • FhuE receptor FhuE receptor, a receptor protein on the outer membrane of gram-negative bacteria. It is a receptor molecule necessary for the absorption of Fe3 + through cobiotics, ferrous amine B and azaric acid.
  • This experiment uses the basic process of proteome identification based on mass spectrometry, that is, after a series of optimization processes on MS/MS mass spectrometry data, the similarity comparison with the database is scored to perform protein identification.
  • Proteome Discoverer 2.4 to search the database. After the search is over, the Reverse and Potential Contaminant proteins are removed, and the results of the search are counted: The total protein (non-redundant) 2302 was detected in this mass spectrometry search, and the total peptides ( Include 20454 redundant), 19006 specific peptides (razor+unique), and 86026 total spectrum (MS/MS Identified).
  • This application calculates the location of the cell where the protein is located, and finds that the protein located in the intracellular area accounts for the largest proportion. The specific results are shown in Figure 48 and Table 6 below:
  • this application also selected 12 representative proteins, and described their protein scores, molecular weights, and functions. The results are shown in the following table:
  • FtsZ Cell division GTPase, a cell division protein, a protein with clear characteristics in bacterial cell division organs. It accumulates in the middle and early stages of bacterial cell division and plays a vital role in the formation of most bacterial membranes. It has also been considered as the bacterial cytoskeleton counterpart of eukaryotic microtubules;
  • YhcB a bacterial inner membrane protein involved in cytoskeleton and peptidoglycan biosynthesis
  • YidC an inner membrane protein translocator, related to the Sec translocator, is a cofactor for the integration, folding and assembly of inner membrane proteins, which can convert small molecules of inner membrane proteins (especially those with small molecule periplasmic domains) The inner membrane protein) is transported into the inner membrane;
  • MsbA an ABC transporter, and also a lipid A export ATP binding/permease protein
  • TatA An inner membrane transport protein, and a protein translocator protein that does not depend on Sec. It is a member of the Tat family.
  • Tat is a system in E. coli that can transport folded proteins across the membrane, and its signal peptide contains a highly conserved double arginine motif.
  • the members of the Tat family include TatA, TatB, TatC and TatE four proteins, and their complexes form a transport channel on the plasma membrane of E. coli.
  • Most of the substrate proteins transported by the E. coli Tat system are components of the respiratory electron transport chain, which are related to many life activities of E. coli;
  • MlaA A lipoprotein in the inter-membrane phospholipid transport system, responsible for the transport and exchange of phospholipids between the inner and outer membranes of bacteria;
  • TolQ an inner membrane protein, which belongs to the Tol-Pal system protein.
  • the Tol-pal inner membrane system is one of the inner membrane protein systems of gram-negative bacteria.
  • the Tol protein in the system is named and resistant to colicin. Sexually related.
  • Tol-pal is composed of five proteins: TolQ, TolA, TolB, TolR and Pal. Their genes are located on the same operon.
  • TolB is a periplasmic protein, and the proteins TolA, Q, and R are three inner membrane proteins. They interact to form an inner membrane protein complex.
  • TolQ protein is related to bacterial cell division;
  • YebE an inner membrane protein, which belongs to the heat shock response molecular tag of Escherichia coli;
  • the signal recognition particle targets the inner membrane protein to the inner membrane by co-translation, while the SecB pathway targets the secreted protein in the later co-translation or post-translational process;
  • FtsY A signal-recognizing granule protein receptor.
  • the E. coli SRP receptor contains only one subunit FtsY, which is a homolog of mammalian SR ⁇ . Escherichia coli FtsY is evenly distributed between the cytoplasm and the inner membrane. The binding of FtsY to the membrane involves lipid and protein membrane factors, which are probably the Sec-translocon component SecY. The interaction with phospholipids stimulates FtsY GTPase activity;
  • Ffh a signal recognition granule protein, homologous to mammalian SRP54. Ffh is necessary for the growth of E. coli. It has been proposed that SRP54 (Ffh), which binds to ribosome targeting signals in an activated, GTP-bound form, can be used for interaction with SR (FtsY). The GTPases of Ffh and FtsY stimulate each other when forming a complex, and it has been proposed that they can act as GTPase activating proteins. It has been proposed that they can act as GTPase activating proteins with each other;
  • YajC A subunit of the Sec translocator accessory complex, which is involved in the biosynthesis of bacterial inner membrane proteins.
  • Escherichia Gram-negative
  • Staphylococcus Staphylococcus, Gram-positive
  • Bacillus Gram-positive
  • Lactobacillus Lactobacillus
  • Gram-positive false single The ability of five Pseudomonas (Gram-negative) bacteria and cell membranes derived from autologous tumor tissues to inhibit tumor recurrence after surgery.
  • HM-NPs formed by encapsulating PLGA in hybrid membranes composed of different bacterial inner membranes and tumor cell membranes were prepared, including Escherichia, Staphylococcus, Bacillus, and Lactobacillus. (Lactobacillus) and Pseudomonas (Pseudomonas).
  • lactobacillus lactobacillus
  • Pseudomonas Pseudomonas
  • the total membrane protein concentration equivalent of the five products is the same.
  • a separate tumor membrane group and a physiological saline group are included as controls.
  • the mouse colon cancer model is used to prove that the different bacteria are The ability of the membrane to inhibit tumor recurrence after surgery. Specific steps are as follows:
  • the specific operation of extracting the inner membrane of different bacteria is: amplify the bacteria, culture the bacteria in the culture medium to an OD600 value of 1.2; add lysozyme (to lyse the cell wall) to a final concentration of 2 mg/mL, and the bacteria at 37 Incubate for 2-3h at °C; centrifuge to remove the supernatant to obtain protoplasts; add extraction buffer, and use density gradient centrifugation to obtain bacterial inner membrane; resuspend and store at -80°C for later use;
  • SM-NPs, TM-NPs, Mix NPs and HM-NPs were subcutaneously injected into each group of mice on the 10th, 12th and 16th days, and the saline group was used as the control group (Control);
  • This application is used for tumor suppression experiments for liver cancer, stomach cancer, kidney cancer, pancreatic cancer, ovarian cancer, lymphoma, osteosarcoma, glioma, prostate cancer and melanoma.
  • C57BL/6J or Balb/C mice aged 6-8 weeks were randomly divided into 2 groups, 8-10 mice in each group, and different tumor cells were inoculated subcutaneously on the right back of each mouse.
  • the tumor cell types and the number of inoculated tumors and the corresponding mouse strains are shown in Table 7.
  • Different mouse tumor recurrence models were constructed to evaluate the inhibitory effect of this application on various tumors.
  • mice with HM-NPs 100 ⁇ g/mouse prepared with corresponding tumor membranes three times on the 3rd, 5th and 9th days after operation, and use the saline group as the control group (Control);
  • mice Observe and record the tumor growth in mice. When the tumor volume grows to about 1000 mm 3 , the mouse is deemed dead, and the specific time of death of each group of mice is recorded. Draw a survival curve.
  • different tumor cells can be prepared with the inner membrane of E. coli to be uniform in size, with the characteristics of core-shell hybrid membrane nanoparticles, which proves that this application can be used to prepare liver cancer cells, gastric cancer cells, and kidney cancer cells.
  • the hybrid membrane nanoparticle vaccines significantly prolonged the survival period of different tumor-bearing mice compared with the control group (Control) without any treatment.
  • a hybrid membrane is used to wrap the PLGA polymer. After passing through a liposome extruder with a pore size of 200 nm, the surface potential of the hybrid membrane shell can reach -50 mV, and after adding cationic liposomes to the hybrid membrane, the The surface potential of the hybrid membrane shell reaches +50mV, and the result is shown in Figure 51.

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Abstract

一种肿瘤疫苗及其制备方法和应用。具体涉及一种药物组合，其包含第一膜组分，所述第一膜组分包含源自细菌的内膜的膜，所述药物组合还包含源自所述细菌以外其它生物体的组分，以及该药物组合的制备方法和应用。

Description

一种肿瘤疫苗及其制备方法和应用 技术领域
本申请涉及生物医药领域，具体的涉及一种肿瘤疫苗及其制备方法和应用。
背景技术
目前对于癌症的治疗，外科手术仍然是首选的治疗方案，其在癌症的诊断、分期、重建和缓解方面具有很重要的作用。传统放化疗和新型辅助放化疗领域的进步在很大程度上也提高了癌症患者的预后。然而，尽管治疗技术在不断发展，局部肿瘤的复发和转移仍然成为了癌症患者发病率和死亡率的主要来源。很多研究发现，手术操作以及对肿瘤切缘的破坏都可能会使个别肿瘤细胞脱落到外周循环中。同时，手术应激反应还会影响神经-内分泌调节、新陈代谢、炎性反应以及肿瘤细胞周围的免疫微环境，导致免疫抑制。在大手术过后，细胞免疫抑制可以持续数天，而这段时间就可能成为肿瘤入侵免疫系统的空窗期。这时期的免疫抑制常表现为对肿瘤细胞监督的缺失、外周血中NK细胞、细胞毒性T淋巴细胞、树突状细胞以及辅助T淋巴细胞的减少。
目前，用于预防术后肿瘤复发的疫苗类型还比较有限。CN104619833A描述了一种针对癌症的肽疫苗，具体为一种引发CTL的衍生自UBE2T的表位肽，进一步提供了含有衍生自UBE2T的此类表位肽或编码该多肽的多核苷酸作为活性成分的药物组合物。此外，该发明提供了用于治疗和/或防范，和/或预防其术后复发的方法，及用于诱导CTL的方法，用于诱导抗肿瘤免疫的方法，其使用该发明的衍生自UBE2T的表位肽，编码肽的多核苷酸，或呈递肽的抗原呈递细胞，或药物组合物进行。CN109481418A公开了一种抗肿瘤纳米颗粒及其制备方法和应用，提供的抗肿瘤纳米颗粒，包括核心、包覆核心的包裹层及包覆包裹层的免疫层。由免疫佐剂包裹纳米颗粒得到的抗肿瘤纳米颗粒能够达到同时具有光动力治疗和免疫治疗的联合治疗效果。然而目前的预防术后肿瘤复发的疫苗种类有限，而且本领域需要效果更佳、安全性更高、制备便捷且适用范围广的疫苗类型。因此，开发一种新型的效果显著的预防癌症术后复发的肿瘤疫苗及其制备方法是非常有意义的。
发明内容
一方面，本申请提供了一种药物组合，其包含第一膜组分，所述第一膜组分包含源自细菌的内膜的膜，所述药物组合还包含源自所述细菌以外其它生物体的组分。
在一种实施方式中，所述其它生物体包含细胞。
在一种实施方式中，所述其它生物体包含哺乳动物细胞。
在一种实施方式中，所述其它生物体包含肿瘤细胞。
在一种实施方式中，所述其它生物体包含实体瘤细胞。
在一种实施方式中，所述其它生物体选自以下组：乳腺癌细胞、结肠癌细胞、肝癌细胞、胃癌细胞、肾癌细胞、胰腺癌细胞、卵巢癌细胞、淋巴瘤细胞、骨肉瘤细胞、胶质瘤细胞、前列腺癌细胞和黑色素瘤细胞，以及上述中的任意组合。
在一种实施方式中，所述其它生物体的所述组分包含具有免疫原性的组分。
在一种实施方式中，所述其它生物体的所述组分能够引发对所述生物体的免疫应答。
在一种实施方式中，所述其它生物体的所述组分包含源自所述生物体的细胞膜的组分。
在一种实施方式中，所述其它生物体的所述组分包含肿瘤抗原或其功能活性片段。
在一种实施方式中，所述其它生物体的所述组分还包含选自以下组的蛋白或其功能活性片段：抗原肽转运体1、H-2Ⅱ类组织相容性抗原γ链、酪氨酸蛋白激酶SYK、高亲和力免疫球蛋白epsilon受体亚单位γ、Ras相关C3肉毒毒素底物2、酪氨酸蛋白激酶BTK、受体型酪氨酸蛋白磷酸酶C、Na +/K +-ATP酶、ATP5A(IM CVa)、泛醌细胞色素C还原酶核心蛋白I(IM Core I)、VDAC1/孔蛋白(OM Porin)、基质亲环素D(Matrix CypD)、膜间隙细胞色素C(IMS Cytc)、基底膜特异性硫酸乙酰肝素蛋白聚糖核心蛋白(HSPG2)、质膜钙转运ATP酶1(ATP2b1)、内质网膜蛋白复合物亚基1(EMC1)、跨膜蛋白43(TMEM43)、囊泡膜蛋白VIP36(LMAN2)、囊泡相关膜蛋白相关蛋白A(VAPA)、跨膜9超家族成员2(TM9SF2)、跨膜emp24域含蛋白10(TMED10)、脂肪细胞质膜相关蛋白(APAMP)、突触小泡膜蛋白VAT(VAT1)、核孔膜糖蛋白210(NUP210)、质膜钙转运ATP酶4(ATP2b4)、质膜钙转运ATPase 2(ATP2b2)、红细胞带7整合膜蛋白(STOM)、跨膜emp24域含蛋白9(TMED9)、线粒体进口内膜转座酶亚基TIM44(TIMM44)、膜相关孕激素受体组分1(PGRMC1)、ER膜蛋白复合物亚基3(EMC3)、囊泡相关膜蛋白相关蛋白B(VAPB)、膜伯胺氧化酶(AOC3)、囊泡相关膜蛋白7(VAMP7)、高尔基体膜蛋白4(GOLIM4)、膜相关孕激素受体组分2(PGRMC2)、跨膜9超家族成员3(TM9SF3)、跨膜9超家族成员4(TM9SF4)、内质网膜蛋白复合物亚基2(EMC2)、线粒体进口内膜转座 酶亚单位TIM50(TIMM50)、过氧化物酶体膜蛋白11B(PEX11b)、跨膜emp24域含蛋白2(TMED2)、分泌载体相关膜蛋白3(SCAMP3)、硫氧还蛋白相关跨膜蛋白4(TMX4)、过氧化物酶体膜蛋白PMP34(SLC25a17)、过氧化物酶体膜蛋白PEX14(PEX14)、内质网膜蛋白复合物亚基8(EMC8)、干扰素诱导的跨膜蛋白3(IFITM3)、溶酶体相关膜糖蛋白2(LAMP2)、硫氧还蛋白相关跨膜蛋白2(TMX2)、囊泡相关膜蛋白3(VAMP3)、溶酶体相关膜糖蛋白1(LAMP1)、线粒体导入内膜转座酶亚单位Tim8A(TIMM8a1)、溶酶体膜蛋白2(SCARB2)、Ig gamma-2A链C区(IGHG2a)、跨膜9超家族成员1(TM9SF1)、核内膜蛋白Man1(LEMD3)、跨膜emp24域含蛋白4(TMED4)、Thy-1膜糖蛋白(Thy1)、线粒体导入内膜转座酶亚基Tim23(TIMM23)、线粒体导入内膜转座酶亚基Tim9(TIMM9)、线粒体导入内膜转座酶亚基Tim13(TIMM13)、分泌载体相关膜蛋白2(SCAMP2)、分泌载体相关膜蛋白1(SCAMP1)、整合膜蛋白2B(ITM2b)、线粒体进口内膜转座酶亚单位Tim10(TIMM10)、液泡膜蛋白1(VMP1)、生长激素诱导的跨膜蛋白(GHITM)、含死亡结构域的膜蛋白(NRADD)、囊泡相关膜蛋白8(VAMP8)、跨膜前后转化蛋白1(TAPT1)、线粒体膜间空间导入和组装蛋白40(CHCHD4)、糖基化溶酶体膜蛋白(GLMP)、跨膜4结构域超家族A成员6D(MS4A6D)、易位链相关膜蛋白1(TRAM1)、干扰素诱导的跨膜蛋白2(IFITM2)、肌膜相关蛋白(SLMAP)、膜镁转运蛋白1(MMGT1)、核包膜孔膜蛋白POM 121(POM121)、跨膜蛋白C16orf54同源蛋白(AI467606)、液泡ATPase组装整合膜蛋白Vma21(VMA21)、上皮膜蛋白1(EMP1)、膜相关磷脂酰肌醇转移蛋白1(PITPNM1)、小整合膜蛋白15(SMIM15)、核膜整合膜蛋白1(NEMP1)、整合膜蛋白GPR180(GPR180)、分泌载体相关膜蛋白4(SCAMP4)、易位链相关膜蛋白2(TRAM2)、跨膜和卷曲螺旋域蛋白3(TMCC3)、基质膜相关蛋白1(SMAP1)、神经元膜糖蛋白M6-b(GPM6b)、上皮膜蛋白2(EMP2)、高尔基体膜蛋白1(GOLM1)、SID1跨膜家族成员2(SIDT2)、跨高尔基体Golgi网络整合膜蛋白2(TGOLN2)、高尔基体膜蛋白TVP23同系物B(FAM18b)、线粒体内膜蛋白OXA1L(OXA1L)、骨化相关的跨膜蛋白1(OSTM1)、过氧化物酶体膜蛋白PEX13(PEX13)、单侧通过膜和卷曲螺旋结构域蛋白1(SMCO1)、远端膜臂组装复合蛋白1(DMAC1)、分泌载体相关膜蛋白5(Scamp5)、囊性纤维化跨膜电导调节剂(CFTR)、破骨细胞刺激性跨膜蛋白(OCSTAMP)、诱导脂肪存储的跨膜蛋白2(FITM2)和跨膜通道样蛋白5(TMC5)，以及上述中的任意组合。
