CN111840528A - Tumor vaccine of exosome combined immune checkpoint blocking agent and preparation method thereof - Google Patents
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Abstract
The invention provides a tumor vaccine of an exosome combined immune checkpoint blocking agent and a preparation method thereof. The tumor vaccine is aPD-L1 modified tumor vaccine Exo-Ag-aPD-L1 which is obtained by modifying a PD-L1 antibody on the surface of a dendritic cell Exo-Ag activated by a specific tumor antigen Ag by using the Exo-Ag as a carrier and using phospholipid-polyethylene glycol-succinimidyl ester DSPE-PEG-NHS as a cross-linking agent. The invention utilizes dendritic cell exosomes as carriers to realize efficient lymph node enrichment and obvious immune activation of tumor vaccines, and simultaneously relieves the brake effect of the tumor vaccines on immune response by blocking immune check point channels so as to achieve the effect that one is added and the other is more than two. The invention takes dendritic cell exosomes as a carrier to be effectively combined with an immune checkpoint blocking agent, cooperatively eliminates tumors and prevents tumor metastasis and recurrence.
Description
Technical Field
The invention relates to the technical field of tumor immunotherapy drugs, in particular to a tumor vaccine of an exosome combined immune checkpoint blocking agent and a preparation method thereof.
Background
At present, the tumor immunotherapy approaches include Chimeric Antigen Receptor (CAR) T cell therapy, Immune Checkpoint Blockade (ICB) therapy, tumor vaccines and the like, and these immunotherapy approaches respectively act on different stages of tumor immune cycle to improve immune response efficiency, and significant improvement of patient survival rate has been achieved in clinical trials. However, the current single immunotherapy approaches under investigation have their own limitations.
CAR-T cells can proliferate exponentially after stimulation, producing a highly amplified T cell response within weeks to eliminate tumor cells. However, extensive research has revealed limitations of CAR-T cell immunotherapy. First, CAR-T cells recognize and act on normal cells expressing the target antigen even at low levels, and this off-target tumor toxicity may lead to death. Second, even with specific tumor targeting, severe side effects such as cytokine release syndrome can occur following infusion of large doses of CAR-T cells. Furthermore, due to differences in T cell responses, persistence and side effects from patient to patient, predicting the optimal number of infused cells remains a challenge. More notably, CAR-T cell therapy is not ideal for the treatment of solid tumors due to the tumor desmoplastic properties and immunosuppressive tumor microenvironment, which makes CAR-T cells generally less efficient to infiltrate in tumors.
Immune checkpoint blockade tumor immunotherapy approaches activate anti-tumor immunity by blocking the inhibitory pathway of tumor-bearing mutant antigen T cells, but are ineffective against poorly immunogenic tumors. It is well known that periodic disturbances in late-stage tumor suppressive receptor signaling often lead to immune escape. Thus, despite considerable success in ICB therapy, there are still a number of problems that need to be solved. For example, lower objective response rates (only about 20%) are due to various immune escape mechanisms that exist locally to the tumor, including lack of tumor associated antigens, infiltration of immunosuppressive cells, epigenetic changes, and limitations of the immunosuppressive tumor microenvironment. In addition, the clinical application of ICB is also related to various side effects of normal organs, and serious adverse reactions related to immunity are easy to occur. Thus, the use of ICB therapy as a monotherapy is often limited to a small fraction of patients.
Tumor vaccines include subunit vaccines, nucleic acid vaccines, DC vaccines, and the like. Generally, tumor vaccines are not sufficient to elicit an effective immune response because of their inefficient administration. Nano-vaccines have been developed to address this problem, delivering vaccines for cancer immunotherapy via synthetic or naturally derived nanoparticles. The nano vaccine can co-deliver adjuvant and multi-epitope antigen to lymphoid organs and antigen presenting cells to a certain extent, and can finely adjust the intracellular release of the vaccine and the cross expression of the antigen through nano vaccine engineering. However, with the intensive research, the pressure gradient between various biological barriers and blood vessels of different tissues in the organism enables only about 5% of nano-vaccines to reach the focus. In DC vaccine therapy, DCs are extracted from a patient's body, then proliferated in large amounts in vitro, activated by specific tumor antigens, and finally injected back into the patient for immunomodulation. Clinical results indicate that the development of protective anti-tumor immunity is dependent on the presentation of tumor antigens by DCs. However, the DC cells after in vitro culture and proliferation have the phenomenon of organism rejection after reinfusion, and the operation is complicated, and the time is long and the cost is high.
