WO2023221833A1 - Active substance for blocking cd93 in pleural mesothelial cells and use - Google Patents

Active substance for blocking cd93 in pleural mesothelial cells and use Download PDF

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WO2023221833A1
WO2023221833A1 PCT/CN2023/093210 CN2023093210W WO2023221833A1 WO 2023221833 A1 WO2023221833 A1 WO 2023221833A1 CN 2023093210 W CN2023093210 W CN 2023093210W WO 2023221833 A1 WO2023221833 A1 WO 2023221833A1
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cells
llc
evs
lung
tumor
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Chinese (zh)
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蔡志坚
曾宪长
南希
章程燕
张根生
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浙江大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to the blocking and application of CD93, in particular to the application of blocking CD93 in pleural mesothelial cells in the treatment of lung cancer or lung infection.
  • Lung cancer ranks second in the world in terms of incidence and mortality, accounting for 18% of malignant tumor deaths.
  • the lung is one of the most common metastatic organs in various cancers, including breast cancer, melanoma and osteosarcoma (Altorki NK, et al.
  • the lung microenvironment an important regulator of tumor growth and metastasis. Nat Rev Cancer 2019,19( 1):9-31; Daw NC, et al. Recurrent osteosarcoma with a single pulmonary metastasis: a multi-institutional review. Br J Cancer 2015,112(2):278-282).
  • lung cancer is mainly treated with surgery, and chemotherapy drugs are mainly used to treat advanced stages.
  • chemotherapy drugs have serious side effects and are prone to drug resistance. Finding new and effective treatment methods is still a research hotspot in the field of cancer treatment.
  • CD93 is a newly revealed tumor treatment target. Studies have shown that blocking the CD93 signal activated by IGFBP7 in endothelial cells can inhibit tumor angiogenesis, normalize tumor blood vessels, and stimulate anti-tumor immunity (Sun, Yi et al. Blockade of the CD93 pathway normalizes tumor vasculature to facilitate drug delivery and immunotherapy.Science translational medicine vol.2021,13(604):8922), but whether CD93 exists on other target cells, and whether blocking CD93 on these target cells can be used to treat cancers such as lung cancer, breast cancer, and melanoma still remains Further research is needed.
  • a first aspect of the present invention provides an active substance that acts on CD93 in pleural mesothelial cells.
  • the substance can inhibit the expression of CD93 gene in pleural mesothelial cells, or the substance can reduce the content of CD93 protein in pleural mesothelial cells, or the substance can inhibit the activity of CD93 protein in pleural mesothelial cells.
  • the substance effectively inhibits lung tumor growth. Further research revealed that the After the substance acts on CD93 of pleural mesothelial cells, it promotes T cell-mediated cellular immunity specific to lung tumors or lung infected cells, and therefore has anti-pulmonary tumors or anti-pulmonary infection effects.
  • the T cells are CD4 + and/or CD8 + T cells.
  • the substance promotes the migration of dendritic cells to lung tumors or lung infection sites, thereby inducing T cell-mediated cellular immunity. Furthermore, after the substance inhibits CD93 of pleural mesothelial cells, it promotes the increase of CCL21 secreted by pleural mesothelial cells. CCL21 acts on dendritic cells and promotes the accumulation of dendritic cells into lung tumors or lung infected cells, thereby further Causes T cell-mediated cellular immunity. Further, the substance specifically inhibits the C1q/CD93 signaling pathway.
  • a second aspect of the present invention provides the use of the active substance in preparing a reagent for treating lung tumors or lung infections.
  • the agent when using the agent to treat lung tumors or lung infections, the agent is injected into the patient's chest via intrapleural injection, or via intravenous injection and targeted to pleural mesothelial cells.
  • a targeted drug carrier delivers the substance to pleural mesothelial cells.
  • a third aspect of the present invention provides a method for treating lung tumors or lung infections.
  • the method is achieved by causing T cell-mediated cellular immunity specific to lung tumors or lung infected cells through CD93 against pleural mesothelial cells.
  • the advantage of the present invention is that: the present invention provides a new target for the treatment of lung cancer or pulmonary infection, and further discloses a new action pathway for the treatment of lung cancer or pulmonary infection, providing a new direction for the treatment of lung cancer.
  • the present invention provides a method for treating lung cancer, and the method includes the following regimen.
  • the invention provides a method of treating lung tumors or pneumonia in a subject in need thereof, comprising administering to the subject an effective amount of C1q/CD93 blockade that specifically inhibits the C1q/CD93 signaling pathway. agent.
  • the C1q/CD93 blocker blocks the interaction between C1q/CD93.
  • the C1q/CD93 blocking agent includes an antibody or antibody fragment that specifically recognizes CD93, or an antibody or antibody fragment that specifically binds to or recognizes C1q.
  • the blocking agent blocks the interaction between C1q/CD93, thereby increasing the expression or activity of CCL21, thereby promoting the recruitment of dendritic cells to lung tumors or lung infected cells.
  • the anti-CD93 antibody binds to the C-type lectin domain region of CD93.
  • the anti-CD93 antibody is an anti-human CD93 antibody.
  • the anti-CD93 antibodies are full-length antibodies, single-chain Fv (scFv), Fab, Fab', F(ab')2, Fv fragments, disulfide bond-stabilized Fv fragments (dsFv), (dsFv)2, VHH, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody or tetrabody.
  • the CD93 is present in pleural mesothelial cells. In some embodiments, the anti-CD93 is included in a fusion protein.
  • the C1q/CD93 blocker is a polypeptide or polypeptide fragment.
  • the polypeptide is a polypeptide that binds to and inhibits CD93 function by binding to a different extracellular domain of CD93.
  • the polypeptide is a soluble polypeptide.
  • the polypeptide has a greater binding affinity to CD93 than to C1q.
  • the polypeptide binds to C1q with greater affinity than to CD93.
  • the present invention provides a method of determining whether a candidate agent can be used to treat lung cancer or lung inflammation, comprising: determining whether the candidate agent disrupts the CD93/C1q interaction, wherein if the candidate agent shows specificity Disrupting the CD93/C1q interaction can be used to treat lung cancer or lung inflammation.
  • the method includes determining whether the candidate agent disrupts the interaction of CD93 with Clq on the cell surface.
  • the method includes determining whether the candidate agent specifically disrupts the interaction of CD93 and Clq in an in vitro assay system.
  • the in vitro system is a yeast two-hybrid system. The system is based on ELISA assay.
  • the candidate drug is one or more of antibodies, peptides, fusion peptides, peptide analogs, polypeptides, aptamers, avimers, anticalins, speigelmers or small molecule compounds.
  • the method includes contacting the candidate agent with a CD93/Clq complex.
  • Figure 1 Flow chart of experimental protocol for EV and tumor cell treatment (EV injection before tumor inoculation).
  • FIG. 1 Size of LLC lung tumors in mice after intrapleural injection of LLC-EVs according to the protocol in Figure 1.
  • Figure 4 Size of B16F10-Luci tumors in mice after intrapleural injection of 5 ⁇ g B16F10-EVs according to the protocol in Figure 1.
  • Figure 5 The size of LLC-luci tumors in mice after intrapleural injection of 5 ⁇ g B16F10-EVs according to the protocol in Figure 1.
  • FIG. 7 On day 0, mice were injected with 4T1 cells subcutaneously. On day 16, the subcutaneous tumors were surgically removed. Then on days 26, 28, 30, 32, and 34, 5 ⁇ g of 4T1-EVs were injected into the chest. On day 37, Mouse lungs were harvested and stained with H&E for quantitative detection.
  • Figure 8 Flowchart of experimental protocol for EV and tumor cell treatment (EV injection after tumor transplantation).
  • FIG. 9 After using LLC-Luci cells to treat mice to construct a lung tumor model, 5 ⁇ g LLC-EVs were injected into the chest according to the protocol in Figure 8. The lung tumor size on the 25th day.
  • FIG. 10 A spontaneous lung tumor model was established by intranasal instillation of adenovirus expressing Cre recombinase into p53fl/flLSL-KrasG12D mice on days 0 and 1, and then on days 30, 32, 34 and 38. The mice were injected with 5 ⁇ g LLC-EVs into the chest cavity, and the lung tumors of the mice were detected on the 41st day.
  • FIG 11 Flow cytometry test results of CD4+T cells, CD8+T cells and DCs in TILs (tumor-infiltrating leukocytes, tumor-infiltrating leukocytes) of mice with LLC lung tumors in Figure 9.
  • Figure 12 Flow cytometry test results of macrophages, neutrophils and B cells in TILs of mice with LLC lung tumors in Figure 9.
  • CD11c-DTR mice were treated with LLC-Luci cells and 5 ⁇ g LLC-EVs according to the protocol in Figure 8, and treated with diphtheria toxin (DT) at the same time. The growth of lung tumors in mice.
  • CD11c-DTR mice were treated with LLC-Luci cells and 5 ⁇ g LLC-EVs according to the protocol in Figure 8, and treated with diphtheria toxin (DT) at the same time.
  • FIG. 15 Using LLC-Luci cells and 5 ⁇ g LLC-EVs to treat mice according to the protocol in Figure 8, while using anti-CD4 antibodies or anti-CD8 antibodies, the growth of mouse lung tumors.
  • FIG 16 Using LLC-Luci cells and 5 ⁇ g LLC-EVs to treat mice according to the protocol in Figure 8, while using anti-CD8 antibodies or anti-CD8 antibodies, flow cytometric detection results of mouse lung tumor TILs.
  • FIG. 17 Schematic diagram of the experimental protocol for injecting LLC and B16F10 cells subcutaneously on both sides of the ventral side of mice with LLC lung tumors, and simultaneously injecting LLC-EVs through the chest cavity.
  • Figure 19 LLC cells were injected subcutaneously into the ventral side of normal mice on day 0, and 5 ⁇ g LLC-EVs were injected into the chest on days 2, 4, 6, 8, and 10, and flow cytometry was performed on day 18. Test results for detecting lung DCs and T cells.
  • Figure 20 shows the size of subcutaneous tumors in mice treated with Figure 19.
  • FIG. 21 Intravenous or intrapleural injection of 100 ⁇ g VivoTrack 680 labeled LLC-EVs, distribution of LLC-EVs in mice.
  • FIG. 22 Intravenous or intrapleural injection of 100 ⁇ g PKH26-labeled LLC-EVs, distribution of LLC-EVs in mice.
  • Figure 23 Fluorescence microscopy of p-pMCs and 40L cells treated with 2.5 ⁇ g ml-1CFSE-labeled LLC-EVs for 24 h. The uptake of EVs by p-pMCs and 40L cells was detected.
  • Figure 24 Inject 20 ⁇ g of CFSE-labeled LLC-EVs into the chest of mice, and 24 hours later use a stereomicroscope to detect the EVs entering the pleura.
  • FIG. 25 Except that 0.25 mg kg-1 Cyto-D is injected into the chest 2 hours before each intrapleural injection of EVs, the other protocols are the same as in Figure 8. Mice are treated with LLC-Luci cells and CFSE-labeled LLC-EVs, and entry into the pleura is detected using a stereomicroscope. EVs situation.
  • Figure 26 is the same as Figure 25. Mice were treated, and flow cytometry was used to detect the proportion of DCs in TILs.
  • Figure 27 is the same as Figure 25. Mice were treated and lung tumor size was measured using IVIS.
  • FIG 28 Except that 0.25 mg kg-1 Cyto-D is injected into the chest 2 hours after each intrapleural injection of EVs, the other protocols are the same as in Figure 8. Mice are treated with LLC-Luci cells and CFSE-labeled LLC-EVs, and entry into the pleura is detected using a stereomicroscope. EVs situation.
  • Figure 29 is the same as Figure 28. Mice were treated, and flow cytometry was used to detect the proportion of DCs in TILs.
  • Figure 30 is the same as Figure 28. Mice were treated and lung tumor size was measured using IVIS.
  • Figure 31 40L cells were stimulated with a certain concentration of LLC-EVs for 24 hours, and cell viability was measured by CCK8 assay.
  • Figure 32 p-pMCs cells or 40L cells were stimulated with 2.5 ⁇ g ml -1 LLC-EV, and the effect of each cell supernatant on the chemotaxis of DCs was detected by Transwell chemotaxis assay 24 hours later.
  • Figure 33 After transfecting BMDCs cells with specific siRNA to silence each chemokine receptor (while using siRNA that has no homology to the target gene as a negative control, denoted as NC siRNA), the silencing effect was verified by Western blotting.
  • FIG. 36 p-pMCs were stimulated with 2.5 ⁇ g ml -1 LLC-EV, and 24 h later, the effect of the cell supernatant on the chemotaxis of DCs (WT) or Ccr7 -/- DCs was detected by Transwell chemotaxis assay.
  • WT chemotaxis of DCs
  • Ccr7 -/- DCs was detected by Transwell chemotaxis assay.
  • Figure 37 p-pMCs or 40L cells were stimulated with 2.5 ⁇ g ml -1 LLC-EV, and ELISA was used to determine the levels of CCL19 and CCL21a in the cell supernatant 24 hours later.
  • Figure 38 p-pMCs or 40L cells were stimulated with 2.5 ⁇ g ml -1 LLC-EV, and 24 h later, real-time PCR was used to determine the CCL19 and CCL21a mRNA levels in the cells.
  • Figure 39 In the presence of specific siRNA to silence CCL19 or CCL21a, 40L cells were stimulated with 2.5 ⁇ g ml -1 LLC-EV, and the effect of the cell supernatant on the chemotaxis of DCs was detected by Transwell chemotaxis assay 24 hours later (NC siRNA was used as a control group, CCL19 and CCL21a were not silenced).
  • Figure 40 After transfecting 40L cells with specific siRNA to silence the target gene, the silencing effect was verified by Western blotting (NC siRNA was the control group and CCL19 or CCL21a was not silenced).
  • Figure 41 Real-time PCR was used to measure pleural Ccl21a mRNA levels 24 hours after pleural injection of 5 ⁇ g ml -1 LLC-EVs into mice.
  • Figure 42 24 h after mice were injected with 5 ⁇ g ml -1 LLC-EVs into the chest, Ccl21a protein levels were determined by Western blotting.
  • Figure 44 uses LLC-Luci cells and LLC-EVs to treat mice according to the protocol in Figure 8, and injects 10 ⁇ g of cholesterol-coupled Ccl21a siRNAs into the chest 24 hours before each injection of EVs, and uses flow cytometry to analyze the DC frequency in TILs.
  • FIG 45 Mice were treated with LLC-Luci cells and LLC-EVs according to the protocol in Figure 8, and 10 ⁇ g of cholesterol-coupled Ccl21a siRNAs was injected into the chest 24 hours before each injection of EVs, and lung tumor size was measured with IVIS.
  • Figure 46 uses LLC-Luci cells and LLC-EVs to treat mice according to the protocol in Figure 8, and uses flow cytometry to analyze the CCR7 + DC frequency in TILs.
  • Figure 47 uses LLC-Luci cells and LLC-EVs to treat Ccr7 -/- mice according to the protocol in Figure 8, and uses flow cytometry to analyze DC frequency in TILs.
  • Figure 48 uses LLC-Luci cells and LLC-EVs to treat Ccr7 -/- mice according to the protocol in Figure 8, and determines lung tumor size using IVIS.
  • FIG. 49 RNA-Seq analysis of mRNA levels in PBS-treated 40L cells (control) and 40L cells treated with LLC-EV.
  • Figure 53 Real-time PCR measured pleural Cd93 and Ccl21a mRNA levels in healthy mice and LCC lung tumor-bearing mice.
  • Figure 54 Western blotting method to verify the silencing effect of CD93 in 40L cells.
  • Figure 56 Western blotting method to detect CD93 in p-pMCs stimulated by LLC-EVs.
  • FIG 57 Immunofluorescence blotting was used to detect CD93 in pMCs 24 hours after pleural injection of 20 ⁇ g LLC-EVs in mice (scale bar: 10 ⁇ m).
  • FIG 58 Real-time PCR detection of Cd93 mRNA levels in mice injected with cholesterol-conjugated Cd93 siRNA into the chest.
  • Figure 59 uses LLC-Luci cells and LLC-EVs to treat mice according to the protocol in Figure 8 and inject 10 ⁇ g of cholesterol-conjugated Cd93 siRNA into the chest before each EV injection, and use real-time PCR to detect pleural Ccl21 mRNA levels.
  • Figure 60 uses LLC-Luci cells and LLC-EVs to treat mice according to the protocol in Figure 8 and injects 10 ⁇ g of cholesterol-conjugated Cd93 siRNA into the chest before each EV injection, and uses ELISA to detect the level of Ccl21 protein secreted by pleural mesothelial cells.
  • Figure 61 Use LLC-Luci cells and LLC-EVs to treat mice according to the protocol in Figure 8 and inject 10 ⁇ g of cholesterol-conjugated Cd93 siRNA into the chest before each EV injection, and use IVIS to detect lung tumor size.
  • Figure 62 Use LLC-Luci cells and LLC-EVs to treat mice according to the protocol in Figure 8, and inject 10 ⁇ g cholesterol-conjugated Cd93 siRNA into the chest before each EV injection, and use flow cytometry to measure DC, CD4+T, and CD8+ in TILs. Proportion of T cells.
  • Figure 64 After digesting LLC-EVs with 10 ⁇ g ml -1 proteinase K for 2 hours, the digestion effect (expressed as protein Alix in the EVs lumen) was determined by Western blotting.
  • Figure 65 40L cells were stimulated with LLC-EVs and/or proteinase K-digested LLC-EVs for 24 h, and the CCL21a level in the cell supernatant was measured by ELISA
  • Figure 66 Measurement of RNA content in LLC-EVs after digestion with 10 ⁇ g ml -1 RNase I for 2 h.
  • Figure 69 The enriched miRNAs obtained by enrichment analysis of miRNAs in MLE-12-EVs and LLC-EVs using the miRNA array method.
  • Figure 70 Prediction of miRNAs upstream of Cd93 through the miRDB and TargetScan databases.
  • Figure 71 Alignment of enriched miRNAs and predicted miRNAs upstream of Cd93.
  • Figure 72 Real-time PCR detection of Cd93mRNA levels in 40L cells 24 hours after transfection with miR-5110 or miR-5107-5p analogues.
  • Figure 73 Western blotting method to detect CD93 protein levels in 40L cells 24 hours after transfection with miR-5110 or miR-5107-5p analogues.
  • Figure 74 Use real-time PCR to detect the overexpression level of miR-5110 or miR-5107-5p analogs in 40L cells 24 hours after transfection with miR-5110 or miR-5107-5p analogs.
  • Figure 76 The luciferase plasmid (Renilla luciferase plasmid) carrying the Cd93 3’-UTR WT or MUT fragment in Figure 75 and the miR-5110 analog were transferred into 40L cells, and the luciferase activity was measured 24 hours later.
  • Figure 78 After 2.5 ⁇ g ml -1 LLC-EVs were used to stimulate 40L cells transfected with miRNA inhibitor NC and miR-5110 inhibitor for 24 hours, CD93 levels in the cells were detected by Western blotting.
  • Figure 80 Real-time PCR measured miRNA-5110 levels in EVs.
  • Figure 81 After stimulating 40L cells with 2.5 ⁇ g ml -1 LLC-EVs, LLC-EVs-miR-5110Ins or LLC-EVs-miR-5110Des for 24 hours, CD93 levels in the cells were measured by Western blotting.
  • FIG 82 Mice were treated with LLC-Luci cells and corresponding EVs according to the protocol in Figure 8. Tumor size measured by IVIS on day 25.
  • FIG 83 Correlation between miR-5110 levels in tumor-derived EVs (TT-EVs) or serum EVs (sEVs) and pleural Cd93 mRNA levels in LLC lung tumor-bearing mice.
  • Figure 84 Cd93 and Ccl21 mRNA levels in human NCI-H2452 cells and HUVECs measured by real-time PCR.
  • Figure 85 ELISA detects CCL21 levels in the supernatant of NCI-H2452 cells (CD93 is silenced: Cd93 siRNA; CD93 is not silenced: NC siRNA).
  • FIG 92 Representative immunofluorescence images of cell pellets from malignant pleural effusions (MPEs).
  • Figure 95 Representative images of representative immunohistochemistry of lung TTs.
  • Figure 96 Overall survival curve of TT-EV/miR-5193 hi and TT-EV/miR-5193 lo lung cancer patients.
  • FIG. 97 Western blot confirms overexpression of C1qA, MMRN2 and IGFBP7 in 40L cells or NCI-H2452 cells.
  • Figure 100 According to the protocol in Figure 8, 5 ⁇ g C1qA was injected into the chest of LLC-Luci tumor-bearing mice on days 14, 16, 18, 20 and 22, and the pleural CCL21a protein level was measured by Western blotting on day 25. .
  • FIG. 101 According to the protocol in Figure 8, 5 ⁇ g C1qA was injected into the chest of LLC-Luci tumor-bearing mice on days 14, 16, 18, 20 and 22, and the lung tumor volume detected by IVIS on day 25.
  • FIG 102 According to the protocol in Figure 8, LLC-Luci tumor-bearing mice were intravenously injected with 100 ⁇ g of cholesterol-conjugated C1qa antisense oligonucleotide on days 14, 16, 18, 20 and 22.
  • pleural CCL21a protein levels were measured by Western blotting on day 25; a cholesterol-coupled negative control gene was also used
  • the antisense oligonucleotide treatment was used as a control (NCASOs, the antisense oligonucleotide sequence is shown in SEQ ID NO: 50).
  • FIG 103 LLC-Luci tumor-bearing mice were intravenously injected with 100 ⁇ g cholesterol-conjugated C1qa ASOs on days 14, 16, 18, 20 and 22 according to the protocol in Figure 8. Lung tumors detected with IVIS on day 25 volume.
  • Figure 104 ELISA measured C1qA levels in serum of LLC lung tumor-bearing mice and lung cancer patients.
  • Figure 105 Schematic representation of full-length and truncated CD93.
  • Figure 106 After the full-length or truncated CD93 fragments shown in Figure 105 were transferred into 40L cells for 48 hours, real-time PCR measured the expression levels of each CD93 fragment in the cells.
  • Figure 107 After the full-length or truncated CD93 fragment shown in Figure 105 was transferred into 40L cells for 48 hours, Ccl21a mRNA was measured by real-time PCR.
  • Figure 108 After the full-length or truncated CD93 fragment shown in Figure 105 was transferred into 40L cells for 48 hours, ELISA measured the CCL21a protein level in the cell supernatant (in the CD93 ⁇ CTLD group, the CTLD domain of CD93 was cleaved).
  • Figure 109 ELISA measured the Kd value of M057 binding to mouse CD93 antigen.
  • Figure 110 Results of staining bone marrow cells of WT mice or Cd93 ⁇ / ⁇ mice using M057.
  • Figure 112 After treating 40L cells and p-pMCs with 10 ⁇ g ml -1 anti-CD93 antibody (M057) and/or 2 ⁇ g ml -1 C1qA for 24 hours, the CCL21a level in the cell supernatant was measured by ELISA.
  • FIG 114 According to the protocol in Figure 8, 100 ⁇ g M057 was intravenously injected into LLC-Luci lung tumor-bearing mice on days 14, 16, 18, 20 and 22. On day 25, pleural CCL21a protein levels were measured by protein immunoblotting.
  • Figure 115 According to the protocol in Figure 8, 100 ⁇ g M057 was intravenously injected into LLC-Luci lung tumor-bearing mice on days 14, 16, 18, 20 and 22, and the lung tumor size was measured with IVIS on day 25.
  • Figure 116 According to the protocol in Figure 8, 100 ⁇ g M057 was intravenously injected into LLC-Luci lung tumor-bearing mice on days 14, 16, 18, 20 and 22. On day 25, flow cytometry measured DC, CD4 + T cell and CD8 + T cell frequencies.
  • FIG 117 LLC-Luci lung tumor-bearing mice were inoculated subcutaneously with LLC and B16F10 tumors on both sides of the mice, followed by treatment with 100 ⁇ g M057 on days 8, 10, 12, 14 and 16, days 10 to 18 Subcutaneous tumor size.
  • FIG 118 According to the protocol in Figure 8, LLC-Luci lung tumor-bearing mice were injected intravenously on days 14, 16, 18, 20 and 22 without CCL21a knockout (NC siRNA) and CCL21a knockout (Ccl21a siRNA). 100 ⁇ g M057, lung tumor size measured by IVIS on day 25.
  • NC siRNA CCL21a knockout
  • Ccl21a siRNA CCL21a knockout
  • Figure 119 Healthy mice were intravenously injected with 100 ⁇ g M057 every other day. After 5 injections, LEISA measured the hourly serum ALT, AST, bilirubin and creatinine levels.
  • Figure 120 Healthy mice were intravenously injected with 100 ⁇ g M057 every other day. After 5 injections, H&E staining (scale bar: 40 ⁇ m) was used to measure the histopathological damage of the mouse heart, liver, spleen, lungs and kidneys.
  • Figure 121 Calcein-AM staining was performed on mouse lung primary endothelial cells after treatment with 10 ⁇ g ml -1 M057 and/or 2 ⁇ g ml -1 IGFBP7 for 24 hours. The angiogenesis and statistical analysis data were detected by fluorescence microscopy.
  • FIG 122 According to the scheme in Figure 8, LLC-Luci lung tumor-bearing mice were intravenously injected with 100 ⁇ g M057 on days 14, 16, 18, 20 and 22. On day 25, tumor tissues were taken and analyzed for NG2 and CD31 or ⁇ SMA and CD31 was stained and quantified.
  • FIG 123 According to the scheme in Figure 8, LLC-Luci lung tumor-bearing mice were intravenously injected with 100 ⁇ g M057 on days 14, 16, 18, 20 and 22, and measured with 5 mg FITC-dextran (70kDa) perfusion on day 25. Tumor endothelial permeability.
  • FIG 124 According to the protocol in Figure 8, LLC-Luci lung tumor-bearing mice were intravenously injected with a certain amount of anti-VEGFR reagent on days 14, 16, 18, 20 and 22, and the size of the lung tumors measured with IVIS on day 25 .
  • FIG. 125 According to the protocol in Figure 8, LLC-Luci lung tumor-bearing WT mice were intravenously injected with 40 ⁇ g of anti-VEGFR reagent on days 13, 15, 17, 19, and 21, and intravenously injected 12 hours after each injection of the anti-VEGFR reagent. 100 ⁇ g M057 or IgG, lung tumor size observed by IVIS on day 25.
  • FIG 126 According to the protocol in Figure 8, LLC-Luci lung tumor-bearing Ccr7 -/- mice were intravenously injected with 40 ⁇ g of anti-VEGFR reagent on days 13, 15, 17, 19 and 21 and 12h after each injection of the anti-VEGFR reagent. After intravenous injection of 100 ⁇ g M057 or IgG, the size of lung tumors observed by IVIS on day 25.
  • Figure 127 LLC cells were injected subcutaneously into the ventral side of normal mice on day 0, 40 ⁇ g of anti-VEGFR reagent was injected intravenously on days 7, 9, 11, and 15, and 100 ⁇ g of M057 or M057 was intravenously injected 12 hours after each injection of anti-VEGFR reagent.
  • IgG subcutaneous tumor size from days 10 to 18.
  • FIG. 128 According to the protocol in Figure 8, LLC-Luci lung tumor-bearing mice were intravenously injected with 40 ⁇ g anti-VEGFR reagent or 100 ⁇ g M057 on days 14, 16, 18, 20 and 22, and the lungs detected by IVIS on day 25 Tumor size.
  • FIG. 129 According to the protocol in Figure 8, LLC-Luci lung tumor-bearing Ccr7 -/- mice were intravenously injected with 40 ⁇ g anti-VEGFR reagent or 100 ⁇ g M057 on days 14, 16, 18, 20 and 22, and IVIS was used on day 25 Detected lung tumor size.
  • Figure 130 According to the scheme in Figure 8, 50 ⁇ g of anti-PD-1 reagent was intravenously injected into B16F10 and 4T1 lung tumor-bearing mice on days 14, 16, 18, 20 and 22, with or without the injection of 100 ⁇ g M057. Lung tumor size detected by IVIS on day 25.
  • FIG 131 B16F10 and 4T1 lung tumor-bearing tumors on days 14, 16, 18, 20 and 22 according to the scheme in Figure 8 Mice were intravenously injected with 50 ⁇ g anti-PD-1 reagent, and simultaneously injected with 100 ⁇ g M057 or without 100 ⁇ g M057. Results of lung H&E staining on the 25th day.
  • Figure 132 PFS of anti-PD-1-treated lung cancer patients at different sEV/miR-5193 and serum C1qA levels.
  • Figure 133 is a schematic diagram of the principle structure of the present invention.
  • Figure 135 Real-time PCR detection of H1N1 infection levels in lung tissue of pleural CD93 knockdown mice.
  • FIG. 136 H&E staining results of lung tissue in mice with and without CD93 knockdown in the pleura after infection with H1N1 (scale bar, 50 ⁇ m).
  • Extracellular vesicles or extracellular vesicles (EVs)
  • EVs refer to vesicle-like bodies with a double-layer phospholipid membrane structure that are shed from the cell membrane or secreted by cells, with diameters ranging from 30nm to 1000nm. They are widely found in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, milk), carrying a variety of proteins, lipids, DNA, mRNA, miRNA, etc. related to cell origin. Processes such as intercellular communication, cell migration, angiogenesis, and immune regulation play an important role in intercellular component exchange, signal transduction, and pathological development.
  • Tumor-derived extracellular vesicles are a type of EVs produced and released extracellularly by tumor cells.
  • TDEVs can be designed as a natural drug carrier with high targeting and high penetration.
  • TT-EVs are extracellular vesicles (EVs from tumor tissues) in tumor tissues.
  • MC Mesothelial cells
  • pMCs pleural mesothelial cells
  • MCs belong to single-layer squamous epithelium in epithelial tissue and develop from mesoderm.
  • the mesothelium is a layer of membrane covering the surface of the pleura, peritoneum and pericardium. It can provide lubrication and protect organs from each other, as well as between organs and the pleura and peritoneum, without causing wear and tear on each other.
  • the lubrication and protection provided by mesothelium rely on substances secreted by mesothelial cells To achieve this, most of these substances are extracellular matrix and hyaluronic acid substances.
  • pMCs are a layer of mesothelial cells covering the pleura.
  • DCs were first isolated from monocytes in 1973 by Steinman and Cohn and were named dendritic cells because they have dendritic or pseudopod-like processes during their maturation.
  • DCs are known to be the most powerful professional antigen-presenting cells in the body and the only ones capable of activating resting T cells. They are the central link in initiating, regulating and maintaining immune responses.
  • tumor immunity DCs cannot directly kill tumor cells, but they can monitor and kill tumors by recognizing tumor cell-specific antigens and presenting their signals to T cells with killing effects.
  • CCL21 also known as exodus-2 and secondary lymphoid tissue chemokine (SLC), is a small cytokine belonging to the CC chemokine family and is located on chromosome 9 in the human genome.
  • CCL21 can be constitutively expressed at the origin of lymphatic vessels and lymphoid organs, is present on several stromal cells and high endothelial venules (HEV), and can bind to glycosaminoglycans (GAGs) and be fixed on the surface of endothelial cells.
  • HEV high endothelial venules
  • GAGs glycosaminoglycans
  • CCL21 can exert biological functions only by binding to the specific receptor CCR7.
  • CCL21 can chemoattract various immune cells such as lymphocytes, macrophages, and T cells, and mediates various physiological and pathological processes such as stress response, infection, vascularization, and dendritic cell maturation.
  • the chemokine receptor CCR7 is a G-protein-linked cell surface receptor containing seven transmembrane domains and participates in signal transduction through heterotrimeric G proteins and its downstream effectors.
  • the exclusive ligands of CCR7 are CCL19 and CCL21, which mediate different physiological functions respectively.
  • CCR7 is mainly expressed on the surface of immune cells such as T cells, B cells, activated NK cells and dendritic cells. In addition, it is also expressed in some non-immune cells such as tumor cells. When maintaining body homeostasis or during infection, it binds to one of its high-affinity functional ligands, CCL21, inducing lymphocyte homing and triggering a series of immune responses.
  • the complement C1q molecule is a heterologous hexamer composed of 6 subunits. Each subunit is composed of 3 polypeptide chains A, B, and C encoded by the C1QA, C1QB, and C1QC genes. That is, C1q is composed of 18 polypeptide chains. . C1q is congenital It is an important recognition molecule of the classical pathway of the immune complement system. It can activate the classical pathway and play an important role in immune regulation, inflammation regulation and maintenance of body balance.
  • C1QA (or C1qA) is one of the sub-chains of complement C1q. It is an important part of the innate immunity and adaptive immune system. It plays an important role in the classical pathway of the complement system. It performs multiple immune functions in a complementary, dependent or independent manner. and non-immune functions that play a promoting or inhibiting role in tumor progression.
  • T cell-mediated cellular immunity uses T cells to eliminate foreign bodies. Effector T cells have antigen recognition receptors and must be stimulated by antigens in order to be activated and play their role as effector cells. It is a type of specific cellular immunity. There are two basic forms of cellular immunity mediated by T cells, which are participated by two different subtypes of T cells. One is delayed-type hypersensitive T cells (CD4 + ), which can secrete cytokines after reacting with antigens. These cytokines then attract and activate macrophages and other types of cells to accumulate at the reaction site and become non-specific effector cells of chronic tissue inflammation. The other is cytotoxic T cells (CD8 + ), which have a specific killing effect on target cells.
  • CD4 + delayed-type hypersensitive T cells
  • CD8 + cytotoxic T cells
  • This application provides an active substance that acts on CD93 in pleural mesothelial cells (pMCs).
  • the active substance can inhibit the expression of CD93 gene in pleural mesothelial cells, reduce the level of CD93 mRNA or inhibit the transcription of CD93 mRNA; or the active substance can Reduce the content of CD93 protein, in addition to inhibiting CD93 gene expression, it may also degrade CD93 protein; or the substance inhibits the activity of CD93 protein in pleural mesothelial cells, such as when the substance is a CD93 antibody, or the modified substance can affect Cq1 and CD93 combination.
  • these active substances can inhibit the expression of C1q gene in pleural mesothelial cells, reduce the level of C1q mRNA or inhibit the transcription of C1q mRNA; or if the active substances can reduce the content of C1q3 protein, in addition to inhibiting the expression of C1q gene, it may also Degrading C1q protein; or the substance inhibits the activity of C1q protein in pleural mesothelial cells, for example, the substance is a C1q antibody. .
  • the active material is TEVs. This application found that only intrathoracic injection of TEVs can inhibit the growth of lung tumors, while subcutaneous, intraperitoneal or intravenous injection of TEVs cannot inhibit the growth of lung tumors.
  • TEVs intrapleural injection of TEVs can act on pMCs and promote systemic tumor-specific T cell immunity, thus having a lung tumor suppressive effect.
  • EVs stimulate pMCs
  • pMCs release CCL21 to recruit DCs
  • DCs present local tumor antigens and promote local anti-tumor T cell immunity.
  • Tumor-specific T cells in the lungs circulate in the blood, where they It is rapidly activated when stimulated by the same tumor antigen in other parts (subcutaneous), and can inhibit the growth of the same tumor in other parts.
  • TEVs intrapleural injection of TEVs can induce systemic tumor-specific T cell immunity only when lung tumors are present. When only subcutaneous tumors and other tumors are present, intrapleural injection of TEVs will not induce systemic tumor-specific T cell immunity.
  • pMCs are the site of action of TEVs in inducing systemic tumor-specific T cell immunity.
  • the T cells here include CD4 + and CD8 + T cells.
  • Systemic tumor-specific T cell immunity is mediated by DCs. After intrapleural injection of TEVs, DCs will accumulate in the lung tumor site, and then create CD4 + and CD8 + T cells. Present the antigen.
  • TEVs acts on CD93 in pMCs, reducing CD93 expression and promoting pMCs to secrete more CCL21.
  • CCL21 binds to the CCR7 receptor of dendritic cells, thereby promoting the accumulation of DCs to lung tumor sites.
  • the receptors of DCs also include other receptors such as CCR1, CCR2, CCR5, CCR6, etc.
  • the ligands of CCR7 also include CCL19. This application found that the active substance acts on pMCs CD93 promotes pMCs to secrete CCL21 and CCL21 binds to the CCR7 receptor of DCs, thereby triggering subsequent systemic tumor-specific T cell immunity.
  • the active agent specifically inhibits the C1q/CD93 signaling pathway.
  • CN202080075436.0 discloses that inhibiting the IGFBP7/CD93 signaling pathway can promote angiogenesis and thus have a tumor suppressive effect.
  • the active substance of the present application inhibits tumors by inducing systemic tumor-specific T cell immunity.
  • the active substance inhibits the C1q/CD93 signaling pathway, thereby inhibiting CD93 of pMCs, and then promotes the secretion of CCL21 by pMCs.
  • CCL21 binds to the CCR7 receptor of DCs, leading to the accumulation of DCs and triggering systemic tumor-specific T cell immunity.
  • the active substance in addition to acting on the C1q/CD93 signaling pathway, can also act on downstream CCL21.
  • the active substance can be a carrier that promotes CCL21 transport, making it easier for CCL21 to reach DCs, or can promote CCL21 Substances that bind to CCR7; the active substances can also act on downstream DCs, such as increasing DCs or increasing CCR7 receptors on DCs.
  • the active substance that inhibits the C1q/CD93 signaling pathway is a CD93/C1q blocker.
  • the blocker can act on CD93, can also act on C1q, or can act on the relationship between the two;
  • the blocking agent can be one or more of antibodies, polypeptides, siRNA, shRNA, miRNA, antisense RNA or gene editing systems, fusion proteins, peptide analogs, aptamers, avimers, anticalin, speigelmer or small molecule compounds, and CN202080075436.0 Similar forms of active substances that can block the C1q/CD93 signaling pathway belong to the scope of blockers in this application.
  • the blocking agent can bind to the C-type lectin domain of CD93 and inhibit CD93 activity.
  • the miRNA described in the present invention is miR-5193 or miR-5110, or a miR-5193 analog or a miR-5110 analog that is homologous to miR-5193 or homologous to miR-5110.
  • Homology here refers to existing miRNAs from different species that are similar to miR-5193 or miR-5110 and have the same function, and also refer to 60% or more (such as 70%, 72%, 80%, 90%, 99%, 100%, etc.) of the same sequence and functionally identical synthetic or naturally derived miRNA.
  • the tumor suppressive effect of the present application is only performed through systemic tumor-specific T cell immunity, specifically by inhibiting the C1q/CD93 signaling pathway, thereby inhibiting CD93 of pMCs, and then promoting the secretion of CCL21 by pMCs, CCL21 and CCR7 of DCs Receptor binding leads to the accumulation of DCs and triggers systemic tumor-specific T cell immunity.
  • the systemic tumor-specific T cell immunity of the present application can be performed simultaneously with the promotion of angiogenesis of CN202080075436.0, that is, the active substance has the ability to inhibit IGFBP7/CD93 signaling while inhibiting the pMCs C1q/CD93 signaling pathway. conduction pathway, so that it can exert a better anti-tumor effect.
  • the active substance can also treat pulmonary infection by inducing specific T cell immunity.
  • the anti-pulmonary infection mode of action is similar to that of anti-tumor, and will not be described in detail one by one.
  • Another aspect of the present application provides the use of the active substance in the preparation of reagents or medicines for treating lung tumors or lung infections.
  • the active substance can treat lung tumors while also treating the same type of tumors in other locations, and is especially suitable for the treatment of individuals with lung tumors and tumor metastasis.
  • the same type of tumor in this application refers to tumors caused by the same or similar types of tumor cells.
  • the location of the tumor can be different. For example, after a person suffers from a lung tumor, the lung tumor metastasizes behind it, such as to the abdomen. This When the abdominal tumor and lung tumor cells are similar, they are recorded as the same type of tumor. Tumor cells of the same type of tumor occurring in different parts of the animal or human body have the same or similar properties as antigens and can therefore produce the same antibodies.
  • the agent when using the agent to treat lung tumors or lung infections, the agent is injected into the patient's chest via intrapleural injection, or via intravenous injection and targeted to pleural mesothelial cells.
  • a targeted drug carrier delivers the substance to pleural mesothelial cells.
  • the reagents or drugs shown are not limited to liquid injection preparations, but can also be other types such as capsules, tablets, freeze-dried powders, and emulsions.
  • the active substance can be prepared into the reagent or medicine together with one or more of the excipients, diluents, sustained-release ingredients, preservatives, suspending agents and other auxiliary ingredients.
  • Example 1 Intrapleural injection of TEV inhibits lung tumor growth by promoting DC aggregation and tumor-specific T cell immunity.
  • TEVs intrathoracic injection of TEVs inhibited the growth of lung tumors in a dose-dependent manner, while neither intraperitoneal injection nor subcutaneous injection inhibited the growth of lung tumors.
  • Intravenous injection of TEVs instead promoted the growth of lung tumors.
  • TEVs when intrapleural injection of TEVs inhibits lung tumors, it promotes the accumulation of DCs to lung tumors and increases CD4 + and CD8 + T cells.
  • Intrapleural injection of TEVs can not only inhibit lung tumors but also inhibit the same type of tumors in other parts.
  • thoracic injection of TEVs cannot inhibit tumors in other parts, confirming that pleural injection of TEVs causes systemic tumor specificity.
  • T cell immunity Lung tumors are divided into two types, spontaneous tumors and tumors that have metastasized to the lungs elsewhere.
