Preparation method of anti-tumor exosome vaccine, exosome vaccine and application thereof

Abstract

This invention relates to a method for preparing an anti-tumor exosome vaccine, the exosome vaccine itself, and its uses, belonging to the field of biomedical technology. The preparation method includes: Step 1, isolating peripheral blood mononuclear cells from human peripheral blood; Step 2, placing the peripheral blood mononuclear cells in a flat-bottomed culture flask and culturing them in an incubator to obtain DC precursor cells; Step 3, administering pomalidomide to the DC precursor cells and culturing them in a cell culture medium containing human GM-CSF and human IL-4; Step 4, adding tumor antigen during the culture process to form DCs that present tumor antigen; Step 5, collecting the supernatant of the culture medium and centrifuging it in fractions to obtain precipitates; Step 6, collecting the obtained precipitates together and resuspending them in PBS; then centrifuging again at 100,000 × g for 60 min to obtain exosomes. This preparation method can significantly increase the proportion of vesicles simultaneously expressing CD63 and CD81 (i.e., exosomes) in the total harvested vesicles and the exosome activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing an anti-tumor exosome vaccine and the exosome vaccine itself. Background Technology

[0002] Multiple myeloma (MM) is a malignant hematologic malignancy originating from plasma cells. It ranks second in incidence among hematologic malignancies and remains incurable, inevitably leading to disease relapse. Studies show that MM patients exhibit significant immunosuppression / immunodeficiency, resulting in the ineffective activation of anti-tumor immune responses and the progression of the disease. Therefore, reversing the immunosuppression / immunodeficiency state in MM patients and effectively activating anti-tumor immune responses is a crucial issue that urgently needs to be addressed.

[0003] Dendritic cells (DCs) are important antigen-presenting cells in the body's immune system. They present antigenic peptides to specific T cells and provide co-stimulatory signals / secondary signals for T cell activation through co-stimulatory molecules, thus becoming a crucial first step in initiating a specific immune response. Normal DC function or enhanced activity determines whether specific T cells can be activated normally and to what extent, thus playing a vital role in the initiation of the body's adaptive immune response. Patients with neoplastic diseases are mostly in a state of significant immunodeficiency, and DC immunodeficiency is one of the key reasons why a specific anti-tumor immune response cannot be properly elicited. Enhancing DC activity to effectively stimulate an anti-tumor immune response is a common strategy in tumor immunotherapy. The conventional method of DC therapy involves inducing the formation of autologous DCs from MM patients in vitro and then reinfusing them into the patient to achieve the purpose of immunotherapy. The common method for in vitro culture and induction of dendritic cells (DCs) is as follows: Peripheral blood mononuclear cells (PBMCs) are isolated from peripheral blood and cultured in 1640 medium at 37°C and 5% CO2 for 2 hours to allow DC precursor cells (monocytes) to adhere. Then, cells that have not adhered are washed away and discarded. The DC precursor cells are then cultured for 7 days in a medium consisting of 1640 medium, 5 vol% human serum, 800 U / mL GM-CSF, 500 U / mL IL-4, 100 U / mL penicillin, and 0.1 mg / mL streptomycin to induce DC formation. The DCs adhere to the bottom of the culture flask / plate and grow. When it is time to collect the DCs, the supernatant is first aspirated to expose the DCs at the bottom, and then the adherent DCs are scraped off to complete the collection.

[0004] Currently, there are still some shortcomings in DC treatment strategies, such as: (1) After DC culture and induction, they can only survive for about 7 days, with a short effective time and no storage. Therefore, each infusion of anti-tumor DC vaccine to patients requires the collection of peripheral blood from the patient and the extraction of PBMCs to induce DC formation in vitro. This results in a high frequency of patient sample collection and high costs for reagents, consumables, and manpower for DC culture. For example, the cost of one infusion of anti-tumor DC vaccine products that have been marketed and put into clinical treatment is as high as RMB 380,000. (2) The preparation process of DC treatment products is difficult to standardize and control in terms of quality, and there is a lack of clearly defined quality parameters. (3) Each induction of DC culture and preparation of DC treatment products must be carried out in a Class 100 laminar flow GMP standard laboratory for cell therapy technology, which has high requirements for laboratory environment and high operating costs.

[0005] Dendritic cell-derived exosomes (Dex) have gradually become a hot topic of research in recent years, and their application as tumor vaccines in the treatment of malignant tumors is a novel immunotherapy strategy that is still in the early stages of research and development. Dex are extracellular vesicles secreted by dendritic cells, usually distributed in the supernatant of culture medium, and have a typical lipid bilayer structure. Dex expresses MHC-I, MHC-II molecules and T cell co-stimulatory molecules. Thus, Dex can present MHC molecules and antigenic peptides to T cells for recognition, and at the same time provide co-stimulatory molecules to provide co-stimulatory signals for T cell activation, which can, to some extent, replace the function of dendritic cells in stimulating T cell activation.

