CN118021941A - Modified exosome preparation, preparation method and application thereof - Google Patents
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- CN118021941A CN118021941A CN202211362440.2A CN202211362440A CN118021941A CN 118021941 A CN118021941 A CN 118021941A CN 202211362440 A CN202211362440 A CN 202211362440A CN 118021941 A CN118021941 A CN 118021941A
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Abstract
The invention relates to an improved exosome preparation, a preparation method and application thereof, wherein the preparation comprises the following components: (1) A concentration greater than 50ug/ml phosphatidylethanolamine distearate-polyethylene glycol (PEG-DSPE); (2) Tumor exosome component with protein concentration greater than 10 ug/ml; (3) An effective amount of an immunoadjuvant molecule for stimulating an immune response; the invention also relates to the application of the modified tumor exosome preparation in preparing antitumor drugs or individual antitumor preparations.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to an improved exosome preparation, a preparation method and application thereof.
Background
Since the advent of tumor immunotherapy age, a variety of immune checkpoint inhibitors (including antibodies and small molecules), CAR-T cell therapies and therapeutic tumor vaccines have entered clinical trials and have played a significant therapeutic role in many solid tumors and hematological malignancies. However, the drug response rate and cure rate of patients are at low levels, and primary or secondary resistance is often produced during treatment, impairing the efficacy of immunotherapy. Studies have shown that limited immunotherapeutic effects are associated with a lack or depletion of tumor antigens, while immunosuppressive properties within tumor tissues are also an obstacle to inhibiting the efficacy of immunotherapy [1-3]. DC cells are professional antigen presenting cells in humans, and can activate helper T cells, which in turn help the activation of CD8 positive T cells, exerting antitumor efficacy [4]. In addition, DCs can also enhance the anti-tumor function of NK cells by secreting antibodies by helper T cells or B cells [5]. Although DC vaccines (which, after isolation of the DC cells, incubate the tumor antigen in vitro and promote maturation and reinfusion into the patient) present some therapeutic advantages clinically, not all patients respond well to this therapy. The reason for this is also factors such as low immunogenicity in tumor antigens, immune escape and immunosuppression by tumors [6,7].
All cells, including both prokaryotic and eukaryotic cells, can release extracellular vesicles. The extracellular vesicles with a diameter of 40-160nm are called exosomes (average diameter 100 nm). Exosomes originate from the endosome of the cell and interact with various organelles prior to secreting the cell, thus containing within it various cytoplasmic and organelle-derived inclusions including DNA, RNA, lipid molecules, metabolites, various proteins on the cytoplasm and cell membrane, etc. [8]. While the function of exosomes has been thought to be a matter of treating cell waste, recent studies have shown that exosomes have very rich functions, and that exosomes produced by different cells carry different contents, this heterogeneity directly leading to differences in the functional orientation of exosomes [8].
Exosomes secreted by tumor cells (simply "tumor exosomes") can be used in immunotherapy of tumors mainly based on several features of their own: firstly, tumor exosomes carry tumor antigens of tumor cells (the blast cells secreting the exosomes) themselves, which are able to activate tumor-specific T cells, eliciting an anti-tumor immune response [9,10]; second, tumor exosomes carry large amounts of histocompatibility complexes (MHC-I and MHC-II), which are structural molecules necessary for antigen peptide binding [11], while being able to avoid immune rejection; in addition, tumor exosomes also have a number of proteins that promote binding to and phagocytosis by receptor cells, such as 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 characteristic of stable structure, and their bilayer lipid membranes can protect their contents from being affected by various nucleases or proteases, suitable for delivery systems of small molecules, nucleic acid drugs [15].
In recent years studies have demonstrated the feasibility of tumor exosomes as therapeutic vaccines for tumors, including preclinical models [16] and clinical trials [17]. Tumor exosomes have limited immunogenicity, but can be immunosuppressive against other immune cell populations in the tumor microenvironment, limiting their efficacy. Furthermore, tumor exosomes can participate in various stages of tumorigenesis by means of inhibiting apoptosis, promoting the development of drug resistance, aiding tumor cell diffusion-migration-colonization, promoting angiogenesis, tumor immune escape, immunosuppression, etc. [8]. Thus, immunotherapy with tumor exosomes alone often fails to produce satisfactory anti-tumor immune activity, which also explains why this therapy has not been used clinically in bulk at the present time.
In order to solve the above problems, many engineering strategies have been developed to the parent cells secreting tumor exosomes, such as increasing the heat shock protein content of tumor exosomes, providing an adjuvant effect [18,19]; increasing the expression level of a tumor-specific antigen, thereby increasing the abundance and immunogenicity of the tumor-specific antigen in the tumor exosomes [20]; adding superantigen into tumor exosomes, and promoting activation of DC cells by the tumor exosomes by utilizing the characteristic that superantigen and MHC II molecules form stable complexes [21 ]; through high expression control of the transcription factor CIITA of MHC II molecules in tumor cells, the abundance of MHC II molecules on tumor exosomes is increased, and the presentation efficiency of antigen peptides and the redundancy of effective tumor antigen peptides are improved [22]; integrating some virus fusion proteins into tumor exosomes, promoting their uptake by DC cells [23]; high expression of cytokines in tumor cells, increasing the content of cytokines in tumor exosomes [24] of immunogenicity of tumor exosomes; some miRNAs with negative regulation effect on genes related to the generation of immunosuppression are highly expressed in tumor cells, so that tumor exosomes also contain the miRNAs, and the tumor exosomes are prevented from generating strong immunosuppression through the mechanism of the miRNAs after being ingested by immune cells, so that the immunogenicity is improved [25].
In addition, there is a strategy: tumor exosomes were presented by co-culturing in vitro with DCs, giving the tumor-specific antigen to DCs, and then treating the tumor in the form of a DC vaccine. This avoids immunosuppression by free tumor exosomes and activates a more potent immune response [7].
In conclusion, tumor exosomes have the potential to be developed as anti-tumor vaccines due to the specific antigen carrying a large number of tumor cells, but their natural properties of being able to inhibit tumor microenvironment and even draining immune cell functions in lymph nodes limit their clinical transformation applications. Although various strategies based on the modification of tumor cells (exosome-secreting blast cells) have been proposed, such schemes are time-consuming, expensive, and difficult to unify in terms of operational procedures, and inconvenient to popularize in clinical practice.
Based on this, the invention is proposed
[ Reference ]
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Disclosure of Invention
The present invention is first directed to an engineered tumor exosome formulation comprising:
(1) A concentration greater than 50ug/ml phosphatidylethanolamine distearate-polyethylene glycol (PEG-DSPE);
(2) Tumor exosome component with protein concentration greater than 10 ug/ml;
(3) An effective amount of an immunoadjuvant molecule for stimulating an immune response;
The purity of the PEG-DSPE is more than or equal to 95%, and the dispersity of PEG chains is less than or equal to 1.1;
Preferably, the chain length of PEG in the PEG-DSPE molecule is 1000-5000 Da; more preferably, the chain length of PEG in the PEG-DSPE molecule is 1500-2500 Da; most preferably, the PEG chain length of the PEG-DSPE molecule is 2000Da (PEG 2000-DSPE);
The PEG-DSPE may also be replaced with other types of pegylated phospholipids, specifically,
In the structure of the polyethylene glycol phospholipid,
The fatty acid chain of the phospholipid moiety contains 10 to 24 carbon atoms, and the fatty acid chain may be saturated or partially saturated, preferably lauric acid (12 carbon), myristic acid (14 carbon), palmitic acid (16 carbon), stearic acid or oleic acid or linoleic acid (18 carbon), arachic acid (20 carbon), behenic acid (22 carbon), and lignoceric acid (lignocerate) (24 carbon);
The phospholipid moiety of the pegylated phospholipid may be Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), diphosphatidylglycerol, lysophosphatidylcholine (LPC), lysoethanolamine Phospholipid (LPE), etc.; preferably phosphatidylethanolamine, in particular distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine.
The protein concentration refers to the protein concentration contained in all tumor exosomes in the preparation, and preferably, the protein concentration of the tumor exosomes is quantified by using a BCA method;
Such immunoadjuvant molecules include, but are not limited to, MPLA (Monophosphoryl lipid A ), QS21 (Quillaja saponaria, quillaja saponaria saponin), polyI:c (polyinosinic acid); preferably MPLA;
Preferably, the amount of said immunoadjuvant molecule is between 0.1 and 10ug/ml.
Further, in the modified tumor exosome preparation, the mass ratio of the three components of PEG-DSPE, an immunoadjuvant molecule and total protein in the tumor exosome is as follows: 100:0.1 to 5:2 to 50 percent; preferably, the mass ratio of the three components of PEG-DSPE, the immunoadjuvant molecule and the total protein in the tumor exosomes is as follows: 100:0.5 to 2:5 to 20.