  • LLC-Luci luciferase-expressing LLC cells
  • LLC-Luci extracellular vesicles secreted by LLC cells
  • Figure 1 in Chapter 1 1 ⁇ 10 6 LLC-Luci tumor cells were intravenously injected on day 5 to construct a lung metastasis model (transfer to the lungs to form lung tumors), and different amounts of ( 2.5 ⁇ g, 5 ⁇ g, and 10 ⁇ g) of LLC-EVs, and mouse lungs were taken for detection on the 30th day.
  • the test results are shown in Figure 2.
  • the lung tumor volume of mice injected with LLC-EVs into the chest was significantly smaller than that of the control group (PBS injected into the chest), and the lung tumor volume injected with 10 ⁇ g LLC-EVs was smaller than that injected with 5 ⁇ g LLC-EVs, and further smaller than that injected with 2.5 ⁇ g. LLC-EVs, indicating that intrapleural injection of LLC-EVs dose-dependently inhibits the growth of LLC lung tumors.
  • 1 ⁇ 10 6 LLC-Luci tumor cells were injected intravenously on day 5 to establish a lung metastasis model, and 5 ⁇ g LLC-EVs were injected intraperitoneally, subcutaneously, or intravenously on days 0, 2, 4, 6, and 8.
  • the lung tumor status measured on the 30th day is shown in Figure 3. It can be seen that intraperitoneal or subcutaneous injection of LLC-EVs does not affect LLC lung tumors, but intravenous injection of LLC-EVs promotes the growth of LLC lung tumors.
  • B16F10-Luci tumor cells were used instead of LLC-Luci
  • B16F10-EVs extracellular vesicles secreted by B16F10 cells
  • the results are shown in Figure 4, indicating that B16F10-EVs can also be injected into the chest cavity. Inhibits lung metastatic B16F10-Luci tumor growth.
  • LLC-Luci tumor cells were injected intravenously on day 5, and 5 ⁇ g B16F10-EVs were injected into the chest on days 0, 2, 4, 6, and 8.
  • the lung tumor status observed on day 30 is shown in Figure 5 ;
  • LLC-Luci tumor cells were injected intravenously on day 5, and 5 ⁇ g 4T1-EVs (EVs secreted by 4T1 breast cancer cells) were injected into the chest on days 0, 2, 4, 6 and 8, and observed on day 30
  • the lung tumor situation is shown in Figure 6.
  • adenovirus expressing Cre recombinase was intranasally instilled in p53fl/flLSL-KrasG12D mice on days 0 and 1 to construct a spontaneous lung tumor model, and then on days 30, 32, 34, and 38
  • 5 ⁇ g LLC-EVs were injected into the chest of the mice, the growth of lung tumors in the mice was shown in Figure 10.
  • the lung tumor area in the test group was significantly smaller than that in the control group, indicating that spontaneous lung tumors occurred in p53fl/flLSL-KrasG12D mice. LLC-EVs also show tumor suppressive effects. Therefore, the above results indicate that intrapleural injection of TEVs can effectively and continuously inhibit the growth of lung tumors and can be applied in the treatment of lung tumors.
  • TILs tumor-infiltrating leukocytes
  • TEVs intrapleural injection of TEVs can promote the recruitment of DCs to lung tumors and subsequently promote the presentation of tumor antigens to CD4 + and CD8 + T cells, thereby increasing the number of CD4 + T cells, CD8 + T cells and DCs.
  • CD11c-diphtheria toxin receptor mice whole body DCs can be selectively deleted when intraperitoneally injected diphtheria toxin to conduct an anti-tumor test of LLC-EVs, using LLC-Luci cells and 5 ⁇ g LLC-EVs.
  • CD11c diphtheria toxin receptor mice were treated according to the protocol in Figure 8, and treated with diphtheria toxin at the same time.
  • the lung tumor situation on the 25th day is shown in Figure 13, and the TILs detection results are shown in Figure 14. It was found that DCs were removed and intrapleural injection of LLC-EVs inhibited lung tumors.
  • mice were treated with LLC-Luci cells and 5 ⁇ g LLC-EVs according to the protocol in Figure 8, and anti-CD4 antibodies or anti-CD8 antibodies were used at the same time.
  • the lung tumor situation on day 25 was as shown in Figure 15, and the TILs detection results were as shown in Figure 15. 16. After blocking CD4 + or CD8 + T cells using CD4 + or CD8 + T cell blocking antibodies, intrapleural injection of LLC-EVs no longer inhibits tumor growth.
  • LLC and B16F10 cells were injected into the ventral sides of mice with LLC lung tumors to construct multiple tumor mouse models, and then LLC-EVs were injected through the chest cavity.
  • the tumor volumes on both ventral sides are shown in Figure 18.
  • Abdominal LLC The tumor volume was significantly smaller than the control group (PBS group). It can be seen that the abdominal B16F10 tumor volume was not significantly different from the control group, indicating that the abdominal LLC tumors were significantly inhibited, while the other ventral B16F10 tumors were not significantly inhibited.
  • LLC cells were injected subcutaneously into the ventral side of normal mice on day 0, and 5 ⁇ g LLC-EVs were intrapleurally injected on days 2, 4, 6, 8, and 10, and flow cytometry was performed on day 18.
  • Example 2 TEV induces pMCs to secrete CCL21a and promotes DCs migration to the lungs
  • TEVs can act on pMCs and cause pMCs to secrete more CCL21.
  • CCL21 acts on CCR7 of DCs, thereby promoting the aggregation of DCs and subsequent tumor-specific T cell immunochemotaxis and subsequent tumor-specific T cells. Immunity, thereby exerting a tumor suppressive effect.
  • the pleural cavity is formed by a single layer of pMCs surrounded by the pleura. Therefore, pMCs have the potential to take up the vast majority of LLC-EVs injected via intrapleural injection.
  • primary pleural mesothelial cells (p-pMCs) and 40L cells were treated with 2.5 ⁇ g ml-1 CFSE-labeled LLC-EVs for 24 h and then the uptake of p-pMCs and 40L cells was detected by fluorescence microscopy.
  • the situation of EVs is shown in Figure 23, indicating that p-pMCs and 40L pleural mesothelioma cells can effectively uptake LLC-EVs in in vitro experiments.
  • mice were treated with LLC-Luci cells and CFSE-labeled LLC-EVs, and 0.25 mg kg-1 cytochalasin D (Cyto-D) was injected into the chest 2 hours before each intrapleural injection of EVs. ), using a stereomicroscope to detect EVs entering the pleura is shown in Figure 25. It can be seen that compared with the control group (DMSO was used instead of Cyto-D), there was no significant difference in the distribution of LLC-EVs in the pleura of mice in the test group. Flow cytometry was used to detect DCs in TILs. The results are shown in Figure 26.
  • mice were treated with LLC-Luci cells and CFSE-labeled LLC-EVs, and 0.25 mg kg-1 cytochalasin D (cytochalasin D, Cyto -D), the detection of EVs entering the pleura using a stereomicroscope is shown in Figure 28, the results of detecting DCs in TILs using flow cytometry are shown in Figure 29, and the results of measuring lung tumor size using IVIS are shown in Figure 30.
  • cytochalasin D cytochalasin D, Cyto -D
  • FIG. 28 mice were treated with LLC-Luci cells and CFSE-labeled LLC-EVs, and 0.25 mg kg-1 cytochalasin D (cytochalasin D, Cyto -D)
  • the detection of EVs entering the pleura using a stereomicroscope is shown in Figure 28
  • the results of detecting DCs in TILs using flow cytometry are shown in Figure 29
  • the results of measuring lung tumor size using IVIS are shown in Figure 30.
  • NC siRNA The NC siRNA sequences involved in this patent are as shown in SEQ ID NO:47 and SEQ ID NO:48).
  • the siRNA sequence of CCR1 is shown in SEQ ID NO:23 and SEQ ID NO:24; the siRNA sequence of CCR2 is shown in SEQ ID NO:25 and SEQ ID NO:26; the siRNA sequence of CCR5 is shown in SEQ ID NO:27 and SEQ ID NO:28; the siRNA sequence of CCR6 is shown in SEQ ID NO:29 and SEQ ID NO:30; the siRNA of CCR7 The sequence is shown in SEQ ID NO:31 and SEQ ID NO:32; the siRNA sequence of CXCR4 is shown in SEQ ID NO:33 and SEQ ID NO:34.
  • the silencing effect was verified by Western blotting, as shown in Figure 33.
  • the silencing effect was good.
  • 40L cells were stimulated with 2.5 ⁇ g ml -1 LLC-EV.
  • the effect of the cell supernatant on the chemotaxis of DCs was detected by Transwell chemotaxis assay.
  • the results are shown in Figure 34.
  • the number of cells in the silencing CCR1, CCR2, CCR5, CCR6 or CXCR4 group increased significantly, while the number of cells in the silencing CCR7 group did not change significantly. This shows that silencing CCR7 rather than other receptors is completely eliminated.
  • the effect of TEV-induced 40L cell supernatant on promoting the chemotaxis of BDMCs was investigated.
  • p-pMCs were stimulated with 2.5 ⁇ g ml -1 LLC-EV under specific siRNA silencing of the CCR7 receptor, and the effect of the cell supernatant on the chemotaxis of DCs was detected by Transwell chemotaxis assay after 24 days.
  • the test results are shown in Figure 35 ;
  • p-pMCs were stimulated with 2.5 ⁇ g ml -1 LLC-EV, and the effect of the cell supernatant on the chemotaxis of DCs (WT) or CCR7 -/- DCs was detected by Transwell chemotaxis assay 24 days later.
  • the test results are shown in Figure 36.
  • TEVs no longer promotes the chemotaxis of the supernatant of p-pMCs to CCR7-silenced or CCR7-deficient BMDCs.
  • CCL19 and CCL21a are known ligands of CCR7.
  • p-pMCs or 40L cells were stimulated with 2.5 ⁇ g ml -1 LLC-EV, and the levels of CCL19 and CCL21a in the cell supernatant were measured by ELISA 24 hours later.
  • the test results are shown in Figure 37; p-pMCs or 40L cells were stimulated with 2.5 ⁇ g ml -1 LLC-EV.
  • siRNA silencing CCL19 or CCL21a (the siRNA sequence of CCL19 is shown in SEQ ID NO:35 and SEQ ID NO:36; the siRNA sequence of CCL21a is shown in SEQ ID NO:37 and SEQ ID NO:38)
  • 40L cells were stimulated with 2.5 ⁇ g ml-1 LLC-EV, and the effect of the cell supernatant on the chemotaxis of DCs was detected by Transwell chemotaxis assay 24 hours later.
  • the silencing effect is shown in Figure 40, indicating that CCL19 or CCL21a is silenced.
  • Transwell chemotaxis assay The results are shown in Figure 39.
  • Example 3 TEVs reduce CD93 levels in pMCs and promote CCL21a secretion
  • CD93 is specifically highly expressed in pMCs, and TEVs promotes the secretion of CCL21 from pMCs by inhibiting the expression of CD93 in pMCs.
  • Cd93 is mostly expressed on platelets, endothelial cells (ECs), hematopoietic progenitors and alveolar epithelial cells.
  • ECs endothelial cells
  • hematopoietic progenitors and alveolar epithelial cells.
  • real-time PCR to detect Cd93 (see SEQ ID NO: 5 and SEQ ID NO: 6 for the Cd93 PCR primer sequence) and Ccl21a mRNA levels in various cells and found that The Cd93 mRNA expression level of p-pMCs was significantly higher than that of normal ECs, bone marrow cells, and MLE-12 mouse lung epithelial cells (Figure 52).
  • Cd93 mRNA decreased significantly, indicating that cholesterol-conjugated Cd93 siRNA can be used to silence CD93.
  • Mice were treated with LLC-Luci cells and LLC-EVs according to the protocol in Figure 8. 10 ⁇ g of cholesterol-conjugated Cd93 siRNA or NC siRNA was injected into the chest before each EV injection. Real-time PCR was used to detect pleural Ccl21 mRNA levels and ELISA was used to detect pleural Ccl21.
  • the enriched miRNAs in exosomes are critical for exosome-mediated intercellular communication.
  • MLE-12 mouse lung epithelial type II cells MLE-12-EVs
  • stimulation with MLE-12-EVs did not inhibit the secretion of CCL21a in 40L cells
  • Figure 68 Compared with MLE-12-EVs, we found 45 enriched miRNAs in LLC-EVs through miRNA array ( Figure 69).
  • the PCR primer sequence of miR-5107-5p is shown in SEQ ID NO:11 and SEQ ID NO:12. From the results, it can be seen that the transfection of miR-5110 or miR-5107-5p was successful); At the same time, set up a control group (Ctrl), transfect 40L cells with the negative control of the miRNA analog (see SEQ ID NO: 19 for the sequence of the analog of the miRNA negative control), and then detect the expression of CD93, as shown in Figure 72-73, and Compared with the control group, the levels of Cd93 mRNA and protein in the miR-5110 analog group were significantly reduced, while the miR-5107-5p analog group had no significant changes, which showed that the miR-5110 analog significantly reduced the mRNA and protein levels of CD93 in 40L cells.
  • CD93 is a direct target of miR-5110 (SEQ ID NO:17: 5'-GGAGGAGGUAGAGGGUGGUGGAAUU-3').
  • TargetScan predicted two miR-5110 action sites of Cd93 3'-UTR. The sequences of the two action sites are the same ( Figure 75).
  • the luciferase reporter assay showed that this sequence (WT: CCUCCUCA) is the target of miR-5110, and the mutated sequence (MUT: GGAGGAGU) eliminates the miR-5110 analog luciferase Activity ( Figure 75-76).
  • LLC-EVs no longer decreased CD93 protein and silencing miR-5110 caused a significant increase in CD93 protein.
  • miR-5110 analogs and inhibitors into LLC cells respectively.
  • the abilities of LLC-EVs-miR-5110 Ins and LLC-EVs-miR-5110 Des to downregulate CD93 in 40L cells were increased and weakened respectively (Fig. 81).
  • mice were treated with LLC-Luci cells and corresponding EVs according to the protocol in Figure 8, and tumor size was detected by IVIS on day 25.
  • the results are shown in Figure 82.
  • the tumor area in the LLC-EVs-miR-5110 Ins group was smaller, and the tumor area in the LLC-EVs-miR-5110 Des group was larger, which shows that LLC-EVs-miR-5110 Ins and LLC-EVs-miR-5110 Des exhibit better and worse inhibitory effects on LLC tumor growth respectively.
  • TEV-derived miR-5110 inhibits lung tumor growth by regulating CD93 in pMCs.
  • Example 5 Decreased CD93 levels in pMCs indicate enhanced human T cell response
  • EV miR-5193 (miR-5110 homolog) in malignant pleural effusions (MPEs) of lung cancer patients was positively correlated with the levels of CCL21 and DC; in tumor tissue, TT-EVs miR-5193 There is a positive correlation with CCL21 levels, DCs, CD4 + T cells and CD8 + T cells levels in TTs. CCL21 levels are also positively correlated with DCs levels, which further confirms that CD93 in pMCs promotes anti-tumor T cells by promoting CCL21-mediated DC recruitment. Cellular immune response.
  • CD93 of human MCs can also regulate anti-tumor immunity in lung cancer patients
  • NCI-H2452 human pleural mesothelioma cells
  • HUVECs human umbilical vein endothelial cells
  • the mRNA levels of Cd93 and Ccl2 in NCI-H2452 are significantly higher than those in HUVECs. This is the first time that the expression levels of CD93 and Ccl21 in NCI-H2452 are higher than those in HUVECs. mRNA expression levels.
  • Cd93 siRNA see SEQ ID NO:41 and SEQ ID NO:42 for the human Cd93 siRNA sequence
  • Ccr7 siRNA see SEQ ID NO:45 and SEQ ID NO:46 for the human Ccr7 siRNA sequence
  • CD93, CCR7 of DCs the Western blot detection results in Figure 87 and Figure 88 confirmed that CD93 or CCR7 protein decreased, indicating that CD93 or CCR7 protein was silenced, and then ELISA was used to detect the CCL21 level in the NCI-H2452 cell supernatant and Transwell Chemotaxis test was used to detect the chemotactic effect of NCI-H2452 cell supernatant on DCs.
  • TT-EV-derived miR-5193 is a negatively correlated indicator of CD93 levels in human pMCs.
  • TT-EVs 73 lung cancer patients were divided into two groups according to the level of miR-5193 in TT-EVs: those with higher levels of miR-5193 (low CD93 in pMCs) and those with lower levels of miR-5193 (high CD93 in pMCs). Recorded as TT-EV/miR-5193 hi (36 people) and TT-EV/miR-5193 lo (37 people), two groups of TT-EV/miR-5193 hi and TT-EV/miR-5193 lo were drawn respectively.
  • the overall survival curve of lung cancer patients is shown in Figure 96. Patients with high levels of TT-EV miR-5193 have a higher overall survival rate than patients with low levels of TT-EV miR-5193.
  • CD93 in pMCs regulates T cell responses by promoting CCL21-mediated DC recruitment.
  • Anti-CD93 inhibits tumor growth by promoting the secretion of CCL21 from pMCs
  • C1q in pMC (rather than IGFBP7) is the ligand for CD93 activation in pMCs.
  • C1qA is overexpressed, Ccl21a mRNA levels increase, and when C1qA is inhibited, pleural CCL21a expression is significantly reduced.
  • CTLD region is the functional region where CD93 inhibits the secretion of CCL21 in pMCs.
  • this embodiment provides an anti-CD93 monoclonal antibody M057. M057 increases the pleural Ccl21a mRNA level and CCL21a protein level.
  • CD4 + T cells, CD8 + T cells and DCs increased, causing systemic tumor-specific immunity.
  • C1q multimerin 2
  • IGFBP7 insulin-like growth factor binding protein 7
  • C1q is mainly synthesized in the liver, and cholesterol-conjugated oligonucleotides have good liver targeting properties.
  • C1qA was knocked out in mouse livers by cholesterol-coupled C1qa antisense oligonucleotide (the antisense oligonucleotide sequence of C1qa is shown in SEQ ID NO:49). Specifically, mice were injected intravenously on day 0.
  • CD93 contains 1 C-type lectin domain (CTLD), 1 Sushi domain, 5 epidermal growth factor (EGF)-like domains and 1 Mucin domain.
  • CTL C-type lectin domain
  • EGF epidermal growth factor
  • Figure 105 we cut CD93 to obtain CD93 truncated fragments containing one or more of these domains. These fragments were transferred into 40L cells, and 48 hours later, real-time PCR was used to detect the expression of each fragment. Expression and detect Ccl21a mRNA levels, as shown in Figure 106-107. Compared with the control group, the Ccl21a mRNA levels in each group decreased significantly.
  • CTLD plasmid but not overexpressing CTLD-deleted CD93 plasmid (CD93 ⁇ CTLD) can significantly inhibit the release of CCL21a from 40L cells, and the inhibitory effect is similar to that of CD93-WT.
  • CTLD of CD93 is associated with C1q-mediated inhibition of CCL21.
  • C1q is the ligand of CD93 when CD93 regulates CCL21 in pMC.
  • an anti-mouse CD93 rabbit monoclonal antibody (M057).
  • the construction method is as follows: immunize experimental rabbits with mouse CD93 antigen, and after verifying the level of serum polyclonal CD93Ab, take spleen plasma cells to amplify the antibody-edited DNA sequence, clone and recombine the DNA sequence into a plasmid vector, and obtain each monoclonal recombinant CD93 antibody through protein purification. For details, see the sequence SEQ ID NO: 1 and SEQ ID NO: 2.
  • the constant region of the antibody is replaced with mouse IgG. .
  • the dissociation constant (Kd) of M057 is 0.24 nM ( Figure 109). M057 can stain WT mouse bone marrow cells, but Cd93 ⁇ / ⁇ mouse bone marrow cells are not stained ( Figure 110). In addition, after intravenous injection of Alexa Fluor 680-labeled M057, a strong signal could be detected in the pleura of WT mice, but no signal was detected in the pleura of Cd93 ⁇ / ⁇ mice ( Figure 111). These results indicate that M057 specifically binds to CD93.
  • the underlined area is the signal peptide
  • the black amino acid sequence without underline is the constant region
  • the lung tumor size was detected, and flow cytometry was used to detect DCs, CD4 + T cells and CD8 + T cells in TILs.
  • the results are shown in Figure 114, Figure 115 and Figure 116 respectively. It can be seen that under the optimal M057 dose, Pleural CCL21a protein levels were significantly increased, LLC tumor growth was significantly inhibited, and M057 significantly increased the proportions of CD4 + T cells, CD8 + T cells, and DCs in TILs.
  • LLC-Luci lung tumor-bearing mice were subcutaneously inoculated with LLC and B16F10 tumors on both sides, respectively, followed by treatment with 100 ⁇ g M057 on days 8, 10, 12, 14, and 16, and on days 10 to 18
  • the size of subcutaneous tumors is shown in Figure 117.
  • Subcutaneous LLC tumors were significantly inhibited, while subcutaneous B16F10 had no significant difference.
  • M057 treatment can inhibit the same type of tumors in other parts of the body besides the lungs.
  • M057 treatment also induces systemic tumor-specific immunity. , has the effect of inhibiting the same type of tumors in different parts.
  • M057 inhibits lung tumor growth by targeting CD93 in pMCs.
  • pleural CCL21a injected 1 ⁇ 10 6 LLC-Luci cells intravenously into CCL21a knockout mice on day 0, day 14 and 16.
  • 100 ⁇ g M057 was injected intravenously on days 18, 20 and 22, and lung tumors were detected with IVIS on day 25.
  • the use of M057 on CCL21a knockout lung tumor-bearing mice can still inhibit the growth of lung tumors, but its inhibition The effect is significantly reduced.
  • I studied the in vivo toxicity of M057 I injected 100 ⁇ g M057 intravenously into healthy mice every other day. After 5 injections, I used LEISA to detect the levels of ALT, AST, bilirubin and creatinine in the serum and conducted experiments on the heart, liver, and creatinine of mice.
  • Anti-CD93 has better anti-lung tumor effect than anti-VEGFR (anti-VEGFR)
  • anti-CD93 can not only promote anti-tumor T cells by promoting CCL21-mediated DC recruitment.
  • the immune response (blocking the C1q/CD93 signaling pathway) can also specifically inhibit tumor growth by normalizing tumor blood vessels (blocking the IGFBP7/CD93 signaling pathway), and the anti-CD93 effect is better.
  • IGFBP7 treatment increased angiogenesis, while treatment with M057+IGFBP7 significantly inhibited angiogenesis, indicating that M057 also inhibits IGFBP7-induced EC angiogenesis in vitro.
  • LLC-Luci lung tumor-bearing mice were intravenously injected with 100 ⁇ g M057 on days 14, 16, 18, 20, and 22. On day 25, tumor tissues were collected and stained and quantified for NG2 and CD31 or ⁇ SMA and CD31. Analysis, as shown in Figure 122, although M057 treatment did not change the density of CD31+ blood vessels in LLC lung tumor mice, the smooth muscle cells [replaced with ⁇ -smooth muscle actin ( ⁇ SMA)] and pericytes [replaced with ⁇ SMA] in the tumor tissue The proportion of blood vessels covered by neural/glial antigen 2 (NG2) was significantly increased.
  • NG2 neural/glial antigen 2
  • anti-VEGFR reagent followed by M057 has a significant tumor inhibitory effect, and compared with the use of anti-VEGFR reagent alone, the use of anti-VEGFR reagent followed by M057 has an enhanced inhibitory effect on lung tumors. . This indicates that although CD93 is located downstream of VEGFR signaling, blocking VEGFR using optimal doses of anti-VEGFR reagents in LLC lung tumor-bearing mice weakens but does not eliminate the anti-tumor effect of M057.
  • LLC-Luci lung tumor-bearing Ccr7 ⁇ / ⁇ mice were intravenously injected with 40 ⁇ g of anti-VEGFR reagent on days 13, 15, 17, 19, and 21, and anti-VEGFR was injected intravenously at each injection.
  • 100 ⁇ g of M057 or IgG was intravenously injected 12 hours after the reagent, and the lung tumor size was measured on the 25th day, as shown in Figure 126.
  • IgG there was no significant difference in the M057 group, indicating that in Ccr7 -/- mice with VEGFR blocked LLC lung tumors , M057 treatment no longer inhibited tumor growth.
  • the LLC subcutaneous tumor-bearing mouse (WT) model was constructed on day 0, and 40 ⁇ g of anti-VEGFR reagent was intravenously injected on days 7, 9, 11, and 15. Reagent or IgG, 100 ⁇ g of M057 or IgG was intravenously injected 12 hours later, and the changes in tumor volume over time were counted, as shown in Figure 127.
  • M057 treatment could not inhibit tumor growth.
  • LLC-Luci lung tumor-bearing Ccr7 -/- mice were intravenously injected with 40 ⁇ g anti-VEGFR reagent or 100 ⁇ g M057 on days 14, 16, 18, 20 and 22, and IVIS was used to detect lung tumor size on day 25.
  • M057 and anti-VEGFR showed comparable abilities to inhibit lung tumor growth. This indicates that increasing tumor infiltration of CCR7 + DCs helps enhance the anti-pulmonary tumor effect of M057.
  • Cold tumors with few or no T cells can be resistant to anti-PD-1 therapy. M057-mediated normalization of tumor blood vessels and enhanced DC migration increase T cells in tumors. Therefore, M057 treatment may reverse the resistance of “cold tumors” to anti-PD-1 therapy.
  • B16F10 and 4T1 lung tumor-bearing mice were intravenously injected with 50 ⁇ g of anti-PD-1 reagent, and simultaneously injected with 100 ⁇ g of M057 or without injection of 100 ⁇ g of M057. IVIS was used for detection on the 18th day. Tumor size and lung H&E staining were performed, as shown in Figure 130-131.
  • the tumor inhibitory effect of the combination of anti-PD-1 and M057 was optimal, that is, both B16F10 and 4T1 lung tumors after anti-PD-1 treatment could be M057 inhibited. More importantly, 30 lung cancer patients with TT-EV/miR-5193 hi and TT-EV/miR-5193 lo , as well as those with higher C1qA levels (denoted as C1qA hi ) and lower C1qA levels (denoted as C1qA lo ) were respectively counted.
  • progression-free survival after anti-PD-1 treatment, as shown in Figure 132, it can be seen that as a negative or positive regulator of CD93, high sEV-derived miRNA-5193 or C1qA is associated with anti-PD in lung cancer patients. -1 is related to the progression-free survival after treatment.
  • C1q and IGFBP7 are upregulated during tumor development.
  • the C1q/CD93 signaling pathway inhibits the production of CCL21 in pMCs, preventing DC migration to the lungs and subsequent activation of T cell immune responses against lung tumors.
  • the IGFBP7/CD93 signaling pathway promotes VEGFR pathway activation in ECs, leading to angiogenesis and tumor growth.
  • anti-CD93 exhibits potent anti-lung tumor immunity.
  • Example 8 Antipleural CD93 has therapeutic effect on pulmonary infection caused by influenza A virus (H1N1)
  • test materials and methods in the above specific examples are as follows:
  • Lung TTs were obtained from the biobank of Zhejiang Cancer Hospital. MPEs were from lung cancer patients, and blood samples were from healthy volunteers and lung cancer patients receiving anti-PD-1 treatment. The above specimens were all from the Second affiliated Hospital of Zhejiang University School of Medicine.
  • mice C57BL/6J, BALB/c and nude mice (6-8 weeks, average weight 20g) were purchased from Shanghai Sipur-Bike Laboratory Animal Co., Ltd.
  • p53 fl/fl LSL-Kras G12D mice were donated by Professor Ying Songmin from Zhejiang University.
  • Ccr 7-/- was donated by Professor Li Jianhua from Fudan University.
  • CD11c-DTR mice were purchased from The Jackson Laboratory (Farmington, USA).
  • Cd93-/- mice were donated by Professor Su Libo of the Chinese Academy of Sciences. The animals were kept under special pathogen-free conditions, and the animal experiment protocol was approved by the Animal Experiment Ethics Committee of Zhejiang University.
  • Mouse 4T1 breast cancer and 3T3 fibroblast cells were purchased from ATCC (American Type Culture Collection).
  • Mouse LLC-Luci lung cancer cells and B16F10-Luci melanoma cells were purchased from PerkinElmer.
  • Mouse pMCs were provided by Li Binghao, Second affiliated Hospital of Zhejiang University School of Medicine.
  • Mouse MLE-12 lung epithelial cells and endothelial cells (HUVECs) were provided by Professor Ke Yuekai of Zhejiang University.
  • Human NCI-H2452 mesothelioma cells were purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (Ningbo, Zhejiang).
  • Antibody information is shown in the table below.
  • FBS was centrifuged at 120000 ⁇ g for 10 h to remove EVs, and then DMEM was added to a final concentration of 10% (v/v). LLC, 4T1 and B16 cells were cultured on 10 cm cell culture dishes to approximately 90% confluence. Lung TTs were sheared and digested with 2 mg/ml collagenase type IV (Worthington Biochemical, USA) and 0.2 mg/ml deoxyribonuclease I (Sigma-Aldrich, USA) in RPMI-1640 medium. The digestion conditions included 37 Shake at °C for 1 hour. Digestion was terminated by adding FBS to remove EVs.
  • collagenase type IV Worthington Biochemical, USA
  • deoxyribonuclease I Sigma-Aldrich, USA
  • a 200-mesh carbon membrane was hydrophilized using a glow discharge mass spectrometer for negative EV staining. Add the EV solution dropwise onto the 200 mesh carbon copper grid and leave it at room temperature (RT) for 1 hour. Use filter paper to absorb excess suspension and rinse the omentum twice with distilled water. EVs are stained with 2% uranyl acetate for 1 min at room temperature. Excess suspension is removed and dried. Imaging was performed with EM (Tecnai G2 Spirit 120 kV, Thermo FEI, Hillsboro, USA).
  • NTA of EVs was performed using a NanoSight NS300 system (Malvern PANalytical, Shanghai) equipped with a 488nm laser and a high-sensitivity sCMOS camera.
  • mice were anesthetized with 1% sodium pentobarbital (Sigma–Aldrich), and 5 ⁇ g of LLC-EVs, B16F10-EVs, or 4T1-EVs were injected into the chest cavity of mice on days 0, 2, 4, 6, and 8, respectively (refer to Stathopoulos GT ,Zhu Z,Everhart MB,Kalomendis I,Lawson WE,Bilaceroglu S,et al.Nuclear factor-kappaB affects tumor progression in a mouse model of malignant pleural effusion.American journal of respiratory celland molecular biology 2006,34(2):142 -150.).
  • mice were intravenously injected with 1 ⁇ 10 6 LLC-Luci cells on day 0, and 5 ⁇ g LLC-EVs were injected intraperitoneally (i.pl. Injection) on days 2, 4, 6, 8, and 10.
  • mice were intraperitoneally injected with 10 ⁇ g siRNAs or 40 ⁇ g anti-VEGFR2 (Bio X Cell, West Riverside, NH, USA) before each treatment.
  • siRNAs or 40 ⁇ g anti-VEGFR2 (Bio X Cell, West Riverside, NH, USA) before each treatment.
  • adenovirus p53 fl/fl LSL-Kras G12D expressing Cre recombinase (2 ⁇ 10 6 PFU ml -1 ) was intranasally instilled in mice on days 0 and 1; then, on days 30 and 32 , intrapleural injection of LLC-EVs on days 34 and 36.
  • mice were anesthetized and intrapleurally injected with 100 ⁇ g/kg body weight of luciferin (Promega, Beijing) on days 14 and 25 to obtain mice carrying luciferase-expressing tumors.
  • luciferin Promega, Beijing
  • IVIS PerkinElmer, Waltham, MA, USA
  • Living Image software PerkinElmer
  • Mice bearing primary or metastatic lung tumors were sacrificed at 41 or 37 days. Then, the lung tissues on both sides were embedded in paraffin, H&E stained, and H&E stained images were acquired using an Olympus BX53 inverted microscope (Olympus, Tokyo, Japan). Metastatic lung tumor burden was calculated by dividing total tumor area by total lung area.
  • Influenza A virus PR8/A/34 (H1N1) was used to intranasally titrate mice, and 24 h later, real-time fluorescence quantitative PCR was used to determine the CD93 mRNA level and H1N1 mRNA expression level in the mouse pleura.
  • 10 ⁇ g of cholesterol-coupled Cd93 siRNA (siRNA was ordered from Shanghai Genepharma) was dissolved in RNase-free double-distilled water and injected into the chest cavity of mice. Injection was performed every other day and the survival of the mice was recorded. Condition. Then, the mice were sacrificed, the lungs were removed, embedded in paraffin, H&E stained, and H&E stained images were acquired using an Olympus BX53 inverted microscope (Olympus, Tokyo, Japan).
  • the lung tissue of the lung metastasis model was cut, cleaned and digested.
  • the digestion process used 2 mg ml -1 type I collagenase (Worthington Biochemical), 2 mg ml -1 type IV collagenase (Worthington Biochemical) and 0.2 mg ml - 1 Deoxyribonuclease I (DNase I, Sigma-Aldrich) was shaken in RPMI-1640 at 37°C for 1 hour, and RPMI-1640 medium containing 10% FBS was added to terminate the digestion process. Then, the digested cell suspension was filtered using a 70 ⁇ m cell strainer, and red blood cells (RBCs) were lysed.
  • RBCs red blood cells
  • BMDCs bone marrow mononuclear cells
  • mouse tibia and femur suspension remove the red blood cells, put it on a 6-well plate at a density of 2 ⁇ 10 6 cells/ml, and use recombinant mouse interleukin GM-CSF added with 10% FBS and 10ng ml -1 Cultured in RPMI1640 medium with 1ng ml -1 mouse IL-4. After 48 hours of culture, the bone marrow hematopoietic cells were removed by washing, and the remaining loosely attached cell clusters were further cultured for 48 hours to obtain mouse BMDCs.
  • DCs human mononuclear cells
  • Peripheral blood mononuclear cells were obtained from heparin-treated blood from healthy volunteers by density centrifugation (Sigma–Aldrich), then resuspended in culture medium and allowed to adhere to the walls of a 6-well plate. After incubation at 37°C for 2 hours, non-adherent cells were removed, and adherent cells were cultured in 3 ml medium containing 10 ng ml-1 GM-CSF and 1 ng ml-1 IL-4. After 3 days, 1.5 ml of medium was removed and fresh same medium was added. After 7 days, human DCs were obtained by washing.
  • mice were treated with LLC cells and LLC-EVs according to the protocol in Figure 8 .
  • CD11c-DTR mice were continuously intraperitoneally injected with 2 ⁇ g DT starting on day 2, once every two days.
  • 60 ⁇ g of anti-CD4 and 16 ⁇ g of anti-CD8 antibodies were continuously injected intraperitoneally starting on day 2, once every two days.
  • 40 ⁇ g of anti-VEGFR2 antibody was continuously injected intravenously starting on day 2, once every two days.
  • VivoTrack 680 Fluorescence, Beijing
  • PKH26 Sigma–Aldrich
  • CFSE Thermo Fisher Scientific
  • VivoTrack 680 markers in 200 ⁇ l 150 ⁇ g EVs were mixed with 42 ⁇ M VivoTrack 680 in PBS for 30 min at room temperature.
  • 150 ⁇ g EVs were suspended in 100 ⁇ l diluent C, 100 ⁇ l diluent C containing 0.4 ⁇ l PKH26 ethanol dye solution was added, and then mixed for 5 min.
  • CFSE labeling 150 ⁇ g EVs were incubated with 7.5 ⁇ M CFSE in 200 ⁇ l PBS for 30 min at 37°C. An equal volume of exosome-depleted FBS (Thermo Fisher Scientific) was added and incubated for 1 min to terminate the labeling reaction. Finally, unbound label was removed by centrifugation at 120,000 ⁇ g for 70 min, and the EV label was suspended in 200 ⁇ l PBS.
  • mice were injected intravenously or intrapleurally with 100 ⁇ g of VivoTrack 680-labeled LLC-EVs. After 24 hours, the mice were euthanized and the brain, heart, lungs, liver, spleen, kidney and gastrointestinal organs were harvested. , and images were acquired using IVIS (PerkinElmer).
  • mice were injected intravenously or intrapleurally with 20 ⁇ g of PKH26-labeled LLC-EVs. After 24 hours, the mouse lungs were collected and embedded with Tissue-Tek TM Cryo-OCT Compound (Thermo Fisher Scientific). Obtain 10 ⁇ m tissue sections. Next, the cells were stained with 0.5 ⁇ g ml -1 DAPI for 20 min at room temperature. Cells and stained tissue sections were observed using a confocal microscope (Olympus IX83-FV3000).
  • CFSE-labeled LLC-EVs were injected into the pleura of mice; in some experiments, 0.25 mg kg -1 Cyto-D was injected intraperitoneally 2 hours before EV injection; the pleura of mice was taken 24 hours later. , observed with a stereomicroscope (Nikon SMZ18, Tokyo, Japan).
  • LLC-EVs were digested with 10 ⁇ g ml-1 proteinase K for 2 h with or without electroporation.
  • LLC-EVs were digested with 10 ⁇ g ml-1 RNase I combined with electroporation for 2 h. Electroporation of EVs was performed using a BTX electroporator (Harvard Biosciences, Cambridge, MA, USA). Briefly, 100 ⁇ g LLC-EVs and 100 ⁇ l electroporation buffer (Harvard Biosciences) were mixed, and a typical procedure was performed at a fixed capacitance of 100 ⁇ F, with optimal efficiency in a 0.2 cm sample tube.
  • mice p-pMCs were isolated and obtained. Briefly, after mice were sacrificed, the chest wall was removed under sterile conditions. Peel off the pleural parietal layer with eye forceps, wash twice with PBS to remove blood stains, and cut into 1 mm 2 Tissue fragments. Then, the pleural tissue was inoculated into a 25 cm 2 culture bottle containing complete DMEM medium. The non-adherent cells were removed the next day and cultured for 72 hours to obtain MCs.
  • Mouse lung and human TT tissue sections were deparaffinized and rehydrated, and 10mM sodium citrate buffer (pH 6.0) was used for antigen retrieval. After blocking with 5% BSA, the tissue sections were incubated with the primary antibody at 4°C overnight, and then incubated with the HRP-conjugated secondary antibody at room temperature for 30 min. Imaging data were randomly acquired and analyzed using ImageJ software (NIH, Bethesda, MD, USA).
  • p-pMCs and 40L cells were stimulated with 2.5 ⁇ g ml -1 LLC-EVs for 24 h, and NCI-H2452 cells were stimulated with 2.5 ⁇ g ml -1 A549-EVs for 24 h. Then, the supernatant was collected and placed in the bottom chamber of a migration chamber with a pore size of 3 ⁇ m (Corning Inc., Corning, NY, USA). DCs were added to the upper chamber of the migration chamber. After 12 h, the migrated cells were counted by flow cytometry. The quantity is used to evaluate the DC migration situation.
  • siRNAs 10 ⁇ g cholesterol-coupled siRNAs were dissolved in RNase-free double-distilled water and injected into the chest cavity of mice with lung metastasis tumors 24 hours before each intrapleural injection of extracellular vesicles.
  • the sequences of siRNAs, miRNA analogs and their inhibitors are shown in Table 2 below.
  • GAPDH was used as an internal control for normalization (for the PCR primers of GAPDH, see SEQ ID NO:7 and SEQ ID NO:8)
  • U6 was used as an internal control for normalization (for the PCR primers of U6, see SEQ ID NO:13 and SEQ ID NO:14).
  • specific miRNAs TaqMan probes (GenePharma) and Universal U+Probe Master Mix V2 (Vazyme) was used for real-time fluorescence quantitative PCR. Primer and probe sequences are shown in the table below.
  • the "limma” package in R 4.1.2 software was used to analyze and screen the differentially expressed genes (DEGs) between PBS and TEVs groups using
  • mice and 40L cells were stimulated with 2.5 ⁇ g ml ⁇ 1 LLC-EVs and NCI-H2452 cells were stimulated with 2.5 ⁇ g ml ⁇ 1 A549-EVs for 24 h. Centrifuge at 300 ⁇ g for 5 minutes to remove cells and debris.
  • Cytokines in the culture supernatant fluid and CCL21 levels in MPE fluid samples were determined by ELISA.
  • mouse and human sera were collected and C1qA levels were determined by ELISA. Specifically, the instructions of mouse CCL19 kit (Absin, Shanghai), mouse CCL21a, human Exodus 2 ELISA kit (Abcam, Cambridge, UK) and human C1qA ELISA kit (Abcam) were carried out.
  • ELISA detects EV miRNA content
  • TT-EVs and MPE-EVs Add equal amounts of TT-EVs and MPE-EVs to the 96-well ELISA plate coated with anti-CD63 antibody, and incubate overnight at 37°C to allow adsorption to the 96-well plate.
  • the mouse Cd93 mRNA 3'-UTR sequence containing the potential miR-5110 binding site was amplified and cloned into the GP-miRGLO vector (a plasmid containing a dual-luciferase reporter gene, Genepharma) to construct a luciferin-containing WTCd93 3'-UTR vector of enzyme reporter gene.
  • a mutant Cd93 3’-UTR vector was constructed through the antisense strand of the WT sequence.
  • 40L cells were transfected with WT Cd93 3’-UTR vector or mutant Cd93 3’-UTR vector and miRNA analogues or reference (final concentration 60nM). After 24 hours, cells were collected and lysed, and luciferase activity was detected using Duo-Lite Luciferase Assay System (Vazyme).
  • C1qA, IGFBP7 and MMRN2 were cloned into the pcDNA3.1 vector with flag tag sequence to construct an expression vector for CD93 ligand.
