WO2024094043A1 - Modified exosome preparation, preparation method therefor, and use thereof - Google Patents

Modified exosome preparation, preparation method therefor, and use thereof Download PDF

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WO2024094043A1
WO2024094043A1 PCT/CN2023/128966 CN2023128966W WO2024094043A1 WO 2024094043 A1 WO2024094043 A1 WO 2024094043A1 CN 2023128966 W CN2023128966 W CN 2023128966W WO 2024094043 A1 WO2024094043 A1 WO 2024094043A1
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tumor
exosomes
modified
cells
preparation
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梁伟
曾文峰
田红健
王子昊
黄峰
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中国科学院生物物理研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor

Definitions

  • the present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to a modified exosome preparation, a preparation method and an application thereof.
  • DC cells are professional antigen-presenting cells in the human body that can activate helper T cells and then help CD8-positive T cells activate and exert anti-tumor effects [4]. In addition, DC can also enhance the anti-tumor function of NK cells by secreting antibodies through helper T cells or B cells [5].
  • DC vaccines DC cells are separated, incubated with tumor antigens in vitro, and promoted to mature, and then returned to the patient) have shown some therapeutic advantages in clinical practice, not all patients can respond well to this therapy. The reasons for this are still the low immunogenicity of tumor antigens, immune escape and tumor-induced immunosuppression[6,7].
  • Exosomes originate from the endosomes of cells and interact with various organelles before being secreted out of the cells. Therefore, they contain a variety of cytoplasmic and organelle-derived contents, including DNA, RNA, lipid molecules, metabolites, various proteins on the cytoplasm and cell membrane, etc. [8]. In the past, it was believed that the function of exosomes was to process cell waste substances, but recent studies have shown that exosomes have very rich functions, and exosomes produced by different cells carry different contents. This heterogeneity directly leads to differences in the functional orientation of exosomes [8].
  • tumor exosomes can be used in tumor immunotherapy mainly based on their own characteristics: first, tumor exosomes carry tumor antigens of tumor cells (the mother cells that secrete the exosomes) themselves, which can activate tumor-specific T cells and induce anti-tumor immune responses [9,10]; second, the surface of tumor exosomes carries a large number of tissue compatibility complexes (MHC-I and MHC-II), which are structural molecules necessary for antigen peptide binding [11] and can also avoid immune rejection reactions; in addition, the surface of tumor exosomes also has a large number of promoting The proteins that bind to receptor cells and are engulfed include MFGE8 (milk fat globulin-E8), rab7, LAMP1 (liposome-associated membrane protein 1), CD9, CD81, Annexin II, CD54 and CD63[12-14]. Finally, tumor exosomes also have the characteristics of structural stability. Their
  • tumor exosomes have only limited immunogenicity, but they can be immunosuppressive against other immune cell populations in the tumor microenvironment, which can also limit their effectiveness.
  • tumor exosomes can participate in various stages of tumor development by inhibiting apoptosis, promoting drug resistance, helping tumor cells spread, migrate, and colonize, promoting angiogenesis, tumor immune escape, and immunosuppression[8]. Therefore, when tumor exosomes are used alone for immunotherapy, they often fail to produce satisfactory anti-tumor immune activity, which explains why this therapy has not yet been approved for clinical use.
  • tumor exosomes have the potential to be developed as anti-tumor vaccines because they carry a large number of tumor cell-specific antigens.
  • the natural properties of tumor exosomes that can inhibit the tumor microenvironment and even drain the immune cell functions in the lymph nodes limit their clinical transformation and application .
  • strategies based on the transformation of tumor cells have been proposed, such schemes are time-consuming, expensive, and difficult to unify in terms of operating procedures, making them inconvenient for promotion in clinical practice.
  • the present invention firstly relates to a modified tumor exosome preparation, which comprises:
  • PEG-DSPE Distearate phosphatidylethanolamine-polyethylene glycol
  • tumor exosome components with protein concentration greater than 10ug/ml
  • the purity of the PEG-DSPE is ⁇ 95%, and the dispersion of the PEG chains is ⁇ 1.1;
  • the PEG chain length in the PEG-DSPE molecule is 1000-5000Da; more preferably, the PEG chain length in the PEG-DSPE molecule is 1500-2500Da; most preferably, the PEG chain length in the PEG-DSPE molecule is 2000Da (PEG2000-DSPE);
  • the PEG-DSPE can also be replaced by other types of PEGylated phospholipids, specifically,
  • the fatty acid chain of the phospholipid part contains 10 to 24 carbon atoms, and the fatty acid chain can be saturated or partially saturated, preferably lauric acid (12 carbons), myristic acid (14 carbons), palmitic acid (16 carbons), stearic acid or oleic acid or linoleic acid (18 carbons), eicosanoic acid (20 carbons), behenic acid (22 carbons), lignocerate (24 carbons);
  • the phospholipid part of the PEGylated phospholipid can be phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), diphosphatidylglycerol, lysophosphatidylcholine (LPC), lysoethanolamine phospholipids (LPE), etc.; preferably phosphatidylethanolamine, especially distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine.
  • PE phosphatidylethanolamine
  • PC phosphatidylcholine
  • PI phosphatidylinositol
  • PS phosphatidylserine
  • diphosphatidylglycerol lysophosphatidylcholine
  • LPE lysoethanolamine phospholipids
  • the protein concentration refers to the protein concentration contained in all tumor exosomes in the preparation.
  • the protein concentration of tumor exosomes is quantified using the BCA method;
  • the immune adjuvant molecules include, but are not limited to, MPLA (Monophosphoryl lipid A), QS21 (Quillaja saponaria), and PolyI:C (polyinosinic acid); preferably, MPLA;
  • the dosage of the immune adjuvant molecule is 0.1-10 ug/ml.
  • the mass ratio of PEG-DSPE, immune adjuvant molecules, and total protein in tumor exosomes is 100: 0.1-5: 2-50; preferably, the mass ratio of PEG-DSPE, immune adjuvant molecules, and total protein in tumor exosomes is 100: 0.5-2: 5-20.
  • the tumor exosomes are derived from tumor lines cultured in vitro or primary tumor cells isolated from tumor tissues;
  • the tumor exosomes are prepared by density gradient centrifugation, differential centrifugation, size exclusion, immunoseparation, polymer precipitation, or by using a commercial kit;
  • the present invention also relates to a method for preparing the modified tumor exosome preparation, which comprises the following steps:
  • the method for preparing the solvent-free mixture of PEG-DSPE and immune adjuvant in step (1) is to dissolve the PEG-DSPE and immune adjuvant molecules in an organic solvent and mix them, and then remove the organic solvent; more preferably, under the protection of an inert gas, use a reduced pressure distillation method to remove the organic solvent at a temperature below 70°C.
  • the present invention also relates to the use of the modified tumor exosome preparation in the preparation of drugs, and the drugs are anti-tumor drugs.
  • the present invention also relates to the use of the modified tumor exosome preparation in the preparation of personalized anti-tumor preparations; preferably, in the personalized anti-tumor preparations, the tumor exosomes are derived from autologous tumor tissue or tumor cells of the patient who needs to receive personalized treatment.
  • the present invention also relates to a medicine or a pharmaceutical composition containing the modified tumor exosome preparation.
  • the present invention also relates to a personalized pharmaceutical preparation containing the modified tumor exosome preparation, wherein the personalized pharmaceutical preparation
  • the tumor exosomes described above are derived from autologous tumor tissue or tumor cells of patients who need personalized treatment.
  • the present invention also relates to the use of the modified tumor exosome preparation for treating tumors; preferably, the modified tumor exosome preparation is prepared using tumor tissue or tumor cells from the patient himself.
  • the tumor exosomes modified by polyethylene glycol phospholipids also have a different entry pathway.
  • PP-modified tumor exosomes can directly enter the endoplasmic reticulum ( Figure 6), which is convenient for inducing stronger tumor antigen-specific T cell responses ( Figures 7, 8, 9, 10 and 11).
  • free tumor exosomes after being internalized by DC cells, free tumor exosomes quickly enter the lysosomes, and the antigens they contain will be presented by the MHC II pathway [14,19,20].
  • PP-modified tumor exosomes In wild-type mice, administration of PP-modified tumor exosomes can induce tumor antigen-specific CTL responses, and in tumor-bearing mice, PP-modified tumor exosomes also show good anti-tumor effects.
  • PP-modified tumor exosomes can also improve the infiltration of immune cells in the tumor microenvironment and increase the proportion of DCs, macrophages, CD4+, and CD8+ T cells with anti-tumor activity, laying a good foundation for combined use with other anti-tumor immunotherapy regimens.
  • the treatment plan has a certain universality: as long as the tumor exosomes used are derived from the same tissue source, they should have a certain therapeutic effect even if the gene mutation frequency or characteristics are inconsistent.
  • FIG. 1 Cytotoxicity of different surfactants on bone marrow-derived DCs [in vitro experiment].
  • the vertical axis shows the activity of DC cells after being treated with different surfactants for 48 hours (MTT method), and the horizontal axis shows the concentration of different surfactants.
  • FIG. 1 Preparation and characterization of PP-modified tumor exosomes.
  • A. Cryo-electron microscopy images show the morphological characteristics of tumor exosomes (left), MPLA-deficient control (middle) and PP-modified tumor exosomes (right);
  • B. Dynamic light scattering results show the average diameter distribution of tumor exosomes (black curve) and PP-modified tumor exosomes (grey curve).
  • FIG. 3 PP-modified tumor exosomes promote the ability of DC cells to present antigens [in vitro experiment].
  • the vertical axis shows the number of complexes formed by OVA protein-specific antigen peptide (SIINFEKL) and MHC-I molecules (H-2K b ) presented on the surface of DC cells treated with different methods, represented by the mean fluorescence intensity (MFI).
  • SIINFEKL OVA protein-specific antigen peptide
  • H-2K b MHC-I molecules
  • FIG. 4 PP-modified tumor exosomes increase the expression level of co-stimulatory molecules on the surface of DC cells [in vitro experiment].
  • the vertical axis shows the expression of co-stimulatory molecules CD80 (left figure) and CD86 (right figure) on the surface of DC cells treated with different methods, which represent activation markers, represented by the mean fluorescence intensity (MFI).
  • MFI mean fluorescence intensity
  • FIG. 5 PP-modified tumor exosomes increase the level of cytokine secretion by DC cells [in vitro experiment].
  • the vertical axis shows the concentrations of TNF- ⁇ , IL-2 and IL-12 (pg/ml) detected by ELISA in the culture supernatant of DC cells treated with different methods, as well as the real-time quantitative PCR results of IFN- ⁇ (relative expression level of ⁇ -actin housekeeping gene).
  • FIG. 6 PP modification can change the path of tumor exosomes after being taken up by DC cells [in vitro experiment].
  • EXO PKH-67-labeled tumor exosomes
  • PP/EXO PP-modified tumor exosomes
  • ER endoplasmic reticulum
  • FIG. 7 PP-modified tumor exosomes promote the proliferation of antigen-specific OT I CD8 + T cells [in vitro experiment].
  • the vertical axis shows the different proportions of OT I CD8 + T cell proliferation activated by DC cells treated differently.
  • FIG. 8 PP-modified tumor exosomes induce strong antigen-specific CTL response in mouse colon cancer model (MC38) [in vivo Experiment ⁇ . Photos of IFN- ⁇ spots (ELLIspot experiment) produced by mouse lymph node T cells after immunization with different groups of exosomes and restimulation with tumor-specific antigen peptides (Rpl18, Reps-1 and Adpgk) in vitro (left) and the counting and statistical results of IFN- ⁇ spots (right).
  • FIG. 9 PP-modified tumor exosomes induce strong antigen-specific CTL response - mouse melanoma model (B16F10) [in vivo experiment]. Photos of IFN- ⁇ spots (ELLIspot experiment) produced by mouse lymph node T cells after immunization with different groups of exosomes and restimulation with tumor-specific antigen peptide (Trp-2) in vitro (left) and the counting and statistical results of IFN- ⁇ spots (right).
  • FIG. 10 PP-modified tumor exosomes induce strong antigen-specific CTL response - mouse triple-negative breast cancer model (4T1) [in vivo experiment]. Photos of IFN- ⁇ spots (ELLIspot experiment) produced by mouse lymph node T cells after immunization with different groups of exosomes and restimulation with tumor-specific antigen peptides (Sptbn and Wdr33) in vitro (left) and the counting and statistical results of IFN- ⁇ spots (right).
  • FIG. 11 PP-modified tumor exosomes induce strong antigen-specific CTL response - mouse cervical cancer model (TC-1, with HPV virus antigens E6 and E7) [in vivo experiment]. Photos of IFN- ⁇ spots (ELLIspot experiment) produced by mouse lymph node T cells after immunization with different groups of exosomes and restimulation with tumor-specific antigen peptides (E7-20) in vitro (left) and the counting and statistical results of IFN- ⁇ spots (right).
  • FIG. 13 Anti-tumor effect of PP-modified tumor exosomes - mouse melanoma model (B16F10) [in vivo experiment]. After mice were implanted with tumors, they were treated with exosomes from different groups, and the tumor growth curve (left) and mouse survival time (right).
  • FIG. 14 Anti-tumor effect of PP-modified tumor exosomes - mouse cervical cancer model (TC-1, with HPV virus antigen E7) [in vivo experiment]. Tumor growth curve of mice after tumor implantation and treatment with different groups of exosomes.
  • FIG. 15 Anti-tumor metastasis effect of PP-modified tumor exosomes - mouse melanoma model (B16F10) [in vivo experiment]. After pre-immunization with exosomes from different groups, mice were implanted with tumors via the tail vein. Representative photos (A) and statistical data (B) of metastatic nodules formed by melanoma cells in the mouse lungs and photos of all mouse lungs in each group (C) were observed 13 days later.
  • Figure 17 PP-modified tumor exosome preparation technology simulates personalized tumor treatment - mouse colon cancer model (MC38) [in vivo experiment].
  • OTI transgenic mouse model The characteristic of this mouse model is that the T cell receptor (TCR) of its CD8 positive T cells has been gene-edited, which can specifically recognize the MHC-I/OVA 257-264 (SIINFEKL) antigen complex on the surface of antigen presenting cells, and be specifically activated and proliferate.
  • TCR T cell receptor
  • SIINFEKL MHC-I/OVA 257-264
  • Example 1 Toxicity of different surfactants to bone marrow-derived DC cells (dendritic cells)
  • surfactants In order to break the lipid vesicle structure of tumor exosomes, surfactants need to be introduced. However, surfactants (Table 1) have the effect of destroying cell membranes. Given that our goal is to use the modified tumor exosomes for the research and application of personalized tumor vaccines, as vaccines Therefore, we then used the MTT assay to investigate whether these surfactants could produce cytotoxicity to bone marrow-derived DC cells.
  • Mouse bone marrow derived DC cells were plated in 96-well plates at 300,000/well, and different concentrations of SDS, TritonX-100, Tween80 and PEG2000-DSPE (PP) solutions were added. After culturing at 37°C for 24 hours, 20 ⁇ l of 5 mg/ml MTT solution was added to the wells and cultured for another 6 hours. Then MTT lysis buffer was added, pipetted and mixed, and then placed in an ELISA reader to detect its absorbance at 570 nm and 630 nm (reference wavelength).
  • Culture of MC38 cell line in mouse colon cancer model Use DMEM medium (containing 10% fetal bovine serum). Take out the cryopreserved tube of MC38 tumor cells from the liquid nitrogen storage tank and immediately put it into a 37°C water bath for rapid dissolution. Then transfer the cell suspension into a centrifuge bucket containing 10ml of culture medium. Centrifuge at 350g for 5 minutes and remove the supernatant. After resuspending with fresh culture medium, transfer the cells to a cell culture bottle, add 10-15ml of culture medium to suspend the precipitated cells, adjust the cell concentration, and culture in a 37°C, 5% CO 2 saturated humidity incubator. During the culture maintenance process, observe the cell status every day and replace the fresh culture medium in time.
  • DMEM medium containing 10% fetal bovine serum
  • Tumor grafting When cells adhere to the wall and grow to 90% confluence, digest them with 0.05% trypsin and subculture them at a ratio of 1:3. On the day of the experiment, digest the cells with good growth and 90% confluence with trypsin, neutralize the trypsin with fresh culture medium, centrifuge at 350g, discard the supernatant, resuspend in sterile PBS, count, and adjust the density of the cell suspension to 2.5 ⁇ 10 6 /ml for later use. 2.5 ⁇ 10 5 cells were subcutaneously grafted on each mouse.
  • Tumor exosome (EXO) collection experiment When the cells adhere to the wall and grow to 90% confluence, they are digested with 0.05% trypsin and subcultured at a ratio of 1:3. During subculture, the culture medium containing 10% fetal bovine serum without exosomes is used for continued culture. After 48 hours, the supernatant is collected and directly used for exosome extraction or frozen in a -80°C refrigerator for later use.
  • tumor cells were cultured in a medium containing 10% exosome-depleted fetal bovine serum (the serum was centrifuged at 100,000 g for 2 hours and the supernatant was retained) for 48 hours, the cell culture supernatant was collected.
  • Density gradient centrifugation Place the crude exosomes in a discontinuous density gradient sucrose solution of 10%, 20%, 40%, and 60%, and perform ultracentrifugation (100,000g) for 2 hours. Then carefully aspirate the white matter in the 10-20% layer, dilute with PBS, and ultracentrifuge again to precipitate the exosomes at the bottom, which is the refined exosomes. After resuspending with an appropriate amount of PBS, the protein concentration was determined by the BCA method, and the exosome content was calibrated with this method, and stored at -80°C for later use.
  • tumor exosomes EXO
  • the tumor exosomes quantified by the BCA protein assay were diluted with sterile water to a 500 ug/ml tumor exosome (EXO) stock solution;
  • PP/MPLA/EXO PP-modified tumor exosomes
  • MPLA powder was dissolved in a methanol/chloroform (1:2, v:v) mixed solution to prepare an MPLA stock solution with a concentration of 1 mg/ml;
  • MPLA/EXO PP-free reference substance
  • MPLA/EXO PP-free control solution
  • the experimental results showed that the average diameter of tumor exosomes was about 100nm, which was consistent with the exosome diameter distribution range reported in the literature (30-150nm).
  • the diameter of PP-modified tumor exosomes was about 10-20nm ( Figure 2A), indicating that the tumor exosomes modified by PP became smaller and more uniform.
  • the sample morphology between the PP/EXO group and the PP/MPLA/EXO group did not show much difference, so MPLA did not affect the modification of the structure of tumor exosomes by PEGylated phospholipids.
  • Example 3 PP-modified tumor exosomes enhance the function of bone marrow-derived DC cells (dendritic cells)
  • the acquisition of bone marrow-derived DCs can refer to Example 1.
  • Bone marrow-derived DC cells were treated with 50ug/ml tumor exosomes (quantified by BCA protein) and PP-modified tumor exosomes, and 0.5mg/ml OVA full-length protein in PBS was added. After 24 hours, flow cytometry was performed using a PE-labeled anti-mouse MHC I-SIINFEKL antigen complex (abbreviated as p-MHC I) fluorescent antibody to examine the fluorescence intensity of DC cells in each treatment group to indicate the number of antigen-MHC-I complexes on the cell surface.
  • p-MHC I PE-labeled anti-mouse MHC I-SIINFEKL antigen complex
  • the levels of p-MHC I in the tumor exosomes alone group, the MPLA-free control group, and the PP-free control group were similar to the untreated group;
  • the p-MHC I level in the PP-modified tumor exosomes treatment group increased significantly (Figure 3), indicating that PP-modified tumor exosomes can enhance the ability of DC cells to process and present antigens .
  • Bone marrow-derived DC cells were treated with tumor exosomes containing 50 ug/ml (quantified by BCA protein) and PP-modified tumor exosomes. After 12 hours, fluorescent antibodies were used to detect the expression levels of CD80 and CD86, molecular markers representing maturity, on the surface of DC cells.
  • the tumor exosomes alone group, the MPLA-free control group, and the PP-free control group were similar to the untreated group, and the expression levels of CD80 and CD86 did not change;
  • the PP-modified tumor exosomes treatment group can significantly increase the expression levels of these two molecular markers ( Figure 4), indicating that PP-modified tumor exosomes can promote the maturation of DC cells and better perform immune functions .
  • Bone marrow-derived DC cells were treated with tumor exosomes containing 50 ug/ml (quantified by BCA protein) and PP-modified tumor exosomes. The cell culture supernatant was collected after 2 hours and 24 hours, respectively. The secretion levels of TNF- ⁇ , IL-2, and IL-12 and the expression of interferon IFN- ⁇ by DC cells in each treatment group were examined by ELISA and q-RT-PCR.
  • the tumor exosomes alone group, the MPLA-free control group, and the PP-free control group showed no changes in the secretion levels of TNF- ⁇ , IL-2, and IL-12, similar to the untreated group. Only the TNF- ⁇ secretion level of the PP-free control group was slightly increased, which may be related to the effect of MPLA itself;
  • the PP-modified tumor exosomes treatment group can significantly increase the levels of TNF- ⁇ , IL-2, and IL-12 secreted by DC cells (Figure 5). At the same time, the expression trend of type I interferon IFN- ⁇ is the same. These results show that PP-modified tumor exosomes can induce DC cells to secrete a large number of cytokines that activate anti-tumor immune responses and help DC cells activate downstream T cells .
  • Example 4 PP modified tumor exosomes to deliver their contents to the endoplasmic reticulum (ER) of DC cells
  • tumor exosomes EXO
  • MC38 cell line The acquisition of tumor exosomes (EXO) derived from MC38 cell line and the preparation of PP-modified tumor exosomes and corresponding reference substances are the same as in Example 2.
  • DC2.4 cell culture Use RPMI1640 medium (containing 10% fetal bovine serum, 1 ⁇ L-glutamine, 1 ⁇ non-essential amino acid solution, 1 ⁇ HEPES buffer and 5uM ⁇ -mercaptoethanol). Take out the tumor cell cryotube from the liquid nitrogen storage tank and immediately put it into a 37°C water bath for rapid dissolution. Then transfer the cell suspension into a centrifuge bucket containing 10ml of culture medium. Centrifuge at 350g for 5 minutes and remove the supernatant. After resuspending with fresh culture medium, transfer the cells to a cell culture bottle, add 10-15ml of culture medium to suspend the precipitated cells, adjust the cell concentration, and place it in a 37°C, 5% CO 2 saturated humidity incubator for culture. During the culture maintenance process, observe the cell status every day and replace the fresh culture medium in time.