[0006] Compared to DC cell products, Dex's membrane stability allows it to be stored at -80°C for 6 months, significantly extending the effective storage and use time of the drug. It also greatly reduces the frequency of peripheral blood collection and in vitro DC induction, thereby significantly lowering treatment costs. Furthermore, Dex preparation is easier to define quality parameters and control, facilitating standardized preparation. Additionally, Dex can be mass-produced and stored in the laboratory in a single operation, significantly reducing laboratory operating costs compared to DC cell product preparation. However, the anti-tumor efficacy of Dex is still not ideal. This is because the function of Dex in stimulating anti-tumor immune effects requires normal or significantly enhanced Dex activity. Dex vaccines are heterogeneous vesicle mixtures secreted by dendritic cells, which are composed of various vesicle subsets. Some of these vesicle subsets cannot express either CD63 or CD81 on their membrane surface. These vesicle subsets are mostly microvesicles, apoptotic bodies, and cell debris, and basically lack the ability to present antigens specifically. Their immune activation is very weak or even ineffective, and they may cause non-specific inflammation and interfere with vaccine efficacy. They are usually considered impurities / ineffective components. Other vesicle subsets can only express CD63 on their membrane surface, belonging to typical exosome subsets, but their maturity is relatively low. Some vesicle subsets have low levels of MHC and co-stimulatory molecules (CD80 / CD86), which can activate immunity, but with moderate efficiency and weak antigen presentation and T cell activation capabilities. Other vesicle subsets express only CD81 on their membrane surface. These subsets have slightly higher membrane fluidity and cellular uptake efficiency, moderate immune regulation and intercellular signal transduction, higher abundance of antigen-presenting related proteins, and moderate to weak immune activation. Still other vesicle subsets can simultaneously express CD63 and CD81, belonging to double-positive vesicle subsets. These are the most mature and typical DC-derived exosomes, highly expressing key immune molecules such as MHC-I / II, CD80, and CD86, exhibiting the strongest antigen presentation ability and efficiently activating CD8⁺. T cells, CD4⁺ T cells, and NK cells have the best anti-tumor immune effects and are the core functional subgroups of vaccines, representing the main components that truly exert their effects. However, Dex vaccines prepared using existing processes have a low number of double-positive vesicles (i.e., exosomes) that simultaneously express CD63 and CD81, resulting in low activity. Consequently, Dex vaccines cannot stimulate an effective anti-tumor immune response, leading to less than ideal anti-tumor efficacy, which urgently needs to be addressed. Summary of the Invention

[0007] In order to solve the above-mentioned technical problems, the first aspect of the present invention provides a method for preparing an anti-tumor exosome vaccine, which can significantly increase the proportion of vesicles with both CD63 and CD81 in the total harvested vesicles and effectively enhance the activity of the exosome vaccine, thereby stimulating a more effective anti-tumor immune effect during treatment and achieving better therapeutic results.

[0008] A method for preparing an anti-tumor exosome vaccine includes the following steps:

[0009] Step 1: Isolate peripheral blood mononuclear cells from human peripheral blood;

[0010] Step 2: Place the peripheral blood mononuclear cells in a flat-bottomed culture flask and incubate in an incubator for 2-3 hours; remove the suspended cells and retain the adherent cells as DC precursor cells;

[0011] Step 3: Administer pomalidomide to DC precursor cells and culture them in cell culture medium;

[0012] Step 4: Add tumor antigen on day 5 of culture and continue culturing in the culture medium for 2 days to form DCs that present tumor antigen;

[0013] Step 5: Collect the supernatant of the above culture medium and centrifuge at 1500×g~2000×g for 8~10 min; collect the supernatant after centrifugation and centrifuge again at 10000×g~12000×g for 25~30 min; collect the supernatant after centrifugation and centrifuge again at 100000×g~120000×g for 60~70 min to obtain the precipitate;

[0014] Step 6: Collect the obtained precipitate and resuspend it in PBS; then centrifuge again at 100,000×g~120,000×g for 60~70 min to obtain exosomes. This preparation method applies pomalidomide to the in vitro culture technology for inducing the formation of DC cell-derived exosomes. This significantly increases the proportion of vesicles simultaneously expressing CD63 and CD81 in the total harvested vesicles and effectively enhances the activity of exosome vaccines, obtaining highly activated DC cell-derived exosomes. This results in more effective anti-tumor immune responses during treatment, leading to better therapeutic effects.

[0015] Preferably, in step 5, the supernatant of the culture medium is collected and centrifuged at 1500×g for 10 min; the supernatant after centrifugation is collected and centrifuged again at 10000×g for 30 min; the supernatant after centrifugation is collected and centrifuged again at 100000×g for 60 min to obtain the precipitate. In this scheme, by controlling the centrifugal force of the three centrifugations to 1500×g, 10000×g, and 100000×g respectively, the precipitate can be effectively separated from the supernatant, and the destruction of DC precursor cells and the introduction of more impurities can be prevented. Thus, while ensuring the structural integrity of exosomes, impurities in the precipitate can be effectively reduced or even eliminated, which is beneficial to obtaining purer exosomes.

[0016] Preferably, in step 6, the obtained precipitate is collected and resuspended in PBS; then it is centrifuged again at 100,000 × g for 60 min to obtain exosomes. This is beneficial for obtaining high concentrations and high purity of target exosomes.

[0017] Furthermore, step 7 involves resuspending the obtained exosomes in buffer solution to between 1 / 1000 and 1 / 200 of the initial sample volume and storing them in a freezer at -80°C.

[0018] Preferably, the amount of pomalidomide is 9–11 μM. When the amount of pomalidomide is less than 9 μM, the effect on increasing the proportion of vesicles with CD63 and CD81 in the total harvested vesicles is not significant, and the improvement in exosome vaccine activity is weak. When the amount of pomalidomide is greater than 11 μM, the drug has stronger or significantly increased cytotoxicity, and the drug residue is high. Exosomes become drug carriers, which can affect downstream cell / animal experiments, interfere with results, and increase toxicity.