  • the expression vector was transfected into CTC cells through JetPEI (Polyplus, Shanghai) to express CD93 ligand, and anti-FLAG M2 magnetic beads (Sigma-Aldrich) and flag-tag polypeptide (Yeasen, Shanghai) were used to isolate the flag tag. binding protein.
  • the variable region of the immune rabbit antibody and the constant region sequence of the mouse IgG1 antibody were cloned into the pcDNA3.1 vector to construct Anti-mouse CD93rabbit/mouse IgG1 chimeric mAb (Sino Biological, Beijing). Cloning M057 is the most effective way to increase the expression of CCL21a in p-pMCs.
  • CD31-positive primary ECs were isolated using anti-mouse CD31-PE antibody (Biolegend, San Diego, CA, USA) and PE positive selection kit (StemCel, Vancouver, BC, Canada) before use in endothelial cell culture medium (Procell, Wuhan, China) for 5 days.
  • murine primary ECs were seeded at 1 ⁇ 10 4 cells per well into ibidi plates coated with endothelial growth factor-reduced matrix (BD Bioscience, Sab Jose, CA , USA).
  • Murine primary ECs were stained with 6.25 ⁇ g/ml calcein AMZ.
  • An inverted fluorescence microscope was used to observe and obtain images of capillary growth, and the ImageJ (NIH) angiogenesis analyzer plug-in was used for analysis.
  • M057 was labeled with a protein labeling kit (Thermo Fisher Scientific) according to the instruction manual.
  • 100 ⁇ g of Alexa Fluor 680-labeled anti-CD93 was intravenously injected into WT and Cd93 ⁇ / ⁇ mice. The mice were sacrificed 24 hours later, and the bilateral chest walls were collected, and the anti-CD93 imaging was observed and recorded with IVIS (PerkinElmer).
  • Nivolumab Anti-Programmed Death 1 Antibody,BMS-936558,ONO-4538

Abstract

The present application provides an active substance for blocking CD93 in pleural mesothelial cells and use thereof. The present application discloses blocking CD93 in pleural mesothelial cells in order to promote a T cell-mediated cellular immune response to lung tumor or lung infection. The invention functions to treat lung tumor or infection while providing a new treatment scheme therefor.

Description

阻断胸膜间皮细胞CD93的活性物质及应用Active substances and applications that block CD93 in pleural mesothelial cells
本申请主张中国在先申请，申请号：202210555701.6；申请日：2022年5月20日的优先权，该申请的说明书、附图、权利要求书与以及摘要作为本申请的一部分全部引用参考。This application claims the priority of China's earlier application, application number: 202210555701.6; filing date: May 20, 2022. The description, drawings, claims and abstract of this application are fully cited as part of this application.
技术领域Technical field
本发明涉及CD93的阻断及应用，特别是涉及阻断胸膜间皮细胞CD93在治疗肺癌或肺部感染方面的应用。The present invention relates to the blocking and application of CD93, in particular to the application of blocking CD93 in pleural mesothelial cells in the treatment of lung cancer or lung infection.
背景技术Background technique
肺癌的发病率和死亡率位居世界第二，占恶性肿瘤死亡人数的18％。肺是多种癌症中最常见的转移器官之一，包括乳腺癌、黑色素瘤和骨肉瘤(Altorki NK,et al.The lung microenvironment:an important regulator of tumour growth and metastasis.Nat Rev Cancer 2019,19(1):9-31；Daw NC,et al.Recurrent osteosarcoma with a single pulmonary metastasis:a multi-institutional review.Br J Cancer 2015,112(2):278-282)。目前，肺癌主要以手术治疗为主，晚期则以化疗药物治疗为主，然而，化疗药物毒副作用大，易产生耐药性，寻找新的有效治疗方法仍是癌症治疗领域的研究热点。Lung cancer ranks second in the world in terms of incidence and mortality, accounting for 18% of malignant tumor deaths. The lung is one of the most common metastatic organs in various cancers, including breast cancer, melanoma and osteosarcoma (Altorki NK, et al. The lung microenvironment: an important regulator of tumor growth and metastasis. Nat Rev Cancer 2019,19( 1):9-31; Daw NC, et al. Recurrent osteosarcoma with a single pulmonary metastasis: a multi-institutional review. Br J Cancer 2015,112(2):278-282). At present, lung cancer is mainly treated with surgery, and chemotherapy drugs are mainly used to treat advanced stages. However, chemotherapy drugs have serious side effects and are prone to drug resistance. Finding new and effective treatment methods is still a research hotspot in the field of cancer treatment.
CD93是新近被揭示的肿瘤治疗靶点。研究表明，阻断内皮细胞中IGFBP7激活的CD93信号，可抑制肿瘤新生血管生成，正常化肿瘤血管，激发抗肿瘤免疫(Sun,Yi et al.Blockade of the CD93 pathway normalizes tumor vasculature to facilitate drug delivery and immunotherapy.Science translational medicine vol.2021,13(604):8922)，但是CD93是否存在于其他靶细胞上，阻断这些靶细胞上的CD93是否能用于治疗肺癌、乳腺癌和黑色素瘤等癌症仍需要进一步研究。CD93 is a newly revealed tumor treatment target. Studies have shown that blocking the CD93 signal activated by IGFBP7 in endothelial cells can inhibit tumor angiogenesis, normalize tumor blood vessels, and stimulate anti-tumor immunity (Sun, Yi et al. Blockade of the CD93 pathway normalizes tumor vasculature to facilitate drug delivery and immunotherapy.Science translational medicine vol.2021,13(604):8922), but whether CD93 exists on other target cells, and whether blocking CD93 on these target cells can be used to treat cancers such as lung cancer, breast cancer, and melanoma still remains Further research is needed.
发明内容Contents of the invention
在研究CD93与肺肿瘤的过程中，本申请意外发现抑制胸膜间皮细胞的CD93能起到抑制肺肿瘤生长的效果。In the process of studying CD93 and lung tumors, the present application unexpectedly discovered that inhibiting CD93 in pleural mesothelial cells can inhibit the growth of lung tumors.
本发明的第一方面，提供了一种作用于胸膜间皮细胞的CD93的活性物质。A first aspect of the present invention provides an active substance that acts on CD93 in pleural mesothelial cells.
所述物质能抑制胸膜间皮细胞的CD93基因表达，或者，所述物质能使胸膜间皮细胞CD93蛋白的含量降低，或者，所述物质能抑制胸膜间皮细胞CD93蛋白活性。通过抑制胸膜间皮细胞CD93的功能，所述物质有效抑制了肺肿瘤生长。进一步研究发现，所述物 质作用于胸膜间皮细胞的CD93后，促进肺部肿瘤或肺部感染细胞特异性的T细胞介导的细胞免疫，因此具有抗肺肿瘤或抗肺感染作用。The substance can inhibit the expression of CD93 gene in pleural mesothelial cells, or the substance can reduce the content of CD93 protein in pleural mesothelial cells, or the substance can inhibit the activity of CD93 protein in pleural mesothelial cells. By inhibiting the function of CD93 on pleural mesothelial cells, the substance effectively inhibits lung tumor growth. Further research revealed that the After the substance acts on CD93 of pleural mesothelial cells, it promotes T cell-mediated cellular immunity specific to lung tumors or lung infected cells, and therefore has anti-pulmonary tumors or anti-pulmonary infection effects.
优选地，所述T细胞为CD4+和/或CD8+T细胞。Preferably, the T cells are CD4 + and/or CD8 + T cells.
进一步地，所述物质促进树突状细胞迁移至肺部肿瘤或肺部感染部位，进而引起T细胞介导的细胞免疫。进一步地，所述物质抑制胸膜间皮细胞的CD93后，促进胸膜间皮细胞分泌的CCL21增加，CCL21作用于树突状细胞，促进树突状细胞向肺部肿瘤或肺部感染细胞聚集，进而引起T细胞介导的细胞免疫。进一步地，所述物质特异性抑制C1q/CD93信号传导途径。Furthermore, the substance promotes the migration of dendritic cells to lung tumors or lung infection sites, thereby inducing T cell-mediated cellular immunity. Furthermore, after the substance inhibits CD93 of pleural mesothelial cells, it promotes the increase of CCL21 secreted by pleural mesothelial cells. CCL21 acts on dendritic cells and promotes the accumulation of dendritic cells into lung tumors or lung infected cells, thereby further Causes T cell-mediated cellular immunity. Further, the substance specifically inhibits the C1q/CD93 signaling pathway.
本发明的第二方面，提供了所述活性物质在制备治疗肺部肿瘤或肺部感染试剂方面的应用。A second aspect of the present invention provides the use of the active substance in preparing a reagent for treating lung tumors or lung infections.
在一些实施方式中，在使用所述试剂治疗肺部肿瘤或肺部感染时，通过胸腔注射的方式将所述试剂注射到患者胸腔中，或者通过静脉注射的方式并由靶向胸膜间皮细胞的靶向药物载体将所述物质输送到胸膜间皮细胞。In some embodiments, when using the agent to treat lung tumors or lung infections, the agent is injected into the patient's chest via intrapleural injection, or via intravenous injection and targeted to pleural mesothelial cells. A targeted drug carrier delivers the substance to pleural mesothelial cells.
本发明的第三方面，提供了一种治疗肺部肿瘤或肺部感染的方法。所述方法通过抗胸膜间皮细胞的CD93引起肺部肿瘤或肺部感染细胞特异性的T细胞介导的细胞免疫来实现。A third aspect of the present invention provides a method for treating lung tumors or lung infections. The method is achieved by causing T cell-mediated cellular immunity specific to lung tumors or lung infected cells through CD93 against pleural mesothelial cells.
本发明的优势在于：本发明提供了肺癌或肺部感染治疗的新靶点，并进一步公开了一条新的肺癌或肺部感染治疗的作用通路，为肺癌治疗提供了新方向。The advantage of the present invention is that: the present invention provides a new target for the treatment of lung cancer or pulmonary infection, and further discloses a new action pathway for the treatment of lung cancer or pulmonary infection, providing a new direction for the treatment of lung cancer.
在另一方面，本发明提供一种能够治疗肺癌的方法，所述的方法包括如下方案。On the other hand, the present invention provides a method for treating lung cancer, and the method includes the following regimen.
一方面，本发明在有需要的受试者中提供了治疗肺肿瘤或者肺炎的方法，其包括向所述受试者施用有效量的特异性抑制C1q/CD93信号传导途径的C1q/CD93阻断剂。In one aspect, the invention provides a method of treating lung tumors or pneumonia in a subject in need thereof, comprising administering to the subject an effective amount of C1q/CD93 blockade that specifically inhibits the C1q/CD93 signaling pathway. agent.
在一些方式中，其中所述C1q/CD93阻断剂阻断C1q/CD93之间的相互作用。In some embodiments, the C1q/CD93 blocker blocks the interaction between C1q/CD93.
在一些方式中，所述的C1q/CD93阻断剂包含特异性识别CD93的抗体或者抗体片段，或者特异结合或者识别C1q的抗体或者抗体片段。In some ways, the C1q/CD93 blocking agent includes an antibody or antibody fragment that specifically recognizes CD93, or an antibody or antibody fragment that specifically binds to or recognizes C1q.
在一些方式中，所述的阻断剂通过阻断C1q/CD93之间的相互作用，从而让CCL21的表达增多，或者活性增强，从而促进树突状细胞向肺部肿瘤或肺部感染细胞聚集。In some ways, the blocking agent blocks the interaction between C1q/CD93, thereby increasing the expression or activity of CCL21, thereby promoting the recruitment of dendritic cells to lung tumors or lung infected cells. .
在一些方式中，所述抗CD93抗体与CD93的C型凝集素结构域区域结合。In some aspects, the anti-CD93 antibody binds to the C-type lectin domain region of CD93.
在一些方式中，所述的抗CD93抗体是抗人CD93抗体。所述的抗CD93抗体是全长抗体、单链Fv(scFv)、Fab、Fab’、F(ab’)2、Fv片段、二硫键稳定化的Fv片段(dsFv)、(dsFv)2、VHH、Fv-Fc融合物、scFv-Fc融合物、scFv-Fv融合物、双抗体、三抗体或四抗体。 In some aspects, the anti-CD93 antibody is an anti-human CD93 antibody. The anti-CD93 antibodies are full-length antibodies, single-chain Fv (scFv), Fab, Fab', F(ab')2, Fv fragments, disulfide bond-stabilized Fv fragments (dsFv), (dsFv)2, VHH, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody or tetrabody.
在一些方式中，所述的CD93是存在于胸膜间皮细胞中。在一些方式中，所述的抗CD93包含在融合蛋白中。In some embodiments, the CD93 is present in pleural mesothelial cells. In some embodiments, the anti-CD93 is included in a fusion protein.
在一些方式中，所述的C1q/CD93阻断剂为多肽或者多肽片段。在一些方式中，所述的多肽是结合并抑制CD93功能的多肽，其结合于CD93的不同胞外结构域。在一些方式中，所述的多肽是可溶性多肽。In some embodiments, the C1q/CD93 blocker is a polypeptide or polypeptide fragment. In some embodiments, the polypeptide is a polypeptide that binds to and inhibits CD93 function by binding to a different extracellular domain of CD93. In some aspects, the polypeptide is a soluble polypeptide.
在一些方式中，其中所述多肽与CD93的结合亲和力大于与C1q的结合亲和力。或者，多肽与C1q的结合亲和力大于与CD93结合的亲和力。In some embodiments, the polypeptide has a greater binding affinity to CD93 than to C1q. Alternatively, the polypeptide binds to C1q with greater affinity than to CD93.
在另外一方面，本发明提供一种确定候选药剂是否可用于治疗肺癌或者肺部炎症的方法，其包括：确定所述候选药剂是否破坏CD93/C1q相互作用，其中如果所述候选药剂显示特异性破坏CD93/C1q相互作用，则其可用于治疗肺癌或者肺部炎症。In another aspect, the present invention provides a method of determining whether a candidate agent can be used to treat lung cancer or lung inflammation, comprising: determining whether the candidate agent disrupts the CD93/C1q interaction, wherein if the candidate agent shows specificity Disrupting the CD93/C1q interaction can be used to treat lung cancer or lung inflammation.
在一些方式中，其中所述方法包括确定所述候选药剂是否破坏细胞表面上CD93与C1q的相互作用。In some aspects, the method includes determining whether the candidate agent disrupts the interaction of CD93 with Clq on the cell surface.
在一些方式中，其中所述方法包括确定所述候选药剂是否在体外测定***中特异性破坏CD93和C1q的相互作用。所述的体外***是是酵母双杂交***。***是基于ELISA的测定。In some embodiments, the method includes determining whether the candidate agent specifically disrupts the interaction of CD93 and Clq in an in vitro assay system. The in vitro system is a yeast two-hybrid system. The system is based on ELISA assay.
所述的候选药物是抗体、肽、融合肽、肽类似物、多肽、适体、avimer、anticalin、speigelmer或小分子化合物中的一种或者多种。所述方法包括将所述候选药剂与CD93/C1q复合物接触。The candidate drug is one or more of antibodies, peptides, fusion peptides, peptide analogs, polypeptides, aptamers, avimers, anticalins, speigelmers or small molecule compounds. The method includes contacting the candidate agent with a CD93/Clq complex.
附图说明Description of the drawings
图1EV和肿瘤细胞处理的试验方案流程图(肿瘤接种前注射EV)。Figure 1 Flow chart of experimental protocol for EV and tumor cell treatment (EV injection before tumor inoculation).
图2按图1方案胸腔注射LLC-EVs后小鼠LLC肺肿瘤大小。Figure 2. Size of LLC lung tumors in mice after intrapleural injection of LLC-EVs according to the protocol in Figure 1.
图3按图1方案腹腔、皮下和注射静脉注射LLC-EVs后小鼠LLC肿瘤大小。Figure 3. LLC tumor size in mice after intraperitoneal, subcutaneous and intravenous injection of LLC-EVs according to the scheme in Figure 1.
图4按图1方案胸腔注射5μgB16F10-EVs后小鼠B16F10-Luci肿瘤大小。Figure 4. Size of B16F10-Luci tumors in mice after intrapleural injection of 5 μg B16F10-EVs according to the protocol in Figure 1.
图5按图1方案胸腔注射5μgB16F10-EVs后小鼠LLC-luci肿瘤大小。Figure 5 The size of LLC-luci tumors in mice after intrapleural injection of 5 μg B16F10-EVs according to the protocol in Figure 1.
图6按图1方案胸腔注射5μg 4T1-EVs小鼠LLC-luci肿瘤大小。Figure 6. LLC-luci tumor size in mice injected with 5 μg 4T1-EVs into the chest according to the scheme in Figure 1.
图7第0天向小鼠皮下注射4T1细胞，第16天手术摘除皮下瘤，再于26天、28天、30天、32天、34天分别于胸腔注射5μg 4T1-EVs，在第37天取小鼠肺用H&E染色并定量检测。 Figure 7: On day 0, mice were injected with 4T1 cells subcutaneously. On day 16, the subcutaneous tumors were surgically removed. Then on days 26, 28, 30, 32, and 34, 5 μg of 4T1-EVs were injected into the chest. On day 37, Mouse lungs were harvested and stained with H&E for quantitative detection.
图8EV和肿瘤细胞处理的试验方案流程图(肿瘤移植后再注射EV)。Figure 8 Flowchart of experimental protocol for EV and tumor cell treatment (EV injection after tumor transplantation).
图9使用LLC-Luci细胞处理小鼠构建肺肿瘤模型后，按图8方案胸腔注射5μgLLC-EVs，第25天时肺肿瘤大小。Figure 9: After using LLC-Luci cells to treat mice to construct a lung tumor model, 5 μg LLC-EVs were injected into the chest according to the protocol in Figure 8. The lung tumor size on the 25th day.
图10第0天和第1天在p53fl/flLSL-KrasG12D小鼠鼻内滴注表达Cre重组酶的腺病毒构建自发性肺肿瘤模型，然后在第30天、32天、34天和38天在在该小鼠胸腔注射5μg LLC-EVs，在第41天检测的小鼠肺肿瘤情况。Figure 10 A spontaneous lung tumor model was established by intranasal instillation of adenovirus expressing Cre recombinase into p53fl/flLSL-KrasG12D mice on days 0 and 1, and then on days 30, 32, 34 and 38. The mice were injected with 5 μg LLC-EVs into the chest cavity, and the lung tumors of the mice were detected on the 41st day.
图11流式细胞仪检测图9LLC肺肿瘤小鼠的TILs(tumor-infiltrating leukocytes，肿瘤浸润白细胞)的CD4+T细胞、CD8+T细胞和DC的试验结果。Figure 11 Flow cytometry test results of CD4+T cells, CD8+T cells and DCs in TILs (tumor-infiltrating leukocytes, tumor-infiltrating leukocytes) of mice with LLC lung tumors in Figure 9.
图12流式细胞仪检测图9LLC肺肿瘤小鼠的TILs的巨噬细胞、中性粒细胞和B细胞的试验结果。Figure 12 Flow cytometry test results of macrophages, neutrophils and B cells in TILs of mice with LLC lung tumors in Figure 9.
图13用LLC-Luci细胞和5μg LLC-EVs按图8的方案处理CD11c-DTR小鼠，同时用白喉毒素(DT)处理，小鼠肺肿瘤生长情况。Figure 13. CD11c-DTR mice were treated with LLC-Luci cells and 5 μg LLC-EVs according to the protocol in Figure 8, and treated with diphtheria toxin (DT) at the same time. The growth of lung tumors in mice.
图14用LLC-Luci细胞和5μg LLC-EVs按图8的方案处理CD11c-DTR小鼠，同时用白喉毒素(DT)处理，小鼠肺肿瘤TILs流式细胞仪检测结果。Figure 14. CD11c-DTR mice were treated with LLC-Luci cells and 5 μg LLC-EVs according to the protocol in Figure 8, and treated with diphtheria toxin (DT) at the same time. Flow cytometric detection results of mouse lung tumor TILs.
图15用LLC-Luci细胞和5μg LLC-EVs按图8的方案处理小鼠，同时使用抗CD4抗体或抗CD8抗体，小鼠肺肿瘤生长情况。Figure 15 Using LLC-Luci cells and 5 μg LLC-EVs to treat mice according to the protocol in Figure 8, while using anti-CD4 antibodies or anti-CD8 antibodies, the growth of mouse lung tumors.
图16用LLC-Luci细胞和5μg LLC-EVs按图8的方案处理小鼠，同时使用抗CD8抗体或抗CD8抗体，小鼠肺肿瘤TILs流式细胞仪检测结果。Figure 16 Using LLC-Luci cells and 5 μg LLC-EVs to treat mice according to the protocol in Figure 8, while using anti-CD8 antibodies or anti-CD8 antibodies, flow cytometric detection results of mouse lung tumor TILs.
图17在LLC肺肿瘤小鼠腹侧两边皮下注入分别注入LLC和B16F10细胞，并同时通过胸腔注射LLC-EVs的试验方案示意图。Figure 17 Schematic diagram of the experimental protocol for injecting LLC and B16F10 cells subcutaneously on both sides of the ventral side of mice with LLC lung tumors, and simultaneously injecting LLC-EVs through the chest cavity.
图18按图17方案处理的小鼠腹侧的LLC和B16F10肿瘤大小。Figure 18. LLC and B16F10 tumor sizes on the ventral side of mice treated according to the protocol of Figure 17.
图19第0天在正常小鼠的腹侧皮下注射LLC细胞，并在第2天、4天、6天、8天和10天胸腔注射5μg LLC-EVs，在第18天用流式细胞仪检测肺部DCs和T细胞的试验结果。Figure 19 LLC cells were injected subcutaneously into the ventral side of normal mice on day 0, and 5 μg LLC-EVs were injected into the chest on days 2, 4, 6, 8, and 10, and flow cytometry was performed on day 18. Test results for detecting lung DCs and T cells.
图20同图19处理小鼠，小鼠皮下肿瘤大小。Figure 20 shows the size of subcutaneous tumors in mice treated with Figure 19.
图21静脉或胸腔注射100μgVivoTrack 680标记的LLC-EVs，LLC-EVs在小鼠体内分布情况。Figure 21 Intravenous or intrapleural injection of 100 μg VivoTrack 680 labeled LLC-EVs, distribution of LLC-EVs in mice.
图22静脉或胸腔注射100μg PKH26标记的LLC-EVs，LLC-EVs在小鼠体内分布情况。Figure 22 Intravenous or intrapleural injection of 100 μg PKH26-labeled LLC-EVs, distribution of LLC-EVs in mice.
图23 2.5μg ml-1CFSE标记的LLC-EVs处理p-pMCs和40L细胞24h后用荧光显微法 检测p-pMCs和40L细胞摄取EVs的情况。Figure 23 Fluorescence microscopy of p-pMCs and 40L cells treated with 2.5 μg ml-1CFSE-labeled LLC-EVs for 24 h. The uptake of EVs by p-pMCs and 40L cells was detected.
图24在小鼠胸腔注射20μg CFSE标记的LLC-EVs，24h后用立体显微镜检测进入胸膜的EVs情况。Figure 24: Inject 20 μg of CFSE-labeled LLC-EVs into the chest of mice, and 24 hours later use a stereomicroscope to detect the EVs entering the pleura.
图25除了在每次胸腔注射EVs前2h胸腔注射0.25mg kg-1Cyto-D，其他同图8的方案，用LLC-Luci细胞和CFSE标记的LLC-EVs处理小鼠，用立体显微镜检测进入胸膜的EVs情况。Figure 25: Except that 0.25 mg kg-1 Cyto-D is injected into the chest 2 hours before each intrapleural injection of EVs, the other protocols are the same as in Figure 8. Mice are treated with LLC-Luci cells and CFSE-labeled LLC-EVs, and entry into the pleura is detected using a stereomicroscope. EVs situation.
图26同图25处理小鼠，用流式细胞仪检测TILs中的DC比例。Figure 26 is the same as Figure 25. Mice were treated, and flow cytometry was used to detect the proportion of DCs in TILs.
图27同图25处理小鼠，用IVIS测定肺肿瘤大小。Figure 27 is the same as Figure 25. Mice were treated and lung tumor size was measured using IVIS.
图28除了在每次胸腔注射EVs后2h胸腔注射0.25mg kg-1Cyto-D，其他同图8的方案，用LLC-Luci细胞和CFSE标记的LLC-EVs处理小鼠，用立体显微镜检测进入胸膜的EVs情况。Figure 28: Except that 0.25 mg kg-1 Cyto-D is injected into the chest 2 hours after each intrapleural injection of EVs, the other protocols are the same as in Figure 8. Mice are treated with LLC-Luci cells and CFSE-labeled LLC-EVs, and entry into the pleura is detected using a stereomicroscope. EVs situation.
图29同图28处理小鼠，用流式细胞仪检测TILs中的DC比例。Figure 29 is the same as Figure 28. Mice were treated, and flow cytometry was used to detect the proportion of DCs in TILs.
图30同图28处理小鼠，用IVIS测定肺肿瘤大小。Figure 30 is the same as Figure 28. Mice were treated and lung tumor size was measured using IVIS.
图31用一定浓度的LLC-EVs刺激40L细胞24h，通过CCK8试验测定细胞活力。Figure 31: 40L cells were stimulated with a certain concentration of LLC-EVs for 24 hours, and cell viability was measured by CCK8 assay.
图32用2.5μg ml-1LLC-EV刺激p-pMCs细胞或40L细胞，24h后通过Transwell趋化试验检测各细胞上清对DCs趋化性的影响。Figure 32: p-pMCs cells or 40L cells were stimulated with 2.5 μg ml -1 LLC-EV, and the effect of each cell supernatant on the chemotaxis of DCs was detected by Transwell chemotaxis assay 24 hours later.
图33使用特定siRNA转染BMDCs细胞使各趋化因子受体沉默(同时使用与目的基因无同源性的siRNA作为阴性对照，记为NC siRNA)后，通过蛋白免疫印迹法验证沉默效果。Figure 33: After transfecting BMDCs cells with specific siRNA to silence each chemokine receptor (while using siRNA that has no homology to the target gene as a negative control, denoted as NC siRNA), the silencing effect was verified by Western blotting.
图34在无siRNA沉默(NC siRNA)或有特定siRNA沉默各受体下，用2.5μg ml-1LLC-EV刺激40L细胞，24h后通过Transwell趋化试验检测细胞上清对DCs趋化性的影响。Figure 34 Without siRNA silencing (NC siRNA) or with specific siRNA silencing each receptor, 40L cells were stimulated with 2.5 μg ml -1 LLC-EV. After 24 hours, the chemotaxis of the cell supernatant to DCs was detected by Transwell chemotaxis assay. Influence.
图35在特定siRNA沉默ccr7受体的情况下，用2.5μg ml-1LLC-EV刺激p-pMCs，24h后通过Transwell趋化试验检测细胞上清对DCs趋化性的影响。Figure 35 Under the condition that specific siRNA silenced the ccr7 receptor, p-pMCs were stimulated with 2.5 μg ml -1 LLC-EV, and the effect of the cell supernatant on the chemotaxis of DCs was detected by Transwell chemotaxis assay 24 hours later.
图36用2.5μg ml-1LLC-EV刺激p-pMCs，24h后通过Transwell趋化试验检测细胞上清对DCs(WT)或Ccr7-/-DCs趋化性的影响。Figure 36: p-pMCs were stimulated with 2.5 μg ml -1 LLC-EV, and 24 h later, the effect of the cell supernatant on the chemotaxis of DCs (WT) or Ccr7 -/- DCs was detected by Transwell chemotaxis assay.
图37用2.5μg ml-1LLC-EV刺激p-pMCs或40L细胞，24h后用ELISA测定细胞上清液中的CCL19和CCL21a水平。Figure 37: p-pMCs or 40L cells were stimulated with 2.5 μg ml -1 LLC-EV, and ELISA was used to determine the levels of CCL19 and CCL21a in the cell supernatant 24 hours later.
图38用2.5μg ml-1LLC-EV刺激p-pMCs或40L细胞，24h后用用real-time PCR测定细胞中的CCL19和CCL21a mRNA水平。 Figure 38: p-pMCs or 40L cells were stimulated with 2.5 μg ml -1 LLC-EV, and 24 h later, real-time PCR was used to determine the CCL19 and CCL21a mRNA levels in the cells.
图39在有特定siRNA沉默CCL19或CCL21a的情况下，用2.5μg ml-1LLC-EV刺激40L细胞，24h后通过Transwell趋化试验检测细胞上清对DCs趋化性的影响(NC siRNA为对照组，未沉默CCL19和CCL21a)。Figure 39 In the presence of specific siRNA to silence CCL19 or CCL21a, 40L cells were stimulated with 2.5 μg ml -1 LLC-EV, and the effect of the cell supernatant on the chemotaxis of DCs was detected by Transwell chemotaxis assay 24 hours later (NC siRNA was used as a control group, CCL19 and CCL21a were not silenced).
图40使用特定siRNA转染40L细胞使目标基因沉默后，通过蛋白免疫印迹法验证沉默效果(NC siRNA为对照组，未沉默CCL19或CCL21a)。Figure 40: After transfecting 40L cells with specific siRNA to silence the target gene, the silencing effect was verified by Western blotting (NC siRNA was the control group and CCL19 or CCL21a was not silenced).
图41小鼠胸腔注射5μg ml-1LLC-EVs后24h，用real-time PCR测定胸膜Ccl21a mRNA水平。Figure 41: Real-time PCR was used to measure pleural Ccl21a mRNA levels 24 hours after pleural injection of 5 μg ml -1 LLC-EVs into mice.
图42小鼠胸腔注射5μg ml-1LLC-EVs后24h，用蛋白免疫印迹法测定Ccl21a蛋白水平。Figure 42: 24 h after mice were injected with 5 μg ml -1 LLC-EVs into the chest, Ccl21a protein levels were determined by Western blotting.
图43胸腔注射10μg胆固醇偶联Ccl21a siRNAs 24h后，用real-time PCR检测小鼠胸膜Ccl21a mRNA水平。Figure 43 After pleural injection of 10 μg cholesterol-conjugated Ccl21a siRNAs for 24 hours, real-time PCR was used to detect mouse pleural Ccl21a mRNA levels.
图44按图8方案使用LLC-Luci细胞和LLC-EVs处理小鼠，并在每次注射EVs前24h在胸腔注射10μg胆固醇偶联Ccl21a siRNAs，用流式细胞仪分析TILs中的DC频率。Figure 44 uses LLC-Luci cells and LLC-EVs to treat mice according to the protocol in Figure 8, and injects 10 μg of cholesterol-coupled Ccl21a siRNAs into the chest 24 hours before each injection of EVs, and uses flow cytometry to analyze the DC frequency in TILs.
图45按图8方案使用LLC-Luci细胞和LLC-EVs处理小鼠，并在每次注射EVs前24h在胸腔注射10μg胆固醇偶联Ccl21a siRNAs，用IVIS测定肺肿瘤大小。Figure 45: Mice were treated with LLC-Luci cells and LLC-EVs according to the protocol in Figure 8, and 10 μg of cholesterol-coupled Ccl21a siRNAs was injected into the chest 24 hours before each injection of EVs, and lung tumor size was measured with IVIS.
图46按图8方案使用LLC-Luci细胞和LLC-EVs处理小鼠，用流式细胞仪分析TILs中的CCR7+DC频率。Figure 46 uses LLC-Luci cells and LLC-EVs to treat mice according to the protocol in Figure 8, and uses flow cytometry to analyze the CCR7 + DC frequency in TILs.
图47按图8方案使用LLC-Luci细胞和LLC-EVs处理Ccr7-/-小鼠，用流式细胞仪分析TILs中的DC频率。Figure 47 uses LLC-Luci cells and LLC-EVs to treat Ccr7 -/- mice according to the protocol in Figure 8, and uses flow cytometry to analyze DC frequency in TILs.
图48按图8方案使用LLC-Luci细胞和LLC-EVs处理Ccr7-/-小鼠，用IVIS测定肺肿瘤大小。Figure 48 uses LLC-Luci cells and LLC-EVs to treat Ccr7 -/- mice according to the protocol in Figure 8, and determines lung tumor size using IVIS.
图49RNA-Seq分析PBS处理40L细胞(对照)和使用LLC-EV处理过的40L细胞的mRNA水平。Figure 49 RNA-Seq analysis of mRNA levels in PBS-treated 40L cells (control) and 40L cells treated with LLC-EV.
图50与对照相比，使用LLC-EV处理过的40L细胞中|Log2FC|≥2的差异表达基因(DEGs)。Figure 50 Differentially expressed genes (DEGs) |Log2FC|≥2 in 40L cells treated with LLC-EV compared with control.
图51 31个DEGs的|Log2FC|和P值(a)以及与Ccl21a表达之间的关联度(b)。Figure 51 |Log2FC| and P value of 31 DEGs (a) and correlation with Ccl21a expression (b).
图52 real-time PCR测得各种细胞中Cd93和Ccl21a mRNA水平。Figure 52 Cd93 and Ccl21a mRNA levels in various cells measured by real-time PCR.
图53 real-time PCR测得健康小鼠和LCC肺荷瘤小鼠胸膜Cd93和Ccl21a mRNA水平。Figure 53 Real-time PCR measured pleural Cd93 and Ccl21a mRNA levels in healthy mice and LCC lung tumor-bearing mice.
图54蛋白免疫印迹法验证40L细胞中CD93的沉默效果。Figure 54 Western blotting method to verify the silencing effect of CD93 in 40L cells.
图55 2.5μg ml-1LLC-EVs刺激CD93沉默的40L细胞24h后，使用ELISA测定细胞上清 液中CCL21水平。Figure 55 After 2.5μg ml -1 LLC-EVs stimulated CD93-silenced 40L cells for 24 hours, the cell supernatant was measured using ELISA CCL21 levels in fluid.
图56蛋白免疫印迹法检测LLC-EVs刺激的p-pMCs中CD93。Figure 56 Western blotting method to detect CD93 in p-pMCs stimulated by LLC-EVs.
图57在小鼠胸腔注射20μg LLC-EVs 24h后，用免疫荧光印记检测pMCs中的CD93(比例尺：10μm)。Figure 57 Immunofluorescence blotting was used to detect CD93 in pMCs 24 hours after pleural injection of 20 μg LLC-EVs in mice (scale bar: 10 μm).
图58real-time PCR检测胸腔注射过胆固醇偶联Cd93 siRNA的小鼠的Cd93 mRNA水平。Figure 58 Real-time PCR detection of Cd93 mRNA levels in mice injected with cholesterol-conjugated Cd93 siRNA into the chest.
图59按图8方案使用LLC-Luci细胞和LLC-EVs处理小鼠并在每次注射EV前胸腔注射10μg胆固醇偶联Cd93 siRNA，用real-time PCR检测胸膜Ccl21 mRNA水平。Figure 59 uses LLC-Luci cells and LLC-EVs to treat mice according to the protocol in Figure 8 and inject 10 μg of cholesterol-conjugated Cd93 siRNA into the chest before each EV injection, and use real-time PCR to detect pleural Ccl21 mRNA levels.
图60按图8方案使用LLC-Luci细胞和LLC-EVs处理小鼠并在每次注射EV前胸腔注射10μg胆固醇偶联Cd93 siRNA，用ELISA检测胸膜间皮细胞分泌的Ccl21蛋白水平。Figure 60 uses LLC-Luci cells and LLC-EVs to treat mice according to the protocol in Figure 8 and injects 10 μg of cholesterol-conjugated Cd93 siRNA into the chest before each EV injection, and uses ELISA to detect the level of Ccl21 protein secreted by pleural mesothelial cells.
图61按图8方案使用LLC-Luci细胞和LLC-EVs处理小鼠并在每次注射EV前胸腔注射10μg胆固醇偶联Cd93 siRNA，用IVIS检测肺肿瘤大小。Figure 61 Use LLC-Luci cells and LLC-EVs to treat mice according to the protocol in Figure 8 and inject 10 μg of cholesterol-conjugated Cd93 siRNA into the chest before each EV injection, and use IVIS to detect lung tumor size.
图62按图8方案使用LLC-Luci细胞和LLC-EVs处理小鼠并在每次注射EV前胸腔注射10μg胆固醇偶联Cd93 siRNA，用流式细胞仪测定TILs中DC、CD4+T、CD8+T细胞的比例。Figure 62 Use LLC-Luci cells and LLC-EVs to treat mice according to the protocol in Figure 8, and inject 10 μg cholesterol-conjugated Cd93 siRNA into the chest before each EV injection, and use flow cytometry to measure DC, CD4+T, and CD8+ in TILs. Proportion of T cells.
图63 10μg ml-1蛋白酶K消化LLC-EVs 2h后，用流式细胞仪测定消化效果(以EVs膜蛋白CD9为代表)。Figure 63 After digesting LLC-EVs with 10 μg ml -1 proteinase K for 2 hours, flow cytometry was used to measure the digestion effect (represented by EVs membrane protein CD9).
图64用10μg ml-1蛋白酶K消化LLC-EVs 2h后，用蛋白免疫印迹法测定消化效果(以EVs囊腔内蛋白Alix表示)。Figure 64: After digesting LLC-EVs with 10 μg ml -1 proteinase K for 2 hours, the digestion effect (expressed as protein Alix in the EVs lumen) was determined by Western blotting.
图65用LLC-EVs和/或蛋白酶K消化后的LLC-EVs刺激40L细胞24h，通过ELISA测定细胞上清中的CCL21a水平Figure 65: 40L cells were stimulated with LLC-EVs and/or proteinase K-digested LLC-EVs for 24 h, and the CCL21a level in the cell supernatant was measured by ELISA
图66用10μg ml-1RNase I消化后LLC-EVs 2h后测量LLC-EVs中RNA含量。Figure 66 Measurement of RNA content in LLC-EVs after digestion with 10 μg ml -1 RNase I for 2 h.
图67 10μg ml-1RNase I消化2h后的LLC-EVs刺激40L细胞24h后用ELISA检测细胞上清中CCL21a水平。Figure 67 LLC-EVs digested with 10 μg ml -1 RNase I for 2 hours stimulated 40L cells for 24 hours and then ELISA was used to detect the level of CCL21a in the cell supernatant.
图68 MLE-12 EVs刺激40L细胞24h后用ELISA检测细胞上清中的CCL21a水平。Figure 68 After stimulating 40L cells with MLE-12 EVs for 24 hours, ELISA was used to detect the level of CCL21a in the cell supernatant.
图69 miRNA array法对MLE-12-EVs和LLC-EVs的miRNA进行富集分析，获得的富集的miRNAs。Figure 69 The enriched miRNAs obtained by enrichment analysis of miRNAs in MLE-12-EVs and LLC-EVs using the miRNA array method.
图70通过miRDB和TargetScan数据库预测Cd93上游的miRNAs。Figure 70 Prediction of miRNAs upstream of Cd93 through the miRDB and TargetScan databases.
图71富集的miRNAs与预测的Cd93上游的miRNAs的比对。 Figure 71 Alignment of enriched miRNAs and predicted miRNAs upstream of Cd93.
图72 real-time PCR检测转染miR-5110或miR-5107-5p类似物24h后，40L细胞中Cd93mRNA水平。Figure 72 Real-time PCR detection of Cd93mRNA levels in 40L cells 24 hours after transfection with miR-5110 or miR-5107-5p analogues.
图73蛋白免疫印迹法检测转染miR-5110或miR-5107-5p类似物24h后，40L细胞中CD93蛋白水平。Figure 73 Western blotting method to detect CD93 protein levels in 40L cells 24 hours after transfection with miR-5110 or miR-5107-5p analogues.
图74用real-time PCR检测转染miR-5110或miR-5107-5p类似物24h后，40L细胞中miR-5110或miR-5107-5p类似物的过表达水平。Figure 74 Use real-time PCR to detect the overexpression level of miR-5110 or miR-5107-5p analogs in 40L cells 24 hours after transfection with miR-5110 or miR-5107-5p analogs.
图75在Cd93 3’-UTR中miR-5110的靶标序列(WT)以及对应的突变序列(MUT)。Figure 75 Target sequence (WT) and corresponding mutant sequence (MUT) of miR-5110 in Cd93 3’-UTR.
图76向40L细胞转入携带有图75中Cd93 3’-UTR WT或MUT片段的荧光素酶质粒(Renilla荧光素酶质粒)和miR-5110类似物，24h后测定荧光素酶活性。Figure 76: The luciferase plasmid (Renilla luciferase plasmid) carrying the Cd93 3’-UTR WT or MUT fragment in Figure 75 and the miR-5110 analog were transferred into 40L cells, and the luciferase activity was measured 24 hours later.
图77 real-time PCR定量检测细胞和EVs中的miR-5110表达。Figure 77 Real-time PCR quantitative detection of miR-5110 expression in cells and EVs.
图78用2.5μg ml-1LLC-EVs刺激转染了miRNA抑制剂NC、miR-5110抑制剂的40L细胞24h后，用蛋白免疫印迹法检测细胞中CD93水平。Figure 78: After 2.5 μg ml -1 LLC-EVs were used to stimulate 40L cells transfected with miRNA inhibitor NC and miR-5110 inhibitor for 24 hours, CD93 levels in the cells were detected by Western blotting.
图79 real-time PCR证实转染了miR-5110抑制剂的40L细胞中，miR-5110水平降低。Figure 79 Real-time PCR confirmed that the level of miR-5110 was reduced in 40L cells transfected with the miR-5110 inhibitor.
图80 real-time PCR测得EVs中miRNA-5110水平。Figure 80 Real-time PCR measured miRNA-5110 levels in EVs.