在一种实施方式中，所述其它生物体的所述组分还包含选自以下组的蛋白或其功能活性 片段：抗原肽转运体1、H-2Ⅱ类组织相容性抗原γ链、酪氨酸蛋白激酶SYK、高亲和力免疫球蛋白epsilon受体亚单位γ、Ras相关C3肉毒毒素底物2、酪氨酸蛋白激酶BTK、受体型酪氨酸蛋白磷酸酶C、Na +/K +-ATP酶、ATP5A(IM CVa)、泛醌细胞色素C还原酶核心蛋白I(IM Core I)、VDAC1/孔蛋白(OM Porin)、基质亲环素D(Matrix CypD)和膜间隙细胞色素C(IMS Cytc)，以及上述中的任意组合。
在一种实施方式中，其包含第二膜组分，所述第二膜组分包含源自所述其它生物体的细胞膜的膜。
在一种实施方式中，所述细菌包含革兰氏阴性菌和/或革兰氏阳性菌。
在一种实施方式中，所述细菌选自以下组的属：大肠杆菌属、葡萄球菌属、芽孢杆菌属、乳杆菌属、克雷伯氏菌属、布氏杆菌、变形杆菌、不动杆菌和假单胞菌属，以及上述中的任意组合。
在一种实施方式中，所述细菌的所述内膜包含选自以下组的蛋白或其功能活性片段：细胞分裂蛋白FtsZ、内膜蛋白YhcB、内膜蛋白易位酶YidC、细胞分裂蛋白NlpI、ABC转运蛋白MsbA、内膜转运蛋白TatA、膜间磷脂转运系统脂蛋白MlaA、内膜蛋白TolQ、膜间转运脂蛋白PqiC、外膜蛋白TolC、外膜引入蛋白FimD、外膜孔蛋白OmpC、膜间转运蛋白PqiB、主要外膜脂蛋白Lpp、膜结合型溶血性壁蛋白转糖基酶MltB、UPF0194膜蛋白YbhG、推定的膜蛋白IgaA同源物、跨膜磷脂转运系统脂蛋白MlaA、内膜蛋白YejM、膜结合型溶血性壁蛋白转糖基酶MltA、跨膜磷脂转运系统结合蛋白MlaC、内膜蛋白YlaC、跨膜转运蛋白YebT、膜结合型溶血性胞壁质转糖基化酶MltC、跨膜磷脂转运系统ATP结合蛋白MlaF、膜间磷脂转运系统结合蛋白MlaD、内膜蛋白YqjE、UPF0053内膜蛋白YfjD、UPF0053内膜蛋白YoaE、内膜型溶胞性胞壁质转糖基酶A、UPF0394内膜蛋白YedE、膜间磷脂转运系统结合蛋白MlaB、内膜蛋白YccF、跨膜磷脂转运系统通透酶蛋白MalE、内膜蛋白YedI、内膜蛋白YgaP、膜间转运蛋白PqiA、UPF0056膜蛋白YhcE、内膜蛋白YbhL、内膜蛋白YhjD、内膜转运蛋白YbaT、内膜蛋白YjjP、内膜蛋白YhaH、内膜蛋白YbjJ、内膜转运蛋白YqeG、UPF0053内膜蛋白YtfL、砷泵膜蛋白ArsB、内膜蛋白YpjD、C型溶菌酶的膜结合溶菌酶抑制剂MliC、UPF0283膜蛋白YcjF、UPF0259膜蛋白YciC、内膜蛋白YgfX、内膜蛋白YbbJ、脂蛋白释放系统跨膜蛋白LolE、内膜蛋白YjcH、蛋白质输出膜蛋白SecG、内膜蛋白YfdC、UPF0324内膜蛋白YeiH、UPF0266膜蛋白YobD、TVP38/TMEM64家族内膜蛋白YdjZ、膜相关蛋白UidC、内膜蛋白YbiR、内膜蛋白YhiM、膜转运蛋白YfcA、内膜蛋白YdgK、膜结合型溶血性壁蛋白转糖基酶F、多药耐药性外膜蛋白 MdtP、UPF0208膜蛋白YfbV、肽聚糖相关脂蛋白、脂蛋白YiaD、脂蛋白YeaY、载脂蛋白N-酰基转移酶、D-蛋氨酸结合脂蛋白MetQ、脂蛋白GfcD、脂蛋白YbjP、脂蛋白YgeR、脂蛋白释放系统ATP结合蛋白LolD、渗透诱导性脂蛋白OsmE、渗透诱导脂蛋白OsmB、脂蛋白YdcL、脂蛋白YajG、脂蛋白NlpE、磷脂酰甘油-脂蛋白二酰甘油基转移酶、脂蛋白YghJ、脂蛋白YedD、脂蛋白YifL、脂蛋白YgdI、脂蛋白YbaY、脂蛋白信号肽酶、脂蛋白YceB、脂蛋白YajI和内膜蛋白YebE，以及上述中的任意组合。
在一种实施方式中，所述细菌的所述内膜包含选自以下组的蛋白或其功能活性片段：FtsZ、YhcB、YidC、NlpI、MsbA、TatA、MlaA、TolQ和YebE，以及上述中的任意组合。
在一种实施方式中，所述药物组合还包含内核。
在一种实施方式中，所述内核包含生物相容性材料。
在一种实施方式中，所述内核包含人工合成材料。
在一种实施方式中，所述内核包含选自以下组的物质：聚乳酸-羟基乙酸共聚物(PLGA)、金属-有机框架材料(MOF)、聚己内酯(PCL)、聚酰胺-胺(PAMAM)、碳纳米管、石墨烯、金纳米颗粒、介孔二氧化硅纳米颗粒、氧化铁纳米颗粒银纳米颗粒、钨纳米颗粒、锰纳米颗粒、铂纳米颗粒、量子点、氧化铝纳米颗粒、羟基磷灰石纳米颗粒、脂质纳米颗粒(LNP)、DNA纳米结构、纳米水凝胶、稀土氟化物纳米晶体和NaYF 4纳米颗粒，以及上述中的任意组合。
在一种实施方式中，所述内核包含选自以下组的物质：免疫佐剂、免疫检查点抑制剂、核酸分子和化学治疗剂，以及上述中的任意组合。
在一种实施方式中，所述内核包含免疫佐剂单磷酰脂质A。
在一种实施方式中，所述内核包含阿霉素、紫杉醇、多西他赛、吉西他滨、卡培他滨、环磷酰胺、氟尿嘧啶、培美曲塞、雷替曲赛、博来霉素、柔红霉素、多柔比星、长春新碱和依托泊苷，以及上述中的任意组合。
在一种实施方式中，所述内核包含吲哚胺2,3-双加氧酶(IDO)抑制剂。
在一种实施方式中，所述内核包含小干扰RNA(siRNA)。
在一种实施方式中，所述内核的直径为约60至约100纳米。
在一种实施方式中，所述内核的直径为约86纳米。
在一种实施方式中，所述药物组合包含外壳，所述外壳包含所述第一膜组分和所述第二膜组分。
在一种实施方式中，所述外壳包含所述第一膜组分和所述第二膜组分融合后的膜。
在一种实施方式中，所述外壳的厚度为约10至约20纳米。
在一种实施方式中，所述外壳的厚度为约14纳米。
在一种实施方式中，所述外壳的直径为约100纳米。
在一种实施方式中，所述外壳的表面电位(Zeta电位)为约+50mV至约-50mV。
在一种实施方式中，所述外壳的表面电位(Zeta电位)为约-21mV。
在一种实施方式中，所述药物组合中的所述第一膜组分与所述第二膜组分的质量比为1:100至100:1。
在一种实施方式中，所述药物组合中的所述第一膜组分与所述第二膜组分的质量比为1:100、1:75、1:50、1:25、1:10、1:5、1:3、1:1、1:0、0:1、3:1、5:1、10:1、25:1、50:1、75:1或100:1。
在一种实施方式中，所述药物组合中的所述第一膜组分与所述第二膜组分的存在和/或比例通过蛋白质印迹法和/或免疫金染色法确认。
在一种实施方式中，所述药物组合包含颗粒，所述颗粒包含所述内核和所述外壳。
在一种实施方式中，所述外壳与内核材料的质量比为约1:1至约1:10。
在一种实施方式中，所述外壳与内核材料的质量比为约1:4至约1:6。
在一种实施方式中，所述颗粒中的脂多糖(LPS)含量与哺乳动物细胞的脂多糖含量相比没有显著差异。
在一种实施方式中，所述颗粒的直径为约70至约120纳米。
在一种实施方式中，所述颗粒的直径为约100纳米。
在一种实施方式中，所述直径通过透射电子显微镜(TEM)测量。
在一种实施方式中，所述颗粒的水合粒径为约150纳米至约250纳米。
在一种实施方式中，所述颗粒的水合粒径为约180纳米。
在一种实施方式中，所述颗粒的表面电位(Zeta电位)为约+50mV至约-50mV。
在一种实施方式中，所述颗粒的表面电位(Zeta电位)为约-21mV。
在一种实施方式中，所述颗粒的水合粒径和/或表面电位通过动态光散射(DLS)仪器测量。
在一种实施方式中，所述颗粒能够在溶液中保持稳定性。
在一种实施方式中，所述稳定性包含所述颗粒保存一段时间之后的水合粒径和/或表面电位与所述一段时间之前相比，没有显著差异。
在一种实施方式中，所述稳定性包含所述颗粒在4摄氏度的磷酸缓冲盐溶液(PBS)中 保存一段时间之后的水合粒径和/或表面电位与所述一段时间之前相比，没有显著差异。
在一种实施方式中，所述一段时间不少于约21天。
在一种实施方式中，其还包含选自以下组的物质：免疫佐剂、免疫检查点抑制剂、核酸分子、化学治疗剂和光敏剂，以及上述中的任意组合。
在一种实施方式中，其还包含免疫佐剂单磷酰脂质A(MPLA)。
在一种实施方式中，其还包含吲哚胺2,3-双加氧酶(IDO)抑制剂。
在一种实施方式中，其还包含小干扰RNA(siRNA)。
另一方面，本申请还提供了一种疫苗，包含本申请药物组合。
另一方面，本申请还提供了一种试剂盒，包含本申请药物组合和/或本申请疫苗。
另一方面，本申请还提供了一种增强目标抗原被免疫细胞摄取的方法，包含提供一种药物组合，所述药物组合包含第一膜组分，所述第一膜组分包含源自细菌的内膜的膜，所述药物组合还包含所述目标抗原。
在一种实施方式中，所述药物组合包含本申请的药物组合。
在一种实施方式中，所述免疫细胞包含免疫呈递细胞。
在一种实施方式中，所述免疫细胞选自以下组：树突细胞(DC)、T淋巴细胞、巨噬细胞和自然杀伤细胞(NK)，以及上述中的任意组合。
在一种实施方式中，所述免疫细胞包含骨髓来源树突状细胞(BMDC)。
在一种实施方式中，所述免疫细胞包含CD8阳性细胞和/或CD4阳性细胞。
在一种实施方式中，本申请的方法可以是体外的或离体的。在一种实施方式中，本申请的方法可以是非预防和非治疗目的的。
另一方面，本申请还提供了一种激活免疫细胞的方法，所述方法包含施用本申请药物组合、本申请疫苗和/或本申请试剂盒。
在一种实施方式中，所述免疫细胞包含免疫呈递细胞。
在一种实施方式中，所述免疫细胞选自以下组：树突细胞(DC)、T淋巴细胞、巨噬细胞和自然杀伤细胞(NK)，以及上述中的任意组合。
在一种实施方式中，所述免疫细胞包含骨髓来源树突状细胞(BMDC)。
在一种实施方式中，所述免疫细胞包含CD8阳性细胞和/或CD4阳性细胞。
在一种实施方式中，所述免疫细胞包含淋巴结和/或脾脏中的所述免疫细胞。
在一种实施方式中，与未施用所述药物组合的免疫细胞相比，施用所述药物组合的所述免疫细胞的所述激活的效果包含选自以下组：增加所述免疫细胞的抗原识别受体的表达水 平、增加的所述免疫细胞的核因子κB(NF-κB)蛋白的表达水平、增加所述免疫细胞的细胞因子的表达和/或分泌水平和增加的成熟的所述免疫细胞的比例，以及上述中的任意组合。
在一种实施方式中，所述抗原识别受体包含模式识别受体(PRR)。
在一种实施方式中，所述抗原识别受体包含Toll样受体(TLR)。
在一种实施方式中，所述抗原识别受体包含TLR1、TLR2和/或TLR6。
在一种实施方式中，与未施用所述药物组合的免疫细胞相比，施用所述药物组合的所述免疫细胞的TLR4的表达基本不变。
在一种实施方式中，所述细胞因子包含促炎细胞因子。
在一种实施方式中，所述细胞因子包含白介素(IL)-6、肿瘤坏死因子(TNF)-α、IL-1β和/或干扰素(IFN)-γ。
在一种实施方式中，成熟的所述免疫细胞包含CD80阳性细胞、CD86阳性细胞和/或效应记忆细胞。
在一种实施方式中，成熟的所述免疫细胞比例包含CD80阳性和/或CD86阳性的所述免疫细胞占CD11c阳性的所述免疫细胞的比例。
在一种实施方式中，成熟的所述免疫细胞比例包含CD44高表达且CD62L低表达的所述免疫细胞占CD8阳性的所述免疫细胞的比例。
在一种实施方式中，本申请的方法可以是体外的或离体的。在一种实施方式中，本申请的方法可以是非预防和非治疗目的的。
另一方面，本申请还提供了本申请药物组合、本申请疫苗和/或本申请试剂盒在制备药物中的应用，所述药物用于增强先天性免疫和/或特异性免疫应答。
在一种实施方式中，与未施用所述药物组合相比，施用所述药物组合促进树突细胞成熟和/或提高淋巴细胞分泌细胞因子。
在一种实施方式中，所述应用基本上不引发全身性炎症反应。
在一种实施方式中，所述应用基本上不引发全身性炎症反应包含，与未施用所述药物组合相比，施用所述药物组合基本上不提高受试者体内血清中的IFN-γ、TNF-α、巨噬细胞趋化蛋白-1(MCP-1)、IL-12p70、IL-10、IL-23、IL-27、IL-17A、IFN-β、粒细胞-巨噬细胞集落刺激因子(GM-CSF)和/或IL-1α的浓度。
在一种实施方式中，所述应用基本上不引发溶血反应、心脏损伤、肝脏损伤、脾脏损伤、肺脏损伤和/或肾脏损伤。
另一方面，本申请还提供了本申请药物组合、本申请疫苗和/或本申请试剂盒，其用于增强先天性免疫和/或特异性免疫应答。
在一种实施方式中，与未施用所述药物组合相比，施用所述药物组合促进树突细胞成熟和/或提高淋巴细胞分泌细胞因子。
在一种实施方式中，所述应用基本上不引发全身性炎症反应。
在一种实施方式中，所述应用基本上不引发全身性炎症反应包含，与未施用所述药物组合相比，施用所述药物组合基本上不提高受试者体内血清中的IFN-γ、TNF-α、巨噬细胞趋化蛋白-1(MCP-1)、IL-12p70、IL-10、IL-23、IL-27、IL-17A、IFN-β、粒细胞-巨噬细胞集落刺激因子(GM-CSF)和/或IL-1α的浓度。
在一种实施方式中，所述应用基本上不引发溶血反应、心脏损伤、肝脏损伤、脾脏损伤、肺脏损伤和/或肾脏损伤。
另一方面，本申请还提供了一种增强先天性免疫和/或特异性免疫应答的方法，包含向有需要的受试者施用本申请药物组合、本申请疫苗和/或本申请试剂盒。
在一种实施方式中，与未施用所述药物组合相比，施用所述药物组合促进树突细胞成熟和/或提高淋巴细胞分泌细胞因子。
在一种实施方式中，所述方法基本上不引发全身性炎症反应。
在一种实施方式中，所述方法基本上不引发全身性炎症反应包含，与未施用所述药物组合相比，施用所述药物组合基本上不提高受试者体内血清中的IFN-γ、TNF-α、巨噬细胞趋化蛋白-1(MCP-1)、IL-12p70、IL-10、IL-23、IL-27、IL-17A、IFN-β、粒细胞-巨噬细胞集落刺激因子(GM-CSF)和/或IL-1α的浓度。
在一种实施方式中，所述方法基本上不引发溶血反应、心脏损伤、肝脏损伤、脾脏损伤、肺脏损伤和/或肾脏损伤。
另一方面，本申请还提供了本申请药物组合、本申请疫苗和/或本申请试剂盒在制备药物中的应用，所述药物用于预防和/或治疗肿瘤。
在一种实施方式中，所述预防和/或治疗肿瘤包含减缓肿瘤的体积增加速度和/或减小所述肿瘤的体积。
在一种实施方式中，所述肿瘤包含肿瘤切除术后未完全切除的所述肿瘤。
在一种实施方式中，抑制肿瘤包含所述肿瘤清除后再次产生的肿瘤。
在一种实施方式中，所述肿瘤包含实体瘤。
在一种实施方式中，所述肿瘤选自以下组：乳腺肿瘤、结肠肿瘤、肝肿瘤、胃肿瘤、肾 肿瘤、胰腺肿瘤、卵巢肿瘤、淋巴瘤、骨肉瘤、胶质瘤、前列腺癌和黑色素瘤以及上述中的任意组合。
另一方面，本申请还提供了本申请药物组合、本申请疫苗和/或本申请试剂盒，其用于预防和/或治疗肿瘤。
在一种实施方式中，所述预防和/或治疗肿瘤包含减缓肿瘤的体积增加速度和/或减小所述肿瘤的体积。
在一种实施方式中，所述肿瘤包含肿瘤切除术后未完全切除的所述肿瘤。
在一种实施方式中，抑制肿瘤包含所述肿瘤清除后再次产生的肿瘤。
在一种实施方式中，所述肿瘤包含实体瘤。
在一种实施方式中，所述肿瘤选自以下组：乳腺肿瘤、结肠肿瘤、肝肿瘤、胃肿瘤、肾肿瘤、胰腺肿瘤、卵巢肿瘤、淋巴瘤、骨肉瘤、胶质瘤、前列腺癌和黑色素瘤以及上述中的任意组合。
另一方面，本申请还提供了一种预防和/或治疗肿瘤的方法，包含向有需要的受试者施用本申请药物组合、本申请疫苗和/或本申请试剂盒。
在一种实施方式中，所述预防和/或治疗肿瘤包含减缓肿瘤的体积增加速度和/或减小所述肿瘤的体积。
在一种实施方式中，所述肿瘤包含肿瘤切除术后未完全切除的所述肿瘤。
在一种实施方式中，抑制肿瘤包含所述肿瘤清除后再次产生的肿瘤。
在一种实施方式中，所述肿瘤包含实体瘤。
在一种实施方式中，所述肿瘤选自以下组：乳腺肿瘤、结肠肿瘤、肝肿瘤、胃肿瘤、肾肿瘤、胰腺肿瘤、卵巢肿瘤、淋巴瘤、骨肉瘤、胶质瘤、前列腺癌和黑色素瘤以及上述中的任意组合。
另一方面，本申请还提供了制备本申请药物组合、本申请疫苗和/或本申请所述试剂盒的方法，包含提供所述源自细菌的内膜的膜。
在一种实施方式中，还包含提供所述源自所述细菌以外其它生物体的组分。
在一种实施方式中，还包含混合所述源自细菌的内膜的膜以及所述源自所述细菌以外其它生物体的组分以提供外壳。
在一种实施方式中，还包含提供内核。
一方面，本发明提供一种预防癌症术后复发的肿瘤疫苗，所述肿瘤疫苗包括杂合细胞膜外壳和内核材料，所述杂合细胞膜外壳包括革兰氏阴性菌内膜和手术切除来源的实体肿瘤细 胞膜。
本发明所涉及的肿瘤疫苗是一种杂合膜包裹内核材料的纳米颗粒，本发明以杂合膜中的手术切除来源的实体肿瘤组织中的细胞膜作为肿瘤抗原，并创造性地以杂合膜中的革兰氏阴性菌内膜作为免疫佐剂，二者可共同递送到同一树突状细胞中，有利用肿瘤抗原的摄取和呈递，能够协同增强机体的先天性免疫和特异性免疫，且具有一定的淋巴结富集能力，具有显著的预防肿瘤切除手术后复发的功效，延长患者的生存期，并提供长效保护机制。
本发明所涉及的肿瘤疫苗杂合膜中包裹的内核材料能够帮助维持杂合膜的刚性结构和稳定性，进而维持杂合膜的免疫增强、淋巴富集、预防肿瘤切除手术后复发的功效；同时可以进一步包载其他生物活性成分例如siRNA或化疗药等，实现更多的功能。
另外，本发明所涉及的肿瘤疫苗生物安全性良好，制备原料易得，制备成本低；且适用范围广，可根据实际需要在疫苗表面修饰各种化学基团，具有广阔的应用前景。
在一种实施方式中，所述细菌内膜与手术切除来源的实体肿瘤细胞膜中蛋白质摩尔量的比例为1:100-100:1，例如1:100、1:75、1:50、1:25、1:10、1:5、1:3、1:1、1:0、0:1、3:1、5:1、10:1、25:1、50:1、75:1或100:1等，例如可以是(2-3):1，上述数值范围内的具体点值均可选择，在此便不再一一赘述。
所述细菌内膜与手术切除来源的实体肿瘤细胞膜中蛋白质摩尔量的比例特定选择为1:100-100:1的数值范围，其中(2-3):1是效果最佳的数值范围。
在一种实施方式中，所述杂合细胞膜外壳与内核材料的质量比为1:(1-10)，例如1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9或1:10等，例如可以是1:(4-6)，上述数值范围内的具体点值均可选择，在此便不再一一赘述。
所述杂合细胞膜外壳与内核材料的质量比特定选择为1:(1-10)的数值范围，因为在此范围内，最终产物的表面电荷较为接近杂合细胞膜外壳的表面电荷。
在一种实施方式中，所述革兰氏阴性菌包括大肠杆菌属、葡萄球菌属、芽孢杆菌属、乳杆菌属、克雷伯氏菌属、布氏杆菌、变形杆菌、不动杆菌或假单胞菌属任意一种或至少两种的组合。
本发明所涉及的革兰氏阴性菌包括但不限于上述类型，其他类型的革兰氏阴性菌的内膜均可获得本发明所涉及的技术效果。革兰氏阴性菌的细胞壁为多层结构，从外到内依次是：外膜层(OM)为磷脂层和脂多糖层；中间为薄薄的肽聚糖层(PGN)；内膜IM(细胞质膜)为脂蛋白层和磷脂层。革兰氏阴性菌内膜基本上不含内毒素(脂多糖LPS)，有好的生物安全性。
在一种实施方式中，所述肿瘤包括乳腺癌、结肠癌、肝癌、胃癌、肾癌、胰腺癌、卵巢癌、淋巴瘤、骨肉瘤、胶质瘤、前列腺癌或黑色素瘤中的任意一种或至少两种的组合。
本发明所涉及的肿瘤包括但不限于上述类型，其他类型的肿瘤均可应用于本发明所涉及的技术方案。
在一种实施方式中，所述内核材料包括PLGA、MOF、PCL、PAMAM、碳纳米管、石墨烯、金纳米颗粒、介孔二氧化硅纳米颗粒或氧化铁纳米颗粒中的任意一种或至少两种的组合。
本发明所涉及的聚合物包括但不限于上述类型，其他具有生物相容性的聚合物均可获得本发明所涉及的技术效果。杂合细胞膜外壳结构为磷脂双分子层，易于包裹在各种内核材料表面，且方法简单，可根据实际需要进行不同组合。
在一种实施方式中，所述预防癌症术后复发的肿瘤疫苗的粒径为100-300nm，例如100nm、150nm、200nm、250nm或300nm等，上述数值范围内的具体点值均可选择，在此便不再一一赘述。
在一种实施方式中，所述肿瘤疫苗的内核材料中还包载免疫检查点抑制剂、IDO抑制剂、siRNA或化疗药中的任意一种或至少两种的组合；所述至少两种的组合例如免疫检查点抑制剂和IDO抑制剂的组合、siRNA和化疗药的组合等，其他任意的组合方式均可选择，在此便不再一一赘述。
另一方面，本发明提供一种如上所述的预防癌症术后复发的肿瘤疫苗的制备方法，其特征在于，所述制备方法包括如下步骤：
(1)分别提取革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜；
(2)将步骤(1)提取到的革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜混合，并用脂质体挤出器挤出，得到杂合膜纳米颗粒；
(3)将步骤(2)得到的杂合膜微粒与纳米颗粒内核混合，并用脂质体挤出器挤出，得到所述预防癌症术后复发的肿瘤疫苗。
在一种实施方式中，步骤(2)所述混合的温度为20-45℃，例如20℃、25℃、30℃、35℃、40℃或45℃等，时间为10-20min，例如10min、12min、15min、18min或20min等，上述数值范围内的具体点值均可选择，在此便不再一一赘述。
在一种实施方式中，步骤(2)所述脂质体挤出器的滤膜孔径为300-500nm，例如300nm、350nm、400nm、450nm或500nm等，上述数值范围内的具体点值均可选择，在此便不再一一赘述。
在一种实施方式中，步骤(2)所述挤出是指累计来回挤压10-15次(一来一回算1次)。
在一种实施方式中，步骤(3)所述混合的温度为20-45℃，例如20℃、25℃、30℃、35℃、40℃或45℃等，时间为10-20min，例如10min、12min、15min、18min或20min等，上述数值范围内的具体点值均可选择，在此便不再一一赘述。
在一种实施方式中，步骤(3)所述脂质体挤出器的滤膜孔径为100-300nm，例如100nm、150nm、200nm、250nm或300nm等，上述数值范围内的具体点值均可选择，在此便不再一一赘述。
在一种实施方式中，步骤(3)所述挤出是指累计来回挤压10-15次(一来一回算1次)。
在一种实施方式中，步骤(3)所述纳米颗粒内核通过双乳法制备得到。具体地，示例性地可以为：
(1)将内核材料溶于有机溶剂，再加入无菌蒸馏水；
(2)混合物超声乳化；
(3)随后与胆酸钠溶液混合，超声乳化；
(4)将乳液逐滴加入胆酸钠溶液，搅拌；
(5)旋蒸；离心；无菌蒸馏水洗涤后重悬得到纳米颗粒内核。
在一种实施方式中，所述有机溶剂包括二氯甲烷、三氯甲烷、丙酮或乙醇中的任意一种或至少两种的组合。
在一种实施方式中，所述有机溶剂与无菌蒸馏水的体积比为(3-7):1，例如3:1、4:1、5:1、6:1或7:1等，上述数值范围内的任意具体点值均可选择，在此便不再一一赘述。
在一种实施方式中，步骤(2)所述超声的温度为0-4℃，例如0℃、1℃、2℃、3℃或4℃等，时间为2-5min，例如2min、3min、4min或5min等，功率为20-30W，例如20W、22W、25W、28W或30W等，上述数值范围内的任意具体点值均可选择，在此便不再一一赘述。
在一种实施方式中，步骤(3)所述胆酸钠溶液的质量分数为1-3％，例如1％、2％或3％等。
在一种实施方式中，步骤(3)所述胆酸钠溶液与有机溶剂的体积比为(1-3):1，例如1:1、2:1或3:1等。
在一种实施方式中，步骤(3)所述超声的温度为0-4℃，例如0℃、1℃、2℃、3℃或4℃等，时间为4-6min，例如4min、5min或6min等，功率为25-35W，例如25W、28W、30W、32W或35W等，上述数值范围内的任意具体点值均可选择，在此便不再一一赘述。
在一种实施方式中，步骤(4)所述胆酸钠溶液的质量分数为0.3-0.7％，例如0.3％、0.5％ 或0.7％等。
在一种实施方式中，步骤(4)所述胆酸钠溶液与有机溶剂的体积比为(8-12):1，例如8:1、10:1或12:1等。
在一种实施方式中，步骤(4)所述搅拌的温度为20-30℃，例如20℃、25℃或30℃等，时间大于10min。
再一方面，本发明提供一种如上所述的预防癌症术后复发的肿瘤疫苗在制备增强机体先天性免疫和特异性免疫应答的药物中的应用。
再一方面，本发明提供一种如上所述的预防癌症术后复发的肿瘤疫苗在制备促进树突状细胞分泌促炎细胞因子的药物中的应用。
再一方面，本发明提供一种如上所述的预防癌症术后复发的肿瘤疫苗在制备促进树突状细胞成熟的药物中的应用。
再一方面，本发明提供一种如上所述的预防癌症术后复发的肿瘤疫苗在制备促进脾脏淋巴细胞分泌IFN-γ的药物中的应用。
再一方面，本发明提供一种如上所述的预防癌症术后复发的肿瘤疫苗在制备刺激全身免疫系统分泌炎症因子和趋化因子的药物中的应用。
相对于现有技术，本发明具有以下有益效果：
(1)本发明所涉及的肿瘤疫苗是一种杂合膜包裹聚合物的纳米颗粒，本发明以杂合膜中的手术切除来源的实体肿瘤组织中的细胞膜作为肿瘤抗原，并创造性地以杂合膜中的革兰氏阴性菌内膜作为免疫佐剂，二者可共同递送到同一树突状细胞中，有利用肿瘤抗原的摄取和呈递，能够增强机体的先天性免疫和特异性免疫，且具有一定的淋巴结富集能力，具有显著的预防肿瘤切除手术后复发的功效，延长患者的生存期，并提供长效保护机制。在动物试验中，其很好地抑制了因患乳腺癌行肿瘤切除术的荷瘤小鼠的术后肿瘤复发，抑制率高达83.3％；完全抑制了因患结肠癌行肿瘤切除术的荷瘤小鼠的术后肿瘤复发，抑制率高达100％。
(2)本发明所涉及的肿瘤疫苗杂合膜中包裹的内核材料能够帮助维持杂合膜的刚性结构和稳定性，进而维持杂合膜的免疫增强、淋巴富集、预防肿瘤切除手术后复发的功效；同时可以进一步包载其他生物活性成分例如siRNA或化疗药等，实现更多的功能。
(3)另外，本发明所涉及的肿瘤疫苗生物安全性良好，制备原料易得，制备成本低，为了获得安全性良好的药物组合或疫苗，本申请应用了细菌内膜的成分作为药物组合的成分之一；且适用范围广，可根据实际需要在疫苗表面修饰各种化学基团，具有广阔的应用前景。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文 的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是EM-NPs，TM-NPs以及HM-NPs杂合膜纳米颗粒的透射电镜图，标尺为100nm；
图2显示的是HM-NPs杂合膜纳米颗粒的动态光散射结果图；
图3A-3C显示的是对照组、EM-NPs组，TM-NPs组，Mix NPs组以及HM-NPs组上清中IL-6、TNF-α以及IL-1β的水平对比图(A为IL-6水平、B为TNF-α水平、C为IL-1β水平)；
图4显示的是对照组、EM-NPs组，TM-NPs组，Mix NPs组以及HM-NPs组上清中CD80和CD86水平对比图(左为CD80水平、右为CD86水平)；