  • RPMI1640 medium containing 10% fetal bovine serum, 1 ⁇ L-glutamine, 1 ⁇ non-essential amino acid solution, 1 ⁇ HEPES buffer and 5
  • PKH67 labeled membrane components of tumor exosomes Take 8 tubes of freshly extracted tumor exosomes (quantified by the BCA method, each tube contains approximately 9 mg of tumor exosomes), add 350ul of the diluent provided in the kit to each EP tube, and gently blow to form a solution of the exosomes. Then, under light-proof conditions, prepare 8 more EP tubes containing 350ul of diluent, add 1.5ul of PKH67 dye to each tube, and mix evenly. Then, add the diluent containing PKH67 dye to the EP tube containing the tumor exosome solution at a volume ratio of 1:1, and gently blow to mix. Stain at room temperature (25°C) for 5 minutes.
  • DC2.4 cells were seeded at 1 ⁇ 10 5 /well in a 15 mm confocal microscopy cell culture dish and allowed to adhere overnight. The next day, DC2.4 cells were treated with the prepared PKH67-labeled tumor exosomes or PP-modified tumor exosomes. Afterwards, 100 nM Lyso-Tracker-Red (37°C, 10 minutes) or 1 uM ER-Tracker-Red (37°C, 30 minutes) dyes were added to stain the lysosomes and endoplasmic reticulum of the cells according to the set time. The final exosome treatment time was maintained at 1 hour. Before going on the machine, the culture medium was replaced with a culture medium without fluorescent dyes, and then observed with a fluorescent confocal microscope;
  • tumor exosomes were mainly co-localized with lysosomes, while PP-modified tumor exosomes were mainly co-localized with the endoplasmic reticulum ( Figure 6A), which preliminarily indicates that PP modification can change the intracellular destination of tumor exosomes.
  • PKH67 marks the membrane structure of tumor exosomes, it is not certain that the contents of PP-modified tumor exosomes (especially antigens) can indeed reach the endoplasmic reticulum.
  • CFSE dye that can mark proteins to mark the contents of tumor exosomes, and still observed their co-localization with lysosomes and endoplasmic reticulum by fluorescence confocal microscopy.
  • CFSE-labeled protein components of tumor exosomes Pipette 20 mg of tumor exosome stock solution (based on BCA protein quantification) into a 1.5 ml EP tube, add PBS to adjust the volume to 500 ul, add 2 ul of 10 mM CFSE stock solution (to make the final concentration reach 4 uM), and stain at room temperature in the dark for 10 minutes. Then add 500 ul of PBS containing 20% FBS to neutralize the reaction, transfer to an ultracentrifuge tube, and centrifuge at 100,000g at 4 degrees for 30 minutes. Discard the supernatant, wash the precipitate with PBS once, resuspend it with an appropriate amount of PBS, quantify it with BCA, and store it at -80°C for later use.
  • DC2.4 cells were seeded at 1 ⁇ 10 5 /well in a 15 mm confocal microscopy cell culture dish and allowed to adhere overnight. The next day, DC2.4 cells were treated with the prepared PKH67-labeled tumor exosomes or PP-modified tumor exosomes. Afterwards, 100 nM Lyso-Tracker-Red (37°C, 10 minutes) or 1 uM ER-Tracker-Red (37°C, 30 minutes) dyes were added to stain the lysosomes and endoplasmic reticulum of the cells according to the set time. The final exosome treatment time was maintained at 1 hour. Before going on the machine, the culture medium was replaced with a culture medium without fluorescent dyes, and then observed with a fluorescent confocal microscope;
  • Example 5 PP-modified tumor exosomes enhance the activation of bone marrow-derived DC on antigen-specific OT I CD8 + T cells
  • the acquisition of bone marrow-derived DCs, the acquisition of tumor exosomes (EXO) derived from the MC38 cell line, and the preparation of PP-modified tumor exosomes and corresponding reference substances refer to Example 2 and Example 3.
  • OT I CD8 + T cells After OT I transgenic mice were sacrificed, spleen cells were obtained, red blood cells were lysed, and CD8 + T cells were then separated using CD8 magnetic beads (Invitrogen).
  • Bone marrow-derived DC cells were treated with 50ug/ml tumor exosomes (quantified by BCA protein) and PP-modified tumor exosomes. After 24 hours, 0.5mg/ml OVA full-length protein PBS solution was added. After another 24 hours, DC cells in each treatment group were collected and counted, and co-cultured with CFSE-stained OTI CD8 + T cells at a ratio of 1:8. After 72 hours, the proliferation level of T cells (CFSE dilution) was detected.
  • the DC cells in the untreated group were able to activate OT I CD8 + T cells well, and the tumor exosome treatment group alone could significantly inhibit this activation effect.
  • Example 6 PP-modified tumor exosomes can induce homologous tumor antigen-specific CTL response
  • DMEM medium containing 10% fetal bovine serum
  • Culture of mouse cervical cancer TC-1 cell line and mouse triple-negative breast cancer 4T1 cell line Use RPMI1640 medium (containing 10% fetal bovine serum). Take out the cryopreserved tube of tumor cells from the liquid nitrogen storage tank and immediately put it into a 37°C water bath for rapid dissolution. Then transfer the cell suspension into a centrifuge bucket containing 10ml of culture medium. Centrifuge at 350g for 5 minutes and remove the supernatant. After resuspending with fresh culture medium, transfer the cells to a cell culture bottle, add 10-15ml of culture medium to suspend the precipitated cells, adjust the cell concentration, and culture in a 37°C, 5% CO 2 saturated humidity incubator. During the culture maintenance process, observe the cell status every day and replace the fresh culture medium in time.
  • RPMI1640 medium containing 10% fetal bovine serum
  • mice were immunized via tail-base injection with MC38-derived tumor exosomes or PP-modified tumor exosomes that elicit homologous tumor antigen-specific CTL responses. Seven days after immunization, the mice were killed, and the proximal lymph nodes were surgically removed. All immune cells in the lymph nodes were obtained after grinding and digestion.
  • the isolated mouse lymph node cells were then added to the plate at 3 ⁇ 10 5 per well and incubated with 20 ⁇ g/ml MC38-specific antigen peptides (including: Rpl-18, Reps-1 and Adpgk) (Identification of a neo-epitope dominating endogenous CD8 T cell responses to MC-38 colorectal cancer.9,1673125(2019); published online EpubOct 13(10.1080/2162402x.2019.1673125)), and cultured in a 37°C incubator for 48 hours. After 48 hours, the culture medium was discarded and deionized water was added to lyse the cells for 5 minutes each time, for a total of 2 times.
  • 20 ⁇ g/ml MC38-specific antigen peptides including: Rpl-18, Reps-1 and Adpgk
  • mice were immunized by tail-base injection with B16F10-derived tumor exosomes or PP-modified tumor exosomes that elicit homologous tumor antigen-specific CTL responses. Seven days after immunization, the mice were killed, and the proximal lymph nodes were surgically removed. All immune cells in the lymph nodes were obtained by grinding and digestion.
  • Wild-type mice were immunized with 4T1-derived tumor exosomes or PP-modified tumor exosomes via tail-base injection. Seven days after immunization, the mice were killed, and the proximal lymph nodes were surgically removed. All immune cells in the lymph nodes were obtained after grinding and digestion.
  • the 4T1-specific antigen peptides used for incubation with lymph node cells are Sptbn4 and Wdr33 (Self-healing microcapsules synergetically modulate immunization microenvironments for potent cancer vaccination. 6, eaay7735 (2020); published online Epub May (10.1126/sciadv.aay7735)).
  • Wild-type mice were immunized with TC-1-derived tumor exosomes or PP-modified tumor exosomes via tail-base injection. Seven days after immunization, the mice were sacrificed, and the proximal lymph nodes were surgically removed. All immune cells in the lymph nodes were obtained after grinding and digestion.
  • the control group without MPLA and the control group without PP could not improve the antigen-specific CTL response.
  • the lymph node cells immunized with PP-modified tumor exosomes could produce a higher proportion of antigen-specific CTL response (specifically manifested as a stronger IFN- ⁇ signal) ( Figure 11) .
  • Example 7 PP-modified tumor exosomes inhibit tumor growth and prolong the survival of tumor-bearing mice
  • Example 4 The cell culture method is referred to Example 4, and the acquisition of tumor exosomes (EXO) and the preparation of PP-modified tumor exosomes and corresponding reference substances are referred to Example 2.
  • EXO tumor exosomes
  • Example 2 the preparation of PP-modified tumor exosomes and corresponding reference substances
  • mice Female wild-type C57BL/6 mice were subcutaneously inoculated with 1ml sterile syringes, 0.1ml per mouse (i.e., 2.5 ⁇ 10 5 MC38 cells were inoculated per mouse). About 30mm 3 tumor formation can be seen in about 4 days. On the same day, 50ug/ml tumor exosomes (based on BCA protein quantification) or tumor exosomes containing the same mass of PP were subcutaneously injected near the tumor for treatment, once a week, for a total of three times. After that, the status of the mice was observed, and the tumor volume changes and deaths of tumor-bearing mice were recorded during this period.
  • mice Female wild-type C57BL/6 mice were subcutaneously inoculated with 1ml sterile syringes, 0.1ml per mouse (i.e., 2.5 ⁇ 10 5 B16F10 cells per mouse). Note that the cells were mixed evenly before each aspiration of the cell suspension. Tumor formation of about 30mm 3 can be seen in about 4 days. On the same day, 50ug/ml tumor exosomes (based on BCA protein quantification) or tumor exosomes modified with PP of equal mass were injected subcutaneously near the tumor for treatment once a week for a total of three times. Afterwards, the condition of the mice was observed, and the changes in tumor volume and the death of tumor-bearing mice were recorded.
  • TC-1 model When the cells adhere to the wall and grow to 90% confluence, they are digested with 0.05% trypsin and subcultured at a ratio of 1:4. On the day of the experiment, the cells with good growth status and 90% confluence are digested with trypsin, and the trypsin is neutralized with fresh culture medium, centrifuged at 350g, the supernatant is discarded, and sterile PBS is added to resuspend, count, and the density of the cell suspension is adjusted to 5 ⁇ 10 5 /ml.
  • mice Female wild-type C57BL/6 mice are subcutaneously inoculated with 1ml sterile syringes, 0.1ml per mouse (i.e., 5 ⁇ 10 4 TC-1 cells are inoculated per mouse). Note that the cells are mixed evenly before each aspiration of the cell suspension. Tumor formation of about 30mm3 can be seen in about 6 days. On the same day, 50ug/ml tumor exosomes (based on BCA protein quantification) or tumor exosomes modified with PP of equal mass are injected subcutaneously near the tumor for treatment once a week for a total of three times. Afterwards, the condition of the mice was observed, and the changes in tumor volume and the death of tumor-bearing mice were recorded.
  • Example 8 PP-modified tumor exosomes inhibit lung metastasis of melanoma
  • Example 4 The cell culture method is referred to Example 4; the acquisition of tumor exosomes (EXO) and the preparation of PP-modified tumor exosomes and corresponding reference substances are referred to Example 2.
  • PP-modified tumor exosomes (derived from mouse melanoma cell line B16F10) was performed by referring to the preparation method in Example 2. Before tumor implantation, each female wild-type C57BL/6 mouse received a tail vein injection of PBS (as a solvent control), 100 ug (calculated by BCA protein quantification) exosomes, or PP-modified tumor exosomes once a week for three weeks.
  • B16F10 cells were cultured and grown to 90% confluence, then digested with 0.05% trypsin and subcultured at a ratio of 1:4. On the day of the experiment, cells with good growth and a confluence of 90% were trypsinized, and the trypsin was neutralized with fresh culture medium, centrifuged at 350g, the supernatant was discarded, and sterile PBS was added to resuspend, counted, and the density of the cell suspension was adjusted to 5 ⁇ 10 4 /ml.
  • a 1ml sterile syringe was used to inject into the tail vein of female wild-type C57BL/6 mice that had been immunized with exosomes or PP-modified tumor exosomes, 0.1ml per mouse (i.e., 5 ⁇ 10 4 B16F10 cells were inoculated per mouse).
  • the exosomes secreted by mouse melanoma are easily enriched in the lungs of mice, turning the lung tissue into an immunosuppressive microenvironment, promoting the colonization of melanoma cells in the circulatory system, and forming metastatic foci.
  • the tumor exosomes modified by PP are likely to change the immunosuppressive microenvironment, enhance the anti-tumor immune response, and thus reduce the metastasis of melanoma.
  • Example 9 PP-modified tumor exosomes improve the immune microenvironment of melanoma
  • Example 4 The cell culture method is referred to Example 4; the acquisition of tumor exosomes (EXO) and the preparation of PP-modified tumor exosomes and corresponding reference substances are referred to Example 2.
  • B16F10 model When the cells adhere to the wall and grow to 90% confluence, they are digested with 0.05% trypsin and subcultured at a ratio of 1:4. On the day of the experiment, the cells with good growth status and a confluence of 90% are trypsinized, and the trypsin is neutralized with fresh culture medium. The cells are centrifuged at 350g, the supernatant is discarded, and sterile PBS is added to resuspend and count. The density of the cell suspension is adjusted to 2.5 ⁇ 10 6 /ml.
  • mice Female wild-type C57BL/6 mice are subcutaneously inoculated with 0.1ml per mouse (i.e., 2.5 ⁇ 10 5 B16F10 cells are inoculated per mouse). Note that the cells should be mixed evenly before aspirating the cell suspension each time. Tumor formation of about 30mm 3 can be seen in about 4 days, and the tumor is inoculated on the same day.
  • the mice were treated with 50ug/ml tumor exosomes (based on BCA protein quantification) or PP-modified tumor exosomes containing an equal mass of PP subcutaneously once a week for a total of three times. Afterwards, the status of the mice was observed, and the changes in tumor volume and death of tumor-bearing mice were recorded during this period (Figure 16A).
  • Tumor exosome treatment was unable to effectively inhibit tumor growth.
  • Tumor exosomes, MPLA-free control and PP-free control had no significant changes in the total immune cell ratio (CD45 + cell ratio) ( Figure 16D), CD8 + T cell ratio (Figure 16E), CD4 + T cell ratio (Figure 16F), DC cell ratio (Figure 16G), macrophage ratio (Figure 16H) and NK cell ratio ( Figure 16I) in tumor tissues.
  • PP-modified tumor exosomes can significantly increase the proportion of the above-mentioned types of immune cells ( Figure 16D-I), indicating that the immunosuppressive microenvironment of tumor tissue has been improved, providing a prerequisite for the function of immunotherapy.
  • Tumor exosomes after treatment with the MPLA-free control and the PP-free control, had a certain enhancing effect on Ki67 (indicating cell proliferation activity) (Figure 16J), IFN- ⁇ and Granzyme B (indicating the tumor killing ability of T cells) ( Figures 16K and L) in CD8 + T cells in tumor tissues.
  • PP-modified tumor exosomes can significantly increase the proliferation and tumor killing ability of CD8 + T cells ( Figure 16J-L ).
  • PP-modified tumor exosomes can significantly increase the infiltration of various types of immune cells, especially CD8 + T cells with proliferation and tumor killing activity, thereby inhibiting the growth of tumor cells and achieving the purpose of treating tumors.
  • Example 10 PP-modified tumor exosomes simulate individualized tumor treatment
  • Example 4 The cell culture method is referred to Example 4; the acquisition of tumor exosomes (EXO) and the preparation of PP-modified tumor exosomes are referred to Example 2.
  • the tumor tissue is surgically removed and the tumor cells in the tissue are sorted out using a Tumor cell isolation kit and continued to be cultured.
  • the tumor cell culture supernatant is collected and tumor exosomes are extracted therefrom.
  • the same batch of tumor cells is used to establish a new tumor model (for specific procedures, refer to the experimental protocol (1) of this example).
  • the exosomes modified by PP show the ability to inhibit tumor growth ( Figure 17B). Furthermore, compared with the original exosomes derived from the MC38 cell line, the tumor cell exosomes in in situ isolated tumor tissues modified by PP have a better tumor growth inhibition effect .
  • Non-synonymous mutations in genes within tumor cells will express variant proteins, which are captured and recognized as foreign substances by the immune system, and can therefore serve as a source of tumor-specific antigens. It is also worth noting that the probability of producing tumor-specific antigens is roughly positively correlated with the probability of somatic mutations in tumor cells. (Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival. Genome Res. 2014; 24: 743-50.; Molecular and genetic properties of tumors associated with local immune cytolytic activity. Cell. 2015; 160: 48-61.). MC38 mouse colon cancer cells are microsatellite unstable cancers with a highly mutable genome. Under certain selection pressure, this characteristic may change the antigen spectrum produced by the cells (Targeting immune checkpoints potentiates immunoediting and changes the dynamics of tumor evolution).
  • mice with complete immune capabilities will also perform immune surveillance on growing tumor cells, kill tumor cells that they can identify, and the surviving tumor cells become resistant to immune killing due to gene mutations.
  • This process is also called immune editing.
  • the original MC38 tumors under the skin of mice have produced some new antigens caused by gene mutations during their growth. Therefore, there is a possibility of producing new antigens in the "new" tumor cells formed by sorting and subculturing from the tumor tissue.
  • the new tumor cell exosomes and the original MC38 exosomes while sharing most of the same antigens, further carry new antigens obtained by gene mutations. Therefore, it is reflected that the PP-modified MC38 exosomes still have a certain tumor inhibitory effect, and the PP-modified tumor cell exosomes have a better tumor growth inhibitory effect.
  • the present invention uses PP molecules to structurally modify tumor exosomes, so that while retaining the original tumor-associated antigens and tumor-specific antigens, it breaks the natural immunosuppressive properties of the original exosomes.
  • Tumor exosomes modified by PP have the ability to induce antigen-specific CD8+T cell responses, effectively reducing the tumor burden of tumor-bearing mice and prolonging their survival.

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Abstract

The present invention relates to a modified exosome preparation, a preparation method therefor, and use thereof. The preparation comprises: (1) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (PEG-DSPE) at a concentration of greater than 50 ug/ml; (2) a tumor exosome component with a protein concentration of greater than 10 ug/ml; and (3) an effective amount of an immunologic adjuvant molecule for stimulating an immune response. The present invention further relates to use of the modified tumor exosome preparation in the preparation of an anti-tumor drug or an individualized anti-tumor preparation.

Description

一种改造的外泌体制剂、其制备方法及应用A modified exosome preparation, preparation method and application thereof 技术领域Technical Field
本发明属于医药生物技术领域，具体的，涉及一种改造的外泌体制剂、其制备方法及应用。The present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to a modified exosome preparation, a preparation method and an application thereof.
背景技术Background technique
进入肿瘤免疫治疗时代以来，多种免疫检查点抑制剂(包括抗体和小分子)、CAR-T细胞治疗和治疗性肿瘤疫苗进入临床试验，并在很多实体瘤和恶性血液癌症中发挥了显著的治疗作用。然而，患者的药物反应率和治愈率均处于较低的水平，而且在治疗过程中还时常产生原发性或继发性抵抗效性，削弱免疫治疗的效果。研究显示，有限的免疫治疗效果与肿瘤抗原的缺失或贫乏相关，同时肿瘤组织内部的免疫抑制性特征也是抑制免疫治疗发挥功效的一道障碍[1-3]。DC细胞是人体内专业的抗原呈递细胞，能够激活辅助T细胞，继而帮助CD8阳性T细胞活化，发挥抗肿瘤功效[4]。此外，DC还能通过辅助性T细胞或B细胞分泌抗体的方式强化NK细胞的抗肿瘤功能[5]。尽管DC疫苗(将DC细胞分离出来后，体外孵育肿瘤抗原，并促进其成熟，再回输到患者体内)在临床上体现出一些治疗优势，但是并非所有的患者都能很好的响应该疗法。究其原因，还是在肿瘤抗原的低免疫原性，免疫逃逸和肿瘤引起的免疫抑制等因素[6,7]。Since the advent of the era of tumor immunotherapy, a variety of immune checkpoint inhibitors (including antibodies and small molecules), CAR-T cell therapy and therapeutic tumor vaccines have entered clinical trials and have played a significant therapeutic role in many solid tumors and malignant blood cancers. However, the drug response rate and cure rate of patients are at a low level, and primary or secondary resistance often occurs during the treatment process, weakening the effect of immunotherapy. Studies have shown that limited immunotherapy effects are related to the absence or deficiency of tumor antigens. At the same time, the immunosuppressive characteristics within tumor tissues are also an obstacle to the effectiveness of immunotherapy [1-3]. DC cells are professional antigen-presenting cells in the human body that can activate helper T cells and then help CD8-positive T cells activate and exert anti-tumor effects [4]. In addition, DC can also enhance the anti-tumor function of NK cells by secreting antibodies through helper T cells or B cells [5]. Although DC vaccines (DC cells are separated, incubated with tumor antigens in vitro, and promoted to mature, and then returned to the patient) have shown some therapeutic advantages in clinical practice, not all patients can respond well to this therapy. The reasons for this are still the low immunogenicity of tumor antigens, immune escape and tumor-induced immunosuppression[6,7].
所有细胞，包括原核细胞和真核细胞均可以释放细胞外囊泡。细胞外囊泡中直径为40-160nm的被称为外泌体(平均直径100nm)。外泌体发源于细胞的内涵体，在分泌出细胞之前与各种细胞器进行相互作用，因此其内部含有多种细胞质及细胞器来源的内含物，包括DNA、RNA、脂类分子、代谢产物、细胞质和细胞膜上的各种蛋白质等等[8]。以往认为外泌体的功能是处理细胞废弃的物质，而近期的研究显示外泌体具有非常丰富的功能，而且不同细胞产生的外泌体携带不同的内容物，这种异质性直接导致外泌体功能导向性的差异[8]。All cells, including prokaryotes and eukaryotic cells, can release extracellular vesicles. Among the extracellular vesicles, those with a diameter of 40-160nm are called exosomes (average diameter 100nm). Exosomes originate from the endosomes of cells and interact with various organelles before being secreted out of the cells. Therefore, they contain a variety of cytoplasmic and organelle-derived contents, including DNA, RNA, lipid molecules, metabolites, various proteins on the cytoplasm and cell membrane, etc. [8]. In the past, it was believed that the function of exosomes was to process cell waste substances, but recent studies have shown that exosomes have very rich functions, and exosomes produced by different cells carry different contents. This heterogeneity directly leads to differences in the functional orientation of exosomes [8].