The tumor exosome is derived from a tumor system cultured in vitro or primary tumor cells separated from tumor tissues;
the tumor exosomes are prepared by density gradient centrifugation, differential centrifugation, size exclusion, immunoseparation, polymer precipitation methods, or by commercial kits;
the invention also relates to a preparation method of the modified tumor exosome preparation, which comprises the following steps:
(1) Preparing a PEG-DSPE and immunoadjuvant mixture without solvent component;
(2) Adding a well-quantified tumor exosome solution into the mixture, and fully and uniformly mixing at normal temperature to obtain the modified tumor exosome preparation;
optionally, the first and second heat exchangers are arranged in a row,
(3) And (3) performing degerming and filtering on the modified tumor exosome preparation.
Preferably, the method for preparing the PEG-DSPE and immunoadjuvant mixture without solvent component in the step (1) comprises the steps of dissolving PEG-DSPE and immunoadjuvant molecules in an organic solvent, uniformly mixing, and then removing the organic solvent; more preferably, the organic solvent is removed at a temperature below 70 ℃ using reduced pressure distillation under the protection of inert gas.
The invention also relates to the application of the modified tumor exosome preparation in preparing medicines, wherein the medicines are anti-tumor medicines.
The invention also relates to the application of the modified tumor exosome preparation in preparing individual anti-tumor preparations; preferably, in the personalized anti-tumor formulation, the tumor exosomes are derived from autologous tumor tissue or tumor cells of the patient in need of personalized treatment.
The invention also relates to a medicament or a pharmaceutical composition containing the modified tumor exosome preparation.
The invention also relates to a personalized medicine preparation containing the modified tumor exosome preparation, wherein the tumor exosome is derived from autologous tumor tissues or tumor cells of a patient in need of personalized treatment.
The invention also relates to the use of the modified tumor exosome system for the treatment of tumors; preferably, the engineered tumor exosome formulation is prepared using tumor tissue or tumor cells from the patient's body.
The invention has the beneficial effects that:
(1) The cell entry pathway of PEGylated phospholipid (PEG 2000-DSPE, hereinafter PP) engineered tumor exosomes was also altered compared to free tumor exosomes, and PP engineered tumor exosomes could enter the endoplasmic reticulum directly (FIG. 6) in order to elicit a stronger tumor antigen-specific T cell response (FIGS. 7,8,9, 10 and 11); in contrast, free tumor exosomes are rapidly taken into lysosomes after endocytosis by DC cells, and the antigen contained therein will be presented by the MHC II pathway [14,19,20].
(2) In wild-type mice, administration of PP-engineered tumor exosomes can elicit tumor antigen-specific CTL responses, and PP-engineered tumor exosomes also exhibit good anti-tumor effects in tumor-bearing mice.
(3) The PP modified tumor exosome can also improve the infiltration condition of immune cells in tumor microenvironment, increase the proportion of DC, macrophages and CD4+ and CD8+ T cells with anti-tumor activity, and lay a good foundation for the combination with other anti-tumor immunotherapy schemes.
(4) The treatment scheme has certain universality: as long as the tumor exosomes are derived from the same tissue source, the tumor exosomes have certain therapeutic effects even if the mutation frequencies or the characteristics of the genes are inconsistent.
(5) Has good curative effect on individual specificity: under the action of in vivo immune editing or after receiving a treatment mode capable of causing gene mutation, including but not limited to radiotherapy, chemotherapy, immunotherapy and the like, part of tumor tissue-derived exosomes are used for preparing PP-modified tumor exosomes which have a therapeutic effect on the residual tumor tissue and can even prevent the metastasis of a distant organ of a tumor.
Drawings
FIG. 1 cytotoxicity of different surfactants against bone marrow derived DC production [ in vitro experiments ]. The ordinate shows the activity of DC cells after 48 hours of treatment with different surfactants (MTT method), and the abscissa shows the concentration of different surfactants.
Figure 2, preparation and characterization of PP-engineered tumor exosomes. A. Frozen electron micrographs show tumor exosomes (left panel), lacking morphological features of MPLA control (middle panel) and PP-engineered tumor exosomes (right panel); B. dynamic light scattering results show the mean diameter distribution of tumor exosomes (black curve) and PP-engineered tumor exosomes (gray curve).
FIG. 3, PP-engineered tumor exosomes promote the ability of DC cells to present antigen [ in vitro experiments ]. The ordinate shows the number of complexes formed by the OVA protein-specific antigenic peptides (SIINFEKL) with MHC-I molecules (H-2K b) presented on the surface of differently treated DC cells, expressed by Mean Fluorescence Intensity (MFI).
Figure 4, PP-engineered tumor exosomes increase the expression level of DC cell surface costimulatory molecules [ in vitro experiments ]. The ordinate shows the expression levels of co-stimulatory molecules CD80 (left panel) and CD86 (right panel) representing activation markers on the surface of differently treated DC cells, expressed as Mean Fluorescence Intensity (MFI).
Fig. 5, PP-engineered tumor exosomes increase the level of DC cell-secreting cytokines [ in vitro experiments ]. The ordinate shows the concentrations (pg/ml) of TNF-. Alpha., IL-2 and IL-12 detected by ELISA in the supernatants of the differently treated DC cells, and the results of real-time quantitative PCR of IFN-. Beta.relative to the relative expression level of the beta-actin housekeeping gene.
Figure 6, PP engineering can alter the pathway of tumor exosomes following uptake by DC cells [ in vitro experiments ]. A. Confocal microscopy photographs show co-localization of PKH-67 labeled tumor Exosomes (EXO) or PP-engineered tumor exosomes (PP/EXO) (Green-Green) with lysosomes (Lysosome) (left) and Endoplasmic Reticulum (ER) (Red-Red) within DC2.4 cells, respectively; B. confocal microscopy photographs showed co-localization of CFSE-labeled tumor Exosomes (EXO) or PP-engineered tumor exosomes (PP/EXO) (Green-Green) with lysosomes (Lysosome) within DC2.4 cells (left side) and Endoplasmic Reticulum (ER) (Red-Red), respectively.
Figure 7, PP-engineered tumor exosomes promote proliferation of antigen-specific OT I CD8 + T cells [ in vitro experiments ]. The ordinate shows the different proportions of OT I cd8+ T cell proliferation activated by differently treated DC cells.
FIG. 8, PP-engineered tumor exosomes elicit a strongly antigen-specific CTL response-mouse colon cancer model (MC 38) [ in vivo experiments ]. Photographs (left panel) of IFN-gamma spots (ELLIspot experiment) and statistical results of counts of IFN-gamma spots (right panel) generated by in vitro re-stimulation of mouse lymph node T cells immunized with different groups of exosomes with tumor specific antigenic peptides (rpi 18, reps-1 and Adpgk).
FIG. 9, PP-engineered tumor exosomes elicit a strongly antigen-specific CTL response-mouse melanoma model (B16F 10) [ in vivo experiments ]. Photographs (left panel) of IFN-gamma spots (ELLIspot experiments) and counts of IFN-gamma spots (right panel) generated by in vitro re-stimulation of mouse lymph node T cells immunized with different groups of exosomes with tumor-specific antigenic peptide (Trp-2).
FIG. 10, PP-engineered tumor exosomes elicit a strong antigen-specific CTL response-mouse triple negative breast cancer model (4T 1) [ in vivo experiments ]. Photographs (left panel) of IFN-gamma spots (ELLIspot experiment) and counts statistics of IFN-gamma spots (right panel) generated by in vitro re-stimulation of mouse lymph node T cells immunized with different groups of exosomes with tumor-specific antigenic peptides (Sptbn and Wdr 33).
FIG. 11, PP-engineered tumor exosomes elicit a strongly antigen-specific CTL response-a mouse cervical cancer model (TC-1, with HPV antigens E6, E7) [ in vivo experiments ]. Photographs (left panel) of IFN-gamma spots (ELLIspot experiment) and counts of IFN-gamma spots (right panel) generated by in vitro re-stimulation of mouse lymph node T cells immunized with different groups of exosomes with tumor-specific antigenic peptides (E7-20).
FIG. 12, antitumor effect of PP-engineered tumor exosomes-mouse colon cancer model (MC 38) [ in vivo experiments ]. Mice were treated with different groups of exosomes following tumor inoculation, tumor growth curves (left panel) and mouse survival (right panel).
FIG. 13, anti-tumor effect of PP-engineered tumor exosomes-mouse melanoma model (B16F 10) [ in vivo experiments ]. Mice were treated with different groups of exosomes following tumor inoculation, tumor growth curves (left panel) and mouse survival (right panel).
FIG. 14, antitumor effect of PP-engineered tumor exosomes-mouse cervical cancer model (TC-1, with HPV antigen E7) [ in vivo experiments ]. Tumor growth curves of mice treated with different groups of exosomes after tumor inoculation.
FIG. 15, anti-tumor metastasis effect of PP-engineered tumor exosomes-mouse melanoma model (B16F 10) [ in vivo experiments ]. Representative photographs (a) and statistical data (B) of metastasis nodules formed by melanoma cells in the lungs of mice were observed after mice were tumor-grafted via tail veins after advanced immunization with different groups of exosomes for 13 days.