[0019] Preferably, the cell culture medium contains human GM-CSF and human IL-4, which is beneficial for achieving better culture results.

[0020] Preferably, the concentration of the human GM-CSF is 720–880 U / mL.

[0021] Preferably, the concentration of human IL-4 is 450–550 U / mL.

[0022] A second aspect of this invention addresses the problem of further improving the activity of exosome vaccines by providing an antitumor exosome vaccine. This exosome vaccine is prepared using the method described above, and its membrane structure simultaneously contains CD63 and CD81 transmembrane proteins. In this solution, by using the above method to prepare the antitumor exosome vaccine, not only can the proportion of vesicles containing both CD63 and CD81 be significantly increased in the total harvested vesicles, but the activity of the exosome vaccine can also be effectively improved, stimulating the release of a greater number of CD81 proteins. + T cells, and the CD8 stimulated + T cells have a stronger killing effect, which helps to achieve better treatment results.

[0023] A third aspect of this invention addresses the problem of effectively promoting T cell activation. Furthermore, the exosome vaccine contains at least one of the following miRNAs loaded within its vesicle lumen:

[0024] (1) miRNA with the sequence shown in SEQ ID NO:1;

[0025] (2) miRNA with the sequence shown in SEQ ID NO:2;

[0026] (3) miRNA with the sequence shown in SEQ ID NO:3;

[0027] The miRNAs in the exosomal vaccine synergistically enhance calcium and Ras-MAPK signaling and fine-tune cAMP signaling, forming a multi-level synergistic regulatory network to jointly relieve the inhibition of the core pathway of T cell activation, thereby promoting T cell activation, proliferation, and functional differentiation. In this protocol, we innovatively discovered that the exosomal vaccine prepared using pomalidomide is loaded with novel miRNAs. Combined with KEGG enrichment analysis, we found that these miRNAs in the exosomal vaccine can synergistically enhance calcium and Ras-MAPK signaling and fine-tune cAMP signaling, forming a multi-level synergistic regulatory network to jointly relieve the inhibition of the core pathway of T cell activation, thereby promoting T cell activation, proliferation, and functional differentiation, providing a new pathway for promoting T cell activation.

[0028] Preferably, the diameter of the vesicles in the exosome vaccine is 50–150 nm.

[0029] The fourth aspect of the present invention provides the use of the above-mentioned exosome vaccine in the preparation of a medicament for treating multiple myeloma, wherein the medicament exosome vaccine simultaneously promotes T cell activation through the MAPK signaling pathway and the PI3K-Akt signaling pathway.

[0030] Compared with the prior art, the method for preparing an anti-tumor exosome vaccine, the exosome vaccine and its uses provided by the present invention can significantly increase the proportion of vesicles that simultaneously express CD63 and CD81 in the total harvested vesicles, and can effectively enhance the activity of the exosome vaccine, obtaining a highly activated DC cell-derived exosome vaccine, thereby stimulating a more effective anti-tumor immune effect during treatment and achieving better therapeutic results. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a flow cytometry data graph of one of the MM patients.

[0033] Figure 2 A bar chart showing the proportion of DEXs from 11 MM patients.

[0034] Figure 3(a) is a flow cytometry data graph of one of the MM patients; (b) is a bar chart showing the proportion of CD80+CD63+CD81+DEXs in CD63+CD81+DEXs.

[0035] Figure 4 (a) is a flow cytometry data graph of one of the MM patients; (b) is a bar chart of the proportion of CD86+CD63+CD81+DEXs in CD63+CD81+DEXs.

[0036] Figure 5 (a) is a flow cytometry data graph of one of the MM patients; (b) is a bar chart showing the proportion of HLA-DR+CD63+CD81+DEXs in CD63+CD81+DEXs.

[0037] Figure 6 In the diagram, A represents CD8+ after DEXs stimulation as observed under a confocal microscope. + An imaging image of T cells killing multiple myeloma cells. From left to right in section B are: statistically analyzed CD8... + Comparison charts of T cell counts, early apoptosis rates, and total apoptosis rates.

[0038] Figure 7 Box plot of protein abundance from DEXs derived from 11 MM patients.

[0039] Figure 8 This is a correlation diagram between samples.

[0040] Figure 9 Volcano plot of differentially expressed proteins (DEPs) in DEXs from the experimental and control groups.

[0041] Figure 10 Expression profiles (cluster heatmaps) of 39 differentially expressed proteins.

[0042] Figure 11 This figure compares the expression levels of 20 of the 39 differentially expressed proteins in DEXs from the experimental and control groups.

[0043] Figure 12 KEGG enrichment analysis of differentially expressed proteins (bubble chart).

[0044] Figure 13 GO enrichment analysis of differentially expressed proteins (bubble chart).

[0045] Figure 14In the diagram, A represents the statistical analysis of miRNA length; B represents the statistical analysis of miRNA types; C represents the differentially expressed miRNAs in the experimental and control DEXs samples (volcano plot); D represents the expression profiles of 17 differentially expressed miRNAs (cluster heatmap); E represents the comparison of the expression levels of 17 differentially expressed miRNAs in the experimental and control DEXs; and F represents the KEGG enrichment analysis of the target genes of differentially expressed miRNAs (bubble plot).