肿瘤细胞分泌的外泌体(简称为“肿瘤外泌体”)能被应用于肿瘤的免疫治疗主要基于其自身的几个特点：首先，肿瘤外泌体携带了肿瘤细胞(分泌该外泌体的母细胞)自身的肿瘤抗原，能够激活肿瘤特异性T细胞，引起抗肿瘤免疫应答[9,10]；其次，肿瘤外泌体表面携带大量组织相容性复合物(MHC-I和MHC-II)，而这些分子是抗原肽结合所必须的结构性分子[11]，同时能够避免产生免疫排斥反应；此外，肿瘤外泌体表面还有大量促进其与受体细胞结合并被吞噬的蛋白，例如MFGE8(milk fat globulin-E8)，rab7，LAMP1(liposome-associated membrane protein 1)，CD9，CD81，Annexin II，CD54和CD63[12-14]；最后，肿瘤外泌体还具有结构稳定的特点，其双层脂膜能够保护其内含物免受各种核酸酶或者蛋白酶的侵扰，适用于小分子、核酸类药物的递送系统[15]。Exosomes secreted by tumor cells (abbreviated as "tumor exosomes") can be used in tumor immunotherapy mainly based on their own characteristics: first, tumor exosomes carry tumor antigens of tumor cells (the mother cells that secrete the exosomes) themselves, which can activate tumor-specific T cells and induce anti-tumor immune responses [9,10]; second, the surface of tumor exosomes carries a large number of tissue compatibility complexes (MHC-I and MHC-II), which are structural molecules necessary for antigen peptide binding [11] and can also avoid immune rejection reactions; in addition, the surface of tumor exosomes also has a large number of promoting The proteins that bind to receptor cells and are engulfed include MFGE8 (milk fat globulin-E8), rab7, LAMP1 (liposome-associated membrane protein 1), CD9, CD81, Annexin II, CD54 and CD63[12-14]. Finally, tumor exosomes also have the characteristics of structural stability. Their double lipid membrane can protect their contents from various nucleases or proteases, making them suitable for the delivery system of small molecules and nucleic acid drugs[15].
近年来已经有研究证实了肿瘤外泌体作为肿瘤治疗性疫苗的可行性，包括临床前模型[16]和临床试验[17]。肿瘤外泌体仅具备有限的免疫原性，但针对肿瘤微环境中其它免疫细胞类群会产生免疫抑制性，也会限制其效能的发挥。而且，肿瘤外泌体能通过抑制凋亡、促进产生药物抗性、帮助肿瘤细胞扩散-迁移-定殖、促进血管新生、肿瘤免疫逃脱以及免疫抑制等手段参与了肿瘤发生发展的各个阶段[8]。因此，单独使用肿瘤外泌体进行免疫治疗时，往往无法产生令人满意的抗肿瘤免疫活性，这也解释了为什么该疗法目前仍未在临床获批使用。In recent years, studies have confirmed the feasibility of tumor exosomes as tumor therapeutic vaccines, including preclinical models[16] and clinical trials[17]. Tumor exosomes have only limited immunogenicity, but they can be immunosuppressive against other immune cell populations in the tumor microenvironment, which can also limit their effectiveness. In addition, tumor exosomes can participate in various stages of tumor development by inhibiting apoptosis, promoting drug resistance, helping tumor cells spread, migrate, and colonize, promoting angiogenesis, tumor immune escape, and immunosuppression[8]. Therefore, when tumor exosomes are used alone for immunotherapy, they often fail to produce satisfactory anti-tumor immune activity, which explains why this therapy has not yet been approved for clinical use.
为了解决上述问题，目前发展出很多针对分泌肿瘤外泌体的母细胞的改造策略，例如增加肿瘤外泌体的热休克蛋白含量，提供佐剂效果[18,19]；增加某种肿瘤特异性抗原的表达量，以此增加肿瘤外泌体中肿瘤特异性抗原的丰度及免疫原性[20]；添加超抗原到肿瘤外泌体中，利用超抗原与MHC II分子形成稳定复合物的特性，促进肿瘤外泌体对DC细胞的激活作用[21)；通过在肿瘤细胞中高表达控制MHC II分子的转录因子CIITA，增加肿瘤外泌体上MHC II分子的丰度，提高抗原肽的呈递效率及有效的肿瘤抗原肽的丰度[22]；整合一些病毒融合蛋白到肿瘤外泌体中，促进DC细胞对其的摄取量[23]；肿瘤细胞中高表达细胞因子，增加肿瘤外泌体中细胞因子的含量以及肿瘤外泌体的免疫原性[24]；肿瘤 细胞中高表达一些对产生免疫抑制性有关基因具有负调控作用的miRNA，那么肿瘤外泌体也会包含这些miRNA，当这些肿瘤外泌体被免疫细胞摄取后将通过miRNA的机制避免产生很强的免疫抑制性，进而提升免疫原性[25]。In order to solve the above problems, many transformation strategies for the mother cells that secrete tumor exosomes have been developed, such as increasing the heat shock protein content of tumor exosomes to provide an adjuvant effect [18,19]; increasing the expression of certain tumor-specific antigens to increase the abundance and immunogenicity of tumor-specific antigens in tumor exosomes [20]; adding superantigens to tumor exosomes, and using the characteristics of superantigens and MHC II molecules to form a stable complex to promote the activation of DC cells by tumor exosomes [21]; by overexpressing the transcription factor CIITA that controls MHC II molecules in tumor cells, increasing the abundance of MHC II molecules on tumor exosomes, improving the presentation efficiency of antigen peptides and the abundance of effective tumor antigen peptides [22]; integrating some viral fusion proteins into tumor exosomes to promote their uptake by DC cells [23]; overexpressing cytokines in tumor cells to increase the content of cytokines in tumor exosomes and the immunogenicity of tumor exosomes [24]; If cells highly express some miRNAs that have a negative regulatory effect on genes related to immunosuppression, then tumor exosomes will also contain these miRNAs. When these tumor exosomes are taken up by immune cells, they will avoid producing strong immunosuppression through the mechanism of miRNA, thereby enhancing immunogenicity[25].
此外，还有一种策略：通过将肿瘤外泌体在体外与DC共培养，将肿瘤特异性抗原交给DC来呈递，之后再以DC疫苗的形式来治疗肿瘤。这样可以避免游离的肿瘤外泌体引起的免疫抑制性以及激活更有效的免疫反应[7]。In addition, there is another strategy: by co-culturing tumor exosomes with DCs in vitro, tumor-specific antigens are presented to DCs, and then the tumor is treated in the form of a DC vaccine. This can avoid the immunosuppression caused by free tumor exosomes and activate a more effective immune response [7].
综上所述，由于携带大量肿瘤细胞的特异性抗原，肿瘤外泌体具有作为抗肿瘤疫苗开发的潜力， 但是肿瘤外泌体能抑制肿瘤微环境，甚至引流淋巴结中免疫细胞功能的天然属性限制了它的临床转化 应用。尽管提出了多种基于改造肿瘤细胞(分泌外泌体的母细胞)的策略，但是此类方案耗时长，价格昂贵且操作流程上难以统一，不便于在临床实践中的推广。In summary, tumor exosomes have the potential to be developed as anti-tumor vaccines because they carry a large number of tumor cell-specific antigens. However, the natural properties of tumor exosomes that can inhibit the tumor microenvironment and even drain the immune cell functions in the lymph nodes limit their clinical transformation and application . Although a variety of strategies based on the transformation of tumor cells (exosome-secreting mother cells) have been proposed, such schemes are time-consuming, expensive, and difficult to unify in terms of operating procedures, making them inconvenient for promotion in clinical practice.
基于此，提出本发明Based on this, the present invention is proposed
[参考文献][references]
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发明内容Summary of the invention
本发明首先涉及一种改造的肿瘤外泌体制剂，所述的制剂包含：The present invention firstly relates to a modified tumor exosome preparation, which comprises:
(1)浓度大于50ug/ml二硬脂酸磷脂酰乙醇胺-聚乙二醇(PEG-DSPE)；(1) Distearate phosphatidylethanolamine-polyethylene glycol (PEG-DSPE) at a concentration greater than 50ug/ml;
(2)蛋白浓度大于10ug/ml的肿瘤外泌体成分； (2) tumor exosome components with protein concentration greater than 10ug/ml;
(3)有效量的用于刺激免疫反应的免疫佐剂分子；(3) an effective amount of an immune adjuvant molecule for stimulating an immune response;
所述的PEG-DSPE的纯度≥95％，其中的PEG链的分散度≤1.1；The purity of the PEG-DSPE is ≥95%, and the dispersion of the PEG chains is ≤1.1;
优选的，所述的PEG-DSPE分子中的PEG链长为1000～5000Da；更优选的，所述的PEG-DSPE分子中的PEG链长为1500～2500Da；最优选的，所述的PEG-DSPE分子中的PEG链长为2000Da(PEG2000-DSPE)；Preferably, the PEG chain length in the PEG-DSPE molecule is 1000-5000Da; more preferably, the PEG chain length in the PEG-DSPE molecule is 1500-2500Da; most preferably, the PEG chain length in the PEG-DSPE molecule is 2000Da (PEG2000-DSPE);
所述的PEG-DSPE也可以使用其他类型的聚乙二醇化磷脂进行代替，具体的，The PEG-DSPE can also be replaced by other types of PEGylated phospholipids, specifically,
聚乙二醇化磷脂结构中，In the structure of PEGylated phospholipids,
磷脂部分的脂肪酸链包含的碳原子数为10～24个，脂肪酸链可以是饱和的，也可以是部分饱和的，优选为为月桂酸(12碳)、肉豆蔻酸(14碳)、棕榈酸(16碳)、硬脂酸或油酸或亚油酸(18碳)、廿酸(20碳)、山俞酸(22碳)、木焦油酸(lignocerate)(24碳)；The fatty acid chain of the phospholipid part contains 10 to 24 carbon atoms, and the fatty acid chain can be saturated or partially saturated, preferably lauric acid (12 carbons), myristic acid (14 carbons), palmitic acid (16 carbons), stearic acid or oleic acid or linoleic acid (18 carbons), eicosanoic acid (20 carbons), behenic acid (22 carbons), lignocerate (24 carbons);
聚乙二醇化磷脂的磷脂部分可以是磷酯酰乙醇胺(PE)、磷脂酰胆碱(PC)、磷脂酰肌醇(PI)、磷脂酰丝氨酸(PS)、二磷脂酰甘油、溶血磷脂胆碱(LPC)、溶血乙醇胺磷脂(LPE)等；优选为磷酯酰乙醇胺，尤其是二硬脂酰磷脂酰乙醇胺、二棕榈酰磷脂酰乙醇胺、二油酰磷脂酰乙醇胺。The phospholipid part of the PEGylated phospholipid can be phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), diphosphatidylglycerol, lysophosphatidylcholine (LPC), lysoethanolamine phospholipids (LPE), etc.; preferably phosphatidylethanolamine, especially distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine.
所述的蛋白浓度指制剂中所有的肿瘤外泌体中所包含的蛋白浓度，优选的，使用BCA法对肿瘤外泌体的蛋白浓度进行定量；The protein concentration refers to the protein concentration contained in all tumor exosomes in the preparation. Preferably, the protein concentration of tumor exosomes is quantified using the BCA method;
所述的免疫佐剂分子包括但不限于，MPLA(Monophosphoryl lipid A，单磷酰脂质A)、QS21(Quillaja saponaria，皂树皂苷)、PolyI:C(聚肌胞苷酸)；优选为MPLA；The immune adjuvant molecules include, but are not limited to, MPLA (Monophosphoryl lipid A), QS21 (Quillaja saponaria), and PolyI:C (polyinosinic acid); preferably, MPLA;
优选的，所述的免疫佐剂分子的用量为0.1～10ug/ml。Preferably, the dosage of the immune adjuvant molecule is 0.1-10 ug/ml.
进一步的，所述的改造的肿瘤外泌体制剂中，PEG-DSPE、免疫佐剂分子、肿瘤外泌体中的总蛋白三种成分的质量比为：100：0.1～5：2～50；优选的，所述的PEG-DSPE、免疫佐剂分子、肿瘤外泌体中的总蛋白三种成分的质量比为：100：0.5～2：5～20。Furthermore, in the modified tumor exosome preparation, the mass ratio of PEG-DSPE, immune adjuvant molecules, and total protein in tumor exosomes is 100: 0.1-5: 2-50; preferably, the mass ratio of PEG-DSPE, immune adjuvant molecules, and total protein in tumor exosomes is 100: 0.5-2: 5-20.
所述的肿瘤外泌体来源于体外培养的肿瘤系或自肿瘤组织中分离的原代肿瘤细胞；The tumor exosomes are derived from tumor lines cultured in vitro or primary tumor cells isolated from tumor tissues;
所述的肿瘤外泌体使用密度梯度离心、差速离心、体积排阻、免疫分离、聚合物沉淀法制备，或使用商品化的试剂盒制备；The tumor exosomes are prepared by density gradient centrifugation, differential centrifugation, size exclusion, immunoseparation, polymer precipitation, or by using a commercial kit;
本发明还涉及所述的改造的肿瘤外泌体制剂的制备方法，其包括如下步骤：The present invention also relates to a method for preparing the modified tumor exosome preparation, which comprises the following steps:
(1)制备不含溶剂成分的PEG-DSPE和免疫佐剂混合物；(1) preparing a mixture of PEG-DSPE and an immune adjuvant without a solvent component;
(2)向上述混合物中加入定量好的肿瘤外泌体溶液，常温下充分混匀即得所述改造的肿瘤外泌体制剂；(2) adding a quantitative tumor exosome solution to the above mixture, and mixing thoroughly at room temperature to obtain the modified tumor exosome preparation;
以及可选的，and optionally,
(3)对所述改造的肿瘤外泌体制剂进行除菌过滤。(3) sterilizing and filtering the modified tumor exosome preparation.
优选的，步骤(1)所述的制备不含溶剂成分的PEG-DSPE和免疫佐剂混合物的方法为，将PEG-DSPE和免疫佐剂分子溶于有机溶剂中并混匀，然后除去有机溶剂；更优选的，在惰性气体的保护下，使用减压蒸馏法，在低于70℃下去除有机溶剂。Preferably, the method for preparing the solvent-free mixture of PEG-DSPE and immune adjuvant in step (1) is to dissolve the PEG-DSPE and immune adjuvant molecules in an organic solvent and mix them, and then remove the organic solvent; more preferably, under the protection of an inert gas, use a reduced pressure distillation method to remove the organic solvent at a temperature below 70°C.
本发明还涉及所述的改造的肿瘤外泌体制剂在制备药物中的应用，所述的药物是抗肿瘤药物。The present invention also relates to the use of the modified tumor exosome preparation in the preparation of drugs, and the drugs are anti-tumor drugs.
本发明还涉及所述的改造的肿瘤外泌体制剂在制备个体化抗肿瘤制剂中的应用；优选的，所述的个体化抗肿瘤制剂中，所述的肿瘤外泌体来自于需要接受个体化治疗的患者自体的肿瘤组织或肿瘤细胞。The present invention also relates to the use of the modified tumor exosome preparation in the preparation of personalized anti-tumor preparations; preferably, in the personalized anti-tumor preparations, the tumor exosomes are derived from autologous tumor tissue or tumor cells of the patient who needs to receive personalized treatment.
本发明还涉及含有所述的改造的肿瘤外泌体制剂的药物或药物组合物。The present invention also relates to a medicine or a pharmaceutical composition containing the modified tumor exosome preparation.
本发明还涉及含有所述的改造的肿瘤外泌体制剂的个体化药物制剂，所述的个体化药物制剂中，所 述的肿瘤外泌体来自于需要接受个体化治疗的患者自体的肿瘤组织或肿瘤细胞。The present invention also relates to a personalized pharmaceutical preparation containing the modified tumor exosome preparation, wherein the personalized pharmaceutical preparation The tumor exosomes described above are derived from autologous tumor tissue or tumor cells of patients who need personalized treatment.
本发明还涉及使用所述的改造的肿瘤外泌体制剂治疗肿瘤的应用；优选的，使用来自于患者自体的肿瘤组织或肿瘤细胞，制备所述的改造的肿瘤外泌体制剂。The present invention also relates to the use of the modified tumor exosome preparation for treating tumors; preferably, the modified tumor exosome preparation is prepared using tumor tissue or tumor cells from the patient himself.
本发明的有益效果在于：The beneficial effects of the present invention are:
(1)聚乙二醇化磷脂(PEG2000-DSPE，以下简称PP)改造的肿瘤外泌体与游离的肿瘤外泌体相比，入胞的途径也发生了改变，PP改造肿瘤外泌体能直接进入内质网(图6)，便于引起更强的肿瘤抗原特异性T细胞应答(图7，8，9，10和11)；而与此对应的，游离的肿瘤外泌体被DC细胞内吞之后，快速进入溶酶体，其所含抗原将由MHC II途径呈递[14,19,20]。(1) Compared with free tumor exosomes, the tumor exosomes modified by polyethylene glycol phospholipids (PEG2000-DSPE, hereinafter referred to as PP) also have a different entry pathway. PP-modified tumor exosomes can directly enter the endoplasmic reticulum (Figure 6), which is convenient for inducing stronger tumor antigen-specific T cell responses (Figures 7, 8, 9, 10 and 11). Correspondingly, after being internalized by DC cells, free tumor exosomes quickly enter the lysosomes, and the antigens they contain will be presented by the MHC II pathway [14,19,20].
(2)在野生型小鼠中，给予PP改造的肿瘤外泌体能引起肿瘤抗原特异性CTL反应，而且在荷瘤小鼠中PP改造的肿瘤外泌体同样展现出良好的抗肿瘤效应。(2) In wild-type mice, administration of PP-modified tumor exosomes can induce tumor antigen-specific CTL responses, and in tumor-bearing mice, PP-modified tumor exosomes also show good anti-tumor effects.
(3)PP改造的肿瘤外泌体还能改善肿瘤微环境中的免疫细胞浸润情况，增加DC、巨噬细胞、具有抗肿瘤活性的CD4+，CD8+T细胞的比例，为与其它抗肿瘤免疫治疗方案的联用奠定了良好的基础。(3) PP-modified tumor exosomes can also improve the infiltration of immune cells in the tumor microenvironment and increase the proportion of DCs, macrophages, CD4+, and CD8+ T cells with anti-tumor activity, laying a good foundation for combined use with other anti-tumor immunotherapy regimens.
(4)治疗方案具有一定普适性：只要使用的肿瘤外泌体来源于同样的组织来源，那么即使基因突变频率或特点不一致，也应具有一定的治疗效果。(4) The treatment plan has a certain universality: as long as the tumor exosomes used are derived from the same tissue source, they should have a certain therapeutic effect even if the gene mutation frequency or characteristics are inconsistent.
(5)针对个体特异性有很好的疗效：在体内免疫编辑的作用下，或者接受了能够引起基因突变的治疗方式，包括并不仅限于放射治疗、化疗、免疫治疗等之后，部分肿瘤组织来源的外泌体用于制备PP改造的肿瘤外泌体对剩余肿瘤组织具有治疗作用，甚至可以预防肿瘤的远端器官转移。(5) Good therapeutic effect for individual specificity: Under the action of in vivo immune editing, or after receiving treatment that can cause gene mutations, including but not limited to radiotherapy, chemotherapy, immunotherapy, etc., some exosomes derived from tumor tissues are used to prepare PP-modified tumor exosomes, which have a therapeutic effect on the remaining tumor tissues and can even prevent the distant organ metastasis of the tumor.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1、不同表面活性剂对骨髓来源DC产生的细胞毒性【体外实验】。纵坐标显示经不同表面活性剂处理48小时后DC细胞的活性(MTT法)，横坐标显示不同表面活性剂的浓度。Figure 1. Cytotoxicity of different surfactants on bone marrow-derived DCs [in vitro experiment]. The vertical axis shows the activity of DC cells after being treated with different surfactants for 48 hours (MTT method), and the horizontal axis shows the concentration of different surfactants.
图2、PP改造的肿瘤外泌体的制备和表征。A、冷冻电子显微镜照片展示肿瘤外泌体(左图)，缺乏MPLA对照品(中图)和PP改造的肿瘤外泌体(右图)的形貌特征；B、动态光散射结果展示肿瘤外泌体(黑色曲线)和PP改造的肿瘤外泌体(灰色曲线)的平均直径分布情况。Figure 2. Preparation and characterization of PP-modified tumor exosomes. A. Cryo-electron microscopy images show the morphological characteristics of tumor exosomes (left), MPLA-deficient control (middle) and PP-modified tumor exosomes (right); B. Dynamic light scattering results show the average diameter distribution of tumor exosomes (black curve) and PP-modified tumor exosomes (grey curve).
图3、PP改造的肿瘤外泌体促进DC细胞对抗原的呈递能力【体外实验】。纵坐标显示经不同处理的DC细胞表面所呈递出的OVA蛋白特异性抗原肽(SIINFEKL)与MHC-I分子(H-2Kb)形成的复合体的数量——由平均荧光强度(MFI)表示。Figure 3. PP-modified tumor exosomes promote the ability of DC cells to present antigens [in vitro experiment]. The vertical axis shows the number of complexes formed by OVA protein-specific antigen peptide (SIINFEKL) and MHC-I molecules (H-2K b ) presented on the surface of DC cells treated with different methods, represented by the mean fluorescence intensity (MFI).