Fig. 16, PP-engineered tumor exosomes enhance infiltration of anti-tumor-associated immune cells—mouse melanoma model (B16F 10) [ in vivo experiments ].
Fig. 17, PP-modified tumor exosome preparation technology simulate to realize personalized tumor therapy—mouse colon cancer model (MC 38) [ in vivo experiments ]. A. Schematic of experimental flow; B. tumor growth curve.
Detailed Description
Mouse model:
OTI transgenic mouse model: the mouse model is characterized in that the T Cell Receptor (TCR) of the CD8 positive T cells can specifically identify the MHC-I/OVA 257-264 (SIINFEKL) antigen complex on the surface of antigen presenting cells through gene editing, and can be specifically activated and proliferated.
Example one toxicity of different surfactants to bone marrow derived DC cells (dendritic cells)
Cell culture and treatment method:
Acquisition of bone marrow derived DCs:
Taking a C57BL/6 mouse with the age of 6-8 weeks, dissecting to obtain long bones and tibia, blowing out bone marrow, treating with ACK 2ml for 1 minute to lyse red blood cells, neutralizing with a serum-free culture medium, centrifuging, and re-suspending to obtain a mouse bone marrow cell suspension, and planting the mouse bone marrow cell suspension in a 10cm culture dish with 3X 10 6 cells/dish. Complete medium (RPMI 1640 with 10% FBS, 50. Mu.M mercaptoethanol) was incubated with 20ng/ml recombinant mouse-derived GM-CSF (calculated on day 0). 10ml of complete medium (containing 20ng/ml of recombinant murine GM-CSF) was supplemented on day 3. Culturing until day 6, collecting non-adherent cells as non-mature bone marrow-derived DC cells. The purity of the DC cells of the CD11c + is over 90 percent after flow identification, and the method can be used for subsequent experiments.
Experimental protocol: cytotoxicity of different surfactants on bone marrow derived DC production
In order to break the lipid vesicle structure of tumor exosomes, a surfactant needs to be introduced. However, the surfactant (Table I) has the effect of destroying the cell membrane, so that the modified tumor exosomes are used for researching and applying the tumor personalized vaccine, and the primary condition of the vaccine is that the safety is needed. Therefore, we next examined whether these surfactants were able to cytotoxicity against bone marrow derived DC cells using MTT experiments.
Mouse bone marrow-derived DC cells were plated at 30 ten thousand per well in 96-well plates, and SDS, triton X-100, tween80, and PEG2000-DSPE (PP) solutions at various concentrations were added. After incubation at 37℃for 24h, 20. Mu.l of 5mg/ml MTT solution was added to the wells and incubation was continued for 6h. And adding MTT lysate, blowing and mixing uniformly, and placing into an enzyme-labeled instrument to detect the absorption values at 570nm and 630nm (reference wavelength).
The results are shown in FIG. 1: first, all doses of PEG2000-DSPE produced substantially no toxicity to DC cells, but SDS and Triton X-100 produced tremendous cytotoxicity to cells at 25 μg/ml. Tween80 started to be toxic to cell fluids at concentrations exceeding 50. Mu.g/ml.
The above results demonstrate that PEG2000-DSPE is the best choice from a safety standpoint. While Tween80 needs to be controlled below a safe dosage (50 mug/ml), SDS and TritonX-100 are not suitable for reconstruction of tumor exosomes due to too large cytotoxicity.
TABLE 1 basic information of different surfactants
Example two preparation and characterization of PP-engineered tumor exosomes
Cell culture and treatment method
Culture of a mouse colon cancer model MC38 cell line: DMEM medium (containing 10% fetal bovine serum) was used. Taking out MC38 tumor cell cryopreservation tube from liquid nitrogen preserving tank, immediately placing into 37 ℃ water bath for rapid dissolution, transferring cell suspension into a centrifuge barrel containing 10ml of culture medium, centrifuging for 5min at 350g, removing supernatant, re-suspending with fresh culture medium, transferring cells into cell culture bottle, adding 10-15ml of culture medium to suspend and precipitate cells, adjusting cell concentration, and culturing in 37 ℃ and 5% CO 2 saturated humidity incubator. During maintenance culture, the cell state was observed daily and fresh medium was replaced in time.
(1) Tumor inoculation: when the cells were grown to 90% confluency by adherence, they were digested with 0.05% pancreatin and subcultured at a ratio of 1:3. On the day of the experiment, cells with good growth state and confluence degree reaching 90% are digested with pancreatin, the pancreatin is neutralized by fresh culture medium, centrifuged at 350g, the supernatant is discarded, sterile PBS is added for resuspension, counting is carried out, and the density of the cell suspension is adjusted to 2.5X10 6/ml for standby. Each mouse was inoculated subcutaneously with 2.5X10 5 cells.
(2) Tumor Exosomes (EXO) experiments were collected: when the cells were grown to 90% confluency by adherence, they were digested with 0.05% pancreatin and subcultured at a ratio of 1:3. When in subculture, the culture medium containing 10% of exosome-free fetal bovine serum is used for continuous culture, and after 48 hours, the supernatant is collected and directly used for exosome extraction or frozen in a refrigerator at-80 ℃ for standby.
Acquisition of tumor Exosomes (EXO)
After culturing tumor cells in a medium containing 10% exosome-removed fetal bovine serum (serum remaining supernatant fraction after centrifugation at 100,000g for 2 hours) for 48 hours, cell culture supernatants were collected.
(1) Centrifugation at 4000rpm for 2 hours removed cell debris from the supernatant.
(2) The fraction of the supernatant above 100kDa was collected using a 100kDa ultrafiltration tube at 4000rpm for 30 minutes/time.
(3) The collected fractions were added to exosome extraction reagent (EXOTC A-1, SBI) at 5:1 (v: v) and left at 4℃for at least 12 hours after thorough mixing.
(4) After 12 hours, precipitation appears, and the precipitate is separated out after centrifugation at 3000rpm for 30 minutes, namely the crude extract exosome. After the PBS is used for resuspension, protein concentration is measured by a BCA method, the content of exosomes is calibrated, and the exosomes are packaged and stored at-80 ℃ for standby.
(5) Density gradient centrifugation: the crude exosomes were placed in 10%, 20%, 40%, 60% discontinuous density gradient sucrose solution, ultracentrifuged (100,000 g) for 2 hours, then white material at 10-20% layer was carefully aspirated, diluted with PBS and ultracentrifuged again to precipitate exosomes at the bottom, i.e. extract exosomes. After being resuspended by using a proper amount of PBS, the protein concentration is measured by a BCA method, and the content of exosomes is calibrated by the protein concentration, and the exosomes are stored at-80 ℃ for standby.
In the following sets of embodiments: except for electron microscopy experiments and dynamic light scattering experiments, the refined extract was used, and the crude extract was used for other experiments. Hereinafter collectively referred to as tumor Exosomes (EXO).
Preparation of Pegylated Phospholipid (PP) engineered tumor exosomes (exemplified by exosomes secreted by the mouse colon cancer MC38 cell line):
(1) Tumor exosomes that have been quantified by BCA protein method are diluted with sterile water to 500ug/ml tumor Exosome (EXO) stock;
(2) Dissolving the MPLA powder in a mixed solution of methanol/chloroform (1:2, v: v) to prepare an MPLA stock solution with a concentration of 1 mg/ml;
(3) Weighing 15mg of PP powder, adding 3ml of chloroform for dissolution, and preparing PP stock solution with the concentration of 5 mg/ml;
(4) 10ml of PP-engineered tumor exosomes (abbreviated as PP/MPLA/EXO) were prepared according to the recipe of Table 2-1, as follows:
a) Taking 1ml of PP stock solution and 50ul of MPLA stock solution, and uniformly mixing;
b) Drying by using nitrogen to remove the organic solvent;
c) Adding 1ml of EXO stock solution, and fully and uniformly mixing to obtain colorless and transparent PP modified tumor exosome solution;
d) Filtering and sterilizing by a 0.22um filter membrane.
Before use, the tumor exosome solution is diluted by sterile water for 10 times to prepare PP modified tumor exosome solution with total volume of 10ml and protein content of 50ug/ml EXO concentration.
Table 2-1: PP modified tumor exosome prescription
| PEG2000-DSPE stock solution concentration | Concentration of MPLA stock solution | Tumor Exosome (EXO) stock solution concentration |
| 5000ug/ml | 50ug/ml | 500ug/ml |
Preparation of a component-deleted control (exemplified by exosomes secreted by the mouse colon cancer MC38 cell line):
The whole PP-engineered tumor exosomes (PP/MPLA/EXO) consist of three components, and a component-deleted control is required in the functional experiments, including a control lacking MPLA and lacking Pegylated Phospholipids (PP).
(1) Preparation of a non-MPLA control (abbreviated as PP/EXO):
a) Tumor exosomes that have been quantified by BCA protein method are diluted with sterile water to 500ug/ml tumor Exosomes (EXO) stock;
b) Weighing 15mg of PP powder, adding 3ml of chloroform for dissolution, and preparing PP stock solution with the concentration of 5 mg/ml;
c) 10ml of a non-MPLA control (abbreviated as PP/EXO) was prepared according to the recipe of Table 2-2, and specific steps were referenced to the preparation of PP-modified tumor exosomes (PP/MPLA/EXO) described above.