图81用2.5μg ml-1LLC-EVs、LLC-EVs-miR-5110Ins或LLC-EVs-miR-5110Des刺激40L细胞24h后，蛋白免疫印迹法测得细胞中的CD93水平。Figure 81 After stimulating 40L cells with 2.5 μg ml -1 LLC-EVs, LLC-EVs-miR-5110Ins or LLC-EVs-miR-5110Des for 24 hours, CD93 levels in the cells were measured by Western blotting.
图82按图8的方案用LLC-Luci细胞和相应的EVs处理小鼠，第25天时IVIS测得的肿瘤大小。Figure 82. Mice were treated with LLC-Luci cells and corresponding EVs according to the protocol in Figure 8. Tumor size measured by IVIS on day 25.
图83 LLC肺荷瘤小鼠中肿瘤来源的EVs(TT-EVs)或血清EVs(sEVs)中的miR-5110水平和胸膜Cd93 mRNA水平的相关性。Figure 83 Correlation between miR-5110 levels in tumor-derived EVs (TT-EVs) or serum EVs (sEVs) and pleural Cd93 mRNA levels in LLC lung tumor-bearing mice.
图84 real-time PCR测得人NCI-H2452细胞和HUVECs中Cd93和Ccl21 mRNA水平。Figure 84 Cd93 and Ccl21 mRNA levels in human NCI-H2452 cells and HUVECs measured by real-time PCR.
图85 ELISA检测NCI-H2452细胞(CD93被沉默：Cd93 siRNA；CD93没有被沉默：NC siRNA)上清中的CCL21水平。Figure 85 ELISA detects CCL21 levels in the supernatant of NCI-H2452 cells (CD93 is silenced: Cd93 siRNA; CD93 is not silenced: NC siRNA).
图86 Transwell趋化试验检测NCI-H2452细胞(CD93被沉默：Cd93 siRNA；CD93没有被沉默：NC siRNA)上清对DCs(CCR7被沉默：Ccr7siRNA；CCR7没有被沉默：NC siRNA)趋化作用。Figure 86 Transwell chemotaxis assay detects the chemotactic effect of NCI-H2452 cell (CD93 is silenced: Cd93 siRNA; CD93 is not silenced: NC siRNA) supernatant on DCs (CCR7 is silenced: Ccr7 siRNA; CCR7 is not silenced: NC siRNA).
图87蛋白免疫印迹证实NCI-H2452细胞中CD93被沉默。Figure 87 Western blot confirms that CD93 is silenced in NCI-H2452 cells.
图88蛋白免疫印迹证实DCs中CCR7被沉默。Figure 88 Western blot confirms that CCR7 is silenced in DCs.
图89 real-time PCR定量检测病人TTs和TT-TVs中miR-5193水平。 Figure 89 Real-time PCR quantitative detection of miR-5193 levels in patient TTs and TT-TVs.
图90 2.5μg ml-1TT-EVs刺激NCI-H2452细胞24h后，蛋白免疫印迹检测细胞中CD93水平。Figure 90 After 2.5μg ml -1 TT-EVs stimulated NCI-H2452 cells for 24 hours, CD93 levels in the cells were detected by Western blotting.
图91图89中测得的TT-EV miR-5193水平与图90中CD93蛋白水平的抑制百分比之间的相关性。Figure 91 Correlation between TT-EV miR-5193 levels measured in Figure 89 and percent inhibition of CD93 protein levels in Figure 90.
图92来自恶性胸腔积液(MPEs)细胞沉淀物代表性的免疫荧光图像。Figure 92 Representative immunofluorescence images of cell pellets from malignant pleural effusions (MPEs).
图93 MPEs中各因子之间的相关性。Figure 93 Correlation between factors in MPEs.
图94 TTs中各因子之间的相关性。Figure 94 Correlation between factors in TTs.
图95肺TTs代表性的免疫组化的代表性图像。Figure 95 Representative images of representative immunohistochemistry of lung TTs.
图96 TT-EV/miR-5193hi和TT-EV/miR-5193lo肺癌患者的总体生存曲线。Figure 96 Overall survival curve of TT-EV/miR-5193 hi and TT-EV/miR-5193 lo lung cancer patients.
图97蛋白免疫印迹证实在40L细胞或NCI-H2452细胞中C1qA、MMRN2和IGFBP7过表达。Figure 97 Western blot confirms overexpression of C1qA, MMRN2 and IGFBP7 in 40L cells or NCI-H2452 cells.
图98 real-time PCR测得C1qA、MMRN2或IGFBP7过表达细胞中Ccl21a mRNA水平。Figure 98 Ccl21a mRNA levels in C1qA, MMRN2 or IGFBP7 overexpressing cells measured by real-time PCR.
图99 2μg ml-1重组C1qA刺激40L细胞或NCI-H2452细胞24h后，ELISA检测细胞上清中的CCL21a或CCL21水平。Figure 99 After 2 μg ml -1 recombinant C1qA stimulated 40L cells or NCI-H2452 cells for 24 hours, ELISA detected the level of CCL21a or CCL21 in the cell supernatant.
图100按图8方案在第14天、16天、18天、20天和22天向LLC-Luci荷瘤小鼠胸腔注射5μg C1qA，在第25天用蛋白免疫印迹测得的胸膜CCL21a蛋白水平。Figure 100 According to the protocol in Figure 8, 5 μg C1qA was injected into the chest of LLC-Luci tumor-bearing mice on days 14, 16, 18, 20 and 22, and the pleural CCL21a protein level was measured by Western blotting on day 25. .
图101按图8方案在第14天、16天、18天、20天和22天向LLC-Luci荷瘤小鼠胸腔注射5μg C1qA，在第25天用IVIS检测的肺肿瘤体积。Figure 101 According to the protocol in Figure 8, 5 μg C1qA was injected into the chest of LLC-Luci tumor-bearing mice on days 14, 16, 18, 20 and 22, and the lung tumor volume detected by IVIS on day 25.
图102按图8方案在第14天、16天、18天、20天和22天向LLC-Luci荷瘤小鼠静脉注射100μg胆固醇偶联的C1qa ASOs(cholesterol-conjugated C1qa antisense oligonucleotide，胆固醇偶联C1qa反义寡核苷酸；C1qa反义寡核苷酸序列如SEQ ID NO:49所示)，在第25天用蛋白免疫印迹测得胸膜CCL21a蛋白水平；同时使用胆固醇偶联的阴性对照基因的反义寡核苷酸处理作为对照(NCASOs，其反义寡核苷酸序列如SEQ ID NO:50所示)。Figure 102 According to the protocol in Figure 8, LLC-Luci tumor-bearing mice were intravenously injected with 100 μg of cholesterol-conjugated C1qa antisense oligonucleotide on days 14, 16, 18, 20 and 22. C1qa antisense oligonucleotide; C1qa antisense oligonucleotide sequence is shown in SEQ ID NO:49), pleural CCL21a protein levels were measured by Western blotting on day 25; a cholesterol-coupled negative control gene was also used The antisense oligonucleotide treatment was used as a control (NCASOs, the antisense oligonucleotide sequence is shown in SEQ ID NO: 50).
图103按图8方案在第14天、16天、18天、20天和22天向LLC-Luci荷瘤小鼠静脉注射100μg胆固醇偶联的C1qa ASOs，在第25天用IVIS检测的肺肿瘤体积。Figure 103. LLC-Luci tumor-bearing mice were intravenously injected with 100 μg cholesterol-conjugated C1qa ASOs on days 14, 16, 18, 20 and 22 according to the protocol in Figure 8. Lung tumors detected with IVIS on day 25 volume.
图104 ELISA测得LLC肺荷瘤小鼠和肺癌病人血清中C1qA水平。Figure 104 ELISA measured C1qA levels in serum of LLC lung tumor-bearing mice and lung cancer patients.
图105全长和截短的CD93示意图。 Figure 105 Schematic representation of full-length and truncated CD93.
图106将图105所示的全长或截短的CD93片段转入40L细胞中48h后，real-time PCR测得各CD93片段在细胞中的表达水平。Figure 106: After the full-length or truncated CD93 fragments shown in Figure 105 were transferred into 40L cells for 48 hours, real-time PCR measured the expression levels of each CD93 fragment in the cells.
图107将图105所示的全长或截短的CD93片段转入40L细胞中48h后，real-time PCR测得Ccl21a mRNA。Figure 107 After the full-length or truncated CD93 fragment shown in Figure 105 was transferred into 40L cells for 48 hours, Ccl21a mRNA was measured by real-time PCR.
图108将图105所示的全长或截短的CD93片段转入40L细胞中48h后，ELISA测得细胞上清中的CCL21a蛋白水平(CD93ΔCTLD组中，CD93的CTLD结构域被切除)。Figure 108: After the full-length or truncated CD93 fragment shown in Figure 105 was transferred into 40L cells for 48 hours, ELISA measured the CCL21a protein level in the cell supernatant (in the CD93ΔCTLD group, the CTLD domain of CD93 was cleaved).
图109ELISA测得M057与小鼠CD93抗原结合的Kd值。Figure 109 ELISA measured the Kd value of M057 binding to mouse CD93 antigen.
图110使用M057染色WT小鼠或Cd93-/-小鼠的骨髓细胞的结果。Figure 110 Results of staining bone marrow cells of WT mice or Cd93 −/− mice using M057.
图111静脉注射Alexa Fluor 680标记的M057后24h，用IVIS检测WT小鼠或Cd93-/-小鼠胸膜中M057的分布。Figure 111 24h after intravenous injection of Alexa Fluor 680-labeled M057, IVIS was used to detect the distribution of M057 in the pleura of WT mice or Cd93 -/- mice.
图112用10μg ml-1抗CD93抗体(M057)和/或2μg ml-1C1qA处理40L细胞和p-pMCs24h后，ELISA测得细胞上清中的CCL21a水平。Figure 112 After treating 40L cells and p-pMCs with 10 μg ml -1 anti-CD93 antibody (M057) and/or 2 μg ml -1 C1qA for 24 hours, the CCL21a level in the cell supernatant was measured by ELISA.
图113静脉注射一定剂量M057至LLC肺荷瘤小鼠24h后，real-time PCR测得胸膜中Ccl21a mRNA水平。Figure 113 After intravenously injecting a certain dose of M057 into LLC lung tumor-bearing mice for 24 hours, real-time PCR measured the level of Ccl21a mRNA in the pleura.
图114按图8方案在第14天、16天、18天、20天和22天静脉注射100μg M057至LLC-Luci肺荷瘤小鼠，在第25天蛋白免疫印迹测得胸膜CCL21a蛋白水平。Figure 114 According to the protocol in Figure 8, 100 μg M057 was intravenously injected into LLC-Luci lung tumor-bearing mice on days 14, 16, 18, 20 and 22. On day 25, pleural CCL21a protein levels were measured by protein immunoblotting.
图115按图8方案在第14天、16天、18天、20天和22天静脉注射100μg M057至LLC-Luci肺荷瘤小鼠，在第25天用IVIS测得肺肿瘤大小。Figure 115 According to the protocol in Figure 8, 100 μg M057 was intravenously injected into LLC-Luci lung tumor-bearing mice on days 14, 16, 18, 20 and 22, and the lung tumor size was measured with IVIS on day 25.
图116按图8方案在第14天、16天、18天、20天和22天静脉注射100μg M057至LLC-Luci肺荷瘤小鼠，在第25天流式细胞仪测得TILs中DC、CD4+T细胞和CD8+T细胞频率。Figure 116 According to the protocol in Figure 8, 100 μg M057 was intravenously injected into LLC-Luci lung tumor-bearing mice on days 14, 16, 18, 20 and 22. On day 25, flow cytometry measured DC, CD4 + T cell and CD8 + T cell frequencies.
图117在LLC-Luci肺荷瘤小鼠的两侧皮下分别接种LLC和B16F10肿瘤，接着在第8天、10天、12天、14天和16天用100μg M057治疗，第10至第18天皮下肿瘤大小情况。Figure 117 LLC-Luci lung tumor-bearing mice were inoculated subcutaneously with LLC and B16F10 tumors on both sides of the mice, followed by treatment with 100 μg M057 on days 8, 10, 12, 14 and 16, days 10 to 18 Subcutaneous tumor size.
图118按图8方案在第14天、16天、18天、20天和22天在没有敲除CCL21a(NC siRNA)和敲除了CCL21a(Ccl21a siRNA)的LLC-Luci肺荷瘤小鼠静脉注射100μg M057，在第25天用IVIS测得的肺肿瘤大小。Figure 118 According to the protocol in Figure 8, LLC-Luci lung tumor-bearing mice were injected intravenously on days 14, 16, 18, 20 and 22 without CCL21a knockout (NC siRNA) and CCL21a knockout (Ccl21a siRNA). 100 μg M057, lung tumor size measured by IVIS on day 25.
图119每隔一天向健康小鼠静脉注射100μg M057，5次注射后LEISA测得小时血清中的ALT、AST、胆红素和肌酐水平。 Figure 119. Healthy mice were intravenously injected with 100 μg M057 every other day. After 5 injections, LEISA measured the hourly serum ALT, AST, bilirubin and creatinine levels.
图120每隔一天向健康小鼠静脉注射100μg M057，5次注射后用H&E染色(比例尺：40μm)测得小鼠心、肝、脾、肺和肾的组织病理学损伤情况。Figure 120 Healthy mice were intravenously injected with 100 μg M057 every other day. After 5 injections, H&E staining (scale bar: 40 μm) was used to measure the histopathological damage of the mouse heart, liver, spleen, lungs and kidneys.
图121 10μg ml-1M057和/或2μg ml-1IGFBP7处理小鼠肺原代内皮细胞24h后进行calcein-AM染色，荧光显微法检测到血管生成情况及统计分析数据。Figure 121 Calcein-AM staining was performed on mouse lung primary endothelial cells after treatment with 10 μg ml -1 M057 and/or 2 μg ml -1 IGFBP7 for 24 hours. The angiogenesis and statistical analysis data were detected by fluorescence microscopy.
图122按图8方案在第14天、16天、18天、20天和22天在LLC-Luci肺荷瘤小鼠静脉注射100μg M057，第25天取肿瘤组织并对NG2和CD31或者αSMA和CD31进行染色和定量分析。Figure 122 According to the scheme in Figure 8, LLC-Luci lung tumor-bearing mice were intravenously injected with 100 μg M057 on days 14, 16, 18, 20 and 22. On day 25, tumor tissues were taken and analyzed for NG2 and CD31 or αSMA and CD31 was stained and quantified.
图123按图8方案在第14天、16天、18天、20天和22天在LLC-Luci肺荷瘤小鼠静脉注射100μg M057，第25天用5mg FITC-dextran(70kDa)灌注测定的肿瘤内皮渗透性。Figure 123 According to the scheme in Figure 8, LLC-Luci lung tumor-bearing mice were intravenously injected with 100 μg M057 on days 14, 16, 18, 20 and 22, and measured with 5 mg FITC-dextran (70kDa) perfusion on day 25. Tumor endothelial permeability.
图124按图8方案在第14天、16天、18天、20天和22天在LLC-Luci肺荷瘤小鼠静脉注射一定量抗VEGFR试剂，第25天用IVIS测得的肺肿瘤大小。Figure 124 According to the protocol in Figure 8, LLC-Luci lung tumor-bearing mice were intravenously injected with a certain amount of anti-VEGFR reagent on days 14, 16, 18, 20 and 22, and the size of the lung tumors measured with IVIS on day 25 .
图125按图8方案在第13天、15天、17天、19天和21天在LLC-Luci肺荷瘤WT小鼠静脉注射40μg抗VEGFR试剂并在每次注射抗VEGFR试剂12h后静脉注射100μg M057或IgG，第25天用IVIS观测到的肺肿瘤大小。Figure 125 According to the protocol in Figure 8, LLC-Luci lung tumor-bearing WT mice were intravenously injected with 40 μg of anti-VEGFR reagent on days 13, 15, 17, 19, and 21, and intravenously injected 12 hours after each injection of the anti-VEGFR reagent. 100μg M057 or IgG, lung tumor size observed by IVIS on day 25.
图126按图8方案在第13天、15天、17天、19天和21天在LLC-Luci肺荷瘤Ccr7-/-小鼠静脉注射40μg抗VEGFR试剂并在每次注射抗VEGFR试剂12h后静脉注射100μg M057或IgG，第25天用IVIS观测到的肺肿瘤大小。Figure 126 According to the protocol in Figure 8, LLC-Luci lung tumor-bearing Ccr7 -/- mice were intravenously injected with 40 μg of anti-VEGFR reagent on days 13, 15, 17, 19 and 21 and 12h after each injection of the anti-VEGFR reagent. After intravenous injection of 100 μg M057 or IgG, the size of lung tumors observed by IVIS on day 25.
图127第0天在正常小鼠的腹侧皮下注射LLC细胞，第7天、9天、11天和15天静脉注射40μg抗VEGFR试剂并在每次注射抗VEGFR试剂12h后静脉注射100μg M057或IgG，第10至第18天皮下肿瘤大小情况。Figure 127 LLC cells were injected subcutaneously into the ventral side of normal mice on day 0, 40 μg of anti-VEGFR reagent was injected intravenously on days 7, 9, 11, and 15, and 100 μg of M057 or M057 was intravenously injected 12 hours after each injection of anti-VEGFR reagent. IgG, subcutaneous tumor size from days 10 to 18.
图128按图8方案在第14天、16天、18天、20天和22天在LLC-Luci肺荷瘤小鼠静脉注射40μg抗VEGFR试剂或100μg M057，第25天用IVIS检测到的肺肿瘤大小。Figure 128 According to the protocol in Figure 8, LLC-Luci lung tumor-bearing mice were intravenously injected with 40 μg anti-VEGFR reagent or 100 μg M057 on days 14, 16, 18, 20 and 22, and the lungs detected by IVIS on day 25 Tumor size.
图129按图8方案在第14天、16天、18天、20天和22天在LLC-Luci肺荷瘤Ccr7-/-小鼠静脉注射40μg抗VEGFR试剂或100μg M057，第25天用IVIS检测到的肺肿瘤大小。Figure 129 According to the protocol in Figure 8, LLC-Luci lung tumor-bearing Ccr7 -/- mice were intravenously injected with 40 μg anti-VEGFR reagent or 100 μg M057 on days 14, 16, 18, 20 and 22, and IVIS was used on day 25 Detected lung tumor size.
图130按图8方案在第14天、16天、18天、20天和22天在B16F10和4T1肺荷瘤小鼠静脉注射50μg抗PD-1试剂，同时注射100μg M057或不注射100μg M057，第25天用IVIS检测的肺肿瘤大小。Figure 130 According to the scheme in Figure 8, 50 μg of anti-PD-1 reagent was intravenously injected into B16F10 and 4T1 lung tumor-bearing mice on days 14, 16, 18, 20 and 22, with or without the injection of 100 μg M057. Lung tumor size detected by IVIS on day 25.
图131按图8方案在第14天、16天、18天、20天和22天在B16F10和4T1肺荷瘤 小鼠静脉注射50μg抗PD-1试剂，同时注射100μg M057或不注射100μg M057，第25天肺H&E染色结果。Figure 131 B16F10 and 4T1 lung tumor-bearing tumors on days 14, 16, 18, 20 and 22 according to the scheme in Figure 8 Mice were intravenously injected with 50 μg anti-PD-1 reagent, and simultaneously injected with 100 μg M057 or without 100 μg M057. Results of lung H&E staining on the 25th day.
图132抗PD-1处理的肺癌病人，在不同sEV/miR-5193和血清C1qA水平下的PFS。Figure 132 PFS of anti-PD-1-treated lung cancer patients at different sEV/miR-5193 and serum C1qA levels.
图133是本发明的原理结构示意图。Figure 133 is a schematic diagram of the principle structure of the present invention.
图134 real-time PCR检测H1N1感染小鼠中胸膜Cd93的表达水平。Figure 134 Real-time PCR detection of pleural Cd93 expression levels in H1N1-infected mice.
图135 real-time PCR检测胸膜CD93敲低小鼠中肺组织H1N1的感染水平。Figure 135 Real-time PCR detection of H1N1 infection levels in lung tissue of pleural CD93 knockdown mice.
图136胸膜敲低和未敲低CD93的小鼠在感染H1N1后，肺组织H&E染色结果(标尺，50μm)。Figure 136 H&E staining results of lung tissue in mice with and without CD93 knockdown in the pleura after infection with H1N1 (scale bar, 50 μm).
图137胸膜敲低和未敲低CD93的小鼠在感染H1N1后，其总体生存率分析。Figure 137 Analysis of the overall survival rate of mice with and without CD93 knockdown in the pleura after infection with H1N1.
详细说明Detailed description
本发明除了属于具有定义外，其它属于按照本发明的技术领域一般人员所理解的含义。以下说明并不构成对本法的任何限制，本发明的范围由本发明的权利要求所限定。In addition to having definitions, the present invention has other meanings as understood by those of ordinary skill in the technical field according to the present invention. The following description does not constitute any limitation on this law, and the scope of the present invention is limited by the claims of the present invention.
定义definition
(1)细胞外囊泡或胞外囊泡(Extracellular vesicles，EVs)(1) Extracellular vesicles or extracellular vesicles (EVs)
EVs是指从细胞膜上脱落或者由细胞分泌的双层磷脂膜结构的囊泡状小体，直径从30nm到1000nm不等。它们广泛存在于细胞培养上清以及各种体液(血液、淋巴液、唾液、尿液、***、乳汁)中，携带有细胞来源相关的多种蛋白质、脂类、DNA、mRNA、miRNA等，参与细胞间通讯、细胞迁移、血管新生和免疫调节等过程，在细胞间成分交换、信号转导和病理发育等方面发挥着重要作用。肿瘤源性胞外囊泡(tumor-derived EVs，TDEVs或TEVs)为肿瘤细胞产生并释放到胞外的一种EVs，被发现与肿瘤微环境和肿瘤发展有关，其与肿瘤微环境中的肿瘤相关成纤维细胞、免疫细胞、内皮细胞及胞外间质等进行信息交换，影响肿瘤进展。TDEVs可设计为一种具有高靶向性和高渗透性的天然药物载体。EVs refer to vesicle-like bodies with a double-layer phospholipid membrane structure that are shed from the cell membrane or secreted by cells, with diameters ranging from 30nm to 1000nm. They are widely found in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, milk), carrying a variety of proteins, lipids, DNA, mRNA, miRNA, etc. related to cell origin. Processes such as intercellular communication, cell migration, angiogenesis, and immune regulation play an important role in intercellular component exchange, signal transduction, and pathological development. Tumor-derived extracellular vesicles (TDEVs or TEVs) are a type of EVs produced and released extracellularly by tumor cells. They have been found to be related to the tumor microenvironment and tumor development. They are related to tumors in the tumor microenvironment. Related fibroblasts, immune cells, endothelial cells and extracellular matrix exchange information to affect tumor progression. TDEVs can be designed as a natural drug carrier with high targeting and high penetration.
本申请中TT-EVs为肿瘤组织中的细胞外囊泡(EVs from tumor tissues)。In this application, TT-EVs are extracellular vesicles (EVs from tumor tissues) in tumor tissues.
(2)间皮细胞(mesothelial cells，MC或MCs)和胸膜间皮细胞(pleural mesothelial cells，pMCs)(2) Mesothelial cells (MC or MCs) and pleural mesothelial cells (pMCs)
MCs属于上皮组织中的单层扁平上皮，由中胚层发育而来。间皮是覆盖于胸膜，腹膜和心包膜表面的一层膜，能提供润滑，使器官与器官、器官与胸膜与腹膜间都能得到良好的保护，不会互相磨损受伤。间皮所提供的润滑和保护作用就是靠间皮细胞所分泌的物质 来达成，这些物质多半为细胞外基质、玻尿酸类物质。pMCs为覆盖于胸膜上的一层间皮细胞，不仅能够提供一个光滑无黏连的表面以利于肺脏的运动，还具有其它许多重要功能，包括胸水的吸收和物质的转运、分泌黏多糖和表面活性物质、参与炎症反应、征集白细胞、抑制肿瘤的生长与播散、溶解纤维素、以及参与胸膜损伤的修复等。MCs belong to single-layer squamous epithelium in epithelial tissue and develop from mesoderm. The mesothelium is a layer of membrane covering the surface of the pleura, peritoneum and pericardium. It can provide lubrication and protect organs from each other, as well as between organs and the pleura and peritoneum, without causing wear and tear on each other. The lubrication and protection provided by mesothelium rely on substances secreted by mesothelial cells To achieve this, most of these substances are extracellular matrix and hyaluronic acid substances. pMCs are a layer of mesothelial cells covering the pleura. They not only provide a smooth and adhesion-free surface to facilitate lung movement, but also have many other important functions, including pleural effusion absorption and material transport, secretion of mucopolysaccharides and surface Active substances, participate in inflammatory reactions, recruit white blood cells, inhibit the growth and spread of tumors, dissolve cellulose, and participate in the repair of pleural damage, etc.
(3)树突状细胞(dendritic cells，DC或DCs)(3) Dendritic cells (DC or DCs)
DCs在1973年第一次由Steinman和Cohn在单核细胞中分离出来，由于其因其成熟过程中有树突状或伪足样的突起而命名为树突状细胞。DCs是已知体内功能最强、惟一能活化静息T细胞的专职抗原提呈细胞，是启动、调控和维持免疫应答的中心环节。通过大量体外活化培养负载肿瘤抗原的DC细胞，当细胞数量达到一定数量后回输给病人，可诱导机体产生强烈的抗肿瘤免疫反应。在肿瘤免疫中，DCs不能直接杀伤肿瘤细胞，但能通过识别肿瘤细胞特异性抗原，将其信号呈递给具杀伤效应的T细胞来达到监测、杀灭肿瘤的功能。DCs were first isolated from monocytes in 1973 by Steinman and Cohn and were named dendritic cells because they have dendritic or pseudopod-like processes during their maturation. DCs are known to be the most powerful professional antigen-presenting cells in the body and the only ones capable of activating resting T cells. They are the central link in initiating, regulating and maintaining immune responses. Through a large number of in vitro activation and culture of DC cells loaded with tumor antigens, when the number of cells reaches a certain number and then infused back to the patient, the body can be induced to produce a strong anti-tumor immune response. In tumor immunity, DCs cannot directly kill tumor cells, but they can monitor and kill tumors by recognizing tumor cell-specific antigens and presenting their signals to T cells with killing effects.
(4)CCL21(C-C motif chemokine ligand 21)和CCR7(C-C Chemokine receptor 7)(4)CCL21(C-C motif chemokine ligand 21) and CCR7(C-C Chemokine receptor 7)
CCL21又称exodus-2、次级淋巴组织趋化因子(SLC)，属于CC趋化因子家族的一种小细胞因子，在人类基因组中位于第9号染色体上。CCL21可以在***起始处和淋巴器官中组成型表达，存在于几种基质细胞和高内皮小静脉(HEV)上，可以与糖胺聚糖(GAG)结合并固定在内皮细胞表面。CCL21与特定的受体CCR7结合才能发挥生物学功能。CCL21可趋化淋巴细胞、巨噬细胞和T细胞等多种免疫细胞，介导应激反应、感染、血管形成及树突状细胞成熟等多种生理及病理过程。CCL21, also known as exodus-2 and secondary lymphoid tissue chemokine (SLC), is a small cytokine belonging to the CC chemokine family and is located on chromosome 9 in the human genome. CCL21 can be constitutively expressed at the origin of lymphatic vessels and lymphoid organs, is present on several stromal cells and high endothelial venules (HEV), and can bind to glycosaminoglycans (GAGs) and be fixed on the surface of endothelial cells. CCL21 can exert biological functions only by binding to the specific receptor CCR7. CCL21 can chemoattract various immune cells such as lymphocytes, macrophages, and T cells, and mediates various physiological and pathological processes such as stress response, infection, vascularization, and dendritic cell maturation.
趋化因子受体CCR7是一种包含7个跨膜结构域的G蛋白连接的细胞表面受体，并通过异三聚体G蛋白及其下游效应器参与信号转导。CCR7的独家配体是CCL19、CCL21，分别介导不同的生理学功能。CCR7主要在T细胞、B细胞、活化的NK细胞和树突状细胞等免疫细胞表面表达。另外在一些如肿瘤细胞等的非免疫细胞也有表达。在维持机体稳态或感染时，与其高亲和力的功能性配体之一CCL21结合，诱导淋巴细胞归巢，引起一系列免疫反应。The chemokine receptor CCR7 is a G-protein-linked cell surface receptor containing seven transmembrane domains and participates in signal transduction through heterotrimeric G proteins and its downstream effectors. The exclusive ligands of CCR7 are CCL19 and CCL21, which mediate different physiological functions respectively. CCR7 is mainly expressed on the surface of immune cells such as T cells, B cells, activated NK cells and dendritic cells. In addition, it is also expressed in some non-immune cells such as tumor cells. When maintaining body homeostasis or during infection, it binds to one of its high-affinity functional ligands, CCL21, inducing lymphocyte homing and triggering a series of immune responses.
(5)C1q(Complement 1)和C1QA(complementcomponent 1,q subcomponent，A chain)(5)C1q(Complement 1) and C1QA(complementcomponent 1,q subcomponent,A chain)
补体C1q分子是由6个亚单位组成的异源六聚体，每个亚单位分别由C1QA、C1QB、C1QC基因编码的A、B、C 3条多肽链组成，即C1q由18条多肽链构成。C1q是先天性 免疫补体***经典途径的重要识别分子，能够启动经典途径从而在免疫调节、炎症调节和维持机体平衡等方面发挥重要作用。The complement C1q molecule is a heterologous hexamer composed of 6 subunits. Each subunit is composed of 3 polypeptide chains A, B, and C encoded by the C1QA, C1QB, and C1QC genes. That is, C1q is composed of 18 polypeptide chains. . C1q is congenital It is an important recognition molecule of the classical pathway of the immune complement system. It can activate the classical pathway and play an important role in immune regulation, inflammation regulation and maintenance of body balance.
C1QA(或C1qA)是补体C1q的其中一条子链，是先天性免疫及适应性免疫***的重要组成部分，在补体***经典通路中起重要作用，其通过互补依赖或独立的方式执行多种免疫和非免疫功能，在肿瘤进展中发挥促进或抑制作用。C1QA (or C1qA) is one of the sub-chains of complement C1q. It is an important part of the innate immunity and adaptive immune system. It plays an important role in the classical pathway of the complement system. It performs multiple immune functions in a complementary, dependent or independent manner. and non-immune functions that play a promoting or inhibiting role in tumor progression.
(6)T细胞介导的细胞免疫(6) T cell-mediated cellular immunity
T细胞介导的细胞免疫通过T细胞发挥效应来清除异物。效应T细胞具有抗原识别受体，必需经抗原激发才能活化发挥其效应细胞的作用，是一种特异性细胞免疫。由T细胞介导的细胞的免疫有二种基本形式，它们分别由二类不同的T细胞亚类参予。一种是迟发型超敏性的T细胞(CD4+)，该细胞和抗原起反应后可分泌细胞因子。这些细胞因子再吸引和活化巨噬细胞和其它类型的细胞在反应部位聚集，成为组织慢性炎症的非特异效应细胞。另一种是细胞毒性T细胞(CD8+)，对靶细胞有特异杀伤作用。T cell-mediated cellular immunity uses T cells to eliminate foreign bodies. Effector T cells have antigen recognition receptors and must be stimulated by antigens in order to be activated and play their role as effector cells. It is a type of specific cellular immunity. There are two basic forms of cellular immunity mediated by T cells, which are participated by two different subtypes of T cells. One is delayed-type hypersensitive T cells (CD4 + ), which can secrete cytokines after reacting with antigens. These cytokines then attract and activate macrophages and other types of cells to accumulate at the reaction site and become non-specific effector cells of chronic tissue inflammation. The other is cytotoxic T cells (CD8 + ), which have a specific killing effect on target cells.
具体实施方式Detailed ways
为了更为具体地描述本发明，下面结合附图及具体实施方式对本发明的技术方案进行详细说明。这些说明仅仅是表明本发明是如何实现的，并不能限定本发明的具体范围。本发明的范围在权利要求中限定。In order to describe the present invention more specifically, the technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. These descriptions merely illustrate how the present invention is implemented and do not limit the specific scope of the present invention. The scope of the invention is defined in the claims.
本申请提供了作用于胸膜间皮细胞(pMCs)中CD93的活性物质，该活性物质能抑制胸膜间皮细胞中CD93基因的表达，使CD93 mRNA水平下降或抑制CD93 mRNA转录；或者该活性物质能使CD93蛋白含量降低，除了抑制CD93基因表达外，也可能使CD93蛋白降解；或者所述物质抑制胸膜间皮细胞CD93蛋白活性，如所述物质为CD93抗体时,或者改物质能够影响Cq1与CD93的结合。可以理解，这些活性物质如果能够抑制胸膜间皮细胞中C1q基因的表达，使C1q mRNA水平下降或抑制C1q mRNA转录；或者该活性物质能使C1q3蛋白含量降低，除了抑制C1q基因表达外，也可能使C1q蛋白降解；或者所述物质抑制胸膜间皮细胞C1q蛋白活性，如所述物质为C1q抗体。。This application provides an active substance that acts on CD93 in pleural mesothelial cells (pMCs). The active substance can inhibit the expression of CD93 gene in pleural mesothelial cells, reduce the level of CD93 mRNA or inhibit the transcription of CD93 mRNA; or the active substance can Reduce the content of CD93 protein, in addition to inhibiting CD93 gene expression, it may also degrade CD93 protein; or the substance inhibits the activity of CD93 protein in pleural mesothelial cells, such as when the substance is a CD93 antibody, or the modified substance can affect Cq1 and CD93 combination. It can be understood that if these active substances can inhibit the expression of C1q gene in pleural mesothelial cells, reduce the level of C1q mRNA or inhibit the transcription of C1q mRNA; or if the active substances can reduce the content of C1q3 protein, in addition to inhibiting the expression of C1q gene, it may also Degrading C1q protein; or the substance inhibits the activity of C1q protein in pleural mesothelial cells, for example, the substance is a C1q antibody. .
在本申请的一个实施方式中，所述活性物质为TEVs。本申请发现只有胸腔注射TEVs能抑制肺肿瘤生长，而皮下、腹腔或静脉注射TEVs均不能抑制肺肿瘤生长。In one embodiment of the present application, the active material is TEVs. This application found that only intrathoracic injection of TEVs can inhibit the growth of lung tumors, while subcutaneous, intraperitoneal or intravenous injection of TEVs cannot inhibit the growth of lung tumors.
进一步研究发现胸腔注射TEVs能作用于pMCs，并促进全身肿瘤特异性T细胞免疫，从而具有肺肿瘤抑制作用。EVs刺激pMCs后，pMCs释放CCL21招募DC，DC对局部肿瘤抗原进行提呈、促进局部抗肿瘤T细胞免疫。肺部的肿瘤特异性T细胞经血液循环，在其 他部位(皮下)遇到相同肿瘤抗原刺激后迅速活化，又能抑制其他部位相同肿瘤的生长。Further studies have found that intrapleural injection of TEVs can act on pMCs and promote systemic tumor-specific T cell immunity, thus having a lung tumor suppressive effect. After EVs stimulate pMCs, pMCs release CCL21 to recruit DCs, and DCs present local tumor antigens and promote local anti-tumor T cell immunity. Tumor-specific T cells in the lungs circulate in the blood, where they It is rapidly activated when stimulated by the same tumor antigen in other parts (subcutaneous), and can inhibit the growth of the same tumor in other parts.
有趣的是，只有在存在肺肿瘤时，胸腔注射TEVs才能引起全身肿瘤特异性T细胞免疫，仅存在皮下肿瘤等其他部位肿瘤时，胸腔注射TEVs不会引起全身肿瘤特异性T细胞免疫，这进一步说明了pMCs是TEVs引起全身肿瘤特异性T细胞免疫的作用位点。另外，在肺部及其他身体部位同时存在同种肿瘤时，例如皮下，使用所述活性物质除了抑制肺肿瘤外也能抑制其他部位的同种肿瘤(同一类别肿瘤但不同位置)。这里的T细胞包括CD4+和CD8+T细胞，全身肿瘤特异性T细胞免疫是由DCs介导的，胸腔注射TEVs后，会使DCs聚集于肺肿瘤部位，进而为CD4+和CD8+T细胞呈递抗原。Interestingly, intrapleural injection of TEVs can induce systemic tumor-specific T cell immunity only when lung tumors are present. When only subcutaneous tumors and other tumors are present, intrapleural injection of TEVs will not induce systemic tumor-specific T cell immunity. This further It illustrates that pMCs are the site of action of TEVs in inducing systemic tumor-specific T cell immunity. In addition, when the same type of tumor exists in the lungs and other body parts, such as subcutaneously, the use of the active substance can not only inhibit lung tumors, but also inhibit the same type of tumors in other parts (same type of tumor but different location). The T cells here include CD4 + and CD8 + T cells. Systemic tumor-specific T cell immunity is mediated by DCs. After intrapleural injection of TEVs, DCs will accumulate in the lung tumor site, and then create CD4 + and CD8 + T cells. Present the antigen.
特别地，TEVs作用于pMCs中的CD93，使CD93表达下降，促进pMCs分泌更多的CCL21。进一步地，CCL21与树突状细胞的CCR7受体结合，从而促进DCs向肺肿瘤部位聚集。需要说明的是，DCs的受体除了CCR7外，还包括CCR1、CCR2、CCR5、CCR6等其他受体，CCR7的配体除了CCL21外，也还有CCL19，本申请发现所述活性物质作用于pMCs中CD93，促进pMCs分泌CCL21且CCL21与DCs的CCR7受体结合，进而引发后续的全身肿瘤特异性T细胞免疫。In particular, TEVs acts on CD93 in pMCs, reducing CD93 expression and promoting pMCs to secrete more CCL21. Furthermore, CCL21 binds to the CCR7 receptor of dendritic cells, thereby promoting the accumulation of DCs to lung tumor sites. It should be noted that in addition to CCR7, the receptors of DCs also include other receptors such as CCR1, CCR2, CCR5, CCR6, etc. In addition to CCL21, the ligands of CCR7 also include CCL19. This application found that the active substance acts on pMCs CD93 promotes pMCs to secrete CCL21 and CCL21 binds to the CCR7 receptor of DCs, thereby triggering subsequent systemic tumor-specific T cell immunity.
在一些实施方式中，所述活性物质特异性抑制C1q/CD93信号传导途径。CN202080075436.0公开了抑制IGFBP7/CD93信号传导途径能促进血管生成从而具有肿瘤抑制作用。不同于CN202080075436.0，本申请的活性物质通过引发全身肿瘤特异性T细胞免疫来抑制肿瘤。尤其是，所述活性物质通过抑制C1q/CD93信号传导途径，进而抑制pMCs的CD93，接着促进pMCs分泌CCL21，CCL21与DCs的CCR7受体结合，导致DCs聚集并引发全身肿瘤特异性T细胞免疫。In some embodiments, the active agent specifically inhibits the C1q/CD93 signaling pathway. CN202080075436.0 discloses that inhibiting the IGFBP7/CD93 signaling pathway can promote angiogenesis and thus have a tumor suppressive effect. Different from CN202080075436.0, the active substance of the present application inhibits tumors by inducing systemic tumor-specific T cell immunity. In particular, the active substance inhibits the C1q/CD93 signaling pathway, thereby inhibiting CD93 of pMCs, and then promotes the secretion of CCL21 by pMCs. CCL21 binds to the CCR7 receptor of DCs, leading to the accumulation of DCs and triggering systemic tumor-specific T cell immunity.
需要说明的是，所述活性物质除了作用于C1q/CD93信号传导途径，也可作用于下游的CCL21，例如所述活性物质可以为促进CCL21转运的载体，使CCL21更易达到DCs，可以是促进CCL21与CCR7结合的物质；所述活性物质也可作用于下游的DCs，如使DCs增加或使DCs上的CCR7受体增加。It should be noted that in addition to acting on the C1q/CD93 signaling pathway, the active substance can also act on downstream CCL21. For example, the active substance can be a carrier that promotes CCL21 transport, making it easier for CCL21 to reach DCs, or can promote CCL21 Substances that bind to CCR7; the active substances can also act on downstream DCs, such as increasing DCs or increasing CCR7 receptors on DCs.
所述抑制C1q/CD93信号传导途径的活性物质为CD93/C1q阻断剂，该阻断剂可以作用于CD93，也可以作用与C1q，也可以是作用与两者之间的相互关系；该阻断剂可以是抗体、多肽、siRNA、shRNA、miRNA、反义RNA或基因编辑***、融合蛋白、肽类似物、适体、avimer、anticalin、speigelmer或小分子化合物等中的一或多种，与CN202080075436.0类似形式的能阻断C1q/CD93信号传导途径的活性物质都属于本申请的阻断剂范围。 The active substance that inhibits the C1q/CD93 signaling pathway is a CD93/C1q blocker. The blocker can act on CD93, can also act on C1q, or can act on the relationship between the two; The blocking agent can be one or more of antibodies, polypeptides, siRNA, shRNA, miRNA, antisense RNA or gene editing systems, fusion proteins, peptide analogs, aptamers, avimers, anticalin, speigelmer or small molecule compounds, and CN202080075436.0 Similar forms of active substances that can block the C1q/CD93 signaling pathway belong to the scope of blockers in this application.
在一些实施方式中，所述阻断剂能与CD93的C型凝集素结构域结合，抑制CD93活性。In some embodiments, the blocking agent can bind to the C-type lectin domain of CD93 and inhibit CD93 activity.