图5显示的是ELISPOT试剂盒检测对照组、EM-NPs组，TM-NPs组，Mix NPs组以及HM-NPs组上清中IFN-γ分泌量的结果图；
图6显示的是利用CTL-ImmunoSpot Plate Reader软件对对照组、EM-NPs组，TM-NPs组，Mix NPs组以及HM-NPs组斑点数目进行统计的结果图；
图7A-7M显示的是对照组、EM-NPs组，TM-NPs组，Mix NPs组以及HM-NPs组小鼠血清中炎症因子和趋化因子分泌水平的结果图(A、B、C、D、E、F、G、H、I、J、K、L、M分别对应于IL-6、IFN-γ、TNF-α、MCP-1、IL-12p70、IL-1β、IL-10、IL-23、IL-27、IL-17A、IFN-β、GM-CSF、IL-1α)；
图8显示的是实施例1制得的产品在体内淋巴结富集效果的活体成像图；
图9显示的是实施例1制得的产品在体内淋巴结富集效果的统计结果图；
图10显示的是HM-NPs杂合膜纳米颗粒的溶血试验结果图；
图11显示的是实施例6的试验操作流程图；
图12显示的是对照组、EM-NPs组，TM-NPs组，Mix NPs组以及HM-NPs组荷瘤小鼠肿瘤荧光强度的活体成像图；
图13显示的是对照组、EM-NPs组，TM-NPs组，Mix NPs组以及HM-NPs组各只荷瘤小鼠肿瘤体积随时间变化图；
图14显示的是对照组、EM-NPs组，TM-NPs组，Mix NPs组以及HM-NPs组荷瘤小鼠肿瘤体积随时间变化图；
图15显示的是对照组、EM-NPs组，TM-NPs组，Mix NPs组以及HM-NPs组荷瘤小鼠的生存曲线图；
图16显示的是实施例7的试验操作流程图；
图17显示的是对照组、Mix NPs组以及HM-NPs组荷瘤小鼠肿瘤随时间变化的照片；
图18显示的是对照组、Mix NPs组以及HM-NPs组荷瘤小鼠的生存曲线图；
图19显示的是术后再激发试验中各组小鼠肿瘤随时间的变化结果图；
图20A-20D显示的是术后再激发试验中各组小鼠血清中IL-6、TNF-α、IL-1β以及IFN-γ表达水平对比图(A、B、C、D分别对应于IL-6、TNF-α、IL-1β以及IFN-γ)；
图21A-21C显示的是杂合膜纳米颗粒中不同膜蛋白浓度比例产品对上清中IL-6、TNF-α以及IL-1β的水平对比图(A、B、C分别对应于IL-6、TNF-α以及IL-1β)。
图22显示的是维恩图展示了来自四个批次的(左)EM和(右)TM的不同蛋白质表达谱。每个大圆圈中数字的总和表示四个生物学独立的重复样本(n＝4)中表达的蛋白质的总数。在EM样品中共检测到2,302个蛋白质，在TM样品中共检测到5,353个蛋白质。圆圈的重叠部分代表共同表达的蛋白质。E1，E2，E3和E4分别代表四批EM样品。T1，T2，T3和T4分别代表四批TM样本。
图23A-23C显示的是用指示的EM与TM的膜蛋白质量比(EM：TM)，1:100、1:75、1:50、1:25、1:10、1:5、1:3、1:1、1:0、0:1、3:1、5:1、10:1、25:1、50:1、75:1或100:1)孵育后，24小时BMDC上清液中促炎细胞因子的浓度结果图。(A至C)使用相应的特异性ELISA试剂盒分析IL-6，TNF-α和IL-1β的产生。n＝3个生物学独立的重复样本。数据表示为平均值±标准差。*p<0.05，**p<0.01。ns表示无显著差异。
图24A-24C显示的是包裹PLGA纳米颗粒内核之前的三种膜囊泡的理化特性。(A)EM，TM和HM囊泡的TEM图像。比例尺，100nm。实验进行了至少三遍(N＝3)。EM，TM和HM囊泡的(B)水合粒径和(C)Zeta电位。n＝3，生物学上独立的重复。数据表示为平均值±标准差。
图25显示的是EM-NP，TM-NP和HM-NP中的LPS含量。在EM的提取过程中，大部分LPS被除去。三个膜NP之间的LPS含量没有显著差异(p>0.9999)(n＝3，生物学上 独立的重复)。数据表示为平均值±标准差。
图26A-26B显示的是核/壳结构的HM-NP示意图。(A)单个HM-NP的代表性TEM图像。(B)基于TEM统计的HM-NP核尺寸(直径)和外壳厚度(n＝26个纳米颗粒)。HM-NP核的直径为86.83±7.41nm(内部线)，壳厚度为14.16±2.07nm(外部线)。实验进行了至少三遍(N＝3)。比例尺，20nm。数据表示为平均值±标准差。
图27A-27B显示的是冻干之前和冻干后重悬在PBS中HM-NP的理化稳定性。(A和B)HM-NPs的大小和Zeta电位在10％重量％的蔗糖中冻干之前和冻干后重悬于PBS后(n＝3，生物学独立的重复)。数据表示为平均值±标准差。
图28A-28B显示的是在4℃条件下不同时间间隔内PBS中EM-NP，TM-NP和HM-NP的粒径和电荷结果图。(A)保存在PBS中3周内，EM-NP，TM-NP和HM-NP的大小和(B)ζ(Zeta)电位(n＝3，生物学独立的重复)。数据表示为平均值±标准差。
图29A-29E显示的是癌症疫苗HM-NP的构建示意图以及EM-NP，TM-NP和HM-NP的表征结果图。(A)制备HM-NP。通过手术从荷瘤小鼠中切除肿瘤以获得TM。用溶菌酶处理扩增的大肠杆菌DH5α以去除细胞壁，低速离心得到原生质体随后使用提取缓冲液制备得到EM。PLGA NP使用双乳液法制备。混合两种膜，并通过400nm滤膜孔径的脂质体挤出器反复物理挤出。然后添加PLGA聚合物纳米颗粒内核，并通过200nm滤膜孔径的脂质体挤出器进行重复的物理挤出，以生成HM-NP。(B)EM-NP，TM-NP和HM-NP的TEM图像。比例尺，100nm。(C)EM-NP，TM-NP和HM-NP的尺寸分布和Zeta电位。(D)EM-NP，TM-NP和HM-NP中特征性蛋白质的蛋白质印迹分析。每个泳道上装有等量的蛋白质(10μg，n＝3个生物学独立的样品)。(E)具有FtsZ(深色箭头，5nm金NPs)和Na +/K +-ATP酶(浅色箭头，10nm金NPs)免疫金标记的EM-NP，TM-NP和HM-NPs的TEM图像用醋酸双氧铀染色。比例尺，100nm。实验进行了至少三遍(N＝3)。
图30显示的是EM-NP，TM-NP和HM-NP的全蛋白质表达谱图。EM-NPs，TM-NPs和HM-NPs的蛋白质谱通过SDS-PAGE解析，然后进行考马斯亮蓝染色。TM矩形：在切除的自体肿瘤组织的肿瘤细胞质膜(TM)上的代表性蛋白质；EM矩形：存在于突出的分子量范围内的蛋白质条带，来自大肠杆菌细胞质膜(EM)。每个泳道上负载等量的蛋白质(10微克)。实验进行了3次，每组3个生物学重复。
图31A-31B显示的是HM-NPs的TEM图像，带有FtsZ(深色箭头，5nm金NPs)和Na +/K +-ATP酶(浅色箭头，10nm金NPs)的免疫金标记，然后用醋酸双氧铀进行负染色。(A)使用三个TEM观察区域计算在HM-NP制剂中同时标记上5nm和10nm金纳米颗粒 (AuNP)的杂合膜纳米颗粒的百分比。如图所示(B)，总共计数了二十个杂合膜纳米颗粒。实验进行了至少三遍。比例尺，100nm。数据表示为平均值±标准差。
图32显示的是EM-NPs-RhoB，TM-NPs-RhoB和HM-NPs-RhoB内化进入BMDC溶酶体区室的激光共聚焦荧光图像。用三种荧光标记的膜纳米颗粒分别与BDMCs共孵育1小时，并通过共聚焦显微镜进行观察。实验进行了3次，每组3个生物学重复。负载罗丹明B的NP：第二列；溶酶体区室：第一列；明场(BF)：第三列；融合图像(Merge)：第四列。比例尺，20μm。
图33显示的是溶酶体区室和膜NP之间的Pearson相关系数分析结果图。n＝20个BMDC，采用Bonferroni的多重比较测试通过单因素方差分析计算统计学显著性。数据表示为平均值±标准差。实验进行了3次，每组3个生物学重复。
图34显示的是用不同浓度(0、0.12、0.25、0.50和1.00mg/mL)的细菌内膜(EM)与BMDCs共孵育，取指定的时间点(0、3、8、24、48小时)的细胞上清液，BMDC中的-NP，通过特异性ELISA试剂盒检测细胞上清液中促炎细胞因子IL-6含量结果图。在所有组中，共孵育24小时后，每组中IL-6的分泌进入平台期。曲线表示48小时内IL-6分泌的变化。IL-6的产生与EM-NPs的浓度呈正相关。数据表示为平均值±标准差。实验进行了3次，每组3个生物学重复。
图35A-35K显示的是HM-NPs增强肿瘤抗原的摄取，激活BMDC表面TLRs表达，同时将抗原和佐剂共递送给BMDC的结果图。(A)通过共聚焦显微镜(孵育1小时)对BMDC中EM-NP，TM-NP和HM-NP(罗丹明B；第二列)与溶酶体区室(第一列)进行共定位分析。第三列，明场。比例尺，5μm。(B)如通过流式细胞术评估的，与BMDC一起孵育8小时后，负载罗丹明B的膜NP的细胞摄取情况(n＝6，生物学独立的样品)。(C)膜包被的纳米颗粒中一系列BMDCs膜TLR蛋白和NF-κB蛋白的蛋白质印迹分析。每个泳道中装有等量的蛋白质。(D)TLR及其下游信号通路激活的示意图。(E-G)与膜NP孵育24小时后，BMDC上清液中三种促炎细胞因子的浓度(n＝6，生物学独立的样品)。(H和I)与膜NP孵育24小时后，CD80 +或CD86 +BMDC的流式细胞仪分析(n＝6，生物学独立的样品)。(J)用混合NP或HM-NP处理8小时的小鼠BMDC的共聚焦激光扫描显微图像的免疫荧光实验。DAPI标记的细胞核：第一列；Na +/K +-ATP酶标记的TM：第二列；FtsZ标记的EM：第三列。比例尺，20μm。(K)样本中TM和EM之间的Pearson相关系数分析(n＝20个细胞)。实验进行了三次。数据表示为平均值±标准差。统计显著性是采用Bonferroni多重比较检验通过单因素方差分析计算得出的，***p<0.001，****p<0.0001，ns表示无显著 性差异。
图36A-36B显示的是HM-NPs在引流淋巴结(LNs)中的积累增强的验证结果图。(A)腹股沟LN的离体光学成像。将IR-780标记的EM-NP，TM-NP或HM-NP皮下注射到Balb/c小鼠体内。LNs(空白组，n＝5只小鼠；EM-NPs，TM-NPs或HM-NPs治疗组，每组n＝6只小鼠)在12小时后收获。(B)图A中平均荧光强度的定量。数据表示为平均值±标准差。实验进行了至少三遍。统计显著性是采用Bonferroni多重比较检验通过双向方差分析计算得出的，****p<0.0001，ns表示无显著差异。
图37A-37G显示的是HM-NPs在体内促进LN中DC的成熟和脾T细胞活化的验证结果图。(A和B)用膜NP皮下接种4T1荷瘤小鼠后腹股沟淋巴结中CD80 +和CD86 +DC的流式细胞术分析。在IFN-γELISPOT分析和流式细胞仪中确定自体膜抗原特异性T细胞的比例。(C-D)IFN-γELISPOT分析的代表性结果以及相应的4T1荷瘤小鼠(每组5只)中斑点的定量数量。(E-F)用膜NP皮下接种4T1荷瘤小鼠后，脾脏中CD3 +CD8 +IFNγ +细胞的百分比(每组5只小鼠)。(G)HM-NPs引起低水平全身炎症反应。使用基于Luminex磁珠的ELISA试剂盒在ProcartaPlex多重免疫测定法中检测血清中的炎性细胞因子和趋化因子(每组n＝5只小鼠)。实验进行了至少三遍。数据表示为平均值±标准差。在图A-D中，采用Bonferroni的多重比较检验通过单因素方差分析计算了统计显著性。在G中，对基于Luminex的ProcartaPlex多重免疫分析的热图进行了分组，并通过双向方差分析和Bonferroni多重比较测试进行了分析，*p<0.05，**p<0.01，***p<0.001，****p<0.0001，ns表示无显著差异。
图38A-38I显示的是HM-NPs引起低水平全身炎症反应的验证结果图。在手术后第3、5和9天对小鼠皮下注射的疫苗制剂(每组n＝5只小鼠)。使用基于Luminex磁珠的ELISA试剂盒检测血清中炎性细胞因子和趋化因子的浓度，并使用BD Accuri C6 FACS流式细胞仪和ProcartaPlex Analysis软件进行分析。数据表示为平均值±标准差。实验进行了三次。统计显著性是采用Bonferroni的多重比较检验通过单因素ANOVA计算得出的。
图39A-39E显示的是在小鼠4T1-Luc小鼠肿瘤模型中，用HM-NPs进行疫苗接种可诱导肿瘤消退的验证结果图。(A)显示动物实验设计的方案。给雌性BALB/c小鼠皮下接种鼠4T1-Luc乳腺肿瘤细胞。当肿瘤的体积达到约300mm 3时，通过手术将其切除。然后在手术后的第3、5和9天用指定的疫苗制剂对小鼠进行免疫，每周通过IVIS成像系统对小鼠肿瘤生长情况进行监测(B)在不同日期携带4T1-Luc肿瘤的小鼠的体内生物发光图像。(C和D)单个和平均肿瘤生长曲线。(complete response，CR)，完整缓解，指所有靶病灶消失，无新病灶出现，且肿瘤标志物正常，至少维持4周。(E)接受每种疫苗处理的小鼠的生存曲 线(每组n＝12只小鼠)。实验进行了至少三遍。数据表示为平均值±标准差。实验的统计显著性是通过双向方差分析(ANOVA)，Bonferroni的生长曲线多重比较测试和用于比较生存曲线的对数秩检验(Mantel-Cox)来计算的，***p<0.001，****p<0.0001。
图40A-40G显示的是HM-NP疫苗接种可在多种小鼠肿瘤模型中抑制肿瘤的复发的验证结果图。(A)显示动物实验设计的方案。给小鼠皮下接种肿瘤细胞。当肿瘤的体积达到约300mm 3时，通过手术将其切除。在手术后第3、5和9天用不同的疫苗制剂免疫小鼠，持续观察60天(B)。对照组，混合NP和HM-NP组中小鼠的图像。CT26肿瘤模型中每组的平均肿瘤生长曲线(C)，单个肿瘤生长曲线(D)和生存曲线(E)(每组n＝12只小鼠)。CR，完整缓解。(F)在B16F10黑色素瘤模型中每组的生存曲线(每组n＝10只小鼠)。(G)在EMT-6肿瘤模型中每组的生存曲线(每组n＝10只小鼠)。数据表示为平均值±标准差。实验进行了至少三遍。通过双向方差分析(ANOVA)，Bonferroni的生长曲线多重比较检验和生存曲线的对数秩检验(Mantel-Cox)来分析统计显著性，***p<0.001，****p<0.0001。
图41A-41B显示的是在小鼠CT-26肿瘤模型中，HM-NP比三种剂型MPLA和肿瘤膜制剂或HM囊泡制剂具有更高的治疗功效验证结果图。给雌性BALB/c小鼠皮下接种CT-26结肠腺癌细胞(每只小鼠2×10 5个)，然后按照上述手术程序和接种时间表进行。MPLA三种疫苗剂型包括TM-NP+游离MPLA，锚定在肿瘤膜上的MPLA(TM-MPLA-NPs)和包含带有肿瘤膜的MPLA的PLGA纳米颗粒(TM-NPs@MPLA)。另外，将不含PLGA的杂合膜囊泡(HM囊泡)皮下接种给术后小鼠，以比较用杂合膜包被的PLGA NP(HM NP)预防肿瘤复发的治疗潜力。(A)每组中单个小鼠的肿瘤生长曲线和(B)平均肿瘤生长曲线和生存曲线。CR：完全缓解。数据表示为平均值±标准差。实验进行了至少三遍。统计显著性是通过双向方差分析(ANOVA)，Bonferroni的生长曲线多重比较检验和对数生存曲线的对数秩(Mantel-Cox)检验计算的，*p<0.05，**p<0.01，****p<0.0001，ns表示无显著差异。
图42A-42I显示的是HM-NP疫苗接种可提供长期的保护性抗肿瘤免疫力；先天性免疫和适应性免疫均对疫苗效力至关重要验证结果图。(A)用HM-NP对术后小鼠进行免疫。在第60天，将接受HM-NP治疗的小鼠随机分为不同的组，并分别接种生理盐水，CT-26或4T1细胞(每组n＝12只小鼠)。60天后用HM-NPs处理并再次接种CT-26或4T1细胞的小鼠的单独肿瘤生长曲线(B)。ELISA测量接种CT-26或4T1细胞的小鼠血清中促炎细胞因子的水平(C)IL-6，(D)IL-1β，(E)TNF-α和(F)IFN-γ的浓度(每组n＝12只小鼠)。从幼稚小鼠或经HM-NPs治疗的，肿瘤根除的小鼠(原发肿瘤接种后90天)中分离脾细胞。(G-H)代表性散点图和CD8 +T细胞中CD44高表达CD62L低表达T细胞亚群的百分比 (％)(每组n＝12只小鼠)。实验进行了至少三遍。(I)消耗特定免疫细胞亚群(NK细胞，巨噬细胞，CD8 +T细胞或CD4 +T细胞)的小鼠平均肿瘤生长曲线，以探索它们对观察到的HM-NPs抑瘤功效的相对贡献(每组n＝5只小鼠)。数据表示为平均值±标准差。统计数据的显著性采用Bonferroni多重比较检验进行了两次方差分析，**p<0.01，***p<0.001，****p<0.0001。
图43显示的是在HM-NP治疗期间消耗特定的免疫细胞亚群结果图。如表3所示，在开始HM-NP治疗前一天开始通过腹腔注射消耗抗体来敲低相应免疫细胞亚群。通过流式细胞术分析小鼠的外周血，确认巨噬细胞(CSF1R抗体)，NK细胞(ASGM1抗体)，CD8 +T细胞(CD8抗体)或CD4 +T细胞(CD4抗体)的耗竭(n＝5，生物学独立的样品)。
图44A-44B显示的是HM-NP治疗后，要有效地抑制肿瘤，先天免疫和适应性免疫都是必需的验证结果图。将雌性BALB/c小鼠皮下接种CT-26结肠腺癌细胞(每只小鼠2×10 5个)。当肿瘤的体积达到约300mm 3时，通过手术将其切除。切除大部分肿瘤组织以制备疫苗制剂，剩余1％以模拟手术床中残留的微肿瘤的存在。在手术后第2天对小鼠进行评估，并随机分为六组(每组n＝5只小鼠)。除对照组外，在手术后第3、5和9天对小鼠进行免疫。在开始HM-NP治疗前一天通过腹腔注射针对指定表面标记的消耗抗体，以消耗小鼠巨噬细胞(CSF1R抗体)，NK细胞(ASGM1抗体)，CD8 +T细胞(CD8抗体)或CD4 +T细胞(CD4抗体)。(A)生存曲线和(B)单个肿瘤生长曲线。CR，完全缓解(每组n＝5只小鼠)。统计显著性是通过双向方差分析(ANOVA)和Bonferroni的生长曲线多重比较测试计算得出的。用于比较生存曲线的对数秩(Mantel-Cox)检验*p<0.05，**p<0.01，ns表示无显著差异。
图45显示的是不同浓度的HM-NP的溶血试验结果图。将一系列浓度的HM-NP加入到红细胞中。3小时后评估并计算溶血百分比。用PBS(0％溶血)和三次水(100％溶血)处理的红细胞用作对照。在所有测试浓度的HM-NP中均未观察到溶血(n＝3，生物学上独立的重复)。
图46显示的是在4T1-Luc模型的肿瘤消退实验终点，来自各组小鼠的主要器官(即心脏，肝脏，脾脏，肺和肾脏)的H&E染色切片的代表性显微图像。实验进行了3次，每组3个生物学重复。比例尺，50μm。
图47A-47D显示的是在4T1-Luc模型的肿瘤消退实验终点，评估所有组小鼠的肝肾功能的结果图。(A至D)在4T1-Luc模型(每组n＝6只小鼠)中，在肿瘤消退实验结束时来自小鼠的血清的生化分析。ALT，丙氨酸转氨酶；AST，天冬氨酸转氨酶；BUN，血液 尿素氮；CREA，肌酐。数据表示为平均值±标准差。使用Bonferroni的多重比较检验，通过单因素方差分析分析统计显著性。
图48显示的是大肠杆菌内膜蛋白的分布情况。
图49A-49B显示的是同属细菌内膜制备HM-NPs杂合膜纳米疫苗抑制结肠癌模型术后复发的结果图。其中(A)不同细菌内膜和肿瘤膜制备的杂合膜纳米颗粒形貌。(B)不同细菌制备的杂合膜纳米颗粒的免疫治疗后小鼠的生存曲线。
图50A-50C显示的是不同肿瘤细胞膜的HM-NPs杂合膜纳米疫苗抑制对应肿瘤模型术后复发的结果图。其中(A)肿瘤模型的构建及免疫程序示意图。(B)不同肿瘤膜细胞膜和细菌内膜制备的杂合膜纳米颗粒形貌。(C)肝癌、胃癌、肾癌、胰腺癌、卵巢癌、淋巴瘤、骨肉瘤、胶质瘤、前列腺癌和黑色素瘤细胞膜制备的杂合膜纳米疫苗HM-NPs免疫治疗后小鼠的生存曲线。
图51显示的是杂合膜包裹PLGA聚合物，通过孔径200nm的脂质体挤出器挤出，通过掺杂不同电荷的聚合物后可使杂合膜外壳的表面电位从+50mV到-50mV。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“细菌内膜”通常是指细菌的一层膜结构。例如，细菌可以多重结构，从外至内依次为细胞壁、细菌外膜、细菌内膜和细胞质。例如，本申请的细菌的内膜可以只是靠近细胞质的内层细菌膜结构。
在本申请中，术语“药物组合”通常是指由一种或多种活性成分的混合或组合产生的产品。术语“药物组合”可以分开、共同或依次地(没有特定时间限制)施用于患者，其中这种施用在患者体内提供治疗有效水平的本申请的第一膜组分和源自所述细菌以外其它生物体的组分。
在本申请中，术语“膜组分”通常是指具有膜结构中成分的组分。例如，本申请的膜组分可以包含天然或人工合成的生物膜组分。例如，本申请的膜组分也可以不包含磷脂组分，本申请的膜组分可以仅包含生物膜的膜蛋白、膜脂质、膜糖分子、膜糖蛋白或膜脂蛋白。例如，本申请的膜组分可以包含膜组分的功能活性片段以及其它任意的结构域。
在本申请中，术语“生物体”通常是指具有边界结构的生物体。例如，本申请的生物体可以是具有生命的物体。例如，本申请的生物体可以是具有细胞结构的细胞生物或不具有细胞结构的病毒生物。例如本申请的生物体可以包含细菌、原核生物、真核生物，以及上述的组织、细胞或非细胞结构。
在本申请中，术语“免疫原性”通常是指能够引起免疫应答(刺激产生特定抗体和/或特定T细胞增殖)。
在本申请中，术语“生物相容性”通常是指材料具有与宿主之间的相容性。例如，生物相容性可以是指材料不引发异常的血液反应，免疫反应和/或组织反应。
在本申请中，术语“革兰氏阳性菌”或“革兰氏阴性菌”通常是指根据革兰氏染色反应的基本特征，细菌可以主要分为两大类：G阳性(G+)和G阴性(G-)。前者经过染色后细菌细胞仍然可以保留初染结晶紫的蓝紫色，后者经过染色后细菌细胞则可以先脱去了初染结晶紫的颜色，带上了复杂蕃红或沙黄的红色。
在本申请中，术语“白介素-6”或“IL-6”通常是指细胞因子的一种。例如，IL-6可以见于GenBank登记号P05231。本申请的IL-6蛋白还可以涵盖其功能活性片段，不限于在细胞中发生的加工和/或修饰后产生的包含IL-6的功能活性片段的物质。例如，本申请的IL-6可以包含IL-6的功能活性片段以及其它任意的结构域。
在本申请中，术语“干扰素-γ”或“IFN-γ”通常是指细胞因子的一种。例如，IFN-γ可以见于GenBank登记号P01579。本申请的IFN-γ蛋白还可以涵盖其功能活性片段，不限于在细胞中发生的加工和/或修饰后产生的包含IFN-γ的功能活性片段的物质。例如，本申请的IFN-γ可以包含IFN-γ的功能活性片段以及其它任意的结构域。
在本申请中，术语“白介素-1β”或“IL-1β”通常是指细胞因子的一种。例如，IL-1β可以见于GenBank登记号P01584。本申请的IL-1β蛋白还可以涵盖其功能活性片段，不限于在细胞中发生的加工和/或修饰后产生的包含IL-1β的功能活性片段的物质。例如，本申请的IL-1β可以包含IL-1β的功能活性片段以及其它任意的结构域。
在本申请中，术语“肿瘤坏死因子-α”或“TNF-α”通常是指细胞因子的一种。例如，TNF-α可以见于GenBank登记号P01375。本申请的TNF-α蛋白还可以涵盖其功能活性片段，不限于在细胞中发生的加工和/或修饰后产生的包含TNF-α的功能活性片段的物质。例如，本申请的TNF-α可以包含TNF-α的功能活性片段以及其它任意的结构域。
在本申请中，术语“脂多糖”通常是指Lipopolysaccharide(英文简写LPS)。例如LPS可以是指革兰氏阴性细菌细胞壁外壁的组成成分，是由脂质和多糖构成的物质(糖脂质)。
在本申请中，术语“FtsZ”通常是指细菌内膜中的一种蛋白。例如，FtsZ可以用于检测杂合膜中是否含有细菌内膜来源的膜组分。例如，FtsZ可以见于GenBank登记号P0A9A6。例如，本申请的FtsZ可以包含FtsZ的功能活性片段以及其它任意的结构域。细菌内膜还可以包含脂多糖(lipopolysaccharide，LPS，俗称内毒素)和磷脂酰乙醇胺(phosphatidylethanolamine，PE)，细胞内膜还可以包含次要脂质类别，例如N-酰化PE(酰基-PE)和O-酰化PG(酰基-PG)等，并可以用于检测杂合膜中是否含有细菌内膜来源的膜组分。脂多糖分子位于细胞外膜外层，可以由亲水的多糖链和疏水的类脂A两部分构成。两亲性分子磷脂酞乙醇胺可以是革兰氏阴性菌细胞内膜中含量最高的脂质分子，约占77％；阴离子脂质磷脂酰甘油(phosphatidylglycerol，PG)约占13％；心磷脂(cardiolipin，CL)约占10％。细菌内膜上富含脂蛋白和脂质。磷脂酰乙醇胺可以是革兰氏阴性菌最重要的磷脂分子，它对于维持细胞的正常形态、细胞的生长分化以及物质合成都起着非常重要的作用。细菌膜可以具有两亲性脂质的多样性，包括常见的磷脂磷脂酰甘油，磷脂酰乙醇胺和心磷脂，较少见的磷脂酰胆碱，磷脂酰肌醇和多种其他膜脂，例如鸟氨酸脂质，糖脂，鞘脂等。
在本申请中，术语“Na+/K+-ATP酶”通常是指细胞膜的一种蛋白。例如，可以是哺乳动物细胞膜的一种蛋白。例如，Na+/K+-ATP酶可以用于检测杂合膜中是否含有细菌内膜以外其它来源的膜组分。例如，Na+/K+-ATP酶可以见于GenBank登记号P13637。例如，本申请的Na+/K+-ATP酶可以包含Na+/K+-ATP酶的功能活性片段以及其它任意的结构域。
发明详述
一方面，本申请提供一种药物组合，其可以包含第一膜组分，所述第一膜组分可以包含源自细菌的内膜的膜，所述药物组合还可以包含源自所述细菌以外其它生物体的组分。例如，本申请的药物组合可以包含源自细菌的内膜的膜，以及源自其它生物体的免疫原性物质。例如，本申请的药物组合可以包含源自细菌的内膜的膜，以及肿瘤抗原。例如，为了制备的便捷本申请的药物组合可以包含源自细菌的内膜的膜，以及抗原所源自的细胞膜和/或非细胞膜结构的外壳。
例如，本申请所述其它生物体可以包含细胞。例如，本申请的其他生物体可以包含非细胞结构。
例如，本申请的其他生物体可以包含哺乳动物细胞。例如，例如，本申请的其他生物体可以包含非哺乳动物细胞，例如可以是原核细胞。
例如，本申请的其他生物体可以包含肿瘤细胞。例如，本申请的其他生物体可以包含实 体瘤细胞。
例如，本申请的其他生物体可以选自以下组：乳腺癌细胞、结肠癌细胞、肝癌细胞、胃癌细胞、肾癌细胞、胰腺癌细胞、卵巢癌细胞、淋巴瘤细胞、骨肉瘤细胞、胶质瘤细胞、前列腺癌细胞和黑色素瘤细胞，以及上述中的任意组合。
例如，所述其它生物体的所述组分可以包含具有免疫原性的组分。例如，所述其它生物体的所述组分可以能够引发对所述生物体的免疫应答。
例如，所述其它生物体的所述组分可以包含源自所述生物体的细胞膜的组分。
例如，所述其它生物体的所述组分可以包含肿瘤抗原或其功能活性片段。例如，所述其它生物体的所述组分可以包含该其它生物体的细胞膜上具有免疫原性的蛋白或其免疫原性片段。
例如，所述其它生物体的所述组分可以包含选自以下组的蛋白或其功能活性片段：抗原肽转运体1、H-2Ⅱ类组织相容性抗原γ链、酪氨酸蛋白激酶SYK、高亲和力免疫球蛋白epsilon受体亚单位γ、Ras相关C3肉毒毒素底物2、酪氨酸蛋白激酶BTK、受体型酪氨酸蛋白磷酸酶C、Na +/K +-ATP酶、ATP5A(IM CVa)、泛醌细胞色素C还原酶核心蛋白I(IM Core I)、VDAC1/孔蛋白(OM Porin)、基质亲环素D(Matrix CypD)和膜间隙细胞色素C(IMS Cytc)，以及上述中的任意组合。
例如本申请的药物组合可以包含第二膜组分，所述第二膜组分包含源自所述其它生物体的细胞膜的膜。例如，本申请的第二膜组分可以是包含具有免疫原性的蛋白的细胞膜。例如，本申请的第二膜组分也可以通过将具有免疫原性的蛋白结合在天然和/或人工的细胞膜上以制备获得。