Table 2-2: prescription of non-MPLA reference (PP/EXO for short)
| PP stock solution concentration | Tumor Exosome (EXO) stock solution concentration |
| 5000ug/ml | 500ug/ml |
Before use, the mixture is diluted by 10 times with sterile water to prepare a reference solution (abbreviated as PP/EXO) without MPLA, wherein the total volume of the reference solution is 10ml, and the protein content of the reference solution is 50ug/ml EXO concentration.
(2) Preparation of PP-free control (MPLA/EXO for short):
Dissolving the MPLA powder in a mixed solution of methanol/chloroform (1:2, v: v) to prepare an MPLA stock solution with a concentration of 1 mg/ml;
A10 ml PP-free control (abbreviated as MPLA/EXO) was prepared by physically mixing 50ul of the MPLA stock solution with 1ml of the EXO stock solution according to the recipe of tables 2-3.
Table 2-3: prescription of PP-free reference substance (MPLA/EXO for short)
| Concentration of MPLA stock solution | Tumor Exosome (EXO) stock solution concentration |
| 50ug/ml | 500ug/ml |
Before use, the solution is diluted by sterile water for 10 times to prepare a PP-free reference substance (called MPLA/EXO for short) solution with the total volume of 10ml and the protein content of 50ug/ml EXO concentration.
Experimental protocol (one) frozen Electron microscopy morphology characterization of PP-engineered tumor exosomes
(1) Preparing a PP modified tumor exosome according to the method;
(2) The experiments were divided into 3 groups, respectively:
a) Tumor exosome group (EXO)
B) PP modified tumor exosome group (PP/MPLA/EXO)
C) No MPLA control group (PP/EXO)
(3) Preparing a frozen electron microscope sample: 2.5 μl of the sample was loaded onto a carbon grid (Quantifoil 300 mesh, R1.2/1.3) pretreated for 30 seconds with H 2/O2 glow discharge. At 23 ℃ and 95% humidity, the excess sample was adsorbed with filter paper for 6 seconds under a level 2 force using a vitro Mark IV (Thermo FISHER SCIENTIFIC), then rapidly frozen in liquid ethane, transferred to liquid nitrogen for storage;
(4) The copper mesh was observed under a transmission electron microscope (Talos L120C 120 kV);
The experimental result shows that the average diameter of tumor exosomes is about 100nm, which accords with the exosome diameter distribution range (30-150 nm) reported in the literature. Whereas PP-engineered tumor exosomes were around 10-20nm in diameter (FIG. 2A), indicating that PP-engineered tumor exosomes became smaller and more uniform. Meanwhile, we found that the sample morphology between the PP/EXO group and the PP/MPLA/EXO group did not show a large difference, so MPLA did not affect the structural modification of tumor exosomes by pegylated phospholipids.
Experimental protocol (II) dynamic light Scattering results of PP-engineered tumor exosomes
(1) Preparing a PP modified tumor exosome according to the method;
(2) The experiments were divided into 2 groups, respectively:
a) Tumor exosome group (EXO)
B) No MPLA control group (PP/EXO)
(3) Each group of samples was diluted with water and placed in a nanoparticle potentiometer (Zetasizer Nano ZS) for detection.
(4) The experimental result shows that the grain size distribution of the tumor exosomes is in the range of 50-200nm, which is basically consistent with the exosome diameter range (30-150 nm) reported in the literature. In view of the fact that MPLA does not affect the structural modification of tumor exosomes by pegylated phospholipids, dynamic light scattering experiments were tested using only a non-MPLA control (PP/EXO), and the results showed that the average particle size distribution of PP/EXO groups was around 10nm (fig. 2B), further demonstrating the results of cryo-electron microscopy.
Experimental protocol (III) PP-engineered tumor exosome stability (4 ℃ C. For one month)
(1) Preparing a PP modified tumor exosome and a single-component default control group according to the method;
(2) The experiments were divided into 3 groups, respectively:
a) No MPLA control group (PP/EXO)
B) PP-free control group (MPLA/EXO)
C) PP modified tumor exosome (PP/MPLA/EXO)
(4) Placing each group of samples into a refrigerator at the temperature of 4 ℃ for preservation for one month, and photographing and observing before and after preservation;
(5) Experimental results show that after one month of placement, each group has no turbidity, which indicates that the tumor exosome prepared by the PP prepared by the prescription has basically stable property and can not deteriorate within one month under the preservation condition of 4 ℃.
Example III, PP-engineered tumor exosomes enhance the function of bone marrow-derived DC cells (dendritic cells)
Cell culture and treatment method:
Acquisition of bone marrow derived DC may be referred to in example one.
Acquisition of tumor Exosomes (EXO), PP-engineered tumor exosomes (MC 38 source) and corresponding controls were prepared as in example two.
Experimental protocol (one): PP engineered tumor exosomes promote the antigen presenting ability of DC cells
(1) Bone marrow-derived DC cells were treated with 50ug/ml tumor exosomes (quantified as BCA protein) and PP-engineered tumor exosomes, while 0.5mg/ml of full-length OVA protein in PBS was added, and after 24 hours, flow cytometry was performed using PE-labeled fluorescent antibodies against the mouse MHC I-SIINFEKL antigen complex (p-MHC I for short), and the intensity of this fluorescence was examined for DC cells in each treatment group to indicate the amount of antigen-MHC-I complex on the cell surface.
(2) The experiments were divided into 5 treatment groups, respectively:
a) Untreated group (CTR)
B) Tumor exosome treatment group (EXO)
C) No MPLA control group (PP/EXO)
D) PP-free control group (MPLA/EXO)
E) PP modified tumor exosome treatment group (PP/MPLA/EXO)
(3) The results show that:
tumor exosomes alone, no MPLA control, no PP control, similar to untreated, the p-MHC I levels were unchanged;
The p-MHC I levels of the PP-engineered tumor exosomes treated group were significantly elevated (fig. 3), demonstrating that PP-engineered tumor exosomes were able to enhance the processing and presentation capacity of DC cells for antigen.
Experimental protocol (two): PP engineered tumor exosomes increase expression levels of DC cell surface costimulatory molecules
(1) Bone marrow-derived DC cells were treated with tumor exosomes (quantified as BCA protein) containing 50ug/ml and PP-engineered tumor exosomes, and after 12 hours, the expression levels of mature molecular markers, CD80 and CD86, were detected on the DC cell surface using fluorescent antibodies.
(2) The experiments were divided into 5 treatment groups, respectively:
a) Untreated group (CTR)
B) Tumor exosome treatment group (EXO)
C) No MPLA control group (PP/EXO)
D) PP-free control group (MPLA/EXO)
E) PP modified tumor exosome treatment group (PP/MPLA/EXO)
(3) The results show that:
tumor exosomes alone, no MPLA control, no PP control, similar to the untreated group, the expression levels of CD80 and CD86 were not altered;
the PP-engineered tumor exosome treatment group can significantly raise the expression level of the two molecular markers (figure 4), which indicates that the PP-engineered tumor exosome can promote the maturation of DC cells and better perform immune functions.
Experimental protocol (iii): PP engineered tumor exosomes increase levels of DC cell-secreting cytokines
(1) Bone marrow-derived DC cells were treated with tumor exosomes (quantified as BCA protein) and PP-modified tumor exosomes containing 50ug/ml, and after 2 hours, 24 hours, cell culture supernatants were collected, and the secretion levels of TNF- α, IL-2, IL-12 and expression of interferon IFN- β by DC cells in each treatment group were examined by ELISA and q-RT-PCR methods, respectively.
(2) The experiments were divided into 5 treatment groups, respectively
A) Untreated group (CTR)
B) Tumor exosome treatment group (EXO)
C) No MPLA control group (PP/EXO)
D) PP-free control group (MPLA/EXO)
E) PP modified tumor exosome treatment group (PP/MPLA/EXO)
(3) The results show that:
The tumor exosomes alone, the MPLA-free control group and the PP-free control group, similar to the untreated group, did not have any change in the secretion levels of TNF- α, IL-2, IL-12. Only the PP-free control group showed slightly elevated levels of TNF- α secretion, possibly related to the actions of MPLA itself;
PP-engineered tumor exosomes treatment groups were able to significantly elevate levels of TNF- α, IL-2, IL-12 secretion by DC cells (FIG. 5). Meanwhile, the expression trend of the interferon type I IFN-beta is the same. These results demonstrate that PP-engineered tumor exosomes can promote DC cells to secrete a large amount of cytokines that activate anti-tumor immune responses, helping DC cells to activate downstream T cells.
Example IV, PP engineering tumor exosomes deliver their contents to the Endoplasmic Reticulum (ER) of DC cells
Cell culture and treatment method:
acquisition of tumor Exosomes (EXO) derived from MC38 cell lines and preparation of PP-engineered tumor exosomes and corresponding controls were as in example two.