Since the immune system of cancer patients is hampered by multiple immunosuppressive mechanisms, a single release of an inhibitory pathway is not effective in activating an immune response. Therefore, the combination therapy of multiple immunotherapy means is more beneficial to relieving a plurality of immunosuppressive mechanisms of cancer, more remarkably activating an immune system, thoroughly eliminating primary tumor and metastatic tumor, and effectively inhibiting tumor recurrence. However, at present, a combined tumor vaccine which is simple to prepare and has a good effect does not exist.
Disclosure of Invention
The invention aims to provide a tumor vaccine of an exosome combined immune checkpoint blocking agent and a preparation method thereof, and aims to solve the problems that the existing tumor immunotherapy means and the tumor vaccine are not ideal in treatment effect.
The purpose of the invention is realized as follows: an exosome-immune checkpoint blocking agent combined tumor vaccine is characterized in that exosome Exo-Ag of dendritic cells activated by specific tumor antigen Ag is used as a carrier, phospholipid-polyethylene glycol-succinimidyl ester DSPE-PEG-NHS is used as a cross-linking agent, and PD-L1 antibody is modified on the surface of exosome Exo-Ag to obtain aPD-L1 modified tumor vaccine Exo-Ag-aPD-L1.
The dendritic cell is at least one of a bone marrow-derived dendritic cell, a DC2.4 cell line and a peripheral blood dendritic cell.
The specific tumor antigen Ag is at least one of OVA antigen, melanoma related antigen MAGE and melanoma specific antigen extracted by cell or tissue disruption.
The preparation method of the tumor vaccine comprises the following steps:
(a) culturing primary dendritic cells: uniformly dispersing bone marrow cells in a cell culture plate for culture, and inducing the bone marrow cells to be differentiated into dendritic cells by taking rmIL-4 and rmGM-CSF as induction factors;
(b) extracting an exosome vaccine: incubating specific tumor antigen Ag and dendritic cells together, collecting cell culture supernatant after incubation is finished, filtering by using a vacuum filter to remove cell debris and large-particle protein impurities, and then concentrating and removing impurities by using an ultrafiltration centrifugal tube to obtain exosome solution Exo-Ag;
(c) PD-L1 antibody modified Exo-Ag: dissolving phospholipid-polyethylene glycol-succinimidyl ester DSPE-PEG-NHS in DMSO, mixing with aPD-L1 which is centrifugally washed in a ratio of 1-5: 1, continuously stirring at 0-4 ℃ for reaction, and connecting aPD-L1 and the DSPE-PEG-NHS through amidation reaction to generate DSPE-PEG-NHS-aPD-L1; after the reaction is finished, removing unreacted DSPE-PEG-NHS by centrifugation, recovering the DSPE-PEG-NHS-aPD-L1 of the upper solution, and reacting the recovered DSPE-PEG-NHS-aPD-L1 with Exo-Ag at the temperature of 0-4 ℃ to insert the DSPE end of the DSPE-PEG-NHS-aPD-L1 into a membrane phospholipid bilayer of an exosome, thereby obtaining the Exo-Ag-aPD-L1 successfully modified aPD-L1.
In step (a), adding RPMI1640 culture medium containing 10-30 ng/mL of rmIL-4 and 10-30 ng/mL of rmGM-CSF into a cell culture plate, and half-replacing every 2 days by using fresh culture medium containing 20-60 ng/mL of rmIL-4 and 20-60 ng/mL of rmGM-CSF to keep the concentration of the induction factor in the cell growth environment unchanged; dendritic cells were used on day 10 or after further maturation.
In step (a), all media contained 100U/mL penicillin and 100U/mL streptomycin.
In the step (b), 0.1-0.5 mg/mL of tumor antigen Ag and dendritic cells are incubated for 12-24h, and then fresh culture medium is replaced for 48 h, and cell culture supernatant is collected and filtered.
In step (c), the aPD-L1 solution was centrifuged using a 10 kDa ultrafiltration tube to remove the solvent, washed with PBS and dissolved before being recovered for use.
In step (c), aPD-L1 and DSPE-PEG-NHS amidation reaction time is 12-24h, and DSPE-PEG-NHS-aPD-L1 and Exo-Ag are reacted in PBS for 3-5 h.
The invention organically combines dendritic cell exosomes, tumor vaccines and immune checkpoint blocking agents, overcomes the pressure gradient between blood vessels and lymph nodes, and solves the problems of low tumor vaccine delivery efficiency, tumor immune escape, easy metastasis and recurrence and the like.