[0046] Figure 15 The enrichment of predicted target genes of three significantly upregulated miRNAs in the KEGG signaling pathway. Detailed Implementation

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0048] Example 1

[0049] This embodiment provides a method for preparing an anti-tumor exosome vaccine, comprising the following steps:

[0050] Step 1: Use the patient's peripheral blood as a specimen and isolate PBMNCs from the peripheral blood specimen; in practice, peripheral blood from 15-20 patients can be collected as specimens.

[0051] Step 2: Place PBMNCs in a flat-bottomed culture flask and incubate for 2-3 hours to allow the DC precursor cells to fully adhere to the bottom wall of the culture flask; remove the suspended cells and retain the adherent cells as DC precursor cells.

[0052] Step 3: Administer 9–11 μM pomalidomide to DC precursor cells and culture them in cell culture medium containing 720–880 U / mL human GM-CSF and 450–550 U / mL human IL-4. When performing this procedure, the preferred amounts of pomalidomide are 9 μM, 10 μM, or 11 μM; human GM-CSF is preferably 750 U / mL, 800 U / mL, or 850 U / mL; and human IL-4 is preferably 480 U / mL, 500 U / mL, or 520 U / mL, to achieve better culture results. During implementation, the amount of pomalidomide should not be too low. For example, when the amount of pomalidomide is less than 9 μM, its effect on increasing the proportion of vesicles with both CD63 and CD81 in the total harvested vesicles is not significant, and the improvement in exosome vaccine activity is weak. At the same time, the amount of pomalidomide should not be too high either. For example, when the amount of pomalidomide is greater than 11 μM, the drug residue is high, and the exosomes become drug carriers, affecting downstream cell / animal experiments, interfering with results, and increasing toxicity. In specific implementation, the amount of pomalidomide can be preferentially controlled at 9.5 μM, 10 μM, or 10.5 μM, which can significantly increase the proportion of vesicles with both CD63 and CD81 in the total harvested vesicles, effectively enhance the activity of the exosome vaccine, stimulate a more effective anti-tumor immune response during treatment, achieve better therapeutic effects, effectively control pomalidomide residue and toxicity, and ensure normal cell growth.

[0053] Step 4: On day 5 of culture, add tumor antigen (e.g., in this embodiment, multiple myeloma-specific antigen is used; of course, in other embodiments, other specific antigens can also be used). Continue culturing for 2 days in a medium containing 720–880 U / mL human GM-CSF and 450–550 U / mL human IL-4 to form tumor antigen-presenting DCs (i.e., DCs loaded with tumor antigens). Similarly, in practice, human GM-CSF can preferably be 750 U / mL, 800 U / mL, or 850 U / mL; human IL-4 can preferably be 480 U / mL, 500 U / mL, or 520 U / mL, which is beneficial for achieving better culture results.

[0054] Step 5: Collect the supernatant of the culture medium and centrifuge at 1500×g~2000×g for 8~10 min to remove intact cells, cell clusters, and large debris. Collect the supernatant again and centrifuge at 10000×g~12000×g for 25~30 min to remove dead cells, cell debris, apoptotic bodies, and large vesicles. Collect the supernatant again and centrifuge at 100000×g~120000×g for 60~70 min to obtain a precipitate, which also helps to remove soluble proteins and small molecule impurities. Because the precipitate is very small, in order to effectively separate the precipitate from the supernatant while avoiding the introduction of more impurities (such as preventing DC precursor cells from being damaged by excessive centrifugation force, thus leading to the introduction of new impurities), this embodiment adopts a three-stage centrifugation method, with the centrifugation force increasing step by step each time. This method can remove impurities and improve purity step by step, which can not only effectively ensure the integrity of the exosome structure, but also effectively reduce or even eliminate impurities in the precipitate, thereby facilitating the obtaining of purer exosomes.

[0055] Understandably, during the process, the centrifugal force for the first centrifugation can be preferentially controlled at 1500×g, 1600×g, or 1700×g, and the centrifugation time can be preferentially controlled at 9 min or 10 min. This effectively prevents cell damage while removing intact cells. The centrifugal force for the second centrifugation can be preferentially controlled at 10000×g or 11000×g, and the centrifugation time can be preferentially controlled at 28 min or 30 min, to more thoroughly remove dead cells, cell debris, apoptotic bodies, and large vesicles. The centrifugal force for the third centrifugation can be preferentially controlled at 100000×g or 110000×g, and the centrifugation time can be preferentially controlled at 60 min or 65 min, to effectively remove soluble proteins and small molecule impurities, thus resulting in a purer precipitate.

[0056] Step 6: Collect the obtained precipitate and resuspend it in PBS to wash away residual impurities, proteins, and culture medium components. Replace the PBS system to further purify the exosomes and avoid interference from the culture medium in subsequent experiments (animal, cell, and assay). Then, ultracentrifuge at 100,000×g to 120,000×g for 60–70 min to obtain high concentrations and high purity exosomes. In practice, the centrifugal force can be preferentially controlled at 100,000×g or 110,000×g, and the centrifugation time can be preferentially controlled at 60 min or 65 min to obtain purer exosomes.

[0057] Step 7: Resuspend the obtained exosomes in buffer solution to between 1 / 1000 and 1 / 200 of the initial sample volume, and store them in a freezer at -80°C for experimental use as the experimental group.

[0058] Comparative Example 1

[0059] Compared with Example 1, in step 3, the DC precursor cells were given the same drug solvent (pomalidomide-free) as the experimental group, and the remaining steps and parameters were the same as in Example 1. Exosomes were prepared and used as the control group.