Culture of DC2.4 cells: RPMI1640 medium (containing 10% fetal bovine serum, 1 XL-glutamine, 1 Xnon-essential amino acid solution, 1 XHEPES buffer and 5uM beta-mercaptoethanol) was used. Taking out the tumor cell cryopreservation tube from the liquid nitrogen preservation tank, immediately putting into a water bath at 37 ℃ for rapid dissolution, transferring the cell suspension into a centrifugal barrel containing 10ml of culture medium, centrifuging for 5 minutes at 350g, removing the supernatant, re-suspending with fresh culture medium, transferring the cells into a cell culture bottle, adding 10-15ml of culture medium to suspend and precipitate the cells, adjusting the cell concentration, and culturing in a culture box with saturated humidity of 5% CO 2 in volume fraction at 37 ℃. During maintenance culture, the cell state was observed daily and fresh medium was replaced in time.
Experimental protocol (one): examination of the pathway of PP-modified tumor exosomes after entry into DC2.4 cells by means of labeling the exosome membrane
(1) PKH67 marks the membrane components of tumor exosomes: 8 tubes of freshly extracted tumor exosomes (quantified as BCA, each tube containing approximately 9mg of tumor exosomes) were taken, and each EP tube was added with 350ul of the dilution prepared with the kit, gently swirled to form a solution. Then, under light-shielding conditions, 8 EP tubes with 350ul of diluent were prepared, and 1.5ul of PKH67 dye was added to each tube and mixed well. Then, the dilution containing PKH67 dye is added into an EP tube containing tumor exosome solution according to the volume ratio of 1:1, and the mixture is gently beaten and mixed. Dyeing at room temperature (25 ℃) for 5 minutes. 400ul of exosome-free fetal bovine serum was added and neutralized for 1 minute. The stained exosomes were then transferred to a new BECKMAN 1.5 ml tube (# 357448). After balancing the 8 EP tubes, the supernatant was discarded after centrifugation at 100,000g for 25 min at room temperature (25 ℃) and after washing once with 1ml PBS, centrifuged again (conditions were kept consistent with before) and the supernatant was discarded. 100ul of sterile water was added to each 2 EP tubes to solubilize the tumor exosome solution (again quantified by BCA, about 12mg, i.e., solution concentration 120 mg/ml) into 1 tube. The PKH67 labeled tumor exosome solution was stored in dark at 4 ℃.
(2) Preparation of PKH 67-labeled PP-engineered tumor exosomes: the formulation according to example 1 is distinguished in that the exosomes used are PKH67 labelled exosomes in step (1) described in this example;
(3) Treatment of DC2.4 cells: DC2.4 cells were seeded at 1X 10 5/well in 15mm confocal microscopy cell culture dishes overnight for cell attachment. The following day, DC2.4 cells were treated with either formulated PKH 67-labeled tumor exosomes or PP-engineered tumor exosomes, after which 100nM Lyso-Tracker-Red (37 ℃,10 min) or 1uM ER-Tracker-Red (37 ℃,30 min) dye was added to stain the lysosomes and endoplasmic reticulum of the cells, respectively, according to the set time, and the final exosome treatment time was maintained at 1 hour. Before the machine is started, the culture medium is changed into a culture medium without fluorescent dye, and then a fluorescent confocal microscope is used for observation;
(4) The experiments were divided into 4 groups, respectively:
a) Tumor exosomes (green) and lysosomes (red) co-localization sets
B) Tumor exosomes (green) and endoplasmic reticulum (red) co-localization group
C) PP engineered tumor exosomes (green) and lysosomes (red) co-localization groups
D) PP-engineered tumor exosomes (green) and endoplasmic reticulum (red) co-localization sets
(5) The experimental results show that:
After 1 hour treatment, tumor exosomes were co-localized mainly with lysosomes, whereas PP-engineered tumor exosomes were co-localized mainly with endoplasmic reticulum (fig. 6A), primarily demonstrating that PP-engineering can alter intracellular destinations of tumor exosomes. However, given that PKH67 marks the membrane structure of tumor exosomes, it is not possible to determine that the contents of PP-engineered tumor exosomes (in particular antigens) are indeed able to reach the endoplasmic reticulum. To solve this problem we further used CFSE dyes capable of labeling proteins to label the contents of tumor exosomes, still observing their co-localization with lysosomes and endoplasmic reticulum by fluorescence confocal microscopy.
Experimental protocol (two): method for marking exosome protein to examine path of PP modified tumor exosome after entering DC2.4 cell
(1) CFSE marks the protein content of tumor exosomes: a20 mg stock solution of tumor exosomes (quantified on BCA protein) was aspirated into a 1.5ml EP tube, adjusted to a volume of 500ul by adding PBS, and stained at room temperature for 10min with 2ul of 10mM CFSE stock solution (to a final concentration of 4 uM). After that, 500ul of PBS containing 20% FBS was added for neutralization reaction, transferred to an ultracentrifuge tube, and centrifuged at 100,000g at 4℃for 30min. The supernatant was discarded, the precipitate was washed once with PBS, resuspended in an appropriate amount of PBS, and the BCA was quantified and stored at-80℃until use.
(2) Preparation of CFSE-labeled PP-engineered tumor exosomes: the formulation according to example 1 is distinguished in that the exosomes used are CFSE-labeled exosomes in step (1) described in this example;
(3) Treatment of DC2.4 cells: DC2.4 cells were seeded at 1X 10 5/well in 15mm confocal microscopy cell culture dishes overnight for cell attachment. The following day, DC2.4 cells were treated with either formulated PKH 67-labeled tumor exosomes or PP-engineered tumor exosomes, after which 100nM Lyso-Tracker-Red (37 ℃,10 min) or 1uM ER-Tracker-Red (37 ℃,30 min) dye was added to stain the lysosomes and endoplasmic reticulum of the cells, respectively, according to the set time, and the final exosome treatment time was maintained at 1 hour. Before the machine is started, the culture medium is changed into a culture medium without fluorescent dye, and then a fluorescent confocal microscope is used for observation;
(4) The experiments were divided into 4 groups, respectively:
a) Tumor exosomes (green) and lysosomes (red) co-localization sets
B) Tumor exosomes (green) and endoplasmic reticulum (red) co-localization group
C) PP engineered tumor exosomes (green) and lysosomes (red) co-localization groups
D) PP-engineered tumor exosomes (green) and endoplasmic reticulum (red) co-localization sets
(5) The experimental results show that:
After 1 hour treatment, CFSE-labeled tumor exosomes were mainly co-localized with lysosomes, similar to PKH67 labeling, whereas PP-engineered CFSE-labeled tumor exosomes were mainly co-localized with the endoplasmic reticulum (fig. 6B). It fully shows that protein molecules (especially antigen molecules carried by the protein molecules) contained in tumor exosomes can reach the endoplasmic reticulum after PP transformation, and provides preconditions for MHCI type presentation of tumor specific antigens.
Example five, PP engineered tumor exosomes enhance activation of bone marrow derived DCs on antigen-specific OTI CD8 + T cells
Cell culture and treatment method:
acquisition of bone marrow-derived DC, acquisition of MC38 cell line-derived tumor Exosomes (EXO), preparation of PP-engineered tumor exosomes and corresponding control, reference example two and example three.
Acquisition of OT I CD8 + T cells: after the OT I transgenic mice were sacrificed, spleen cells were taken, red blood cells were lysed, and CD8 + T cells were isolated using CD8 magnetic beads (Invitrogen).
Experimental protocol:
(1) Bone marrow-derived DC cells were treated with tumor exosomes (quantified as BCA protein) containing 50ug/ml and PP-engineered tumor exosomes, and after 24 hours, 0.5mg/ml of OVA full-length protein PBS solution was added. After 24 hours more, DC cells from each treatment group were collected and counted to 1 with CFSE stained OT I CD8 + T cells: 8, and detecting proliferation level of T cells after 72 hours (CFSE dilution).
(2) The experiments are divided into 5 groups respectively
A) Untreated group (CTR)
B) Tumor exosome treatment group (EXO)
C) No MPLA control group (PP/EXO)
D) PP-free control group (MPLA/EXO)
E) PP modified tumor exosome treatment group (PP/MPLA/EXO)
(3) The experimental result shows that the method has the advantages of high yield,
DC cells in the untreated group can well activate OTI CD8 + T cells, and the independent tumor exosome treated group can remarkably inhibit the activation effect.
Compared to the MPLA-free control group and the PP-free control group, the PP-engineered tumor exosome treatment group did not inhibit the activation ability of DCs on T cells, whereas there was some promotion (fig. 7).
Example six tumor exosomes engineered with PP can elicit homologous tumor antigen-specific CTL responses
Cell culture and treatment method:
acquisition of tumor Exosomes (EXO), preparation of PP-engineered tumor exosomes and corresponding controls reference example two.