The tumor vaccine and the immune checkpoint blocking agent are reasonably combined, the dendritic cell exosomes are used as carriers to realize efficient lymph node enrichment and obvious immune activation of the tumor vaccine, and meanwhile, the 'braking' effect of the tumor vaccine on immune response is relieved by blocking an immune checkpoint channel, so that the effect that one is added and the effect that the other is more than two is achieved, and the limitation of a single immunotherapy is overcome. The invention takes dendritic cell exosomes as carriers, achieves effective antigen presentation and activation immunity, is effectively combined with an immune checkpoint blocking agent, synergistically eliminates tumors, and prevents tumor metastasis and recurrence.
The preparation method is simple, aPD-L1 modified Exo-Ag is obtained through a specific cross-linking agent, the preparation cost of the vaccine is reduced, and toxic and side effects are reduced.
Drawings
FIG. 1 is a schematic diagram of the preparation steps and therapeutic principle of the tumor vaccine according to the embodiment of the present invention.
FIG. 2 is a diagram of gel electrophoresis of aPD-L1 linked to DSPE-PEG-NHS in example of the present invention.
FIG. 3 is a diagram showing the results of Western blot qualitative analysis of Exo-OVA-aPD-L1 according to example of the present invention.
FIG. 4 is an immuno-gold TEM image of Exo-OVA-aPD-L1 of example of the present invention.
FIG. 5 is a graph showing the particle size distribution of Exo-OVA-aPD-L1 in example of the present invention.
FIG. 6 is a flow cytometer analyzing Exo-OVA-aPD-L1 in vitro immune activation capacity.
FIG. 7 shows the results of Exo-OVA-aPD-L1 inhibiting tumor growth in examples of the present invention.
FIG. 8 is a graph showing survival rates of tumor-bearing mice treated with Exo-OVA-aPD-L1 in accordance with an example of the present invention.
FIG. 9 is a pathological analysis diagram of an organ and tumor tissue according to an embodiment of the present invention.
FIG. 10 is a graph of the efficiency of local tumor immune activation by flow cytometry after Exo-OVA-aPD-L1 treatment in accordance with an embodiment of the present invention.
FIG. 11 is a graph showing the results of measurement of relevant immunocytokines in mouse serum after treatment in the example of the present invention.
FIG. 12 is a graph showing the results of measurement of tumor recurrence in mice in the example of the present invention.
FIG. 13 is a graph showing the results of measurement of metastatic nodules in lung tissue of tumor-bearing mice in the examples of the present invention.
FIG. 14 is a graph comparing tumor growth inhibition by Exo-OVA-aPD-L1 and Exo-OVA + aPD-L1 over time.
FIG. 15 is a graph comparing the local CD8+ T cell infiltration of tumors from Exo-OVA-aPD-L1 and Exo-OVA + aPD-L1 immunohistochemical analyses.
Detailed Description
The technical solution of the present invention will be described in detail with reference to specific examples. The test conditions and procedures not mentioned in the examples of the present invention were carried out according to the conventional methods in the art or the conditions suggested by the manufacturer.
Example 1:
the following method for preparing Exo-OVA-aPD-L1 is described in detail by taking a widely used antigen model OVA as an example, and as shown in FIG. 1, the method comprises the following steps:
(1) culturing primary Dendritic Cells (DCs)
C57BL/6 female mice are killed by removing neck, soaked in 75% alcohol for 5-10 min, and primarily disinfected. In a clean bench, the experiment was performed using clean and sterilized instruments such as scissors and tweezers. The bones of the limbs of the mouse were taken out under sterile conditions, immersed in PBS containing 10% penicillin & streptomycin (double antibody), and the muscle and connective tissues on the surface of the bones of the limbs were removed as much as possible with a surgical blade. The treated leg Bone was placed in an incomplete medium RPMI1640, and the joint at both ends of the leg Bone was excised to expose Bone Marrow Cells (BMC). Using a prepared 1 mL syringe, inserting a needle into two ends of the leg bone, repeatedly blowing and sucking until bone marrow cells completely enter the culture medium, and discarding the leg bone. The collected BMC was centrifuged at 13523 g at 4 ℃ for 5 min, the cells were pelleted and the supernatant was discarded, followed by addition of cell lysate and standing. The mixture was centrifuged to discard the supernatant to remove the enriched red blood cells and suspension cells and resuspended in cold complete medium RPMI 1640. Cells were cultured in 12-well plate cell culture plates, and RPMI1640 medium (10% FBS) containing rmIL-4 (20 ng/mL) and rmGM-CSF (20 ng/mL) was added. Every 2 days, half the volume was changed and replaced with fresh medium containing 40 ng/mL of rmIL-4 and 40 ng/mL of rmGM-CSF to maintain the concentration of the inducing factor in the cell growth environment. Dendritic cells can be used on day 10 or can be further matured. All media contained 100U/mL penicillin & streptomycin.