在一些实施方式中，本申请发明所述miRNA为miR-5193或miR-5110，或者与miR-5193同源或与miR-5110同源的miR-5193类似物或miR-5110类似物。这里的同源是指现有来源于不同物种中与miR-5193或miR-5110相似且具有相同功能的miRNA，也指具有60％及以上(如70％、72％、80％、90％、99％、100％等)的相同序列且功能相同的人工合成或天然来源的miRNA。In some embodiments, the miRNA described in the present invention is miR-5193 or miR-5110, or a miR-5193 analog or a miR-5110 analog that is homologous to miR-5193 or homologous to miR-5110. Homology here refers to existing miRNAs from different species that are similar to miR-5193 or miR-5110 and have the same function, and also refer to 60% or more (such as 70%, 72%, 80%, 90%, 99%, 100%, etc.) of the same sequence and functionally identical synthetic or naturally derived miRNA.
在一些实施方式中，本申请抑制肿瘤的作用仅通过全身肿瘤特异性T细胞免疫进行，具体通过抑制C1q/CD93信号传导途径，进而抑制pMCs的CD93，接着促进pMCs分泌CCL21，CCL21与DCs的CCR7受体结合，导致DCs聚集并引发全身肿瘤特异性T细胞免疫。In some embodiments, the tumor suppressive effect of the present application is only performed through systemic tumor-specific T cell immunity, specifically by inhibiting the C1q/CD93 signaling pathway, thereby inhibiting CD93 of pMCs, and then promoting the secretion of CCL21 by pMCs, CCL21 and CCR7 of DCs Receptor binding leads to the accumulation of DCs and triggers systemic tumor-specific T cell immunity.
在一些实施方式中，本申请的全身肿瘤特异性T细胞免疫可以与CN202080075436.0的促进血管生成同时进行，即所述活性物质在抑制pMCs C1q/CD93信号传导途径的同时具有抑制IGFBP7/CD93信号传导途径的作用，这样可以发挥更好的抗肿瘤作用。In some embodiments, the systemic tumor-specific T cell immunity of the present application can be performed simultaneously with the promotion of angiogenesis of CN202080075436.0, that is, the active substance has the ability to inhibit IGFBP7/CD93 signaling while inhibiting the pMCs C1q/CD93 signaling pathway. conduction pathway, so that it can exert a better anti-tumor effect.
除了上述的肿瘤抑制作用外，本申请发现所述活性物质也能通过引起特异性的T细胞免疫来治疗肺部感染，抗肺部感染的作用方式与抗肿瘤类似，不再一一赘述。In addition to the above-mentioned tumor inhibitory effect, the present application found that the active substance can also treat pulmonary infection by inducing specific T cell immunity. The anti-pulmonary infection mode of action is similar to that of anti-tumor, and will not be described in detail one by one.
本申请的另一方面提供了所述活性物质在制备治疗肺部肿瘤或肺部感染试剂或药物方面的应用。特别地，所述活性物质在治疗肺部肿瘤的同时也能治疗其他部位的同种肿瘤，尤其适用于发生肺部肿瘤并有肿瘤转移的个体的治疗。Another aspect of the present application provides the use of the active substance in the preparation of reagents or medicines for treating lung tumors or lung infections. In particular, the active substance can treat lung tumors while also treating the same type of tumors in other locations, and is especially suitable for the treatment of individuals with lung tumors and tumor metastasis.
本申请中的同种肿瘤是指相同或相似类型肿瘤细胞引起的肿瘤，肿瘤的位置可以不同，例如在人患有肺部肿瘤后，该肺部肿瘤后面发生了转移，如转移到腹部，这时的腹部肿瘤和肺部肿瘤细胞是类似的，记为同种肿瘤。发生于动物或人体不同部位的同种肿瘤的肿瘤细胞作为抗原时具有相同或相似的性质，因而能产生相同的抗体。The same type of tumor in this application refers to tumors caused by the same or similar types of tumor cells. The location of the tumor can be different. For example, after a person suffers from a lung tumor, the lung tumor metastasizes behind it, such as to the abdomen. This When the abdominal tumor and lung tumor cells are similar, they are recorded as the same type of tumor. Tumor cells of the same type of tumor occurring in different parts of the animal or human body have the same or similar properties as antigens and can therefore produce the same antibodies.
在一些实施方式中，在使用所述试剂治疗肺部肿瘤或肺部感染时，通过胸腔注射的方式将所述试剂注射到患者胸腔中，或者通过静脉注射的方式并由靶向胸膜间皮细胞的靶向药物载体将所述物质输送到胸膜间皮细胞。In some embodiments, when using the agent to treat lung tumors or lung infections, the agent is injected into the patient's chest via intrapleural injection, or via intravenous injection and targeted to pleural mesothelial cells. A targeted drug carrier delivers the substance to pleural mesothelial cells.
当然，所示试剂或药物不限于液体注射制剂，也可以是胶囊、片剂、冻干粉、乳剂等其他类型。所述活性物质可以与赋形剂、稀释剂、缓释成分、防腐剂、助悬剂等其他辅助成分中的一种或多种共同制备成所述试剂或药物。Of course, the reagents or drugs shown are not limited to liquid injection preparations, but can also be other types such as capsules, tablets, freeze-dried powders, and emulsions. The active substance can be prepared into the reagent or medicine together with one or more of the excipients, diluents, sustained-release ingredients, preservatives, suspending agents and other auxiliary ingredients.
实施例子 Implementation example
本发明的实施例子仅仅是对如何实现本发明的技术方案的具体示例性的列举，并不能对本发明构成任何的限制。The implementation examples of the present invention are only specific examples of how to implement the technical solution of the present invention, and do not constitute any limitation on the present invention.
实施例1胸腔注射TEV通过促进DC聚集及肿瘤特异性T细胞免疫从而抑制肺肿瘤生长Example 1 Intrapleural injection of TEV inhibits lung tumor growth by promoting DC aggregation and tumor-specific T cell immunity.
本实施例研究发现胸腔注射TEVs剂量依赖性的抑制肺肿瘤生长，而腹腔注射或皮下注射均不会抑制肺肿瘤生长，静脉注射TEVs反而促进肺肿瘤生长。The study in this example found that intrathoracic injection of TEVs inhibited the growth of lung tumors in a dose-dependent manner, while neither intraperitoneal injection nor subcutaneous injection inhibited the growth of lung tumors. Intravenous injection of TEVs instead promoted the growth of lung tumors.
此外，胸腔注射TEVs抑制肺肿瘤时，促进DCs向肺肿瘤聚集，使CD4+和CD8+T细胞增加。胸腔注射TEVs不仅能抑制肺肿瘤也能抑制其他部位的同种肿瘤，而仅存在其他部位肿瘤无肺肿瘤时，胸腔注射TEVs并不能抑制其他部位肿瘤，证实了胸腔注射TEVs引起***的肿瘤特异性T细胞免疫。肺部肿瘤分为两种，自发性的肿瘤以及别的地方转移到肺部的肿瘤。In addition, when intrapleural injection of TEVs inhibits lung tumors, it promotes the accumulation of DCs to lung tumors and increases CD4 + and CD8 + T cells. Intrapleural injection of TEVs can not only inhibit lung tumors but also inhibit the same type of tumors in other parts. However, when there are only tumors in other parts but no lung tumors, thoracic injection of TEVs cannot inhibit tumors in other parts, confirming that pleural injection of TEVs causes systemic tumor specificity. T cell immunity. Lung tumors are divided into two types, spontaneous tumors and tumors that have metastasized to the lungs elsewhere.
为了研究TEVs对胸腔肺转移瘤的作用，我们将能表达荧光素酶的LLC细胞(LLC-Luci)静脉注经胸膜腔注射(尾静脉回输肿瘤细胞模拟经血液转移并定位至肺的肿瘤；摘除4T1皮下瘤促进4T1向肺转移、基因鼠中滴加Ad Cre病毒，模拟自发转移性肺肿瘤)过LLC-EVs(LLC细胞分泌的胞外囊泡)的小鼠，如图1，在第5天静脉注射1×106LLC-Luci肿瘤细胞构建肺转移瘤模型(转移至肺部形成肺肿瘤)，并在第0天、2天、4天、6天和8天胸腔注射不同量(2.5μg、5μg、10μg)的LLC-EVs，在第30天时取小鼠肺进行检测。检测结果如图2，胸腔注射LLC-EVs的小鼠肺部肿瘤体积明显小于控制组(胸腔注射PBS)，并且注射10μgLLC-EVs的肺肿瘤体积小于注射5μg LLC-EVs的，进一步小于注射2.5μg LLC-EVs的，说明胸腔注射LLC-EVs剂量依赖性地抑制LLC肺肿瘤生长。然而，在第5天静脉注射1×106LLC-Luci肿瘤细胞构建肺转移瘤模型，并在第0天、2天、4天、6天和8天经腹腔或皮下或静脉注射5μgLLC-EVs，第30天时测得肺部肿瘤情况如图3，可知腹腔注射或皮下注射LLC-EVs并不会影响LLC肺肿瘤，静脉注射LLC-EVs反而促进LLC肺肿瘤增长。同理，使用B16F10-Luci肿瘤细胞代替LLC-Luci，并用B16F10-EVs(B16F10细胞分泌的胞外囊泡)代替LLC-EVs进行上述试验，结果如图4，说明在胸腔注射B16F10-EVs也可以抑制肺转移B16F10-Luci肿瘤生长。除此以外，在第5天静脉注射LLC-Luci肿瘤细胞，并在第0天、2天、4天、6天和8天胸腔注射5μgB16F10-EVs，第30天观察的肺肿瘤情况如图5；同时，在5天静脉注射LLC-Luci肿瘤细胞，并在第0天、2天、4天、6天和8天胸腔注射5μg4T1-EVs(4T1乳腺癌细胞分泌的EVs)，第30天观察的肺肿瘤情况如图6，由图5-6的结果可知胸腔注射B16F10-EVs或4T1-EVs能抑制LLC-Luci引起的肺转移瘤，这说明这种TEVs抑制 肺肿瘤生长的现象并非依赖于肿瘤特异性抗原和MHC I类分子。In order to study the effect of TEVs on thoracic and pulmonary metastases, we injected luciferase-expressing LLC cells (LLC-Luci) intravenously through the pleural cavity (tail vein infusion of tumor cells simulates tumors that metastasize through the blood and localize to the lungs; The 4T1 subcutaneous tumors were removed to promote 4T1 metastasis to the lungs, and Ad Cre virus was added dropwise to the genetic mice to simulate spontaneous metastatic lung tumors) mice that passed LLC-EVs (extracellular vesicles secreted by LLC cells), as shown in Figure 1, in Chapter 1 1×10 6 LLC-Luci tumor cells were intravenously injected on day 5 to construct a lung metastasis model (transfer to the lungs to form lung tumors), and different amounts of ( 2.5 μg, 5 μg, and 10 μg) of LLC-EVs, and mouse lungs were taken for detection on the 30th day. The test results are shown in Figure 2. The lung tumor volume of mice injected with LLC-EVs into the chest was significantly smaller than that of the control group (PBS injected into the chest), and the lung tumor volume injected with 10 μg LLC-EVs was smaller than that injected with 5 μg LLC-EVs, and further smaller than that injected with 2.5 μg. LLC-EVs, indicating that intrapleural injection of LLC-EVs dose-dependently inhibits the growth of LLC lung tumors. However, 1 × 10 6 LLC-Luci tumor cells were injected intravenously on day 5 to establish a lung metastasis model, and 5 μg LLC-EVs were injected intraperitoneally, subcutaneously, or intravenously on days 0, 2, 4, 6, and 8. , The lung tumor status measured on the 30th day is shown in Figure 3. It can be seen that intraperitoneal or subcutaneous injection of LLC-EVs does not affect LLC lung tumors, but intravenous injection of LLC-EVs promotes the growth of LLC lung tumors. In the same way, B16F10-Luci tumor cells were used instead of LLC-Luci, and B16F10-EVs (extracellular vesicles secreted by B16F10 cells) were used instead of LLC-EVs to conduct the above experiments. The results are shown in Figure 4, indicating that B16F10-EVs can also be injected into the chest cavity. Inhibits lung metastatic B16F10-Luci tumor growth. In addition, LLC-Luci tumor cells were injected intravenously on day 5, and 5 μg B16F10-EVs were injected into the chest on days 0, 2, 4, 6, and 8. The lung tumor status observed on day 30 is shown in Figure 5 ; At the same time, LLC-Luci tumor cells were injected intravenously on day 5, and 5 μg 4T1-EVs (EVs secreted by 4T1 breast cancer cells) were injected into the chest on days 0, 2, 4, 6 and 8, and observed on day 30 The lung tumor situation is shown in Figure 6. From the results in Figure 5-6, it can be seen that intrapleural injection of B16F10-EVs or 4T1-EVs can inhibit lung metastases caused by LLC-Luci, which shows that this TEVs inhibit The phenomenon of lung tumor growth is not dependent on tumor-specific antigens and MHC class I molecules.
为了检测胸腔注射TEVs对自发性肺转移瘤的作用，我们在小鼠皮下植入易转移到肺部的4T1细胞，具体为第0天向小鼠皮下注射4T1细胞，第16天手术摘除肿瘤，再于26天、28天、30天、32天、34天分别于胸腔注射5μg 4T1-EVs，在第37天取小鼠肺进行检测，结果如图7，可知注射4T1-EVs组肺肿瘤面积明显小于控制组(PBS组，用PBS缓冲液代替4T1-EVs),说明在胸腔注射4T1-EVs也能抑制4T1自发性肺转移瘤的生长。In order to test the effect of intrapleural injection of TEVs on spontaneous lung metastases, we subcutaneously implanted 4T1 cells that can easily metastasize to the lungs into mice. Specifically, 4T1 cells were subcutaneously injected into mice on day 0, and the tumors were surgically removed on day 16. Then, 5 μg 4T1-EVs was injected into the chest cavity on days 26, 28, 30, 32, and 34 respectively. On the 37th day, mouse lungs were taken for detection. The results are shown in Figure 7, which shows the lung tumor area of the 4T1-EVs injected group. It was significantly smaller than the control group (PBS group, using PBS buffer instead of 4T1-EVs), indicating that pleural injection of 4T1-EVs can also inhibit the growth of 4T1 spontaneous lung metastases.
为了检测胸腔注射TEVs是否能治疗肺肿瘤，在肿瘤移植后胸腔注射LLC-EVs，观察肺肿瘤生长情况。具体实验方案如图8，在第0天静脉注射肿瘤细胞，在第14天、16天、18天、20天和22天胸腔注射TEVs，在第25天的时候检测肿瘤生长情况。图9肿瘤细胞为LLC-Luci，TEVs为5μg LLC-EVs时，按图8方案测得的肺肿瘤生长情况，可知胸腔注射TEVs显著抑制肺肿瘤生长，能用于肺肿瘤的治疗。此外，第0天和第1天在p53fl/flLSL-KrasG12D小鼠鼻内滴注表达的Cre重组酶的腺病毒构建自发性肺肿瘤模型，然后在第30天、32天、34天和38天在在该小鼠胸腔注射5μg LLC-EVs，小鼠肺肿瘤生长情况如图10，试验组肺肿瘤面积显著小于控制组，说明自发性肺肿瘤在p53fl/flLSL-KrasG12D小鼠上时，胸腔注射LLC-EVs也显示肿瘤抑制作用。因此，上述这些结果说明胸腔注射TEVs能有效持续抑制肺肿瘤生长，能应用于肺肿瘤的治疗。In order to test whether intrapleural injection of TEVs can treat lung tumors, LLC-EVs were injected into the intrapleural cavity after tumor transplantation and the growth of lung tumors was observed. The specific experimental plan is shown in Figure 8. Tumor cells were injected intravenously on day 0, TEVs were injected into the chest on days 14, 16, 18, 20 and 22, and tumor growth was detected on day 25. Figure 9: When the tumor cells are LLC-Luci and TEVs are 5 μg LLC-EVs, the growth of lung tumors is measured according to the protocol in Figure 8. It can be seen that intrapleural injection of TEVs significantly inhibits the growth of lung tumors and can be used for the treatment of lung tumors. In addition, adenovirus expressing Cre recombinase was intranasally instilled in p53fl/flLSL-KrasG12D mice on days 0 and 1 to construct a spontaneous lung tumor model, and then on days 30, 32, 34, and 38 When 5 μg LLC-EVs were injected into the chest of the mice, the growth of lung tumors in the mice was shown in Figure 10. The lung tumor area in the test group was significantly smaller than that in the control group, indicating that spontaneous lung tumors occurred in p53fl/flLSL-KrasG12D mice. LLC-EVs also show tumor suppressive effects. Therefore, the above results indicate that intrapleural injection of TEVs can effectively and continuously inhibit the growth of lung tumors and can be applied in the treatment of lung tumors.
为了研究胸腔注射TEVs是怎样抑制肺肿瘤生长的，我们检测了LLC肺肿瘤的肿瘤浸润白细胞(tumor-infiltrating leukocytes，TILs)。进一步地，对上述静脉注射LLC-Luci肿瘤细胞并胸腔注射5μg LLC-EVs治疗的小鼠的TILs进行检测，实验结果如图11-12，发现胸腔注射LLC-EVs使CD4+T细胞、CD8+T细胞和DCs的比例显著升高，而巨噬细胞、NK细胞、中性粒细胞以及B细胞的比例没有改变。因此，我们猜测胸腔注射TEVs能促进DC向肺肿瘤聚集，随后促进肿瘤抗原呈递给CD4+和CD8+T细胞，从而使CD4+T细胞、CD8+T细胞和DCs数量增加。To investigate how intrapleural injection of TEVs inhibits lung tumor growth, we examined tumor-infiltrating leukocytes (TILs) in LLC lung tumors. Furthermore, the TILs of mice treated with intravenous injection of LLC-Luci tumor cells and intrapleural injection of 5 μg LLC-EVs were detected. The experimental results are shown in Figure 11-12. It was found that intrapleural injection of LLC-EVs increased CD4 + T cells, CD8 + The proportions of T cells and DCs increased significantly, while the proportions of macrophages, NK cells, neutrophils, and B cells did not change. Therefore, we speculate that intrapleural injection of TEVs can promote the recruitment of DCs to lung tumors and subsequently promote the presentation of tumor antigens to CD4 + and CD8 + T cells, thereby increasing the number of CD4 + T cells, CD8 + T cells and DCs.
我们使用CD11c白喉毒素受体(CD11c-diphtheria toxin receptor，DTR)小鼠(腹腔注射白喉毒素时可以选择性删除全身DCs)进行LLC-EVs的抗肿瘤试验，用LLC-Luci细胞和5μg LLC-EVs按图8的方案处理CD11c白喉毒素受体小鼠，同时用白喉毒素处理，第25天时肺部肿瘤情况如图13，TILs检测结果如图14，发现剔除DCs后胸腔注射LLC-EVs抑制肺肿瘤生长的现象被显著逆转，胸腔注射LLC-EVs后小鼠TILs中的CD4+和CD8+T细胞比例和数目也没有增加。此外，用LLC-Luci细胞和5μg LLC-EVs按图8的方案处理小鼠，同时使用抗CD4抗体或抗CD8抗体，第25天时肺部肿瘤情况如图15，TILs检测结果如图 16，使用CD4+或CD8+T细胞阻断抗体阻断CD4+或CD8+T细胞后，胸腔注射LLC-EVs也不再抑制肿瘤生长,，然而，不管是阻断CD4+T细胞还是阻断CD8+T细胞，胸腔注射LLC-EVs依旧可以使TILs中DCs增加。上述结果说明DCs数目的变化导致了CD4+T细胞和CD8+T细胞数目变化，即DCs的变化是在CD4+T细胞和CD8+T细胞之前的。We used CD11c-diphtheria toxin receptor (DTR) mice (whole body DCs can be selectively deleted when intraperitoneally injected diphtheria toxin) to conduct an anti-tumor test of LLC-EVs, using LLC-Luci cells and 5 μg LLC-EVs. CD11c diphtheria toxin receptor mice were treated according to the protocol in Figure 8, and treated with diphtheria toxin at the same time. The lung tumor situation on the 25th day is shown in Figure 13, and the TILs detection results are shown in Figure 14. It was found that DCs were removed and intrapleural injection of LLC-EVs inhibited lung tumors. The growth phenomenon was significantly reversed, and the proportion and number of CD4 + and CD8 + T cells in mouse TILs did not increase after intrapleural injection of LLC-EVs. In addition, mice were treated with LLC-Luci cells and 5 μg LLC-EVs according to the protocol in Figure 8, and anti-CD4 antibodies or anti-CD8 antibodies were used at the same time. The lung tumor situation on day 25 was as shown in Figure 15, and the TILs detection results were as shown in Figure 15. 16. After blocking CD4 + or CD8 + T cells using CD4 + or CD8 + T cell blocking antibodies, intrapleural injection of LLC-EVs no longer inhibits tumor growth. However, regardless of blocking CD4 + T cells or For CD8 + T cells, intrapleural injection of LLC-EVs can still increase DCs in TILs. The above results indicate that changes in the number of DCs lead to changes in the numbers of CD4 + T cells and CD8 + T cells, that is, the changes in DCs precede those of CD4 + T cells and CD8 + T cells.
如图17，在LLC肺肿瘤的小鼠两边腹侧分别注射LLC和B16F10细胞构建多处肿瘤小鼠模型，再通过胸腔注射LLC-EVs，两边腹侧的肿瘤体积如图18所示，腹部LLC肿瘤体积显著小于对照组(PBS组)，可知腹部B16F10肿瘤体积与对照组无显著差异，说明腹部LLC肿瘤均被显著抑制，另一腹侧的B16F10肿瘤未被明显抑制。此外，第0天在正常小鼠的腹侧皮下注射LLC细胞，并在第2天、4天、6天、8天和10天胸腔注射5μg LLC-EVs，在第18天用流式细胞仪检测肺部DCs和T细胞试验结果如图19，皮下肿瘤大小变化如图20，与对照相比DCs显著升高，CD4+T细胞、CD8+T细胞以及肿瘤体积无显著变化，这些结果说明在不存在肺肿瘤的时候，即使观察到肺部DCs增加，胸腔注射LLC-EVs也不能抑制LLC皮下肿瘤生长，同时，胸腔注射LLC-EVs也没有增加CD4+T细胞和CD8+T细胞在肺部的比例。综上，上述结果说明胸腔注射TEVs促进DCs聚集到肺部肿瘤并随后引起***的肿瘤特异性T细胞免疫。As shown in Figure 17, LLC and B16F10 cells were injected into the ventral sides of mice with LLC lung tumors to construct multiple tumor mouse models, and then LLC-EVs were injected through the chest cavity. The tumor volumes on both ventral sides are shown in Figure 18. Abdominal LLC The tumor volume was significantly smaller than the control group (PBS group). It can be seen that the abdominal B16F10 tumor volume was not significantly different from the control group, indicating that the abdominal LLC tumors were significantly inhibited, while the other ventral B16F10 tumors were not significantly inhibited. In addition, LLC cells were injected subcutaneously into the ventral side of normal mice on day 0, and 5 μg LLC-EVs were intrapleurally injected on days 2, 4, 6, 8, and 10, and flow cytometry was performed on day 18. The results of the test for detecting DCs and T cells in the lungs are shown in Figure 19. The changes in subcutaneous tumor size are shown in Figure 20. Compared with the control, DCs increased significantly, but there were no significant changes in CD4 + T cells, CD8 + T cells, and tumor volume. These results show that in In the absence of lung tumors, intrapleural injection of LLC-EVs did not inhibit LLC subcutaneous tumor growth even if an increase in lung DCs was observed. At the same time, intrapleural injection of LLC-EVs did not increase CD4 + T cells and CD8 + T cells in the lungs. proportion. Taken together, the above results indicate that intrapleural injection of TEVs promotes the recruitment of DCs to lung tumors and subsequently induces systemic tumor-specific T cell immunity.
实施例2 TEV诱导pMCs分泌CCL21a促进DCs向肺部迁移Example 2 TEV induces pMCs to secrete CCL21a and promotes DCs migration to the lungs
本实施例研究发现TEVs能作用于pMCs，并使pMCs分泌更多的CCL21，CCL21作用于DCs的CCR7，从而促进DCs聚集和后续的肿瘤特异性T细胞免疫趋化和后续的肿瘤特异性T细胞免疫，从而发挥肿瘤抑制作用。The study in this example found that TEVs can act on pMCs and cause pMCs to secrete more CCL21. CCL21 acts on CCR7 of DCs, thereby promoting the aggregation of DCs and subsequent tumor-specific T cell immunochemotaxis and subsequent tumor-specific T cells. Immunity, thereby exerting a tumor suppressive effect.
为了研究胸腔注射TEVs怎样促进DCs的迁移，我们分别通过静脉注射和胸腔注射100μg VivoTrack 680标记的LLC-EVs，观察LLC-EVs在体内分布情况，如图21，两种方式下LLC-EVs在体内的分布情况无明显不同。另外，我们分别通过静脉注射和胸腔注射100μg PKH26标记的LLC-EVs，观察LLC-EVs在体内分布情况，如图22，发现静脉注射的LLC-EVs主要分布在肺部内部，同时不少胸腔注射的LLC-EVs位于肺部边缘。胸膜腔由单层pMCs的胸膜环绕形成。因此，pMCs有可能摄取绝大多数通过胸腔注射的LLC-EVs。实际上，用2.5μg ml-1 CFSE标记的LLC-EVs处理原代胸膜间皮细胞(primary pleural mesothelial cells，p-pMCs)和40L细胞24h后用荧光显微法检测p-pMCs和40L细胞摄取EVs的情况如图23所示，说明p-pMCs和40L胸膜间皮瘤细胞在体外试验中能有效的摄取LLC-EVs。此外，在小鼠胸腔注射20μg CFSE标记的LLC-EVs，24h后用立体显微镜检测进入胸膜的EVs情况，如图24 LLC-EVs也在胸膜聚集。 In order to study how intrapleural injection of TEVs promotes the migration of DCs, we observed the distribution of LLC-EVs in the body through intravenous injection and intrapleural injection of 100 μg VivoTrack 680-labeled LLC-EVs, as shown in Figure 21. LLC-EVs in vivo in the two ways There is no significant difference in distribution. In addition, we injected 100 μg of PKH26-labeled LLC-EVs through intravenous injection and intrathoracic injection respectively to observe the distribution of LLC-EVs in the body, as shown in Figure 22. It was found that the intravenously injected LLC-EVs were mainly distributed inside the lungs, and many intrapleural injections were also LLC-EVs are located at the edge of the lungs. The pleural cavity is formed by a single layer of pMCs surrounded by the pleura. Therefore, pMCs have the potential to take up the vast majority of LLC-EVs injected via intrapleural injection. In fact, primary pleural mesothelial cells (p-pMCs) and 40L cells were treated with 2.5 μg ml-1 CFSE-labeled LLC-EVs for 24 h and then the uptake of p-pMCs and 40L cells was detected by fluorescence microscopy. The situation of EVs is shown in Figure 23, indicating that p-pMCs and 40L pleural mesothelioma cells can effectively uptake LLC-EVs in in vitro experiments. In addition, 20 μg of CFSE-labeled LLC-EVs were injected into the thoracic cavity of mice, and the EVs entering the pleura were detected using a stereomicroscope 24 hours later. As shown in Figure 24, LLC-EVs also accumulated in the pleura.
按图8的试验方案，使用LLC-Luci细胞和CFSE标记的LLC-EVs处理小鼠，并在每次胸腔注射EVs前2h胸腔注射0.25mg kg-1细胞松弛素D(cytochalasin D，Cyto-D)，用立体显微镜检测进入胸膜的EVs情况如25所示，可知与对照组相比(用DMSO代替Cyto-D)，试验组小鼠LLC-EVs在胸膜的分布无明显差异。用流式细胞仪检测TILs中DCs，结果如图26，与控制组相比(用PBS代替CFSE标记的LLC-EVs)试验组DCs无显著差异。用IVIS测定肺肿瘤大小情况，结果如图27，与控制组相比(用PBS代替CFSE标记的LLC-EVs)试验组肿瘤大小无明显差异。这些结果说明在每次LLC-EVs注射前注射Cyto-D，抑制了LLC-EVs向胸膜的聚集，并使肺肿瘤处的DCs不再增加、且肿瘤抑制作用消失。此外，按图8的试验方案，使用LLC-Luci细胞和CFSE标记的LLC-EVs处理小鼠，并在每次胸腔注射EVs后2h胸腔注射0.25mg kg-1细胞松弛素D(cytochalasin D，Cyto-D)，用立体显微镜检测进入胸膜的EVs情况如28所示，用流式细胞仪检测TILs中DCs的结果如图29所示，用IVIS测定肺肿瘤大小的结果如图30所示，可见LLC-EVs在胸膜聚集、TILs中DCs显著增加以及肺肿瘤明显被抑制，这说明在每次LLC-EVs注射后再注射Cyto-D则仍存在DCs招募增多、肿瘤生长抑制的现象。上述结果说明pMCs摄取TEVs是胸腔注射TEVs介导的肺肿瘤抑制所必需的。According to the experimental protocol in Figure 8, mice were treated with LLC-Luci cells and CFSE-labeled LLC-EVs, and 0.25 mg kg-1 cytochalasin D (Cyto-D) was injected into the chest 2 hours before each intrapleural injection of EVs. ), using a stereomicroscope to detect EVs entering the pleura is shown in Figure 25. It can be seen that compared with the control group (DMSO was used instead of Cyto-D), there was no significant difference in the distribution of LLC-EVs in the pleura of mice in the test group. Flow cytometry was used to detect DCs in TILs. The results are shown in Figure 26. Compared with the control group (PBS was used instead of CFSE-labeled LLC-EVs), there was no significant difference in DCs in the test group. Lung tumor size was measured using IVIS. The results are shown in Figure 27. Compared with the control group (PBS was used instead of CFSE-labeled LLC-EVs), there was no significant difference in tumor size in the test group. These results indicate that injection of Cyto-D before each injection of LLC-EVs inhibits the accumulation of LLC-EVs into the pleura, prevents the increase of DCs in lung tumors, and eliminates the tumor inhibitory effect. In addition, according to the experimental protocol in Figure 8, mice were treated with LLC-Luci cells and CFSE-labeled LLC-EVs, and 0.25 mg kg-1 cytochalasin D (cytochalasin D, Cyto -D), the detection of EVs entering the pleura using a stereomicroscope is shown in Figure 28, the results of detecting DCs in TILs using flow cytometry are shown in Figure 29, and the results of measuring lung tumor size using IVIS are shown in Figure 30. It can be seen that LLC-EVs accumulated in the pleura, DCs were significantly increased in TILs, and lung tumors were significantly inhibited. This shows that after each LLC-EVs injection and then injection of Cyto-D, there is still an increase in DCs recruitment and tumor growth inhibition. The above results indicate that the uptake of TEVs by pMCs is necessary for lung tumor inhibition mediated by intrapleural injection of TEVs.
进一步，我们研究了TEVs对pMCs的作用。现有研究表明TEVs引起腹膜MCs的凋亡。然而，我们用一定浓度的LLC-EVs刺激40L细胞24h，通过CCK8试验测定细胞活力，实验结果如图31所示，发现LLC-EVs在5μg ml-1的高浓度下仅能略微增加40L细胞的活性。接着，我们检测了TEVs是否通过MCs影响骨髓源性DCs(bone marrow-derived DCs，BMDCs)的趋化作用，在无mRNA沉默下，用2.5μg ml-1LLC-EV刺激p-pMCs细胞或40L细胞，24h后通过Transwell趋化试验检测各细胞上清对DCs趋化性的影响，结果见图32，与控制组(用PBS代替LLC-EV)相比，试验组的细胞数量均显著增加，这说明LLC-EVs刺激的p-pMCs或40L细胞的上清能够增强BMDCs的趋化能力。Further, we studied the effect of TEVs on pMCs. Existing studies have shown that TEVs cause apoptosis of peritoneal MCs. However, we stimulated 40L cells with a certain concentration of LLC-EVs for 24 hours and measured cell viability through CCK8 assay. The experimental results are shown in Figure 31. It was found that LLC-EVs could only slightly increase the viability of 40L cells at a high concentration of 5 μg ml -1 . active. Next, we examined whether TEVs affects the chemotaxis of bone marrow-derived DCs (BMDCs) through MCs. In the absence of mRNA silencing, p-pMCs cells or 40L were stimulated with 2.5 μg ml -1 LLC-EV. cells, and the effect of each cell supernatant on the chemotaxis of DCs was tested by Transwell chemotaxis assay 24 hours later. The results are shown in Figure 32. Compared with the control group (using PBS instead of LLC-EV), the number of cells in the test group increased significantly. This shows that p-pMCs or the supernatant of 40L cells stimulated by LLC-EVs can enhance the chemotactic ability of BMDCs.
已有报道DCs表达的受体有CCR1、CCR2、CCR5、CCR6、CCR7和CXCR4，因此，我们分别使用特定siRNA转染BMDCs细胞使目标受体沉默(同时使用与目的基因无同源性的siRNA作为阴性对照，记为NC siRNA，本专利所涉及NC siRNA序列均如SEQ ID NO:47和SEQ ID NO:48所示)。It has been reported that the receptors expressed by DCs include CCR1, CCR2, CCR5, CCR6, CCR7 and CXCR4. Therefore, we used specific siRNA to transfect BMDCs cells to silence the target receptors (while using siRNA that has no homology with the target gene as The negative control is recorded as NC siRNA. The NC siRNA sequences involved in this patent are as shown in SEQ ID NO:47 and SEQ ID NO:48).
CCR1的siRNA序列如SEQ ID NO:23和SEQ ID NO:24所示；CCR2的siRNA序列如SEQ ID NO:25和SEQ ID NO:26所示；CCR5的siRNA序列如SEQ ID NO:27和SEQ ID NO:28所示；CCR6的siRNA序列如SEQ ID NO:29和SEQ ID NO:30所示；CCR7的siRNA 序列如SEQ ID NO:31和SEQ ID NO:32所示；CXCR4的siRNA序列如SEQ ID NO:33和SEQ ID NO:34所示。The siRNA sequence of CCR1 is shown in SEQ ID NO:23 and SEQ ID NO:24; the siRNA sequence of CCR2 is shown in SEQ ID NO:25 and SEQ ID NO:26; the siRNA sequence of CCR5 is shown in SEQ ID NO:27 and SEQ ID NO:28; the siRNA sequence of CCR6 is shown in SEQ ID NO:29 and SEQ ID NO:30; the siRNA of CCR7 The sequence is shown in SEQ ID NO:31 and SEQ ID NO:32; the siRNA sequence of CXCR4 is shown in SEQ ID NO:33 and SEQ ID NO:34.
通过蛋白免疫印迹法验证沉默效果，如图33，沉默效果良好。在无mRNA沉默(NC siRNA)或有特定siRNA沉默各受体下，用2.5μg ml-1LLC-EV刺激40L细胞，24后通过Transwell趋化试验检测细胞上清对DCs趋化性的影响，结果如图34，与控制组相比，沉默CCR1、CCR2、CCR5、CCR6或CXCR4组，细胞数量均显著增加，而沉默CCR7，细胞数量无明显变化，这说明沉默CCR7而不是其他受体完全消除了TEV诱导40L细胞上清促进BDMCs趋化的作用。The silencing effect was verified by Western blotting, as shown in Figure 33. The silencing effect was good. In the absence of mRNA silencing (NC siRNA) or specific siRNA silencing of each receptor, 40L cells were stimulated with 2.5 μg ml -1 LLC-EV. After 24 days, the effect of the cell supernatant on the chemotaxis of DCs was detected by Transwell chemotaxis assay. The results are shown in Figure 34. Compared with the control group, the number of cells in the silencing CCR1, CCR2, CCR5, CCR6 or CXCR4 group increased significantly, while the number of cells in the silencing CCR7 group did not change significantly. This shows that silencing CCR7 rather than other receptors is completely eliminated. The effect of TEV-induced 40L cell supernatant on promoting the chemotaxis of BDMCs was investigated.
特别地，在特定siRNA沉默CCR7受体下，用2.5μg ml-1LLC-EV刺激p-pMCs，24后通过Transwell趋化试验检测细胞上清对DCs趋化性的影响，试验结果如图35；同时，在无mRNA沉默下，用2.5μg ml-1LLC-EV刺激p-pMCs，24后通过Transwell趋化试验检测细胞上清对DCs(WT)或CCR7-/-DCs趋化性的影响,试验结果如图36。由图35和36的结果可知TEVs不再促进p-pMCs的上清趋化CCR7沉默的或CCR7缺失的BMDCs。CCL19和CCL21a是CCR7的已知配体。用2.5μg ml-1LLC-EV刺激p-pMCs或40L细胞，24h后用ELISA测定细胞上清液中的CCL19和CCL21a水平，试验结果见图37；用2.5μg ml-1LLC-EV刺激p-pMCs或40L细胞，24h后用用real-time PCR测定细胞中的CCL19和CCL21a mRNA水平(CCL21 PCR引物序列见SEQ ID NO:3和SEQ ID NO:4)，试验结果见图38。可知，LLC-EVs显著增加了p-pMCs和40L细胞中的CCL19和CCL21a的蛋白和mRNA水平。Specifically, p-pMCs were stimulated with 2.5 μg ml -1 LLC-EV under specific siRNA silencing of the CCR7 receptor, and the effect of the cell supernatant on the chemotaxis of DCs was detected by Transwell chemotaxis assay after 24 days. The test results are shown in Figure 35 ; At the same time, without mRNA silencing, p-pMCs were stimulated with 2.5 μg ml -1 LLC-EV, and the effect of the cell supernatant on the chemotaxis of DCs (WT) or CCR7 -/- DCs was detected by Transwell chemotaxis assay 24 days later. ,The test results are shown in Figure 36. It can be seen from the results of Figures 35 and 36 that TEVs no longer promotes the chemotaxis of the supernatant of p-pMCs to CCR7-silenced or CCR7-deficient BMDCs. CCL19 and CCL21a are known ligands of CCR7. p-pMCs or 40L cells were stimulated with 2.5 μg ml -1 LLC-EV, and the levels of CCL19 and CCL21a in the cell supernatant were measured by ELISA 24 hours later. The test results are shown in Figure 37; p-pMCs or 40L cells were stimulated with 2.5 μg ml -1 LLC-EV. -pMCs or 40L cells, 24 hours later, real-time PCR was used to measure the CCL19 and CCL21a mRNA levels in the cells (see SEQ ID NO: 3 and SEQ ID NO: 4 for the CCL21 PCR primer sequence). The test results are shown in Figure 38. It can be seen that LLC-EVs significantly increased the protein and mRNA levels of CCL19 and CCL21a in p-pMCs and 40L cells.
然而，在有特定siRNA沉默CCL19或CCL21a下(CCL19的siRNA序列如SEQ ID NO:35和SEQ ID NO:36所示；CCL21a的siRNA序列如SEQ ID NO:37和SEQ ID NO:38所示)，用2.5μg ml-1 LLC-EV刺激40L细胞，24h后通过Transwell趋化试验检测细胞上清对DCs趋化性的影响，沉默效果见图40，说明CCL19或CCL21a被沉默，Transwell趋化试验结果见图39，由图中结果可知敲除CCL21a后LLC-EVs刺激的40L细胞上清不再促进BDMCs的趋化作用，敲除CCL19并没有该影响(39-40)。因此，TEVs通过体外pMCs引起CCL21a分泌来促进BMDCs的趋化作用。However, under specific siRNA silencing CCL19 or CCL21a (the siRNA sequence of CCL19 is shown in SEQ ID NO:35 and SEQ ID NO:36; the siRNA sequence of CCL21a is shown in SEQ ID NO:37 and SEQ ID NO:38) , 40L cells were stimulated with 2.5 μg ml-1 LLC-EV, and the effect of the cell supernatant on the chemotaxis of DCs was detected by Transwell chemotaxis assay 24 hours later. The silencing effect is shown in Figure 40, indicating that CCL19 or CCL21a is silenced. Transwell chemotaxis assay The results are shown in Figure 39. From the results, it can be seen that the supernatant of 40L cells stimulated by LLC-EVs after knocking out CCL21a no longer promotes the chemotaxis of BDMCs, and knocking out CCL19 has no such effect (39-40). Therefore, TEVs promotes the chemotaxis of BMDCs by causing CCL21a secretion from pMCs in vitro.