例如本申请的药物组合，所述细菌可以包含革兰氏阴性菌和/或革兰氏阳性菌。
例如本申请的药物组合，所述细菌选自以下组的属：大肠杆菌属、葡萄球菌属、芽孢杆菌属、乳杆菌属、克雷伯氏菌属、布氏杆菌、变形杆菌、不动杆菌和假单胞菌属，以及上述中的任意组合。例如本申请的药物组合，所述细菌选自以下组：大肠杆菌(Escherichia)、葡萄球菌(Staphylococcus)、芽孢杆菌(Bacillus)、乳杆菌属(Lactobacillus)和假单胞菌(Pseudomonas)。例如本申请的药物组合，所述细菌可以包含大肠杆菌和/或金黄葡萄球菌。
例如本申请的药物组合，所述细菌的所述内膜可以包含选自以下组的蛋白或其功能活性片段：FtsZ、YhcB、YidC、NlpI、MsbA、TatA、MlaA、TolQ和YebE，以及上述中的任意组合。
例如，本申请的药物组合还可以包含内核。例如，本申请的药物组合的内核可以用于支撑本申请的膜组分。
例如，本申请的药物组合的内核还可以包含生物相容性材料。例如，本申请的药物组合的内核还可以包含人工合成材料。例如，本领域公知的可以被本申请的膜覆盖或部分覆盖的颗粒和/或非颗粒结构的材料可以用于本申请药物组合的内核。
例如，本申请的药物组合的内核还可以包含选自以下组的物质：聚乳酸-羟基乙酸共聚物(PLGA)、金属-有机框架材料(MOF)、聚己内酯(PCL)、聚酰胺-胺(PAMAM)、碳纳米管、石墨烯、金纳米颗粒、介孔二氧化硅纳米颗粒、氧化铁纳米颗粒银纳米颗粒、钨纳米颗粒、锰纳米颗粒、铂纳米颗粒、量子点、氧化铝纳米颗粒、羟基磷灰石纳米颗粒、脂质纳米颗粒(LNP)、DNA纳米结构、纳米水凝胶、稀土氟化物纳米晶体和NaYF 4纳米颗粒，以及上述中的任意组合。
例如，本申请的药物组合的内核还可以包含选自以下组的物质：免疫佐剂、免疫检查点抑制剂、核酸分子和化学治疗剂，以及上述中的任意组合。
例如，本申请的药物组合的内核还可以包含单磷酰脂质A(MPLA)、R848、CpG、poly(I:C)、以及上述中的任意组合。
例如，本申请的药物组合的内核还可以包含阿霉素、紫杉醇、多西他赛、吉西他滨、卡培他滨、环磷酰胺、氟尿嘧啶、培美曲塞、雷替曲赛、博来霉素、柔红霉素、多柔比星、长春新碱和依托泊苷，以及上述中的任意组合。
例如，本申请的药物组合的内核还可以包含吲哚胺2,3-双加氧酶(IDO)抑制剂。
例如，本申请的药物组合的内核还可以包含小干扰RNA(siRNA)。
例如，本申请的药物组合的内核的直径可以为约60至约100纳米。例如，本申请的药物组合的内核的尺寸大小可以基本不影响本申请药物组合的效果。例如，本申请的药物组合的内核的直径可以为约60至约100纳米、约70至约100纳米、约80至约100纳米、约90至约100纳米、约60至约90纳米、约70至约90纳米、或约80至约90纳米。例如，本申请的药物组合的内核的直径可以为约86纳米。例如，本申请的内核的直径可以通过透射电子显微镜测量。
例如，本申请的药物组合可以包含外壳，所述外壳可以包含所述第一膜组分和所述第二膜组分。
例如，本申请的外壳可以包含所述第一膜组分和所述第二膜组分融合后的膜。例如，本申请的外壳可以包含天然和/或人工合成的生物膜，以及细菌内膜的蛋白和其它生物体的生 物膜的蛋白。例如，本申请的外壳可以包含天然或人工合成的生物膜，以及细菌内膜的具有免疫原性的蛋白和其它生物体的具有免疫原性的蛋白。例如，为了制备方便，本申请的外壳可以由以下组构成：包含具有免疫原性的蛋白的细菌内膜和包含具有免疫原性的蛋白的其它生物膜。例如，其中来源于细菌内膜的成分可以用于增强受试者的免疫应答；例如，其中来源于其它生物体的成分可以用于引起受试者对该其它生物体的免疫应答。例如，以肿瘤细胞膜作为来源于其它生物体的成分可以用于引起和/或增强受试者对该肿瘤的免疫反应。
例如，本申请的外壳的厚度可以为约10至约20纳米。例如，本申请的外壳的厚度可以为约10至约20纳米、约11至约20纳米、约12至约20纳米、约13至约20纳米、约14至约20纳米、约15至约20纳米、约16至约20纳米、约17至约20纳米、约18至约20纳米、约19至约20纳米、约10至约15纳米、约11至约15纳米、约12至约15纳米、约13至约15纳米、或约14至约15纳米。例如，本申请的外壳的厚度可以为约14纳米。
例如，本申请的外壳的直径可以为约100纳米。例如，本申请的外壳的厚度或直径可以通过透射电子显微镜测量。
例如，本申请的外壳的表面电位(Zeta电位)可以为约+50mV至约-50mV。
例如，本申请的外壳的表面电位(Zeta电位)可以为约-21mV。例如，所述外壳的表面电位(Zeta电位)可以为约+50mV至约-50mV、约-15至约-50mV、约-21至约-50mV、约-25至约-50mV、约+50mV至约-25mV、约+50mV至约-21mV、约-15至约-25mV、约-17至约-25mV、约-19至约-25mV、约-21至约-25mV、约-23至约-25mV、约-15至约-21mV、约-17至约-21mV、或约-19至约-21mV。
例如，本申请的外壳的所述第一膜组分与所述第二膜组分的质量比可以为1：100至100:1。
例如，本申请的外壳的所述第一膜组分与所述第二膜组分的质量比可以为1:100-100:1，例如1:100、1:75、1:50、1:25、1:10、1:5、1:3、1:1、1:0、0:1、3:1、5:1、10:1、25:1、50:1、75:1或100:1。例如，本申请的外壳的所述第一膜组分与所述第二膜组分的质量比可以为1:100、1:90、1:80、1:70、1:60、1:50、1:40、1:20、1:10、1:9、1:8、1:7、1:6、1:5、1:4、1:3、1:2、1:1、0:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1、20:1、40:1、50:1、60:1、70:1、80:1、90:1或100:1。
例如，本申请药物组合中的所述第一膜组分与所述第二膜组分的存在和/或比例可以通过蛋白质印迹法和/或免疫金染色法确认。
例如，本申请药物组合可以包含颗粒，所述颗粒可以包含所述内核和所述外壳。
例如，本申请药物组合的所述外壳与内核材料的质量比可以为约1:1至约1:10。
例如，本申请药物组合的所述外壳与内核材料的质量比可以为约1:4至约1:6。例如，本申请药物组合的所述外壳与内核材料的质量比可以为约1:1至约1:10、约1:2至约1:10、约1:4至约1:10、约1:6至约1:10、约1:8至约1:10、约1:1至约1:6、约1:2至约1:6、或约1:4至约1:6。例如，本申请药物组合的所述外壳与内核材料的质量比可以为约1:1、约1:2、约1:3、约1:4、约1:5、约1:6、约1:7、约1:8、约1:9或约1:10。
例如，本申请药物组合的脂多糖(LPS)含量与哺乳动物细胞的脂多糖含量相比可以没有显著差异。例如，本申请药物组合可以基本不包含来自细菌的脂多糖。
例如，本申请的药物组合的颗粒的直径可以为约70至约120纳米。
例如，本申请的药物组合的颗粒的直径可以为约100纳米。例如，本申请的药物组合的颗粒的直径可以为约70至约120纳米、约80至约120纳米、约90至约120纳米、约100至约120纳米、约110至约120纳米、约70至约100纳米、约80至约100纳米、或约90至约100纳米。例如，所述直径可以通过透射电子显微镜(TEM)测量。
例如，所述颗粒的水合粒径可以为约150纳米至约250纳米。例如，所述颗粒的水合粒径可以为约180纳米。例如，所述颗粒的水合粒径可以为约150纳米至约250纳米、160纳米至约250纳米、180纳米至约250纳米、200纳米至约250纳米、220纳米至约250纳米、240纳米至约250纳米、约150纳米至约180纳米、或160纳米至约180纳米。
例如，本申请的颗粒的表面电位(Zeta电位)可以为约+50mV至约-50mV。例如，本申请的颗粒的表面电位(Zeta电位)可以为约-21mV。例如，所述颗粒的表面电位(Zeta电位)可以为约+50mV至约-50mV、约-15至约-50mV、约-21至约-50mV、约-25至约-50mV、约+50mV至约-25mV、约+50mV至约-21mV、约-15至约-25mV、约-17至约-25mV、约-19至约-25mV、约-21至约-25mV、约-23至约-25mV、约-15至约-21mV、约-17至约-21mV、或约-19至约-21mV。
例如，所述颗粒的水合粒径和/或表面电位可以通过动态光散射(DLS)仪器测量。
例如，所述颗粒可以能够在溶液中保持稳定性。例如，所述颗粒的稳定性可以包含所述颗粒保存一段时间之后的水合粒径和/或表面电位与所述一段时间之前相比，没有显著差异。
例如，所述颗粒的稳定性可以包含所述颗粒在4摄氏度的磷酸缓冲盐溶液(PBS)中保存一段时间之后的水合粒径和/或表面电位与所述一段时间之前相比，没有显著差异。
例如，所述颗粒的稳定性可以包含所述颗粒在4摄氏度的磷酸缓冲盐溶液(PBS)中保 存一段时间之后的水合粒径和/或表面电位与所述一段时间之前相比，没有显著差异。例如，所述一段时间可以不少于约21天。例如，所述一段时间可以不少于约21天、不少于约20天、不少于约15天、不少于约10天、不少于约5天、不少于约4天、不少于约3天、不少于约2天、或不少于约1天。
例如，本申请的药物组合还可以包含选自以下组的物质：免疫佐剂、免疫检查点抑制剂、核酸分子、化学治疗剂和光敏剂，以及上述中的任意组合。例如，本申请的药物组合中的上述组分可以同时施用和/或分别施用。
例如，本申请的药物组合还可以包含单磷酰脂质A(MPLA)。例如，本申请的药物组合还可以包含吲哚胺2,3-双加氧酶(IDO)抑制剂。例如，本申请的药物组合还可以包含小干扰RNA(siRNA)。
另一方面，本申请提供了一种疫苗，可以包含本申请的药物组合。
另一方面，本申请提供了一种试剂盒，可以包含本申请药物组合和/或本申请疫苗。
另一方面，本申请还提供了制备本申请药物组合、本申请疫苗和/或本申请试剂盒的方法，可以包含提供所述源自细菌的内膜的膜。
例如，本申请的方法中，还可以包含提供所述源自所述细菌以外其它生物体的组分。
例如，本申请的方法中，还可以包含混合所述源自细菌的内膜的膜以及所述源自所述细菌以外其它生物体的组分以提供外壳。
例如，本申请的方法中，还可以包含提供内核。
另一方面，本申请提供了一种增强目标抗原被免疫细胞摄取的方法，可以包含提供一种药物组合，所述药物组合可以包含第一膜组分，所述第一膜组分可以包含源自细菌的内膜的膜，所述药物组合还可以包含所述目标抗原。例如，本申请的方法可以是体外的或离体的。例如，本申请的方法可以是非预防、非治疗和/或非诊断目的的。
例如，本申请的方法中，所述药物组合可以包含本申请的药物组合。
例如，本申请的方法中，所述免疫细胞可以包含免疫呈递细胞。
例如，本申请的方法中，所述免疫细胞可以包含以下组：树突细胞(DC)、T淋巴细胞、巨噬细胞和自然杀伤细胞(NK)，以及上述中的任意组合。
例如，本申请的方法中，所述免疫细胞可以包含骨髓来源树突状细胞(BMDC)。
例如，本申请的方法中，所述免疫细胞可以包含CD8阳性细胞和/或CD4阳性细胞。
另一方面，本申请提供了一种激活免疫细胞的方法，所述方法可以包含施用本申请药物组合、本申请所述疫苗和/或本申请所述试剂盒。例如，本申请的方法可以是体外的或离体 的。例如，本申请的方法可以是非预防、非治疗和/或非诊断目的的。
例如，本申请的方法中，所述免疫细胞可以包含免疫呈递细胞。
例如，本申请的方法中，所述免疫细胞可以包含以下组：树突细胞(DC)、T淋巴细胞、巨噬细胞和自然杀伤细胞(NK)，以及上述中的任意组合。
例如，本申请的方法中，所述免疫细胞可以包含骨髓来源树突状细胞(BMDC)。
例如，本申请的方法中，所述免疫细胞可以包含CD8阳性细胞和/或CD4阳性细胞。
例如，本申请的方法中，所述免疫细胞可以包含淋巴结和/或脾脏中的所述免疫细胞。
例如，本申请的方法中，与未施用所述药物组合的免疫细胞相比，施用所述药物组合的所述免疫细胞的所述激活的效果可以选自以下组：增加所述免疫细胞的抗原识别受体的表达水平、增加的所述免疫细胞的核因子κB(NF-κB)蛋白的表达水平、增加所述免疫细胞的细胞因子的表达和/或分泌水平和增加的成熟的所述免疫细胞的比例，以及上述中的任意组合。例如，所述增加包含与未施用所述药物组合的免疫细胞相比，施用所述药物组合的所述免疫细胞的选自上述组的效果提高约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约10％、约5％、约4％、约3％、约2％、或约1％。例如，所述增加包含与未施用所述药物组合的免疫细胞相比，施用所述药物组合的所述免疫细胞的抗原识别受体的表达水平提高约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约10％、约5％、约4％、约3％、约2％、或约1％。例如，所述增加包含与未施用所述药物组合的免疫细胞相比，施用所述药物组合的所述免疫细胞的核因子κB(NF-κB)蛋白的表达水平提高约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约10％、约5％、约4％、约3％、约2％、或约1％。例如，所述增加包含与未施用所述药物组合的免疫细胞相比，施用所述药物组合的所述免疫细胞的增加所述免疫细胞的细胞因子的表达和/或分泌水平提高约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约10％、约5％、约4％、约3％、约2％、或约1％。例如，所述增加包含与未施用所述药物组合的免疫细胞相比，施用所述药物组合的所述免疫细胞的增加的成熟的所述免疫细胞的比例提高约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约10％、约5％、约4％、约3％、约2％、或约1％。
例如，本申请的方法中，所述抗原识别受体可以包含模式识别受体(PRR)。
例如，本申请的方法中，所述抗原识别受体可以包含Toll样受体(TLR)。
例如，本申请的方法中，所述抗原识别受体可以包含TLR1、TLR2和/或TLR6。
例如，本申请的方法中，与未施用所述药物组合的免疫细胞相比，施用所述药物组合的 所述免疫细胞的TLR4的表达可以基本不变。
例如，本申请的方法中，所述细胞因子可以包含促炎细胞因子。
例如，本申请的方法中，所述细胞因子可以包含白介素(IL)-6、肿瘤坏死因子(TNF)-α、IL-1β和/或干扰素(IFN)-γ。
例如，本申请的方法中，成熟的所述免疫细胞可以包含CD80阳性细胞、CD86阳性细胞和/或效应记忆细胞。
例如，本申请的方法中，成熟的所述免疫细胞比例可以包含CD80阳性和/或CD86阳性的所述免疫细胞占CD11c阳性的所述免疫细胞的比例。
例如，本申请的方法中，成熟的所述免疫细胞比例可以包含CD44高表达且CD62L低表达的所述免疫细胞占CD8阳性的所述免疫细胞的比例。
另一方面，本申请提供了一种增强先天性免疫和/或特异性免疫应答的方法，可以包含向有需要的受试者施用本申请药物组合、本申请疫苗和/或本申请试剂盒。
例如，本申请的方法中，与未施用所述药物组合相比，施用所述药物组合可以促进树突细胞成熟和/或可以提高淋巴细胞分泌细胞因子。
例如，本申请的方法中，所述应用可以基本上不引发全身性炎症反应。
例如，本申请的方法中，所述应用基本上不引发全身性炎症反应可以包含与未施用所述药物组合相比，施用所述药物组合基本上不提高受试者体内血清中的IFN-γ、TNF-α、巨噬细胞趋化蛋白-1(MCP-1)、IL-12p70、IL-10、IL-23、IL-27、IL-17A、IFN-β、粒细胞-巨噬细胞集落刺激因子(GM-CSF)和/或IL-1α的浓度。
例如，本申请的方法中，所述应用可以基本上不引发溶血反应、心脏损伤、肝脏损伤、脾脏损伤、肺脏损伤和/或肾脏损伤。
另一方面，本申请提供了本申请药物组合、本申请疫苗和/或本申请试剂盒，其可以用于增强先天性免疫和/或特异性免疫应答。
例如，本申请的应用中，与未施用所述药物组合相比，施用所述药物组合可以促进树突细胞成熟和/或可以提高淋巴细胞分泌细胞因子。
例如，本申请的应用中，所述应用可以基本上不引发全身性炎症反应。
例如，本申请的应用中，所述应用基本上不引发全身性炎症反应可以包含与未施用所述药物组合相比，施用所述药物组合基本上不提高受试者体内血清中的IFN-γ、TNF-α、巨噬细胞趋化蛋白-1(MCP-1)、IL-12p70、IL-10、IL-23、IL-27、IL-17A、IFN-β、粒细胞-巨噬细胞集落刺激因子(GM-CSF)和/或IL-1α的浓度。
例如，本申请的应用中，所述应用可以基本上不引发溶血反应、心脏损伤、肝脏损伤、脾脏损伤、肺脏损伤和/或肾脏损伤。
另一方面，本申请提供了本申请药物组合、本申请疫苗和/或本申请试剂盒在制备药物中的应用，所述药物可以用于增强先天性免疫和/或特异性免疫应答。
例如，本申请的应用中，与未施用所述药物组合相比，施用所述药物组合可以促进树突细胞成熟和/或可以提高淋巴细胞分泌细胞因子。
例如，本申请的应用中，所述应用可以基本上不引发全身性炎症反应。
例如，本申请的应用中，所述应用基本上不引发全身性炎症反应可以包含与未施用所述药物组合相比，施用所述药物组合基本上不提高受试者体内血清中的IFN-γ、TNF-α、巨噬细胞趋化蛋白-1(MCP-1)、IL-12p70、IL-10、IL-23、IL-27、IL-17A、IFN-β、粒细胞-巨噬细胞集落刺激因子(GM-CSF)和/或IL-1α的浓度。
例如，本申请的应用中，所述应用可以基本上不引发溶血反应、心脏损伤、肝脏损伤、脾脏损伤、肺脏损伤和/或肾脏损伤。
另一方面，本申请还提供了一种预防和/或治疗肿瘤的方法，可以包含向有需要的受试者施用本申请药物组合、本申请疫苗和/或本申请试剂盒。
例如，本申请的方法中，所述预防和/或治疗肿瘤可以包含减缓肿瘤的体积增加速度和/或减小所述肿瘤的体积。
例如，本申请的方法中，所述肿瘤可以包含肿瘤切除术后未完全切除的所述肿瘤。例如，当通过外科手术切除患者的肿瘤后，未完全切除的肿瘤细胞和/或肿瘤组织也可以再次发展成肿瘤，这种肿瘤也可以通过本申请的方法预防和/或治疗。例如，本申请的方法中，所述肿瘤可以包含所述肿瘤清除后再次产生的肿瘤。例如，当通过外科手术切除患者的肿瘤后，即使完全将肿瘤切除，患者也有可能再次生产出肿瘤，比如该患者容易发生肿瘤，这种再次生产出的新肿瘤也可以通过本申请的方法预防和/或治疗。例如，这种再次生产出的新肿瘤与原有的肿瘤具有至少一种相同的抗原。
例如，本申请的方法中，所述肿瘤可以包含实体瘤。
例如，本申请的方法中，所述肿瘤可以选自以下组：乳腺肿瘤、结肠肿瘤、肝肿瘤、胃肿瘤、肾肿瘤、胰腺肿瘤、卵巢肿瘤、淋巴瘤、骨肉瘤、胶质瘤、前列腺癌和黑色素瘤，以及上述中的任意组合。
另一方面，本申请还提供了本申请药物组合、本申请疫苗和/或本申请试剂盒，其可以用于预防和/或治疗肿瘤。
例如，本申请的应用中，所述预防和/或治疗肿瘤可以包含减缓肿瘤的体积增加速度和/或减小所述肿瘤的体积。
例如，本申请的应用中，所述肿瘤可以包含肿瘤切除术后未完全切除的所述肿瘤。
例如，本申请的应用中，抑制肿瘤可以包含所述肿瘤清除后再次产生的肿瘤。
例如，本申请的应用中，所述肿瘤可以包含实体瘤。
例如，本申请的应用中，所述肿瘤可以选自以下组：乳腺肿瘤、结肠肿瘤、肝肿瘤、胃肿瘤、肾肿瘤、胰腺肿瘤、卵巢肿瘤、淋巴瘤、骨肉瘤、胶质瘤、前列腺癌和黑色素瘤，以及上述中的任意组合。
另一方面，本申请还提供了本申请药物组合、本申请疫苗和/或本申请试剂盒在制备药物中的应用，所述药物可以用于预防和/或治疗肿瘤。
例如，本申请的应用中，所述预防和/或治疗肿瘤可以包含减缓肿瘤的体积增加速度和/或减小所述肿瘤的体积。
例如，本申请的应用中，所述肿瘤可以包含肿瘤切除术后未完全切除的所述肿瘤。
例如，本申请的应用中，抑制肿瘤可以包含所述肿瘤清除后再次产生的肿瘤。
例如，本申请的应用中，所述肿瘤可以包含实体瘤。
例如，本申请的应用中，所述肿瘤可以选自以下组：乳腺肿瘤、结肠肿瘤、肝肿瘤、胃肿瘤、肾肿瘤、胰腺肿瘤、卵巢肿瘤、淋巴瘤、骨肉瘤、胶质瘤、前列腺癌和黑色素瘤，以及上述中的任意组合。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的产品、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
以下实施例所涉及的Elisa试剂盒购自美国Abcam公司；CD80和CD86抗体购自美国BioLegend公司；ELISPOT试剂盒购自北京达科为生物技术有限公司；ProcartaPlex multiplex免疫分析试剂盒购自美国Thermo Fisher Scientific公司。
以下实施例所涉及的试验动物小鼠型号为BALB/c，鼠龄6-8周，体重16-18g，雌性，购自北京维通利华实验动物技术有限公司，饲养在国家纳米科学中心无病原体动物房内，所有动物实验都严格按照国家机构动物护理和使用委员会(IACUC)批准的指导方针进行。
以下实施例使用大肠杆菌(E.coli DH5α)内膜作为革兰氏阴性菌内膜的研究模型，使用手术切除来源的4T1-luciferase三阴性乳腺癌组织中细胞的细胞膜或CT26结肠癌组织中细胞的细胞膜作为手术切除来源的实体肿瘤细胞膜的研究模型，使用PLGA作为内核材料的研究 模型。
本申请的药物组合使用了来自切除肿瘤组织的混合大肠杆菌胞质膜(EM)和自体肿瘤膜(TM)来生成用于抗原和佐剂的杂合膜纳米颗粒疫苗(HM-NPs)递送至抗原呈递细胞(APC)。这种个性化的肿瘤疫苗是经过量身设计的，可以安全地增强先天免疫反应，在避免副作用的同时最大程度地发挥抗肿瘤作用。在当前的研究中，本申请证明了EM的存在可显著提高DC摄取(p<0.0001)肿瘤抗原，激活其质膜上的Toll样受体(TLR)并促进其体外成熟。本申请证实了淋巴结(LNs)中DC的刺激性标记的上调和体内肿瘤膜抗原特异性T细胞的后续激活。在术后复发预防模型中，HM-NP在4T1-Luc，B16F10，EMT-6和CT-26肿瘤模型中获得了显著的抗肿瘤免疫作用。此外，在CT26结肠肿瘤模型中，HM-NPs可引起强烈的肿瘤特异性免疫反应，不仅延长了术后动物的存活时间，而且对肿瘤再激发具有长期保护作用(长达3个月)。本申请的HM-NPs是一种有效的自体肿瘤疫苗，可以能够成功激活先天免疫和适应性免疫反应，从而显著抑制肿瘤复发，且副作用可忽略不计。目前的研究表明，混合膜疫苗技术可能会转化为术后癌症治疗的有效临床治疗策略。
实施例1
本实施例制备四种膜纳米颗粒体系，分别为①大肠杆菌内膜包裹PLGA形成的纳米颗粒(后面以代号EM-NPs表示)体系；②4T1-luciferase三阴性乳腺癌组织中细胞的细胞膜包裹PLGA形成的纳米颗粒(后面以代号TM-NPs表示)体系；③将①和②的产品混合得到的纳米颗粒(后面以代号Mix NPs表示)体系；④大肠杆菌内膜和肿瘤细胞膜组成的杂合膜包裹PLGA形成的纳米颗粒(后面以代号HM-NPs表示)体系。四种产品中总膜蛋白浓度当量均保持一致，Mix NPs和HM-NPs中的两种膜蛋白的浓度当量各自保持一致。
其中HM-NPs的制备方法为如下步骤：
(1)提取大肠杆菌内膜EM，操作为：将大肠杆菌接种在LB液体培养基中进行培养，过夜培养扩增至OD600值为1.2；加入终浓度为2mg/mL的溶菌酶，与大肠杆菌在37℃下共孵育1h，以裂解细胞壁；4度条件下3000g离心5分钟，弃上清得到原生质体；加入extraction buffer(提取buffer)，利用密度梯度离心得到大肠杆菌内膜；重悬后保存在-80℃备用；
(2)提取4T1-luciferase三阴性乳腺癌组织中细胞的细胞膜TM，操作为：每只BALB/c小鼠皮下接种20万个肿瘤细胞，等肿瘤体积达到300mm 3左右，无菌条件下手术切除肿瘤组织；将得到的肿瘤组织用剪刀初步剪碎，然后加入含有胶原酶IV(1.0mg·mL -1)、脱氧核糖核酸酶DNase(0.1mg·mL -1)以及透明质酸酶(0.1mg·mL -1)的GBSS溶液，37℃放置15min用来消化肿瘤组织；将消化的组织置于70μm的滤网上进行研磨，然后通过孔径为70μm的滤网 进行过滤，滤液离心重悬后得到单细胞悬液；加入含有甘露醇、蔗糖、牛血清白蛋白(BSA)、Tris盐酸、EGTA以及磷酸酶-蛋白酶抑制剂混合物的提取缓冲液，然后在冰浴条件下利用细胞破碎仪30W超声破碎细胞5分钟，依次通过3000g、10000g以及100000g离心速率，最终得到肿瘤组织来源的细胞膜；将细胞膜重悬并冻存于-80℃备用；
(3)将3mg提取的大肠杆菌内膜EM与1mg提取的手术切除肿瘤细胞膜TM混合于PBS中，37℃恒温摇床摇15min，之后将混合膜溶液通过滤膜孔径为400nm的脂质体挤出器，累计来回挤压13次(一来一回算1次)，得到HM杂合膜微粒；
(4)将10mg PLGA溶于1mL二氯甲烷，再加入0.2mL无菌蒸馏水；混合物冰上25W超声乳化3min；随后与2mL 1％胆酸钠溶液(表面活性剂)混合，冰上30W超声乳化5min；将乳液逐滴加入10mL 0.5％胆酸钠溶液中，室温搅拌15min；利用旋转蒸发仪37℃旋蒸10min；将乳液11000g室温离心15min；用无菌蒸馏水洗两次，重悬得到PLGA聚合物纳米颗粒溶液；
(5)将50μL步骤(3)制备的2mg/mL HM杂合膜微粒与500μL步骤(4)制备的1mg/mL PLGA聚合物纳米颗粒溶液混合后通过滤膜孔径为200nm的脂质体挤出器，累计来回挤压13次，得到HM-NPs杂合膜纳米颗粒。
其中EM-NPs的制备方法为如下步骤：
(1)将4mg制备好的细菌内膜EM溶液置于37℃摇床震荡10min；然后通过滤膜孔径为400nm的脂质体挤出器，累计来回挤压13次(一来一回算1次)；
(2)参照HM-NPs的制备方法中步骤(4)利用双乳法制备PLGA纳米颗粒，将50μL制备的2mg/mL细菌内膜EM与500μL制备的1mg/mL PLGA聚合物纳米颗粒溶液混合，然后通过滤膜孔径为200nm的脂质体挤出器，累计来回挤压13次(一来一回算1次)，最终得到EM-NPs。
其中TM-NPs的制备方法为如下步骤：
(1)将4mg制备好的手术切除肿瘤细胞膜TM溶液置于37℃摇床震荡10min；然后通过滤膜孔径为400nm的脂质体挤出器，累计来回挤压13次(一来一回算1次)；
(2)参照HM-NPs的制备方法中步骤(4)利用双乳法制备PLGA纳米颗粒，将50μL制备的2mg/mL手术切除肿瘤细胞膜TM与500μL制备的1mg/mL PLGA聚合物纳米颗粒溶液混合，然后通过滤膜孔径为200nm的脂质体挤出器，累计来回挤压13次(一来一回算1次)，最终得到TM-NPs。
其中Mix NPs的制备方法为如下步骤：
(1)将制备得到的EM-NPs与TM-NPs在37℃下进行混合，其中两种膜蛋白的浓度分别与HM-NPs中的浓度保持一致。
对制备得到的EM-NPs，TM-NPs以及HM-NPs杂合膜纳米颗粒用透射电子显微镜(美国FEI，Tecnai G2 20S-TWIN，200KV)进行观察，结果如图1所示，结果显示上述三种膜纳米颗粒形状均呈规则圆形，且大小较均一，图中标尺为100nm。
对制备得到的HM-NPs杂合膜纳米颗粒用动态光散射(Zetasizer NanoZS)进行表征，结果如图2所示，由图可知：制备而成的HM-NPs杂合膜纳米颗粒粒径主要分布在100-300nm之间，并且在164.2nm处达到峰值(占15.8％)，说明制备的杂合膜纳米颗粒粒径分布好，有利于进入细胞发挥生物学作用。
实施例2