(2) Extraction of exosome vaccines
After the 0.1mg/mL OVA and the DC are incubated for 24h, the fresh culture medium is replaced for 48 h, and then cell culture supernatant is collected and filtered by a 0.22 mu m vacuum filter to remove impurities such as cell debris, large granular proteins and the like. Then, the culture solution is centrifuged for 5 min at 4000 g by using a 100 KD ultrafiltration centrifugal tube, and the culture solution is concentrated while removing small-particle impurities. And obtaining the exosome solution (Exo-OVA) with better purity after concentration. Can be stored in a refrigerator at-80 ℃ for a long time or at 4 ℃ for at most one week.
(3) PD-L1 antibody modified Exo-OVA
Exo-OVA was isolated and purified, and then surface-modified with an anti-mouse PD-L1 antibody (aPD-L1). In order to prevent the solvent of aPD-L1 from interfering the binding reaction of the antibody and the linking agent, a 10 KDa ultrafiltration centrifugal tube is used for centrifuging at 2000 g for 5 min to remove the solvent, and PBS is used for washing once and recycling for later use. Phospholipid-polyethylene glycol-succinimidyl ester (DSPE-PEG-NHS) is selected as a cross-linking agent. DSPE-PEG-NHS is dissolved in DMSO, and is reacted with aPD-L1 after centrifugation for 24 hours under the conditions of 2:1 ratio and 4 ℃, and aPD-L1 is connected with the DSPE-PEG-NHS through amidation reaction. 4500 g, ultrafiltering and centrifuging for 5 min, removing unreacted DSPE-PEG-NHS, recovering upper layer solution DSPE-PEG-NHS-aPD-L1, reacting with purified Exo-OVA at 4 ℃ for 3 h, inserting the DSPE end of DSPE-PEG-NHS-aPD-L1 into membrane phospholipid bilayer of exosome, and successfully modifying aPD-L1 to obtain Exo-OVA-aPD-L1.
As shown in FIG. 1, the anti-tumor principle of Exo-OVA-aPD-L1 is: MHC II molecules on the surface of Exo-OVA-aPD-L1 present antigen peptides to Th cells to stimulate B cells to produce antibodies, and simultaneously activate cytotoxic T lymphocytes through the cross presentation of antigens, so that the generation of anti-OVA specific antibodies of melanoma cells (B16-OVA) is triggered, and the response of CD8+ T cells is activated. In addition, the introduced PD-L1 antibody blocks the immune escape of tumor cells, and further improves the efficiency of vaccine treatment. The combined treatment of the vaccine and the immune check point blocking treatment can enhance the activation and recognition effects of immune cells, further inhibit the metastasis of tumors and achieve the effects of treating the tumors and inhibiting the metastasis. In addition, Exo-OVA can be used as a nano-carrier of aPD-L1, and the stability and accumulation in tumor tissues of aPD-L1 are enhanced. Therefore, the combination of DC-derived exosome vaccines and ICB provides a synergistic therapeutic strategy, significantly improving immune efficacy and reducing toxic side effects.
Exo-OVA-aPD-L1 was characterized and shown in FIGS. 2-5.
FIG. 2 shows gel electrophoresis results of aPD-L1 linked with DSPE-PEG-NHS (DSPE-PEG-NHS-aPD-L1) electrophoresis bands are located on pure aPD-L1 bands, which shows that aPD-L1 linked with a linker has larger molecular weight and dispersed bands, and proves that aPD-L1 and DSPE-PEG-NHS are successfully linked through amidation reaction.
The Western blot qualitative analysis result of the Exo-OVA-aPD-L1 in FIG. 3 shows that the extracted vesicle expression marker proteins such as CD63, CD81, Tsg101 and the like are exosomes. And the exosomes secreted by the DC after OVA stimulation successfully express OVA antigen, which indicates that Exo-OVA-aPD-L1 has the function of activating the immune system, and the expression of aPD-L1 indicates that the modification is successful.
In the observation of an immune gold-labeled electron microscope, the secondary antibody marked by 5 nm gold particles can be combined with aPD-L1 modified on the surface of an exosome, and can also prove that the secondary antibody is effectively modified by aPD-L1 and is still a spherical hollow vesicle with the shape of about 150 nm.