图4、PP改造的肿瘤外泌体增加DC细胞表面共刺激分子的表达水平【体外实验】。纵坐标显示经不同处理的DC细胞表面代表激活标志的共刺激分子CD80(左图)和CD86(右图)的表达量——由平均荧光强度(MFI)表示。Figure 4. PP-modified tumor exosomes increase the expression level of co-stimulatory molecules on the surface of DC cells [in vitro experiment]. The vertical axis shows the expression of co-stimulatory molecules CD80 (left figure) and CD86 (right figure) on the surface of DC cells treated with different methods, which represent activation markers, represented by the mean fluorescence intensity (MFI).
图5、PP改造的肿瘤外泌体增加DC细胞分泌细胞因子的水平【体外实验】。纵坐标显示经不同处理的DC细胞培养上清中经ELISA法检测得到的TNF-α、IL-2和IL-12的浓度(pg/ml)，以及IFN-β的实时定量PCR结果(相对于β-actin管家基因的相对表达量)。Figure 5. PP-modified tumor exosomes increase the level of cytokine secretion by DC cells [in vitro experiment]. The vertical axis shows the concentrations of TNF-α, IL-2 and IL-12 (pg/ml) detected by ELISA in the culture supernatant of DC cells treated with different methods, as well as the real-time quantitative PCR results of IFN-β (relative expression level of β-actin housekeeping gene).
图6、PP改造能改变肿瘤外泌体被DC细胞摄取后的路径【体外实验】。A、共聚焦显微镜照片展示PKH-67标记的肿瘤外泌体(EXO)或PP改造的肿瘤外泌体(PP/EXO)(绿色-Green)分别与DC2.4细胞内的溶酶体(Lysosome)(左侧)以及内质网(ER)(红色-Red)的共定位情况；B、共聚焦显微镜照片展示CFSE标记的肿瘤外泌体(EXO)或PP改造的肿瘤外泌体(PP/EXO)(绿色-Green)分别与DC2.4细胞内的溶酶体(Lysosome)(左侧)以及内质网(ER)(红色-Red)的共定位情况。Figure 6. PP modification can change the path of tumor exosomes after being taken up by DC cells [in vitro experiment]. A. Confocal microscopy photos show the co-localization of PKH-67-labeled tumor exosomes (EXO) or PP-modified tumor exosomes (PP/EXO) (green-Green) with lysosomes (Lysosome) (left) and endoplasmic reticulum (ER) (red-Red) in DC2.4 cells; B. Confocal microscopy photos show the co-localization of CFSE-labeled tumor exosomes (EXO) or PP-modified tumor exosomes (PP/EXO) (green-Green) with lysosomes (Lysosome) (left) and endoplasmic reticulum (ER) (red-Red) in DC2.4 cells.
图7、PP改造的肿瘤外泌体促进抗原特异性OT I CD8+T细胞的增殖【体外实验】。纵坐标显示经不同处理的DC细胞激活OT I CD8+T细胞增殖的不同比例。Figure 7. PP-modified tumor exosomes promote the proliferation of antigen-specific OT I CD8 + T cells [in vitro experiment]. The vertical axis shows the different proportions of OT I CD8 + T cell proliferation activated by DC cells treated differently.
图8、PP改造的肿瘤外泌体引起强烈抗原特异性的CTL反应——小鼠结肠癌模型(MC38)【体内 实验】。经不同组别的外泌体免疫后的小鼠淋巴结T细胞经肿瘤特异性抗原肽(Rpl18，Reps-1和Adpgk)体外再刺激产生的IFN-γ斑点(ELLIspot实验)的照片(左图)和IFN-γ斑点的计数统计结果(右图)。Figure 8. PP-modified tumor exosomes induce strong antigen-specific CTL response in mouse colon cancer model (MC38) [in vivo Experiment】. Photos of IFN-γ spots (ELLIspot experiment) produced by mouse lymph node T cells after immunization with different groups of exosomes and restimulation with tumor-specific antigen peptides (Rpl18, Reps-1 and Adpgk) in vitro (left) and the counting and statistical results of IFN-γ spots (right).
图9、PP改造的肿瘤外泌体引起强烈抗原特异性的CTL反应——小鼠黑色素瘤模型(B16F10)【体内实验】。经不同组别的外泌体免疫后的小鼠淋巴结T细胞经肿瘤特异性抗原肽(Trp-2)体外再刺激产生的IFN-γ斑点(ELLIspot实验)的照片(左图)和IFN-γ斑点的计数统计结果(右图)。Figure 9. PP-modified tumor exosomes induce strong antigen-specific CTL response - mouse melanoma model (B16F10) [in vivo experiment]. Photos of IFN-γ spots (ELLIspot experiment) produced by mouse lymph node T cells after immunization with different groups of exosomes and restimulation with tumor-specific antigen peptide (Trp-2) in vitro (left) and the counting and statistical results of IFN-γ spots (right).
图10、PP改造的肿瘤外泌体引起强烈抗原特异性的CTL反应——小鼠三阴性乳腺癌模型(4T1)【体内实验】。经不同组别的外泌体免疫后的小鼠淋巴结T细胞经肿瘤特异性抗原肽(Sptbn和Wdr33)体外再刺激产生的IFN-γ斑点(ELLIspot实验)的照片(左图)和IFN-γ斑点的计数统计结果(右图)。Figure 10. PP-modified tumor exosomes induce strong antigen-specific CTL response - mouse triple-negative breast cancer model (4T1) [in vivo experiment]. Photos of IFN-γ spots (ELLIspot experiment) produced by mouse lymph node T cells after immunization with different groups of exosomes and restimulation with tumor-specific antigen peptides (Sptbn and Wdr33) in vitro (left) and the counting and statistical results of IFN-γ spots (right).
图11、PP改造的肿瘤外泌体引起强烈抗原特异性的CTL反应——小鼠宫颈癌模型(TC-1，带有HPV病毒抗原E6、E7)【体内实验】。经不同组别的外泌体免疫后的小鼠淋巴结T细胞经肿瘤特异性抗原肽(E7-20)体外再刺激产生的IFN-γ斑点(ELLIspot实验)的照片(左图)和IFN-γ斑点的计数统计结果(右图)。Figure 11. PP-modified tumor exosomes induce strong antigen-specific CTL response - mouse cervical cancer model (TC-1, with HPV virus antigens E6 and E7) [in vivo experiment]. Photos of IFN-γ spots (ELLIspot experiment) produced by mouse lymph node T cells after immunization with different groups of exosomes and restimulation with tumor-specific antigen peptides (E7-20) in vitro (left) and the counting and statistical results of IFN-γ spots (right).
图12、PP改造的肿瘤外泌体的抗肿瘤作用——小鼠结肠癌模型(MC38)【体内实验】。小鼠接瘤后经不同组别的外泌体治疗，肿瘤生长曲线(左图)和小鼠生存期(右图)。Figure 12. Anti-tumor effect of PP-modified tumor exosomes - mouse colon cancer model (MC38) [in vivo experiment]. After mice were implanted with tumors, they were treated with exosomes from different groups, and the tumor growth curve (left) and mouse survival time (right).
图13、PP改造的肿瘤外泌体的抗肿瘤作用——小鼠黑色素瘤模型(B16F10)【体内实验】。小鼠接瘤后经不同组别的外泌体治疗，肿瘤生长曲线(左图)和小鼠生存期(右图)。Figure 13. Anti-tumor effect of PP-modified tumor exosomes - mouse melanoma model (B16F10) [in vivo experiment]. After mice were implanted with tumors, they were treated with exosomes from different groups, and the tumor growth curve (left) and mouse survival time (right).
图14、PP改造的肿瘤外泌体的抗肿瘤作用——小鼠宫颈癌模型(TC-1，带有HPV病毒抗原E7)【体内实验】。小鼠接瘤后经不同组别的外泌体治疗，肿瘤生长曲线。Figure 14. Anti-tumor effect of PP-modified tumor exosomes - mouse cervical cancer model (TC-1, with HPV virus antigen E7) [in vivo experiment]. Tumor growth curve of mice after tumor implantation and treatment with different groups of exosomes.
图15、PP改造的肿瘤外泌体的抗肿瘤转移作用——小鼠黑色素瘤模型(B16F10)【体内实验】。经不同组别的外泌体提前免疫后，小鼠经尾静脉接瘤，13天后观察小鼠肺部黑色素瘤细胞形成的转移结节的代表性照片(A)及统计数据(B)和各组所有小鼠肺脏的照片(C)。Figure 15. Anti-tumor metastasis effect of PP-modified tumor exosomes - mouse melanoma model (B16F10) [in vivo experiment]. After pre-immunization with exosomes from different groups, mice were implanted with tumors via the tail vein. Representative photos (A) and statistical data (B) of metastatic nodules formed by melanoma cells in the mouse lungs and photos of all mouse lungs in each group (C) were observed 13 days later.
图16、PP改造的肿瘤外泌体增强抗肿瘤相关免疫细胞的浸润——小鼠黑色素瘤模型(B16F10)【体内实验】。Figure 16. PP-modified tumor exosomes enhance the infiltration of anti-tumor-related immune cells - mouse melanoma model (B16F10) [in vivo experiment].
图17、PP改造的肿瘤外泌体制备技术模拟实现个体化肿瘤治疗——小鼠结肠癌模型(MC38)【体内实验】。A、实验流程示意图；B、肿瘤生长曲线。Figure 17. PP-modified tumor exosome preparation technology simulates personalized tumor treatment - mouse colon cancer model (MC38) [in vivo experiment]. A, schematic diagram of the experimental process; B, tumor growth curve.
具体实施方式Detailed ways
小鼠模型：Mouse Model:
OTI转基因小鼠模型：该小鼠模型的特点是其CD8阳性T细胞的T细胞受体(TCR)经基因编辑，能特异性识别抗原呈递细胞表面MHC-I/OVA257-264(SIINFEKL)抗原复合物，并被特异性激活，增殖。OTI transgenic mouse model: The characteristic of this mouse model is that the T cell receptor (TCR) of its CD8 positive T cells has been gene-edited, which can specifically recognize the MHC-I/OVA 257-264 (SIINFEKL) antigen complex on the surface of antigen presenting cells, and be specifically activated and proliferate.
实施例一、不同表面活性剂对骨髓来源DC细胞(树突状细胞)的毒性Example 1: Toxicity of different surfactants to bone marrow-derived DC cells (dendritic cells)
细胞培养及处理方法：Cell culture and treatment methods:
骨髓来源DC的获取：Obtaining bone marrow-derived DCs:
取6-8周龄C57BL/6小鼠，解剖获取长骨和胫骨，将骨髓吹出，ACK2ml处理1分钟裂解红细胞，无血清培养基中和后离心，重悬获得小鼠骨髓细胞悬液，以3×106细胞/皿种于10cm培养皿中。完全培养基(含10％FBS、50μM巯基乙醇的RPMI 1640)加入20ng/ml重组小鼠源GM-CSF培养(按第0天计算)。第3天补加10ml完全培养基(含20ng/ml重组小鼠源GM-CSF)。培养至第6天，收集未贴壁细胞作为未成熟的骨髓来源DC细胞。经流式鉴定CD11c+的DC细胞纯度达90％以上，可用于后续实验。Take 6-8 week old C57BL/6 mice, dissect and obtain long bones and tibiae, blow out the bone marrow, treat with ACK2ml for 1 minute to lyse red blood cells, neutralize with serum-free medium and centrifuge, resuspend to obtain mouse bone marrow cell suspension, and plant in 10 cm culture dish at 3×10 6 cells/dish. Add 20ng/ml recombinant mouse GM-CSF to complete medium (RPMI 1640 containing 10% FBS and 50μM mercaptoethanol) for culture (calculated as day 0). On the 3rd day, add 10ml complete medium (containing 20ng/ml recombinant mouse GM-CSF). Culture to day 6, collect non-adherent cells as immature bone marrow-derived DC cells. The purity of CD11c + DC cells identified by flow cytometry is more than 90%, which can be used for subsequent experiments.
实验方案：不同表面活性剂对骨髓来源DC产生的细胞毒性Experimental protocol: Cytotoxicity of different surfactants on bone marrow-derived DCs
为了打破肿瘤外泌体的脂质囊泡结构，需要引入表面活性剂。但是，表面活性剂(表一)具有破坏细胞膜的作用，鉴于我们的目的是将改造后的肿瘤外泌体用于肿瘤个性化疫苗的研究和应用，而作为疫 苗的首要条件就是需要安全。因此，我们接下来利用MTT实验考察了这些表面活性剂是否能够对骨髓来源的DC细胞产生细胞毒性。In order to break the lipid vesicle structure of tumor exosomes, surfactants need to be introduced. However, surfactants (Table 1) have the effect of destroying cell membranes. Given that our goal is to use the modified tumor exosomes for the research and application of personalized tumor vaccines, as vaccines Therefore, we then used the MTT assay to investigate whether these surfactants could produce cytotoxicity to bone marrow-derived DC cells.
小鼠骨髓来源的DC细胞以30万/孔铺于96孔板中，加入不同浓度的SDS、TritonX-100、Tween80和PEG2000-DSPE(PP)溶液。37℃培养24h后，向孔中加入20μl的5mg/ml的MTT溶液，继续培养6h。之后加入MTT裂解液，吹打混匀后，放入酶标仪中检测其在570nm和630nm(参比波长)处的吸收值。Mouse bone marrow derived DC cells were plated in 96-well plates at 300,000/well, and different concentrations of SDS, TritonX-100, Tween80 and PEG2000-DSPE (PP) solutions were added. After culturing at 37°C for 24 hours, 20 μl of 5 mg/ml MTT solution was added to the wells and cultured for another 6 hours. Then MTT lysis buffer was added, pipetted and mixed, and then placed in an ELISA reader to detect its absorbance at 570 nm and 630 nm (reference wavelength).
结果如图1所示：首先，所有剂量的PEG2000-DSPE对DC细胞基本没有产生毒性，但SDS和TritonX-100在25μg/ml时便对细胞产生了巨大的细胞毒性。Tween80在浓度超过50μg/ml时对细胞液开始产生毒性。The results are shown in Figure 1: First, all doses of PEG2000-DSPE had little toxicity to DC cells, but SDS and TritonX-100 had great cytotoxicity to cells at 25μg/ml. Tween80 began to be toxic to cell fluid at a concentration of more than 50μg/ml.
以上结果说明，从安全性角度出发PEG2000-DSPE是最佳选择。而Tween80需要控制在安全用量(50μg/ml)以下，SDS和TritonX-100则由于细胞毒性太大，不适用于肿瘤外泌体的改造。The above results show that PEG2000-DSPE is the best choice from the perspective of safety. Tween80 needs to be controlled below the safe dosage (50μg/ml), and SDS and TritonX-100 are not suitable for the modification of tumor exosomes due to their high cytotoxicity.
表1、不同表面活性剂的基本信息
Table 1. Basic information of different surfactants
实施例二、PP改造的肿瘤外泌体的制备和表征Example 2. Preparation and characterization of PP-modified tumor exosomes
细胞培养及处理方法Cell culture and treatment methods
小鼠结肠癌模型MC38细胞系的培养：使用DMEM培养基(含10％胎牛血清)。将MC38肿瘤细胞冻存管从液氮保存罐中取出，立即放入37℃水浴中快速溶解，然后将细胞悬液移入含10ml培养基的离心桶中，350g离心5分钟后去除上清，用新鲜培养基重悬后，将细胞转移到细胞培养瓶中，加入10-15ml培养基混悬沉淀细胞，调整细胞浓度后，置于37℃、体积分数5％CO2饱和湿度培养箱中培养。在维持培养过程中，每天观察细胞状态并及时更换新鲜培养基。Culture of MC38 cell line in mouse colon cancer model: Use DMEM medium (containing 10% fetal bovine serum). Take out the cryopreserved tube of MC38 tumor cells from the liquid nitrogen storage tank and immediately put it into a 37°C water bath for rapid dissolution. Then transfer the cell suspension into a centrifuge bucket containing 10ml of culture medium. Centrifuge at 350g for 5 minutes and remove the supernatant. After resuspending with fresh culture medium, transfer the cells to a cell culture bottle, add 10-15ml of culture medium to suspend the precipitated cells, adjust the cell concentration, and culture in a 37°C, 5% CO 2 saturated humidity incubator. During the culture maintenance process, observe the cell status every day and replace the fresh culture medium in time.
(1)接瘤：当细胞贴壁生长至90％汇合度时，用0.05％胰酶消化，按照1:3的比例进行传代培养。实验当天，将生长状态良好，汇合度达到90％的细胞胰酶消化后，用新鲜培养基中和胰酶，350g离心，弃上清后加入无菌PBS重悬，计数，将细胞悬液的密度调整为2.5×106/ml待用。每只小鼠皮下接瘤2.5×105细胞。(1) Tumor grafting: When cells adhere to the wall and grow to 90% confluence, digest them with 0.05% trypsin and subculture them at a ratio of 1:3. On the day of the experiment, digest the cells with good growth and 90% confluence with trypsin, neutralize the trypsin with fresh culture medium, centrifuge at 350g, discard the supernatant, resuspend in sterile PBS, count, and adjust the density of the cell suspension to 2.5×10 6 /ml for later use. 2.5×10 5 cells were subcutaneously grafted on each mouse.
(2)收集肿瘤外泌体(EXO)实验：当细胞贴壁生长至90％汇合度时，用0.05％胰酶消化，按照1:3的比例进行传代培养。传代培养时，换用含有10％无外泌体的胎牛血清的培养基继续培养，48小时后，收取上清，直接用于外泌体提取或者冻存于-80℃冰箱备用。(2) Tumor exosome (EXO) collection experiment: When the cells adhere to the wall and grow to 90% confluence, they are digested with 0.05% trypsin and subcultured at a ratio of 1:3. During subculture, the culture medium containing 10% fetal bovine serum without exosomes is used for continued culture. After 48 hours, the supernatant is collected and directly used for exosome extraction or frozen in a -80℃ refrigerator for later use.
肿瘤外泌体(EXO)的获取Obtaining tumor exosomes (EXO)
肿瘤细胞培养在含有10％已去除外泌体的胎牛血清(血清经100,000g，2小时离心后保留上清部分)的培养基中48小时后，收集细胞培养上清。After tumor cells were cultured in a medium containing 10% exosome-depleted fetal bovine serum (the serum was centrifuged at 100,000 g for 2 hours and the supernatant was retained) for 48 hours, the cell culture supernatant was collected.
(1)4000rpm离心2小时去除上清中的细胞碎片。(1) Centrifuge at 4000 rpm for 2 hours to remove cell debris in the supernatant.
(2)利用100kDa的超滤管以4000rpm、30分钟/次的速度收集上清中大于100kDa的组份。 (2) Use a 100 kDa ultrafiltration tube at 4000 rpm, 30 minutes/time to collect the components larger than 100 kDa in the supernatant.
(3)收集的组份以5:1(v:v)加入外泌体提取试剂(EXOTC10A-1，SBI)，充分混合后4℃放置至少12小时。(3) The collected fractions were added to exosome extraction reagent (EXOTC10A-1, SBI) at a ratio of 5:1 (v:v), mixed thoroughly, and placed at 4°C for at least 12 h.
(4)待12小时后沉淀出现，3000rpm离心30分钟，分离出沉淀物，即为粗提外泌体。使用的PBS重悬后，经BCA法测定蛋白质浓度，并以此标定外泌体的含量，分装后置于-80℃保存，备用。(4) After 12 hours, a precipitate appeared. Centrifuge at 3000 rpm for 30 minutes to separate the precipitate, which is the crude exosome. Resuspend in PBS, measure the protein concentration by BCA method, and use it to calibrate the exosome content. After packaging, store at -80°C for later use.
(5)密度梯度离心：将粗体外泌体置于10％、20％、40％、60％的不连续密度梯度蔗糖溶液中，进行2小时的超速离心(100，000g)，之后小心吸取位于10-20％层的白色物质，用PBS稀释后再次超速离心将外泌体沉淀于底部，即为精提外泌体。使用适量PBS重悬后，经BCA法测定蛋白质浓度，并以此标定外泌体的含量，-80℃保存备用。(5) Density gradient centrifugation: Place the crude exosomes in a discontinuous density gradient sucrose solution of 10%, 20%, 40%, and 60%, and perform ultracentrifugation (100,000g) for 2 hours. Then carefully aspirate the white matter in the 10-20% layer, dilute with PBS, and ultracentrifuge again to precipitate the exosomes at the bottom, which is the refined exosomes. After resuspending with an appropriate amount of PBS, the protein concentration was determined by the BCA method, and the exosome content was calibrated with this method, and stored at -80°C for later use.
以下各组实施例中：除电子显微镜实验和动态光散射实验使用精提外泌体之外，其它实验均使用粗提外泌体。后续文中将统一称为肿瘤外泌体(EXO)。In the following examples, except for the electron microscopy experiment and the dynamic light scattering experiment, which used refined exosomes, all other experiments used crude exosomes, which will be collectively referred to as tumor exosomes (EXO) in the following text.
聚乙二醇化磷脂(PP)改造的肿瘤外泌体的制备(以小鼠结肠癌MC38细胞系分泌的外泌体为例)：Preparation of tumor exosomes modified by PEGylated phospholipids (PP) (taking exosomes secreted by mouse colon cancer MC38 cell line as an example):
(1)将已经通过BCA蛋白法定量的肿瘤外泌体，用无菌水稀释成500ug/ml的肿瘤外泌体(EXO)储液；(1) The tumor exosomes quantified by the BCA protein assay were diluted with sterile water to a 500 ug/ml tumor exosome (EXO) stock solution;
(2)将MPLA粉末溶解于甲醇/氯仿(1：2，v:v)混合溶液中，配制成浓度为1mg/ml的MPLA储液；(2) Dissolve MPLA powder in a methanol/chloroform (1:2, v:v) mixed solution to prepare an MPLA stock solution with a concentration of 1 mg/ml;
(3)称取15mg PP粉末加入3ml氯仿溶解，配制成浓度为5mg/ml的PP储液；(3) Weigh 15 mg of PP powder and add 3 ml of chloroform to dissolve it to prepare a PP stock solution with a concentration of 5 mg/ml;
(4)按照表2-1的处方制备10ml PP改造的肿瘤外泌体(简称为PP/MPLA/EXO)，具体步骤如下：(4) Prepare 10 ml of PP-modified tumor exosomes (abbreviated as PP/MPLA/EXO) according to the prescription in Table 2-1. The specific steps are as follows:
a)取1ml PP储液，50ul MPLA储液混合均匀；a) Take 1ml PP stock solution and 50ul MPLA stock solution and mix them evenly;
b)使用氮气吹干的方法，除去有机溶剂；b) using nitrogen to blow dry and remove the organic solvent;
c)加入1ml EXO储液，充分混匀，获得无色透明的PP改造的肿瘤外泌体溶液；c) Add 1 ml of EXO stock solution and mix thoroughly to obtain a colorless and transparent PP-modified tumor exosome solution;
d)经0.22um滤膜过滤除菌。d) Sterilize by filtration through a 0.22 um filter membrane.