本实施例对实施例1制得的四种产品增强先天性免疫和特异性免疫的效果进行探究，具体内容包括：
1、促进BMDC分泌促炎细胞因子实验：
(1)提取BALB/c小鼠骨髓来源的树突状细胞(BMDCs)；
(2)将BMDC培养至第5天，用1640完全培养基重悬，每孔4万个细胞接种在96孔板中，再分别加入终浓度为1mg/mL的EM-NPs、TM-NPs、Mix NPs以及HM-NPs溶液进行共孵育，每组3个平行孔，另外加入等体积的1640完全培养基作为对照组(Control，Ctrl)；
(3)37℃共孵育24h后，分别取出各组培养基上清；
(4)利用Elisa试剂盒检测上清中IL-6、TNF-α以及IL-1β的水平。
结果如图3A-3C所示，可知：HM-NPs杂合膜纳米颗粒与其他组相比具有最佳的增强机体先天性免疫的潜力。
2、促进BMDC成熟实验：
(1)提取BALB/c小鼠骨髓来源的树突状细胞(BMDC)；
(2)将BMDC培养至第5天，用1640完全培养基重悬，每孔4万个细胞接种在96孔板中，分别加入终浓度为1mg/mL EM-NPs、TM-NPs、Mix NPs以及HM-NPs溶液进行共孵育，每组3个平行孔，另外加入等体积的1640完全培养基作为对照组(Control，Ctrl)；
(3)37℃共孵育24h后，收集细胞，用PBS洗3次；
(4)用FITC-CD11c,PE-CD80以及PE-CD86流式抗体对细胞避光染色20分钟；
(5)利用BD-C6流式细胞仪检测各组细胞表面表达的CD80和CD86水平，每组采集10000个细胞。
结果如图4所示，可知：HM-NPs杂合膜纳米颗粒与其他组相比具有最强刺激DC细胞成熟的潜力。
3、促进小鼠脾脏淋巴细胞分泌IFN-γ实验：
(1)将BALB/c小鼠随机分为5组，每组3只；
(2)分别在第1、3和第7天对各组小鼠右后背皮下接种EM-NPs，TM-NPs，Mix NPs以及HM-NPs疫苗(100μg/只)；以等体积的PBS溶液作为对照组(Control，Ctrl)；
(3)第8天分别取各组小鼠脾脏细胞进行研磨，过70μm滤网后得到混合脾脏淋巴细胞悬液；
(4)将各组小鼠脾脏淋巴细胞与HM-NPs疫苗中相同来源的肿瘤细胞膜(作为肿瘤抗原)在37℃5％CO 2培养箱内共孵育24h；
(5)将各组细胞进行裂解，用ELISPOT(酶联免疫斑点)试剂盒检测其中IFN-γ分泌量；
(6)利用CTL-ImmunoSpot Plate Reader软件对各组斑点数目进行统计，分析数据。
结果如图5和图6所示，可知：HM-NPs杂合膜纳米颗粒与其他组相比具有最强刺激脾脏淋巴细胞分泌IFN-γ的潜力。
综合上述三个试验结果，表明HM-NPs杂合膜纳米颗粒具有提高的增强先天性和特异性免疫的潜力。
实施例3
本实施例对实施例1制得的四种产品刺激小鼠全身免疫系统分泌炎症因子和趋化因子的效果进行探究，具体步骤如下：
(1)将BALB/c小鼠随机分为5组，每组5只；
(2)分别在第1、3和第7天对各组小鼠右后背皮下接种EM-NPs，TM-NPs，Mix NPs以及HM-NPs疫苗(100μg/只)；以等体积的生理盐水液作为对照组(Control，Ctrl)；
(3)第7天皮下注射疫苗12小时后，分别取各组小鼠血清，用ProcartaPlex multiplex免疫分析试剂盒检测其中炎症因子和趋化因子分泌水平；
(4)利用BD-C6流式细胞仪进行数据分析，利用ProcartaPlex Analysis软件进行数据处理。
结果如图7A-7M所示，可知：HM-NPs杂合膜纳米颗粒对全身免疫系统分泌炎症因子和趋化因子具有一定影响，与空白对照组相比，只有促炎细胞因子IL-6和IL-1β上升较明显，其他11种炎症因子和趋化因子基本保持不变。表明HM-NPs杂合膜纳米疫苗具有好的生物 安全性。
实施例4
本实施例对实施例1制得的四种产品在体内淋巴结富集的效果进行探究，具体步骤如下：
(1)包载荧光染料IR780的各组膜纳米颗粒构建：利用双乳法将亲水性荧光染料IR780包载在PLGA中，具体为：取200μL浓度为1mg/mL亲水性荧光染料IR780，加入1mL浓度为10mg/mL的PLGA二氯甲烷溶液中，初乳3min；加入1％表面活性剂胆酸钠，复乳5min；避光旋蒸10min去除有机溶剂二氯甲烷；11000g离心15min，并用三次水洗两次，得到包载荧光染料IR780的PLGA纳米颗粒。然后与提前制备好的各组膜微粒混合。具体步骤及投料比例同实施例1；
(2)将BALB/c小鼠随机分为4组，每组4只；
(3)各组小鼠右后背皮下注射各组荧光标记的膜纳米颗粒，12h后取各组小鼠淋巴结，利用小动物光学3D活体成像系统进行荧光成像，并用LivingImage软件进行分析。
结果如图8和图9所示，可知：HM-NPs杂合膜纳米颗粒与其他组相比在淋巴结荧光信号最强，表明HM-NPs杂合膜纳米颗粒有较强淋巴结富集潜力。
实施例5
本实施例对实施例1制得的HM-NPs杂合膜纳米颗粒的生物安全性进行探究，采用溶血试验评价，具体步骤如下：
(1)BALB/c小鼠取血液1mL，溶于2mL PBS溶液中，3000rpm离心10min；
(2)用PBS洗三次，轻轻吹打至血液上清无颜色；
(3)将离心出的红细胞加2mL PBS稀释，分别取0.1mL血液稀释液加入到0.9mL待测样品(不同浓度HM-NPs杂合膜纳米颗粒、PBS作为阴性对照、三次水作为阳性对照)中；
(4)所有样品置于摇床，150rpm，37℃摇晃3h；
(5)将摇晃后的混合物12000rpm离心10min，摆好拍照；用酶标仪检测上清液在541nm处的吸收值，计算溶血率。
结果如图10所示，可知：各浓度的HM-NPs杂合膜纳米颗粒与红细胞混合后，均未出现溶血现象，说明本发明所涉及的肿瘤疫苗具有好的生物安全性。
实施例6
本实施例对实施例1制得的四种产品抑制行肿瘤切除术的4T1-luciferase乳腺癌荷瘤小鼠 术后肿瘤复发的效果进行探究，本实施例的试验操作流程图如图11所示。具体步骤如下：
(1)将雌性BALB/c小鼠随机分为5组，每组6只，每只小鼠右后背皮下接种20万个4T1-luciferase乳腺癌细胞，构建小鼠乳腺癌模型(第0天)；
(2)每隔一天观察并记录小鼠肿瘤生长情况，并且每隔一周采用小动物荧光成像系统检测荷瘤小鼠肿瘤荧光强度如图12所示；
(3)当平均肿瘤体积达到300mm 3左右时，手术切除所有老鼠肿瘤，然后对伤口进行缝合(第7天)；
(4)将切下来的瘤块研磨破碎，用胶原酶、DNA酶以及透明质酸酶混合溶液在37℃条件下对肿瘤组织进行消化，消化完的细胞悬液过70μm滤网然后离心，离心弃上清后加入提取buffer，重悬得到细胞悬液；用细胞破碎仪对细胞悬液进行冰浴超声破碎(35W，5min)；将破碎后的细胞悬液进行离心(3000g，5min，4℃)；取上一步的上清再次进行离心(10000g，10min，4℃)；取离心后的细胞上清加入到超离管中进行超速离心(100000g，2h，4℃)；超速离心后弃上清，用1mL PBS重悬，得到手术切除来源的肿瘤细胞膜碎片TM，置于-80℃保存；
(5)参照实施例1中方法制备各种膜纳米颗粒；
(6)对术后小鼠腹腔注射荧光素钾，进行小动物荧光成像，观察并统计术后小鼠残余肿瘤体积(第9天)；
(7)根据小动物荧光成像结果对小鼠重新随机分组，共分5组，每组6只；
(8)分别在第10、12以及16天给各组小鼠皮下注射EM-NPs，TM-NPs，Mix NPs以及HM-NPs(100μg/只)，生理盐水组作为对照组(Control，Ctrl)；
(9)每隔一天观察并记录小鼠肿瘤生长情况，当小鼠肿瘤体积生长达到1000mm 3左右，即视为小鼠死亡。记录每组小鼠死亡的具体时间。
结果如图13-15所示，可知：本发明所涉及的HM-NPs杂合膜纳米颗粒与其他组相比具有更明显抑制乳腺癌术后小鼠肿瘤复发的潜力，抑制率达到83.3％。
实施例7
本实施例对实施例1制得的四种产品抑制行肿瘤切除术的CT26结肠癌荷瘤小鼠术后肿瘤复发以及肿瘤细胞再激发实验的效果进行探究，本实施例的试验操作流程图如图16所示。具体步骤如下：
1、预防结肠癌术后复发实验：
(1)将雄性BALB/c小鼠随机分为5组，每组6只，每只小鼠右后背皮下接种20万个 CT26结肠癌细胞，构建小鼠结肠癌模型(第0天)；
(2)每隔一天观察并记录小鼠肿瘤生长情况；
(3)当平均肿瘤体积达到300mm 3左右时，手术切除所有小鼠肿瘤，然后对伤口进行缝合(第7天)；
(4)将切下来的瘤块研磨破碎，用胶原酶、DNA酶以及透明质酸酶混合溶液在37℃条件下对肿瘤组织进行消化，消化完的细胞悬液过70μm滤网然后离心，离心弃上清后加入提取buffer，重悬得到细胞悬液；用细胞破碎仪对细胞悬液进行冰浴超声破碎(35W，5min)；将破碎后的细胞悬液进行离心(3000g，5min，4℃)；取上一步的上清再次进行离心(10000g，10min，4℃)；取离心后的细胞上清加入到超离管中进行超速离心(100000g，2h，4℃)；超速离心后弃上清，用1mL PBS重悬，得到手术切除来源的肿瘤细胞膜碎片TM，置于-80℃保存；
(5)参照实施例1中方法制备各种膜纳米颗粒；
(6)对小鼠重新随机分组，共分5组，每组15只；
(7)分别在第10、12以及16天给各组小鼠皮下注射EM-NPs，TM-NPs，Mix NPs以及HM-NPs(100μg/只)，生理盐水组作为对照组(Control，Ctrl)；
(8)每隔一天观察并记录小鼠肿瘤生长情况，当小鼠肿瘤体积生长达到1000mm 3左右，即视为小鼠死亡。记录每组小鼠死亡的具体时间。
结果如图17-18所示，可知：本发明所涉及的HM-NPs杂合膜纳米颗粒与其他组相比具有更明显抑制结肠癌术后小鼠肿瘤复发的潜力，抑制率达到100％。
2、术后肿瘤再挑战试验：
(1)将1中接种HM-NPs疫苗的小鼠在60天后重新随机分为3组，每组5只；
(2)其中一组小鼠右后背皮下接种20万个4T1乳腺癌细胞，另一组小鼠右后背皮下接种20万个CT26结肠癌细胞，剩下一组小鼠不接种肿瘤细胞(接种生理盐水作为对照Control，Ctrl)；
(3)每隔一天观察并记录各组小鼠肿瘤生长情况，当小鼠肿瘤体积生长达到1000mm 3左右，即视为小鼠死亡，记录每组小鼠死亡的具体时间。
结果如图19，可知：当CT26结肠癌细胞第二次“入侵”小鼠体内后，此前接种过结肠癌组织来源的肿瘤细胞膜制备的杂合膜疫苗组小鼠具有完全抵御肿瘤的能力；而当4T1乳腺癌细胞“入侵”小鼠体内后，由于小鼠此前未接触4T1乳腺癌细胞，未获得4T1乳腺癌细胞相关肿瘤抗原，因此不具有对4T1乳腺癌细胞的豁免能力。并在第90天对各组小鼠血清中的 IL-6、TNF-α、IL-1β和IFN-γ进行测定，结果如图20A-20D所示，可知：二次接种CT26结肠癌细胞的小鼠分泌的促炎因子(IL-6，TNF-α以及IL-1β)明显高于另外两组，其分泌的IFN-γ与接种4T1乳腺癌细胞的小鼠相比也有一定程度升高。
实施例8
本实施例对本发明所涉及的HM-NPs杂合膜纳米颗粒中革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜的用量配比进行探究，具体包括如下内容：
(1)根据实施例1的方法制备不同膜蛋白浓度比例的杂合膜纳米颗粒，具体设置为：单独的革兰氏阴性菌内膜EM、单独的手术切除来源的实体肿瘤细胞膜TM、以及革兰氏阴性菌内膜EM与手术切除来源的实体肿瘤细胞膜TM的质量比为1:100、1:75、1:50、1:25、1:10、1:5、1:3、1:1、1:0、0:1、3:1、5:1、10:1、25:1、50:1、75:1或100:1的杂合膜，以等体积的PBS溶液作为对照组(Control)；
(2)根据实施例2的方法提取并培养BALB/c小鼠骨髓来源的树突状细胞(BMDC)；将BMDC培养至第5天，用1640完全培养基重悬，均分为6份每孔5万个细胞接种在96孔板中，分别加入不同膜蛋白比例的杂合膜纳米颗粒进行共孵育，每组3个平行；
(3)37℃共孵育24h后，分别取出各组培养基上清；利用Elisa试剂盒检测上清中IL-6、TNF-α以及IL-1β的水平。
结果如图21A-21C所示，可知：与其他组相比，杂合膜比例中EM:TM为3:1时有最强刺激促炎细胞因子(IL-1β，TNF-α、IL-6)的能力，说明其有最强刺激机体先天性免疫的潜力。
实施例9
本实施例制备一种膜纳米颗粒体系，为克雷白氏肺炎杆菌(CG43菌株)内膜和HepG2肝癌组织中细胞的细胞膜组成的杂合膜包裹的基于锆(Zr)的MOF(金属有机框架)形成的纳米颗粒(后面以代号HM-NPs-2表示)体系。其制备方法为如下步骤：
(1)提取肺炎杆菌内膜EM，具体操作同实施例1，包括：细菌培养与扩增；加溶菌酶破裂细胞壁并低速离心得到原生质体；加入提取buffer并利用密度梯度离心法最终得到肺炎杆菌内膜EM；
(2)提取HepG2肝癌组织中细胞的细胞膜TM，具体操作同实施例1，包括：BALB/c小鼠皮下接瘤；等肿瘤长到一定体积时手术切除；剪刀剪碎、加三种酶进行消化裂解肿瘤组织、研磨过70μm滤网；加入提取buffer并利用超声细胞破碎仪进行破碎；最终离心得到肝癌组织来源的肝癌细胞膜；
(3)将3mg提取的肺炎杆菌内膜EM与1mg提取的手术切除肿瘤细胞膜TM(具有相同的膜蛋白浓度)混合于PBS中，37℃恒温摇床摇15min，之后将混合膜溶液通过滤膜孔径为400nm的脂质体挤出器，累计来回挤压13次(一来一回算1次)，得到HM杂合膜微粒。
(4)合成MOF：将300mg ZrOCl 2·8H 2O，100mg H 2TCPP以及2.8mg苯甲酸(BA)溶于100mL二甲基甲酰胺(DMF)溶剂中，氮气保护条件下90℃搅拌5h；冷却至25℃后，10000g离心30min收集MOF产物并用DMF洗三次，合成好的MOF溶于DMF并储存在4℃条件下，实验使用前需用去离子水洗三次。
(5)将50μL步骤(3)制备的2mg/mL杂合膜微粒与500μL步骤(4)制备的1mg/mL MOF混合，将混合物在浴用超声仪(超声水浴锅)中超声30min，产物离心收集，得到HM-NPs-2杂合膜纳米颗粒。
实施例10
本实施例制备一种膜纳米颗粒体系，为布氏杆菌PBP 39内膜和SN12-PM6SN12-PM6肾癌组织中细胞的细胞膜组成的杂合膜包裹MSN(介孔二氧化硅)形成的纳米颗粒(后面以代号HM-NPs-3表示)体系。其制备方法为如下步骤：
(1)提取布氏杆菌内膜EM，具体操作同实施例1，包括：细菌培养与扩增；加溶菌酶破裂细胞壁并低速离心得到原生质体；加入提取buffer并利用密度梯度离心法最终得到布氏杆菌内膜EM；
(2)提取SN12-PM6肾癌组织中细胞的细胞膜TM，具体操作同实施例1，包括：BALB/c小鼠皮下接瘤；等肿瘤长到一定体积时手术切除；剪刀剪碎、加三种酶进行消化裂解肿瘤组织、研磨过70μm滤网；加入提取buffer并利用超声细胞破碎仪进行破碎；最终离心得到肝癌组织来源的肝癌细胞膜；
(3)将3mg提取的布氏杆菌内膜EM与1mg提取的手术切除肿瘤细胞膜TM(具有相同的膜蛋白浓度)混合于PBS中，37℃恒温摇床摇15min，之后将混合膜溶液通过滤膜孔径为400nm的脂质体挤出器，累计来回挤压13次(一来一回算1次)，得到HM杂合膜微粒；
(4)合成MSN：1.5mL正硅酸乙酯(TEOS)和5mL丙酮作为油相，随后加入40mL溶有0.15g十六烷基三甲基溴化铵(CTAB)的水溶液；将混合物以10000rpm的速度搅拌剪切5min，形成乳液；在乳液中加入50μL氨水，随后以400rpm的速度磁力搅拌2h以蒸发有机溶剂；将悬液离心，沉淀物用去离子水洗三次然后冷冻干燥；将正硅酸乙酯(TEOS)被水解并在油/水界面浓缩形成二氧化硅壳；最后在反应或干燥过程中通过蒸发(挥发)除去液滴，获得介孔二氧化硅MSN。
(5)将50μL步骤(3)制备的2mg/mL HM杂合膜微粒与500μL步骤(4)制备的1mg/mL MSN混合，将混合物在浴用超声仪(超声水浴锅)中超声30min，产物离心收集，得到HM-NPs-3杂合膜纳米颗粒。
实施例11
本实施例对实施例9和实施例10制得的两种产品增强先天性免疫和特异性免疫的效果进行探究，具体内容包括：
1、促进BMDC分泌促炎细胞因子实验：
具体操作方法参见实施例2。
结果显示HM-NPs-2和HM-NPs-3杂合膜纳米颗粒促进BMDC分泌IL-6、TNF-α、IL-1β的水平与HM-NPs相似，具有显著的增强机体先天性免疫的潜力。
2、促进BMDC成熟实验：
具体操作方法参见实施例2。
结果显示HM-NPs-2和HM-NPs-3杂合膜纳米颗粒促进BMDC细胞表面表达的CD80和CD86水平与HM-NPs相似，具有显著的刺激DC细胞成熟的潜力。
3、促进小鼠脾脏淋巴细胞分泌IFN-γ实验：
具体操作方法参见实施例2。
结果显示HM-NPs-2和HM-NPs-3杂合膜纳米颗粒组的斑点数与HM-NPs相似，具有显著的刺激脾脏淋巴细胞分泌IFN-γ的潜力。
综合上述三个试验结果，表明HM-NPs-2和HM-NPs-3杂合膜纳米颗粒具有显著的增强先天性和特异性免疫的潜力。
实施例12
研究方案
分离切除的自体肿瘤组织的大肠杆菌内膜和肿瘤细胞膜，并利用液相色谱-质谱法(Liquid chromatography-mass spectrometry，LC-MS)对四个批次EM和TM样品进行全蛋白质组学分析。使用双乳液法制备了PLGA聚合物纳米颗粒(NP)。骨髓来源树突状细胞(BMDC)是从小鼠骨髓间充质干细胞中诱导分化而产生的，而骨髓间充质干细胞是从6-8周雌性C57BL/6J小鼠中新鲜提取的。本申请研究了不同膜纳米颗粒体内和体外的先天性免疫刺激应答和特异性免疫刺激应答，不同膜纳米颗粒的理化性质表征以及蛋白质组学表征。对于动物实验，使用电子游标卡尺测量肿瘤体积。当肿瘤的体积达到约300mm 3时，通过手术将其切 除。然后在手术后第3、5和9天用不同的疫苗制剂对小鼠进行免疫。对于CT-26肿瘤再挑战模型，将来自HM-NP疫苗接种组的小鼠随机分为三组，并在60天后接种CT-26结肠腺癌细胞或4T1乳腺癌细胞。免疫细胞消耗模型中，在开始进行HM-NP治疗之前的一天，针对指定表面标记的消耗抗体进行腹膜内注射以消耗巨噬细胞(CSF1R抗体)，NK细胞(ASGM1抗体)，CD8 +T细胞(CD8抗体)或CD4 +T细胞(CD4抗体)。除未治疗组外，所有小鼠均在手术后第3、5和9天接种HM-NP。为了进行体内生物安全性评估，在4T1-Luc模型的肿瘤消退实验终点，对各组小鼠的主要器官进行了苏木精-伊红(H&E)染色切片，并检测了各组小鼠血清中的肝肾功能指标。
HM-NPs的体外BMDC摄取检测
在合成过程中通过将罗丹明B加入到PLGA核中来制备荧光标记的纳米颗粒。BMDCs是通过诱导分化从C57BL/6J小鼠的新鲜骨髓间充质干细胞中产生的。将细胞培养在含有10％FBS和青霉素-链霉素的RPMI 1640培养基中。将细胞加入1.5mL离心管中(40,000个细胞/管)，再加入终浓度为0.4mg·mL -1的不同的疫苗制剂，在37℃下共培养8小时。然后500×g离心3分钟收集细胞，并重悬于FACS缓冲液(补充2％FBS的RPMI 1640培养基)中。使用BD Accuri C6 FACS流式细胞仪(BD Biosciences，美国圣何塞)评估荧光信号，并使用BD Accuri C6软件进行分析。
HM-NP的共聚焦显微镜检测
为了评估内化进入树突状细胞的过程，制备了带有荧光标记溶酶体的Cy5.5标记的膜包被NP和BMDC。EM-NP，TM-NP和HM-NP与BMDC共孵育，并在共聚焦激光扫描显微镜(Zeiss 710，Zeiss Microsystems，德国)下成像。为了进一步比较HM-NP和Mix NP的共定位效率，将BMDC用HM-NP或Mix NP处理，并在共聚焦激光扫描显微镜下成像。用细菌内膜特异的一抗FtsZ和抗兔IgG的山羊二抗(Alexa Fluor@633山羊，Abcam，剑桥，英国)标记EM-NP。用肿瘤细胞膜特异性的一抗Na +/K +-ATP酶和对小鼠IgG的山羊二抗(Alexa Fluor@488山羊，Abcam，剑桥，英国)标记TM-NP。
体外先天免疫实验
为了研究不同膜NPs体外刺激先天免疫反应的能力，将BMDCs加入到96孔板中(40,000个细胞/孔)，并与终浓度为0.4mg·mL -1的不同的纳米制剂一起在37℃下共孵育24小时。收集上清液，并使用相应的特异性ELISA试剂盒分析IL-6，TNF-α和IL-1β的浓度。
体外和体内DC成熟实验
为了研究不同膜NPs在体外促进BMDC成熟的能力，将BMDCs加入到1.5mL离心管 中(40,000个细胞/管)，并与终浓度为0.4mg·mL -1的不同纳米制剂在37℃下共孵育24小时。然后通过离心收集细胞，并重悬于FACS缓冲液中。FITC-CD11c，PE-CD80和PE-CD86用于荧光标记，并使用BD Accuri C6 FACS流式细胞仪和BD Accuri C6软件进行分析。为了评估体内DC成熟，将雌性BALB/c小鼠皮下接种4T1乳腺癌细胞。当肿瘤的体积达到约300mm 3时，通过手术将其切除。随后将术后小鼠随机分为五组(每组n＝5只小鼠)。在手术后第3、5和9天用不同的疫苗制剂对小鼠进行免疫。
T细胞反应测量
为了检查HM-NP是否可以有效激活T细胞，雌性BALB/c小鼠皮下接种了4T1乳腺癌细胞。手术步骤和接种时间表与上述研究方案相同。最后一次疫苗接种12小时后收集脾细胞，并与4T1细胞膜(作为抗原)共孵育24小时上。预包被IFN-γ捕获抗体的ELISPOT试剂盒用于评估由不同膜NP诱导的T细胞IFN-γ释放。使用CTL-ImmunoSpot板读取器扫描板，并使用CTL ImmunoSpot软件分析数据。
血清细胞因子测定
为了评估用不同疫苗制剂治疗后的全身炎症反应程度，将雌性BALB/c小鼠皮下接种4T1乳腺癌细胞。如上所述进行外科手术和疫苗接种程序。根据制造商的说明，使用基于Luminex磁珠的ELISA试剂盒对血清中的炎症细胞因子和趋化因子进行检测，捕获磁珠和检测抗体的使用量均为推荐量的1/5。
肿瘤消退实验
雌性BALB/c或C57BL/6小鼠皮下接种4T1-Luc，CT26，B16F10或EMT-6肿瘤细胞(每只小鼠2×10 5个)。如上所述进行外科手术和疫苗接种程序。IVIS(Spectrum CT，Perkin Elmer，英国)用于监测4T1-Luc细胞产生的生物发光信号。每隔一天用电子卡尺测量所有肿瘤的体积。根据以下公式计算肿瘤体积：
V＝(L×W×W)/2(L，最长尺寸；W，最短尺寸)
CT-26肿瘤与再挑战模型
为建立CT-26肿瘤再挑战模型，经HM-NPs治疗的术后小鼠在60天后随机分为三组，分别接种CT-26结肠腺癌细胞或4T1乳腺肿瘤细胞(n＝12)。每隔一天用电子卡尺测量所有肿瘤的体积。用特异性ELISA试剂盒检测再挑战小鼠血清中细胞因子的分泌。
体内巨噬细胞，NK细胞，CD8 +T细胞或CD4 +T细胞的耗竭检测
将雌性BALB/c小鼠皮下接种CT-26结肠腺癌细胞(每只小鼠2×10 5个)。手术程序和疫苗接种时间表与上述相同。腹腔内注射抗体消耗了免疫细胞亚群。在HM-NP疫苗治疗开 始前一天开始。免疫细胞消耗抗体种类及使用方式如下：具有抗小鼠CSF1R的巨噬细胞(CD115，BioXCell；每隔一天注射300微克)，具有抗ASGM1的NK细胞(抗小鼠Asialo-GM1，BioLegend；50μL/次注射，每周两次)，带有抗小鼠CD8的CD8 +T细胞(Lyt 2.1，BioXCell；400微克/次注射，每周两次)和带有抗小鼠CD4的CD4 +T细胞(每周两次，GK1.5，BioXCell；200微克/次注射，每周一次)。每隔一天用电子卡尺测量所有肿瘤的体积。通过PBMC的流式细胞术证实巨噬细胞，NK细胞，CD8 +T细胞和CD4 +T细胞的细胞耗竭。
统计分析
使用GraphPad Prism软件5.0版进行统计分析。使用Bonferroni的多重比较测试，通过单因素方差分析或两因素方差分析评估两个实验组之间的差异。基于Luminex的ProcartaPlex多重免疫分析的热图通过双向方差分析与Bonferroni的多重比较测试进行了分组分析。用对数秩(Mantel-Cox)检验评估生存曲线。数据以平均值±标准差表示，如图中的图例所示。统计显著性设定如下：，*p＜0.05。**，p<0.01；***，p<0.001，****，p<0.0001，并且ns表示无显著差异。
材料来源
聚(丙交酯-乙醇酸共聚物)-OH(75:25，相对分子质量[Mr]20,000Da)购自Jinandaigang Biotechnology Company(中国北京)。磷酸盐缓冲液(PBS)，DMEM，RPMI 1640培养基和胎牛血清(FBS)购自Wisent Bio-Products(加拿大蒙特利尔)。BCA蛋白测定试剂盒购自Thermo Fisher Scientific(美国马萨诸塞州沃尔瑟姆)。用于LPS检测的预涂ELISA试剂盒购自eBioscience(加利福尼亚州圣地亚哥)。抗FtsZ的兔抗体购自Agrisera(
Figure PCTCN2021094037-appb-000001
瑞典)。小鼠抗Na +/K +-ATP酶，ATP5A(IM CVa)，泛醇-细胞色素C还原酶核心蛋白I(IM Core I)，VDAC1/孔蛋白(OM Porin)，基质亲环蛋白D(Matrix CypD)和IMS细胞色素C(IMS)的抗体Cytc购自Abcam(英国剑桥)。抗兔IgG-金结合物(金纳米大小为5nm)和抗小鼠IgG-金结合物(金纳米大小为10nm)购自Abcam(英国剑桥)。鼠IL-4和GM-CSF购自Beyotime Biotechnology(中国上海)。用于IL-6，TNF-α，IL-1β和IFN-γ分析的ELISA试剂盒购自eBioscience(加利福尼亚州圣地亚哥)。针对FITC-CD11c，PE-CD80和PE-CD86的小鼠抗体购自BioLegend(美国圣地亚哥)。用于IFN-γ的预涂ELISPOT试剂盒购自大克威生物技术有限公司(中国北京)。基于Luminex珠的ELISA试剂盒购自Thermo Fisher Scientific(美国马萨诸塞州沃尔瑟姆)。D-荧光素钾购自bide Pharmatech Co.Ltd(中国上海)。巨噬细胞(CD115，抗小鼠CSF1R)，CD8 +T细胞(Lyt 2.1，抗小鼠CD8)和CD4 +T细胞(GK1.5，抗小鼠CD4)的耗竭抗体购自Bio X Cell公司，美国。NK细胞的耗竭抗体(抗小鼠Asialo- GM1)购自BioLegend(美国圣地亚哥)。除非另有说明，否则其他化学品是从华立德科技有限公司(中国北京)购买的。
细胞系和动物
小鼠4T1乳腺癌细胞，4T1-荧光素酶乳腺癌细胞，CT-26结肠肿瘤细胞，EMT-6乳腺癌细胞和B16F10黑色素瘤细胞均购自美国典型培养物保藏中心(ATCC，Manassas，USA)。在含有10％FBS，2.5g·L -1葡萄糖和0.11g·L -1丙酮酸钠的RPMI 1640培养基中培养4T1、4T1-Luc，EMT-6和B16F10细胞。将CT-26细胞维持在补充有10％FBS的DMEM培养基中。将所有细胞在37℃，5％CO 2条件下孵育。
雌性BALB/c或C57BL/6小鼠购自维通利华实验动物技术有限公司(中国北京)，并保持在无病原体的条件下。所有动物实验均在中国科学院国家纳米科学与技术中心(中国北京)的机构动物护理和使用委员会(IACUC)批准的指导下进行。
骨髓来源树突状细胞(BMDC)的生成