[0060] Example 2

[0061] In this embodiment, exosomes (DEXs) were first prepared from peripheral blood samples of 11 MM patients using the preparation method in Example 1, serving as the experimental group (POMA group); simultaneously, exosomes (DEXs) were prepared from peripheral blood samples of these 11 MM patients using the preparation method in Comparative Example 1, serving as the control group (DMSO group); then, flow cytometry was performed on each exosome in the experimental and control groups. The flow cytometry process was implemented using existing technology, and a blank group was configured during the flow cytometry process (using conventional flow cytometry configuration). The results of the flow cytometry experiment showed:

[0062] (1) The proportion of CD63+CD81+DEXs in the total harvested vesicles was significantly higher in the experimental group than in the control group (83.64%±10.25% vs 77.55%±15.41%, p=0.045, paired samples t test), such as Figure 1 As shown in the figure, flow cytometry data showed that the proportions of CD63+CD81+DEXs in the total harvested vesicles were as follows: blank group: 0%, POMA group (experimental group): 82.6%, DMSO group (control group): 74.8%. Figure 2 As shown, the proportion of CD63+CD81+DEXs in the total harvested vesicles was significantly higher in the experimental group than in the control group (n=11, 83.64%±10.25% vs 77.55%±15.41%, p=0.045), demonstrating that pomalidomide can significantly increase the proportion of CD63+CD81+DEXs in the total harvested vesicles.

[0063] (2) Figure 3 The expression levels of CD80 on the surface of CD63+CD81+DEXs were compared between the experimental group and the control group. Figure 3The flow cytometry data in Figure (a) show the proportion of CD80+CD63+CD81+DEXs in CD63+CD81+DEXs: blank group (0%), POMA group (47.0%) and DMSO group (27.5%). Figure 3 Figure (b) shows that the proportion of CD80+CD63+CD81+DEXs in CD63+CD81+DEXs was significantly higher in the experimental group than in the control group (n=11, 25.51%±13.96% vs. 21.21%±9.17%, p=0.029, paired-samples t-test). Furthermore, the MFI of CD80 expression on the surface of DEXs was also significantly higher in the experimental group than in the control group (n=11, 125.50%±38.14% vs. 109.62%±32.38%, p=0.049, paired-samples t-test), demonstrating that pomalidomide can significantly increase the expression level of CD80 on the surface of CD63+CD81+DEXs (i.e., the exosome surface) in MM patients.

[0064] (3) Figure 4 The expression levels of CD86 on the surface of CD63+CD81+DEXs were compared between the experimental group and the control group. Figure 4 The flow cytometry data in Figure (a) show the proportion of CD86+CD63+CD81+DEXs in CD63+CD81+DEXs: blank group (0%), POMA group (73.1%) and DMSO group (48.3%). Figure 4 Figure (b) shows that the proportion of CD86+CD63+CD81+DEXs in CD63+CD81+DEXs was significantly higher in the experimental group than in the control group (n=11, 49.70%±11.18% vs. 44.50±8.26%, p =0.021, paired-samples t-test). Furthermore, the MFI of CD86 expression on the surface of DEXs was also significantly higher in the pomalidomide group than in the control group (n=11, 176.19%±80.21% vs. 145.64%±49.93%, p=0.016, paired-samples t-test), demonstrating that pomalidomide can significantly increase the expression level of CD86 on the surface of CD63+CD81+DEXs (i.e., the surface of exosomes) in MM patients.

[0065] (4) Figure 5 The expression levels of HLA-DR on the surface of CD63+CD81+DEXs were compared between the experimental group and the control group. Figure 5 The flow cytometry data in Figure (a) show the proportion of HLA-DR+CD63+CD81+DEXs in CD63+CD81+DEXs: blank group (0%), POMA group (31.3%) and DMSO group (25.3%). Figure 5Figure (b) shows that the proportion of HLA-DR+CD63+CD81+DEXs in CD63+CD81+DEXs was significantly higher in the pomalidomide group than in the control group (n=11, 23.93%±12.38% vs. 18.42%±12.99%, p =0.003, paired-samples t-test). Furthermore, the MFI of HLA-DR expression on the surface of DEXs was also significantly higher in the pomalidomide group than in the control group (n=11, 176.35%±91.76% vs. 143.53%±84.07%, p=0.026, paired-samples t-test), demonstrating that pomalidomide can significantly increase the expression level of HLA-DR on the surface of CD63+CD81+DEXs (i.e., the surface of exosomes) in MM patients.

[0066] In summary, this application is the first to demonstrate that pomalidomide significantly enhances the expression of MHC molecules (HLA-DR) and co-stimulatory molecules (CD80 / CD86) on DC-derived exosomes from MM patients. Simultaneously, pomalidomide significantly enhances the tumor-killing immune response stimulated by DC-derived exosomes from MM patients, confirming that pomalidomide significantly improves the activity and function of DC-derived exosomes from MM patients. In the in vitro culture and induction of DC-derived exosomes from MM patients, using pomalidomide as an excipient reduces the number of drugs used compared to other adjuvants. Furthermore, even if the excipients are administered to the patient along with the DC-derived exosomes, the safety and efficacy impact on MM patients is significantly reduced.