FIG. 5 Nanoparticle Tracking Analyzer (NTA) for Exo-OVA-aPD-L1 particle size of 154.0. + -. 0.9 nm, concentration of 3-5X 106one/mL.
Example 2
The anti-tumor effect of Exo-OVA-aPD-L1 was studied, and the results were as follows:
(1) Exo-OVA-aPD-L1 in vitro immune activation capability evaluation
The mouse spleen lymphocyte is separated, the proliferation and differentiation quantity of the lymphocyte is detected, and the in-vitro activation immunity capability of the material is verified. After incubation of mouse spleen lymphocytes with Exo, Exo-OVA-aPD-L1, PBS was centrifuged to resuspend the cells and label T cells, DCs, B cells, and macrophages with fluorescent antibodies, respectively. The results are shown in FIG. 6, where panel a is a first row of CD8+ T cells and a second row of CD4+ T cells. Panel B is a dendritic cell, panel c is a macrophage, and panel d is a B cell. The results showed that Exo was essentially identical to the control group and had no ability to activate the immune system. And Exo-OVA-aPD-L1 can activate immune cells to different degrees and promote the proliferation and differentiation of the immune cells. The Exo-OVA-aPD-L1 can remarkably induce 31.31% of CD4+ T cell proliferation and 12.81% of B cell proliferation to differentiate, and proves that the antigen carried by the material can effectively activate CD4+ T cells, so that the B cells are promoted to differentiate into plasma cells and then secrete corresponding antibodies. Meanwhile, immune cells such as CD8+ T cells and macrophages are activated by antigen cross-presentation. In-vitro immune cell activation experiments show that the exosome carrying the OVA antigen can activate an immune system and promote the proliferation and differentiation of related immune cells.
(2) Exo-OVA-aPD-L1 in vivo evaluation of tumor suppression levels
To verify whether the OVA-specific tumor vaccine in combination with ICB treatment could enhance the tumor growth-inhibiting effect, different materials, PBS, Exo-OVA, Exo-aPD-L1, Exo-OVA-aPD-L1 (Exo 10)10aPD-L140 mug/mouse) was administered to the tail vein of B16-OVA tumor-bearing mice. Meanwhile, the tumor size of each group of mice is measured every two days, and the tumor growth condition is detected. As shown in fig. 7, the inhibition effect of Exo-OVA-aPD-L1 treatment group on tumor growth was most significant, and there was a very significant difference compared to the PBS control group. The treatment groups such as Exo, Exo-OVA, Exo-aPD-L1 and the like have limited inhibition effect on tumor growth, wherein the Exo-OVA-aPD-L1 group and the Exo-OVA group have obvious difference in tumor volume. The combined action of the ICB and the tumor vaccine is proved to be beneficial to the immunotherapy of the tumor. In addition, the tumor volume growth rate of the Exo-OVA-aPD-L1-treated group was still slow until 40 days after treatment, showing significant antitumor effects.
The results in FIG. 8 show that ingestion of Exo-OVA-aPD-L1 effectively extended the mean survival time of mice, with 4 mice surviving until the end of the 40 day experiment.
FIG. 9 shows H & E stained heart, liver, spleen, lung, kidney, and no apparent damage was observed to the organs of each group. The tumor tissue staining results show that tumor cells treated by Exo-OVA-aPD-L1 are obviously necrotic.
Tumor samples from tumor-bearing mice were collected 21 days after treatment and analyzed by flow cytometry. As shown in FIG. 10, infiltration of B cells, CD4 + T cells and CD8+ T cells was significantly increased in Exo-OVA-aPD-L1 group compared to untreated group. Notably, the Exo-OVA-aPD-L1 component was more favorable for infiltration of immune cells than the Exo-OVA component, probably due to the immune checkpoint blockade of the PD-L1 antibody. The infiltration of local immune cells of the tumor is increased, the immunosuppressive microenvironment of the local tumor can be effectively improved, and the tumor immunotherapy effect is obviously enhanced. Studies have shown that an increased proportion of Tumour Infiltrating Lymphocytes (TILs) can effectively reverse the inhibitory microenvironment.