Culture of a mouse colon cancer model MC38 cell line and a mouse melanoma B16F10 cell line: DMEM medium (containing 10% fetal bovine serum) was used. Taking out the tumor cell cryopreservation tube from the liquid nitrogen preservation tank, immediately putting into a 37 ℃ water bath for rapid dissolution, transferring the cell suspension into a centrifugal barrel containing 10ml of culture medium, centrifuging for 5 minutes at 350g, removing the supernatant, re-suspending with fresh culture medium, transferring the cells into a cell culture bottle, adding 10-15ml of culture medium to suspend and precipitate the cells, adjusting the cell concentration, and culturing in a 37 ℃ and 5% CO 2 saturated humidity incubator. During maintenance culture, the cell state was observed daily and fresh medium was replaced in time.
Culture of mouse cervical cancer TC-1 cell line and mouse triple negative breast cancer 4T1 cell line: RPMI1640 medium (10% fetal bovine serum) was used. Taking out the tumor cell cryopreservation tube from the liquid nitrogen preservation tank, immediately putting into a 37 ℃ water bath for rapid dissolution, transferring the cell suspension into a centrifugal barrel containing 10ml culture medium, centrifuging for 5 minutes by 350g, removing the supernatant, re-suspending by using fresh culture medium, transferring the cells into a cell culture bottle, adding 10-15ml of culture medium to suspend the precipitated cells, adjusting the cell concentration, and culturing in a 37 ℃ and 5% CO 2 saturated humidity culture box. During maintenance culture, the cell state was observed daily and fresh medium was replaced in time.
Experimental protocol (one): PP-engineered tumor exosomes are capable of eliciting homologous tumor antigen-specific CTL responses (MC 38 cell lines)
(1) Wild mice were immunized by tail root injection with tumor exosomes derived from MC38 or PP-engineered tumor exosomes, respectively, which were able to elicit a homeotumor antigen-specific CTL response. On day 7 after the immunization, mice were sacrificed, proximal lymph nodes were surgically removed, and all immune cells in the lymph nodes were obtained after grinding and digestion.
In vitro treatment: the anti-mouse IFN-gamma antibody was pre-incubated in Ellispot plates the day before isolation of the mouse lymph node cells at an effect concentration of 5 μg/ml overnight at 4 ℃. The following day the antibodies were discarded and blocked for 2 hours at room temperature by adding blocking solution (RPMI 1640 medium containing 10% fetal calf serum, 1% penicillin streptomycin and beta-mercaptoethanol). The isolated mouse lymph node cells were then added to the plate at 3X 10 5 wells per well and incubated with 20. Mu.g/ml MC38 specific antigen peptide (including: rpl-18, reps-1 and Adpgk)(Identification of a neo-epitope dominating endogenous CD8 T cell responses to MC-38colorectal cancer.9,1673125(2019);published online EpubOct 13 (10.1080/2162402x.2019.1673125)),37℃ incubator for 48 hours. 48 hours post-incubation broth, lysed with deionized water for 5 minutes/time for 2 times. Washing 3 times with PBST (1 XPBS containing 0.05% Tween-20) solution followed by biotin-labeled anti-mouse IFN-gamma antibody, incubated 2 hours at room temperature. PBST solution for 4 times, followed by streptavidin-labeled horseradish peroxidase (strepitavidin-HRP), incubated 1 hour at room temperature. Finally 1 XPBS was washed 3 times and then with AEC substrate for 5-60 minutes, the reaction was stopped with deionized water and the ELISPot plate was blow dried after continued washing.
(2) The experiments were divided into 4 groups, respectively:
a) Tumor exosome treatment group (EXO)
B) No MPLA control group (PP/EXO)
C) PP-free control group (MPLA/EXO)
D) PP modified tumor exosome treatment group (PP/MPLA/EXO)
(3) The detection method comprises the following steps: ELISPot results were scanned and counted by a luciferase spot analyzer CTL analyzer LLC.
The results showed that, compared to the tumor exosome-treated group,
Neither the MPLA-free group nor the PP-free control group were able to increase the antigen-specific CTL response, and a higher proportion of antigen-specific CTL responses (embodied as stronger IFN- γ signals) were generated in the lymph node cells after PP-engineered tumor exosome immunization (fig. 8).
Experimental protocol (two): PP-engineered tumor exosomes can elicit homologous tumor antigen-specific CTL responses (B16F 10 cell lines)
(1) Wild mice were immunized by tail root injection with tumor exosomes derived from B16F10 or PP-engineered tumor exosomes, respectively, which were able to elicit homologous tumor antigen-specific CTL response-engineered tumor exosomes. On day 7 after the immunization, mice were sacrificed, proximal lymph nodes were surgically removed, and all immune cells in the lymph nodes were obtained after grinding and digestion.
In vitro treatment: the experimental scheme (I) of this example was the same. Wherein 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 EpubMar 1(10.1158/0008-5472.can-11-3722)).
(2) The experiments were divided into 2 groups, respectively:
a) Tumor exosome treatment group (EXO)
B) PP modified tumor exosome treatment group (PP/MPLA/EXO)
The results showed that a higher proportion of antigen-specific CTL responses were generated in PP-engineered tumor exosomes immunized lymph node cells compared to tumor exosome treated groups (fig. 9).
Experimental protocol (iii): PP-engineered tumor exosomes are capable of eliciting a cognate tumor antigen-specific CTL response (cell line 4T 1)
(1) Wild mice were immunized by tail root injection with tumor exosomes derived from 4T1 or PP-engineered tumor exosomes, respectively. On day 7 after the immunization, mice were sacrificed, proximal lymph nodes were surgically removed, and all immune cells in the lymph nodes were obtained after grinding and digestion.
In vitro treatment: the experimental scheme (I) of this example was the same. Wherein the 4T 1-specific antigen peptides used for incubation with lymph node cells are Sptbn and Wdr33(Self-healing microcapsules synergetically modulate immunization microenvironments for potent cancer vaccination.6,eaay7735(2020);published online EpubMay(10.1126/sciadv.aay7735)).
(2) The experiments were divided into 4 groups, respectively:
a) Tumor exosome treatment group (EXO)
B) No MPLA control group (PP/EXO)
C) PP-free control group (MPLA/EXO)
D) PP modified tumor exosome treatment group (PP/MPLA/EXO)
The results showed that, compared to the tumor exosome-treated group,
Neither the MPLA-free control nor the PP-free control were able to increase the antigen-specific CTL response, producing a higher proportion of antigen-specific CTL responses (embodied as stronger IFN- γ signals) in the lymph node cells following PP-engineered tumor exosome immunization (fig. 10).
Experimental protocol (four): PP-engineered tumor exosomes are capable of eliciting a cognate tumor antigen-specific CTL response (cell line TC-1)
(1) Wild mice were immunized by tail root injection with tumor exosomes derived from TC-1 or PP-engineered tumor exosomes, respectively. On day 7 after the immunization injection, mice were sacrificed, proximal lymph nodes were surgically removed, and all immune cells in the lymph nodes were obtained after grinding and digestion.
In vitro treatment: refer to experimental scheme (one) of this example. Wherein 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) The experiments were divided into 4 groups, respectively:
a) Tumor exosome treatment group (EXO)
B) No MPLA control group (PP/EXO)
C) PP-free control group (MPLA/EXO)
D) PP modified tumor exosome treatment group (PP/MPLA/EXO)
The results show that, compared with the tumor exosome immune group,
Neither the MPLA-free control nor the PP-free control were able to increase the antigen-specific CTL response, and the PP-engineered tumor exosome immunized lymph node cells were able to produce a higher proportion of antigen-specific CTL responses (as embodied by a stronger IFN- γ signal) (fig. 11).
Example seven PP engineered tumor exosomes inhibit tumor growth and extend survival of tumor-bearing mice
Cell culture and treatment method:
cell culture methods refer to example four, acquisition of tumor Exosomes (EXOs) and preparation of PP-engineered tumor exosomes and corresponding controls refer to example two.
Experimental protocol (one): PP-engineered tumor exosomes (derived from the mouse colon cancer cell line MC 38) inhibit tumor growth
(1) Construction of MC38 model: when the cells were grown to 90% confluency by adherence, they were digested with 0.05% pancreatin and subcultured at a ratio of 1:3. On the day of the experiment, cells with good growth state and confluence degree reaching 90% are digested with pancreatin, the pancreatin is neutralized by fresh culture medium, centrifuged at 350 g, the supernatant is discarded, sterile PBS is added for resuspension, and the cell suspension density is adjusted to 2.5X10 6/ml. Female wild-type C57BL/6 mice were inoculated subcutaneously using a 1ml sterile syringe, 0.1ml per mouse (i.e., 2.5X10 5 MC38 cells per mouse). Tumor formation of about 30mm 3 was seen on about 4 days, and 50ug/ml tumor exosomes (based on BCA protein quantification) were subcutaneously injected near the tumor on the same day or tumor exosomes containing an equal mass of PP-engineered were treated three times a week. Thereafter, the status of the mice was observed at all times, during which tumor volume changes and death of tumor-bearing mice were recorded.