[0067] Example 3

[0068] In this embodiment, exosomes (DEXs) were first prepared from peripheral blood samples of 11 MM patients using the preparation method in Example 1. These were used as the experimental group (POMA group). The tumor antigen added during the exosome preparation process was the multiple myeloma-specific antigen MART-1 polypeptide, resulting in tumor antigen-presenting DC-derived exosomes. Similarly, exosomes (DEXs) were prepared from peripheral blood samples of the same 11 MM patients using the preparation method in Comparative Example 1. These were used as the control group (DMSO group). The tumor antigen added during the exosome preparation process was the multiple myeloma-specific antigen MART-1 polypeptide, resulting in tumor antigen-presenting DC-derived exosomes. Then, the exosomes from the experimental and control groups were co-cultured with autologous PBMCs to stimulate T cell activation and proliferation. After 10 days, CD8+ cells were sorted out. + T cells were incubated with the U266 multiple myeloma cell line for 2 hours, and the efficacy of the stimulated anti-tumor T cell immune response was evaluated.

[0069] Figure 6 The tumor-killing effect stimulated by DEXs was demonstrated, among which, Figure 6 Figure A shows CD8 after DEXs stimulation as observed under a confocal microscope. + T cells kill multiple myeloma cells. Figure 6 Figure B shows that DEXs treated with pomalidomide (experimental group) stimulated a greater number of CD8 cells than DEXs not treated with pomalidomide (control group). + The early apoptosis rate (n=3, P=0.029) and total apoptosis rate (n=3, P=0.048) of T cells and myeloma cells were higher in the experimental group than in the control group. The results showed that: (1) Exosomes treated with pomalidomide (experimental group) were able to stimulate a greater number of CD8 cells than exosomes not treated with pomalidomide (control group). + T cells; (2) CD8 cells stimulated by exosomes (experimental group) after pomalidomide treatment. + T cells have a stronger ability to kill and lyse multiple myeloma cells.

[0070] Example 4

[0071] This embodiment first performed rigorous data quality control analysis on all the aforementioned samples, and the results showed that the experimental system was stable and reliable. Specifically: First, the protein composition of the exosomes prepared in the experimental group was measured, and the normalized protein abundance distribution was visualized using box plots, such as... Figure 7 As shown, the medians of each group are highly aligned, and there is no significant difference in data dispersion (box range), indicating that batch effects have been effectively corrected and the data quality meets the requirements for subsequent analysis. The correlation between samples is as follows: Figure 8 As shown, the correlation within the groups is high, indicating high sample repeatability within the groups; the correlation between the groups is low, indicating a large difference between the experimental group and the control group, proving that the data quality meets the requirements for subsequent analysis.

[0072] Based on a strict screening threshold (|log2FC| > 1 and corrected p-value < 0.05), a total of 39 differentially expressed proteins (DEPs) were detected. Among them, the experimental group showed significantly upregulated proteins in 4 proteins and significantly downregulated proteins in 35 proteins compared to the control group. Figure 9 As shown. Figure 10 The heatmap showed that the expression of proteins associated with T cell activation (such as DHPS, PSMD1, EPHB6, FOXO1, CCND1, IFNB1, WASF3, MAVS, EVL, CLEC6A, GSDMD, SIGLEC9, IL7, CCL7, IRAK1BP1, EVI5, CXCL9, LRRC25, TNFSF12, and TNFRSF13C) changed most significantly. Figures 9-11As shown. Specifically, compared with the control group, PSMD1 and EPHB6 were upregulated (log2FC > 2) in the experimental group, while DHPS, FOXO1, CCND1, WASF3, MAVS, EVL, CLEC6A, GSDMD, SIGLEC9, IL7, CCL7, IRAK1BP1, EVI5, CXCL9, LRRC25, TNFSF12, and TNFRSF13C were downregulated; among them, NBEAL2 and IFNB1 (proteins related to T cell activation) showed significant downregulation.

[0073] The NBEAL2 protein in the experimental group was downregulated by more than 80% compared to the control group, suggesting that under pomalidomide stimulation, the exosome generation rate of multivesicular bodies (MVBs) is significantly accelerated. NBEAL2, as a scaffold protein of the MVB membrane structure, is efficiently assembled into the luminal vesicles and released with the exosomes, resulting in a decrease in intracellular residual levels (i.e., "utilization consumption"). At the same time, the cell may initiate negative feedback regulation to inhibit the resynthesis of NBEAL2 in order to avoid overloading. Echoing this explanation, the proteomic volcano plot showed that several proteins related to immune recognition, vesicle transport, and cytoskeleton regulation (such as MAVS, SIGLEC9, CCND1, and CLEC6A) were also significantly downregulated (log2FC ≤ -1.7), indicating that pomalidomide induced a global "non-classical activation mode"—many positive immune regulatory factors were not increased but decreased, while exosome membrane integrity, the enrichment of marker proteins (CD63 / CD81), the loading efficiency of functional proteins (VWF, PDGF, etc.), and the biological activities promoting repair and angiogenesis were significantly enhanced, achieving unexpected technical effects. This suggests that pomalidomide can bypass or compensate for the downregulation of NBEAL2 and other common immune-activating proteins, driving exosome maturation and functional loading more efficiently through alternative pathways (such as the ESCRT system, Rab family proteins, or other MVB regulators).