Serum was analyzed for relevant immunocytokines during treatment. As shown in FIGS. 11 a-c, the secretion of cytokines including interferon-gamma, tumor necrosis factor-alpha, interleukin-6, etc., further confirmed that Exo-OVA-aPD-L1 was effective in activating innate immunity and adaptive immune response, resulting in effective killing. OVA antibodies were detected in mouse sera by ELISA. FIG. 11 d results show that Exo-OVA-aPD-L1 treated mice produced large amounts of OVA antibodies that specifically recognized B18-OVA tumors that highly expressed OVA antigen, resulting in tumor-specific killing. The improved anti-cancer effect of Exo-OVA-aPD-L1 compared to Exo-aPD-L1 may be attributed to the effective activation of immunity by the antigen, an increase in local concentration of antibody around cancer cells, resulting in specific killing of the tumor by the produced antibody.
(3) Evaluation of Exo-OVA-aPD-L1 Effect in inhibiting tumor metastasis and recurrence
To test the therapeutic potential of Exo-OVA-aPD-L1, a model of relapsing metastatic mice was developed and tested. Based on the tumor recurrence volume after mouse surgery, it was found that the single drug treatment group could not effectively prevent local tumor recurrence at the surgical site. In contrast, Exo-OVA-aPD-L1 treatment was significantly effective in preventing local tumor recurrence, as shown in fig. 12.
In a mouse tumor recurrence and metastasis model, metastatic lung tissues are collected, each group of lung tissues is analyzed to count the number of lung nodules, and a blank group is found to have obvious metastasis points, while a group treated by Exo-OVA-aPD-L1 has obviously reduced average number of lung metastasis and has extremely obvious difference with the blank group. The results of the Exo-OVA-aPD-L1 group are shown in FIG. 13, which is different from the Exo-OVA group.
Example 3
The anti-tumor effect of the cross-linked Exo-OVA-aPD-L1 in combination with the exosomal Exo-OVA alone and the immune checkpoint drug aPD-L1 was studied, and the results are shown in FIGS. 14 and 15.
Through the tumor inhibition effect of the mouse living body and the tumor local immunohistochemical analysis, the cross-linked material can be proved to be more effective in activating immunity, enhancing the tumor local immune cell infiltration and effectively inhibiting the tumor growth compared with the simple drug combination treatment.
DC cells are antigen presenting cells which express PD-L1 and secrete exosomes which also express PD-L1, and the DC and exosomes can influence the efficiency of activating immune cells by forming an immunosuppressive pathway with PD-1 on the surface of T cells in the antigen presenting process. And the PD-L1 of the antibody can be blocked by modifying the PD-L1 antibody, and the PD-L1 antibody is modified on the surface of the antibody, so that the antigen presentation is enhanced, and the immune activation efficiency is improved.
2. The combination application of a pure immune checkpoint medicament and an exosome, wherein an immune checkpoint blocking agent is free, has been proved by researches that the free PD-L1 antibody can generate serious immune adverse reaction.
3. The crosslinked material can be enriched at the tumor site according to the EPR effect on one hand, and can be transported to the tumor site together with activated T cells on the other hand. After being combined with PD-L1 on the surface of tumor cells, the composition can relieve braking effect and enhance immunotherapy.
Claims (9)
1. The tumor vaccine is characterized in that the tumor vaccine is aPD-L1 modified tumor vaccine Exo-Ag-aPD-L1 which is obtained by modifying PD-L1 antibody to the surface of exosome Exo-Ag by using exosome Exo-Ag of dendritic cells activated by specific tumor antigen Ag as a carrier and using phospholipid-polyethylene glycol-succinimidyl ester DSPE-PEG-NHS as a cross-linking agent.
2. The exosome-immune checkpoint blocker-combined tumor vaccine according to claim 1, characterized in that the dendritic cells are at least one of bone marrow-derived dendritic cells, DC2.4 cell line, peripheral blood dendritic cells.
3. The exosome-immune checkpoint blocker combined tumor vaccine according to claim 1, characterized in that the specific tumor antigen Ag is at least one of OVA antigen, melanoma-associated antigen MAGE and melanoma-specific antigen extracted by cell or tissue disruption.