使用前用无菌水稀释10倍，制成总体积为10ml，蛋白含量为50ug/ml EXO浓度的PP改造的肿瘤外泌体溶液。Before use, dilute 10 times with sterile water to prepare a PP-modified tumor exosome solution with a total volume of 10 ml and a protein content of 50 ug/ml EXO concentration.
表2-1：PP改造的肿瘤外泌体处方
Table 2-1: PP modified tumor exosome prescription
成分缺失对照品的制备(以小鼠结肠癌MC38细胞系分泌的外泌体为例)：Preparation of component deficiency control (taking exosomes secreted by mouse colon cancer MC38 cell line as an example):
完整的PP改造的肿瘤外泌体(PP/MPLA/EXO)由三个成分组成，功能实验中需要成分缺失的对照，包括缺失MPLA和缺失聚乙二醇化磷脂(PP)的对照品。The complete PP-modified tumor exosomes (PP/MPLA/EXO) are composed of three components, and functional experiments require controls with missing components, including controls lacking MPLA and controls lacking PEGylated phospholipids (PP).
(1)无MPLA对照品(简称为PP/EXO)的制备：(1) Preparation of MPLA-free reference substance (referred to as PP/EXO):
a)将已经通过BCA蛋白法定量的肿瘤外泌体，用无菌水稀释成500ug/ml的肿瘤外泌体(EXO)储液；a) diluting the tumor exosomes quantified by BCA protein assay with sterile water to a 500ug/ml tumor exosome (EXO) stock solution;
b)称取15mg PP粉末加入3ml氯仿溶解，配制成浓度为5mg/ml的PP储液；b) Weigh 15 mg PP powder and add 3 ml chloroform to dissolve it to prepare a PP stock solution with a concentration of 5 mg/ml;
c)按照表2-2的处方制备10ml无MPLA对照品(简称为PP/EXO)，具体步骤参考上述PP改造的肿瘤外泌体(PP/MPLA/EXO)的制备。c) Prepare 10 ml of MPLA-free control substance (referred to as PP/EXO) according to the prescription in Table 2-2. The specific steps refer to the preparation of the above-mentioned PP-modified tumor exosomes (PP/MPLA/EXO).
表2-2：无MPLA对照品(简称为PP/EXO)处方
Table 2-2: Prescriptions for reference substances without MPLA (referred to as PP/EXO)
使用前用无菌水稀释10倍制成总体积为10ml，蛋白含量为50ug/ml EXO浓度的无MPLA的对照品(简称为PP/EXO)溶液。 Before use, dilute 10 times with sterile water to prepare a control solution without MPLA (abbreviated as PP/EXO) with a total volume of 10 ml and a protein content of 50 ug/ml EXO concentration.
(2)无PP对照品(简称MPLA/EXO)的制备：(2) Preparation of PP-free reference substance (MPLA/EXO):
将MPLA粉末溶解于甲醇/氯仿(1：2，v:v)混合溶液中，配制成浓度为1mg/ml的MPLA储液；MPLA powder was dissolved in a methanol/chloroform (1:2, v:v) mixed solution to prepare an MPLA stock solution with a concentration of 1 mg/ml;
按照表2-3的处方将50ul MPLA储液和1ml EXO储液进行物理混合制备10ml无PP对照品(简称MPLA/EXO)。According to the prescription in Table 2-3, 50ul of MPLA stock solution and 1ml of EXO stock solution were physically mixed to prepare 10ml of PP-free reference substance (abbreviated as MPLA/EXO).
表2-3：无PP对照品(简称MPLA/EXO)处方
Table 2-3: Prescription of PP-free reference substance (MPLA/EXO for short)
使用前用无菌水稀释10倍，制成总体积为10ml，蛋白含量为50ug/ml EXO浓度的无PP对照品(简称为MPLA/EXO)溶液。Before use, dilute 10 times with sterile water to prepare a PP-free control solution (abbreviated as MPLA/EXO) with a total volume of 10 ml and a protein content of 50ug/ml EXO concentration.
实验方案(一)冷冻电镜观察PP改造的肿瘤外泌体的形态表征Experimental plan (I) Morphological characterization of PP-modified tumor exosomes observed by cryo-electron microscopy
(1)按照前述方法制备PP改造的肿瘤外泌体；(1) preparing PP-modified tumor exosomes according to the aforementioned method;
(2)实验共分为3组，分别为：(2) The experiment was divided into three groups:
a)肿瘤外泌体组(EXO)a) Tumor exosomes (EXO)
b)PP改造的肿瘤外泌体组(PP/MPLA/EXO)b) PP-modified tumor exosome group (PP/MPLA/EXO)
c)无MPLA对照组(PP/EXO)c) Control group without MPLA (PP/EXO)
(3)冷冻电镜样品制备：将2.5μl样品加载到H2/O2辉光放电预处理30秒的碳网格(Quantifoil 300目，R1.2/1.3)上。在23℃和95％湿度下，使用Vitrobot Mark IV(Thermo Fisher Scientific)将多余样品在2级力下用滤纸吸附6秒，之后迅速放入液体乙烷中快速冷冻，转移到液氮中储存；(3) Cryo-EM sample preparation: 2.5 μl of sample was loaded onto a carbon grid (Quantifoil 300 mesh, R1.2/1.3) pretreated with H 2 /O 2 glow discharge for 30 seconds. The excess sample was adsorbed on filter paper at level 2 force for 6 seconds using a Vitrobot Mark IV (Thermo Fisher Scientific) at 23°C and 95% humidity, then quickly placed in liquid ethane for rapid freezing and transferred to liquid nitrogen for storage;
(4)将铜网置于透射电子显微镜(Talos L120C 120kV)下观察；(4) Place the copper mesh under a transmission electron microscope (Talos L120C 120 kV) for observation;
实验结果显示，肿瘤外泌体的平均直径在100nm左右，符合文献报道的外泌体直径分布范围(30-150nm)。而PP改造的肿瘤外泌体直径在10-20nm左右(图2A)，说明经过PP改造的肿瘤外泌体会变得更小且更均匀。同时，我们发现PP/EXO组和PP/MPLA/EXO组之间的样品形态并未表现出很大的差别，因此MPLA不影响聚乙二醇化磷脂对肿瘤外泌体结构上的改造。The experimental results showed that the average diameter of tumor exosomes was about 100nm, which was consistent with the exosome diameter distribution range reported in the literature (30-150nm). The diameter of PP-modified tumor exosomes was about 10-20nm (Figure 2A), indicating that the tumor exosomes modified by PP became smaller and more uniform. At the same time, we found that the sample morphology between the PP/EXO group and the PP/MPLA/EXO group did not show much difference, so MPLA did not affect the modification of the structure of tumor exosomes by PEGylated phospholipids.
实验方案(二)PP改造的肿瘤外泌体的动态光散射结果Experimental plan (II) Dynamic light scattering results of PP-modified tumor exosomes
(1)按照前述方法制备PP改造的肿瘤外泌体；(1) preparing PP-modified tumor exosomes according to the aforementioned method;
(2)实验共分为2组，分别为：(2) The experiment was divided into two groups:
a)肿瘤外泌体组(EXO)a) Tumor exosomes (EXO)
b)无MPLA对照组(PP/EXO)b) Control group without MPLA (PP/EXO)
(3)用水将各组样品稀释后置于纳米粒度电位仪(Zetasizer Nano ZS)中进行检测。(3) Dilute each group of samples with water and place them in a Zetasizer Nano ZS for testing.
(4)实验结果显示，肿瘤外泌体的粒径分布在50-200nm范围内，与文献报道的外泌体直径范围(30-150nm)基本一致。鉴于MPLA不影响聚乙二醇化磷脂对肿瘤外泌体结构上的改造，动态光散射实验仅采用无MPLA对照品(PP/EXO)进行测试，结果显示PP/EXO组的平均粒径分布在10nm附近(图2B)，进一步映证了冷冻电镜的结果。(4) The experimental results showed that the particle size distribution of tumor exosomes was in the range of 50-200 nm, which is basically consistent with the exosome diameter range reported in the literature (30-150 nm). Given that MPLA does not affect the structural modification of tumor exosomes by PEGylated phospholipids, the dynamic light scattering experiment was tested only with the MPLA-free control (PP/EXO). The results showed that the average particle size distribution of the PP/EXO group was around 10 nm (Figure 2B), which further confirmed the results of cryo-EM.
实验方案(三)PP改造的肿瘤外泌体稳定性(4℃保存一个月)Experimental plan (III) Stability of PP-modified tumor exosomes (stored at 4°C for one month)
(1)按照前述方法制备PP改造的肿瘤外泌体以及单一成分缺省对照组；(1) PP-modified tumor exosomes and a single-component default control group were prepared according to the aforementioned method;
(2)实验共分为3组，分别为：(2) The experiment was divided into three groups:
a)无MPLA对照组(PP/EXO)a) Control group without MPLA (PP/EXO)
b)无PP对照组(MPLA/EXO)b) Control group without PP (MPLA/EXO)
c)PP改造的肿瘤外泌体(PP/MPLA/EXO)c) PP modified tumor exosomes (PP/MPLA/EXO)
(4)将各组样品放入4℃冰箱保存一个月，并于保存前和保存后进行拍照观察； (4) Each group of samples was placed in a 4°C refrigerator for one month, and photographed before and after storage;
(5)实验结果显示，经过一个月的放置，各组均未出现溶液变浑浊的现象，说明该处方制备的PP改造的肿瘤外泌体性质基本稳定，在4℃的保存条件下，一个月之内不会变质。(5) The experimental results showed that after one month of storage, none of the solutions in each group became turbid, indicating that the properties of the PP-modified tumor exosomes prepared by this prescription were basically stable and would not deteriorate within one month when stored at 4°C.
实施例三、PP改造的肿瘤外泌体增强骨髓来源DC细胞(树突状细胞)的功能Example 3: PP-modified tumor exosomes enhance the function of bone marrow-derived DC cells (dendritic cells)
细胞培养及处理方法：Cell culture and treatment methods:
骨髓来源DC的获取可参考实施例一。The acquisition of bone marrow-derived DCs can refer to Example 1.
肿瘤外泌体(EXO)的获取，PP改造的肿瘤外泌体(MC38来源)及相应对照品的制备同实施例二。The acquisition of tumor exosomes (EXO), the preparation of PP-modified tumor exosomes (MC38 source) and corresponding reference substances were the same as in Example 2.
实验方案(一)：PP改造的肿瘤外泌体促进DC细胞对抗原的呈递能力Experimental plan (I): PP-modified tumor exosomes promote the antigen presentation ability of DC cells
(1)使用含有50ug/ml的肿瘤外泌体(以BCA蛋白定量)及PP改造的肿瘤外泌体处理骨髓来源的DC细胞，同时加入0.5mg/ml OVA全长蛋白的PBS溶液，24小时后，使用PE标记的抗小鼠的MHC I-SIINFEKL抗原复合物(简称p-MHC I)的荧光抗体进行流式细胞术检测，考察各处理组中DC细胞该荧光的强度，以指示抗原-MHC-I复合物在细胞表面的数量。(1) Bone marrow-derived DC cells were treated with 50ug/ml tumor exosomes (quantified by BCA protein) and PP-modified tumor exosomes, and 0.5mg/ml OVA full-length protein in PBS was added. After 24 hours, flow cytometry was performed using a PE-labeled anti-mouse MHC I-SIINFEKL antigen complex (abbreviated as p-MHC I) fluorescent antibody to examine the fluorescence intensity of DC cells in each treatment group to indicate the number of antigen-MHC-I complexes on the cell surface.
(2)实验共分为5个处理组，分别为：(2) The experiment was divided into five treatment groups:
a)不处理组(CTR)a) No treatment group (CTR)
b)肿瘤外泌体处理组(EXO)b) Tumor exosomes treatment group (EXO)
c)无MPLA对照组(PP/EXO)c) Control group without MPLA (PP/EXO)
d)无PP对照组(MPLA/EXO)d) Control group without PP (MPLA/EXO)
e)PP改造的肿瘤外泌体处理组(PP/MPLA/EXO)e) PP modified tumor exosomes treatment group (PP/MPLA/EXO)
(3)结果显示：(3) The results show that:
单独肿瘤外泌体组，无MPLA对照组，无PP对照组与不处理组类似，p-MHC I的水平并未改变；The levels of p-MHC I in the tumor exosomes alone group, the MPLA-free control group, and the PP-free control group were similar to the untreated group;
PP改造的肿瘤外泌体处理组的p-MHC I水平发生了显著升高(图3)，说明PP改造的肿瘤外泌体能提 升DC细胞对于抗原的处理和呈递能力。The p-MHC I level in the PP-modified tumor exosomes treatment group increased significantly (Figure 3), indicating that PP-modified tumor exosomes can enhance the ability of DC cells to process and present antigens .
实验方案(二)：PP改造的肿瘤外泌体增加DC细胞表面共刺激分子的表达水平Experimental plan (II): PP-modified tumor exosomes increase the expression level of co-stimulatory molecules on the surface of DC cells
(1)使用含有50ug/ml的肿瘤外泌体(以BCA蛋白定量)及PP改造的肿瘤外泌体处理骨髓来源的DC细胞，12小时后，使用荧光抗体检测DC细胞表面代表成熟的分子标记物，CD80和CD86的表达水平。(1) Bone marrow-derived DC cells were treated with tumor exosomes containing 50 ug/ml (quantified by BCA protein) and PP-modified tumor exosomes. After 12 hours, fluorescent antibodies were used to detect the expression levels of CD80 and CD86, molecular markers representing maturity, on the surface of DC cells.
(2)实验共分为5个处理组，分别为：(2) The experiment was divided into five treatment groups:
a)不处理组(CTR)a) No treatment group (CTR)
b)肿瘤外泌体处理组(EXO)b) Tumor exosomes treatment group (EXO)
c)无MPLA对照组(PP/EXO)c) Control group without MPLA (PP/EXO)
d)无PP对照组(MPLA/EXO)d) Control group without PP (MPLA/EXO)
e)PP改造的肿瘤外泌体处理组(PP/MPLA/EXO)e) PP modified tumor exosomes treatment group (PP/MPLA/EXO)
(3)结果显示：(3) The results show that:
单独肿瘤外泌体组，无MPLA对照组，无PP对照组与不处理组类似，CD80和CD86的表达水平并未改变；The tumor exosomes alone group, the MPLA-free control group, and the PP-free control group were similar to the untreated group, and the expression levels of CD80 and CD86 did not change;
PP改造的肿瘤外泌体处理组能显著升高这两个分子标记物的表达水平(图4)，说明PP改造的肿瘤 外泌体能促进DC细胞的成熟，更好的执行免疫功能。The PP-modified tumor exosomes treatment group can significantly increase the expression levels of these two molecular markers (Figure 4), indicating that PP-modified tumor exosomes can promote the maturation of DC cells and better perform immune functions .
实验方案(三)：PP改造的肿瘤外泌体增加DC细胞分泌细胞因子的水平Experimental plan (III): PP-modified tumor exosomes increase the level of cytokine secretion by DC cells
(1)使用含有50ug/ml的肿瘤外泌体(以BCA蛋白定量)及PP改造的肿瘤外泌体处理骨髓来源的DC细胞，分别于2小时，24小时后，收集细胞培养上清，利用ELISA和q-RT-PCR的方法考察各处理组中DC细胞分泌TNF-α，IL-2、IL-12的分泌水平以及干扰素IFN-β的表达情况。(1) Bone marrow-derived DC cells were treated with tumor exosomes containing 50 ug/ml (quantified by BCA protein) and PP-modified tumor exosomes. The cell culture supernatant was collected after 2 hours and 24 hours, respectively. The secretion levels of TNF-α, IL-2, and IL-12 and the expression of interferon IFN-β by DC cells in each treatment group were examined by ELISA and q-RT-PCR.
(2)实验共分为5个处理组，分别为(2) The experiment was divided into five treatment groups:
a)不处理组(CTR)a) No treatment group (CTR)
b)肿瘤外泌体处理组(EXO) b) Tumor exosomes treatment group (EXO)
c)无MPLA对照组(PP/EXO)c) Control group without MPLA (PP/EXO)
d)无PP对照组(MPLA/EXO)d) Control group without PP (MPLA/EXO)
e)PP改造的肿瘤外泌体处理组(PP/MPLA/EXO)e) PP modified tumor exosomes treatment group (PP/MPLA/EXO)
(3)结果显示：(3) The results show that:
单独肿瘤外泌体组，无MPLA对照组和无PP对照组，与不处理组类似，TNF-α，IL-2、IL-12的分泌水平没有任何改变。仅无PP对照组的TNF-α的分泌水平略有升高，可能与MPLA自身的作用有关；The tumor exosomes alone group, the MPLA-free control group, and the PP-free control group showed no changes in the secretion levels of TNF-α, IL-2, and IL-12, similar to the untreated group. Only the TNF-α secretion level of the PP-free control group was slightly increased, which may be related to the effect of MPLA itself;
PP改造的肿瘤外泌体处理组能显著提升DC细胞分泌TNF-α，IL-2、IL-12的水平(图5)。同时，一型干扰素IFN-β的表达趋势也一样。这些结果说明，PP改造的肿瘤外泌体能促使DC细胞分泌大量激活 抗肿瘤免疫反应的细胞因子，帮助DC细胞活化下游的T细胞。The PP-modified tumor exosomes treatment group can significantly increase the levels of TNF-α, IL-2, and IL-12 secreted by DC cells (Figure 5). At the same time, the expression trend of type I interferon IFN-β is the same. These results show that PP-modified tumor exosomes can induce DC cells to secrete a large number of cytokines that activate anti-tumor immune responses and help DC cells activate downstream T cells .
实施例四、PP改造肿瘤外泌体将其内容物递送至DC细胞的内质网(ER)Example 4: PP modified tumor exosomes to deliver their contents to the endoplasmic reticulum (ER) of DC cells
细胞培养及处理方法：Cell culture and treatment methods:
MC38细胞系来源的肿瘤外泌体(EXO)的获取和PP改造肿瘤外泌体及相应对照品的制备同实施例二。The acquisition of tumor exosomes (EXO) derived from MC38 cell line and the preparation of PP-modified tumor exosomes and corresponding reference substances are the same as in Example 2.
DC2.4细胞的培养：使用RPMI1640培养基(含10％胎牛血清，1×L-谷氨酰胺，1×非必需氨基酸溶液，1×HEPES缓冲液和5uMβ-巯基乙醇)。将肿瘤细胞冻存管从液氮保存罐中取出，立即放入37℃水浴中快速溶解，然后将细胞悬液移入含10ml培养基的离心桶中，350g离心5分钟后去除上清，用新鲜培养基重悬后，将细胞转移到细胞培养瓶中，加入10-15ml培养基混悬沉淀细胞，调整细胞浓度后，置于37℃、体积分数5％CO2饱和湿度培养箱中培养。在维持培养过程中，每天观察细胞状态并及时更换新鲜培养基。DC2.4 cell culture: Use RPMI1640 medium (containing 10% fetal bovine serum, 1×L-glutamine, 1×non-essential amino acid solution, 1×HEPES buffer and 5uMβ-mercaptoethanol). Take out the tumor cell cryotube from the liquid nitrogen storage tank and immediately put it into a 37°C water bath for rapid dissolution. Then transfer the cell suspension into a centrifuge bucket containing 10ml of culture medium. Centrifuge at 350g for 5 minutes and remove the supernatant. After resuspending with fresh culture medium, transfer the cells to a cell culture bottle, add 10-15ml of culture medium to suspend the precipitated cells, adjust the cell concentration, and place it in a 37°C, 5% CO 2 saturated humidity incubator for culture. During the culture maintenance process, observe the cell status every day and replace the fresh culture medium in time.
实验方案(一)：标记外泌体膜的方式考察PP改造的肿瘤外泌体进入DC2.4细胞后的路径Experimental plan (I): Labeling the exosome membrane to investigate the path of PP-modified tumor exosomes after entering DC2.4 cells
(1)PKH67标记肿瘤外泌体的膜成分：取8管新鲜提取的肿瘤外泌体(以BCA法定量，每管约含有9mg肿瘤外泌体)，向每个EP管中加入350ul试剂盒配备的稀释液，轻轻吹打，使外泌体形成溶液。之后在避光条件下，再准备8个装有350ul稀释液的EP管，每管加入1.5ul PKH67染料，混合均匀。之后将含有PKH67染料的稀释液按照体积比1:1加入到含有肿瘤外泌体溶液的EP管中，轻轻吹打混匀。室温(25℃)染色5分钟。再加入400ul无外泌体的胎牛血清，中和1分钟。之后，将染色完成的外泌体转移到新的BECKMAN 1.5ml管中(#357448)。将8个EP管配平后，置于超高速离心机中，100,000g，室温(25℃)离心25分钟后，弃去上清，用1ml PBS洗一遍后，再次离心(条件与之前保持一致)并弃去上清。每2个EP管加入100ul无菌水溶解合为1管肿瘤外泌体溶液(经再次BCA定量，约12mg，即溶液浓度为120mg/ml)。PKH67标记成功的肿瘤外泌体溶液4℃避光保存。(1) PKH67 labeled membrane components of tumor exosomes: Take 8 tubes of freshly extracted tumor exosomes (quantified by the BCA method, each tube contains approximately 9 mg of tumor exosomes), add 350ul of the diluent provided in the kit to each EP tube, and gently blow to form a solution of the exosomes. Then, under light-proof conditions, prepare 8 more EP tubes containing 350ul of diluent, add 1.5ul of PKH67 dye to each tube, and mix evenly. Then, add the diluent containing PKH67 dye to the EP tube containing the tumor exosome solution at a volume ratio of 1:1, and gently blow to mix. Stain at room temperature (25°C) for 5 minutes. Then add 400ul of exosome-free fetal bovine serum and neutralize for 1 minute. Then, transfer the stained exosomes to a new BECKMAN 1.5ml tube (#357448). After balancing the 8 EP tubes, place them in an ultra-high speed centrifuge, centrifuge at 100,000g at room temperature (25°C) for 25 minutes, discard the supernatant, wash once with 1ml PBS, centrifuge again (conditions remain the same as before) and discard the supernatant. Add 100ul of sterile water to every 2 EP tubes to dissolve into 1 tube of tumor exosome solution (after BCA quantification again, about 12mg, that is, the solution concentration is 120mg/ml). The tumor exosome solution successfully labeled with PKH67 is stored at 4°C away from light.