[0074] The IFNB1 protein level in the experimental group was only 1 / 45th that in the control group (downregulated by more than 97%), suggesting that pomalidomide may enhance exosome function through non-type I interferon-dependent pathways or alternative protective signaling networks. Specifically, under pomalidomide stimulation, exosomes may compensate for the downregulation of IFNB1 and MAVS through other type I interferon subtypes (such as the IFN α family), STING-independent pathways, or metabolic reprogramming, while avoiding inflammatory damage or immune tolerance that may be caused by excessive elevation of these factors. Nevertheless, the antigen presentation efficiency and CD8+ of exosomes remained unchanged. +The activation capacity of T cells and NK cells, as well as the in vivo anti-tumor and anti-infection immune surveillance functions, were significantly enhanced, resulting in unexpected technical effects. This indicates that pomalidomide can bypass the classic IFNB1-JAK-STAT axis and MAVS-RIG-I pathway, and achieve the same or even stronger biological effects through optimization of exosome membrane stability, rebalancing of other pattern recognition receptors, or alteration of nucleic acid load in exosomes.

[0075] Based on the results of KEGG pathway and GO functional enrichment analysis, we identified pathways and biological processes significantly associated with T cell activation. The analysis showed that several highly enriched and statistically significant pathways directly participate in regulating T cell activation, proliferation, and function. In the KEGG pathway analysis, a total of 11 pathways were associated with T cell activation, proliferation, and migration, including significantly enriched pathways such as cytokine-cytokinereceptor interaction (hsa04060), the PI3K-Akt signaling pathway (hsa04151), and the Toll-like receptor signaling pathway (hsa04620). In GO pathway analysis, 115 pathways across Molecular Function, Biological Process, and Cellular Component were significantly associated with T cell activation, proliferation, and migration, including significantly enriched pathways such as the cytokine-mediated signaling pathway (GO:0019221), positive regulation of T cell proliferation (GO:0042102), and receptor signaling pathway via JAK-STAT (GO:0007259). Figure 12 , Figure 13 And as shown in Table 1.

[0076] Table 1. Significantly enriched GO pathways identified by proteomics analysis

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[0085] Example 5

[0086] This study used miRNA transcriptomics to explore the effects of pomalidomide on DC-derived exosomes. A total of 10 DC-derived exosomes (DEXs) from healthy donors were included in the analysis. Each DC-derived exosome was divided into an experimental group treated with pomalidomide (i.e., POMA group) and a control group not treated with pomalidomide (i.e., DMSO group), for a total of 20 DC-derived exosome specimens.

[0087] First, miRNA sequencing was performed on 20 DC-derived exosomes, retaining sequences with a base length of 18-26 nt, such as... Figure 14 As shown in Figure A.

[0088] Secondly, statistical analysis was performed on the species of the identified miRNAs, such as... Figure 14 As shown in Figure B, biological analysis detected 3987 miRNAs in 20 exosome samples. Seventeen significantly different miRNAs were found between the experimental and control groups. Specifically, the experimental group showed significant upregulation of 13 miRNAs (red dots) and significant downregulation of 4 miRNAs (blue dots) compared to the control group. Figure 14 As shown in Figure C. Simultaneously, the expression levels of 17 differentially expressed miRNAs in each exosome specimen were analyzed, such as... Figure 14 As shown in Figure D, the expression levels of 17 differentially expressed miRNAs in exosomes (green dots) from 10 healthy donor pomalidomide groups and exosomes (orange dots) from the control group were compared using a paired-samples t-test. The results showed that 13 upregulated miRNAs were significantly higher in the experimental group than in the control group, while 4 downregulated miRNAs were significantly lower in the experimental group than in the control group. Figure 14 As shown in Figure E.

[0089] In addition, among the 13 significantly upregulated miRNAs, 3 miRNAs were unique to the experimental group, representing a change from none to presence in DC-derived exosomes that were not treated with pomalidomide. Detailed information on the 3 miRNAs is shown in Table 2.

[0090] Table 2 shows the sequences of three miRNAs.

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[0092] Downstream target genes of differentially expressed miRNAs from DC-derived exosomes were predicted using TargetScan and miRanda algorithms, and KEGG was used for bioinformatics annotation of these downstream target genes. In KEGG enrichment analysis, we identified 21 pathways involved in the direct or indirect regulation of T cell activation (all P < 0.05). Among these, the experimental group (i.e., the pomalidomide group) showed significant enrichment in seven key signaling pathways: calcium signaling, PI3K-Akt, MAPK, actin cytoskeleton regulation, mTOR, TGF-β, and Wnt (Rich Factor > 0.5 and p < 0.01). Furthermore, previous studies further performed KEGG enrichment analysis on the three newly identified miRNAs individually, such as... Figure 15 As shown, the results indicate that the target genes of the three upregulated miRNAs are significantly enriched in the Ras signaling pathway, calcium reabsorption-related pathway, and cAMP signaling pathway. This suggests that these miRNAs can promote T cell activation through multi-level signal regulation. The three upregulated miRNAs form a synergistic regulatory network by targeting and inhibiting negative regulators in their respective pathways: First, by regulating calcium channels or endoplasmic reticulum calcium release mechanisms (such as STIM / Orai), they enhance intracellular calcium flux and activate downstream transcription factors (such as key transcription factors like NFAT); second, by affecting the activation state of Ras protein, they drive the MAPK cascade reaction, enhance the Ras-MAPK signaling axis, and promote T cell proliferation and clonal expansion; simultaneously, by fine-tuning the cAMP pathway, they reshape the T cell activation threshold. These three pathways together relieve the inhibition of the core pathways of T cell activation, thereby promoting T cell activation, proliferation, and functional differentiation. Together, they constitute the core signaling basis for the enhancement of T cell function by pomalidomide-treated DEXs, ultimately achieving the goal of effectively promoting T cell activation.