4. The method for preparing a tumor vaccine of claim 1, comprising the steps of:
(a) culturing primary dendritic cells: uniformly dispersing bone marrow cells in a cell culture plate for culture, and inducing the bone marrow cells to be differentiated into dendritic cells by taking rmIL-4 and rmGM-CSF as induction factors;
(b) extracting an exosome vaccine: incubating specific tumor antigen Ag and dendritic cells together, collecting cell culture supernatant after incubation is finished, filtering by using a vacuum filter to remove cell debris and large-particle protein impurities, and then concentrating and removing impurities by using an ultrafiltration centrifugal tube to obtain exosome solution Exo-Ag;
(c) PD-L1 antibody modified Exo-Ag: dissolving phospholipid-polyethylene glycol-succinimidyl ester DSPE-PEG-NHS in DMSO, mixing with aPD-L1 which is centrifugally washed in a ratio of 1-5: 1, continuously stirring at 0-4 ℃ for reaction, and connecting aPD-L1 and the DSPE-PEG-NHS through amidation reaction to generate DSPE-PEG-NHS-aPD-L1; after the reaction is finished, removing unreacted DSPE-PEG-NHS by centrifugation, recovering the DSPE-PEG-NHS-aPD-L1 of the upper solution, and reacting the recovered DSPE-PEG-NHS-aPD-L1 with Exo-Ag at the temperature of 0-4 ℃ to insert the DSPE end of the DSPE-PEG-NHS-aPD-L1 into a membrane phospholipid bilayer of an exosome, thereby obtaining the Exo-Ag-aPD-L1 successfully modified aPD-L1.
5. The method according to claim 4, wherein in step (a), the cell culture plate is supplemented with RPMI1640 medium containing 10-30 ng/mL of rmIL-4 and 10-30 ng/mL of rmGM-CSF, and half-replaced every 2 days with fresh medium containing 20-60 ng/mL of rmIL-4 and 20-60 ng/mL of rmGM-CSF to maintain the concentration of the inducer in the cell growth environment; dendritic cells were used on day 10 or after further maturation.
6. The process according to claim 5, wherein in step (a), all the culture media contain 100U/mL of penicillin and 100U/mL of streptomycin.
7. The method according to claim 4, wherein in the step (b), the tumor antigen Ag is incubated with the dendritic cells at a concentration of 0.1 to 0.5 mg/mL for 12 to 24 hours, and then the culture medium is replaced with fresh one for 48 hours, and then the cell culture supernatant is collected and filtered.
8. The method according to claim 4, wherein in the step (c), the aPD-L1 solution is centrifuged by a 10 kDa ultrafiltration centrifugal tube to remove the solvent, washed with PBS and dissolved, and then recovered for use.
9. The method of claim 8, wherein in the step (c), the amidation reaction time of aPD-L1 with DSPE-PEG-NHS is 12-24h, and the reaction time of DSPE-PEG-NHS-aPD-L1 with Exo-Ag in PBS is 3-5 h.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113403276A (en) * | 2021-06-23 | 2021-09-17 | 河北大学 | Antibody functionalized exosome preparation and preparation method and application thereof |
| CN113398258A (en) * | 2021-06-23 | 2021-09-17 | 河北大学 | M1 type macrophage exosome vaccine as well as preparation method and application thereof |
| CN113648429A (en) * | 2021-07-06 | 2021-11-16 | 中山大学 | Polymer for activating immunity by targeting tumor peripheral cells and preparation method and application thereof |
| CN114349845A (en) * | 2021-12-15 | 2022-04-15 | 深圳先进技术研究院 | Exosome for promoting tumor infiltration of T lymphocytes and preparation method thereof |
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| CN114887075A (en) * | 2022-06-08 | 2022-08-12 | 南京涵台余生物科技有限公司 | Pharmaceutical composition containing immune cell exosomes and application thereof |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101085349A (en) * | 2006-06-09 | 2007-12-12 | 项雯华 | Vesicle guiding immunocyte and application of the same in preparing antineoplastic medicine |
| CN102470167A (en) * | 2009-07-02 | 2012-05-23 | Ith免疫治疗控股公司 | Exosome based treatment of cancer |
| WO2013181746A1 (en) * | 2012-06-04 | 2013-12-12 | National Health Research Institutes | Immunosuppressive cells and methods of making and using thereof |
| CN108379569A (en) * | 2018-05-11 | 2018-08-10 | 北京百益宁医学科技有限责任公司 | The DC vaccines of efficient load tumour antigen and its method of induced amplification specific for tumour antigen CTL |