(2)制备PKH67标记的PP改造的肿瘤外泌体：参照实施例1的处方配制，区别在于使用的外泌体为本实施例前述步骤(1)中的PKH67标记的外泌体；(2) Preparation of PKH67-labeled PP-modified tumor exosomes: Prepared according to the prescription of Example 1, except that the exosomes used are the PKH67-labeled exosomes in the aforementioned step (1) of this example;
(3)处理DC2.4细胞：将DC2.4细胞以1×105/孔种于15mm共聚焦显微镜用细胞培养小皿，过夜待细胞贴壁。第二天，用配制好的PKH67标记的肿瘤外泌体或PP改造的肿瘤外泌体处理DC2.4细胞，之后，按照设定时间分别加入100nM Lyso-Tracker-Red(37℃，10分钟)或1uM ER-Tracker-Red(37℃，30分钟)染料对细胞的溶酶体和内质网进行染色，最终外泌体处理时间保持在1小时。上机前，将培养基换为无荧光染料的培养基，之后用荧光共聚焦显微镜观察；(3) Treatment of DC2.4 cells: DC2.4 cells were seeded at 1×10 5 /well in a 15 mm confocal microscopy cell culture dish and allowed to adhere overnight. The next day, DC2.4 cells were treated with the prepared PKH67-labeled tumor exosomes or PP-modified tumor exosomes. Afterwards, 100 nM Lyso-Tracker-Red (37°C, 10 minutes) or 1 uM ER-Tracker-Red (37°C, 30 minutes) dyes were added to stain the lysosomes and endoplasmic reticulum of the cells according to the set time. The final exosome treatment time was maintained at 1 hour. Before going on the machine, the culture medium was replaced with a culture medium without fluorescent dyes, and then observed with a fluorescent confocal microscope;
(4)实验共分为4组，分别为：(4) The experiment was divided into 4 groups:
a)肿瘤外泌体(绿色)与溶酶体(红色)共定位组a) Co-localization of tumor exosomes (green) and lysosomes (red)
b)肿瘤外泌体(绿色)与内质网(红色)共定位组b) Tumor exosomes (green) and endoplasmic reticulum (red) co-localization group
c)PP改造的肿瘤外泌体(绿色)与溶酶体(红色)共定位组c) Co-localization of PP-modified tumor exosomes (green) and lysosomes (red)
d)PP改造的肿瘤外泌体(绿色)与内质网(红色)共定位组d) PP-modified tumor exosomes (green) co-localized with the endoplasmic reticulum (red)
(5)实验结果显示： (5) Experimental results show that:
经过1小时的处理，肿瘤外泌体主要与溶酶体共定位，而PP改造的肿瘤外泌体则主要与内质网共定位(图6A)，初步说明PP的改造能够改变肿瘤外泌体的细胞内目的地。但是鉴于PKH67标记的是肿瘤外 泌体的膜结构，因此无法确定PP改造的肿瘤外泌体的内容物(特别是抗原)的确能够到达内质网。为 了解决这个问题，我们进一步使用能够标记蛋白的CFSE染料来标记肿瘤外泌体的内含物，仍然通过荧 光共聚焦显微镜观察其与溶酶体和内质网的共定位情况。 After 1 hour of treatment, tumor exosomes were mainly co-localized with lysosomes, while PP-modified tumor exosomes were mainly co-localized with the endoplasmic reticulum (Figure 6A), which preliminarily indicates that PP modification can change the intracellular destination of tumor exosomes. However, since PKH67 marks the membrane structure of tumor exosomes, it is not certain that the contents of PP-modified tumor exosomes (especially antigens) can indeed reach the endoplasmic reticulum. To solve this problem, we further used CFSE dye that can mark proteins to mark the contents of tumor exosomes, and still observed their co-localization with lysosomes and endoplasmic reticulum by fluorescence confocal microscopy.
实验方案(二)：标记外泌体蛋白的方式考察PP改造的肿瘤外泌体进入DC2.4细胞后的路径Experimental plan (II): Labeling exosome proteins to investigate the path of PP-modified tumor exosomes after entering DC2.4 cells
(1)CFSE标记肿瘤外泌体的蛋白成分：吸取20mg肿瘤外泌体储液(基于BCA蛋白定量)于1.5ml EP管中，加入PBS调整体积至500ul，加入2ul 10mM CFSE储液(使终浓度达到4uM)，室温避光染色10分钟。之后加入500ul含20％FBS的PBS中和反应，转移到超速离心管中，100,000g，4度离心30min。弃上清，沉淀用PBS洗一遍后，用适量PBS重悬，BCA定量后，-80℃保存，备用。(1) CFSE-labeled protein components of tumor exosomes: Pipette 20 mg of tumor exosome stock solution (based on BCA protein quantification) into a 1.5 ml EP tube, add PBS to adjust the volume to 500 ul, add 2 ul of 10 mM CFSE stock solution (to make the final concentration reach 4 uM), and stain at room temperature in the dark for 10 minutes. Then add 500 ul of PBS containing 20% FBS to neutralize the reaction, transfer to an ultracentrifuge tube, and centrifuge at 100,000g at 4 degrees for 30 minutes. Discard the supernatant, wash the precipitate with PBS once, resuspend it with an appropriate amount of PBS, quantify it with BCA, and store it at -80°C for later use.
(2)制备CFSE标记的PP改造的肿瘤外泌体：参照实施例1的处方配制，区别在于使用的外泌体为本实施例前述步骤(1)中的CFSE标记的外泌体；(2) Preparation of CFSE-labeled PP-modified tumor exosomes: Prepared according to the prescription of Example 1, except that the exosomes used are the CFSE-labeled exosomes in the aforementioned step (1) of this example;
(3)处理DC2.4细胞：将DC2.4细胞以1×105/孔种于15mm共聚焦显微镜用细胞培养小皿，过夜待细胞贴壁。第二天，用配制好的PKH67标记的肿瘤外泌体或PP改造的肿瘤外泌体处理DC2.4细胞，之后，按照设定时间分别加入100nM Lyso-Tracker-Red(37℃，10分钟)或1uM ER-Tracker-Red(37℃，30分钟)染料对细胞的溶酶体和内质网进行染色，最终外泌体处理时间保持在1小时。上机前，将培养基换为无荧光染料的培养基，之后用荧光共聚焦显微镜观察；(3) Treatment of DC2.4 cells: DC2.4 cells were seeded at 1×10 5 /well in a 15 mm confocal microscopy cell culture dish and allowed to adhere overnight. The next day, DC2.4 cells were treated with the prepared PKH67-labeled tumor exosomes or PP-modified tumor exosomes. Afterwards, 100 nM Lyso-Tracker-Red (37°C, 10 minutes) or 1 uM ER-Tracker-Red (37°C, 30 minutes) dyes were added to stain the lysosomes and endoplasmic reticulum of the cells according to the set time. The final exosome treatment time was maintained at 1 hour. Before going on the machine, the culture medium was replaced with a culture medium without fluorescent dyes, and then observed with a fluorescent confocal microscope;
(4)实验共分为4组，分别为：(4) The experiment was divided into 4 groups:
a)肿瘤外泌体(绿色)与溶酶体(红色)共定位组a) Co-localization of tumor exosomes (green) and lysosomes (red)
b)肿瘤外泌体(绿色)与内质网(红色)共定位组b) Tumor exosomes (green) and endoplasmic reticulum (red) co-localization group
c)PP改造的肿瘤外泌体(绿色)与溶酶体(红色)共定位组c) Co-localization of PP-modified tumor exosomes (green) and lysosomes (red)
d)PP改造的肿瘤外泌体(绿色)与内质网(红色)共定位组d) PP-modified tumor exosomes (green) co-localized with the endoplasmic reticulum (red)
(5)实验结果显示：(5) Experimental results show that:
经过1小时的处理，与PKH67标记类似，CFSE标记的肿瘤外泌体主要与溶酶体共定位，而PP改造的CFSE标记的肿瘤外泌体则主要与内质网共定位(图6B)。充分说明经过PP改造，肿瘤外泌体内包含的蛋白分子(特别是其携带的抗原分子)能够到达内质网，为肿瘤特异性抗原的MHC I型呈递途径提供了 前提条件。After 1 hour of treatment, similar to PKH67 labeling, CFSE-labeled tumor exosomes were mainly co-localized with lysosomes, while PP-modified CFSE-labeled tumor exosomes were mainly co-localized with the endoplasmic reticulum (Figure 6B). This fully demonstrates that after PP modification, the protein molecules contained in tumor exosomes (especially the antigen molecules they carry) can reach the endoplasmic reticulum, providing the prerequisite for the MHC type I presentation pathway of tumor-specific antigens.
实施例五、PP改造的肿瘤外泌体增强骨髓来源DC对抗原特异性OT I CD8+T细胞的激活作用Example 5: PP-modified tumor exosomes enhance the activation of bone marrow-derived DC on antigen-specific OT I CD8 + T cells
细胞培养及处理方法：Cell culture and treatment methods:
骨髓来源DC的获取、MC38细胞系来源的肿瘤外泌体(EXO)的获取，PP改造的肿瘤外泌体及相应对照品的制备参考实施例二和实施例三。The acquisition of bone marrow-derived DCs, the acquisition of tumor exosomes (EXO) derived from the MC38 cell line, and the preparation of PP-modified tumor exosomes and corresponding reference substances refer to Example 2 and Example 3.
OT I CD8+T细胞的获取：OT I转基因小鼠处死后，取脾脏细胞，裂解红细胞，之后用CD8磁珠(Invitrogen公司)分离CD8+T细胞。Acquisition of OT I CD8 + T cells: After OT I transgenic mice were sacrificed, spleen cells were obtained, red blood cells were lysed, and CD8 + T cells were then separated using CD8 magnetic beads (Invitrogen).
实验方案：Experimental program:
(1)使用含有50ug/ml的肿瘤外泌体(以BCA蛋白定量)及PP改造的肿瘤外泌体处理骨髓来源的DC细胞，24小时后，再加入0.5mg/ml OVA全长蛋白PBS溶液。再24小时后，收集各处理组的DC细胞，经计数后与CFSE染色的OT I CD8+T细胞以1：8的比例共培养，72小时后检测T细胞的增殖水平(CFSE稀释情况)。(1) Bone marrow-derived DC cells were treated with 50ug/ml tumor exosomes (quantified by BCA protein) and PP-modified tumor exosomes. After 24 hours, 0.5mg/ml OVA full-length protein PBS solution was added. After another 24 hours, DC cells in each treatment group were collected and counted, and co-cultured with CFSE-stained OTI CD8 + T cells at a ratio of 1:8. After 72 hours, the proliferation level of T cells (CFSE dilution) was detected.
(2)实验共分为5组，分别为(2) The experiment was divided into 5 groups:
a)不处理组(CTR)a) No treatment group (CTR)
b)肿瘤外泌体处理组(EXO)b) Tumor exosomes treatment group (EXO)
c)无MPLA对照组(PP/EXO) c) Control group without MPLA (PP/EXO)
d)无PP对照组(MPLA/EXO)d) Control group without PP (MPLA/EXO)
e)PP改造的肿瘤外泌体处理组(PP/MPLA/EXO)e) PP modified tumor exosomes treatment group (PP/MPLA/EXO)
(3)实验结果显示，(3) Experimental results show that
不处理组的DC细胞能够很好的活化OT I CD8+T细胞，而且单独肿瘤外泌体处理组能显著抑制该活化效应。The DC cells in the untreated group were able to activate OT I CD8 + T cells well, and the tumor exosome treatment group alone could significantly inhibit this activation effect.
相较于无MPLA对照组和无PP对照组，PP改造的肿瘤外泌体处理组并未抑制DC对T细胞的活化能 力，反之，还有一定的促进作用(图7)。Compared with the control group without MPLA and the control group without PP, the group treated with PP-modified tumor exosomes did not inhibit the activation ability of DCs on T cells, but on the contrary, it had a certain promoting effect (Figure 7) .
实施例六、PP改造的肿瘤外泌体能引起同源肿瘤抗原特异性CTL反应Example 6: PP-modified tumor exosomes can induce homologous tumor antigen-specific CTL response
细胞培养及处理方法：Cell culture and treatment methods:
肿瘤外泌体(EXO)的获取，PP改造的肿瘤外泌体及相应对照品的制备参考实施例二。The acquisition of tumor exosomes (EXO), the preparation of PP-modified tumor exosomes and corresponding reference substances refer to Example 2.
小鼠结肠癌模型MC38细胞系和小鼠黑色素瘤B16F10细胞系的培养：使用DMEM培养基(含10％胎牛血清)。将肿瘤细胞冻存管从液氮保存罐中取出，立即放入37℃水浴中快速溶解，然后将细胞悬液移入含10ml培养基的离心桶中，350g离心5分钟后去除上清，用新鲜培养基重悬后，将细胞转移到细胞培养瓶中，加入10-15ml培养基混悬沉淀细胞，调整细胞浓度后，置于37℃、体积分数5％CO2饱和湿度培养箱中培养。在维持培养过程中，每天观察细胞状态并及时更换新鲜培养基。Culture of mouse colon cancer model MC38 cell line and mouse melanoma B16F10 cell line: Use DMEM medium (containing 10% fetal bovine serum). Take out the tumor cell cryotube from the liquid nitrogen storage tank and immediately put it into a 37°C water bath for rapid dissolution. Then transfer the cell suspension into a centrifuge bucket containing 10ml of culture medium. Centrifuge at 350g for 5 minutes and remove the supernatant. After resuspending with fresh culture medium, transfer the cells to a cell culture bottle, add 10-15ml of culture medium to suspend the precipitated cells, adjust the cell concentration, and place it in a 37°C, 5% CO 2 saturated humidity incubator for culture. During the culture maintenance process, observe the cell status every day and replace the fresh culture medium in time.
小鼠宫颈癌TC-1细胞系和小鼠三阴性乳腺癌4T1细胞系的培养：使用RPMI1640培养基(含10％胎牛血清)。将肿瘤细胞冻存管从液氮保存罐中取出，立即放入37℃水浴中快速溶解，然后将细胞悬液移入含10ml培养基的离心桶中，350g离心5分钟后去除上清，用新鲜培养基重悬后，将细胞转移到细胞培养瓶中，加入10-15ml培养基混悬沉淀细胞，调整细胞浓度后，置于37℃、体积分数5％CO2饱和湿度培养箱中培养。在维持培养过程中，每天观察细胞状态并及时更换新鲜培养基。Culture of mouse cervical cancer TC-1 cell line and mouse triple-negative breast cancer 4T1 cell line: Use RPMI1640 medium (containing 10% fetal bovine serum). Take out the cryopreserved tube of tumor cells from the liquid nitrogen storage tank and immediately put it into a 37°C water bath for rapid dissolution. Then transfer the cell suspension into a centrifuge bucket containing 10ml of culture medium. Centrifuge at 350g for 5 minutes and remove the supernatant. After resuspending with fresh culture medium, transfer the cells to a cell culture bottle, add 10-15ml of culture medium to suspend the precipitated cells, adjust the cell concentration, and culture in a 37°C, 5% CO 2 saturated humidity incubator. During the culture maintenance process, observe the cell status every day and replace the fresh culture medium in time.
实验方案(一)：PP改造的肿瘤外泌体能引起同源肿瘤抗原特异性CTL反应(MC38细胞系)Experimental plan (I): PP-modified tumor exosomes can induce homologous tumor antigen-specific CTL response (MC38 cell line)
(1)野生型小鼠分别经尾根部注射用来源于MC38的肿瘤外泌体或PP改造的肿瘤外泌体能引起同源肿瘤抗原特异性CTL反应改造的肿瘤外泌体进行免疫。免疫注射后第7天，处死小鼠，手术取出近端淋巴结，研磨、消化后获得淋巴结中的所有免疫细胞。(1) Wild-type mice were immunized via tail-base injection with MC38-derived tumor exosomes or PP-modified tumor exosomes that elicit homologous tumor antigen-specific CTL responses. Seven days after immunization, the mice were killed, and the proximal lymph nodes were surgically removed. All immune cells in the lymph nodes were obtained after grinding and digestion.
体外处理：在分离小鼠淋巴结细胞的前一天在Ellispot板中预孵育anti-mouse IFN-γ抗体，作用浓度为5μg/ml，4℃孵育过夜。次日弃去抗体，加入封闭液(含10％胎牛血清、1％青霉素链霉素和β-巯基乙醇的RPMI 1640培养基)室温封闭2小时。随后将分离得到的小鼠淋巴结细胞按照3×105每孔加入板中，并孵育20μg/ml MC38特异性抗原肽(包括：Rpl-18，Reps-1和Adpgk)(Identification of a neo-epitope dominating endogenous CD8 T cell responses to MC-38 colorectal cancer.9,1673125(2019)；published online EpubOct 13(10.1080/2162402x.2019.1673125))，37℃培养箱培养48小时。48小时后弃培养液，加入去离子水裂解细胞，5分钟/次，共2次。用PBST(含0.05％Tween-20的1×PBS)溶液洗3遍，随后加入生物素标记的anti-mouse IFN-γ抗体，室温孵育2小时。PBST溶液洗4遍，随后加入链亲和素标记的辣根过氧化物酶(streptavidin-HRP)，室温孵育1小时。最后1×PBS洗3遍后加入AEC底物显色5-60分钟，加入去离子水终止反应，继续冲洗干净后将ELISpot板子吹干。In vitro treatment: The day before mouse lymph node cells were isolated, anti-mouse IFN-γ antibody was pre-incubated in Ellispot plates at a concentration of 5 μg/ml and incubated overnight at 4°C. The next day, the antibody was discarded and blocking solution (RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin-streptomycin and β-mercaptoethanol) was added for blocking at room temperature for 2 hours. The isolated mouse lymph node cells were then added to the plate at 3×10 5 per well and incubated with 20 μg/ml MC38-specific antigen peptides (including: Rpl-18, Reps-1 and Adpgk) (Identification of a neo-epitope dominating endogenous CD8 T cell responses to MC-38 colorectal cancer.9,1673125(2019); published online EpubOct 13(10.1080/2162402x.2019.1673125)), and cultured in a 37°C incubator for 48 hours. After 48 hours, the culture medium was discarded and deionized water was added to lyse the cells for 5 minutes each time, for a total of 2 times. Wash three times with PBST (1×PBS containing 0.05% Tween-20) solution, and then add biotin-labeled anti-mouse IFN-γ antibody and incubate at room temperature for 2 hours. Wash 4 times with PBST solution, then add streptavidin-HRP and incubate at room temperature for 1 hour. Finally, wash 3 times with 1×PBS and add AEC substrate for color development for 5-60 minutes, add deionized water to terminate the reaction, continue to rinse and blow dry the ELISpot plate.
(2)实验共分为4组，分别为：(2) The experiment was divided into 4 groups:
a)肿瘤外泌体处理组(EXO)a) Tumor exosomes treatment group (EXO)
b)无MPLA对照组(PP/EXO)b) Control group without MPLA (PP/EXO)
c)无PP对照组(MPLA/EXO)c) Control group without PP (MPLA/EXO)
d)PP改造的肿瘤外泌体处理组(PP/MPLA/EXO)d) PP-modified tumor exosomes treatment group (PP/MPLA/EXO)
(3)检测方法：ELISpot结果通过荧光酶斑点分析仪CTL analyzer LLC进行扫描和计数分析。(3) Detection method: ELISpot results were scanned and counted using the fluorescent enzyme spot analyzer CTL analyzer LLC.
结果显示，与肿瘤外泌体处理组相比， The results showed that compared with the tumor exosome-treated group,
无MPLA组和无PP对照组组均不能提高抗原特异性CTL反应，PP改造的肿瘤外泌体免疫后的淋巴结 细胞中产生更高比例的抗原特异性CTL反应(具体表现为更强的IFN-γ信号)(图8)。Neither the MPLA-free group nor the PP-free control group could improve antigen-specific CTL responses. PP-modified tumor exosomes produced a higher proportion of antigen-specific CTL responses in lymph node cells after immunization (specifically, a stronger IFN-γ signal) (Figure 8) .
实验方案(二)：PP改造的肿瘤外泌体能引起同源肿瘤抗原特异性CTL反应(B16F10细胞系)Experimental plan (II): PP-modified tumor exosomes can induce homologous tumor antigen-specific CTL response (B16F10 cell line)
(1)野生型小鼠分别经尾根部注射用来源于B16F10的肿瘤外泌体或PP改造的肿瘤外泌体能引起同源肿瘤抗原特异性CTL反应改造的肿瘤外泌体进行免疫。免疫注射后第7天，处死小鼠，手术取出近端淋巴结，研磨、消化后获得淋巴结中的所有免疫细胞。(1) Wild-type mice were immunized by tail-base injection with B16F10-derived tumor exosomes or PP-modified tumor exosomes that elicit homologous tumor antigen-specific CTL responses. Seven days after immunization, the mice were killed, and the proximal lymph nodes were surgically removed. All immune cells in the lymph nodes were obtained by grinding and digestion.
体外处理：同本实施例的实验方案(一)。其中，与淋巴结细胞孵育所用的B16F10特异性抗原肽为Trp2(Exploiting the mutanome for tumor vaccination.Cancer Res 72,1081-1091(2012)；published online EpubMar 1(10.1158/0008-5472.can-11-3722))。In vitro treatment: Same as the experimental scheme (I) of this example. The B16F10 specific antigen peptide used for incubation with lymph node cells is Trp2 (Exploiting the mutanome for tumor vaccination. Cancer Res 72, 1081-1091 (2012); published online Epub Mar 1 (10.1158/0008-5472.can-11-3722)).