BMDC通过诱导分化从雌性BALB/c小鼠(6至8周龄)的BMSC中产生。简而言之，对小鼠实施安乐死后，收集骨骼并将其放入70％的乙醇中，然后用PBS洗涤。切开骨头的两个远端，并通过用RPMI 1640培养基轻轻冲洗来收集骨髓。收集的细胞中的红细胞用ACK缓冲液裂解，并以300×g离心5分钟收集剩余的细胞，然后在6孔板中的RPMI 1640培养基中培养(1×10 6细胞/孔)。10％FBS，2mM L-谷氨酰胺，100μg·mL -1青霉素，100μg·mL -1链霉素，0.05mMβ-巯基乙醇(β-ME)，10ng·mL -1鼠IL-4和20ng·mL -1鼠GM-CSF。在第2天和第4天添加新鲜培养基。在第6天，吸出非贴壁细胞，并在6孔板中与新鲜培养基一起孵育以进行进一步的实验。
分离大肠杆菌来源的细胞质膜(EM)
从大肠杆菌菌株DH5α分离出细胞质膜。简而言之，将冷冻保存的大肠杆菌DH5α细胞在液体LB培养基中于37℃在200rpm摇动下培养过夜。当OD 600为1.2时，通过离心(3000×g，20min，4℃)收集细菌，并用PBS洗涤3次。将细胞沉淀重悬于10mL缓冲液A(1M蔗糖，0.2M Tris-HCl，pH 8.0)中。然后加入溶菌酶至终浓度为2mg·mL -1，并将细胞在37℃下以120rpm摇动孵育1小时。加入补充有DNase(10μg·mL -1)的无菌水(90mL)，并将试管轻轻混合20次。通过在4℃下以3000×g离心20分钟收集原生质体(Spheroplasts)，并重悬于含有20％w/v的10mL冰冷的缓冲液B(20mM Tris-HCl，pH 7.2，50mM NaCl，5mM EDTA)中。蔗糖裂解细胞。通过在4℃以10,000×g离心30分钟来澄清裂解物的细胞碎片。将上清液的样品(5mL)放在不连续的蔗糖梯度上(底部至顶部：50％时10mL，46％ 时5mL，42％时10mL，36％时10mL，32时5mL)和10mL(27％)溶液)，并在Beckman Optima XPN-100超速离心机(Brea，CA，USA)中使用70Ti转子在4℃下以113,000×g离心2小时。随后，提取1.5mL的含细胞质膜的级分，并用冰冷的缓冲液B稀释，以将蔗糖浓度降低至10％(w/v)。通过在4℃下以113,000×g离心1小时收集膜，将其重悬于PBS中，并保存在-80℃下进行后续实验。使用BCA蛋白测定试剂盒定量最终膜制品的蛋白浓度。为了分析EM的蛋白质组成，进行了蛋白质组学分析(H·Wayen生物技术有限公司，中国上海)，共检测到2,302种蛋白质。
从切除的肿瘤组织中分离肿瘤细胞膜(TM)
为了从肿瘤组织获得细胞膜，将雌性BALB/c小鼠皮下接种鼠类4T1乳腺癌细胞，4T1-Luc乳腺癌细胞或CT-26结肠癌细胞。接种肿瘤后第7天，将肿瘤(约300mm 3)切除并切成小块，然后用GBSS缓冲液(2mL)处理，该缓冲液包含胶原酶IV(1.0mg·mL -1)，DNase(0.1mg·mL -1)和透明质酸酶(0.1mg·mL -1)于37℃消化组织。刮下细胞并通过以1000×g离心5分钟来收集。将沉淀物在2mL分离缓冲液(225mM甘露醇，75mM蔗糖，0.5％BSA，0.5mM EGTA，30mM Tris和磷酸酶和蛋白酶抑制剂混合物)中匀浆，然后在冰中通过超声处理(30W)分配浴3分钟，以充分破坏细胞。将细胞匀浆在4℃下以3000×g离心5分钟，并收集上清液。将上清液在4℃下以10,000×g进一步离心10分钟，并将得到的上清液在4℃下以100,000×g超离心2小时。最后，将源自肿瘤组织的细胞膜重悬于5mM Bis-Tris缓冲液(pH 6.0)中，并保存在-80℃下。使用BCA蛋白测定试剂盒确定膜提取物的蛋白浓度。为了分析TMs的蛋白质组成，进行了蛋白质组学分析(H·Wayen生物技术有限公司，中国上海)，共检测到5,353种蛋白质。
HM-NPs的产生及其理化性质
为了制备杂合膜(HM)囊泡，在膜融合之前，使用干浴培养箱在37℃下轻轻摇动混合的EM和TM 15分钟。HM囊泡是通过400nm截止挤出机(Hamilton Company，Reno，Nevada，USA)重复物理挤出而形成的。使用双乳液法制备了聚(丙交酯-乙醇酸共聚物)-OH(PLGA)纳米颗粒。简而言之，将PLGA以10mg·mL -1的浓度溶解在二氯甲烷中。然后，将1mL PLGA溶液与0.2mL无菌水混合。将混合物通过在冰浴中超声处理(25W)3分钟进行乳化，然后添加2mL 1％胆酸钠，并通过在冰浴中超声处理(30W)5分钟进行乳化。将乳液滴加到10mL的0.5％胆酸钠溶液中，并在室温下搅拌30分钟。旋转蒸发15分钟后，通过将乳液以10,000×g离心15分钟并在无菌水中洗涤两次来收获PLGA NP。为了工程化HM-NP，再次将PLGA NP和HM囊泡以200：1的截止挤出机(Hamilton Company)以5：1的聚合物与膜蛋白的质量 比重复地共挤出。
为了检查纳米颗粒和囊泡的形态，将10μL样品沉积在碳涂层的铜网上并孵育15分钟。使用滤纸吸收残留的液体，并用1％(v/v)乙酸铀酰对网格上的样品进行8分钟的负染色，然后风干，以使用透射电子显微镜(TEM；HT7700，HITACHI，东京，日本)。使用Zetasizer Nano ZS动态光散射(DLS)仪器(英国Malvern)测量流体动力学粒度分布，ζ电位和多分散指数(PDI)。使用类似的制备方法来产生EM-NP和TM-NP。
HM-NPs的蛋白质表征
为了研究HM-NPs的蛋白质谱，采用SDS-PAGE，所得凝胶用考马斯亮蓝(Solarbio，北京，中国)染色。所有样品均在上样缓冲液(Invitrogen，卡尔斯巴德，加利福尼亚州，美国)中制备，并在MOPAGE运行缓冲液(Invitrogen)中的NuPAGE Novex 12％分离凝胶(Invitrogen)上装载等量的蛋白质(10μg)。进行蛋白质印迹分析以鉴定每种组分的特异性蛋白质标记物的存在。如上所述将蛋白质分解并转移到硝酸纤维素膜上(Thermo Fisher Scientific，沃尔瑟姆，马萨诸塞州，美国)，并用对FtsZ有特异性的一抗进行探测，用于表征细菌的细胞质膜。小鼠抗Na +/K +-ATP酶，ATP5A(IM CVa)，泛醇-细胞色素C还原酶核心蛋白I(IM Core I)，VDAC1/Porin(OM Porin)，基质亲环蛋白D(Matrix CypD)和IMS细胞色素C(IMS Cytc)的抗体也同样用于检测；这些是肿瘤来源的膜的标志物。对于FtsZ，使用的第二抗体是抗兔IgG的HRP偶联抗体，而对于其他第一抗体，第二抗体是HRP的抗小鼠IgG偶联抗体。为了进一步表征HM-NP，进行了免疫金染色。将样品沉积到碳涂层的铜栅板上。用PBS中的1wt％牛血清白蛋白(BSA)封闭网格。EM-NP网格用10μL抗兔FtsZ(0.5mg·mL -1)染色，TM-NP网格用10μL抗小鼠Na +/K +-ATP酶(0.5mg·mL -1)染色。孵育一个小时后，将样品在1％BSA中洗涤6次。EM-NPs网格随后用10μL抗兔IgG-金结合物(金纳米大小为5nm)染色，用1％BSA以1:20稀释。TM-NP网格用10μL抗小鼠IgG-金结合物(金纳米大小为10nm)染色，并用1％BSA以1:20稀释。在黑暗中孵育一个小时后，将网格用PBS洗涤8次。然后将样品固定在PBS中的50μL1％戊二醛中，并用无菌水洗涤8次，每次2分钟。最后，将样品网格用10μL1％乙酸铀酰染色，并使用TEM(HT7700，HITACHI，日本)进行检查。HM-NP网格是按照相同的程序制备的。针对FtsZ和Na +/K +-ATP酶的抗体混合物被用作一抗。结合抗兔IgG-金结合物(金纳米大小为5nm)和抗小鼠IgG-金结合物(金纳米大小为10nm)以标记HM-NP。
HM-NP的设计与表征
HM-NP的工程设计包括三个步骤：1)制备EM-TM杂合膜(HM)囊泡，2)合成丙交 酯-乙醇酸聚合物纳米颗粒(PLGA NPs)和3)HM涂层到PLGA NP上(图29A)。从大肠杆菌菌株DH5α中分离出EM，从自体肿瘤细胞膜中分离出TM，并通过液相色谱-质谱(LC-MS)鉴定了大肠杆菌内膜(表1A-1B)和手术切除的4T1肿瘤细胞质膜(表2A-2B)的蛋白质表达谱。图22中的维恩图说明EM(图22左；E1，E2，E3和E4)和TM(图22右；T1，T2，T3和T4)的四批不同样品之间的膜蛋白差异很小。以EM与TM之间的比例1:100、1:75、1:50、1:25、1:10、1:5、1:3、1:1、1:0、0:1、3:1、5:1、10:1、25:1、50:1、75:1或100:1的蛋白质质量比，将不同比例的膜进行融合，反复通过400nm滤膜孔径的脂质体挤出器来制备HM。为了确定激活DC的最佳膜比例，将不同膜比例的HM与BMDC共孵育24小时，并测量释放的促炎细胞因子。结果表明，随着EM量的增加，BMDC促炎性细胞因子释放增加(图23A-23C)。EM与TM的蛋白质质量比为3：1的组诱导了水平最高的促炎细胞因子产生。
表1A LC-MS鉴定大肠杆菌DH5α细胞质膜的蛋白质表达谱
Figure PCTCN2021094037-appb-000002
表1B LC-MS鉴定革兰氏阴性菌内膜中的蛋白质表达谱
Figure PCTCN2021094037-appb-000003
Figure PCTCN2021094037-appb-000004
Figure PCTCN2021094037-appb-000005
使用Proteome Discoverer 2.4软件(Thermo Fisher Scientific，美国)分析了EM中的蛋白 质。登记号(蛋白登录号)：默认显示用于生成报告的FASTA数据库分配给蛋白质的唯一标识符。丰度：显示缩放和归一化之前样本的丰度值。数据表示为大肠杆菌DH5α的4个生物学独立的细胞质膜样品的平均值±标准差。
通过质谱分析，还鉴定出一些特征蛋白，这些特征蛋白可以用于确认药物组合中具有来源于细菌内膜的组分。例如，这些细菌内膜特征蛋白可以包含选自以下组的蛋白或其功能活性片段：细胞分裂蛋白FtsZ、内膜蛋白YhcB、内膜蛋白易位酶YidC、细胞分裂蛋白NlpI、ABC转运蛋白MsbA、内膜转运蛋白TatA、膜间磷脂转运系统脂蛋白MlaA、内膜蛋白TolQ、膜间转运脂蛋白PqiC、外膜蛋白TolC、外膜引入蛋白FimD、外膜孔蛋白OmpC、膜间转运蛋白PqiB、主要外膜脂蛋白Lpp、膜结合型溶血性壁蛋白转糖基酶MltB、UPF0194膜蛋白YbhG、推定的膜蛋白IgaA同源物、跨膜磷脂转运系统脂蛋白MlaA、内膜蛋白YejM、膜结合型溶血性壁蛋白转糖基酶MltA、跨膜磷脂转运系统结合蛋白MlaC、内膜蛋白YlaC、跨膜转运蛋白YebT、膜结合型溶血性胞壁质转糖基化酶MltC、跨膜磷脂转运系统ATP结合蛋白MlaF、膜间磷脂转运系统结合蛋白MlaD、内膜蛋白YqjE、UPF0053内膜蛋白YfjD、UPF0053内膜蛋白YoaE、内膜型溶胞性胞壁质转糖基酶A、UPF0394内膜蛋白YedE、膜间磷脂转运系统结合蛋白MlaB、内膜蛋白YccF、跨膜磷脂转运系统通透酶蛋白MalE、内膜蛋白YedI、内膜蛋白YgaP、膜间转运蛋白PqiA、UPF0056膜蛋白YhcE、内膜蛋白YbhL、内膜蛋白YhjD、内膜转运蛋白YbaT、内膜蛋白YjjP、内膜蛋白YhaH、内膜蛋白YbjJ、内膜转运蛋白YqeG、UPF0053内膜蛋白YtfL、砷泵膜蛋白ArsB、内膜蛋白YpjD、C型溶菌酶的膜结合溶菌酶抑制剂MliC、UPF0283膜蛋白YcjF、UPF0259膜蛋白YciC、内膜蛋白YgfX、内膜蛋白YbbJ、脂蛋白释放系统跨膜蛋白LolE、内膜蛋白YjcH、蛋白质输出膜蛋白SecG、内膜蛋白YfdC、UPF0324内膜蛋白YeiH、UPF0266膜蛋白YobD、TVP38/TMEM64家族内膜蛋白YdjZ、膜相关蛋白UidC、内膜蛋白YbiR、内膜蛋白YhiM、膜转运蛋白YfcA、内膜蛋白YdgK、膜结合型溶血性壁蛋白转糖基酶F、多药耐药性外膜蛋白MdtP、UPF0208膜蛋白YfbV、肽聚糖相关脂蛋白、脂蛋白YiaD、脂蛋白YeaY、载脂蛋白N-酰基转移酶、D-蛋氨酸结合脂蛋白MetQ、脂蛋白GfcD、脂蛋白YbjP、脂蛋白YgeR、脂蛋白释放系统ATP结合蛋白LolD、渗透诱导性脂蛋白OsmE、渗透诱导脂蛋白OsmB、脂蛋白YdcL、脂蛋白YajG、脂蛋白NlpE、磷脂酰甘油-脂蛋白二酰甘油基转移酶、脂蛋白YghJ、脂蛋白YedD、脂蛋白YifL、脂蛋白YgdI、脂蛋白YbaY、脂蛋白信号肽酶、脂蛋白YceB、脂蛋白YajI和内膜蛋白YebE，以及上述中的任意组合。
表2A LC-MS鉴定来源于手术切除的肿瘤的肿瘤膜的蛋白表达谱
Figure PCTCN2021094037-appb-000006
表2B LC-MS鉴定肿瘤组织来源细胞膜中的蛋白质表达谱
Figure PCTCN2021094037-appb-000007
Figure PCTCN2021094037-appb-000008
Figure PCTCN2021094037-appb-000009
Figure PCTCN2021094037-appb-000010
使用Proteome Discoverer 2.4软件(Thermo Fisher Scientific，美国)分析TM中的蛋白质。蛋白登录号：默认显示用于生成报告的FASTA数据库分配给蛋白质的唯一标识符。丰度：显示缩放和归一化之前样本的丰度值。数据表示为来自手术切除的肿瘤的4个生物学独立的肿瘤膜样品的平均值±标准差。
通过质谱分析，还鉴定出一些特征蛋白，这些特征蛋白可以用于确认药物组合中具有来源于其它生物体的组分。例如，这些其它生物体的特征蛋白可以包含选自以下组的蛋白或其功能活性片段：抗原肽转运体1、H-2Ⅱ类组织相容性抗原γ链、酪氨酸蛋白激酶SYK、高亲和力免疫球蛋白epsilon受体亚单位γ、Ras相关C3肉毒毒素底物2、酪氨酸蛋白激酶BTK、受体型酪氨酸蛋白磷酸酶C、Na +/K +-ATP酶、ATP5A(IM CVa)、泛醌细胞色素C还原酶核心蛋白I(IM Core I)、VDAC1/孔蛋白(OM Porin)、基质亲环素D(Matrix CypD)、膜间隙细胞色素C(IMS Cytc)、基底膜特异性硫酸乙酰肝素蛋白聚糖核心蛋白(HSPG2)、质膜钙转运ATP酶1(ATP2b1)、内质网膜蛋白复合物亚基1(EMC1)、跨膜蛋白43(TMEM43)、囊泡膜蛋白VIP36(LMAN2)、囊泡相关膜蛋白相关蛋白A(VAPA)、跨膜9超家族成员2(TM9SF2)、跨膜emp24域含蛋白10(TMED10)、脂肪细胞质膜相关蛋白(APAMP)、突触小泡膜蛋白VAT(VAT1)、核孔膜糖蛋白210(NUP210)、质膜钙转运ATP酶4(ATP2b4)、质膜钙转运ATPase 2(ATP2b2)、红细胞带7整合膜蛋白(STOM)、跨膜emp24域含蛋白9(TMED9)、线粒体进口内膜转座酶亚基TIM44(TIMM44)、膜相关孕激素受体组分1(PGRMC1)、ER膜蛋白复合物亚基3(EMC3)、囊泡相关膜蛋白相关蛋白B(VAPB)、膜伯胺氧化酶(AOC3)、囊泡相关膜蛋白7(VAMP7)、高尔基体膜蛋白4(GOLIM4)、膜相关孕激素受体组分2(PGRMC2)、跨膜9超家族成员3(TM9SF3)、跨膜9超家族成员4(TM9SF4)、内质网膜蛋白复合物亚基2(EMC2)、线粒体进口内膜转座酶亚单位TIM50(TIMM50)、过氧化物酶体膜蛋白11B(PEX11b)、跨膜emp24域含蛋白2(TMED2)、分泌载体相关膜蛋白3(SCAMP3)、硫氧还蛋白相关跨膜蛋白4(TMX4)、过氧化物酶体膜蛋白PMP34(SLC25a17)、 过氧化物酶体膜蛋白PEX14(PEX14)、内质网膜蛋白复合物亚基8(EMC8)、干扰素诱导的跨膜蛋白3(IFITM3)、溶酶体相关膜糖蛋白2(LAMP2)、硫氧还蛋白相关跨膜蛋白2(TMX2)、囊泡相关膜蛋白3(VAMP3)、溶酶体相关膜糖蛋白1(LAMP1)、线粒体导入内膜转座酶亚单位Tim8A(TIMM8a1)、溶酶体膜蛋白2(SCARB2)、Ig gamma-2A链C区(IGHG2a)、跨膜9超家族成员1(TM9SF1)、核内膜蛋白Man1(LEMD3)、跨膜emp24域含蛋白4(TMED4)、Thy-1膜糖蛋白(Thy1)、线粒体导入内膜转座酶亚基Tim23(TIMM23)、线粒体导入内膜转座酶亚基Tim9(TIMM9)、线粒体导入内膜转座酶亚基Tim13(TIMM13)、分泌载体相关膜蛋白2(SCAMP2)、分泌载体相关膜蛋白1(SCAMP1)、整合膜蛋白2B(ITM2b)、线粒体进口内膜转座酶亚单位Tim10(TIMM10)、液泡膜蛋白1(VMP1)、生长激素诱导的跨膜蛋白(GHITM)、含死亡结构域的膜蛋白(NRADD)、囊泡相关膜蛋白8(VAMP8)、跨膜前后转化蛋白1(TAPT1)、线粒体膜间空间导入和组装蛋白40(CHCHD4)、糖基化溶酶体膜蛋白(GLMP)、跨膜4结构域超家族A成员6D(MS4A6D)、易位链相关膜蛋白1(TRAM1)、干扰素诱导的跨膜蛋白2(IFITM2)、肌膜相关蛋白(SLMAP)、膜镁转运蛋白1(MMGT1)、核包膜孔膜蛋白POM 121(POM121)、跨膜蛋白C16orf54同源蛋白(AI467606)、液泡ATPase组装整合膜蛋白Vma21(VMA21)、上皮膜蛋白1(EMP1)、膜相关磷脂酰肌醇转移蛋白1(PITPNM1)、小整合膜蛋白15(SMIM15)、核膜整合膜蛋白1(NEMP1)、整合膜蛋白GPR180(GPR180)、分泌载体相关膜蛋白4(SCAMP4)、易位链相关膜蛋白2(TRAM2)、跨膜和卷曲螺旋域蛋白3(TMCC3)、基质膜相关蛋白1(SMAP1)、神经元膜糖蛋白M6-b(GPM6b)、上皮膜蛋白2(EMP2)、高尔基体膜蛋白1(GOLM1)、SID1跨膜家族成员2(SIDT2)、跨高尔基体Golgi网络整合膜蛋白2(TGOLN2)、高尔基体膜蛋白TVP23同系物B(FAM18b)、线粒体内膜蛋白OXA1L(OXA1L)、骨化相关的跨膜蛋白1(OSTM1)、过氧化物酶体膜蛋白PEX13(PEX13)、单侧通过膜和卷曲螺旋结构域蛋白1(SMCO1)、远端膜臂组装复合蛋白1(DMAC1)、分泌载体相关膜蛋白5(Scamp5)、囊性纤维化跨膜电导调节剂(CFTR)、破骨细胞刺激性跨膜蛋白(OCSTAMP)、诱导脂肪存储的跨膜蛋白2(FITM2)和跨膜通道样蛋白5(TMC5)，以及上述中的任意组合。
透射电子显微镜(TEM)图像显示，所有三种膜囊泡制剂(EM，TM和HM)具有相似的大小，直径约100nm，而与TM和HM囊泡相比，EM囊泡的Zeta电位更负(图24A-24C)。本申请评估了所有三个膜NP(EM-NP，TM-NP和HM-NP)中脂多糖(LPS)的含量，并且发现HM-NP与无LPS对照组之间无显著差异(p＝0.2977)(图25)，表明从大肠杆菌内膜在制备过程中去除了大部分LPS。使用双乳液法制备了PLGA NP。通过将PLGA核和HM囊泡 通过200nm滤膜孔径的脂质体挤出器重复共挤出来得到HM-NP。类似地，通过将PLGA核与EM囊泡或TM囊泡通过200nm滤膜孔径的脂质体挤出器重复共挤出，即可生产出EM涂层PLGA NP(EM-NP)和TM涂层PLGA NP(TM-NP)。TEM图像显示HM-NPs具有均匀的球形纳米结构，具有内核和外壳(图29B和图26A-26B)。更具体地说，HM-NP的PLGA内核和膜外壳的直径分别为86.83±7.41nm和14.16±2.07nm(图26A-26B)。并且在纳米颗粒与杂合膜的质量比为5：1时，HM-NPs的表面电荷为-21.3mV(图29C)，与HM囊泡的电荷相比基本不变(图24C)，表明在该比例下，HM表面是完整的并且已达到饱和。动态光散射(DLS)分析表明，HM-NP的水合粒径约为180nm(图29C)，与EM-NP和TM-NP相似。TEM和DLS结果之间的粒径大小差异归因于以下原因：TEM测量的是样品干燥状态下的直径，而DLS测定的是样品的水合粒径，同时考虑了颗粒表面的水合层。在稳定性研究中，HM-NPs的大小和Zeta电位在冷冻干燥之前和重悬于磷酸盐缓冲盐水(PBS；图27A-27B)后几乎保持不变。此外，HM-NP的这些物理性质在将它们悬浮于4℃的PBS中超过3周后仍保持稳定(图28A-28B)。
为了鉴定膜包被的NPs所包含的蛋白质，本申请通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)解析了蛋白质，并用考马斯亮蓝对所得的凝胶进行了染色。HM-NP的蛋白质特征类似于来自两个单独的膜NP的蛋白质组合(图30，EM-NP和TM-NP)。为了进一步表征HM-NP的特性，本申请检查了EM-NP，TM-NP和HM-NP的特定蛋白(图29D)。FtsZ是一种细菌细胞分裂蛋白，同时存在于EM-NP和HM-NP上(图29D上)，而一系列哺乳动物细胞蛋白，包括Na +/K +-ATP酶，ATP5A(IM CVa)，泛醇-细胞色素C还原酶核心蛋白I(IM Core I)，VDAC1/Porin(OM Porin)，基质亲环蛋白D(Matrix CypD)和IMS细胞色素C(IMS Cytc)都很明显存在于TM-NP和HM-NP(图29D下)。此外，对三种膜纳米颗粒进行免疫金染色，然后利用TEM进行观察，提供了直接的视觉证据，即HM-NP上分别存在FtsZ和Na +/K +-ATP酶(分别为EM和TM的特异性标志物)(图29E)。
与HM-NPs中的10nm金颗粒(与Na +/K +-ATP酶结合，标记TM)相比，HM-NPs表面的5nm金颗粒(与FtsZ结合，标记EM)的丰度更大(图31A-31B)，对应的事实是EM的投料比比TM大。综上所述，本申请已经成功构建了从细菌内膜和肿瘤细胞膜继承了关键成分的杂合膜包被的纳米颗粒。
实施例13
HM-NPs增强肿瘤抗原的摄取，激活BMDC表面TLRs表达，同时将抗原和佐剂共递送给BMDC的检测
内化进入DC是肿瘤相关抗原(TAA)加工和呈递的第一步。因此，本申请研究了BMDCs对三种膜NP的内化作用。将装载罗丹明B的膜制剂与BMDCs孵育一个小时后，发现罗丹明B标记的三种膜纳米颗粒均内化进入BMDCs中，并定位于溶酶体结构内(图35A和图32)。由于大多数纳米颗粒都被捕获进溶酶体区室中，因此三种膜NP与溶酶体的共定位效率没有统计学差异(图33)。我们还利用流式细胞分析仪研究小鼠BMDCs摄取各组膜纳米颗粒的能力，，合成了NP(Mix NP，即物理混合EM-NP和TM-NP)或HM-NP的组合罗丹明B负载的膜样品，并与BMDC共孵育8小时。然后，本申请通过流式细胞仪评估了各组细胞内的罗丹明B荧光强度(图35B)。本申请观察到BMDC仅摄取了很少部分TM-NP(5.16％)，这表明了仅来自TM的疫苗不足以有效地将抗原呈递给APCs。相反，BMDC有效地摄取了两种细菌内膜衍生的制剂。含EM的样品(EM-NP，Mix NP和HM-NP)比TM-NP组显示出20倍的细胞相关荧光。分析原因，DCs表面展示有大量PRRs，它有助于识别EM表面PAMPs，有利于增加DCs的摄取水平。此外，HM-NPs的吸收率几乎与EM-NPs的吸收率相同(图35B)。这些结果表明，细菌内膜的引入可以促进BMDC对纳米颗粒的摄取，并且通过膜融合技术赋予HM-NP以刺激DC摄取肿瘤抗原的的特性。研究表明，TLRs可以识别细菌表面PAMPs，启动先天性免疫应答。为了研究EM佐剂刺激DCs表面TLRs表达情况，我们将不同组膜纳米颗粒(EM-NPs，TM-NPs，Mix NPs以及HM-NPs)与BMDCs在37℃条件下共孵育24小时。随后提取各组细胞表面全蛋白，利用一系列特异性膜表面TLRs蛋白抗体TLR1，TLR2，TLR4和TLR6检测各组蛋白表达水平。，本申请观察到HM-NP治疗组的BMDC中TLR1，TLR2和TLR6的表达高于对照组(图35C)，证明了HM-NPs能够刺激APCs表面多种膜TLRs表达，显示出很好的先天性免疫刺激潜力。本申请发现所有组的TLR4表达均无明显差异，这可能是由于在HM-NPs制备过程中移除了绝大部分LPS，导致其下游TLR4受体表达无明显变化(图25)。TLR刺激通过下游促炎信号传导调节促炎细胞因子的产生。与对照组相比，本申请观察到了HM-NPs组的NF-κB分子表达明显上调(图35C)。图35D中示出了可能的已知信号转导机制。
为了评估EM对BMDCs的先天性免疫刺激水平活，本申请首先检查了单独EM-NPs与BMDCs共孵育一定时间后刺激其分泌促炎细胞因子的能力及变化情况。我们选取了一系列浓度梯度(0.12mg/mL，0.25mg/mL，0.50mg/mL，1.00mg/mL)的EM-NPs与BMDCs共孵育，并选取不同的时间点(0h，3h，8h，24h，48h)收集各组细胞上清液，利用试剂盒检测上清液中促炎细胞因子IL-6的分泌水平。观察到IL-6的分泌水平呈现出明显的浓度和时间依赖性，表明细菌来源的膜囊泡可以刺激BMDC活化，，激活先天性免疫反应(图34)。为了 进一步了研究不同组膜纳米颗粒刺激BMDCs分泌促炎细胞因子的能力，将不同的膜NP与BMDC共孵育24小时，然后测量特定的细胞因子(图35E-35G)。与TM-NP组相比，本申请观察到HM-NP处理的BMDC中大量IL-6，TNF-α和IL-1β释放。HM-NPs中这种引人注目的细胞因子释放曲线归因于细菌细胞质膜成分的佐剂性，因为TM-NPs缺乏刺激BMDC的能力。此外，HM-NPs在刺激IL-6和IL-1β分泌方面比Mix NPs更有效(p<0.0001)。相比于单个成分的混合一起递送，肿瘤自体抗原和免疫佐剂在同一纳米颗粒上具有更强提高免疫刺激强度的潜力。
接下来，本申请研究了HM-NPs促进BMDC成熟的能力。将BMDC用不同的样品处理24小时，然后用流式抗体CD11c(DC的标记)，CD80(成熟DC的刺激标记)和CD86(成熟DC的刺激标记)对细胞进行染色，然后测量流式细胞仪检测细胞的荧光强度(图35H-35I)。与BMDC活化结果一致(图35E-35G)，所有含EM的纳米颗粒(EM-NP，Mix NP和HM-NP)中成熟BMDC的百分比均显著增加(p<0.0001)。如CD80和CD86的表达增强所表明的。本申请还观察到，当与TM包被的纳米颗粒一起孵育时，BMDCs未能实现有效成熟(图35H-35I)。这些结果表明细菌内膜赋予了HM-NPs促进DC成熟的能力。再次，与DC激活结果一样(图35E-35G)，与Mix NP组相比，HM-NPs处理过的BMDCs成熟比例更大(p<0.0001)，进一步证实了HM-NPs在增强先天性免疫应答上的优势(图35H-35I)。值得注意的是，HM-NP和Mix NP的BMDC内化率之间没有显著差异(p>0.9999)，但二者在先天性免疫刺激能力方面却出现了显著性差异(图35B)。为了解释这一现象，我们猜想：与单独的抗原和佐剂物理混合相比，利用同一个膜包裹的纳米体系可以实现更高水平的抗原-佐剂共递送，产生更强的协同刺激免疫效果。
为了验证上述假设，本申请利用免疫荧光实验进一步研究了HM-NP和Mix NP处理的BMDC中EM和TM组分的共定位效率。将BMDC与HM-NP或Mix NP孵育8小时。红色荧光标记的FtsZ抗体和绿色荧光标记的Na +/K +-ATP酶抗体分别用于鉴定EM和TM(图35J)。免疫荧光图像显示，当用HM-NPs处理BMDC时，共定位显著大于Mix NP组(p<0.0001)(平均81.53％vs 52.39％；图35K)。这些结果表明，与混合样品相比，HM-NP具有增强的将EM和TM输送到同一DC的能力。将抗原和佐剂共同递送至DC可增强肿瘤抗原的免疫原性和癌症疫苗在免疫疗法中的功效。本申请的结果验证了HM-NP可以促进这种共同呈递。