[0093] In this embodiment, calcium signaling participates in T cell activation by triggering the activation of transcription factors such as NFAT, promoting the expression of cytokines such as IL-2. Furthermore, calcium signaling may also interact with other pathways (such as NF-κB) to regulate T cell function.

[0094] PI3K-Akt enhances T cell survival and proliferation by inhibiting pro-apoptotic proteins (such as BAD) and promoting metabolism-related proteins (such as mTOR). In addition, it is associated with T cell metabolic reprogramming, supporting its activation and function.

[0095] The MAPK pathway regulates cell proliferation and the production of inflammatory factors, and also participates in the regulation of T cell differentiation and effector function. The actin cytoskeleton regulatory pathway mediates immune synapse formation and promotes signal transduction.

[0096] The mTOR pathway integrates nutrient and energy signals during T cell activation, supporting protein synthesis and metabolic needs. The TGF-β pathway may promote the differentiation of specific T cell subsets through non-canonical pathways.

[0097] The Wnt pathway promotes T cell survival and clonal expansion by regulating T cell homeostasis-related genes (such as c-Myc).

[0098] These pathways work synergistically at multiple levels during the early activation, metabolic maintenance, and differentiation homeostasis phases to jointly promote T cell activation, proliferation, and function. Among them, the MAPK and PI3K-Akt pathways, due to their high enrichment of factors and gene counts, have become core target pathways for moDCs-derived exosomes to promote T cell activation. Specifically, significantly downregulated miRNAs relieve the inhibition of positive regulatory factors, thereby activating the MAPK and PI3K-Akt pathways.

[0099] In summary, pomalidomide-treated DEXs miRNAs can synergistically regulate T cell proliferation, inflammatory responses, and metabolic reprogramming-related pathways, ultimately promoting T cell activation and enhancing immune responses, providing a multi-dimensional theoretical basis for subsequent mechanism verification and targeted intervention strategies.

[0100] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an anti-tumor exosome vaccine, characterized by, Includes the following steps: Step 1: Isolate peripheral blood mononuclear cells from human peripheral blood; Step 2: Place the peripheral blood mononuclear cells in a flat-bottomed culture flask and incubate in an incubator for 2-3 hours; remove the suspended cells and retain the adherent cells as DC precursor cells; Step 3: Administer pomalidomide to DC precursor cells and culture them in cell culture medium; Step 4: Add tumor antigen on day 5 of culture and continue culturing in the culture medium for 2 days to form DCs that present tumor antigen; Step 5: Collect the supernatant of the above culture medium and centrifuge it at 1500×g~2000×g for 8~10 min; Collect the supernatant after centrifugation and centrifuge again at 10000×g~12000×g for 25~30min; collect the supernatant after centrifugation and centrifuge again at 100000×g~120000×g for 60~70min to obtain the precipitate; Step 6: Collect the resulting precipitates together and resuspend them in PBS solution; Then, centrifuge again at 100,000×g to 120,000×g for 60 to 70 minutes to obtain exosomes.

2. The method of claim 1, wherein the anti-tumor exosome vaccine is prepared by, In step 5, the supernatant of the above culture medium is collected and centrifuged at 1500×g for 10 min; Collect the supernatant after centrifugation and centrifuge again at 10000×g for 30 min; Collect the supernatant after centrifugation and centrifuge again at 100,000 × g for 60 min to obtain the precipitate.

3. The method for preparing the antitumor exosome vaccine according to claim 1, characterized in that, In step 6, the resulting precipitates were collected and resuspended in PBS; then centrifuged again at 100,000 × g for 60 min to obtain exosomes.

4. The method for preparing the antitumor exosome vaccine according to claim 1, characterized in that, The procedure also includes step 7, resuspending the obtained exosomes in buffer to between 1 / 1000 and 1 / 200 of the initial sample volume and storing them in a freezer at -80°C.

5. The method for preparing the antitumor exosome vaccine according to claim 1, characterized in that, The amount of pomalidomide is 9–11 μM.

6. The method for preparing the antitumor exosome vaccine according to claim 1, characterized in that, The cell culture medium contains human GM-CSF and human IL-4.

7. An anti-tumor exosome vaccine, characterized by, The exosome vaccine is prepared by any of the preparation methods described in claims 1-6, and the membrane structure of the exosome vaccine simultaneously contains CD63 and CD81 transmembrane proteins.

8. The anti-tumor exosome vaccine of claim 7, characterized in that, The vesicles of exosome vaccines are loaded with at least one of the following miRNAs: (1) miRNA with the sequence shown in SEQ ID NO:1; (2) miRNA with the sequence shown in SEQ ID NO:2; (3) miRNA with the sequence shown in SEQ ID NO:3; The miRNAs in the exosome vaccine form a multi-level synergistic regulatory network by synergistically enhancing calcium signaling, Ras-MAPK signaling, and fine-tuning cAMP signaling. This network jointly relieves the inhibition of the core pathway for T cell activation, thereby promoting the activation, proliferation, and functional differentiation of T cells.

9. The anti-tumor exosome vaccine of claim 7, characterized in that, The diameter of exosome vesicles is 50–150 nm.

10. Use of the exosome vaccine according to any one of claims 7-9 in the preparation of a medicament for treating multiple myeloma, wherein the exosome vaccine promotes T cell activation through the MAPK signaling pathway and the PI3K-Akt signaling pathway.

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