随后，我们进行了胸腔注射TEVs是否诱使pMCs分泌CCL21a的体内验证试验。小鼠胸腔注射5μg ml-1 LLC-EVs后24h，测定胸膜Ccl21a mRNA和蛋白水平，如图41-42，胸腔注射LLC-EVs使胸膜中CCL21a的mRNA和蛋白水平显著升高。进一步，我们在小鼠胸腔注射10μg胆固醇偶联Ccl21a siRNAs 24h后，用real-time PCR检测小鼠胸膜Ccl21a mRNA水平，如图43，证实胸膜内Ccl21a mRNA水平下降。按图8方案使用LLC-Luci细胞和 LLC-EVs处理小鼠，并在每次注射EVs前24h在胸腔注射10μg胆固醇偶联Ccl21a siRNAs，流式细胞仪检测TILs中的DCs并IVIS测定肺肿瘤大小，如图44-45，与控制组相比，试验组DCs水平和肺肿瘤大小均无显著差异，这说明一旦沉默了胸膜中的CCL21a，胸腔注射LLC-EVs不再使肺肿瘤中的DC数目增加或不再有肿瘤抑制作用。然后，我们研究确认了CCR7在胸腔注射LLC-EVs后引起的DC聚集上的作用。首先，按图8方案使用LLC-Luci细胞和LLC-EVs处理小鼠，用流式细胞仪检测TILs中的CCR7+DC，结果如图46，该结果证实胸腔注射LLC-EVs增加了肺肿瘤中CCR7+DCs的比例。接着，我们按图8方案使用LLC-Luci细胞和LLC-EVs处理Ccr7-/-小鼠，用流式细胞仪检测TILs中的DC并用IVIS测定肺肿瘤大小，如图47-48，发现胸腔注射LLC-EVs不会增加Ccr7-/-小鼠肺肿瘤中DCs比例或者抑制其肺肿瘤。总的来说，这些结果表明胸腔注射TEVs使pMCs分泌的CCL21a增加，进而促进CCR7+DCs向肺肿瘤聚集。Subsequently, we conducted an in vivo verification test to determine whether intrapleural injection of TEVs induces pMCs to secrete CCL21a. The pleural Ccl21a mRNA and protein levels were measured 24 hours after mice were injected with 5 μg ml-1 LLC-EVs into the chest cavity, as shown in Figure 41-42. The intrapleural injection of LLC-EVs significantly increased the mRNA and protein levels of CCL21a in the pleura. Furthermore, 24 hours after injecting 10 μg of cholesterol-conjugated Ccl21a siRNAs into the thoracic cavity of mice, we used real-time PCR to detect the pleural Ccl21a mRNA levels of mice, as shown in Figure 43, which confirmed that the intrapleural Ccl21a mRNA levels decreased. Use LLC-Luci cells and Mice were treated with LLC-EVs, and 10 μg cholesterol-coupled Ccl21a siRNAs were injected into the chest 24 hours before each injection of EVs. DCs in TILs were detected by flow cytometry and lung tumor size was determined by IVIS, as shown in Figure 44-45, compared with the control group. In comparison, there was no significant difference in DCs levels and lung tumor size in the experimental group, indicating that once CCL21a in the pleura was silenced, intrapleural injection of LLC-EVs no longer increased the number of DCs in lung tumors or had a tumor suppressive effect. Then, we confirmed the role of CCR7 in DC accumulation after intrapleural injection of LLC-EVs. First, mice were treated with LLC-Luci cells and LLC-EVs according to the protocol in Figure 8, and flow cytometry was used to detect CCR7 + DC in TILs. The results are shown in Figure 46. This result confirms that intrapleural injection of LLC-EVs increases the number of lung tumors. Ratio of CCR7 + DCs. Next, we used LLC-Luci cells and LLC-EVs to treat Ccr7 -/- mice according to the protocol in Figure 8, used flow cytometry to detect DCs in TILs and used IVIS to measure lung tumor size. As shown in Figure 47-48, it was found that intrapleural injection LLC-EVs did not increase the proportion of DCs in the lung tumors of Ccr7 -/- mice or inhibit their lung tumors. Collectively, these results indicate that intrapleural injection of TEVs increases CCL21a secretion from pMCs, thereby promoting the recruitment of CCR7 + DCs to lung tumors.
实施例3 TEVs使pMCs中CD93水平下降进而促进CCL21a分泌Example 3 TEVs reduce CD93 levels in pMCs and promote CCL21a secretion
本实施例研究发现pMCs中CD93特异性高表达，TEVs通过抑制pMCs中CD93的表达来促进pMCS分泌CCL21。The study in this example found that CD93 is specifically highly expressed in pMCs, and TEVs promotes the secretion of CCL21 from pMCs by inhibiting the expression of CD93 in pMCs.
为了研究TEV怎样促进pMCs分泌CCL21a，我们对LLC-EVs处理和未处理的40L细胞进行了RNA-Seq分析，结果在两组间发现109个差异表达基因(differentially expressed genes，DEGs)(图49)。接着，我们分析了其中|Log2fold change(FC)|≥2的31个DEGs(图50)与Ccl21a的关联并发现Gm13054(上调)和Cd93(下调)与Ccl21a高度相关(相关系数≥0.90)，此外，Cd93的Log2FC值大于Gm13054的Log2FC值(图51)。Cd93多表达于血小板、内皮细胞(endothelial cells，ECs)、造血祖细胞(hematopoietic progenitors)和肺上皮细胞(alveolar epithelial cells)。除了血小板及已知的肿瘤ECs中Cd93 mRNA水平较高，我们用real-time PCR检测各种细胞中Cd93(Cd93PCR引物序列见SEQ ID NO:5和SEQ ID NO:6)和Ccl21a mRNA水平，发现p-pMCs的Cd93 mRNA表达水平显著高于正常ECs、骨髓细胞(bone marrow cells)、MLE-12鼠肺上皮细胞(图52)。荷瘤小鼠胸膜Cd93 mRNA表达水平较正常小鼠上调而Ccl21a mRNA表达没有上调(图53)。使用2.5μg ml-1LLC-EVs刺激CD93沉默的40L细胞(使用siRNA沉默CD93，CD93 siRNA的序列如SEQ ID NO:39和SEQ ID NO:40所示；沉默效果见图54)24h后，使用ELISA测定细胞上清液中CCL21水平，如图55，CCL21a蛋白水平显著增加，且LLC-EVs不再促进该细胞中CCL21a表达,。因此，这些结果表明CD93负向调节CCL21a，并且CD93在pMCs中显著高表达。In order to study how TEV promotes the secretion of CCL21a from pMCs, we performed RNA-Seq analysis on LLC-EVs-treated and untreated 40L cells, and found 109 differentially expressed genes (DEGs) between the two groups (Figure 49) . Next, we analyzed the association between 31 DEGs (Figure 50) with |Log2fold change(FC)|≥2 and Ccl21a and found that Gm13054 (up-regulation) and Cd93 (down-regulation) were highly correlated with Ccl21a (correlation coefficient ≥ 0.90). In addition, , the Log2FC value of Cd93 is greater than the Log2FC value of Gm13054 (Figure 51). Cd93 is mostly expressed on platelets, endothelial cells (ECs), hematopoietic progenitors and alveolar epithelial cells. In addition to the higher Cd93 mRNA levels in platelets and known tumor ECs, we used real-time PCR to detect Cd93 (see SEQ ID NO: 5 and SEQ ID NO: 6 for the Cd93 PCR primer sequence) and Ccl21a mRNA levels in various cells and found that The Cd93 mRNA expression level of p-pMCs was significantly higher than that of normal ECs, bone marrow cells, and MLE-12 mouse lung epithelial cells (Figure 52). The expression level of Cd93 mRNA in the pleura of tumor-bearing mice was up-regulated compared with that of normal mice, but the expression of Ccl21a mRNA was not up-regulated (Figure 53). Use 2.5 μg ml -1 LLC-EVs to stimulate CD93-silenced 40L cells (use siRNA to silence CD93, the sequences of CD93 siRNA are shown in SEQ ID NO: 39 and SEQ ID NO: 40; the silencing effect is shown in Figure 54). After 24 hours, use The CCL21 level in the cell supernatant was measured by ELISA. As shown in Figure 55, the CCL21a protein level increased significantly, and LLC-EVs no longer promoted CCL21a expression in the cells. Therefore, these results indicate that CD93 negatively regulates CCL21a and that CD93 is significantly highly expressed in pMCs.
进一步，我们使用LLC-EVs刺激p-pMCs，发现LLC-EVs能使p-pMCs中CD93蛋白的表 达减少(图56)，MC标记间皮素试验结果如图57，在小鼠胸腔注射20μg LLC-EVs 24h后，用免疫荧光印记检测pMCs中的CD93，CD93明显减少，这显示胸腔注射LLC-EVs使CD93的原位表达减少。real-time PCR检测胸腔注射过胆固醇偶联Cd93 siRNA的小鼠的Cd93 mRNA水平，如图58，Cd93 mRNA显著下降，说明胆固醇偶联Cd93 siRNA能用于沉默CD93。按图8方案使用LLC-Luci细胞和LLC-EVs处理小鼠，在每次注射EV前胸腔注射10μg胆固醇偶联Cd93 siRNA或NC siRNA，用real-time PCR检测胸膜Ccl21 mRNA水平并用ELISA检测胸膜Ccl21蛋白水平，结果如图59-60；当CD93被沉默后，LLC-EVs处理后胸膜Ccl21 mRNA和蛋白水平无明显变化，这说明在荷瘤小鼠胸腔注射胆固醇偶联Cd93 siRNA后，胸腔注射LLC-EVs不再诱导胸膜CCL21a的mRNA和蛋白表达。同时，用IVIS检测肺肿瘤大小，结果见图61，用流式细胞仪检测TILs中DC，结果见图62，LLC-EVs抑制肺肿瘤现象消失，并且DCs、CD4+T细胞和CD8+T细胞比例不再升高。值得注意的是，仅进行CD93沉默就能显著引起CCL21a分泌和抗肿瘤免疫。因此，这些结果表明TEVs通过减低CD93表达来促进CCL21a分泌。Furthermore, we used LLC-EVs to stimulate p-pMCs and found that LLC-EVs could increase the expression of CD93 protein in p-pMCs. The results of the MC-labeled mesothelin test are shown in Figure 57. After 20 μg LLC-EVs were injected into the chest of mice for 24 hours, immunofluorescence blotting was used to detect CD93 in pMCs. CD93 was significantly reduced, which showed that LLC-EVs were injected into the chest. EVs reduce the in situ expression of CD93. Real-time PCR detected Cd93 mRNA levels in mice injected with cholesterol-conjugated Cd93 siRNA into the chest. As shown in Figure 58, Cd93 mRNA decreased significantly, indicating that cholesterol-conjugated Cd93 siRNA can be used to silence CD93. Mice were treated with LLC-Luci cells and LLC-EVs according to the protocol in Figure 8. 10 μg of cholesterol-conjugated Cd93 siRNA or NC siRNA was injected into the chest before each EV injection. Real-time PCR was used to detect pleural Ccl21 mRNA levels and ELISA was used to detect pleural Ccl21. Protein level, the results are shown in Figure 59-60; when CD93 was silenced, there was no significant change in pleural Ccl21 mRNA and protein levels after treatment with LLC-EVs, which shows that after intrathoracic injection of cholesterol-conjugated Cd93 siRNA in tumor-bearing mice, LLC was injected into the intrathoracic cavity. -EVs no longer induce pleural CCL21a mRNA and protein expression. At the same time, IVIS was used to detect the size of lung tumors. The results are shown in Figure 61. DCs in TILs were detected using flow cytometry. The results were shown in Figure 62. The phenomenon of LLC-EVs inhibiting lung tumors disappeared, and DCs, CD4 + T cells and CD8 + T cells The ratio is no longer rising. Notably, CD93 silencing alone significantly induced CCL21a secretion and anti-tumor immunity. Therefore, these results indicate that TEVs promote CCL21a secretion by reducing CD93 expression.
实施例4通过TEV-derived miR-5110下调pMCs的CD93Example 4 Down-regulation of CD93 in pMCs by TEV-derived miR-5110
本实施例研究发现作用于CD93的特异性miRNA-miR-5110，其能显著降低细胞中CD93的mRNA和蛋白水平。使用miR-5110水平更高的TEV具有更好的抗肺肿瘤效果，pMCs中的CD93 mRNA水平更低。The study in this example found that specific miRNA-miR-5110 acts on CD93, which can significantly reduce the mRNA and protein levels of CD93 in cells. TEV using higher levels of miR-5110 had better anti-lung tumor effects and lower CD93 mRNA levels in pMCs.
我们探究了TEVs如何下调pMCs中CD93的相关机制。首先，我们通过电穿孔法将LLC-EVs与蛋白酶K共孵育以消化LLC-EVs的总蛋白，具体地，使用10μg ml-1蛋白酶K消化LLC-EVs 2h，用流式细胞仪和蛋白免疫印迹法检测消化效果，分别见图63和图64，说明消化效果好，使用消化好的LLC-EVs刺激40L细胞24h，通过ELISA测定细胞上清中的CCL21a水平，结果见图65，蛋白酶K消化组与未消化组间CCL21a蛋白水平无明显差异，这说明TEV蛋白不参与40L细胞对CCL21a分泌的诱导。接着，我们将LLC-EVs与RNase共孵育并使用电穿孔方式降解囊泡内RNAs，具体地，用10μg ml-1RNase I消化LLC-EVs 2h后测量LLC-EVs中RNA含量，如图66，RNA浓度显著下降，证实LLC-EVs中RNA被消化。使用RNase I消化过的LLC-EVs刺激40L细胞24h后用ELISA检测细胞上清中CCL21a水平，结果见图67，CCL21a无显著升高，证实LLC-EVs不再促进40L细胞的CCL21a分泌。上述结果说明LLC-EVs中的RNAs而不是蛋白在升高CCL21a中发挥着重要作用。We explored the relevant mechanisms of how TEVs downregulate CD93 in pMCs. First, we co-incubated LLC-EVs with proteinase K by electroporation to digest the total protein of LLC-EVs. Specifically, we used 10 μg ml-1 proteinase K to digest LLC-EVs for 2 h, followed by flow cytometry and Western blotting. Method to detect the digestion effect, shown in Figure 63 and Figure 64 respectively, indicating that the digestion effect is good, the digested LLC-EVs were used to stimulate 40L cells for 24h, and the CCL21a level in the cell supernatant was measured by ELISA. The results are shown in Figure 65, Proteinase K digestion group There was no significant difference in CCL21a protein levels between the undigested group and the undigested group, which indicated that TEV protein was not involved in the induction of CCL21a secretion by 40L cells. Next, we incubated LLC-EVs with RNase and used electroporation to degrade the RNAs in the vesicles. Specifically, we digested LLC-EVs with 10 μg ml -1 RNase I for 2 hours and then measured the RNA content in LLC-EVs, as shown in Figure 66. The RNA concentration decreased significantly, confirming the digestion of RNA in LLC-EVs. RNase I-digested LLC-EVs were used to stimulate 40L cells for 24 hours and then ELISA was used to detect the CCL21a level in the cell supernatant. The results are shown in Figure 67. There was no significant increase in CCL21a, confirming that LLC-EVs no longer promote CCL21a secretion in 40L cells. The above results indicate that RNAs rather than proteins in LLC-EVs play an important role in increasing CCL21a.
外泌体中富集的miRNAs对于外泌体介导的细胞间通讯至关重要。为了确定miRNAs在TEV诱导pMCs分泌CCL21a中的作用，我们收集了来自MLE-12小鼠肺II型上皮细胞 (MLE-12mouse lung epithelial type II cells)的EVs(MLE-12-EVs)，发现使用MLE-12-EVs刺激并不能抑制40L细胞分泌CCL21a(图68)。相较于MLE-12-EVs，我们通过miRNA array发现LLC-EVs中有45个富集的miRNAs(图69)。然后，利用miRDB和TargetScan数据库预测Cd93的上游miRNA，通过两个数据库识别出123个miRNA(图70)。将富集的miRNA与预测的Cd93上游miRNA进行比对，我们发现miR-5110和miR-5107-5p为两组miRNAs的重叠部分(图71)。因此，我们进一步研究这两个miRNAs对CD93表达的作用。我们使用miR-5110或miR-5107-5p类似物(miR-5110类似物和miR-5107-5p类似物的序列分别见SEQ ID NO:17和SEQ ID NO:18)转染40L细胞(图74为检测转染miR-5110或miR-5107-5p类似物24h后使用real-time PCR检测40L细胞中miR-5110或miR-5107-5p类似物的表达情况，miR-5110的PCR引物序列见SEQ ID NO:9和SEQ ID NO:10，miR-5107-5p的PCR引物序列见SEQ ID NO:11和SEQ ID NO:12，由结果可知miR-5110或miR-5107-5p转染成功)；同时设置控制组(Ctrl)，使用miRNA类似物的阴性对照转染40L细胞(miRNA阴性对照的类似物的序列见SEQ ID NO:19)，再检测CD93的表达情况，如图72-73，与控制组相比，miR-5110类似物组Cd93 mRNA和蛋白水平均明显下降，miR-5107-5p类似物组没有显著变化，这说明miR-5110类似物显著降低40L细胞的CD93的mRNA和蛋白水平，而miR-5107-5p类似物没有该作用。然后，我们研究了CD93是否是miR-5110(SEQ ID NO:17：5’-GGAGGAGGUAGAGGGUGGUGGAAUU-3’)的直接靶标，TargetScan预测了Cd93 3’-UTR的两个miR-5110作用位点，这两个作用位点的序列相同(图75)，荧光素酶报告试验显示这个序列(WT：CCUCCUCA)是miR-5110的靶点，突变序列(MUT：GGAGGAGU)消除了miR-5110类似物荧光素酶活性(图75-76)。The enriched miRNAs in exosomes are critical for exosome-mediated intercellular communication. To determine the role of miRNAs in TEV-induced CCL21a secretion from pMCs, we collected type II epithelial cells from MLE-12 mouse lungs (MLE-12 mouse lung epithelial type II cells) EVs (MLE-12-EVs), it was found that stimulation with MLE-12-EVs did not inhibit the secretion of CCL21a in 40L cells (Figure 68). Compared with MLE-12-EVs, we found 45 enriched miRNAs in LLC-EVs through miRNA array (Figure 69). Then, the miRDB and TargetScan databases were used to predict the upstream miRNA of Cd93, and 123 miRNAs were identified through the two databases (Figure 70). Comparing the enriched miRNAs with the predicted Cd93 upstream miRNA, we found that miR-5110 and miR-5107-5p were the overlapping parts of the two groups of miRNAs (Figure 71). Therefore, we further studied the effects of these two miRNAs on CD93 expression. We transfected 40L cells (Figure 74 To detect the expression of miR-5110 or miR-5107-5p analogs in 40L cells using real-time PCR 24 hours after transfection with miR-5110 or miR-5107-5p analogs, the PCR primer sequence of miR-5110 is shown in SEQ. ID NO:9 and SEQ ID NO:10, the PCR primer sequence of miR-5107-5p is shown in SEQ ID NO:11 and SEQ ID NO:12. From the results, it can be seen that the transfection of miR-5110 or miR-5107-5p was successful); At the same time, set up a control group (Ctrl), transfect 40L cells with the negative control of the miRNA analog (see SEQ ID NO: 19 for the sequence of the analog of the miRNA negative control), and then detect the expression of CD93, as shown in Figure 72-73, and Compared with the control group, the levels of Cd93 mRNA and protein in the miR-5110 analog group were significantly reduced, while the miR-5107-5p analog group had no significant changes, which showed that the miR-5110 analog significantly reduced the mRNA and protein levels of CD93 in 40L cells. , while the miR-5107-5p analogue has no such effect. Then, we studied whether CD93 is a direct target of miR-5110 (SEQ ID NO:17: 5'-GGAGGAGGUAGAGGGUGGUGGAAUU-3'). TargetScan predicted two miR-5110 action sites of Cd93 3'-UTR. The sequences of the two action sites are the same (Figure 75). The luciferase reporter assay showed that this sequence (WT: CCUCCUCA) is the target of miR-5110, and the mutated sequence (MUT: GGAGGAGU) eliminates the miR-5110 analog luciferase Activity (Figure 75-76).
为了检测LLC-EV-derived miR-5110是否下调CD93，我们首先使用real-time PCR定量检测亲代细胞及其对应EVs中的miR-5110水平，结果见图77(miR-5110的PCR引物序列见SEQ ID NO:9和SEQ ID NO:10，miR-5110的Taqman探针序列见SEQ ID NO:51)，由图可知EVs中的miR-5110水平明显高于各自对应的亲代细胞，该结果证实与亲代细胞相比，miR-5110显著富集在TEVs中。进一步，我们用2.5μg ml-1LLC-EVs刺激转染了miRNA抑制剂的阴性对照(序列如SEQ ID NO:22)、miR-5110抑制剂(序列如SEQ ID NO:20所示)的40L细胞24h，real-time PCR检测40L细胞的miR-5110，结果见图79，证实40L细胞中的miR-5110被阻断，用蛋白免疫印迹法检测40L细胞中CD93水平，如图78，可知miR-5110阻断或敲除的40L细胞中，LLC-EVs不再使CD93蛋白下降且miR-5110沉默引起了CD93蛋白明显上升。通过分别转染miR-5110类似物和抑制剂至LLC细胞，我们分别得到 miR-5110水平升高的LLC-EV(LLC-EVs with an increased miR-5110level，LLC-EVs-miR-5110Ins)和miR-5110水平下降的LLC-EV(LLC-EVs with a decreased miR-5110level，LLC-EVs-miR-5110Des)(图80)。与LLC-EVs相比，LLC-EVs-miR-5110Ins和LLC-EVs-miR-5110Des在40L细胞中下调CD93的能力分别增加和减弱(图81)。此外，按图8的方案用LLC-Luci细胞和相应的EVs处理小鼠，第25天时IVIS检测肿瘤大小，结果见图82，相比LLC-EVs，LLC-EVs-miR-5110Ins组肿瘤面积更小，LLC-EVs-miR-5110Des组肿瘤面积更大，这说明LLC-EVs-miR-5110Ins和LLC-EVs-miR-5110Des分别展现更好的和更差的抑制LLC肿瘤生长作用。因此，TEV-derived miR-5110通过调节pMCs中的CD93来抑制肺肿瘤生长。随后，我们对LLC肺荷瘤小鼠中TT-EVs或EVs中的miR-5110水平与胸膜Cd93 mRNA水平进行了相关性分析，LLC肺荷瘤小鼠的肿瘤组织的EVs和血清EVs(sEVs)中miR-5110水平与这些小鼠pMCs中的Cd93 mRNA水平均呈负相关(图83)。上述结果说明TEV-5110水平与pMCs中的CD93表达呈负相关。In order to detect whether LLC-EV-derived miR-5110 downregulates CD93, we first used real-time PCR to quantitatively detect the levels of miR-5110 in parental cells and their corresponding EVs. The results are shown in Figure 77 (the PCR primer sequence of miR-5110 is shown in SEQ ID NO:9 and SEQ ID NO:10, the Taqman probe sequence of miR-5110 is shown in SEQ ID NO:51). It can be seen from the figure that the level of miR-5110 in EVs is significantly higher than that of the corresponding parental cells. This result confirms that Compared with parental cells, miR-5110 was significantly enriched in TEVs. Further, we used 2.5 μg ml-1LLC-EVs to stimulate 40L cells transfected with the negative control of the miRNA inhibitor (sequence shown in SEQ ID NO:22) and miR-5110 inhibitor (sequence shown in SEQ ID NO:20). At 24h, real-time PCR was used to detect miR-5110 in 40L cells. The results are shown in Figure 79, which confirmed that miR-5110 in 40L cells was blocked. Western blotting was used to detect the level of CD93 in 40L cells, as shown in Figure 78. It can be seen that miR-5110 in 40L cells was blocked. In 40L cells blocked or knocked out by 5110, LLC-EVs no longer decreased CD93 protein and silencing miR-5110 caused a significant increase in CD93 protein. By transfecting miR-5110 analogs and inhibitors into LLC cells respectively, we obtained LLC-EVs with an increased miR-5110level, LLC-EVs-miR-5110 Ins and LLC-EVs with a decreased miR-5110level , LLC-EVs-miR-5110 Des ) (Figure 80). Compared with LLC-EVs, the abilities of LLC-EVs-miR-5110 Ins and LLC-EVs-miR-5110 Des to downregulate CD93 in 40L cells were increased and weakened respectively (Fig. 81). In addition, mice were treated with LLC-Luci cells and corresponding EVs according to the protocol in Figure 8, and tumor size was detected by IVIS on day 25. The results are shown in Figure 82. Compared with LLC-EVs, the tumor area in the LLC-EVs-miR-5110 Ins group was smaller, and the tumor area in the LLC-EVs-miR-5110 Des group was larger, which shows that LLC-EVs-miR-5110 Ins and LLC-EVs-miR-5110 Des exhibit better and worse inhibitory effects on LLC tumor growth respectively. . Therefore, TEV-derived miR-5110 inhibits lung tumor growth by regulating CD93 in pMCs. Subsequently, we performed a correlation analysis between TT-EVs or miR-5110 levels in EVs and pleural Cd93 mRNA levels in LLC lung tumor-bearing mice, EVs in tumor tissues and serum EVs (sEVs) in LLC lung tumor-bearing mice. There was a negative correlation between miR-5110 levels and Cd93 mRNA levels in pMCs of these mice (Figure 83). The above results indicate that TEV-5110 levels are negatively correlated with CD93 expression in pMCs.
实施例5 pMCs中CD93水平下降表明人体T细胞应答增强Example 5 Decreased CD93 levels in pMCs indicate enhanced human T cell response
本实施例发现肺癌病人恶性肿瘤胸腔积液(malignant pleural effusions，MPEs)中EV miR-5193(miR-5110同源物)水平与CCL21和DC水平呈正相关；肿瘤组织中，TT-EVs miR-5193与CCL21水平、TTs的DCs、CD4+T细胞以及CD8+T细胞水平呈正相关，CCL21水平与DCs水平也呈正相关，这进一步证实pMCs中CD93通过促进CCL21介导的DC招募来促进抗肿瘤的T细胞免疫应答。This example found that the level of EV miR-5193 (miR-5110 homolog) in malignant pleural effusions (MPEs) of lung cancer patients was positively correlated with the levels of CCL21 and DC; in tumor tissue, TT-EVs miR-5193 There is a positive correlation with CCL21 levels, DCs, CD4 + T cells and CD8 + T cells levels in TTs. CCL21 levels are also positively correlated with DCs levels, which further confirms that CD93 in pMCs promotes anti-tumor T cells by promoting CCL21-mediated DC recruitment. Cellular immune response.
为了确认人MCs的CD93是否也能调控肺癌患者的抗肿瘤免疫，我们用real-time PCR检测人胸膜间皮瘤细胞(NCI-H2452)和人脐静脉内皮细胞(human umbilical vein endothelial cells，HUVECs)中Cd93和Ccl21 mRNA水平，如图84，NCI-H2452中的Cd93和Ccl2的mRNA水平均显著高于HUVEC的，这是首次证明NCI-H2452的CD93和Ccl21 mRNA表达水平高于HUVECs的CD93和Ccl21 mRNA表达水平。进一步，我们用Cd93 siRNA(人Cd93 siRNA序列见SEQ ID NO:41和SEQ ID NO:42)、Ccr7 siRNA(人Ccr7 siRNA序列见SEQ ID NO:45和SEQ ID NO:46)分别沉默NCI-H2452的CD93、DCs的CCR7，图87和图88的蛋白免疫印迹检测结果证实CD93或CCR7蛋白下降，说明CD93或CCR7蛋白被沉默，再用ELISA检测NCI-H2452细胞上清中的CCL21水平以及用Transwell趋化试验检测NCI-H2452细胞上清对DCs的趋化作用，试验结果如图85-86所示，CD93被沉默后CCL21水平显著升高，细胞上清对DCs的趋化作用明显增加，而当DCs的CCR7也被沉默时，细胞上清对DCs的趋化作用无明显变化，这些结果说明沉默NCI-H2452细 胞中的CD93会显著促进CCL21分泌；同时，这些细胞上清增加了人DCs的趋化能力，并且当DCs中的CCR7被敲除或沉默时趋化能力不再增加。此外，我们real-time PCR定量检测肺癌病人TTs(tumortissues)和TT-TVs中miR-5193水平(miR-5193的引物序列如SEQ ID NO:15和SEQ ID NO:16所示，miR-5193的Taqman探针序列见SEQ ID NO:52)，13名病人的检测结果见图89，可知TT-EVs中miR-5193RNA水平均显著高于TTs的。进一步我们用2.5μg ml-1各病人的TT-EVs分别刺激NCI-H2452细胞，24h后用蛋白免疫印迹检测的细胞中CD93水平，结果见图90，CD93均明显下降。上述结果说明miR-5193(人的miR-5110同源物)在肺癌病人的TT-EVs中富集，且TT-EVs显著抑制NCI-H2452细胞中CD93的表达。特别地，进一步分析TT-EVs中的miR-5193水平与TT-EV抑制CD93表达的能力的相关性，如图91TT-EVs中的miR-5193水平与TT-EV抑制CD93表达的能力呈正相关，这表明TT-EV-derived miR-5193是人pMCs中CD93水平的一个负相关指标。In order to confirm whether CD93 of human MCs can also regulate anti-tumor immunity in lung cancer patients, we used real-time PCR to detect human pleural mesothelioma cells (NCI-H2452) and human umbilical vein endothelial cells (HUVECs). As shown in Figure 84, the mRNA levels of Cd93 and Ccl2 in NCI-H2452 are significantly higher than those in HUVECs. This is the first time that the expression levels of CD93 and Ccl21 in NCI-H2452 are higher than those in HUVECs. mRNA expression levels. Furthermore, we used Cd93 siRNA (see SEQ ID NO:41 and SEQ ID NO:42 for the human Cd93 siRNA sequence) and Ccr7 siRNA (see SEQ ID NO:45 and SEQ ID NO:46 for the human Ccr7 siRNA sequence) to silence NCI-H2452 respectively. CD93, CCR7 of DCs, the Western blot detection results in Figure 87 and Figure 88 confirmed that CD93 or CCR7 protein decreased, indicating that CD93 or CCR7 protein was silenced, and then ELISA was used to detect the CCL21 level in the NCI-H2452 cell supernatant and Transwell Chemotaxis test was used to detect the chemotactic effect of NCI-H2452 cell supernatant on DCs. The test results are shown in Figure 85-86. After CD93 was silenced, the level of CCL21 increased significantly, and the chemotactic effect of the cell supernatant on DCs increased significantly. When CCR7 of DCs was also silenced, the chemotactic effect of the cell supernatant on DCs did not change significantly. These results indicate that silencing NCI-H2452 cells CD93 in cells significantly promoted CCL21 secretion; at the same time, the supernatant of these cells increased the chemotactic ability of human DCs, and the chemotactic ability no longer increased when CCR7 in DCs was knocked out or silenced. In addition, we quantitatively detected the levels of miR-5193 in TTs (tumortissues) and TT-TVs of lung cancer patients using real-time PCR (the primer sequences of miR-5193 are shown in SEQ ID NO:15 and SEQ ID NO:16, and the primer sequences of miR-5193 are as follows: The Taqman probe sequence is shown in SEQ ID NO: 52), and the test results of 13 patients are shown in Figure 89. It can be seen that the levels of miR-5193 RNA in TT-EVs are significantly higher than those in TTs. Further, we used 2.5 μg ml-1 of each patient's TT-EVs to stimulate NCI-H2452 cells, and 24 hours later we used Western blot to detect the CD93 levels in the cells. The results are shown in Figure 90, and CD93 levels were significantly reduced. The above results indicate that miR-5193 (human miR-5110 homolog) is enriched in TT-EVs from lung cancer patients, and TT-EVs significantly inhibit the expression of CD93 in NCI-H2452 cells. In particular, the correlation between the level of miR-5193 in TT-EVs and the ability of TT-EV to inhibit CD93 expression was further analyzed, as shown in Figure 91 The level of miR-5193 in TT-EVs was positively correlated with the ability of TT-EV to inhibit the expression of CD93, This suggests that TT-EV-derived miR-5193 is a negatively correlated indicator of CD93 levels in human pMCs.
我们进一步评价了肺癌病人的pMCs中CD93水平和T细胞应答的关系。由于肺癌病人的MCs难以获得，我们检测了EVs中的miR-5193水平，作为pMCs中CD93水平的替代指标。首先，我们测定了肺癌病人恶性肿瘤胸腔积液(malignant pleural effusions，MPEs)中的CCL21，CD11c+DCs和EV miR-5193浓度(miR-5193检测探针序列见SEQ ID NO:54)，如图92，发现EV miR-5193浓度与CCL21和DC浓度呈正相关，此外，CCL21和DC浓度呈正相关(图93)。接着，我们测定了来自73个肺癌病人的TT-EVs miR-5193水平，发现TT-EVs miR-5193水平(miR-5193检测探针序列见SEQ ID NO:54)与CCL21水平、TTs的CD11c+DCs、CD4+T细胞和CD8+T细胞呈正相关，CCL21水平与DC数量也呈正相关(图94-95)。除此以外，根据TT-EVs miR-5193水平的高低将73个肺癌病人分为miR-5193水平较高(pMCs中CD93低)和miR-5193水平较低(pMCs中CD93高)的两组，分别记为TT-EV/miR-5193hi(36人)和TT-EV/miR-5193lo(37人)，分别绘制TT-EV/miR-5193hi和TT-EV/miR-5193lo两组肺癌患者的总生存曲线，如图96，TT-EV miR-5193水平高的病人比TT-EV miR-5193水平低的病人的总存活率更高。综上，pMCs中CD93通过促进CCL21介导的DC招募来调节T细胞应答。We further evaluated the relationship between CD93 levels and T cell responses in pMCs from lung cancer patients. Since MCs from lung cancer patients are difficult to obtain, we detected the level of miR-5193 in EVs as a surrogate for CD93 levels in pMCs. First, we measured the concentrations of CCL21, CD11c + DCs and EV miR-5193 in malignant pleural effusions (MPEs) of lung cancer patients (miR-5193 detection probe sequence, see SEQ ID NO: 54), as shown in the figure 92, it was found that EV miR-5193 concentration was positively correlated with CCL21 and DC concentrations, and in addition, CCL21 and DC concentrations were positively correlated (Figure 93). Next, we measured the levels of miR-5193 in TT-EVs from 73 lung cancer patients and found that the level of miR-5193 in TT-EVs (see SEQ ID NO: 54 for the sequence of the miR-5193 detection probe) is closely related to the level of CCL21 and the CD11c + level of TTs. There is a positive correlation between DCs, CD4 + T cells and CD8 + T cells, and there is also a positive correlation between CCL21 levels and the number of DCs (Figure 94-95). In addition, 73 lung cancer patients were divided into two groups according to the level of miR-5193 in TT-EVs: those with higher levels of miR-5193 (low CD93 in pMCs) and those with lower levels of miR-5193 (high CD93 in pMCs). Recorded as TT-EV/miR-5193 hi (36 people) and TT-EV/miR-5193 lo (37 people), two groups of TT-EV/miR-5193 hi and TT-EV/miR-5193 lo were drawn respectively. The overall survival curve of lung cancer patients is shown in Figure 96. Patients with high levels of TT-EV miR-5193 have a higher overall survival rate than patients with low levels of TT-EV miR-5193. In summary, CD93 in pMCs regulates T cell responses by promoting CCL21-mediated DC recruitment.
实施例6抗CD93(Anti-CD93)通过促进pMCs分泌CCL21抑制肿瘤生长Example 6 Anti-CD93 (Anti-CD93) inhibits tumor growth by promoting the secretion of CCL21 from pMCs
本实施例研究发现pMC中C1q(而不是IGFBP7)是pMCs中CD93激活的配体。C1qA过表达时，Ccl21a mRNA水平增加，C1qA被抑制时，胸膜CCL21a表达显著降低。本实施例还发现CTLD区域是CD93抑制pMCs中分泌CCL21的功能区域。另外，本实施例提供了一种抗CD93的单克隆抗体M057，M057使胸膜Ccl21a mRNA水平和CCL21a蛋白水平升高， CD4+T细胞、CD8+T细胞和DCs升高，引起了全身肿瘤特异性免疫。This example found that C1q in pMC (rather than IGFBP7) is the ligand for CD93 activation in pMCs. When C1qA is overexpressed, Ccl21a mRNA levels increase, and when C1qA is inhibited, pleural CCL21a expression is significantly reduced. This example also found that the CTLD region is the functional region where CD93 inhibits the secretion of CCL21 in pMCs. In addition, this embodiment provides an anti-CD93 monoclonal antibody M057. M057 increases the pleural Ccl21a mRNA level and CCL21a protein level. CD4 + T cells, CD8 + T cells and DCs increased, causing systemic tumor-specific immunity.
由于pMC CD93对调节抗肿瘤免疫至关重要，我们试图找出负责pMCs中CD93激活的配体。C1q、多聚蛋白(multimerin 2，MMRN2)和***结合蛋白7(insulin-like growth factor binding protein 7，IGFBP7)是CD93的已知配体。使40L和NCI-H2452细胞中C1qA、MMRN2或IGFBP7过表达，用蛋白免疫印迹检测各基因过表达情况。结果如图97，证实两种细胞中C1qA、MMRN2或IGFBP7过表达。进一步用real-time PCR检测C1qA、MMRN2或IGFBP7过表达的40L或NCI-H2452细胞中Ccl21a mRNA水平，结果如图98所示，可知C1qA过表达的细胞(40L或NCI-H2452)中Ccl21amRNA水平显著低于其他各组，其他各组间Ccl21a mRNA水平无明显差别，这说明过表达(Overexpression，OE)C1qA能显著抑制Ccl21a mRNA水平，而过表达MMRN2或IGFBP7对Ccl21a mRNA水平无明显影响。此外，用2μg ml-1重组C1qA刺激40L细胞或NCI-H2452细胞，24h后用ELISA检测细胞上清中的CCL21a或CCL21水平，结果见图99，CCL21a或CCL21均显著下降，这说明重组C1qA刺激显著降低40L和NCI-H2452细胞中CCL21a或CCL21的分泌。在0天向小鼠静脉注射1×106LLC-Luci细胞，并在14、16、18、20和22天胸腔注射5μg C1qA，在第25天用蛋白免疫印迹检测胸膜CCL21a蛋白水平且用IVIS检测的肺肿瘤体积，结果分别如图100-101，可知CCL21a蛋白明显下降，注射C1qA后小鼠肺肿瘤面积变大，这说明胸腔注射重组C1qA也使胸膜CCL21a蛋白下降并促进肺肿瘤生长。Since pMC CD93 is critical for regulating anti-tumor immunity, we sought to identify the ligands responsible for CD93 activation in pMCs. C1q, multimerin 2 (MMRN2) and insulin-like growth factor binding protein 7 (IGFBP7) are known ligands of CD93. C1qA, MMRN2 or IGFBP7 were overexpressed in 40L and NCI-H2452 cells, and Western blotting was used to detect the overexpression of each gene. The results are shown in Figure 97, confirming the overexpression of C1qA, MMRN2 or IGFBP7 in both cells. Real-time PCR was further used to detect Ccl21a mRNA levels in 40L or NCI-H2452 cells overexpressing C1qA, MMRN2 or IGFBP7. The results are shown in Figure 98. It can be seen that the Ccl21amRNA level in C1qA overexpressing cells (40L or NCI-H2452) is significantly higher. Lower than other groups, there was no significant difference in Ccl21a mRNA levels among other groups, which shows that overexpression (OE) C1qA can significantly inhibit Ccl21a mRNA levels, while overexpression of MMRN2 or IGFBP7 has no significant effect on Ccl21a mRNA levels. In addition, 40L cells or NCI-H2452 cells were stimulated with 2 μg ml -1 recombinant C1qA, and ELISA was used to detect the CCL21a or CCL21 levels in the cell supernatant 24 hours later. The results are shown in Figure 99. Both CCL21a or CCL21 decreased significantly, which shows that recombinant C1qA stimulated Significantly reduced the secretion of CCL21a or CCL21 in 40L and NCI-H2452 cells. Mice were intravenously injected with 1 × 106 LLC-Luci cells on day 0, and 5 μg C1qA was intrapleurally injected on days 14, 16, 18, 20, and 22. On day 25, pleural CCL21a protein levels were detected by Western blotting and IVIS. Lung tumor volume, the results are shown in Figures 100-101 respectively. It can be seen that CCL21a protein significantly decreased, and the lung tumor area of mice became larger after C1qA injection. This shows that intrapleural injection of recombinant C1qA also reduces pleural CCL21a protein and promotes lung tumor growth.