(2)实验共分为2组，分别为：(2) The experiment was divided into two groups:
a)肿瘤外泌体处理组(EXO)a) Tumor exosomes treatment group (EXO)
b)PP改造的肿瘤外泌体处理组(PP/MPLA/EXO)b) PP-modified tumor exosomes treatment group (PP/MPLA/EXO)
结果显示，与肿瘤外泌体处理组相比，PP改造的肿瘤外泌体免疫后的淋巴结细胞中产生更高比例 的抗原特异性CTL反应(图9)。The results showed that compared with the tumor exosome-treated group, PP-modified tumor exosomes produced a higher proportion of antigen-specific CTL responses in lymph node cells after immunization (Figure 9) .
实验方案(三)：PP改造的肿瘤外泌体能引起同源肿瘤抗原特异性CTL反应(细胞系4T1)Experimental plan (III): PP-modified tumor exosomes can induce homologous tumor antigen-specific CTL response (cell line 4T1)
(1)野生型小鼠分别经尾根部注射用来源于4T1的肿瘤外泌体或PP改造的肿瘤外泌体进行免疫。免疫注射后第7天，处死小鼠，手术取出近端淋巴结，研磨、消化后获得淋巴结中的所有免疫细胞。(1) Wild-type mice were immunized with 4T1-derived tumor exosomes or PP-modified tumor exosomes via tail-base injection. Seven days after immunization, the mice were killed, and the proximal lymph nodes were surgically removed. All immune cells in the lymph nodes were obtained after grinding and digestion.
体外处理：同本实施例的实验方案(一)。其中，与淋巴结细胞孵育所用的4T1特异性抗原肽为Sptbn4和Wdr33(Self-healing microcapsules synergetically modulate immunization microenvironments for potent cancer vaccination.6,eaay7735(2020)；published online EpubMay(10.1126/sciadv.aay7735))。In vitro treatment: Same as the experimental scheme (I) of this embodiment. Among them, the 4T1-specific antigen peptides used for incubation with lymph node cells are Sptbn4 and Wdr33 (Self-healing microcapsules synergetically modulate immunization microenvironments for potent cancer vaccination. 6, eaay7735 (2020); published online Epub May (10.1126/sciadv.aay7735)).
(2)实验共分为4组，分别为：(2) The experiment was divided into 4 groups:
a)肿瘤外泌体处理组(EXO)a) Tumor exosomes treatment group (EXO)
b)无MPLA对照组(PP/EXO)b) Control group without MPLA (PP/EXO)
c)无PP对照组(MPLA/EXO)c) Control group without PP (MPLA/EXO)
d)PP改造的肿瘤外泌体处理组(PP/MPLA/EXO)d) PP-modified tumor exosomes treatment group (PP/MPLA/EXO)
结果显示，与肿瘤外泌体处理组相比，The results showed that compared with the tumor exosome-treated group,
无MPLA对照组和无PP对照组均不能提高抗原特异性CTL反应，PP改造的肿瘤外泌体免疫后的淋巴 结细胞中产生更高比例的抗原特异性CTL反应(具体表现为更强的IFN-γ信号)(图10)。Neither the MPLA-free control group nor the PP-free control group could improve the antigen-specific CTL response. A higher proportion of antigen-specific CTL responses (specifically, stronger IFN-γ signals) were produced in lymph node cells after immunization with PP- modified tumor exosomes (Figure 10) .
实验方案(四)：PP改造的肿瘤外泌体能引起同源肿瘤抗原特异性CTL反应(细胞系TC-1)Experimental plan (IV): PP-modified tumor exosomes can induce homologous tumor antigen-specific CTL response (cell line TC-1)
(1)野生型小鼠分别经尾根部注射用来源于TC-1的肿瘤外泌体或PP改造的肿瘤外泌体进行免疫。免疫注射后第7天，处死小鼠，手术取出近端淋巴结，研磨、消化后获得淋巴结中的所有免疫细胞。(1) Wild-type mice were immunized with TC-1-derived tumor exosomes or PP-modified tumor exosomes via tail-base injection. Seven days after immunization, the mice were sacrificed, and the proximal lymph nodes were surgically removed. All immune cells in the lymph nodes were obtained after grinding and digestion.
体外处理：参考本实施例实验方案(一)。其中，与淋巴结细胞孵育所用的TC-1特异性抗原肽为E7(Coordinating antigen cytosolic delivery and danger signaling to program potent cross-priming by micelle-based nanovaccine.Cell Discovery 3,17007(2017)；published online Epub2017/04/04(10.1038/celldisc.2017.7))。In vitro treatment: refer to the experimental scheme (I) of this example. Among them, the TC-1 specific antigen peptide used for incubation with lymph node cells is E7 (Coordinating antigen cytosolic delivery and danger signaling to program potent cross-priming by micelle-based nanovaccine. Cell Discovery 3,17007(2017); published online Epub2017/04/04(10.1038/celldisc.2017.7)).
(2)实验共分为4组，分别为：(2) The experiment was divided into 4 groups:
a)肿瘤外泌体处理组(EXO)a) Tumor exosomes treatment group (EXO)
b)无MPLA对照组(PP/EXO)b) Control group without MPLA (PP/EXO)
c)无PP对照组(MPLA/EXO)c) Control group without PP (MPLA/EXO)
d)PP改造的肿瘤外泌体处理组(PP/MPLA/EXO)d) PP-modified tumor exosomes treatment group (PP/MPLA/EXO)
结果显示，与肿瘤外泌体免疫组相比，The results showed that compared with the tumor exosome immunization group,
无MPLA对照组和无PP对照组均不能提高抗原特异性CTL反应，PP改造的肿瘤外泌体免疫后的淋巴 结细胞能产生更高比例的抗原特异性CTL反应(具体表现为更强的IFN-γ信号)(图11)。 The control group without MPLA and the control group without PP could not improve the antigen-specific CTL response. The lymph node cells immunized with PP-modified tumor exosomes could produce a higher proportion of antigen-specific CTL response (specifically manifested as a stronger IFN-γ signal) (Figure 11) .
实施例七、PP改造的肿瘤外泌体抑制肿瘤生长并延长荷瘤小鼠的生存期Example 7: PP-modified tumor exosomes inhibit tumor growth and prolong the survival of tumor-bearing mice
细胞培养及处理方法：Cell culture and treatment methods:
细胞培养方法参考实施例四，肿瘤外泌体(EXO)的获取和PP改造的肿瘤外泌体及相应对照品的制备参考实施例二。The cell culture method is referred to Example 4, and the acquisition of tumor exosomes (EXO) and the preparation of PP-modified tumor exosomes and corresponding reference substances are referred to Example 2.
实验方案(一)：PP改造的肿瘤外泌体(来源于小鼠结肠癌细胞系MC38)抑制肿瘤生长Experimental plan (I): PP-modified tumor exosomes (derived from mouse colon cancer cell line MC38) inhibit tumor growth
(1)MC38模型的构建：当细胞贴壁生长至90％汇合度时，用0.05％胰酶消化，按照1:3的比例进行传代培养。实验当天，将生长状态良好，汇合度达到90％的细胞胰酶消化后，用新鲜培养基中和胰酶，350g离心，弃上清后加入无菌PBS重悬，计数，将细胞悬液的密度调整为2.5×106/ml。利用1ml无菌注射器皮下接种雌性野生型C57BL/6小鼠，每只小鼠0.1ml(即每只小鼠接种2.5×105个MC38细胞)。约在4天左右可看到约30mm3的肿瘤形成，当日在肿瘤附近的皮下注射50ug/ml肿瘤外泌体(基于BCA蛋白定量)或者含有等质量的PP改造的肿瘤外泌体进行治疗，每周一次，共三次。之后，一直观察小鼠的状态，期间记录荷瘤小鼠的肿瘤体积变化和死亡情况。(1) Construction of MC38 model: When the cells adhered to the wall and grew to 90% confluence, they were digested with 0.05% trypsin and subcultured at a ratio of 1:3. On the day of the experiment, the cells with good growth status and 90% confluence were digested with trypsin, and the trypsin was neutralized with fresh culture medium, centrifuged at 350g, the supernatant was discarded, and sterile PBS was added to resuspend, counted, and the density of the cell suspension was adjusted to 2.5×10 6 /ml. Female wild-type C57BL/6 mice were subcutaneously inoculated with 1ml sterile syringes, 0.1ml per mouse (i.e., 2.5×10 5 MC38 cells were inoculated per mouse). About 30mm 3 tumor formation can be seen in about 4 days. On the same day, 50ug/ml tumor exosomes (based on BCA protein quantification) or tumor exosomes containing the same mass of PP were subcutaneously injected near the tumor for treatment, once a week, for a total of three times. After that, the status of the mice was observed, and the tumor volume changes and deaths of tumor-bearing mice were recorded during this period.
(2)实验组共有5组，分别为(2) There are 5 experimental groups:
a)PBS溶剂对照组(CTR)a) PBS solvent control group (CTR)
b)肿瘤外泌体处理组(EXO)b) Tumor exosomes treatment group (EXO)
c)无MPLA对照组(PP/EXO)c) Control group without MPLA (PP/EXO)
d)无PP对照组(MPLA/EXO)d) Control group without PP (MPLA/EXO)
e)PP改造的肿瘤外泌体处理组(PP/MPLA/EXO)e) PP modified tumor exosomes treatment group (PP/MPLA/EXO)
实验结果显示，与PBS溶剂对照组相比，无MPLA对照组以及无PP对照组均不能有效抑制肿瘤生长，PP改造的肿瘤外泌体能够显著延缓肿瘤的生长，并延长荷瘤小鼠的生存期(图12)。The experimental results showed that compared with the PBS solvent control group, the control group without MPLA and the control group without PP could not effectively inhibit tumor growth. The PP-modified tumor exosomes could significantly delay tumor growth and prolong the survival of tumor-bearing mice (Figure 12) .
实验方案(二)：PP改造的肿瘤外泌体(来源于小鼠黑色素瘤细胞系B16F10)抑制肿瘤生长Experimental plan (II): PP-modified tumor exosomes (derived from mouse melanoma cell line B16F10) inhibit tumor growth
(1)B16F10模型的构建：当细胞贴壁生长至90％汇合度时，用0.05％胰酶消化，按照1:4的比例进行传代培养。实验当天，将生长状态良好，汇合度达到90％的细胞胰酶消化后，用新鲜培养基中和胰酶，350g离心，弃上清后加入无菌PBS重悬，计数，将细胞悬液的密度调整为2.5×106/ml。利用1ml无菌注射器皮下接种雌性野生型C57BL/6小鼠，每只小鼠0.1ml(即每只小鼠接种2.5×105个B16F10细胞)。注意每次吸取细胞悬液前将细胞混合均匀。约在4天左右可看到约30mm3的肿瘤形成，当日在肿瘤附近的皮下注射50ug/ml肿瘤外泌体(基于BCA蛋白定量)或者含有等质量的PP改造的肿瘤外泌体进行治疗，每周一次，共三次。之后，一直观察小鼠的状态，期间记录荷瘤小鼠的肿瘤体积变化和死亡情况。(1) Construction of B16F10 model: When the cells adhered to the wall and grew to 90% confluence, they were digested with 0.05% trypsin and subcultured at a ratio of 1:4. On the day of the experiment, the cells with good growth status and 90% confluence were digested with trypsin, and the trypsin was neutralized with fresh culture medium, centrifuged at 350g, the supernatant was discarded, and sterile PBS was added to resuspend, counted, and the density of the cell suspension was adjusted to 2.5×10 6 /ml. Female wild-type C57BL/6 mice were subcutaneously inoculated with 1ml sterile syringes, 0.1ml per mouse (i.e., 2.5×10 5 B16F10 cells per mouse). Note that the cells were mixed evenly before each aspiration of the cell suspension. Tumor formation of about 30mm 3 can be seen in about 4 days. On the same day, 50ug/ml tumor exosomes (based on BCA protein quantification) or tumor exosomes modified with PP of equal mass were injected subcutaneously near the tumor for treatment once a week for a total of three times. Afterwards, the condition of the mice was observed, and the changes in tumor volume and the death of tumor-bearing mice were recorded.
(2)实验组共有3组，分别为(2) There are 3 experimental groups:
a)PBS溶剂对照组(CTR)a) PBS solvent control group (CTR)
b)肿瘤外泌体处理组(EXO)b) Tumor exosomes treatment group (EXO)
c)PP改造的肿瘤外泌体处理组(PP/MPLA/EXO)c) PP modified tumor exosomes treatment group (PP/MPLA/EXO)
实验结果显示，与PBS溶剂对照组相比，肿瘤外泌体处理组不能有效抑制肿瘤生长，PP改造的肿瘤 外泌体能够显著延缓肿瘤的生长，并延长荷瘤小鼠的生存期(图13)。The experimental results showed that compared with the PBS solvent control group, the tumor exosome treatment group could not effectively inhibit tumor growth, while the PP-modified tumor exosomes could significantly delay tumor growth and prolong the survival of tumor-bearing mice (Figure 13) .
实验方案(三)：PP改造的肿瘤外泌体(来源于小鼠宫颈癌细胞系TC-1)抑制肿瘤生长Experimental plan (III): PP-modified tumor exosomes (derived from mouse cervical cancer cell line TC-1) inhibit tumor growth
(1)TC-1模型的构建：当细胞贴壁生长至90％汇合度时，用0.05％胰酶消化，按照1:4的比例进行传代培养。实验当天，将生长状态良好，汇合度达到90％的细胞胰酶消化后，用新鲜培养基中和胰酶，350g离心，弃上清后加入无菌PBS重悬，计数，将细胞悬液的密度调整为5×105/ml。利用1ml无菌注射器皮下接种雌性野生型C57BL/6小鼠，每只小鼠0.1ml(即每只小鼠接种5×104个TC-1细胞)。注意每次吸取细胞悬液前将细胞混合均匀。约在6天左右可看到约30mm3的肿瘤形成，当日在肿瘤附近的皮下注射50ug/ml肿瘤外泌体(基于BCA蛋白定量)或者含有等质量的PP改造的肿瘤外泌体进行治疗，每周一次，共三次。之后，一直观察小鼠的状态，期间记录荷瘤小鼠的肿瘤体积变化和死亡情况。 (1) Construction of TC-1 model: When the cells adhere to the wall and grow to 90% confluence, they are digested with 0.05% trypsin and subcultured at a ratio of 1:4. On the day of the experiment, the cells with good growth status and 90% confluence are digested with trypsin, and the trypsin is neutralized with fresh culture medium, centrifuged at 350g, the supernatant is discarded, and sterile PBS is added to resuspend, count, and the density of the cell suspension is adjusted to 5×10 5 /ml. Female wild-type C57BL/6 mice are subcutaneously inoculated with 1ml sterile syringes, 0.1ml per mouse (i.e., 5×10 4 TC-1 cells are inoculated per mouse). Note that the cells are mixed evenly before each aspiration of the cell suspension. Tumor formation of about 30mm3 can be seen in about 6 days. On the same day, 50ug/ml tumor exosomes (based on BCA protein quantification) or tumor exosomes modified with PP of equal mass are injected subcutaneously near the tumor for treatment once a week for a total of three times. Afterwards, the condition of the mice was observed, and the changes in tumor volume and the death of tumor-bearing mice were recorded.
(2)实验组共有5组，分别为(2) There are 5 experimental groups:
a)PBS溶剂对照组(CTR)a) PBS solvent control group (CTR)
b)肿瘤外泌体处理组(EXO)b) Tumor exosomes treatment group (EXO)
c)无MPLA对照组(PP/EXO)c) Control group without MPLA (PP/EXO)
d)无PP对照组(MPLA/EXO)d) Control group without PP (MPLA/EXO)
e)PP改造的肿瘤外泌体处理组(PP/MPLA/EXO)e) PP modified tumor exosomes treatment group (PP/MPLA/EXO)
实验结果显示，与PBS溶剂对照组相比，无MPLA对照组和无PP对照组均不能有效抑制肿瘤生长，PP改造的肿瘤外泌体能够显著延缓肿瘤的生长(图14)。The experimental results showed that compared with the PBS solvent control group, the control group without MPLA and the control group without PP could not effectively inhibit tumor growth, and the tumor exosomes modified by PP could significantly delay tumor growth (Figure 14) .
实施例八、PP改造的肿瘤外泌体抑制黑色素瘤的肺转移Example 8: PP-modified tumor exosomes inhibit lung metastasis of melanoma
细胞培养及处理方法：Cell culture and treatment methods:
细胞培养方法参考实施例四；肿瘤外泌体(EXO)的获取和PP改造的肿瘤外泌体及相应对照品的制备参考实施例二。The cell culture method is referred to Example 4; the acquisition of tumor exosomes (EXO) and the preparation of PP-modified tumor exosomes and corresponding reference substances are referred to Example 2.
实验方案：PP改造的肿瘤外泌体(来源于小鼠黑色素瘤细胞系B16F10)抑制肿瘤的肺转移Experimental plan: PP-modified tumor exosomes (derived from the mouse melanoma cell line B16F10) inhibit tumor lung metastasis
(1)制备PP改造的肿瘤外泌体(来源于小鼠黑色素瘤细胞系B16F10)，参考之前实施例二中的制备方法制备。在接瘤前，每只雌性野生型C57BL/6小鼠每次接受PBS(作为溶剂对照)、含有100ug的(以BCA蛋白定量计算)外泌体或者PP改造的肿瘤外泌体尾静脉注射。每周一次，共三周。(1) Preparation of PP-modified tumor exosomes (derived from mouse melanoma cell line B16F10) was performed by referring to the preparation method in Example 2. Before tumor implantation, each female wild-type C57BL/6 mouse received a tail vein injection of PBS (as a solvent control), 100 ug (calculated by BCA protein quantification) exosomes, or PP-modified tumor exosomes once a week for three weeks.
(2)小鼠黑色素瘤B16F10肺转移模型的构建：培养B16F10细胞生长，至90％汇合度时，用0.05％胰酶消化，按照1:4的比例进行传代培养。实验当天，将生长状态良好，汇合度达到90％的细胞胰酶消化后，用新鲜培养基中和胰酶，350g离心，弃上清后加入无菌PBS重悬，计数，将细胞悬液的密度调整为5×104/ml。利用1ml无菌注射器尾静脉注射到已接受过外泌体或者PP改造的肿瘤外泌体免疫的雌性野生型C57BL/6小鼠体内，每只小鼠0.1ml(即每只小鼠接种5×104个B16F10细胞)。(2) Construction of a mouse melanoma B16F10 lung metastasis model: B16F10 cells were cultured and grown to 90% confluence, then digested with 0.05% trypsin and subcultured at a ratio of 1:4. On the day of the experiment, cells with good growth and a confluence of 90% were trypsinized, and the trypsin was neutralized with fresh culture medium, centrifuged at 350g, the supernatant was discarded, and sterile PBS was added to resuspend, counted, and the density of the cell suspension was adjusted to 5×10 4 /ml. A 1ml sterile syringe was used to inject into the tail vein of female wild-type C57BL/6 mice that had been immunized with exosomes or PP-modified tumor exosomes, 0.1ml per mouse (i.e., 5×10 4 B16F10 cells were inoculated per mouse).
(3)实验共分为3组，分别为：(3) The experiment was divided into three groups:
a)PBS溶剂对照组(CTR)a) PBS solvent control group (CTR)
b)肿瘤外泌体处理组(EXO)b) Tumor exosomes treatment group (EXO)
c)PP改造的肿瘤外泌体(PP/MPLA/EXO)c) PP modified tumor exosomes (PP/MPLA/EXO)
结果显示，无论肺部转移的器官照片还是肺部黑色素瘤转移结节数量的统计数据皆表明，接瘤后13天，与溶剂对照组对比，肿瘤外泌体具有促进黑色素瘤向肺组织转移的功能，而PP改造的肿瘤外泌体 成功的抑制了黑色素瘤的肺转移(图15)。同时，依据该结果可合理推断，小鼠黑色素瘤分泌的外泌体本身容易富集到小鼠的肺部，使肺组织转变成免疫抑制性微环境，促进循环系统中的黑色素瘤细胞定殖，形成转移灶。而PP改造的肿瘤外泌体很有可能改变了免疫抑制性微环境，增强抗肿瘤免疫反应，进而 降低黑色素瘤的转移。 The results showed that both the organ photos of lung metastasis and the statistical data of the number of lung melanoma metastatic nodules showed that 13 days after tumor implantation, compared with the solvent control group, tumor exosomes had the function of promoting the metastasis of melanoma to lung tissue, and PP-modified tumor exosomes successfully inhibited the lung metastasis of melanoma (Figure 15) . At the same time, based on this result, it can be reasonably inferred that the exosomes secreted by mouse melanoma are easily enriched in the lungs of mice, turning the lung tissue into an immunosuppressive microenvironment, promoting the colonization of melanoma cells in the circulatory system, and forming metastatic foci. The tumor exosomes modified by PP are likely to change the immunosuppressive microenvironment, enhance the anti-tumor immune response, and thus reduce the metastasis of melanoma.
实施例九、PP改造的肿瘤外泌体改善黑色素瘤的免疫微环境Example 9: PP-modified tumor exosomes improve the immune microenvironment of melanoma
细胞培养及处理方法：Cell culture and treatment methods:
细胞培养方法参考实施例四；肿瘤外泌体(EXO)的获取和PP改造的肿瘤外泌体及相应对照品的制备参考实施例二。The cell culture method is referred to Example 4; the acquisition of tumor exosomes (EXO) and the preparation of PP-modified tumor exosomes and corresponding reference substances are referred to Example 2.