实施例14
HM-NPs促进LNs中的DC成熟并激活肿瘤膜抗原特异性T细胞的检测
为了评估体内DC成熟，将雌性BALB/c小鼠皮下接种鼠类4T1乳腺癌细胞。当肿瘤的体积达到约300mm 3时，通过手术将其切除。切除每只小鼠的大部分肿瘤组织以制备疫苗制剂，剩下的大约1％用来模拟诊所手术床中残留的微肿瘤的存在。在手术后第2天给小鼠成像，测量肿瘤并将其随机分为五组。在手术后第3、5和9天用不同的疫苗制剂对小鼠进行免疫。在最后一次疫苗接种12小时后，本申请评估了腹股沟淋巴结中DC上刺激分子的存在。如图37A-37B所示，DC刺激后两种刺激标记(CD80和CD86)的表达均显著升高。用HM-NP组中表达最高的所有三种膜包被的纳米颗粒(包括TM-NP)治疗小鼠(HM-NP，EM-NP和Mix组中CD80和CD86的表达分别为14.38％，12.36％和13.82％；分别为14.46％，9.76％和11.92％)。与体外(图35H-35I)相比，体内TM-NP的功效增强(图37A-37B)，可能是因为TM-NPs进入小鼠体内后，作为病原体损伤相关分子模式(Damage-associated molecular patterns,DAMPs)被免疫系统中致敏的免疫细胞(如DCs)所识别，在一定程度上刺激了DCs成熟。HM-NP治疗组表现出最高的共刺激标志物表达，这与体外成熟结果一致(图35H-35I)。
要通过激活的DC将与肿瘤膜相关抗原呈递给初始T细胞，抗原必须迁移至淋巴结(LN)。为了确定HM-NP是否可以在LN中积累，本申请将荧光染料IR-780标记的膜NP皮下注射到BALB/c小鼠中。12小时后，切除腹股沟LN并使用体内成像系统(IVIS；图36A)成像。在所有组中均观察到荧光信号，而HM-NP组的荧光强度是EM-NP组的3.3倍，是TM-NP组的12.4倍(图36B)，表明HM-NPs最有效地迁移至LNs进行肿瘤抗原呈递。
为了进一步验证HM-NP是否可以有效激活初始T细胞，本申请用鼠4T1乳腺癌细胞皮下接种了雌性BALB/c小鼠。如上所述进行外科手术和疫苗接种程序。为了评估抗原特异性CD8 +T细胞，在最终疫苗接种12小时后收集脾细胞，并与4T1细胞膜(作为抗原)一起孵育24小时。结果表明，在TM-NP治疗组中几乎观察不到阳性斑点，表明仅肿瘤膜制剂对T细胞的激活有限(图37C-37D)。同时，在EM-NP治疗组中激活了适量的T细胞。这种弱的免疫反应可能是由于缺乏肿瘤膜抗原。既包含抗原又包含佐剂的Mix NP和HM-NP组显示出比其他组更多的阳性斑点。这些结果反映了肿瘤膜的弱免疫原性，而EMs的存在可以增强这种免疫原性。与Mix NP治疗组相比，抗原的共同输送和佐剂策略引起T细胞释放的IFN-γ大约增加了两倍。此外，通过流式细胞术评估，T细胞中肿瘤膜抗原特异性细胞因子的产生证实，HM-NPs增强特异性T细胞反应的能力比其他制剂要强得多(图37E-37F)。这些结果表明，HM-NP可以通过肿瘤抗原和佐剂的共同转运来实现DC的成熟和脾T细胞的活化。
HM-NPs引起低全身炎症反应的检测
在癌症免疫治疗过程中增强对患者免疫系统的刺激，以及在制剂中整合细菌膜成分，可 导致危及生命的副作用，例如细胞因子风暴。为了评估疫苗制剂刺激的全身炎症反应强度，本申请用鼠4T1乳腺癌细胞皮下接种了雌性BALB/c小鼠。如上所述进行外科手术和疫苗接种程序。在ProcartaPlex多重免疫分析中，通过基于Luminex磁珠的ELISA试剂盒检测血清中炎性细胞因子和趋化因子的浓度。总体而言，经HM-NP治疗的患者血清IL-6，IFN-γ，TNF-α，MCP-1，IL-12p70，IL-1β，IL-23，IL-27和IL-17A的血清浓度均升高小鼠(图37G和图38A-38I)。然而，与对照小鼠相比，在HM-NP治疗的小鼠中仅两种促炎细胞因子IL-6和IL-1β的增加达到统计学显著性(p＝0.0269；p＝0.0139)。其余11种细胞因子或趋化因子在所有组中表达相似，表明由HM-NP诱导的炎症反应可控(图37G和图38A-38I)。两者合计，HM-NPs既可以增强先天免疫又具有适应性免疫，且刺激全身性炎症反应低，表明该制剂具有安全引发抗肿瘤免疫治疗疗效的潜力。
实施例15
HM-NP疫苗接种可在鼠4T1-Luc肿瘤模型中诱导肿瘤消退的检测
确认HM-NP可以在体内激活DC和T细胞后，本申请着手评估HM-NP疫苗抑制肿瘤复发的能力(图39A)。为此，本申请按照外科手术和疫苗接种程序，对雌性BALB/c小鼠皮下注射了鼠4T1-Luc乳腺癌细胞，手术前后使用IVIS对小鼠皮下肿瘤进行监测(图39B)。IVIS图像显示在所有非HM-NP疫苗接种组中肿瘤体积逐渐增加。如图39C-39E，肿瘤生长不受EM-NP或TM-NP疫苗接种的影响。尽管Mix NPs制剂延迟了肿瘤的复发，但抗肿瘤作用仍很温和，在60天内的存活率为25％(图39E，n＝12)。相反，用HM-NP疫苗治疗的小鼠表现出更强的肿瘤抑制能力，存活率高达约92％，进一步证实了使用杂合膜纳米平台共同递送肿瘤抗原和佐剂的好处。重要的是，在存活至实验终点(第60天)的小鼠中未观察到肿瘤复发，表明动物存活率明显高于其他组。
实施例16
HM-NP疫苗可抑制多种鼠类肿瘤模型的肿瘤消退的检测
为了证明HM-NP在术后免疫治疗中的普适性，本申请将雌性BALB/c小鼠皮下接种了CT26结肠肿瘤细胞。手术程序和疫苗接种时间表(图40A)与上述相同。用电子卡尺测量肿瘤体积，并在60天内监测三个不同组的存活。如图40B-40D所示，在HM-NP组中，手术治疗后的疫苗接种有效地抑制了CT26肿瘤在手术部位的复发。与上述4T1-Luc模型的结果相似(图39A-39E)，与未治疗组相比，Mix NP制剂仅将肿瘤复发延迟了八天。Mix NP组中没有小鼠存活至第60天的时间点。接种HM-NP的小鼠在60天内未出现肿瘤复发，而Mix  NP组的中位生存时间约为37天(图40E)。值得注意的是，本实验中HM-NPs免疫后的所有小鼠均实现了有效的肿瘤复发抑制，抑制率高达100％。
CT26和4T1-luc模型具有更高的免疫原性，并且对免疫疗法反应更强。为了进一步证明本申请基于HM的疫苗的治疗潜力，本申请在两种免疫原性较低的肿瘤模型B16F10黑色素瘤和EMT-6乳腺肿瘤模型中测试了该制剂。与本申请在CT26和4T1-Luc模型中的结果相似，接种HM-NP的小鼠中有90％以上的小鼠在60天之内未见肿瘤复发，而在Mix NP和对照组中观察到40％-100％的肿瘤复发。B16F10黑色素瘤(图40F)和EMT-6乳腺肿瘤(图40G)模型。同样，本申请的结果表明，基于杂合膜策略的疫苗可应用于多种实体瘤模型，具有显著的治疗功效。
本申请还在小鼠CT-26肿瘤模型中比较了用作疫苗佐剂的细菌内膜与市售佐剂单磷酰脂质A(MPLA)的作用(图41A-41B)。同时，本申请还研究了没有PLGA核心的杂交膜囊泡的抗肿瘤功效。基于EM的癌症疫苗制剂始终比包括HM囊泡和三个含MPLA的组(TM-NP+游离MPLA，TM-MPLA-NP和TM-NP)的其他含肿瘤膜抗原的制剂表现出更高的治疗功效，图41A-41B。本申请观察到，HM-NP组的完全缓解率(CR)为100％，而HM囊泡组为62.5％，三个含MPLA的组为50.0％-62.5％(图41A-41B)，结果表明在实验组中具有杂合膜包被的PLGA NP的疫苗制剂是最佳制剂。
实施例17
HM-NP疫苗接种可保护CT26肿瘤模型中的肿瘤再调整的检测
本申请进一步调查了HM-NPs是否可以在CT-26肿瘤再激发模型中提供超过60天的长期保护。在该模型中，将所有来自CT-26肿瘤模型的HM-NPs疫苗接种组的小鼠随机分为三组，并分别接种生理盐水，CT-26结肠腺癌细胞或4T1乳腺癌细胞。如图42A-42B所示，接种CT-26肿瘤细胞的小鼠显示出完全的肿瘤消除和100％的肿瘤抑制率。此外，生理盐水组中的小鼠在90天内仍未出现肿瘤复发。但是，接种4T1细胞的小鼠的肿瘤体积在3周后达到1000cm 3，这表明HM-NP提供的保护作用是CT-26特异性免疫反应。此外，本申请发现，与4T1接种组相比，CT-26细胞接种的小鼠中促炎细胞因子(包括IL-6，IL-1β和TNF-α)的血清浓度增加了。CT26肿瘤再激发(挑战)模型(图42C-42E)。此外，在CT-26接种组中特异性免疫细胞因子IFN-γ的分泌也增加了(图42F)。在T细胞亚群中，HM-NPs与初始的T细胞相比也增加了效应记忆T细胞(图42G-42H)。由于存在相同的肿瘤抗原，这些结果与更强的免疫应答是一致的。因此，HM-NP疫苗不仅提供了针对肿瘤复发的免疫力，而且还提供了特定的长效保护。
HM-NP疫苗接种后肿瘤抑制需要先天性和适应性免疫的分析
NK细胞和巨噬细胞在先天免疫中起着重要作用，而CD4 +T细胞和CD8 +T细胞对于适应性免疫应答至关重要。为了探索HM-NPs引发抗肿瘤反应增强的潜在机制，本申请用CT-26鼠结肠腺癌细胞接种给小鼠，并如上所述切除了肿瘤。给雌性BALB/c小鼠皮下接种CT-26细胞。当肿瘤的体积达到约300mm 3时，通过手术将其切除。然后，本申请将小鼠随机分为六个不同的组。腹腔内注射针对免疫细胞表面标志物的消耗抗体(表3)。在开始进行HM-NP治疗以耗消耗巨噬细胞(CSF1R抗体)，NK细胞(ASGM1抗体)，CD8 +T细胞(CD8抗体)或CD4 +T细胞(CD4抗体)之前的一天。通过外周血单核细胞(PBMC；图43)的流式细胞术证实了特定细胞类型的消耗。然后在手术后的第3、5和9天给小鼠接种HM-NP或生理盐水(作为对照)3次。如图42I和图44A所示，接种HM-NP的小鼠的免疫系统保持完整时，其肿瘤缓解率达到100％。但是，尽管接种了HM-NPs，所有CD8耗竭的小鼠仍会复发肿瘤，这揭示了CD8 +T细胞在肿瘤消退中的主要作用(图42I和图44B)。NK细胞耗竭也导致总体存活率显著降低，而CD4 +T细胞阻断并未显著影响(p＝0.3173)HM-NP疫苗接种的效果(图44B)。巨噬细胞的限制导致在HM-NP接种的小鼠中一定程度的肿瘤复发，肿瘤复发率高达40％(图42I和图44B)。综上，抗体耗竭研究表明CD8 +T细胞和NK细胞均对肿瘤抑制至关重要，其他免疫细胞亚群也在肿瘤消退中发挥了一定作用。
表3 HM-NP治疗期间的选择性细胞类型耗竭
Figure PCTCN2021094037-appb-000011
通过流式细胞仪分析小鼠的外周血，确认了巨噬细胞(CSF1R抗体)，NK细胞(ASGM1抗体)，CD8 +T细胞(CD8抗体)或CD4 +T细胞(CD4抗体)的耗竭(图43)。
实施例18
HM-NP疫苗安全性检测
由于细菌成分存在于疫苗制剂中，因此本申请评估了其生物安全性。由于产生溶血素，许多细菌具有致病性。因此，本申请进行了溶血试验，其中用一定范围的HM-NP浓度处理了红细胞。在任何测试浓度的HM-NP中均未观察到明显的溶血现象(图45)。接下来，在4T1-Luc模型的肿瘤消退实验终点，本申请收获并用苏木精-伊红(H&E)染色处理各组的小鼠的主要器官。在所检查的小鼠任何器官中均未观察到明显的组织学变化(图46)。此外，有研究表明细菌膜中的病原体源性分子可以通过多种病因引发肝脏和肾脏损伤，因此对小鼠组的肝和肾功能进行了评估。在HM-NP治疗的小鼠中，肝酶(ALT和AST)的血清浓度无统计学差异(图47A-47B)。肾功能标记物(BUN和CREA)的血清浓度在所有组中均大致相同(图47C-47D)。综上所述，这些数据表明HM-NP具有良好的生物安全性，可作为一种高效安全的实体瘤患者术后治疗性疫苗，具备临床转化的潜力。
实施例19
细菌外膜囊泡(Bacterial outer membrane vesicle，OMV)与细菌内膜的差异分析
OMV蛋白质组学分析：
(1)提取大肠杆菌外膜囊泡，并冷冻保存；
(2)采用BCA法测定蛋白浓度；
(3)过滤器辅助蛋白质组制备(Filter Aided Proteome Preparation，FASP)酶解；
(4)将离心浓缩干燥后的多肽，ziptip C18柱脱盐，干燥后准备质谱上机分析；
(5)干燥的样品加入30μL上样Buffer(乙腈：水：甲酸＝2：98：0.1)，震荡溶解，移至样品瓶，上机质谱分析。
搜库及结果：本次实验采用基于质谱方法的蛋白质组鉴定基本流程，即对MS/MS质谱数据经过系列优化处理后与数据库进行相似性比较打分从而进行蛋白鉴定。分别将每一个原始数据文件和相应的数据库上传至Maxquant 1.5.8.3软件，搜库结束后，去掉Reverse和Potential Contaminant蛋白，对搜库结果进行统计：此次质谱搜库共检测出总蛋白(non-redundant)218个，总肽段(include redundant)441个，特异肽段(razor+unique)430个，总谱图(MS/MS Identified)1230个。本申请对蛋白所在细胞位置进行统计，发现位于胞质(cytosol)中的蛋白占比最大，高达34.63％，具体结果如表4和表5所示：
表4 大肠杆菌外膜囊泡中蛋白分布情况
蛋白 ％
胞质 34.63
胞浆大核糖体亚基 10.51
细胞质 10.12
外膜边界周质空间 7.39
胞浆小核糖体亚基 7.00
膜 6.23
质膜 5.06
周质空间 3.89
细胞外膜 3.89
膜的组成部分 3.50
质膜的组成部分 1.56
细胞外膜的组成部分 1.17
孔复合体 1.17
GroEL-GroES复合物 0.78
外脱氧核糖核酸酶VII复合物 0.78
外膜 0.78
核苷 0.78
ATP结合盒(ABC)转运蛋白复合体 0.78
此外，本申请还选取了具有代表性的10个关键蛋白，并对其分子量以及功能进行描述。结果如下表所示：
表5 大肠杆菌外膜囊泡中主要蛋白表达情况
Figure PCTCN2021094037-appb-000012
功能描述:
OmpA：即外膜蛋白A，普遍存在于革兰氏阴性菌表面，具有β-桶状结构；功能上，它们具有为外膜提供通透性，维持外膜结构稳定的作用；在细菌生命活动中发挥至关重要的作用：它可以作为细菌的黏附素和侵袭素，参与生物膜的形成，也可作为多种噬菌体的受体,是先天免疫系统的重要靶位。
OmpC：即外膜蛋白C，作为革兰氏阴性菌外膜孔道蛋白，允许小分子在外膜上被动扩散。
OmpX：即外膜蛋白X，是一种重要的细菌外膜蛋白,属于Ail蛋白家族。该蛋白在其他细菌中参与细菌对细胞的黏附和侵袭,对抗宿主的免疫防御等,且与细菌的毒力有密切关系。因此,被认为有可能作为疫苗开发的候选靶标。
OsmE：即渗透诱导脂蛋白E，与渗透压响应有关；
LamB：即麦芽糖孔蛋白，涉及麦芽糖和麦芽糊精的运输，这对于包含三个以上葡糖基部分的糊精的转运是必不可少的。芳香族残基的疏水路径(“油脂滑”)用于引导和选择糖类，使其通过通道运输。也可作为几种噬菌体(包括λ)的受体；
MipA：即MltA互作蛋白，可以作为与MrcB/PonB和MltA形成复合物所需的支架蛋白，该复合物可能在壁蛋白囊泡的扩大和分离中发挥作用；
Lpp：即外膜脂蛋白Lpp，与肽聚糖共价和非共价地相互作用。这种相互作用有助于维持细胞包膜的结构和功能完整性；
SlyB：即外膜脂蛋白SlyB，由主要参与调控细菌毒力基因的表达的PhoP基因直接调控；
OmpW：即外膜蛋白W，作为革兰氏阴性菌外膜孔道蛋白，是大肠菌素S4的受体，可以作为药物筛选靶点；
FhuE：即FhuE受体，是革兰氏阴性菌外膜上的受体蛋白，是通过辅生素，亚铁胺B和杜鹃酸吸收Fe3 +所必需的受体分子。
大肠杆菌内膜蛋白质组学分析：
(1)提取四个批次大肠杆菌内膜，并冷冻保存；
(2)采用BCA法测定蛋白浓度；
(3)还原烷基化；
(4)过滤器辅助蛋白质组制备(Filter Aided Proteome Preparation，FASP)酶解；
(5)将离心浓缩干燥后的多肽，ziptip C18柱脱盐，干燥后准备质谱上机分析；
(6)将离心浓缩干燥后的多肽，ziptip C18柱脱盐，干燥后准备质谱上机分析。
搜库及结果：本次实验采用基于质谱方法的蛋白质组鉴定基本流程，即对MS/MS质谱数据经过一系列优化处理后，与数据库进行相似性比较打分，从而进行蛋白鉴定。我们使用Proteome Discoverer 2.4进行搜库，搜库结束后，去掉Reverse和Potential Contaminant蛋白，对搜库结果进行统计：此次质谱搜库共检测出总蛋白(non-redundant)2302个，总肽段(include redundant)20454个，特异肽段(razor+unique)19006个，总谱图(MS/MS Identified)86026个。本申请对蛋白所在细胞位置进行统计，发现位于细胞内(intracellular)中的蛋白占比最大，具体结果如下图48和表6所示：
此外，本申请还选取了具有代表性的12个蛋白质，并对其蛋白打分、分子量以及功能进行描述。结果如下表所示：
表6 不同批次大肠杆菌内膜主要蛋白表达情况
蛋白名称 蛋白登录号 蛋白搜库打分 分子量[KDa] 类型
FtsZ P0A9A6 991 40.3 细胞分裂蛋白
YhcB P0ADW3 125 15.0 内膜蛋白
YidC P25714 109 61.5 内膜蛋白易位酶
MsbA P60752 69 64.4 ABC转运蛋白
TatA P69428 58 9.7 内膜转运蛋白
MlaA P76506 43 28.0 膜间磷脂转运系统脂蛋白
TolQ P0ABU9 26 25.6 内膜蛋白
YebE P33218 24 23.7 内膜蛋白
SecB C4ZXK1 63 17.3 蛋白输出蛋白
FtsY P10121 103 54.5 信号识别颗粒蛋白受体
Ffh P0AGD8 174 49.8 信号识别颗粒蛋白
YajC P0ADZ8 70 11.9 Sec易位子附件复合物亚基
功能描述:
FtsZ：即细胞分裂GTP酶，是一种细胞分裂蛋白，是细菌细胞分裂器官中一个特征明确的蛋白质。它在细菌细胞分裂的中早期积累，并且在大多数细菌的隔膜形成过程中起着至关重要的作用。它也已经被认为是真核微管的细菌细胞骨架对应物；
YhcB：一种细菌内膜蛋白，参与细胞骨架以及肽聚糖生物合成；
YidC：一种一种内膜蛋白转位酶，与Sec易位子有关，是内膜蛋白整合、折叠和组装的辅助因子，可将小分子内膜蛋白(尤其是具有小分子细胞周质结构域的内膜蛋白)转运进内膜；
MsbA：一种ABC转运蛋白，也是一种脂质A出口ATP结合/渗透酶蛋白；
TatA：一种内膜转运蛋白，也是一种不依赖于Sec的蛋白转位酶蛋白，属于Tat家族成员之一。Tat是大肠杆菌中能够将折叠蛋白质跨膜转运的体系，其信号肽中含有一个高度保守的双精氨酸模体。Tat家族成员包括TatA、TatB、TatC和TatE4种蛋白质，它们的复合物在大肠杆菌质膜上形成转运通道。大肠杆菌Tat体系转运的底物蛋白质多为呼吸电子传递链组分，与大肠杆菌的许多生命活动有关；
MlaA：一种膜间磷脂转运系统脂蛋白，负责细菌内外膜之间磷脂转运交换；
TolQ：一种内膜蛋白，属于Tol-Pal系统蛋白，Tol-pal内膜系统是革兰氏阴性细菌内膜蛋白质系统之一，系统中Tol蛋白质的命名与对大肠杆菌素(colicin)的抗性有关。Tol-pal由TolQ、TolA、TolB、TolR以及Pal这五个蛋白质组成。它们的基因位于同一操纵子(operon) 上。TolB是一个周质蛋白，蛋白质TolA，Q，R则是三个内膜蛋白，他们之间相互作用，形成一个内膜蛋白复合体。TolQ蛋白与细菌细胞分裂有关；
YebE：一种内膜蛋白，属于大肠杆菌的热激响应分子标签；
SecB，一种蛋白输出蛋白。信号识别颗粒(SRP)以共翻译的方式将内膜蛋白靶向到内膜，而SecB途径以后期的共翻译或翻译后过程靶向分泌蛋白；
FtsY：一种信号识别颗粒蛋白受体。大肠杆菌SRP受体仅包含一个亚基FtsY，即哺乳动物SRα的同源物。大肠杆菌FtsY均匀分布在细胞质和内膜之间。FtsY与膜的结合涉及脂质和蛋白质膜因子，这很可能是Sec-translocon成分SecY。与磷脂的相互作用刺激FtsY GTPase活性；
Ffh：一种信号识别颗粒蛋白，与哺乳动物SRP54同源。Ffh是大肠杆菌生长所必需的。有人提出，以激活的、GTP结合形式结合于核糖体靶向信号的SRP54(Ffh)可以用于与SR(FtsY)的相互作用。Ffh和FtsY的GTPases在形成复合物时相互刺激，并提出可以起到GTPase激活作用，已经提出它们可以互相充当GTPase激活蛋白；
YajC：一种Sec易位子附件复合物亚基，与细菌内膜蛋白生物合成有关。
实施例20
大肠杆菌属(Escherichia，革兰氏阴性)、葡萄球菌属(Staphylococcus，革兰氏阳性)、芽孢杆菌属(Bacillus，革兰氏阳性)、乳杆菌属(Lactobacillus，革兰氏阳性)和假单胞菌属(Pseudomonas，革兰氏阴性)五种细菌与自体肿瘤组织来源细胞膜联用后抑制肿瘤术后复发的能力。
本实施例制备五种不同细菌内膜和肿瘤细胞膜组成的杂合膜包裹PLGA形成的纳米颗粒HM-NPs，包括大肠杆菌(Escherichia)、葡萄球菌(Staphylococcus)、芽孢杆菌(Bacillus)、乳杆菌属(Lactobacillus)和假单胞菌(Pseudomonas)属，五种产品中总膜蛋白浓度当量均保持一致，同时还包括单独肿瘤膜组和生理盐水组作为对照，使用小鼠结肠癌模型证明不同细菌内膜抑制肿瘤术后复发的能力。具体步骤如下：
(1)提取不同细菌的内膜具体操作为：扩增细菌，在培养基中将细菌分别培养至OD600值为1.2；加溶菌酶(裂解细胞壁)至终浓度为2mg/mL，与细菌在37℃下共孵育2-3h；离心去上清得到原生质体；加入extraction buffer(提取buffer)，利用密度梯度离心得到细菌内膜；重悬后保存在-80℃备用；
(2)将雌性BALB/c小鼠随机分为7组，每组8-10只，每只小鼠右后背皮下接种20万个CT26结肠癌细胞，构建小鼠结肠癌模型(第0天)；
(3)每隔一天观察并记录小鼠肿瘤生长情况，当平均肿瘤体积达到300mm 3左右时，手术切除所有小鼠肿瘤，然后对伤口进行缝合(第7天)；
(4)提取CT26结直肠癌组织中肿瘤细胞的细胞膜，将(3)得到的肿瘤组织用剪刀初步剪碎，然后加入含有胶原酶IV(1.0mg·mL -1)、脱氧核糖核酸酶DNase(0.1mg·mL -1)以及透明质酸酶(0.1mg·mL -1)的GBSS溶液，37℃放置15min用来消化肿瘤组织；将消化的组织进行研磨，然后通过孔径为0.22μm的滤网进行过滤，滤液进行离心；将离心细胞重悬，加入含有甘露醇、蔗糖和EGTA的提取buffer，然后在冰浴条件下利用细胞破碎仪破碎细胞，最终通过离心得到肿瘤组织来源的肿瘤细胞膜；将细胞膜重悬并冻存于-80℃备用；
(5)将不同的细菌内膜去3mg分别与提取的手术切除肿瘤细胞膜TM 1mg混合于PBS中，37℃恒温摇床摇15min，之后将混合膜溶液通过滤膜孔径为400nm的脂质体挤出器，累计来回挤压13次(一来一回算1次)，得到HM杂合膜微粒；
(6)将10mg PLGA溶于1mL二氯甲烷，再加入0.2mL无菌蒸馏水；混合物冰上25W超声乳化3min；随后与2mL 1％胆酸钠溶液(表面活性剂)混合，冰上30W超声乳化5min；将乳液逐滴加入10mL 0.5％胆酸钠溶液中，25℃搅拌15min；37℃旋蒸10min；将乳液10000g离心15min；用无菌蒸馏水洗两次，重悬得到PLGA聚合物溶液；
(7)将50μL步骤(5)制备的2mg/mL HM杂合膜微粒与500μL步骤(6)制备的1mg/mL PLGA混合后通过滤膜孔径为200nm的脂质体挤出器，累计来回挤压13次(一来一回算1次)，得到HM-NPs杂合膜纳米颗粒。
(8)分别在第10、12以及16天给各组小鼠皮下注射SM-NPs，TM-NPs，Mix NPs以及HM-NPs(100μg/只)，生理盐水组作为对照组(Control)；
(9)每隔一天观察并记录小鼠肿瘤生长情况，当小鼠肿瘤体积生长达到1000mm 3左右，即视为小鼠死亡，记录每组小鼠死亡的具体时间。
结果可知，本申请不同细菌内膜制备的HM-NPs杂合膜纳米颗粒粒径均一(图49A)，抑制结肠癌术后小鼠肿瘤复发的潜力，抑制率显著提高(图49B)。
实施例21
本申请用于肝癌、胃癌、肾癌、胰腺癌、卵巢癌、淋巴瘤、骨肉瘤、胶质瘤、前列腺癌和黑色素瘤的抑瘤实验。
实验流程如图50A所示：
(1)将6-8周龄的C57BL/6J或Balb/C小鼠随机分为2组，每组8-10只，在每只小鼠右后背皮下接种不同的肿瘤细胞。接种肿瘤细胞类型和接种数目及对应小鼠品系如表7所示， 构建不同的小鼠肿瘤术后复发模型用于评估本申请对多种肿瘤的抑制效果。
(2)当平均肿瘤体积达到300mm 3左右时，手术切除99％肿瘤，并残留1％的肿瘤，然后对伤口进行缝合，用于模拟肿瘤术后复发(第0天)；
(3)将切下来的瘤块研磨破碎，用胶原酶、DNA酶以及透明质酸酶混合溶液在37℃条件下对肿瘤组织进行消化，消化完的细胞悬液过滤网然后离心，离心弃上清后加入提取buffer，重悬得到细胞悬液；用细胞破碎仪对细胞悬液进行冰浴超声破碎(35W，5min)；将破碎后的细胞悬液进行离心(3000g，5min，4℃)；取上一步的上清再次进行离心(10000g，10min，4℃)；取离心后的细胞上清加入到超离管中进行超速离心(100000g，2h，4℃)；超速离心后弃上清，用1mL PBS重悬，得到手术切除来源的肿瘤细胞膜碎片TM，置于-80℃保存；
(5)参照本申请实施例中方法制备各种膜纳米颗粒，并拍摄电镜图片，对不同肿瘤细胞制备的杂合膜纳米颗粒进行表征；
(6)分别在术后第3、5以及9天给使用对应肿瘤膜制备HM-NPs(100μg/只)免疫小鼠三次，使用生理盐水组作为对照组(Control)；
(7)观察并记录小鼠肿瘤生长情况，当小鼠肿瘤体积生长达到1000mm 3左右，即视为小鼠死亡，记录每组小鼠死亡的具体时间。绘制生存曲线。
表7 不同肿瘤模型构建使用细胞和小鼠情况
Figure PCTCN2021094037-appb-000013
如图50B所示，在不同的肿瘤细胞均可以和大肠杆菌内膜制备成尺寸均一，具备核壳杂合膜纳米颗粒特征，证明本申请可以用于制备含有肝癌细胞、胃癌细胞、肾癌细胞、胰腺癌细胞、卵巢癌细胞、淋巴瘤细胞、骨肉瘤细胞、胶质瘤细胞、前列腺癌细胞和黑色素瘤细胞等杂合膜纳米颗粒。
如图50C所示，在对应的肿瘤模型中，与未经任何处理的对照组(Control)相比，杂合膜纳米颗粒疫苗(HM-NPs)均显著延长了不同荷瘤小鼠生存期。
实施例22
本申请用杂合膜包裹PLGA聚合物，通过孔径200nm的脂质体挤出器后可使杂合膜外壳的表面电位达到-50mV，而将杂合膜中加入阳离子脂质体后，可使杂合膜外壳的表面电位达到+50mV，结果如图51所示。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (115)

  1. 一种药物组合，其包含第一膜组分，所述第一膜组分包含源自细菌的内膜的膜，所述药物组合还包含源自所述细菌以外其它生物体的组分。
  2. 如权利要求1所述的药物组合，所述其它生物体包含细胞。
  3. 如权利要求1-2中任一项所述的药物组合，所述其它生物体包含哺乳动物细胞。
  4. 如权利要求1-3中任一项所述的药物组合，所述其它生物体包含肿瘤细胞。
  5. 如权利要求1-4中任一项所述的药物组合，所述其它生物体包含实体瘤细胞。
  6. 如权利要求1-5中任一项所述的药物组合，所述其它生物体选自以下组：乳腺癌细胞、结肠癌细胞、肝癌细胞、胃癌细胞、肾癌细胞、胰腺癌细胞、卵巢癌细胞、淋巴瘤细胞、骨肉瘤细胞、胶质瘤细胞、前列腺癌细胞和黑色素瘤细胞，以及上述中的任意组合。