(2) The experimental groups are 5 groups respectively
A) PBS solvent control group (CTR)
B) Tumor exosome treatment group (EXO)
C) No MPLA control group (PP/EXO)
D) PP-free control group (MPLA/EXO)
E) PP modified tumor exosome treatment group (PP/MPLA/EXO)
Experimental results showed that neither the MPLA-free control nor the PP-free control was able to effectively inhibit tumor growth compared to the PBS solvent control, PP-engineered tumor exosomes were able to significantly delay tumor growth and extend survival of tumor-bearing mice (fig. 12).
Experimental protocol (two): PP-engineered tumor exosomes (derived from the mouse melanoma cell line B16F 10) inhibit tumor growth
(1) Construction of B16F10 model: when the cells were grown to 90% confluency by adherence, subcultured with 0.05% pancreatin at a ratio of 1:4. On the day of the experiment, cells with good growth state and confluence degree reaching 90% are digested with pancreatin, the pancreatin is neutralized by fresh culture medium, centrifuged at 350g, the supernatant is discarded, sterile PBS is added for resuspension, and the cell suspension density is adjusted to 2.5X10 6/ml. Female wild-type C57BL/6 mice were inoculated subcutaneously using a 1ml sterile syringe, 0.1ml per mouse (i.e., 2.5X10 5 B16F10 cells per mouse). Note that the cells were mixed well before each aspiration of the cell suspension. Tumor formation of about 30mm 3 was seen on about 4 days, and 50ug/ml tumor exosomes (based on BCA protein quantification) were subcutaneously injected near the tumor on the same day or tumor exosomes containing an equal mass of PP-engineered were treated three times a week. Thereafter, the status of the mice was observed at all times, during which tumor volume changes and death of tumor-bearing mice were recorded.
(2) The experimental groups were 3 groups, respectively
A) PBS solvent control group (CTR)
B) Tumor exosome treatment group (EXO)
C) PP modified tumor exosome treatment group (PP/MPLA/EXO)
Experimental results showed that the tumor exosome treated group was not able to effectively inhibit tumor growth compared to the PBS solvent control group, PP-engineered tumor exosome was able to significantly delay tumor growth and extend survival of tumor-bearing mice (fig. 13).
Experimental protocol (iii): PP-engineered tumor exosomes (derived from the mouse cervical cancer cell line TC-1) inhibit tumor growth
(1) Construction of TC-1 model: when cells were grown to 90% confluency by adherence, subcultured with 0.05% pancreatin at a ratio of 1:4. On the day of the experiment, cells with good growth state and confluence degree reaching 90% are digested with pancreatin, the pancreatin is neutralized by fresh culture medium, centrifuged at 350 g, the supernatant is discarded, sterile PBS is added for resuspension, and the density of the cell suspension is adjusted to 5X 10 5/ml. Female wild-type C57BL/6 mice were inoculated subcutaneously using a 1ml sterile syringe, 0.1ml per mouse (i.e., 5X 10 4 TC-1 cells per mouse). Note that the cells were mixed well before each aspiration of the cell suspension. About 30mm3 of tumor formation was seen on about 6 days, and 50ug/ml tumor exosomes (based on BCA protein quantification) were subcutaneously injected near the tumor on the same day or tumor exosomes containing an equal mass of PP-engineered were treated three times per week. Thereafter, the status of the mice was observed at all times, during which tumor volume changes and death of tumor-bearing mice were recorded.
(2) The experimental groups are 5 groups respectively
A) PBS solvent control group (CTR)
B) Tumor exosome treatment group (EXO)
C) No MPLA control group (PP/EXO)
D) PP-free control group (MPLA/EXO)
E) PP modified tumor exosome treatment group (PP/MPLA/EXO)
Experimental results showed that neither the MPLA-free control group nor the PP-free control group was able to effectively inhibit tumor growth compared to the PBS solvent control group, and PP-engineered tumor exosomes were able to significantly delay tumor growth (fig. 14).
EXAMPLE eight PP-engineered tumor exosomes inhibit lung metastasis of melanoma
Cell culture and treatment method:
Cell culture methods refer to example four; acquisition of tumor Exosomes (EXO) and preparation of PP-engineered tumor exosomes and corresponding controls reference example two.
Experimental protocol: PP-engineered tumor exosomes (derived from the mouse melanoma cell line B16F 10) inhibit lung metastasis of tumors
(1) Preparation of PP-engineered tumor exosomes (derived from the mouse melanoma cell line B16F 10) were prepared by reference to the preparation method in example two above. Prior to tumor inoculation, each female wild type C57BL/6 mouse received PBS (as a solvent control), 100ug (calculated as BCA protein quantification) exosomes or PP-engineered tumor exosomes by tail vein injection at a time. Once a week for three weeks.
(2) Construction of a murine melanoma B16F10 lung metastasis model: B16F10 cells were grown in culture, digested with 0.05% pancreatin to 90% confluency, and subcultured at a ratio of 1:4. On the day of the experiment, cells with good growth state and confluence degree reaching 90% are digested with pancreatin, the pancreatin is neutralized by fresh culture medium, centrifuged at 350g, the supernatant is discarded, sterile PBS is added for resuspension, and the density of the cell suspension is adjusted to 5X 10 4/ml. Female wild-type C57BL/6 mice immunized with exosomes or PP-engineered tumor exosomes were injected tail-vein by means of a 1ml sterile syringe, 0.1ml per mouse (i.e.5X 10 4 B16F10 cells per mouse).
(3) The experiments were divided into 3 groups, respectively:
a) PBS solvent control group (CTR)
B) Tumor exosome treatment group (EXO)
C) PP modified tumor exosome (PP/MPLA/EXO)
The results show that both organ photographs of lung metastasis and statistics of the number of lung melanoma metastasis nodules indicate that tumor exosomes have the function of promoting metastasis of melanoma to lung tissue in comparison to the solvent control group 13 days after tumor inoculation, while PP-engineered tumor exosomes successfully inhibit lung metastasis of melanoma (fig. 15). Meanwhile, according to the result, it can be reasonably deduced that exosomes secreted by the melanoma of the mice are easily enriched in the lungs of the mice, so that lung tissues are converted into an immunosuppressive microenvironment, and the colonization of melanoma cells in the circulatory system is promoted to form metastasis. Whereas PP-engineered tumor exosomes are likely to alter the immunosuppressive microenvironment, enhancing anti-tumor immune response, and thus reducing metastasis of melanoma.
Example nine PP-engineered tumor exosomes improve the immune microenvironment of melanoma
Cell culture and treatment method:
Cell culture methods refer to example four; acquisition of tumor Exosomes (EXO) and preparation of PP-engineered tumor exosomes and corresponding controls reference example two.
Experimental protocol: PP-modified tumor exosomes improve the immune microenvironment of melanoma
(1) Construction of B16F10 model: when the cells were grown to 90% confluency by adherence, subcultured with 0.05% pancreatin at a ratio of 1:4. On the day of the experiment, cells with good growth state and confluence degree reaching 90% are digested with pancreatin, the pancreatin is neutralized by fresh culture medium, centrifuged at 350g, the supernatant is discarded, sterile PBS is added for resuspension, and the cell suspension density is adjusted to 2.5X10 6/ml. Female wild-type C57BL/6 mice were inoculated subcutaneously using a 1ml sterile syringe, 0.1ml per mouse (i.e., 2.5X10 5 B16F10 cells per mouse). Note that the cells were mixed well before each aspiration of the cell suspension. Tumor formation of about 30mm 3 was seen on about 4 days, and 50ug/ml tumor exosomes (based on BCA protein quantification) were subcutaneously injected near the tumor on the same day or tumor exosomes containing an equal mass of PP-engineered were treated three times a week. Thereafter, the status of the mice was observed at all times, during which tumor volume changes and death of tumor-bearing mice were recorded (fig. 16A).
(2) The experimental groups are 5 groups respectively
A) PBS solvent control group (CTR)
B) Tumor exosome treatment group (EXO)
C) No MPLA control group (PP/EXO)
D) PP-free control group (MPLA/EXO)
E) PP modified tumor exosome treatment group (PP/MPLA/EXO)
The experimental results show that, compared with the PBS solvent control group,
Tumor exosome treatment, neither the MPLA control nor the PP control were effective in inhibiting tumor growth,
PP-engineered tumor exosomes were able to significantly delay tumor growth (fig. 16B and C).
In addition, compared with the PBS solvent control group, the flow type detection of various immune cell surface markers in tumor tissues shows that,
Tumor exosomes, no MPLA control and no PP control had no significant changes to the total immune cell fraction (CD 45 + cell fraction) (fig. 16D), CD8 + T cell fraction (fig. 16E), CD4 + T cell fraction (fig. 16F), DC cell fraction (fig. 16G), macrophage fraction (fig. 16H) and NK cell fraction (fig. 16I) in tumor tissue,
The PP-engineered tumor exosomes were able to significantly increase the duty cycle of the various types of immune cells described above (FIGS. 16D-I), indicating that the immunosuppressive microenvironment of the tumor tissue was improved, providing a prerequisite for the functioning of immunotherapy.