实验方案：PP改造的肿瘤外泌体改善黑色素瘤的免疫微环境Experimental plan: PP-modified tumor exosomes improve the immune microenvironment of melanoma
(1)B16F10模型的构建：当细胞贴壁生长至90％汇合度时，用0.05％胰酶消化，按照1:4的比例进行传代培养。实验当天，将生长状态良好，汇合度达到90％的细胞胰酶消化后，用新鲜培养基中和胰酶，350g离心，弃上清后加入无菌PBS重悬，计数，将细胞悬液的密度调整为2.5×106/ml。利用1ml无菌注射器皮下接种雌性野生型C57BL/6小鼠，每只小鼠0.1ml(即每只小鼠接种2.5×105个B16F10细胞)。注意每次吸取细胞悬液前将细胞混合均匀。约在4天左右可看到约30mm3的肿瘤形成，当日在肿瘤 附近的皮下注射50ug/ml肿瘤外泌体(基于BCA蛋白定量)或者含有等质量的PP改造的肿瘤外泌体进行治疗，每周一次，共三次。之后，一直观察小鼠的状态，期间记录荷瘤小鼠的肿瘤体积变化和死亡情况(图16A)。(1) Construction of B16F10 model: When the cells adhere to the wall and grow to 90% confluence, they are digested with 0.05% trypsin and subcultured at a ratio of 1:4. On the day of the experiment, the cells with good growth status and a confluence of 90% are trypsinized, and the trypsin is neutralized with fresh culture medium. The cells are centrifuged at 350g, the supernatant is discarded, and sterile PBS is added to resuspend and count. The density of the cell suspension is adjusted to 2.5×10 6 /ml. Female wild-type C57BL/6 mice are subcutaneously inoculated with 0.1ml per mouse (i.e., 2.5×10 5 B16F10 cells are inoculated per mouse). Note that the cells should be mixed evenly before aspirating the cell suspension each time. Tumor formation of about 30mm 3 can be seen in about 4 days, and the tumor is inoculated on the same day. The mice were treated with 50ug/ml tumor exosomes (based on BCA protein quantification) or PP-modified tumor exosomes containing an equal mass of PP subcutaneously once a week for a total of three times. Afterwards, the status of the mice was observed, and the changes in tumor volume and death of tumor-bearing mice were recorded during this period (Figure 16A).
(2)实验组共有5组，分别为(2) There are 5 experimental groups:
a)PBS溶剂对照组(CTR)a) PBS solvent control group (CTR)
b)肿瘤外泌体处理组(EXO)b) Tumor exosomes treatment group (EXO)
c)无MPLA对照组(PP/EXO)c) Control group without MPLA (PP/EXO)
d)无PP对照组(MPLA/EXO)d) Control group without PP (MPLA/EXO)
e)PP改造的肿瘤外泌体处理组(PP/MPLA/EXO)e) PP modified tumor exosomes treatment group (PP/MPLA/EXO)
实验结果显示，与PBS溶剂对照组相比，The experimental results showed that compared with the PBS solvent control group,
肿瘤外泌体处理，无MPLA对照和无PP对照均不能有效抑制肿瘤生长，Tumor exosome treatment, MPLA-free control and PP-free control were unable to effectively inhibit tumor growth.
PP改造的肿瘤外泌体能够显著延缓肿瘤的生长(图16B和C)。PP-modified tumor exosomes were able to significantly delay tumor growth ( Figure 16B and C ).
另外，通过对肿瘤组织中各类免疫细胞表面标志物的流式检测发现，与PBS溶剂对照组相比，In addition, flow cytometry analysis of various immune cell surface markers in tumor tissues revealed that compared with the PBS solvent control group,
肿瘤外泌体，无MPLA对照和无PP对照对肿瘤组织中总的免疫细胞比例(CD45+细胞占比)(图16D)、CD8+T细胞占比(图16E)、CD4+T细胞占比(图16F)、DC细胞占比(图16G)、巨噬细胞占比(图16H)以及NK细胞占比(图16I)均无明显改变，Tumor exosomes, MPLA-free control and PP-free control had no significant changes in the total immune cell ratio (CD45 + cell ratio) (Figure 16D), CD8 + T cell ratio (Figure 16E), CD4 + T cell ratio (Figure 16F), DC cell ratio (Figure 16G), macrophage ratio (Figure 16H) and NK cell ratio (Figure 16I) in tumor tissues.
PP改造的肿瘤外泌体能够显著增加上述各类型免疫细胞的占比(图16D-I)，说明肿瘤组织的免疫抑制性微环境得到改善，为免疫治疗发挥功能提供了先决条件。PP-modified tumor exosomes can significantly increase the proportion of the above-mentioned types of immune cells (Figure 16D-I), indicating that the immunosuppressive microenvironment of tumor tissue has been improved, providing a prerequisite for the function of immunotherapy.
通过胞内细胞因子染色法考察具有肿瘤杀伤能力的CD8+T细胞的活性和功能指标发现，与PBS溶剂对照组相比，The activity and functional indicators of CD8 + T cells with tumor killing ability were examined by intracellular cytokine staining method, and it was found that compared with the PBS solvent control group,
肿瘤外泌体，无MPLA对照和无PP对照处理后对肿瘤组织中CD8+T细胞胞内的Ki67(表征细胞增殖活力)(图16J)，IFN-γ和Granzyme B(表征T细胞的肿瘤杀伤能力)(图16K和L)具有一定的增强作用，Tumor exosomes, after treatment with the MPLA-free control and the PP-free control, had a certain enhancing effect on Ki67 (indicating cell proliferation activity) (Figure 16J), IFN-γ and Granzyme B (indicating the tumor killing ability of T cells) (Figures 16K and L) in CD8 + T cells in tumor tissues.
PP改造的肿瘤外泌体能够显著增加CD8+T细胞的增殖能力和肿瘤杀伤能力(图16J-L)。PP-modified tumor exosomes can significantly increase the proliferation and tumor killing ability of CD8 + T cells ( Figure 16J-L ).
综上，PP改造的肿瘤外泌体能够显著增加各个类型的免疫细胞浸润，特别是具有增殖和肿瘤杀伤活力的CD8+T细胞，进而抑制肿瘤细胞的生长，达到治疗肿瘤的目的。In summary, PP-modified tumor exosomes can significantly increase the infiltration of various types of immune cells, especially CD8 + T cells with proliferation and tumor killing activity, thereby inhibiting the growth of tumor cells and achieving the purpose of treating tumors.
实施例十、PP改造的肿瘤外泌体模拟个体化肿瘤治疗Example 10: PP-modified tumor exosomes simulate individualized tumor treatment
细胞培养及处理方法：Cell culture and treatment methods:
细胞培养方法参考实施例四；肿瘤外泌体(EXO)的获取和PP改造的肿瘤外泌体的制备参考实施例二。The cell culture method is referred to Example 4; the acquisition of tumor exosomes (EXO) and the preparation of PP-modified tumor exosomes are referred to Example 2.
实验方案：PP改造的肿瘤外泌体(来源于小鼠结肠癌细胞系MC38)模拟个体化肿瘤治疗Experimental plan: PP-modified tumor exosomes (derived from mouse colon cancer cell line MC38) simulate personalized tumor therapy
(1)MC38模型的构建：当细胞贴壁生长至90％汇合度时，用0.05％胰酶消化，按照1:3的比例进行传代培养。实验当天，将生长状态良好，汇合度达到90％的细胞胰酶消化后，用新鲜培养基中和胰酶，350g离心，弃上清后加入无菌PBS重悬，计数，将细胞悬液的密度调整为2.5×106/ml。利用1ml无菌注射器皮下接种雌性野生型C57BL/6小鼠，每只小鼠0.1ml(即每只小鼠接种2.5×105个MC38细胞)。(1) Construction of MC38 model: When the cells adhered to the wall and grew to 90% confluence, they were digested with 0.05% trypsin and subcultured at a ratio of 1:3. On the day of the experiment, the cells with good growth and a confluence of 90% were digested with trypsin, and the trypsin was neutralized with fresh culture medium. The cells were centrifuged at 350g, the supernatant was discarded, and sterile PBS was added for resuspending. The cells were counted and the density of the cell suspension was adjusted to 2.5×10 6 /ml. Female wild-type C57BL/6 mice were subcutaneously inoculated with 0.1 ml per mouse using a 1 ml sterile syringe (i.e., 2.5×10 5 MC38 cells were inoculated per mouse).
(2)待肿瘤生长到200mm3左右，手术剥离肿瘤组织，使用Tumor cell分离试剂盒将组织中的肿瘤细胞分选出来，继续培养。(2) When the tumor grows to about 200 mm3 , the tumor tissue is surgically removed and the tumor cells in the tissue are sorted out using a Tumor cell isolation kit and continued to be cultured.
(3)肿瘤细胞传代一定代数后，收集肿瘤细胞培养上清，并从中提取肿瘤外泌体。同时使用同批次肿瘤细胞用于新的肿瘤模型建立(具体方案参考本实施例实验方案(1)中所述)。(3) After a certain number of tumor cell passages, the tumor cell culture supernatant is collected and tumor exosomes are extracted therefrom. The same batch of tumor cells is used to establish a new tumor model (for specific procedures, refer to the experimental protocol (1) of this example).
(4)接种新的肿瘤4天后，分别使用原始MC38细胞系所提取的外泌体和第(3)步所提取的肿瘤组织来源的外泌体制备PP改造的外泌体，对新的肿瘤模型进行治疗。(具体方案见图17A)(4) Four days after the new tumor was inoculated, the exosomes extracted from the original MC38 cell line and the exosomes from the tumor tissue extracted in step (3) were used to prepare PP-modified exosomes to treat the new tumor model. (See Figure 17A for the specific scheme)
(5)实验组共有3组，分别为(5) There are 3 experimental groups:
a)PBS溶剂对照组(CTR) a) PBS solvent control group (CTR)
b)PP改造的MC38细胞系外泌体处理组(PP/MPLA/EXO-MC38)b) PP-modified MC38 cell line exosome treatment group (PP/MPLA/EXO-MC38)
c)PP改造的肿瘤细胞外泌体处理组(PP/MPLA/EXO-Tumor)c) PP-modified tumor cell exosome treatment group (PP/MPLA/EXO-Tumor)
实验结果显示，与PBS溶剂对照组相比，The experimental results showed that compared with the PBS solvent control group,
无论肿瘤外泌体的来源于传代培养的细胞系还是原位分离的肿瘤组织中的肿瘤细胞，经PP改造的外泌体均显示出抑制肿瘤生长的能力(图17B)。更进一步的，相较于原初的MC38细胞系来源的外泌体， PP改造的原位分离的肿瘤组织中的肿瘤细胞外泌体具有更好的肿瘤生长抑制效果。Regardless of whether the tumor exosomes are derived from cell lines cultured in succession or tumor cells in in situ isolated tumor tissues, the exosomes modified by PP show the ability to inhibit tumor growth (Figure 17B). Furthermore, compared with the original exosomes derived from the MC38 cell line, the tumor cell exosomes in in situ isolated tumor tissues modified by PP have a better tumor growth inhibition effect .
肿瘤细胞内部基因的非同义突变将表达出变种蛋白，被免疫系统捕获并识别成外源物质，因此可作为肿瘤特异性抗原的来源。而且值得注意的是，产生肿瘤特异性抗原的概率与肿瘤细胞体细胞突变概率大致成呈正相关。(Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival.Genome Res.2014；24:743-50.；Molecular and genetic properties of tumors associated with local immune cytolytic activity.Cell.2015；160:48-61.)。MC38小鼠结肠癌细胞属于微卫星不稳定型癌种，其基因组具有高突变性特点，在一定的选择压力下，此特性将可能改变该细胞所产生的抗原谱(Targeting immune checkpoints potentiates immunoediting and changes the dynamics of tumor evolution)。Non-synonymous mutations in genes within tumor cells will express variant proteins, which are captured and recognized as foreign substances by the immune system, and can therefore serve as a source of tumor-specific antigens. It is also worth noting that the probability of producing tumor-specific antigens is roughly positively correlated with the probability of somatic mutations in tumor cells. (Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival. Genome Res. 2014; 24: 743-50.; Molecular and genetic properties of tumors associated with local immune cytolytic activity. Cell. 2015; 160: 48-61.). MC38 mouse colon cancer cells are microsatellite unstable cancers with a highly mutable genome. Under certain selection pressure, this characteristic may change the antigen spectrum produced by the cells (Targeting immune checkpoints potentiates immunoediting and changes the dynamics of tumor evolution).
在本实施例中，具备完整免疫能力的小鼠也会对生长的肿瘤细胞进行免疫监视，将其能够识别的肿瘤细胞杀死，而生还的肿瘤细胞则因为基因突变变得具备抵抗免疫杀伤的能力，该过程也被称为免疫编辑。在基因组突变和免疫编辑压力的共同作用下，相信小鼠皮下原初的MC38肿瘤在生长过程中已经产生了一些由基因突变引起的新抗原。因此，从该肿瘤组织中分选、传代培养形成的“新”肿瘤细胞中存在产生新抗原的可能性。在此基础上，可以合理推断，新的肿瘤细胞外泌体与原始MC38外泌体在分享了大部分相同抗原的前提下，进一步携带了由基因突变获得的新抗原。所以，才体现出PP改造的MC38外泌体仍然具有一定的肿瘤抑制作用，而PP改造的肿瘤细胞外泌体具有更好的肿瘤生长抑制效果。In this embodiment, mice with complete immune capabilities will also perform immune surveillance on growing tumor cells, kill tumor cells that they can identify, and the surviving tumor cells become resistant to immune killing due to gene mutations. This process is also called immune editing. Under the combined effects of genomic mutations and immune editing pressure, it is believed that the original MC38 tumors under the skin of mice have produced some new antigens caused by gene mutations during their growth. Therefore, there is a possibility of producing new antigens in the "new" tumor cells formed by sorting and subculturing from the tumor tissue. On this basis, it can be reasonably inferred that the new tumor cell exosomes and the original MC38 exosomes, while sharing most of the same antigens, further carry new antigens obtained by gene mutations. Therefore, it is reflected that the PP-modified MC38 exosomes still have a certain tumor inhibitory effect, and the PP-modified tumor cell exosomes have a better tumor growth inhibitory effect.
综上，本发明利用PP分子对肿瘤外泌体进行结构改造，使其在保留原有肿瘤相关抗原和肿瘤特异性抗原的同时，打破原初状态的外泌体免疫抑制的天然属性。PP改造后的肿瘤外泌体，具备引起抗原特异性CD8+T细胞反应的能力，有效降低荷瘤小鼠的肿瘤负荷，延长生存期。In summary, the present invention uses PP molecules to structurally modify tumor exosomes, so that while retaining the original tumor-associated antigens and tumor-specific antigens, it breaks the natural immunosuppressive properties of the original exosomes. Tumor exosomes modified by PP have the ability to induce antigen-specific CD8+T cell responses, effectively reducing the tumor burden of tumor-bearing mice and prolonging their survival.
最后需要说明的是，以上实施例仅用于帮助本领域技术人员理解本发明的实质，不用于限定本发明的保护范围。 Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the protection scope of the present invention.

Claims (13)

  1. 一种改造的肿瘤外泌体制剂，所述的制剂包含：A modified tumor exosome preparation, the preparation comprising:
    (1)浓度大于50ug/ml二硬脂酸磷脂酰乙醇胺-聚乙二醇(PEG-DSPE)组分；(1) Distearate phosphatidylethanolamine-polyethylene glycol (PEG-DSPE) component with a concentration greater than 50ug/ml;
    (2)蛋白浓度大于10ug/ml的肿瘤外泌体组分；(2) tumor exosome components with protein concentration greater than 10ug/ml;
    (3)有效量的用于刺激免疫反应的免疫佐剂分子。(3) An effective amount of an immune adjuvant molecule for stimulating an immune response.
  2. 根据权利要求1所述的改造的肿瘤外泌体制剂，其特征在于，The modified tumor exosome preparation according to claim 1, characterized in that
    所述的PEG-DSPE的纯度≥95％，其中的PEG链的分散度≤1.1；The purity of the PEG-DSPE is ≥95%, and the dispersion of the PEG chains is ≤1.1;
    优选的，所述的PEG-DSPE分子中的PEG链长为1000～5000Da；更优选的，所述的PEG-DSPE分子中的PEG链长为1500～2500Da；最优选的，所述的PEG-DSPE分子中的PEG链长为2000Da(PEG2000-DSPE)。Preferably, the PEG chain length in the PEG-DSPE molecule is 1000-5000Da; more preferably, the PEG chain length in the PEG-DSPE molecule is 1500-2500Da; most preferably, the PEG chain length in the PEG-DSPE molecule is 2000Da (PEG2000-DSPE).
  3. 根据权利要求1或2所述的改造的肿瘤外泌体制剂，其特征在于，所述的蛋白浓度指制剂中所有的肿瘤外泌体中所包含的蛋白浓度，优选的，使用BCA法对肿瘤外泌体的蛋白浓度进行定量。The modified tumor exosome preparation according to claim 1 or 2, characterized in that the protein concentration refers to the protein concentration contained in all tumor exosomes in the preparation, and preferably, the protein concentration of tumor exosomes is quantified using the BCA method.
  4. 根据权利要求1-3任一所述的改造的肿瘤外泌体制剂，其特征在于，所述的免疫佐剂分子包括但不限于，MPLA、QS21、PolyI:C；优选为MPLA；The modified tumor exosome preparation according to any one of claims 1 to 3, characterized in that the immune adjuvant molecules include but are not limited to MPLA, QS21, PolyI:C; preferably MPLA;
    优选的，所述的免疫佐剂分子的用量为0.1～10ug/ml。Preferably, the dosage of the immune adjuvant molecule is 0.1-10 ug/ml.
  5. 根据权利要求1-4任一所述的改造的肿瘤外泌体制剂，其特征在于，所述的改造的肿瘤外泌体制剂中，The modified tumor exosome preparation according to any one of claims 1 to 4, characterized in that, in the modified tumor exosome preparation,
    PEG-DSPE、免疫佐剂分子、肿瘤外泌体中的总蛋白三种成分的质量比为：100：0.1～5：2～50；The mass ratio of PEG-DSPE, immune adjuvant molecules, and total protein in tumor exosomes is 100: 0.1-5: 2-50;
    优选的，所述的PEG-DSPE、免疫佐剂分子、肿瘤外泌体中的总蛋白三种成分的质量比为：100：0.5～2：5～20。Preferably, the mass ratio of the three components of PEG-DSPE, immune adjuvant molecules, and total protein in tumor exosomes is: 100:0.5~2:5~20.
  6. 根据权利要求1-5任一所述的改造的肿瘤外泌体制剂，其特征在于，The modified tumor exosome preparation according to any one of claims 1 to 5, characterized in that
    所述的肿瘤外泌体来源于体外培养的肿瘤系或自肿瘤组织中分离的原代肿瘤细胞；The tumor exosomes are derived from tumor lines cultured in vitro or primary tumor cells isolated from tumor tissues;
    所述的肿瘤外泌体使用密度梯度离心、差速离心、体积排阻、免疫分离、聚合物沉淀法制备，或使用商品化的试剂盒制备。The tumor exosomes are prepared by density gradient centrifugation, differential centrifugation, size exclusion, immunoseparation, polymer precipitation, or by using a commercial kit.
  7. 权利要求1-6任一所述的改造的肿瘤外泌体制剂的制备方法，其包括如下步骤：The method for preparing the modified tumor exosome preparation according to any one of claims 1 to 6 comprises the following steps:
    (1)将PEG-DSPE和免疫佐剂分子混匀后去除溶剂，制成PEG-DSPE和免疫佐剂混合物；(1) mixing PEG-DSPE and immune adjuvant molecules and removing the solvent to prepare a mixture of PEG-DSPE and immune adjuvant;
    (2)向上述混合物中加入定量好的肿瘤外泌体溶液，常温下充分混匀即得所述改造的肿瘤外泌体制剂。(2) Add a predetermined amount of tumor exosome solution to the above mixture, and mix thoroughly at room temperature to obtain the modified tumor exosome preparation.
  8. 根据权利要求7所述的方法，其特征在于，还包括，The method according to claim 7, further comprising:
    (3)对所述改造的肿瘤外泌体制剂进行除菌过滤。(3) sterilizing and filtering the modified tumor exosome preparation.
  9. 根据权利要求7或8所述的方法，其特征在于，The method according to claim 7 or 8, characterized in that
    步骤(1)所述的制备不含溶剂成分的PEG-DSPE和免疫佐剂混合物的方法为，将PEG-DSPE和免疫佐剂分子溶于有机溶剂中并混匀，然后除去有机溶剂；The method for preparing the solvent-free mixture of PEG-DSPE and immune adjuvant described in step (1) is to dissolve PEG-DSPE and immune adjuvant molecules in an organic solvent and mix them evenly, and then remove the organic solvent;
    优选的，在惰性气体的保护下，使用减压蒸馏法，在低于70℃下去除有机溶剂。Preferably, the organic solvent is removed at a temperature below 70° C. using reduced pressure distillation under the protection of an inert gas.
  10. 权利要求1-6任一所述的改造的肿瘤外泌体制剂在制备抗肿瘤药物中的应用。Use of the modified tumor exosome preparation according to any one of claims 1 to 6 in the preparation of anti-tumor drugs.
  11. 权利要求1-6任一所述的改造的肿瘤外泌体制剂在制备个体化抗肿瘤制剂中的应用；优选的，所述的个体化抗肿瘤制剂中，所述的肿瘤外泌体来自于需要接受个体化治疗的患者自体的肿瘤组织或肿瘤细胞。Use of the modified tumor exosome preparation according to any one of claims 1 to 6 in the preparation of a personalized anti-tumor preparation; preferably, in the personalized anti-tumor preparation, the tumor exosomes are derived from autologous tumor tissue or tumor cells of a patient who needs to receive personalized treatment.
  12. 含有权利要求1-6任一所述的改造的肿瘤外泌体制剂的药物或药物组合物。A drug or pharmaceutical composition containing the modified tumor exosome preparation according to any one of claims 1 to 6.
  13. 含有权利要求1-6任一所述的改造的肿瘤外泌体制剂的个体化药物制剂，优选的，所述的个体化药物制剂中，所述的肿瘤外泌体来自于需要接受个体化治疗的患者自体的肿瘤组织或肿瘤细胞。 A personalized pharmaceutical preparation containing the modified tumor exosome preparation according to any one of claims 1 to 6, preferably, in the personalized pharmaceutical preparation, the tumor exosomes are derived from autologous tumor tissue or tumor cells of a patient who needs to receive personalized treatment.
PCT/CN2023/128966 2022-11-02 2023-11-01 Modified exosome preparation, preparation method therefor, and use thereof WO2024094043A1 (en)

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