C1q主要在肝脏中合成，且胆固醇偶联寡核苷酸具有很好的肝脏靶向性。通过胆固醇偶联C1qa反义寡核苷酸(C1qa的反义寡核苷酸序列如SEQ ID NO:49所示)敲除小鼠肝脏中的C1qA，具体地，在0天向小鼠静脉注射1×106LLC-Luci细胞，并在14、16、18、20和22天静脉注射100μg C1qa ASOs(沉默C1qA)，在第25天用蛋白免疫印迹检测胸膜CCL21a蛋白水平以及用IVIS检测的肺肿瘤体积，如图102-103，CCL21a蛋白明显增加同时肿瘤面积明显小于对照组(阴性对照的反义寡核苷酸序列如SEQ ID NO:50所示)，这说明胸膜CCL21a表达显著增加，同时肺肿瘤增长变慢。此外，用LEISA检测LLC肺荷瘤小鼠和肺癌病人血清中C1qA水平，如图104，肺肿瘤小鼠和肺肿瘤病人血清C1qA水平也明显高于正常个体的。CD93含有1个C型凝集素结构域(C-type lectin domain，CTLD)、1个Sushi结构域、5个表皮生长因子(epidermal growth factor，EGF)样结构域和1个Mucin结构域。接着，我们对CD93进行了剪切，获得含有这些结构域中一个或多个的CD93截短体片段(图105)，将这些片段转入40L细胞，48h后用real-time PCR检测各片段的表达情况并检测Ccl21a mRNA水平，如图106-107，与控制组相比，各组Ccl21a mRNA水平均显著下降，该结果说 明同转染CD93-WT质粒一样，过表达含CTLD片段的CD93质粒均能有效抑制40L细胞中的Ccl21a mRNA表达。此外，切除CD93的CTLD部分获得CD93ΔCTLD，同上转入40L细胞，48h后用ELISA检测细胞上清中的CCL21a蛋白水平，如图108，CTLD被切除后，CCL21a蛋白水平与对照组无显著差异。因此，过表达CTLD质粒，而不是过表达CTLD缺失的CD93质粒(CD93ΔCTLD)，能显著抑制40L细胞释放CCL21a，并且抑制效果与CD93-WT相似。这些结果表明CD93的CTLD与C1q介导的CCL21抑制相关。综上所述，在CD93调控pMC中CCL21时，C1q是CD93的配体。C1q is mainly synthesized in the liver, and cholesterol-conjugated oligonucleotides have good liver targeting properties. C1qA was knocked out in mouse livers by cholesterol-coupled C1qa antisense oligonucleotide (the antisense oligonucleotide sequence of C1qa is shown in SEQ ID NO:49). Specifically, mice were injected intravenously on day 0. 1 × 10 6 LLC-Luci cells and intravenous injection of 100 μg C1qa ASOs (silencing C1qA) on days 14, 16, 18, 20, and 22, and pleural CCL21a protein levels were detected by Western blotting and lung by IVIS on day 25 Tumor volume, as shown in Figure 102-103, CCL21a protein significantly increased and the tumor area was significantly smaller than the control group (the antisense oligonucleotide sequence of the negative control is shown in SEQ ID NO: 50), which shows that the expression of pleural CCL21a significantly increased, and at the same time Lung tumors grow more slowly. In addition, LEISA was used to detect C1qA levels in the serum of LLC lung tumor-bearing mice and lung cancer patients. As shown in Figure 104, the serum C1qA levels of lung tumor mice and lung tumor patients were also significantly higher than those of normal individuals. CD93 contains 1 C-type lectin domain (CTLD), 1 Sushi domain, 5 epidermal growth factor (EGF)-like domains and 1 Mucin domain. Next, we cut CD93 to obtain CD93 truncated fragments containing one or more of these domains (Figure 105). These fragments were transferred into 40L cells, and 48 hours later, real-time PCR was used to detect the expression of each fragment. Expression and detect Ccl21a mRNA levels, as shown in Figure 106-107. Compared with the control group, the Ccl21a mRNA levels in each group decreased significantly. The results said It was found that, like transfection of CD93-WT plasmid, overexpression of CD93 plasmid containing CTLD fragment could effectively inhibit Ccl21a mRNA expression in 40L cells. In addition, the CTLD part of CD93 was removed to obtain CD93ΔCTLD, which was transferred into 40L cells as above. 48 hours later, ELISA was used to detect the CCL21a protein level in the cell supernatant, as shown in Figure 108. After the CTLD was removed, there was no significant difference between the CCL21a protein level and the control group. Therefore, overexpressing CTLD plasmid, but not overexpressing CTLD-deleted CD93 plasmid (CD93ΔCTLD), can significantly inhibit the release of CCL21a from 40L cells, and the inhibitory effect is similar to that of CD93-WT. These results indicate that CTLD of CD93 is associated with C1q-mediated inhibition of CCL21. In summary, C1q is the ligand of CD93 when CD93 regulates CCL21 in pMC.
因此，我们推测如果抗CD93能消除pMCs中C1q介导的CD93激活，则可以获得治疗肺肿瘤的效果。因而，我们构建了一种抗鼠CD93的兔单克隆抗体(M057)，构建方法为：小鼠CD93抗原免疫实验兔子，其血清多克隆CD93Ab验证水平后，取脾脏浆细胞扩增其抗体编辑的DNA序列，将DNA序列克隆、重组至质粒载体，通过蛋白纯化方式获取各单克隆的重组CD93抗体，具体见序列SEQ IDNO:1和SEQ ID NO:2，该抗体的恒定区用小鼠IgG替代。M057的解离常数(Kd)是0.24nM(图109)。M057能染色WT小鼠骨髓细胞，而Cd93-/-小鼠骨髓细胞不会被染色(图110)。此外，静脉注射Alexa Fluor 680标记的M057，在WT小鼠胸膜可检测到强信号，但Cd93-/-小鼠胸膜未检测到信号(图111)。这些结果表明M057与CD93特异性结合。Therefore, we speculated that if anti-CD93 could eliminate C1q-mediated CD93 activation in pMCs, therapeutic effects on lung tumors could be obtained. Therefore, we constructed an anti-mouse CD93 rabbit monoclonal antibody (M057). The construction method is as follows: immunize experimental rabbits with mouse CD93 antigen, and after verifying the level of serum polyclonal CD93Ab, take spleen plasma cells to amplify the antibody-edited DNA sequence, clone and recombine the DNA sequence into a plasmid vector, and obtain each monoclonal recombinant CD93 antibody through protein purification. For details, see the sequence SEQ ID NO: 1 and SEQ ID NO: 2. The constant region of the antibody is replaced with mouse IgG. . The dissociation constant (Kd) of M057 is 0.24 nM (Figure 109). M057 can stain WT mouse bone marrow cells, but Cd93 −/− mouse bone marrow cells are not stained (Figure 110). In addition, after intravenous injection of Alexa Fluor 680-labeled M057, a strong signal could be detected in the pleura of WT mice, but no signal was detected in the pleura of Cd93 −/− mice (Figure 111). These results indicate that M057 specifically binds to CD93.
L(轻链):(SEQ IDNO:1)
L(light chain):(SEQ IDNO:1)
H(重链):(SEQ IDNO:2)
H (heavy chain): (SEQ IDNO: 2)
说明：illustrate:
下划线区域是信号肽；The underlined area is the signal peptide;
灰色区域可变区；gray area variable area;
黑色氨基酸序列没有下划线的是恒定区；The black amino acid sequence without underline is the constant region;
灰色区域内下划线的是CDR区域或者序列。 Underlined in the gray area are CDR regions or sequences.
为验证M057功能，我们用10μg ml-1M057和/或2μg ml-1C1qA处理40L细胞或p-pMCs，24h后ELISA检测细胞上清中的CCL21a水平，如图112，与控制组相比(IgG组)，使用M057后CCL21a水平显著升高，同时使用M057和C1qA，CCL21a水平也显著升高，但是单独使用M057时CCL21a水平升高更加显著。这说明即使在存在重组C1q的情况下，用M057处理也能显著促进40L细胞和p-pMCs分泌CCL21a，但效果较差。连续给LLC肺肿瘤小鼠一定剂量的M057，发现M057显著提高胸膜Ccl21a mRNA水平且呈剂量依赖性，同时，100μg M057的剂量下效果最优(图113)。在0天向小鼠静脉注射1×106LLC-Luci细胞，并在14、16、18、20和22天静脉注射100μg M057，在第25天通过蛋白免疫印迹检测胸膜CCL21a蛋白水平，用IVIS检测肺肿瘤大小，同时用流式细胞仪检测TILs中的DC、CD4+T细胞和CD8+T细胞，结果分别如图114、图115和图116所示，可知在该最优M057剂量下，胸膜CCL21a蛋白水平显著升高，LLC肿瘤生长被显著抑制，并且M057显著增加了TILs中CD4+T细胞、CD8+T细胞和DCs的比例。此外，在LLC-Luci肺荷瘤小鼠的两侧皮下分别接种LLC和B16F10肿瘤，接着在第8天、10天、12天、14天和16天用100μg M057治疗，第10至第18天皮下肿瘤大小如图117所示，皮下LLC肿瘤被显著抑制，而皮下B16F10无明显差异，这说明M057处理能抑制肺部以外的其他部位的同种肿瘤，M057处理也诱导了全身肿瘤特异性免疫，具有抑制不同部位同种肿瘤的作用。为了研究M057是否通过靶向pMCs中的CD93来抑制肺部肿瘤生长，我们敲除胸膜CCL21a，在0天向敲除了CCL21a小鼠静脉注射1×106LLC-Luci细胞，第14天、16天、18天、20天和22天静脉注射100μg M057，在第25天用IVIS检测肺肿瘤，如图118，对敲除CCL21a肺荷瘤小鼠使用M057仍能抑制其肺肿瘤生长，但是其抑制作用明显明显下降。这些结果说明M057主要通过抑制胸膜CCL21分泌来抑制肺肿瘤生长。To verify the function of M057, we treated 40L cells or p-pMCs with 10 μg ml -1 M057 and/or 2 μg ml -1 C1qA. After 24 hours, ELISA detected the CCL21a level in the cell supernatant, as shown in Figure 112, compared with the control group ( IgG group), the level of CCL21a was significantly increased after using M057, and the level of CCL21a was also significantly increased when M057 and C1qA were used simultaneously, but the increase in CCL21a level was more significant when M057 was used alone. This shows that even in the presence of recombinant C1q, treatment with M057 can significantly promote the secretion of CCL21a in 40L cells and p-pMCs, but the effect is poor. A certain dose of M057 was continuously given to LLC lung tumor mice, and it was found that M057 significantly increased the pleural Ccl21a mRNA level in a dose-dependent manner. At the same time, the best effect was achieved at the dose of 100 μg M057 (Figure 113). Mice were intravenously injected with 1 × 10 6 LLC-Luci cells on day 0 and 100 μg M057 on days 14, 16, 18, 20, and 22. Pleural CCL21a protein levels were detected by Western blotting on day 25 with IVIS. The lung tumor size was detected, and flow cytometry was used to detect DCs, CD4 + T cells and CD8 + T cells in TILs. The results are shown in Figure 114, Figure 115 and Figure 116 respectively. It can be seen that under the optimal M057 dose, Pleural CCL21a protein levels were significantly increased, LLC tumor growth was significantly inhibited, and M057 significantly increased the proportions of CD4 + T cells, CD8 + T cells, and DCs in TILs. In addition, LLC-Luci lung tumor-bearing mice were subcutaneously inoculated with LLC and B16F10 tumors on both sides, respectively, followed by treatment with 100 μg M057 on days 8, 10, 12, 14, and 16, and on days 10 to 18 The size of subcutaneous tumors is shown in Figure 117. Subcutaneous LLC tumors were significantly inhibited, while subcutaneous B16F10 had no significant difference. This shows that M057 treatment can inhibit the same type of tumors in other parts of the body besides the lungs. M057 treatment also induces systemic tumor-specific immunity. , has the effect of inhibiting the same type of tumors in different parts. To study whether M057 inhibits lung tumor growth by targeting CD93 in pMCs, we knocked out pleural CCL21a and injected 1×10 6 LLC-Luci cells intravenously into CCL21a knockout mice on day 0, day 14 and 16. , 100 μg M057 was injected intravenously on days 18, 20 and 22, and lung tumors were detected with IVIS on day 25. As shown in Figure 118, the use of M057 on CCL21a knockout lung tumor-bearing mice can still inhibit the growth of lung tumors, but its inhibition The effect is significantly reduced. These results indicate that M057 inhibits lung tumor growth mainly by inhibiting pleural CCL21 secretion.
进一步，我研究了M057的体内毒性，每隔一天向健康小鼠静脉注射100μg M057，5次注射后用LEISA检测血清中的ALT、AST、胆红素和肌酐水平并进行小鼠心、肝、脾、肺和肾的的组织病理学检测，如图119-120，与对照组相比(IgG组)，发现M057治疗没有增加血清中谷丙转氨酶(alanine transaminase，ALT)、天门冬氨酸转氨酶(aspartate aminotransferaseAST、胆红素(bilirubin)和肌酐(creatinine)的水平；M057治疗也未对主要器官(包括心、肝、脾、肺和肾)造成明显的组织病理学损害(图120)，这些结果表明M057具有较好的生物安全性。Further, I studied the in vivo toxicity of M057. I injected 100 μg M057 intravenously into healthy mice every other day. After 5 injections, I used LEISA to detect the levels of ALT, AST, bilirubin and creatinine in the serum and conducted experiments on the heart, liver, and creatinine of mice. Histopathological examination of spleen, lung and kidney, as shown in Figure 119-120, compared with the control group (IgG group), it was found that M057 treatment did not increase serum alanine transaminase (ALT) and aspartate aminotransferase ( aspartate aminotransferase AST, bilirubin and creatinine levels; M057 treatment also did not cause obvious histopathological damage to major organs (including heart, liver, spleen, lung and kidney) (Figure 120). These results It shows that M057 has good biological safety.
实施例7抗CD93比抗VEGFR(anti-VEGFR)拥有更好的抗肺肿瘤作用Example 7 Anti-CD93 has better anti-lung tumor effect than anti-VEGFR (anti-VEGFR)
本实施例证实抗CD93不仅能通过促进CCL21介导的DC招募来促进抗肿瘤的T细胞 免疫应答(阻断C1q/CD93信号通路)，还能通过使肿瘤血管正常化来特异性地抑制肿瘤生长(阻断IGFBP7/CD93信号通路)，且抗CD93的效果更优。This example demonstrates that anti-CD93 can not only promote anti-tumor T cells by promoting CCL21-mediated DC recruitment. The immune response (blocking the C1q/CD93 signaling pathway) can also specifically inhibit tumor growth by normalizing tumor blood vessels (blocking the IGFBP7/CD93 signaling pathway), and the anti-CD93 effect is better.
已有报道通过阻断IGFBP7和CD93的相互作用可以通过正常化肿瘤血管来抑制肿瘤生长。鉴于CTLD结构域是CD93促进肿瘤血管生成和诱导MCs分泌CCL21的功能所必需的，并且沉默胸膜CCL21并不能完全消除M057的抗肿瘤作用(图118)，我们猜测M057或许也能通过正常肿瘤血管来抗肺肿瘤。首先，我们用10μg ml-1 M057和/或2μg ml-1 IGFBP7处理小鼠原发性内皮细胞，24h后进行calcein-AM染色，用荧光显微法检测到血管生成情况，如图121，可知IGFBP7处理血管生成增加，而使用M057+IGFBP7处理血管生成明显被抑制，这说明M057还抑制IGFBP7诱导的体外EC血管生成。It has been reported that blocking the interaction of IGFBP7 and CD93 can inhibit tumor growth by normalizing tumor blood vessels. Given that the CTLD domain is required for the function of CD93 in promoting tumor angiogenesis and inducing MCs to secrete CCL21, and silencing pleural CCL21 does not completely eliminate the anti-tumor effect of M057 (Figure 118), we speculate that M057 may also be able to function through normal tumor blood vessels. Anti-lung tumors. First, we treated mouse primary endothelial cells with 10 μg ml-1 M057 and/or 2 μg ml-1 IGFBP7. After 24 hours, calcein-AM staining was performed, and angiogenesis was detected using fluorescence microscopy, as shown in Figure 121. IGFBP7 treatment increased angiogenesis, while treatment with M057+IGFBP7 significantly inhibited angiogenesis, indicating that M057 also inhibits IGFBP7-induced EC angiogenesis in vitro.
此外，第14天、16天、18天、20天和22天在LLC-Luci肺荷瘤小鼠静脉注射100μg M057，第25天取肿瘤组织并对NG2和CD31或者αSMA和CD31进行染色和定量分析，如图122，尽管M057处理未改变LLC肺肿瘤小鼠CD31+血管密度，但肿瘤组织中平滑肌细胞【用α-平滑肌肌动蛋白(α-smooth muscle actin，αSMA)代替】和周细胞【用神经/胶质抗原2(neural/glial antigen 2，NG2)代替】覆盖的血管比例明显升高。第14天用静脉注射5mg葡聚糖(FITC-dextran，70kDa)以测定肿瘤内皮渗透性，如图124，相反地，与对照组相比，M057处理的小鼠静脉注射FITC-dextran后，肿瘤的泄漏面积(葡聚糖阳性)显著减小。第14天、16天、18天、20天和22天在LLC-Luci肺荷瘤小鼠静脉注射不同量(20μg、40μg、60μg)的抗VEGFR试剂，第25天检测肺肿瘤大小，结果如图124所示，可知注射抗VEGFR试剂抑制肺肿瘤生长且40μg剂量为最佳作用剂量。此外，第13天、15天、17天、19天和21天在LLC-Luci肺荷瘤小鼠(WT)静脉注射40μg抗VEGFR试剂或IgG，并在12h后静脉注射100μg M057或IgG，第25天检测肺肿瘤大小，如图125，可知使用抗VEGFR试剂后使用M057，具有显著的肿瘤抑制作用，且相对于单独使用抗VEGFR试剂，使用抗VEGFR试剂后再使用M057，肺肿瘤抑制作用增强。这说明虽然CD93位于VEGFR信号下游，但在LLC肺荷瘤小鼠中使用最佳剂量的抗VEGFR试剂阻断VEGFR会减弱M057的抗肿瘤作用，但不能消除M057的抗肿瘤作用。然而，第13天、15天、17天、19天和21天在LLC-Luci肺荷瘤Ccr7-/-小鼠(Ccr7敲除小鼠)静脉注射40μg抗VEGFR试剂并在每次注射抗VEGFR试剂12h后静脉注射100μg M057或IgG，第25天检测肺肿瘤大小，如图126，与注射IgG相比，M057组无显著差异，说明在VEGFR阻断的LLC肺肿瘤Ccr7-/-小鼠中，M057处理不再抑制肿瘤生长。此外，第0天构建LLC皮下荷瘤小鼠(WT)模型，在第7天、9天、11天和15天静脉注射40μg抗VEGFR试剂并在每次注射抗VEGFR 试剂或IgG，12h后静脉注射100μg M057或IgG，统计肿瘤体积随时间的变化情况，如图127，在VEGFR阻断的皮下LLC荷瘤WT小鼠中，M057治疗也不能抑制肿瘤生长。这些结果表明M057不仅通过促进CCL21介导的DC迁移，也通过使肿瘤血管正常化来特异性地抑制肿瘤生长。In addition, LLC-Luci lung tumor-bearing mice were intravenously injected with 100 μg M057 on days 14, 16, 18, 20, and 22. On day 25, tumor tissues were collected and stained and quantified for NG2 and CD31 or αSMA and CD31. Analysis, as shown in Figure 122, although M057 treatment did not change the density of CD31+ blood vessels in LLC lung tumor mice, the smooth muscle cells [replaced with α-smooth muscle actin (αSMA)] and pericytes [replaced with αSMA] in the tumor tissue The proportion of blood vessels covered by neural/glial antigen 2 (NG2) was significantly increased. On day 14, 5 mg of dextran (FITC-dextran, 70 kDa) was intravenously injected to determine tumor endothelial permeability, as shown in Figure 124. On the contrary, compared with the control group, the tumor of M057-treated mice after intravenous injection of FITC-dextran The leakage area (dextran positive) was significantly reduced. On days 14, 16, 18, 20 and 22, LLC-Luci lung tumor-bearing mice were intravenously injected with different amounts (20 μg, 40 μg, 60 μg) of anti-VEGFR reagents. On day 25, the lung tumor size was measured. The results are as follows As shown in Figure 124, it can be seen that the injection of anti-VEGFR agent inhibits the growth of lung tumors and the dose of 40 μg is the optimal dose. In addition, LLC-Luci lung tumor-bearing mice (WT) were intravenously injected with 40 μg of anti-VEGFR reagent or IgG on days 13, 15, 17, 19, and 21, and 100 μg of M057 or IgG was intravenously injected 12 h later. The lung tumor size was detected on 25 days, as shown in Figure 125. It can be seen that the use of anti-VEGFR reagent followed by M057 has a significant tumor inhibitory effect, and compared with the use of anti-VEGFR reagent alone, the use of anti-VEGFR reagent followed by M057 has an enhanced inhibitory effect on lung tumors. . This indicates that although CD93 is located downstream of VEGFR signaling, blocking VEGFR using optimal doses of anti-VEGFR reagents in LLC lung tumor-bearing mice weakens but does not eliminate the anti-tumor effect of M057. However, LLC-Luci lung tumor-bearing Ccr7 −/− mice (Ccr7 knockout mice) were intravenously injected with 40 μg of anti-VEGFR reagent on days 13, 15, 17, 19, and 21, and anti-VEGFR was injected intravenously at each injection. 100 μg of M057 or IgG was intravenously injected 12 hours after the reagent, and the lung tumor size was measured on the 25th day, as shown in Figure 126. Compared with the injection of IgG, there was no significant difference in the M057 group, indicating that in Ccr7 -/- mice with VEGFR blocked LLC lung tumors , M057 treatment no longer inhibited tumor growth. In addition, the LLC subcutaneous tumor-bearing mouse (WT) model was constructed on day 0, and 40 μg of anti-VEGFR reagent was intravenously injected on days 7, 9, 11, and 15. Reagent or IgG, 100 μg of M057 or IgG was intravenously injected 12 hours later, and the changes in tumor volume over time were counted, as shown in Figure 127. In VEGFR-blocked subcutaneous LLC tumor-bearing WT mice, M057 treatment could not inhibit tumor growth. These results indicate that M057 specifically inhibits tumor growth not only by promoting CCL21-mediated DC migration but also by normalizing tumor blood vessels.
抗CD93和抗VEGFR均通过正常肿瘤血管抑制皮下肿瘤生长，因此具有相似的抗肿瘤效果。然而，将M057用于肺肿瘤治疗时，其能正常化肿瘤血管***并促进CCL21介导DC向肿瘤迁移。第14天、16天、18天、20天和22天在LLC-Luci肺荷瘤小鼠静脉注射40μg抗VEGFR试剂或100μg M057，第25天用IVIS检测肺肿瘤大小，如图128，正如预期的那样，我们观察到M057比抗VEGFR试剂具有更好的抑制肺肿瘤生长作用。然而，第14天、16天、18天、20天和22天在LLC-Luci肺荷瘤Ccr7-/-小鼠静脉注射40μg抗VEGFR试剂或100μg M057，第25天用IVIS检测肺肿瘤大小，如图129，在Ccr7-/-小鼠中，M057和anti-VEGFR显示出相当的抑制肺肿瘤生长的能力。这说明增加CCR7+DCs的肿瘤浸润有助于增强M057的抗肺肿瘤作用。Both anti-CD93 and anti-VEGFR inhibit subcutaneous tumor growth through normal tumor blood vessels and therefore have similar anti-tumor effects. However, when M057 was used for lung tumor treatment, it normalized tumor vasculature and promoted CCL21-mediated DC migration into tumors. LLC-Luci lung tumor-bearing mice were intravenously injected with 40 μg anti-VEGFR reagent or 100 μg M057 on days 14, 16, 18, 20, and 22, and IVIS was used to detect lung tumor size on day 25, as shown in Figure 128, as expected. , we observed that M057 had a better inhibitory effect on lung tumor growth than anti-VEGFR agents. However, LLC-Luci lung tumor-bearing Ccr7 -/- mice were intravenously injected with 40 μg anti-VEGFR reagent or 100 μg M057 on days 14, 16, 18, 20 and 22, and IVIS was used to detect lung tumor size on day 25. As shown in Figure 129, in Ccr7 −/− mice, M057 and anti-VEGFR showed comparable abilities to inhibit lung tumor growth. This indicates that increasing tumor infiltration of CCR7 + DCs helps enhance the anti-pulmonary tumor effect of M057.
T细胞很少或不存在的“冷肿瘤”会存在抵抗抗PD-1治疗。M057介导的肿瘤血管正常化和DC迁移增强使肿瘤中的T细胞增加。因此，M057治疗可能逆转“冷肿瘤”对抗PD-1治疗的耐药性。第14天、16天、18天、20天和22天在B16F10和4T1肺荷瘤小鼠静脉注射50μg抗PD-1试剂，同时注射100μg M057或不注射100μg M057，第18天用用IVIS检测肿瘤大小并进行肺H&E染色，如图130-131，正如预期的那样，抗PD-1和M057组合使用的肿瘤抑制作用最优，即抗PD-1治疗后的B16F10和4T1肺肿瘤均能被M057抑制。更重要的是，分别统计30名TT-EV/miR-5193hi和TT-EV/miR-5193lo以及C1qA水平较高(记为C1qAhi)和C1qA水平较低(记为C1qAlo)肺癌患者在抗PD-1治疗后无进展生存期(progression-free survival，PFS)，如图132，可知作为CD93的负向或正向调控因子，高sEV衍生的miRNA-5193或C1qA与肺癌患者抗PD-1治疗后无进展生存期的好坏有关。"Cold tumors" with few or no T cells can be resistant to anti-PD-1 therapy. M057-mediated normalization of tumor blood vessels and enhanced DC migration increase T cells in tumors. Therefore, M057 treatment may reverse the resistance of “cold tumors” to anti-PD-1 therapy. On days 14, 16, 18, 20 and 22, B16F10 and 4T1 lung tumor-bearing mice were intravenously injected with 50 μg of anti-PD-1 reagent, and simultaneously injected with 100 μg of M057 or without injection of 100 μg of M057. IVIS was used for detection on the 18th day. Tumor size and lung H&E staining were performed, as shown in Figure 130-131. As expected, the tumor inhibitory effect of the combination of anti-PD-1 and M057 was optimal, that is, both B16F10 and 4T1 lung tumors after anti-PD-1 treatment could be M057 inhibited. More importantly, 30 lung cancer patients with TT-EV/miR-5193 hi and TT-EV/miR-5193 lo , as well as those with higher C1qA levels (denoted as C1qA hi ) and lower C1qA levels (denoted as C1qA lo ) were respectively counted. In the progression-free survival (PFS) after anti-PD-1 treatment, as shown in Figure 132, it can be seen that as a negative or positive regulator of CD93, high sEV-derived miRNA-5193 or C1qA is associated with anti-PD in lung cancer patients. -1 is related to the progression-free survival after treatment.
综上所述，C1q和IGFBP7在肿瘤发展过程中上调。如图133，一方面，C1q/CD93信号通路在pMCs中抑制CCL21的产生，阻止DC向肺迁移和随后的抗肺肿瘤的T细胞免疫应答的激活。另一方面，IGFBP7/CD93信号通路在ECs中促进VEGFR途径激活，导致血管生成和肿瘤生长。在抗CD93存在时，pMCs中的C1q/CD93信号通路和ECs中IGFBP7/CD93信号通路被阻断，分别导致CCL21介导的T细胞免疫应答激活和VEGFR介导的肿瘤血管生成的抑制，从而抑制肿瘤生长。因此，抗CD93表现出强大的抗肺肿瘤免疫。 In summary, C1q and IGFBP7 are upregulated during tumor development. As shown in Figure 133, on the one hand, the C1q/CD93 signaling pathway inhibits the production of CCL21 in pMCs, preventing DC migration to the lungs and subsequent activation of T cell immune responses against lung tumors. On the other hand, the IGFBP7/CD93 signaling pathway promotes VEGFR pathway activation in ECs, leading to angiogenesis and tumor growth. In the presence of anti-CD93, the C1q/CD93 signaling pathway in pMCs and the IGFBP7/CD93 signaling pathway in ECs are blocked, respectively leading to the activation of CCL21-mediated T cell immune responses and the inhibition of VEGFR-mediated tumor angiogenesis, thereby inhibiting Tumor growth. Therefore, anti-CD93 exhibits potent anti-lung tumor immunity.
实施例8抗胸膜CD93对甲型流感病毒(H1N1)引起的肺部感染具有治疗作用Example 8 Antipleural CD93 has therapeutic effect on pulmonary infection caused by influenza A virus (H1N1)
为了研究抗胸膜CD93对肺部感染是否有治疗作用，我们构建了肺部H1N1感染小鼠模型，并在小鼠胸腔注射抗CD93试剂(胆固醇偶联Cd93 siRNA)，发现感染H1N1的小鼠，其胸膜CD93表达显著升高(图134)，敲低胸膜中的Cd93能抑制病毒H1N1的肺部感染(图135)，小鼠肺损伤明显减轻(图136)，生存率明显提高(图137)。In order to study whether anti-pleural CD93 has a therapeutic effect on pulmonary infection, we constructed a mouse model of pulmonary H1N1 infection and injected anti-CD93 reagent (cholesterol-conjugated Cd93 siRNA) into the chest cavity of mice. We found that mice infected with H1N1 The expression of CD93 in the pleura was significantly increased (Figure 134). Knocking down Cd93 in the pleura can inhibit the lung infection of virus H1N1 (Figure 135). The lung damage of mice was significantly reduced (Figure 136), and the survival rate was significantly improved (Figure 137).
上述具体实施例中的试验材料和方法如下：The test materials and methods in the above specific examples are as follows:
1.人源样本来源1. Source of human samples
肺TTs来自浙江肿瘤医院的生物样本库。MPEs来自肺癌病人，血液样本来自健康志愿者和接受抗PD-1治疗的肺癌病人，以上标本均来自浙江大学医学院第二附属医院。Lung TTs were obtained from the biobank of Zhejiang Cancer Hospital. MPEs were from lung cancer patients, and blood samples were from healthy volunteers and lung cancer patients receiving anti-PD-1 treatment. The above specimens were all from the Second Affiliated Hospital of Zhejiang University School of Medicine.
2.小鼠和细胞株信息2. Mouse and cell line information
C57BL/6J、BALB/c和裸鼠(6-8周，平均体重20g)购买自上海西普尔-必凯实验动物有限公司。p53fl/flLSL-KrasG12D小鼠由浙江大学的应颂敏教授捐助。Ccr7-/-由复旦大学的李健华教授捐助。CD11c-DTR小鼠购买自杰克逊实验室(美国，法明顿)。Cd93-/-小鼠由中国科学院苏立波教授捐助。动物饲养在特殊的无病原体调节下，动物试验方案由浙江大学动物实验伦理委员批准。C57BL/6J, BALB/c and nude mice (6-8 weeks, average weight 20g) were purchased from Shanghai Sipur-Bike Laboratory Animal Co., Ltd. p53 fl/fl LSL-Kras G12D mice were donated by Professor Ying Songmin from Zhejiang University. Ccr 7-/- was donated by Professor Li Jianhua from Fudan University. CD11c-DTR mice were purchased from The Jackson Laboratory (Farmington, USA). Cd93-/- mice were donated by Professor Su Libo of the Chinese Academy of Sciences. The animals were kept under special pathogen-free conditions, and the animal experiment protocol was approved by the Animal Experiment Ethics Committee of Zhejiang University.
小鼠4T1乳腺癌和3T3纤维母细胞购自美国ATCC(American Type CultureCollection)。小鼠LLC-Luci肺癌细胞和B16F10-Luci黑色素瘤细胞购自珀金埃尔默。小鼠pMCs由浙江大学医学院附属第二医院李冰皓提供。小鼠MLE-12肺上皮细胞和内皮细胞(HUVECs)由浙江大学柯越凯教授提供。人NCI-H2452间皮瘤细胞购自宁波明舟生物科技有限公司(浙江，宁波)。Mouse 4T1 breast cancer and 3T3 fibroblast cells were purchased from ATCC (American Type Culture Collection). Mouse LLC-Luci lung cancer cells and B16F10-Luci melanoma cells were purchased from PerkinElmer. Mouse pMCs were provided by Li Binghao, Second Affiliated Hospital of Zhejiang University School of Medicine. Mouse MLE-12 lung epithelial cells and endothelial cells (HUVECs) were provided by Professor Ke Yuekai of Zhejiang University. Human NCI-H2452 mesothelioma cells were purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (Ningbo, Zhejiang).
3.抗体3. Antibodies
抗体信息如下表所示。Antibody information is shown in the table below.
表1抗体来源及相关信息表


Table 1 Antibody sources and related information table


4.EV分离4. EV isolation
将FBS在120000×g下离心10h移除EVs，然后加入DMEM至终浓度为10％(v/v)。LLC、4T1和B16细胞在10cm细胞培养皿上培养至大约90％的汇合。肺TTs剪切后于RPMI-1640培养基中用2mg/ml胶原酶IV型(Worthington Biochemical，美国)和0.2mg/ml脱氧核糖核酸酶I(Sigma–Aldrich，美国)进行消化，消化条件包括37℃下振摇1h。加入移除EVs的FBS终止消化。收集培养基、消化液和MPE并在4℃下300×g离心5min(移除细胞)，2000×g离心20min(移除碎屑和凋亡小体)和10000×g离心30min(移除大的EVs)。用0.22μm滤膜进行过滤，取滤液在4℃120000×g下离心70min。EVs中的蛋白含量通过BCA蛋白定量试剂盒(Thermo Fisher Scientific)进行测定。FBS was centrifuged at 120000 × g for 10 h to remove EVs, and then DMEM was added to a final concentration of 10% (v/v). LLC, 4T1 and B16 cells were cultured on 10 cm cell culture dishes to approximately 90% confluence. Lung TTs were sheared and digested with 2 mg/ml collagenase type IV (Worthington Biochemical, USA) and 0.2 mg/ml deoxyribonuclease I (Sigma-Aldrich, USA) in RPMI-1640 medium. The digestion conditions included 37 Shake at ℃ for 1 hour. Digestion was terminated by adding FBS to remove EVs. Collect culture medium, digestion fluid, and MPE and centrifuge at 4°C at 300 × g for 5 min (to remove cells), 2000 × g for 20 min (to remove debris and apoptotic bodies), and 10000 × g for 30 min (to remove large cells). EVs). Filter with a 0.22 μm filter membrane, and centrifuge the filtrate at 120,000×g for 70 min at 4°C. The protein content in EVs was determined by BCA protein quantification kit (Thermo Fisher Scientific).
5.EM(Electron Microscope，电子显微镜)5.EM (Electron Microscope, electron microscope)
使用辉光放电质谱仪(glow discharge instrument)对200目碳膜进行亲水化处理，以用于阴性EV染色。取EV溶液滴加至200目碳铜网上并室温(RT)放置1h。用滤纸吸取过量得悬浊液并用蒸馏水冲洗网膜两次，EVs用2％乙酸双氧铀(uranyl acetate)在室温下染色1min，移除过量悬浊液并晾干。用EM(Tecnai G2 Spirit 120kV，Thermo FEI,Hillsboro，USA)成像。A 200-mesh carbon membrane was hydrophilized using a glow discharge mass spectrometer for negative EV staining. Add the EV solution dropwise onto the 200 mesh carbon copper grid and leave it at room temperature (RT) for 1 hour. Use filter paper to absorb excess suspension and rinse the omentum twice with distilled water. EVs are stained with 2% uranyl acetate for 1 min at room temperature. Excess suspension is removed and dried. Imaging was performed with EM (Tecnai G2 Spirit 120 kV, Thermo FEI, Hillsboro, USA).
6.NTA(Nanoparticle Tracking Analysis，纳米颗粒追踪分析)6.NTA (Nanoparticle Tracking Analysis, nanoparticle tracking analysis)
为了测量粒度和浓度，用配备有488nm激光和高灵敏度sCMOS相机的NanoSight NS300***(Malvern PANalytical，上海)对EVs进行NTA。To measure particle size and concentration, NTA of EVs was performed using a NanoSight NS300 system (Malvern PANalytical, Shanghai) equipped with a 488nm laser and a high-sensitivity sCMOS camera.
7.蛋白免疫印迹(Western blotting)7. Western blotting
用冰PBS缓冲液清洗总细胞和EVs，在SDS缓冲液中裂解，置于冰上和100℃煮10min。然后，用SDS-PAGE将样品中的蛋白进行分离，并将蛋白转移到PVDF膜(Millipore)上， 用相应的一抗和HRP(horseradish peroxidase，辣根过氧化物酶)偶联的二抗进行检测。用ECL试剂盒(MultiSciences，浙江杭州)对条带进行显色。Wash total cells and EVs with ice-cold PBS buffer, lyse in SDS buffer, place on ice and boil at 100°C for 10 min. Then, SDS-PAGE was used to separate the proteins in the sample, and the proteins were transferred to PVDF membrane (Millipore). Use the corresponding primary antibody and HRP (horseradish peroxidase, horseradish peroxidase)-coupled secondary antibody for detection. The bands were developed using an ECL kit (MultiSciences, Hangzhou, Zhejiang).
8.肺荷瘤小鼠的处理8. Treatment of lung tumor-bearing mice
注射1％戊巴比妥钠(Sigma–Aldrich)麻醉小鼠，在0、2、4、6和8天分别注射5μg LLC-EVs、B16F10-EVs或4T1-EVs至小鼠胸腔(参照Stathopoulos GT,Zhu Z,Everhart MB,Kalomenidis I,Lawson WE,Bilaceroglu S,et al.Nuclear factor-kappaB affects tumor progression in a mouse model of malignant pleural effusion.American journal of respiratory celland molecular biology 2006,34(2):142-150.)。然后，在第5天注射1×106LLC-Luci或B16F10-Luci细胞构建肺转移性肿瘤。为了构建继发性肺转移瘤模型，0天，在小鼠皮下植入1×106 4T1细胞并在16天进行手术移除。然后，在26、28、30、32和34天，在这些小鼠的胸膜内注入5μg 4T1-EVs。为了评价治疗效果，小鼠在0天静脉注射1×106LLC-Luci细胞，并在2、4、6、8和10天腹腔注射(i.pl.Injection)5μg LLC-EVs。部分试验中，在每次处理前会在这些小鼠腹腔注射10μg siRNAs或40μg anti-VEGFR2(Bio X Cell，West Lebanon，NH，USA)。为了引起继发性肺肿瘤，0和1天在小鼠鼻内滴表达Cre重组酶(2×106PFU ml-1)的腺病毒p53fl/flLSL-KrasG12D；然后，在30、32、34和36天胸膜内注射LLC-EVs。为了检测肿瘤负荷，在14天和25天对小鼠进行麻醉处理并在胸膜内注射100μg/每kg体重的荧光素(Promega，北京)获得携带表达荧光素酶的肿瘤的小鼠。注射荧光素10min后，用IVIS(PerkinElmer,Waltham,MA,USA)获取发光图像，并用Living Image软件(PerkinElmer)分析整个肺区域的光通量。在41天或37天时，将携带原发或转移性肺肿瘤的小鼠处死。然后，用石蜡包埋两侧肺组织，进行H&E染色并用Olympus BX53倒置显微镜(Olympus,Tokyo,Japan)获取H&E染色图像。通过肿瘤总面积除以肺总面积计算转移性肺肿瘤负荷。Mice were anesthetized with 1% sodium pentobarbital (Sigma–Aldrich), and 5 μg of LLC-EVs, B16F10-EVs, or 4T1-EVs were injected into the chest cavity of mice on days 0, 2, 4, 6, and 8, respectively (refer to Stathopoulos GT ,Zhu Z,Everhart MB,Kalomendis I,Lawson WE,Bilaceroglu S,et al.Nuclear factor-kappaB affects tumor progression in a mouse model of malignant pleural effusion.American journal of respiratory celland molecular biology 2006,34(2):142 -150.). Then, 1 × 10 6 LLC-Luci or B16F10-Luci cells were injected on day 5 to construct lung metastatic tumors. To construct a secondary lung metastasis model, 1 × 10 6 4T1 cells were subcutaneously implanted in mice on day 0 and surgically removed on day 16. Then, on days 26, 28, 30, 32, and 34, these mice were injected intrapleurally with 5 μg of 4T1-EVs. To evaluate the therapeutic effect, mice were intravenously injected with 1 × 10 6 LLC-Luci cells on day 0, and 5 μg LLC-EVs were injected intraperitoneally (i.pl. Injection) on days 2, 4, 6, 8, and 10. In some experiments, these mice were intraperitoneally injected with 10 μg siRNAs or 40 μg anti-VEGFR2 (Bio X Cell, West Lebanon, NH, USA) before each treatment. To induce secondary lung tumors, adenovirus p53 fl/fl LSL-Kras G12D expressing Cre recombinase (2×10 6 PFU ml -1 ) was intranasally instilled in mice on days 0 and 1; then, on days 30 and 32 , intrapleural injection of LLC-EVs on days 34 and 36. To detect tumor burden, mice were anesthetized and intrapleurally injected with 100 μg/kg body weight of luciferin (Promega, Beijing) on days 14 and 25 to obtain mice carrying luciferase-expressing tumors. Ten minutes after injection of fluorescein, luminescence images were acquired using IVIS (PerkinElmer, Waltham, MA, USA), and Living Image software (PerkinElmer) was used to analyze the luminous flux in the entire lung area. Mice bearing primary or metastatic lung tumors were sacrificed at 41 or 37 days. Then, the lung tissues on both sides were embedded in paraffin, H&E stained, and H&E stained images were acquired using an Olympus BX53 inverted microscope (Olympus, Tokyo, Japan). Metastatic lung tumor burden was calculated by dividing total tumor area by total lung area.
9.肺部感染小鼠的处理9. Treatment of mice with lung infection
用甲型流感病毒PR8/A/34(H1N1)鼻滴定小鼠，24h后用实时荧光定量PCR测定小鼠胸膜中CD93 mRNA水平以及H1N1 mRNA的表达水平。第5天开始使用10μg胆固醇偶联Cd93 siRNA(siRNA订购自上海吉玛生物有限公司，Genepharma))溶解在无RNase的双蒸水中，注射至小鼠胸腔中，隔天注射一次，记录小鼠生存情况。然后，将小鼠处死，取肺用石蜡包埋，进行H&E染色并用Olympus BX53倒置显微镜(Olympus,Tokyo,Japan)获取H&E染色图像。Influenza A virus PR8/A/34 (H1N1) was used to intranasally titrate mice, and 24 h later, real-time fluorescence quantitative PCR was used to determine the CD93 mRNA level and H1N1 mRNA expression level in the mouse pleura. Starting from the 5th day, 10 μg of cholesterol-coupled Cd93 siRNA (siRNA was ordered from Shanghai Genepharma) was dissolved in RNase-free double-distilled water and injected into the chest cavity of mice. Injection was performed every other day and the survival of the mice was recorded. Condition. Then, the mice were sacrificed, the lungs were removed, embedded in paraffin, H&E stained, and H&E stained images were acquired using an Olympus BX53 inverted microscope (Olympus, Tokyo, Japan).