The activity and the function index of the CD8 + T cells with tumor killing capacity are inspected by an intracellular cytokine staining method, compared with a PBS solvent control group,
Tumor exosomes, no MPLA control and no PP control treatment had a certain enhancement effect on Ki67 (characterizing cell proliferation activity) in CD8 + T cells (FIG. 16J), IFN-gamma and Granzyme B (characterizing tumor killing ability of T cells) (FIGS. 16K and L) in tumor tissues,
PP-engineered tumor exosomes were able to significantly increase the proliferative capacity and tumor killing capacity of CD8 + T cells (fig. 16J-L).
In conclusion, the PP modified tumor exosome can obviously increase the infiltration of various types of immune cells, especially CD8 + T cells with proliferation and tumor killing activity, so as to inhibit the growth of tumor cells and achieve the purpose of treating tumors.
Example ten, PP-engineered tumor exosome mimetic personalized tumor therapy
Cell culture and treatment method:
Cell culture methods refer to example four; acquisition of tumor Exosomes (EXO) and preparation of PP-engineered tumor exosomes reference example two.
Experimental protocol: PP-engineered tumor exosomes (derived from the mouse colon cancer cell line MC 38) mimic personalized tumor therapy
(1) Construction of MC38 model: when the cells were grown to 90% confluency by adherence, they were digested with 0.05% pancreatin and subcultured at a ratio of 1:3. On the day of the experiment, cells with good growth state and confluence degree reaching 90% are digested with pancreatin, the pancreatin is neutralized by fresh culture medium, centrifuged at 350 g, the supernatant is discarded, sterile PBS is added for resuspension, and the cell suspension density is adjusted to 2.5X10 6/ml. Female wild-type C57BL/6 mice were inoculated subcutaneously using a 1ml sterile syringe, 0.1ml per mouse (i.e., 2.5X10 5 MC38 cells per mouse).
(2) And (3) after the Tumor grows to about 200mm 3, removing Tumor tissues by operation, separating Tumor cells in the tissues by using a Tumor cell separation kit, and continuing culturing.
(3) After passage of tumor cells for a certain number of passages, the tumor cell culture supernatant was collected, and tumor exosomes were extracted therefrom. The same lot of tumor cells were used simultaneously for new tumor model establishment (specific protocol is described in experimental protocol (1) of this example).
(4) And (3) preparing PP modified exosomes by using exosomes extracted from the original MC38 cell line and exosomes derived from tumor tissues extracted in the step (3) respectively 4 days after inoculating the new tumor, and treating the new tumor model. (for details, see FIG. 17A)
(5) The experimental groups were 3 groups, respectively
A) PBS solvent control group (CTR)
B) PP-engineered MC38 cell line exosome treatment group (PP/MPLA/EXO-MC 38)
C) PP modified Tumor cell exosome treatment group (PP/MPLA/EXO-Tumor)
The experimental results show that, compared with the PBS solvent control group,
PP-engineered exosomes show the ability to inhibit tumor growth, whether derived from subcultured cell lines or tumor cells in situ isolated tumor tissue (fig. 17B). Furthermore, compared with the original exosomes derived from the MC38 cell line, the tumor cell exosomes in the PP-modified in-situ separated tumor tissue have better tumor growth inhibition effect.
Non-synonymous mutations in genes within tumor cells will express variant proteins, which are captured by the immune system and recognized as foreign and thus can serve as a source of tumor-specific antigens. It is also notable that the probability of producing tumor-specific antigen and the probability of mutation of tumor cells are approximately correlated to form .(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 belonging to microsatellite-unstable carcinoma species, the genome of which has high mutation characteristics, and under a certain selection pressure, the characteristics can change the antigen spectrum produced by the cells (Targeting immune checkpoints potentiates immunoediting and changes the dynamics of tumor evolution).
In this example, mice with complete immunity will also monitor the immunity of the growing tumor cells, killing the tumor cells that they can recognize, while the surviving tumor cells become immune-killing resistant due to the genetic mutation, a process also known as immune editing. Under the combined action of genomic mutation and immune editing pressure, it is believed that the mouse subcutaneous primary MC38 tumor has developed some new antigens caused by the genetic mutation during growth. Thus, the possibility exists of producing a neoantigen in "new" tumor cells formed by sorting, subculturing from the tumor tissue. Based on this, it can be reasonably inferred that the new tumor cell exosomes and the original MC38 exosomes further carry the new antigens obtained by the gene mutation on the premise of sharing most of the same antigens. Therefore, the MC38 exosomes modified by PP still have a certain tumor inhibition effect, and the tumor cell exosomes modified by PP have a better tumor growth inhibition effect.
In conclusion, the invention utilizes PP molecules to carry out structural transformation on tumor exosomes, so that the natural attribute of original exosome immunosuppression is broken while original tumor-related antigens and tumor-specific antigens are maintained. The tumor exosome after PP modification has the capability of inducing antigen-specific CD8+ T cell reaction, effectively reduces the tumor load of tumor-bearing mice and prolongs the survival period.
Finally, it should be noted that the above embodiments are only for helping the person skilled in the art to understand the essence of the present invention, and are not intended to limit the protection scope of the present invention.
Claims (13)
1. An engineered tumor exosome formulation, said formulation comprising:
(1) A phosphatidylethanolamine-polyethylene glycol (PEG-DSPE) component at a concentration greater than 50 ug/ml;
(2) Tumor exosome fraction with protein concentration greater than 10 ug/ml;
(3) An effective amount of an immunoadjuvant molecule for stimulating an immune response.
2. The modified tumor exosome formulation of claim 1, wherein,
The purity of the PEG-DSPE is more than or equal to 95%, and the dispersity of PEG chains is less than or equal to 1.1;
Preferably, the chain length of PEG in the PEG-DSPE molecule is 1000-5000 Da; more preferably, the chain length of PEG in the PEG-DSPE molecule is 1500-2500 Da; most preferably, the PEG chain length of the PEG-DSPE molecule is 2000Da (PEG 2000-DSPE).
3. The engineered tumor exosome formulation according to claim 1 or 2, wherein the protein concentration refers to the protein concentration contained in all tumor exosomes in the formulation, preferably the protein concentration of tumor exosomes is quantified using BCA method.
4. The engineered tumor exosome formulation of any one of claims 1-3, wherein the immunoadjuvant molecule includes, but is not limited to, MPLA, QS21, polyI:c; preferably MPLA;
Preferably, the amount of said immunoadjuvant molecule is between 0.1 and 10ug/ml.
5. The modified tumor exosome formulation according to any one of claims 1-4, wherein in the modified tumor exosome formulation,
The mass ratio of the three components of the PEG-DSPE, the immunoadjuvant molecule and the total protein in the tumor exosome is as follows: 100:0.1 to 5:2 to 50 percent;
Preferably, the mass ratio of the three components of PEG-DSPE, the immunoadjuvant molecule and the total protein in the tumor exosomes is as follows: 100:0.5 to 2:5 to 20.
6. The modified tumor exosome formulation according to any one of claims 1-5,
The tumor exosome is derived from a tumor system cultured in vitro or primary tumor cells separated from tumor tissues;
The tumor exosomes are prepared using density gradient centrifugation, differential centrifugation, size exclusion, immunoseparation, polymer precipitation methods, or using commercial kits.
7. A method of preparing the engineered tumor exosome formulation of any one of claims 1-6, comprising the steps of:
(1) Uniformly mixing PEG-DSPE and immune adjuvant molecules, and removing a solvent to prepare a PEG-DSPE and immune adjuvant mixture;
(2) And adding a well-quantified tumor exosome solution into the mixture, and fully and uniformly mixing at normal temperature to obtain the modified tumor exosome preparation.
8. The method of claim 7, further comprising,
(3) And (3) performing degerming and filtering on the modified tumor exosome preparation.
9. The method according to claim 7 or 8, wherein,
The method for preparing the PEG-DSPE and immunoadjuvant mixture without solvent component in the step (1) comprises the steps of dissolving PEG-DSPE and immunoadjuvant molecules in an organic solvent, uniformly mixing, and then removing the organic solvent;
Preferably, the organic solvent is removed at a temperature below 70 ℃ using reduced pressure distillation under the protection of inert gas.
10. Use of the modified tumor exosome formulation of any one of claims 1-6 in the preparation of an anti-tumor medicament.
11. Use of the engineered tumor exosome formulation of any one of claims 1-6 in the preparation of an individualized anti-tumor formulation; preferably, in the personalized anti-tumor formulation, the tumor exosomes are derived from autologous tumor tissue or tumor cells of the patient in need of personalized treatment.
12. A medicament or pharmaceutical composition comprising the modified tumor exosome formulation of any one of claims 1-6.
13. A personalized medicine formulation comprising the modified tumor exosome formulation of any one of claims 1-6, preferably wherein the tumor exosome is derived from autologous tumor tissue or tumor cells of a patient in need of personalized treatment.
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| PCT/CN2023/128966 WO2024094043A1 (en) | 2022-11-02 | 2023-11-01 | Modified exosome preparation, preparation method therefor, and use thereof |
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