  7. 如权利要求1-6中任一项所述的药物组合，所述其它生物体的所述组分包含具有免疫原性的组分。
  8. 如权利要求1-7中任一项所述的药物组合，所述其它生物体的所述组分能够引发对所述生物体的免疫应答。
  9. 如权利要求1-8中任一项所述的药物组合，所述其它生物体的所述组分包含源自所述生物体的细胞膜的组分。
  10. 如权利要求1-9中任一项所述的药物组合，所述其它生物体的所述组分包含肿瘤抗原或其功能活性片段。
  11. 如权利要求1-10中任一项所述的药物组合，所述其它生物体的所述组分还包含选自以下组的蛋白或其功能活性片段：抗原肽转运体1、H-2Ⅱ类组织相容性抗原γ链、酪氨酸蛋白激酶SYK、高亲和力免疫球蛋白epsilon受体亚单位γ、Ras相关C3肉毒毒素底物2、酪氨酸蛋白激酶BTK、受体型酪氨酸蛋白磷酸酶C、Na +/K +-ATP酶、ATP5A(IM CVa)、泛醌细胞色素C还原酶核心蛋白I(IM Core I)、VDAC1/孔蛋白(OM Porin)、基质亲环素D(Matrix CypD)、膜间隙细胞色素C(IMS Cytc)、基底膜特异性硫酸乙酰肝素蛋白聚糖核心蛋白(HSPG2)、质膜钙转运ATP酶1(ATP2b1)、内质网膜蛋白复合物亚基1(EMC1)、跨膜蛋白43(TMEM43)、囊泡膜蛋白VIP36(LMAN2)、囊泡相关膜蛋白相关蛋白A(VAPA)、跨膜9超家族成员2(TM9SF2)、跨膜emp24域含蛋白10(TMED10)、脂肪细胞质膜相关蛋白(APAMP)、突触小泡膜蛋白VAT(VAT1)、核孔膜糖蛋白210(NUP210)、质膜钙转运ATP酶4(ATP2b4)、质膜钙转运ATPase 2(ATP2b2)、红细胞带7整合膜蛋白(STOM)、跨膜emp24域含蛋白9(TMED9)、线粒体进口内膜转座酶亚基TIM44(TIMM44)、膜相关孕激素受体组分1(PGRMC1)、ER膜蛋白复合物亚基3 (EMC3)、囊泡相关膜蛋白相关蛋白B(VAPB)、膜伯胺氧化酶(AOC3)、囊泡相关膜蛋白7(VAMP7)、高尔基体膜蛋白4(GOLIM4)、膜相关孕激素受体组分2(PGRMC2)、跨膜9超家族成员3(TM9SF3)、跨膜9超家族成员4(TM9SF4)、内质网膜蛋白复合物亚基2(EMC2)、线粒体进口内膜转座酶亚单位TIM50(TIMM50)、过氧化物酶体膜蛋白11B(PEX11b)、跨膜emp24域含蛋白2(TMED2)、分泌载体相关膜蛋白3(SCAMP3)、硫氧还蛋白相关跨膜蛋白4(TMX4)、过氧化物酶体膜蛋白PMP34(SLC25a17)、过氧化物酶体膜蛋白PEX14(PEX14)、内质网膜蛋白复合物亚基8(EMC8)、干扰素诱导的跨膜蛋白3(IFITM3)、溶酶体相关膜糖蛋白2(LAMP2)、硫氧还蛋白相关跨膜蛋白2(TMX2)、囊泡相关膜蛋白3(VAMP3)、溶酶体相关膜糖蛋白1(LAMP1)、线粒体导入内膜转座酶亚单位Tim8 A(TIMM8a1)、溶酶体膜蛋白2(SCARB2)、Ig gamma-2A链C区(IGHG2a)、跨膜9超家族成员1(TM9SF1)、核内膜蛋白Man1(LEMD3)、跨膜emp24域含蛋白4(TMED4)、Thy-1膜糖蛋白(Thy1)、线粒体导入内膜转座酶亚基Tim23(TIMM23)、线粒体导入内膜转座酶亚基Tim9(TIMM9)、线粒体导入内膜转座酶亚基Tim13(TIMM13)、分泌载体相关膜蛋白2(SCAMP2)、分泌载体相关膜蛋白1(SCAMP1)、整合膜蛋白2B(ITM2b)、线粒体进口内膜转座酶亚单位Tim10(TIMM10)、液泡膜蛋白1(VMP1)、生长激素诱导的跨膜蛋白(GHITM)、含死亡结构域的膜蛋白(NRADD)、囊泡相关膜蛋白8(VAMP8)、跨膜前后转化蛋白1(TAPT1)、线粒体膜间空间导入和组装蛋白40(CHCHD4)、糖基化溶酶体膜蛋白(GLMP)、跨膜4结构域超家族A成员6D(MS4A6D)、易位链相关膜蛋白1(TRAM1)、干扰素诱导的跨膜蛋白2(IFITM2)、肌膜相关蛋白(SLMAP)、膜镁转运蛋白1(MMGT1)、核包膜孔膜蛋白POM 121(POM121)、跨膜蛋白C16orf54同源蛋白(AI467606)、液泡ATPase组装整合膜蛋白Vma21(VMA21)、上皮膜蛋白1(EMP1)、膜相关磷脂酰肌醇转移蛋白1(PITPNM1)、小整合膜蛋白15(SMIM15)、核膜整合膜蛋白1(NEMP1)、整合膜蛋白GPR180(GPR180)、分泌载体相关膜蛋白4(SCAMP4)、易位链相关膜蛋白2(TRAM2)、跨膜和卷曲螺旋域蛋白3(TMCC3)、基质膜相关蛋白1(SMAP1)、神经元膜糖蛋白M6-b(GPM6b)、上皮膜蛋白2(EMP2)、高尔基体膜蛋白1(GOLM1)、SID1跨膜家族成员2(SIDT2)、跨高尔基体Golgi网络整合膜蛋白2(TGOLN2)、高尔基体膜蛋白TVP23同系物B(FAM18b)、线粒体内膜蛋白OXA1L(OXA1L)、骨化相关的跨膜蛋白1(OSTM1)、过氧化物酶体膜蛋白PEX13(PEX13)、单侧通过膜和卷曲螺旋结构域蛋白1(SMCO1)、远端膜臂组装复合蛋白1(DMAC1)、分泌载体相关膜蛋白5(Scamp5)、囊性纤维化跨膜电导调节剂(CFTR)、破 骨细胞刺激性跨膜蛋白(OCSTAMP)、诱导脂肪存储的跨膜蛋白2(FITM2)和跨膜通道样蛋白5(TMC5)，以及上述中的任意组合。
  12. 如权利要求1-11中任一项所述的药物组合，所述其它生物体的所述组分还包含选自以下组的蛋白或其功能活性片段：抗原肽转运体1、H-2Ⅱ类组织相容性抗原γ链、酪氨酸蛋白激酶SYK、高亲和力免疫球蛋白epsilon受体亚单位γ、Ras相关C3肉毒毒素底物2、酪氨酸蛋白激酶BTK、受体型酪氨酸蛋白磷酸酶C、Na +/K +-ATP酶、ATP5A(IM CVa)、泛醌细胞色素C还原酶核心蛋白I(IM Core I)、VDAC1/孔蛋白(OM Porin)、基质亲环素D(Matrix CypD)和膜间隙细胞色素C(IMS Cytc)，以及上述中的任意组合。
  13. 如权利要求1-12中任一项所述的药物组合，其包含第二膜组分，所述第二膜组分包含源自所述其它生物体的细胞膜的膜。
  14. 如权利要求1-13中任一项所述的药物组合，所述细菌包含革兰氏阴性菌和/或革兰氏阳性菌。
  15. 如权利要求1-14中任一项所述的药物组合，所述细菌选自以下组的属：大肠杆菌属、葡萄球菌属、芽孢杆菌属、乳杆菌属、克雷伯氏菌属、布氏杆菌、变形杆菌、不动杆菌和假单胞菌属，以及上述中的任意组合。
  16. 如权利要求1-15中任一项所述的药物组合，所述细菌的所述内膜包含选自以下组的蛋白或其功能活性片段：细胞分裂蛋白FtsZ、内膜蛋白YhcB、内膜蛋白易位酶YidC、细胞分裂蛋白NlpI、ABC转运蛋白MsbA、内膜转运蛋白TatA、膜间磷脂转运系统脂蛋白MlaA、内膜蛋白TolQ、膜间转运脂蛋白PqiC、外膜蛋白TolC、外膜引入蛋白FimD、外膜孔蛋白OmpC、膜间转运蛋白PqiB、主要外膜脂蛋白Lpp、膜结合型溶血性壁蛋白转糖基酶MltB、UPF0194膜蛋白YbhG、推定的膜蛋白IgaA同源物、跨膜磷脂转运系统脂蛋白MlaA、内膜蛋白YejM、膜结合型溶血性壁蛋白转糖基酶MltA、跨膜磷脂转运系统结合蛋白MlaC、内膜蛋白YlaC、跨膜转运蛋白YebT、膜结合型溶血性胞壁质转糖基化酶MltC、跨膜磷脂转运系统ATP结合蛋白MlaF、膜间磷脂转运系统结合蛋白MlaD、内膜蛋白YqjE、UPF0053内膜蛋白YfjD、UPF0053内膜蛋白YoaE、内膜型溶胞性胞壁质转糖基酶A、UPF0394内膜蛋白YedE、膜间磷脂转运系统结合蛋白MlaB、内膜蛋白YccF、跨膜磷脂转运系统通透酶蛋白MalE、内膜蛋白YedI、内膜蛋白YgaP、膜间转运蛋白PqiA、UPF0056膜蛋白YhcE、内膜蛋白YbhL、内膜蛋白YhjD、内膜转运蛋白YbaT、内膜蛋白YjjP、内膜蛋白YhaH、内膜蛋白YbjJ、内膜转运蛋白YqeG、UPF0053内膜蛋白YtfL、砷泵膜蛋白ArsB、内膜蛋白YpjD、C型溶菌酶的膜结合溶菌酶抑制剂MliC、UPF0283膜蛋白 YcjF、UPF0259膜蛋白YciC、内膜蛋白YgfX、内膜蛋白YbbJ、脂蛋白释放系统跨膜蛋白LolE、内膜蛋白YjcH、蛋白质输出膜蛋白SecG、内膜蛋白YfdC、UPF0324内膜蛋白YeiH、UPF0266膜蛋白YobD、TVP38/TMEM64家族内膜蛋白YdjZ、膜相关蛋白UidC、内膜蛋白YbiR、内膜蛋白YhiM、膜转运蛋白YfcA、内膜蛋白YdgK、膜结合型溶血性壁蛋白转糖基酶F、多药耐药性外膜蛋白MdtP、UPF0208膜蛋白YfbV、肽聚糖相关脂蛋白、脂蛋白YiaD、脂蛋白YeaY、载脂蛋白N-酰基转移酶、D-蛋氨酸结合脂蛋白MetQ、脂蛋白GfcD、脂蛋白YbjP、脂蛋白YgeR、脂蛋白释放系统ATP结合蛋白LolD、渗透诱导性脂蛋白OsmE、渗透诱导脂蛋白OsmB、脂蛋白YdcL、脂蛋白YajG、脂蛋白NlpE、磷脂酰甘油-脂蛋白二酰甘油基转移酶、脂蛋白YghJ、脂蛋白YedD、脂蛋白YifL、脂蛋白YgdI、脂蛋白YbaY、脂蛋白信号肽酶、脂蛋白YceB、脂蛋白YajI和内膜蛋白YebE，以及上述中的任意组合。
  17. 如权利要求1-16中任一项所述的药物组合，所述细菌的所述内膜包含选自以下组的蛋白或其功能活性片段：FtsZ、YhcB、YidC、NlpI、MsbA、TatA、MlaA、TolQ和YebE，以及上述中的任意组合。
  18. 如权利要求1-17中任一项所述的药物组合，所述药物组合还包含内核。
  19. 如权利要求18所述的药物组合，所述内核包含生物相容性材料。
  20. 如权利要求18-19中任一项所述的药物组合，所述内核包含人工合成材料。
  21. 如权利要求18-20中任一项所述的药物组合，所述内核包含选自以下组的物质：聚乳酸-羟基乙酸共聚物(PLGA)、金属-有机框架材料(MOF)、聚己内酯(PCL)、聚酰胺-胺(PAMAM)、碳纳米管、石墨烯、金纳米颗粒、介孔二氧化硅纳米颗粒、氧化铁纳米颗粒银纳米颗粒、钨纳米颗粒、锰纳米颗粒、铂纳米颗粒、量子点、氧化铝纳米颗粒、羟基磷灰石纳米颗粒、脂质纳米颗粒(LNP)、DNA纳米结构、纳米水凝胶、稀土氟化物纳米晶体和NaYF 4纳米颗粒，以及上述中的任意组合。
  22. 如权利要求18-21中任一项所述的药物组合，所述内核包含选自以下组的物质：免疫佐剂、免疫检查点抑制剂、核酸分子和化学治疗剂，以及上述中的任意组合。
  23. 如权利要求22所述的药物组合，所述内核包含免疫佐剂单磷酰脂质A。
  24. 如权利要求18-23中任一项所述的药物组合，所述内核包含阿霉素、紫杉醇、多西他赛、吉西他滨、卡培他滨、环磷酰胺、氟尿嘧啶、培美曲塞、雷替曲赛、博来霉素、柔红霉素、多柔比星、长春新碱和依托泊苷，以及上述中的任意组合。
  25. 如权利要求18-24中任一项所述的药物组合，所述内核包含吲哚胺2,3-双加氧酶 (IDO)抑制剂。
  26. 如权利要求18-25中任一项所述的药物组合，所述内核包含小干扰RNA(siRNA)。
  27. 如权利要求18-26中任一项所述的药物组合，所述内核的直径为约60至约100纳米。
  28. 如权利要求18-27中任一项所述的药物组合，所述内核的直径为约86纳米。
  29. 如权利要求13-28中任一项所述的药物组合，所述药物组合包含外壳，所述外壳包含所述第一膜组分和所述第二膜组分。
  30. 如权利要求29所述的药物组合，所述外壳包含所述第一膜组分和所述第二膜组分融合后的膜。
  31. 如权利要求29-30中任一项所述的药物组合，所述外壳的厚度为约10至约20纳米。
  32. 如权利要求29-31中任一项所述的药物组合，所述外壳的厚度为约14纳米。
  33. 如权利要求29-32中任一项所述的药物组合，所述外壳的直径为约100纳米。
  34. 如权利要求29-33中任一项所述的药物组合，所述外壳的表面电位(Zeta电位)为约+50mV至约-50mV。
  35. 如权利要求29-34中任一项所述的药物组合，所述外壳的表面电位(Zeta电位)为约-21mV。
  36. 如权利要求13-35中任一项所述的药物组合，所述药物组合中的所述第一膜组分与所述第二膜组分的质量比为1:100至100:1。
  37. 如权利要求13-36中任一项所述的药物组合，所述药物组合中的所述第一膜组分与所述第二膜组分的质量比为1:100、1:75、1:50、1:25、1:10、1:5、1:3、1:1、1:0、0:1、3:1、5:1、10:1、25:1、50:1、75:1或100:1。
  38. 如权利要求13-37中任一项所述的药物组合，所述药物组合中的所述第一膜组分与所述第二膜组分的存在和/或比例通过蛋白质印迹法和/或免疫金染色法确认。
  39. 如权利要求29-38中任一项所述的药物组合，所述药物组合包含颗粒，所述颗粒包含所述内核和所述外壳。
  40. 如权利要求39所述的药物组合，所述外壳与内核材料的质量比为约1:1至约1:10。
  41. 如权利要求39-40中任一项所述的药物组合，所述外壳与内核材料的质量比为约1:4至约1:6。
  42. 如权利要求39-41中任一项所述的药物组合，所述颗粒中的脂多糖(LPS)含量与哺 乳动物细胞的脂多糖含量相比没有显著差异。
  43. 如权利要求39-42中任一项所述的药物组合，所述颗粒的直径为约70至约120纳米。
  44. 如权利要求39-43中任一项所述的药物组合，所述颗粒的直径为约100纳米。
  45. 如权利要求43-44中任一项所述的药物组合，所述直径通过透射电子显微镜(TEM)测量。
  46. 如权利要求39-45中任一项所述的药物组合，所述颗粒的水合粒径为约150纳米至约250纳米。
  47. 如权利要求39-46中任一项所述的药物组合，所述颗粒的水合粒径为约180纳米。
  48. 如权利要求39-47中任一项所述的药物组合，所述颗粒的表面电位(Zeta电位)为约+50mV至约-50mV。
  49. 如权利要求39-48中任一项所述的药物组合，所述颗粒的表面电位(Zeta电位)为约-21mV。
  50. 如权利要求46-49中任一项所述的药物组合，所述颗粒的水合粒径和/或表面电位通过动态光散射(DLS)仪器测量。
  51. 如权利要求39-50中任一项所述的药物组合，所述颗粒能够在溶液中保持稳定性。
  52. 如权利要求51所述的药物组合，所述稳定性包含所述颗粒保存一段时间之后的水合粒径和/或表面电位与所述一段时间之前相比，没有显著差异。
  53. 如权利要求51-52中任一项所述的药物组合，所述稳定性包含所述颗粒在4摄氏度的磷酸缓冲盐溶液(PBS)中保存一段时间之后的水合粒径和/或表面电位与所述一段时间之前相比，没有显著差异。
  54. 如权利要求52-53中任一项所述的药物组合，所述一段时间不少于约21天。
  55. 如权利要求1-54中任一项所述的药物组合，其还包含选自以下组的物质：免疫佐剂、免疫检查点抑制剂、核酸分子、化学治疗剂和光敏剂，以及上述中的任意组合。
  56. 如权利要求55所述的药物组合，其还包含免疫佐剂单磷酰脂质A(MPLA)。
  57. 如权利要求1-56中任一项所述的药物组合，其还包含吲哚胺2,3-双加氧酶(IDO)抑制剂。
  58. 如权利要求1-57中任一项所述的药物组合，其还包含小干扰RNA(siRNA)。
  59. 一种疫苗，包含权利要求1-58中任一项所述药物组合。
  60. 一种试剂盒，包含权利要求1-58中任一项所述药物组合和/或权利要求59所述疫 苗。
  61. 一种增强目标抗原被免疫细胞摄取的方法，包含提供一种药物组合，所述药物组合包含第一膜组分，所述第一膜组分包含源自细菌的内膜的膜，所述药物组合还包含所述目标抗原。
  62. 如权利要求61所述的方法，所述药物组合包含权利要求1-58中任一项所述药物组合。
  63. 如权利要求61-62中任一项所述的方法，所述免疫细胞包含免疫呈递细胞。
  64. 如权利要求61-63中任一项所述的方法，所述免疫细胞选自以下组：树突细胞(DC)、T淋巴细胞、巨噬细胞和自然杀伤细胞(NK)，以及上述中的任意组合。
  65. 如权利要求61-64中任一项所述的方法，所述免疫细胞包含骨髓来源树突状细胞(BMDC)。
  66. 如权利要求61-65中任一项所述的方法，所述免疫细胞包含CD8阳性细胞和/或CD4阳性细胞。
  67. 一种激活免疫细胞的方法，所述方法包含施用权利要求1-58中任一项所述药物组合、权利要求59所述疫苗和/或权利要求60所述试剂盒。
  68. 如权利要求67所述的方法，所述免疫细胞包含免疫呈递细胞。
  69. 如权利要求67-68中任一项所述的方法，所述免疫细胞选自以下组：树突细胞(DC)、T淋巴细胞、巨噬细胞和自然杀伤细胞(NK)，以及上述中的任意组合。
  70. 如权利要求67-69中任一项所述的方法，所述免疫细胞包含骨髓来源树突状细胞(BMDC)。
  71. 如权利要求67-70中任一项所述的方法，所述免疫细胞包含CD8阳性细胞和/或CD4阳性细胞。
  72. 如权利要求67-71中任一项所述的方法，所述免疫细胞包含淋巴结和/或脾脏中的所述免疫细胞。
  73. 如权利要求67-72中任一项所述的方法，与未施用所述药物组合的免疫细胞相比，施用所述药物组合的所述免疫细胞的所述激活的效果包含选自以下组：增加所述免疫细胞的抗原识别受体的表达水平、增加的所述免疫细胞的核因子κB(NF-κB)蛋白的表达水平、增加所述免疫细胞的细胞因子的表达和/或分泌水平和增加的成熟的所述免疫细胞的比例，以及上述中的任意组合。
  74. 如权利要求73所述的方法，所述抗原识别受体包含模式识别受体(PRR)。
  75. 如权利要求73-74中任一项所述的方法，所述抗原识别受体包含Toll样受体(TLR)。
  76. 如权利要求73-75中任一项所述的方法，所述抗原识别受体包含TLR1、TLR2和/或TLR6。
  77. 如权利要求67-76中任一项所述的方法，与未施用所述药物组合的免疫细胞相比，施用所述药物组合的所述免疫细胞的TLR4的表达基本不变。
  78. 如权利要求73-77中任一项所述的方法，所述细胞因子包含促炎细胞因子。
  79. 如权利要求73-78中任一项所述的方法，所述细胞因子包含白介素(IL)-6、肿瘤坏死因子(TNF)-α、IL-1β和/或干扰素(IFN)-γ。
  80. 如权利要求73-79中任一项所述的方法，成熟的所述免疫细胞包含CD80阳性细胞、CD86阳性细胞和/或效应记忆细胞。
  81. 如权利要求73-80中任一项所述的方法，成熟的所述免疫细胞比例包含CD80阳性和/或CD86阳性的所述免疫细胞占CD11c阳性的所述免疫细胞的比例。
  82. 如权利要求73-81中任一项所述的方法，成熟的所述免疫细胞比例包含CD44高表达且CD62L低表达的所述免疫细胞占CD8阳性的所述免疫细胞的比例。
  83. 权利要求1-58中任一项所述药物组合、权利要求59所述疫苗和/或权利要求60所述试剂盒在制备药物中的应用，所述药物用于增强先天性免疫和/或特异性免疫应答。
  84. 如权利要求83所述的应用，与未施用所述药物组合相比，施用所述药物组合促进树突细胞成熟和/或提高淋巴细胞分泌细胞因子。
  85. 如权利要求83-84中任一项所述的应用，所述应用基本上不引发全身性炎症反应。
  86. 如权利要求83-85中任一项所述的应用，与未施用所述药物组合相比，施用所述药物组合基本上不提高受试者体内血清中的IFN-γ、TNF-α、巨噬细胞趋化蛋白-1(MCP-1)、IL-12p70、IL-10、IL-23、IL-27、IL-17A、IFN-β、粒细胞-巨噬细胞集落刺激因子(GM-CSF)和/或IL-1α的浓度。
  87. 如权利要求83-86中任一项所述的应用，所述应用基本上不引发溶血反应、心脏损伤、肝脏损伤、脾脏损伤、肺脏损伤和/或肾脏损伤。
  88. 权利要求1-58中任一项所述药物组合、权利要求59所述疫苗和/或权利要求60所述试剂盒在制备药物中的应用，所述药物用于预防和/或治疗肿瘤。
  89. 如权利要求88所述的应用，所述预防和/或治疗肿瘤包含减缓肿瘤的体积增加速度和/或减小所述肿瘤的体积。
  90. 如权利要求88-89中任一项所述的应用，所述肿瘤包含肿瘤切除术后未完全切除的所述肿瘤。
  91. 如权利要求88-90中任一项所述的应用，所述肿瘤包含所述肿瘤清除后再次产生的肿瘤。
  92. 如权利要求88-91中任一项所述的应用，所述肿瘤包含实体瘤。
  93. 如权利要求88-92中任一项所述的应用，所述肿瘤选自以下组：乳腺肿瘤、结肠肿瘤、肝肿瘤、胃肿瘤、肾肿瘤、胰腺肿瘤、卵巢肿瘤、淋巴瘤、骨肉瘤、胶质瘤、前列腺癌和黑色素瘤以及上述中的任意组合。
  94. 制备权利要求1-58中任一项所述药物组合、权利要求59所述疫苗和/或权利要求60所述试剂盒的方法，包含提供所述源自细菌的内膜的膜。
  95. 如权利要求94所述的方法，还包含提供所述源自所述细菌以外其它生物体的组分。
  96. 如权利要求94-95中任一项所述的方法，还包含混合所述源自细菌的内膜的膜以及所述源自所述细菌以外其它生物体的组分以提供外壳。
  97. 如权利要求94-96中任一项所述的方法，还包含提供内核。
  98. 一种预防癌症术后复发的肿瘤疫苗，其特征在于，所述肿瘤疫苗包括杂合细胞膜外壳和内核材料，所述杂合细胞膜外壳包括革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜。
  99. 如权利要求98所述的预防癌症术后复发的肿瘤疫苗，其特征在于，所述革兰氏阴性菌内膜与手术切除来源的实体肿瘤细胞膜中蛋白质摩尔量的比例为1:100-100:1。
  100. 如权利要求98-99中任一项所述的预防癌症术后复发的肿瘤疫苗，其特征在于，所述革兰氏阴性菌内膜与手术切除来源的实体肿瘤细胞膜中蛋白质摩尔量的比例为1:100、1:75、1:50、1:25、1:10、1:5、1:3、1:1、1:0、0:1、3:1、5:1、10:1、25:1、50:1、75:1或100:1。
  101. 如权利要求98-100中任一项所述的预防癌症术后复发的肿瘤疫苗，其特征在于，所述革兰氏阴性菌内膜与手术切除来源的实体肿瘤细胞膜中蛋白质摩尔量的比例为(2-3):1。
  102. 如权利要求98-101中任一项所述的预防癌症术后复发的肿瘤疫苗，其特征在于，所述杂合细胞膜外壳与内核材料的质量比为1:(1-10)。
  103. 如权利要求98-102中任一项所述的预防癌症术后复发的肿瘤疫苗，其特征在于，所述杂合细胞膜外壳与内核材料的质量比为1:(4-6)。
  104. 如权利要求98-103中任一项所述的预防癌症术后复发的肿瘤疫苗，其特征在于，所述革兰氏阴性菌包括大肠杆菌属、葡萄球菌属、芽孢杆菌属、乳杆菌属、克雷伯氏菌属、布氏杆菌、变形杆菌、不动杆菌或假单胞菌属中的任意一种或至少两种的组合。
  105. 如权利要求98-104中任一项所述的预防癌症术后复发的肿瘤疫苗，其特征在于，所述肿瘤包括乳腺肿瘤、结肠肿瘤、肝肿瘤、胃肿瘤、肾肿瘤、胰腺肿瘤、卵巢肿瘤、淋巴瘤、骨肉瘤、胶质瘤、前列腺癌和黑色素瘤中的任意一种或至少两种的组合。
  106. 如权利要求98-105中任一项所述的预防癌症术后复发的肿瘤疫苗，其特征在于，所述内核材料包括PLGA、MOF、PCL、PAMAM、碳纳米管、石墨烯、金纳米颗粒、介孔二氧化硅纳米颗粒或氧化铁纳米颗粒中的任意一种或至少两种的组合。
  107. 如权利要求98-106中任一项所述的预防癌症术后复发的肿瘤疫苗，其特征在于，所述预防癌症术后复发的肿瘤疫苗的粒径为100-300nm。
  108. 如权利要求98-107中任一项所述的预防癌症术后复发的肿瘤疫苗，其特征在于，所述肿瘤疫苗的内核材料中还包载免疫检查点抑制剂、IDO抑制剂、siRNA或化疗药中的任意一种或至少两种的组合。
  109. 如权利要求98-108中任一项所述的预防癌症术后复发的肿瘤疫苗的制备方法，其特征在于，所述制备方法包括如下步骤：
    (1)分别提取革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜；
    (2)将步骤(1)提取到的革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜混合，并用脂质体挤出器挤出，得到杂合膜纳米颗粒；
    (3)将步骤(2)得到的杂合膜微粒与纳米颗粒内核混合，并用脂质体挤出器挤出，得到所述预防癌症术后复发的肿瘤疫苗。
  110. 如权利要求109所述的预防癌症术后复发的肿瘤疫苗的制备方法，其特征在于，步骤(2)所述混合的温度为20-45℃，时间为10-20min。
  111. 如权利要求109-110中任一项所述的预防癌症术后复发的肿瘤疫苗的制备方法，其特征在于，步骤(2)所述脂质体挤出器的滤膜孔径为300-500nm。
  112. 如权利要求109-111中任一项所述的预防癌症术后复发的肿瘤疫苗的制备方法，其特征在于，步骤(3)所述混合的温度为20-45℃，时间为10-20min。
  113. 如权利要求109-112中任一项所述的预防癌症术后复发的肿瘤疫苗的制备方法，其特征在于，步骤(3)所述脂质体挤出器的滤膜孔径为100-300nm。
  114. 如权利要求109-113中任一项所述的预防癌症术后复发的肿瘤疫苗的制备方法， 其特征在于，步骤(3)所述纳米颗粒内核通过双乳法制备得到。
  115. 如权利要求98-108中任一项所述的预防癌症术后复发的肿瘤疫苗在制备增强机体先天性免疫和特异性免疫应答的药物中的应用。
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WO2023115676A1 (zh) * 2021-12-24 2023-06-29 苏州尔生生物医药有限公司 一种树突状细胞癌症疫苗及其应用
CN114668742A (zh) * 2022-03-17 2022-06-28 中国科学技术大学 仿细菌纳米药物递送系统及其制备方法和应用
CN114903870A (zh) * 2022-04-01 2022-08-16 浙江大学 一种工程化细胞膜纳米颗粒及其制备方法和应用
CN114903870B (zh) * 2022-04-01 2023-08-22 浙江大学 一种工程化细胞膜纳米颗粒及其制备方法和应用
CN115068438A (zh) * 2022-04-28 2022-09-20 浙江大学医学院附属邵逸夫医院 破骨细胞前体同源靶向的细胞膜纳米囊泡制备方法及应用
CN115068438B (zh) * 2022-04-28 2023-09-22 浙江大学医学院附属邵逸夫医院 破骨细胞前体同源靶向的细胞膜纳米囊泡制备方法及应用
CN115252782A (zh) * 2022-07-28 2022-11-01 重庆医科大学 一种携氧仿生分子探针及其制备方法和在hifu及免疫协同治疗癌症中的应用
CN115252782B (zh) * 2022-07-28 2023-05-19 重庆医科大学 一种携氧仿生分子探针及其制备方法和在hifu及免疫协同治疗癌症中的应用

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