10.TILs分离和流式细胞术(Isolation of TILs and flow cytometry) 10.Isolation of TILs and flow cytometry (Isolation of TILs and flow cytometry)
取肺转移瘤模型的肺组织切开、清洗并进行消化处理，消化处理使用2mg ml-1I型胶原酶(Worthington Biochemical)，2mg ml-1IV型胶原酶(Worthington Biochemical)和0.2mg ml-1脱氧核糖核酸酶I(DNase I，Sigma–Aldrich)在RPMI-1640中、37℃下振摇1h，添加含10％FBS的RPMI-1640培养基终止消化过程。然后，用70μm细胞过滤网对消化后的细胞悬浊液进行过滤，红细胞(RBCs)是被裂解的。The lung tissue of the lung metastasis model was cut, cleaned and digested. The digestion process used 2 mg ml -1 type I collagenase (Worthington Biochemical), 2 mg ml -1 type IV collagenase (Worthington Biochemical) and 0.2 mg ml - 1 Deoxyribonuclease I (DNase I, Sigma-Aldrich) was shaken in RPMI-1640 at 37°C for 1 hour, and RPMI-1640 medium containing 10% FBS was added to terminate the digestion process. Then, the digested cell suspension was filtered using a 70 μm cell strainer, and red blood cells (RBCs) were lysed.
进行流式细胞术时，首先将单细胞悬浊液和FcR封闭抗体在4℃下孵育30min，接着用偶联荧光基团的第一抗体在4℃下孵育30min。通过Fixable Viability Dye eFluorTM(Thermo Fisher Scientific)染色排除掉死细胞。将样本注入Deflex流式细胞仪(Beckman Coulter，Brea，CA，USA)进行检测，通过FlowJo软件(Tree Star,Ashland,OR,USA)进行数据分析。When performing flow cytometry, first incubate the single cell suspension and FcR blocking antibody at 4°C for 30 min, and then incubate with the primary antibody coupled to a fluorescent group for 30 min at 4°C. Dead cells were excluded by staining with Fixable Viability Dye eFluor ™ (Thermo Fisher Scientific). The samples were injected into a Deflex flow cytometer (Beckman Coulter, Brea, CA, USA) for detection, and data analysis was performed by FlowJo software (Tree Star, Ashland, OR, USA).
11.BMDC的获得11. Obtaining BMDC
按已有方法(Shen Y,Guo D,Weng L,Wang S,Ma Z,Yang Y,et al.Tumor-derived exosomes educate dendritic cells topromote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway.Oncoimmunology 2017,6(12):e1362527.)获取BMDCs(bone marrow mononuclear cells)。简单来说，取小鼠胫骨和股骨悬浮液，除去红细胞，按2×106个细胞/ml的密度在6孔板上，使用添加有10％FBS、10ng ml-1重组鼠白介素GM-CSF和1ng ml-1鼠IL-4的RPMI1640培养基进行培养。培养48h后，通过清洗移去骨髓造血细胞，剩下松散附着的细胞簇进一步培养48h后获得小鼠BMDCs。为了获取人单核细胞(mononuclear cells，DCs)，使用(Sigma–Aldrich)密度离心肝素处理的来自健康志愿者的血液获取外周血单核细胞，然后重悬于培养基中并允许黏附于6孔板壁。37℃孵育2h后，移除未黏附细胞，取黏附细胞于含10ng ml-1 GM-CSF和1ng ml-1 IL-4的3ml培养基中培养。3天后，移除1.5ml培养基并加入新鲜的相同的培养基。7天后，清洗获得人DCs。According to existing methods (Shen Y, Guo D, Weng L, Wang S, Ma Z, Yang Y, et al. Tumor-derived exosomes educate dendritic cells topromote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway. Oncoimmunology 2017, 6 (12):e1362527.) Obtain BMDCs (bone marrow mononuclear cells). Briefly, take the mouse tibia and femur suspension, remove the red blood cells, put it on a 6-well plate at a density of 2×10 6 cells/ml, and use recombinant mouse interleukin GM-CSF added with 10% FBS and 10ng ml -1 Cultured in RPMI1640 medium with 1ng ml -1 mouse IL-4. After 48 hours of culture, the bone marrow hematopoietic cells were removed by washing, and the remaining loosely attached cell clusters were further cultured for 48 hours to obtain mouse BMDCs. To obtain human mononuclear cells (DCs), use Peripheral blood mononuclear cells were obtained from heparin-treated blood from healthy volunteers by density centrifugation (Sigma–Aldrich), then resuspended in culture medium and allowed to adhere to the walls of a 6-well plate. After incubation at 37°C for 2 hours, non-adherent cells were removed, and adherent cells were cultured in 3 ml medium containing 10 ng ml-1 GM-CSF and 1 ng ml-1 IL-4. After 3 days, 1.5 ml of medium was removed and fresh same medium was added. After 7 days, human DCs were obtained by washing.
12.免疫细胞亚群的移除和VEGFR阻断12. Removal of Immune Cell Subpopulations and VEGFR Blockade
按图8方案用LLC细胞和LLC-EVs处理小鼠。为了移除DCs，在2天开始对CD11c-DTR小鼠连续腹腔注射2μg DT，每两天一次。为了移除CD4+或CD8+T细胞，在2天开始连续腹腔注射60μg抗CD4和16μg抗CD8抗体(Bio X Cell)，每两天一次。为了阻断VEGFR，在2天开始连续静脉注射40μg抗VEGFR2抗体(anti-VEGFR2)，每两天一次。Mice were treated with LLC cells and LLC-EVs according to the protocol in Figure 8 . To remove DCs, CD11c-DTR mice were continuously intraperitoneally injected with 2 μg DT starting on day 2, once every two days. To remove CD4 + or CD8 + T cells, 60 μg of anti-CD4 and 16 μg of anti-CD8 antibodies (Bio X Cell) were continuously injected intraperitoneally starting on day 2, once every two days. To block VEGFR, 40 μg of anti-VEGFR2 antibody (anti-VEGFR2) was continuously injected intravenously starting on day 2, once every two days.
13.EV标记13.EV mark
按使用说明书，用VivoTrack 680(Fluorescence，北京)、PKH26(Sigma–Aldrich)或CFSE(Thermo Fisher Scientific)对EVs进行标记。对于VivoTrack 680标记，在200μl  PBS中使150μg EVs与42μMVivoTrack 680在室温下混合30min。对于PKH26标记，将150μg EVs悬浮于100μl稀释液C中，加入含0.4μl PKH26乙醇染料溶液的100μl稀释液C，然后，混合5min。对于CFSE标记，在200μl PBS中使150μg EVs与7.5μMCFSE在37℃下孵育30min。加入等体积的无外泌体的FBS(exosome-depleted FBS，Thermo Fisher Scientific)并孵育1min终止标记反应。最终，在120000×g下离心70min移除未结合的标记物，并使EV标记物悬浮于200μl PBS中EVs were labeled with VivoTrack 680 (Fluorescence, Beijing), PKH26 (Sigma–Aldrich) or CFSE (Thermo Fisher Scientific) according to the instructions for use. For VivoTrack 680 markers, in 200 μl 150 μg EVs were mixed with 42 μM VivoTrack 680 in PBS for 30 min at room temperature. For PKH26 labeling, 150 μg EVs were suspended in 100 μl diluent C, 100 μl diluent C containing 0.4 μl PKH26 ethanol dye solution was added, and then mixed for 5 min. For CFSE labeling, 150 μg EVs were incubated with 7.5 μM CFSE in 200 μl PBS for 30 min at 37°C. An equal volume of exosome-depleted FBS (Thermo Fisher Scientific) was added and incubated for 1 min to terminate the labeling reaction. Finally, unbound label was removed by centrifugation at 120,000 × g for 70 min, and the EV label was suspended in 200 μl PBS.
14.体外和体内EV摄取情况的检测(Detection of EV uptake in vitro and in vivo)14. Detection of EV uptake in vitro and in vivo
为了检测MCs摄取TEVs情况，用2.5μg ml-1CFSE标记的LLC-EVs处理p-pMCs和40L细胞24h。为了检测TEVs在体内的分布情况，在小鼠静脉或胸膜内注射100μg VivoTrack 680-标记的LLC-EVs，24h后将小鼠安乐死，取脑、心、肺、肝、脾、肾和胃肠器官，并用IVIS(PerkinElmer)获取图像。为了检测TEVs在肺部的分布情况，在小鼠静脉或胸膜内注射20μg PKH26标记的LLC-EVs，24h后收集小鼠肺并用Tissue-TekTM Cryo-O.C.T.Compound(Thermo Fisher Scientific)进行包埋处理，获取10μm的组织切片。接着，用0.5μg ml-1DAPI在室温下处理20min，进行细胞核染色。通过共聚焦显微镜(Olympus IX83-FV3000)观测细胞和染色后的组织切片。为了检测胸膜TEV摄取情况，在小鼠胸膜内注射了20μg CFSE标记的LLC-EVs；部分试验中，在EV注射前2h通过腹腔注射0.25mg kg-1Cyto-D；24h后取小鼠的胸膜，用立体显微镜(Nikon SMZ18，Tokyo，Japan)进行观察。In order to detect the uptake of TEVs by MCs, p-pMCs and 40L cells were treated with 2.5 μg ml -1 CFSE-labeled LLC-EVs for 24 h. In order to detect the distribution of TEVs in the body, mice were injected intravenously or intrapleurally with 100 μg of VivoTrack 680-labeled LLC-EVs. After 24 hours, the mice were euthanized and the brain, heart, lungs, liver, spleen, kidney and gastrointestinal organs were harvested. , and images were acquired using IVIS (PerkinElmer). In order to detect the distribution of TEVs in the lungs, mice were injected intravenously or intrapleurally with 20 μg of PKH26-labeled LLC-EVs. After 24 hours, the mouse lungs were collected and embedded with Tissue-Tek TM Cryo-OCT Compound (Thermo Fisher Scientific). Obtain 10 μm tissue sections. Next, the cells were stained with 0.5 μg ml -1 DAPI for 20 min at room temperature. Cells and stained tissue sections were observed using a confocal microscope (Olympus IX83-FV3000). In order to detect pleural TEV uptake, 20 μg CFSE-labeled LLC-EVs were injected into the pleura of mice; in some experiments, 0.25 mg kg -1 Cyto-D was injected intraperitoneally 2 hours before EV injection; the pleura of mice was taken 24 hours later. , observed with a stereomicroscope (Nikon SMZ18, Tokyo, Japan).
15.EV蛋白和RNAs的降解15. Degradation of EV proteins and RNAs
为了降解EV蛋白，用10μg ml-1蛋白酶K在有电穿孔(electroporation)或无电穿孔情况下分别消化LLC-EVs 2h。为了降解EV RNAs，用10μg ml-1RNase I结合电穿孔(electroporation)消化LLC-EVs 2h。用BTX电穿孔仪(Harvard Biosciences,Cambridge,MA,USA)进行EVs的电穿孔。简单来说，将100μg LLC-EVs和100μl电穿孔缓冲液(Harvard Biosciences)进行混合，在固定电容为100μF的条件下进行典型程序，在0.2cm样品管中效率最佳。To degrade EV proteins, LLC-EVs were digested with 10 μg ml-1 proteinase K for 2 h with or without electroporation. To degrade EV RNAs, LLC-EVs were digested with 10 μg ml-1 RNase I combined with electroporation for 2 h. Electroporation of EVs was performed using a BTX electroporator (Harvard Biosciences, Cambridge, MA, USA). Briefly, 100 μg LLC-EVs and 100 μl electroporation buffer (Harvard Biosciences) were mixed, and a typical procedure was performed at a fixed capacitance of 100 μF, with optimal efficiency in a 0.2 cm sample tube.
16.p-pMCs的分离16. Isolation of p-pMCs
在已有方法(Gilmer J,Serve K,Davis C,Anthony M,Hanson R,Harding T,et al.Libby amphibole-induced mesothelial cell autoantibodies promote collagen deposition in mice.American journal of physiology Lung cellular and molecular physiology 2016,310(11):L1071-1077)的基础上进行了改进，分离获得鼠p-pMCs。简单来说，小鼠处死后，在无菌条件下移取胸壁。用眼钳将胸膜壁层剥离，用PBS清洗两次以移除血迹，并剪切成1mm2的 组织碎片。然后，将胸膜组织接种于含含完全DMEM培养基的25cm2的培养瓶中。次日将未黏附的细胞移除，再培养至72h即可获得MCs。In existing methods (Gilmer J, Serve K, Davis C, Anthony M, Hanson R, Harding T, et al. Libby amphibole-induced mesothelial cell autoantibodies promote collagen deposition in mice. American journal of physiology Lung cellular and molecular physiology 2016, 310(11):L1071-1077), mouse p-pMCs were isolated and obtained. Briefly, after mice were sacrificed, the chest wall was removed under sterile conditions. Peel off the pleural parietal layer with eye forceps, wash twice with PBS to remove blood stains, and cut into 1 mm 2 Tissue fragments. Then, the pleural tissue was inoculated into a 25 cm 2 culture bottle containing complete DMEM medium. The non-adherent cells were removed the next day and cultured for 72 hours to obtain MCs.
17.免疫组织化学着色17. Immunohistochemical staining
将鼠肺和人TT组织切片进行脱蜡和再水化处理，用10mM柠檬酸钠缓冲液(pH 6.0)进行抗原修复。用5％BSA阻断处理后，将组织切片与第一抗体再4℃下孵育过夜，再在室温下与HRP偶联的第二抗体孵育30min。随机获取成像数据并用ImageJ软件(NIH，Bethesda，MD，USA)进行分析。Mouse lung and human TT tissue sections were deparaffinized and rehydrated, and 10mM sodium citrate buffer (pH 6.0) was used for antigen retrieval. After blocking with 5% BSA, the tissue sections were incubated with the primary antibody at 4°C overnight, and then incubated with the HRP-conjugated secondary antibody at room temperature for 30 min. Imaging data were randomly acquired and analyzed using ImageJ software (NIH, Bethesda, MD, USA).
18.趋化性检测(Chemotaxis assay)18. Chemotaxis assay
为了进行DC趋化性检测，用2.5μg ml-1LLC-EVs刺激p-pMCs和40L细胞24h，用2.5μg ml-1A549-EVs刺激NCI-H2452细胞24h。然后，收集上清液，置于3μm孔隙大小的迁移室(Corning Inc.，Corning，NY，USA)的底部小室中，迁移室的上部小室加有DCs，12h后通过流式细胞仪计算迁移细胞数量进而评价DC迁移情况。For DC chemotaxis assay, p-pMCs and 40L cells were stimulated with 2.5 μg ml -1 LLC-EVs for 24 h, and NCI-H2452 cells were stimulated with 2.5 μg ml -1 A549-EVs for 24 h. Then, the supernatant was collected and placed in the bottom chamber of a migration chamber with a pore size of 3 μm (Corning Inc., Corning, NY, USA). DCs were added to the upper chamber of the migration chamber. After 12 h, the migrated cells were counted by flow cytometry. The quantity is used to evaluate the DC migration situation.
19.siRNAs、miRNA类似物和抑制剂的转染19. Transfection of siRNAs, miRNA analogs and inhibitors
按说明书使用TransIT-TKO转染试剂盒(Mirus Bio，Madison，WI，USA)将Scrambled NC或目标siRNA和miRNA类似物，抑制剂或相应对照(GenePharma，上海)进行转染。Scrambled NC or target siRNA and miRNA analogs, inhibitors or corresponding controls (GenePharma, Shanghai) were transfected using the TransIT-TKO transfection kit (Mirus Bio, Madison, WI, USA) according to the instructions.
体内实验：将10μg胆固醇偶联siRNAs溶解在无RNase的双蒸水中，并于每次胞外囊泡胸腔注射前24h注射至肺转移性肿瘤小鼠的胸腔中。siRNAs、miRNA类似物及其抑制剂序列见下表2。In vivo experiments: 10 μg cholesterol-coupled siRNAs were dissolved in RNase-free double-distilled water and injected into the chest cavity of mice with lung metastasis tumors 24 hours before each intrapleural injection of extracellular vesicles. The sequences of siRNAs, miRNA analogs and their inhibitors are shown in Table 2 below.
20.实时荧光定量PCR(Real-time PCR)20. Real-time PCR
按Trizol试剂说明提取总RNA，并按cDNA合成试剂盒(Toyobo，Osaka，Japan)进行逆转录。对于miRNA逆转录，特异性引物由GenePharma合成。进行相对定量分析时，用SYBR Green(Vazyme，南京)在罗氏480实时荧光定量PCR仪进行实时荧光定量PCR。对于细胞和组织样品以GAPDH作为内参进行归一化(GAPDH的PCR引物见SEQ ID NO:7和SEQ ID NO:8)，对于EV样品以U6作为内参进行归一化(U6的PCR引物见SEQ ID NO:13和SEQ ID NO:14)。进行绝对定量时，使用特异性的miRNAs TaqMan探针(GenePharma)和Universal U+Probe Master Mix V2(Vazyme)进行实时荧光定量PCR。引物和探针序列见下表。Total RNA was extracted according to the instructions of Trizol reagent, and reverse transcribed according to the cDNA synthesis kit (Toyobo, Osaka, Japan). For miRNA reverse transcription, specific primers were synthesized by GenePharma. For relative quantitative analysis, SYBR Green (Vazyme, Nanjing) was used at Roche 480 real-time fluorescence quantitative PCR instrument for real-time fluorescence quantitative PCR. For cell and tissue samples, GAPDH was used as an internal control for normalization (for the PCR primers of GAPDH, see SEQ ID NO:7 and SEQ ID NO:8), for EV samples, U6 was used as an internal control for normalization (for the PCR primers of U6, see SEQ ID NO:13 and SEQ ID NO:14). For absolute quantification, specific miRNAs TaqMan probes (GenePharma) and Universal U+Probe Master Mix V2 (Vazyme) was used for real-time fluorescence quantitative PCR. Primer and probe sequences are shown in the table below.
表2序列表


Table 2 Sequence List


21.RNA-Seq分析21.RNA-Seq analysis
提取总RNA并反转录，构建cDNA文库，该文库送至杭州联科生物公司。在Illumina HiSeq2000平台上进行测序，通过Bowtie2将各reads序列比对至小鼠基因组参考序列(GRCm38)。利用R 4.1.2软件中的“limma”包，以|Log2FC|>1和P值<0.05为标准，分析并筛选PBS和TEVs组之间的差异表达基因(DEGs)。随后，用“gplot”包中的heatmap.2功能绘制DEGs热图，用“corrplot”包计算DEG两两之间的Pearson相关系数。Total RNA was extracted and reverse transcribed to construct a cDNA library, which was sent to Hangzhou Lianke Biotechnology Company. Sequencing was performed on the Illumina HiSeq2000 platform, and each read sequence was aligned to the mouse genome reference sequence (GRCm38) through Bowtie2. The "limma" package in R 4.1.2 software was used to analyze and screen the differentially expressed genes (DEGs) between PBS and TEVs groups using |Log2FC|>1 and P value <0.05. Subsequently, the heatmap.2 function in the "gplot" package was used to draw the DEGs heat map, and the "corrplot" package was used to calculate the Pearson correlation coefficient between two DEGs.
22.miRNA微阵列测序分析(MiRNA array)22.miRNA microarray sequencing analysis (MiRNA array)
收集LLC-EVs和MLE-12-EVs样本，在安捷伦SurePrint Mouse miRNA 8×60K v.21.0芯片上进行两样本中miRNA的微阵列分析。用特征提取软件v.11.5.1.1(安捷伦)进行数据采集、数据提取和质量控制分析，再用用Gene Spring GX软件对各样本间的信号进行标准化处理。用R 4.1.2软件中的“limma”包，以|Log2FC|>1和P值<0.05为标准，鉴定组间差异表达的miRNA。LLC-EVs and MLE-12-EVs samples were collected, and microarray analysis of miRNA in the two samples was performed on the Agilent SurePrint Mouse miRNA 8×60K v.21.0 chip. Feature extraction software v.11.5.1.1 (Agilent) was used for data acquisition, data extraction and quality control analysis, and Gene Spring GX software was used to standardize the signals between each sample. Use the "limma" package in R 4.1.2 software to identify differentially expressed miRNAs between groups, using |Log2FC|>1 and P value <0.05 as standards.
23.细胞因子的测量 23. Measurement of Cytokine
用2.5μg ml-1LLC-EVs刺激p-pMCs和40L细胞以及用2.5μg ml-1A549-EVs刺激NCI-H2452细胞24h。300×g离心5min移除细胞和残渣。为了检测p-pMCs细胞分泌的细胞因子，将小鼠处死后，取等面积胸膜按上述方法培养24h。通过ELISA测定培养基上层液体中的细胞因子和MPE液体样本中的CCL21水平。为了检测体内的细胞因子，收集小鼠和人体血清，通过ELISA测定C1qA水平。具体按鼠CCL19试剂盒(Absin，上海)，鼠CCL21a、人Exodus 2 ELISA试剂盒(Abcam，Cambridge，UK)以及人C1qA ELISA试剂盒(Abcam)的说明书进行。p-pMCs and 40L cells were stimulated with 2.5 μg ml −1 LLC-EVs and NCI-H2452 cells were stimulated with 2.5 μg ml −1 A549-EVs for 24 h. Centrifuge at 300×g for 5 minutes to remove cells and debris. In order to detect the cytokines secreted by p-pMCs cells, the mice were sacrificed, and equal areas of pleura were taken and cultured according to the above method for 24 hours. Cytokines in the culture supernatant fluid and CCL21 levels in MPE fluid samples were determined by ELISA. To detect cytokines in vivo, mouse and human sera were collected and C1qA levels were determined by ELISA. Specifically, the instructions of mouse CCL19 kit (Absin, Shanghai), mouse CCL21a, human Exodus 2 ELISA kit (Abcam, Cambridge, UK) and human C1qA ELISA kit (Abcam) were carried out.
24.ELISA检测EV miRNA含量24. ELISA detects EV miRNA content
在anti-CD63抗体包被的96孔ELISA板中加入等量的TT-EVs和MPE-EVs，在37℃下孵育过夜使其被96孔板吸附。用无RNase的Intracellular Fixation and Permeabilization Buffer(Thermo Fisher Scientific)固定囊泡、破囊泡膜，再用20nM生物素标记抗CD9抗体或miR-5193探针在37℃下孵育1h。洗涤后，每孔加入25μl avidin-HRP并室温孵育1h。最后，加入TMB显色，2M硫酸溶液终止显色，并在450nm波长处测量吸光度。Add equal amounts of TT-EVs and MPE-EVs to the 96-well ELISA plate coated with anti-CD63 antibody, and incubate overnight at 37°C to allow adsorption to the 96-well plate. Use RNase-free Intracellular Fixation and Permeabilization Buffer (Thermo Fisher Scientific) to fix the vesicles and rupture the vesicle membrane, and then incubate with 20nM biotin-labeled anti-CD9 antibody or miR-5193 probe at 37°C for 1 hour. After washing, 25 μl avidin-HRP was added to each well and incubated at room temperature for 1 h. Finally, TMB was added to develop color, 2M sulfuric acid solution was used to terminate the color development, and the absorbance was measured at a wavelength of 450 nm.
25.荧光素酶报告基因分析25.Luciferase reporter gene assay
扩增包含潜在miR-5110结合位点的鼠Cd93 mRNA 3’-UTR序列并将其克隆到GP-miRGLO载体(一种含双荧光素酶报告基因的质粒，Genepharma)，构建成含萤光素酶报告基因的WTCd93 3’-UTR载体。同理，通过WT序列的反义链构建突变型Cd93 3’-UTR载体。将WT Cd93 3’-UTR载体或突变型Cd93 3’-UTR载体和miRNA类似物或参照物(终浓度60nM)转染40L细胞。24h后收集细胞并裂解，用Duo-Lite Luciferase Assay System(Vazyme)检测荧光素酶活性。The mouse Cd93 mRNA 3'-UTR sequence containing the potential miR-5110 binding site was amplified and cloned into the GP-miRGLO vector (a plasmid containing a dual-luciferase reporter gene, Genepharma) to construct a luciferin-containing WTCd93 3'-UTR vector of enzyme reporter gene. In the same way, a mutant Cd93 3’-UTR vector was constructed through the antisense strand of the WT sequence. 40L cells were transfected with WT Cd93 3’-UTR vector or mutant Cd93 3’-UTR vector and miRNA analogues or reference (final concentration 60nM). After 24 hours, cells were collected and lysed, and luciferase activity was detected using Duo-Lite Luciferase Assay System (Vazyme).
26.重组蛋白和抗体的产生26. Production of recombinant proteins and antibodies
将C1qA，IGFBP7和MMRN2克隆到带flag标签序列的pcDNA3.1载体中，构建成CD93配体的表达载体。将该表达载体通过JetPEI(Polyplus，上海)转染到CTC细胞中并使CD93配体表达，用anti-FLAG M2磁珠(Sigma-Aldrich)和flag-tag多肽(Yeasen，上海)分离带flag标签的结合蛋白。克隆免疫兔抗体的可变区和鼠IgG1抗体的恒定区序列至pcDNA3.1载体中，构建Anti-mouse CD93rabbit/mouse IgG1 chimeric mAb(Sino Biological，北京)。克隆M057是增加CCL21a在p-pMCs表达最有效的方式。C1qA, IGFBP7 and MMRN2 were cloned into the pcDNA3.1 vector with flag tag sequence to construct an expression vector for CD93 ligand. The expression vector was transfected into CTC cells through JetPEI (Polyplus, Shanghai) to express CD93 ligand, and anti-FLAG M2 magnetic beads (Sigma-Aldrich) and flag-tag polypeptide (Yeasen, Shanghai) were used to isolate the flag tag. binding protein. The variable region of the immune rabbit antibody and the constant region sequence of the mouse IgG1 antibody were cloned into the pcDNA3.1 vector to construct Anti-mouse CD93rabbit/mouse IgG1 chimeric mAb (Sino Biological, Beijing). Cloning M057 is the most effective way to increase the expression of CCL21a in p-pMCs.
27.内皮血管生成试验27. Endothelial angiogenesis assay
用2mg ml-1I型胶原酶(Worthington Biochemical)，2mg ml-1IV型胶原酶(Worthington  Biochemical)和0.2mg ml-1脱氧核糖核酸酶I(DNase I，Sigma–Aldrich)在37℃下消化肺组织1h，用含10％FBS的培养基终止反应。用70μm细胞筛过滤获得单细胞悬浊液。用抗鼠CD31-PE抗体(Biolegend，San Diego，CA，USA)和PE阳性选择试剂盒(StemCel，Vancouver，BC，Canada)分离CD31阳性的原发ECs，使用前在内皮细胞培养基(Procell,Wuhan,China)中培养5天。Use 2 mg ml -1 type I collagenase (Worthington Biochemical), 2 mg ml -1 type IV collagenase (Worthington Biochemical) Biochemical) and 0.2 mg ml -1 deoxyribonuclease I (DNase I, Sigma-Aldrich) to digest lung tissue at 37°C for 1 h, and the reaction was terminated with culture medium containing 10% FBS. Filter through a 70 μm cell sieve to obtain a single cell suspension. CD31-positive primary ECs were isolated using anti-mouse CD31-PE antibody (Biolegend, San Diego, CA, USA) and PE positive selection kit (StemCel, Vancouver, BC, Canada) before use in endothelial cell culture medium (Procell, Wuhan, China) for 5 days.
用2μgml-1IGFBP7和/或2μgml-1M057处理24h后，将鼠原发性ECs按每孔1×104细胞接种至包被内皮生长因子减少基质的ibidi板(BD Bioscience，Sab Jose，CA，USA)中。用6.25μg/ml钙黄绿素AMZ对鼠原发性ECs进行染色。用倒置荧光显微镜观察获取毛细管生长情况的图像，用ImageJ(NIH)血管生成分析仪插件进行分析。After treatment with 2 μg ml -1 IGFBP7 and/or 2 μg ml -1 M057 for 24 h, murine primary ECs were seeded at 1 × 10 4 cells per well into ibidi plates coated with endothelial growth factor-reduced matrix (BD Bioscience, Sab Jose, CA , USA). Murine primary ECs were stained with 6.25 μg/ml calcein AMZ. An inverted fluorescence microscope was used to observe and obtain images of capillary growth, and the ImageJ (NIH) angiogenesis analyzer plug-in was used for analysis.
28.蛋白标记和检测28. Protein labeling and detection
按使用说明书用蛋白标记试剂盒(Thermo Fisher Scientific)对M057进行标记。将100μg Alexa Fluor 680-labeled anti-CD93静脉注射至WT和Cd93-/-小鼠，24h后将小鼠处死，收集双侧胸壁，用IVIS(PerkinElmer)观察记录anti-CD93成像情况。M057 was labeled with a protein labeling kit (Thermo Fisher Scientific) according to the instruction manual. 100 μg of Alexa Fluor 680-labeled anti-CD93 was intravenously injected into WT and Cd93 −/− mice. The mice were sacrificed 24 hours later, and the bilateral chest walls were collected, and the anti-CD93 imaging was observed and recorded with IVIS (PerkinElmer).
下述列出的为本申请的参考文献，本申请所提供的所有参考文献(例如CN202080075436.0)的内容都属于本申请内容的一部分。The following are the references of this application. The contents of all references provided in this application (for example, CN202080075436.0) are part of the content of this application.
本发明说明书中提到的所有专利和出版物都表示这些是本领域的公开技术，本发明可以使用。这里所引用的所有专利和出版物都被同样列在参考文献中，跟每一个出版物具体的单独被参考引用一样。这里所述的本发明可以在缺乏任何一种元素或多种元素，一种限制或多种限制的情况下实现，这里这种限制没有特别说明。例如这里每一个实例中术语“包含”，“实质由……组成”和“由……组成”可以用两者之一的其余2个术语代替。这里的所谓的“一个”仅仅表示“一”的意思，而不排除仅仅只是包括一个，也可以表示包括2个以上。这里采用的术语和表达方式所为描述方式，而不受其限制，这里也没有任何意图来指明此书描述的这些术语和解释排除了任何等同的特征，但是可以知道，可以在本发明和权利要求的范围内做任何合适的改变或修改。可以理解，本发明所描述的实施例子都是一些优选的实施例子和特点，任何本领域的一般技术人员都可以根据本发明描述的精髓下做一些更改和变化，这些更改和变化也被认为属于本发明的范围和独立权利要求以及附属权利要求所限制的范围内。All patents and publications mentioned in the description of the present invention indicate that they are published techniques in the field and can be used by the present invention. All patents and publications cited herein are similarly incorporated by reference to the same extent as if each publication was specifically and individually cited by reference. The invention described herein may be practiced in the absence of any element or elements, limitation or limitations, no such limitation is specifically stated herein. For example, in each instance herein the terms "comprising", "consisting essentially of" and "consisting of" may be replaced by one of the remaining two terms. The so-called "a" here only means "one", and it does not exclude that it only includes one, and can also mean including two or more. The terms and expressions used herein are by way of description without limitation, nor is there any intention that these terms and expressions described in this book exclude any equivalent features, but it will be understood that the invention and rights may be Make any appropriate changes or modifications within the scope required. It can be understood that the implementation examples described in the present invention are some preferred implementation examples and features. Any person of ordinary skill in the art can make some modifications and changes based on the essence of the description of the present invention. These modifications and changes are also considered to belong to The scope of the invention is limited by the independent claims and the appended claims.
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Claims (34)

  1. 一种阻断剂，所述的阻断剂是一种能够特异性抑制C1q/CD93信号传导途径的试剂。A blocking agent is a reagent that can specifically inhibit the C1q/CD93 signaling pathway.
  2. 根据权利要求1所述的阻断剂，其中，所述的阻断剂能够阻断C1q/CD93之间的相互作用。The blocking agent according to claim 1, wherein the blocking agent can block the interaction between C1q/CD93.
  3. 根据权利要求2所述的阻断剂，其中，所述的阻断剂包括特异性识别CD93的抗体或者抗体片段，或者特异结合或者识别C1q的抗体或者抗体片段；或者能够降低CD93表达的siRNA、shRNA、miRNA、反义RNA或基因编辑***。The blocking agent according to claim 2, wherein the blocking agent includes an antibody or antibody fragment that specifically recognizes CD93, or an antibody or antibody fragment that specifically binds or recognizes C1q; or siRNA that can reduce the expression of CD93, shRNA, miRNA, antisense RNA or gene editing systems.
  4. 根据权利要求3所述的阻断剂，其中，所述的阻断剂通过阻断C1q/CD93之间的相互作用，从而让CCL21的表达增多，或者活性增强，从而让树突状细胞向肺部肿瘤或肺部感染细胞聚集。The blocking agent according to claim 3, wherein the blocking agent increases the expression or activity of CCL21 by blocking the interaction between C1q/CD93, thereby allowing dendritic cells to move to the lungs. Local tumors or infected cells accumulate in the lungs.
  5. 根据权利要求3所述的阻断剂，其中，所述的CD93位于胸膜间皮细胞中。The blocking agent according to claim 3, wherein the CD93 is located in pleural mesothelial cells.
  6. 根据权利要求5所述的阻断剂，其中，所述特异性识别CD93的CD93抗体是抗人CD93抗体。The blocking agent of claim 5, wherein the CD93 antibody that specifically recognizes CD93 is an anti-human CD93 antibody.
  7. 根据权利要求6所述的阻断剂，其中，所述的抗人CD93抗体是全长抗体、单链Fv(scFv)、Fab、Fab’、F(ab’)2、Fv片段、二硫键稳定化的Fv片段(dsFv)、(dsFv)2、VHH、Fv-Fc融合物、scFv-Fc融合物、scFv-Fv融合物、双抗体、三抗体或四抗体。The blocking agent according to claim 6, wherein the anti-human CD93 antibody is a full-length antibody, single chain Fv (scFv), Fab, Fab', F(ab')2, Fv fragment, disulfide bond Stabilized Fv fragment (dsFv), (dsFv)2, VHH, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody or tetrabody.
  8. 根据权利要求6所述的阻断剂，其中，所述的CD93抗体包含在融合蛋白中。The blocking agent according to claim 6, wherein the CD93 antibody is included in a fusion protein.
  9. 根据权利要求3所述的阻断剂，其中，所述的抗体与CD93结合的亲和力大于C1q与CD93结合的亲和力。The blocking agent according to claim 3, wherein the affinity of the antibody for binding to CD93 is greater than the affinity of C1q for binding to CD93.
  10. 根据权利要求2所述的阻断剂，其中，阻断剂为多肽或者多肽片段。The blocking agent according to claim 2, wherein the blocking agent is a polypeptide or a polypeptide fragment.
  11. 根据权利要求10所述的阻断剂，其中，所述的多肽是结合并抑制CD93功能的多肽，其结合于CD93的不同胞外结构域。The blocking agent according to claim 10, wherein the polypeptide is a polypeptide that binds to and inhibits the function of CD93 and binds to different extracellular domains of CD93.
  12. 根据权利要求11所述的阻断剂，其中，所述的多肽是可溶性多肽。The blocking agent according to claim 11, wherein said polypeptide is a soluble polypeptide.
  13. 根据权利要求11所述的阻断剂，所述多肽与CD93的结合亲和力大于与C1q的结合亲和力。According to the blocking agent of claim 11, the binding affinity of the polypeptide to CD93 is greater than the binding affinity to C1q.
  14. 一种能够特异性抑制C1q/CD93信号传导途径的阻断剂在用于制备肺癌或者肺部感染试剂上的用途。A blocker that can specifically inhibit the C1q/CD93 signaling pathway is used for preparing a lung cancer or lung infection reagent.
  15. 根据权利要求14所述的用途，其中，所述的阻断剂能够阻断C1q/CD93之间的相互作用。The use according to claim 14, wherein the blocking agent can block the interaction between C1q/CD93.
  16. 根据权利要求14所述的用途，其中，所述的阻断剂至少具有如下功能之一：(1) 降低C1q或者CD93的基因表达量；(2)降低C1q或者CD93的蛋白的活性；(3)降低C1q或者CD93的识别或者结合能力。The use according to claim 14, wherein the blocking agent has at least one of the following functions: (1) Reduce the gene expression of C1q or CD93; (2) Reduce the protein activity of C1q or CD93; (3) Reduce the recognition or binding ability of C1q or CD93.
  17. 根据权利要求16所述的用途，其中，所述的阻断剂是特异结合或者识别CD93的抗体或者抗体片段，或者特异结合或者识别C1q的抗体或者抗体片段；或者能够降低CD93表达的siRNA、shRNA、miRNA、反义RNA或基因编辑***；或者降低C1q表达的siRNA、shRNA、miRNA、反义RNA或基因编辑***。The use according to claim 16, wherein the blocking agent is an antibody or antibody fragment that specifically binds or recognizes CD93, or an antibody or antibody fragment that specifically binds or recognizes C1q; or siRNA or shRNA that can reduce the expression of CD93 , miRNA, antisense RNA or gene editing system; or siRNA, shRNA, miRNA, antisense RNA or gene editing system that reduces C1q expression.
  18. 根据权利要求16所述的用途，其中，所述的阻断剂通过阻断C1q/CD93之间的相互作用，从而让CCL21的表达增多，或者活性增强，从而让树突状细胞向肺部肿瘤或肺部感染细胞的区域聚集。The use according to claim 16, wherein the blocking agent increases the expression or activity of CCL21 by blocking the interaction between C1q/CD93, thereby allowing dendritic cells to contribute to lung tumors. or areas of collection of infected cells in the lungs.
  19. 根据权利要求16所述的用途，其中，所述的CD93是胸膜间皮细胞中的。The use according to claim 16, wherein said CD93 is in pleural mesothelial cells.
  20. 根据权利要求17所述的用途，其中，CD93抗体是抗人CD93抗体。The use according to claim 17, wherein the CD93 antibody is an anti-human CD93 antibody.
  21. 根据权利要求20所述的用途，其中，所述的抗人CD93抗体是全长抗体、单链Fv(scFv)、Fab、Fab’、F(ab’)2、Fv片段、二硫键稳定化的Fv片段(dsFv)、(dsFv)2、VHH、Fv-Fc融合物、scFv-Fc融合物、scFv-Fv融合物、双抗体、三抗体或四抗体。The use according to claim 20, wherein the anti-human CD93 antibody is a full-length antibody, single chain Fv (scFv), Fab, Fab', F(ab')2, Fv fragment, disulfide bond stabilized Fv fragment (dsFv), (dsFv)2, VHH, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody or tetrabody.
  22. 根据权利要求21所述的用途，其中，所述的CD93抗体包含在融合蛋白中。The use according to claim 21, wherein the CD93 antibody is included in a fusion protein.
  23. 根据权利要求20所述的用途，其中，所述的抗体与CD93结合的亲和力大于C1q与CD93结合的亲和力。The use according to claim 20, wherein the affinity of the antibody for binding to CD93 is greater than the affinity of C1q for binding to CD93.
  24. 根据权利要求16所述的用途，其中，阻断剂为多肽或者多肽片段。The use according to claim 16, wherein the blocking agent is a polypeptide or a polypeptide fragment.
  25. 根据权利要求24所述的用途，其中，所述的多肽是结合并抑制CD93功能的多肽，其结合于CD93的不同胞外结构域。The use according to claim 24, wherein the polypeptide is a polypeptide that binds to and inhibits the function of CD93, and binds to different extracellular domains of CD93.
  26. 根据权利要求25所述的用途，其中，所述的多肽是可溶性多肽。The use according to claim 25, wherein said polypeptide is a soluble polypeptide.
  27. 根据权利要求26所述的用途，所述多肽与CD93的结合亲和力大于与C1q的结合亲和力。According to the use of claim 26, the binding affinity of the polypeptide to CD93 is greater than the binding affinity to C1q.
  28. 根据权利要求26所述的用途，所述的阻断剂能促进全身肿瘤特异性的T细胞介导的细胞免疫，在抗肺部肿瘤的同时具有抗其他部位同种肿瘤的作用。According to the use of claim 26, the blocking agent can promote systemic tumor-specific T cell-mediated cellular immunity, and has the effect of resisting the same type of tumors in other parts of the body while resisting lung tumors.
  29. 根据权利要求28所述的用途,所述的肺部肿瘤包括肺部原生肿瘤或者其他肿瘤转移到肺部的肿瘤。According to the use of claim 28, the lung tumors include primary tumors of the lungs or tumors metastasized to the lungs from other tumors.
  30. 一种确定候选药剂是否可用于治疗肺癌或者肺部炎症的方法，其包括：确定所述候选药剂是否破坏CD93/C1q相互作用，其中，如果所述候选药剂显示特异性破坏 CD93/C1q相互作用，则其可用于治疗肺癌或者肺部炎症。A method of determining whether a candidate agent can be used to treat lung cancer or lung inflammation, comprising: determining whether the candidate agent disrupts CD93/C1q interaction, wherein if the candidate agent shows specific disruption CD93/C1q interacts, so it can be used to treat lung cancer or lung inflammation.
  31. 根据权利要求30所述的方法，其中，所述方法包括确定所述候选药剂是否破坏细胞表面上CD93与C1q的相互作用。The method of claim 30, wherein the method includes determining whether the candidate agent disrupts the interaction of CD93 with Clq on the cell surface.
  32. 根据权利要求30所述的方法，其中，所述方法包括确定所述候选药剂是否在体外测定***中特异性破坏CD93和C1q的相互作用。The method of claim 30, wherein the method includes determining whether the candidate agent specifically disrupts the interaction of CD93 and Clq in an in vitro assay system.
  33. 根据权利要求30所述的方法，其中，所述的候选药物是抗体、肽、融合肽、肽类似物、多肽、适体、avimer、anticalin、speigelmer或小分子化合物中的一种或者多种。The method according to claim 30, wherein the candidate drug is one or more of an antibody, a peptide, a fusion peptide, a peptide analog, a polypeptide, an aptamer, an avimer, anticalin, a speigelmer or a small molecule compound.
  34. 根据权利要求30所述的方法，其中，所述方法包括将所述候选药剂与CD93/C1q复合物接触。 The method of claim 30, wherein the method includes contacting the candidate agent with a CD93/Clq complex.
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