| CN108938568A (en) * | 2018-08-07 | 2018-12-07 | 中国医学科学院生物医学工程研究所 | DCs vaccine and the preparation method and application thereof based on the phosphatide hybridized polymer vesica for containing antigen and double immune agonists altogether |
| WO2019161192A1 (en) * | 2018-02-15 | 2019-08-22 | North Carolina State University | Engineered nanovesicles as checkpoint blockade for cancer immunotherapy |
| CN110227162A (en) * | 2019-05-15 | 2019-09-13 | 清华-伯克利深圳学院筹备办公室 | Target excretion body and preparation method, application, drug delivery system and drug |
| WO2019178334A1 (en) * | 2018-03-14 | 2019-09-19 | The Regents Of The University Of California | Suppressive exosomes in cancer and for immunosuppression |
| CN110540960A (en) * | 2019-09-11 | 2019-12-06 | 苏州大学 | T lymphocyte combined with immune check point blocking agent and application thereof in preparing anti-tumor medicine |
| CN110846281A (en) * | 2018-08-20 | 2020-02-28 | 中国科学院过程工程研究所 | Exosome-based anti-tumor vaccine |
| CN111117964A (en) * | 2019-07-30 | 2020-05-08 | 段海峰 | Tumor-derived exosome and preparation method and application thereof |
-
2020
- 2020-06-17 CN CN202010554387.0A patent/CN111840528A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101085349A (en) * | 2006-06-09 | 2007-12-12 | 项雯华 | Vesicle guiding immunocyte and application of the same in preparing antineoplastic medicine |
| CN102470167A (en) * | 2009-07-02 | 2012-05-23 | Ith免疫治疗控股公司 | Exosome based treatment of cancer |
| WO2013181746A1 (en) * | 2012-06-04 | 2013-12-12 | National Health Research Institutes | Immunosuppressive cells and methods of making and using thereof |
| CN104955941A (en) * | 2012-06-04 | 2015-09-30 | 财团法人卫生研究院 | Immunosuppressive cells and methods of making and using thereof |
| WO2019161192A1 (en) * | 2018-02-15 | 2019-08-22 | North Carolina State University | Engineered nanovesicles as checkpoint blockade for cancer immunotherapy |
| WO2019178334A1 (en) * | 2018-03-14 | 2019-09-19 | The Regents Of The University Of California | Suppressive exosomes in cancer and for immunosuppression |
| CN108379569A (en) * | 2018-05-11 | 2018-08-10 | 北京百益宁医学科技有限责任公司 | The DC vaccines of efficient load tumour antigen and its method of induced amplification specific for tumour antigen CTL |
| CN108938568A (en) * | 2018-08-07 | 2018-12-07 | 中国医学科学院生物医学工程研究所 | DCs vaccine and the preparation method and application thereof based on the phosphatide hybridized polymer vesica for containing antigen and double immune agonists altogether |
| CN110846281A (en) * | 2018-08-20 | 2020-02-28 | 中国科学院过程工程研究所 | Exosome-based anti-tumor vaccine |
| CN110227162A (en) * | 2019-05-15 | 2019-09-13 | 清华-伯克利深圳学院筹备办公室 | Target excretion body and preparation method, application, drug delivery system and drug |
| CN111117964A (en) * | 2019-07-30 | 2020-05-08 | 段海峰 | Tumor-derived exosome and preparation method and application thereof |
| CN110540960A (en) * | 2019-09-11 | 2019-12-06 | 苏州大学 | T lymphocyte combined with immune check point blocking agent and application thereof in preparing anti-tumor medicine |
Non-Patent Citations (6)
| Title |
|---|
| CASPER J.E.WAHLUND等: "Exosomes from antigen-pulsed dendritic cells induce stronger antigen-specific immune responses than microvesicles in vivo", 《SCIENTIFIC REPORTS》 * |
| JONATHAN M.PITT等: "Dendritic cell-derived exosomes for cancer therapy.", 《THE JOURNAL OF CLINICAL INVERSTIGATION》 * |
| MARIE N"DIAYE等: "Rat bone marrow-derived dendritic cells generated with GM-CSF/IL-4 or FLT3L exhibit distinct phenotypical and functional characteristics.", 《JOURNAL OF LEUKOCYTE BIOLOGY》 * |
| 孟松等: "大鼠骨髓来源树突状细胞外泌体的提取及功能研究", 《南京医科大学学报（自然科学版）》 * |
| 张正: "PD-1抗体和外泌体激活的树突状细胞联合治疗前列腺癌的实验研究", 《中国优秀博士论文全文数据库 医药卫生科技辑》 * |
| 时圣彬: "负载Exosome的树突状细胞联合PD-1抗体在小鼠肝癌治疗中的应用", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113403276A (en) * | 2021-06-23 | 2021-09-17 | 河北大学 | Antibody functionalized exosome preparation and preparation method and application thereof |
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| CN113648429A (en) * | 2021-07-06 | 2021-11-16 | 中山大学 | Polymer for activating immunity by targeting tumor peripheral cells and preparation method and application thereof |
| CN113648429B (en) * | 2021-07-06 | 2023-11-10 | 中山大学 | Polymer for activating immunity by targeting tumor surrounding cells, and preparation method and application thereof |
| CN114349845A (en) * | 2021-12-15 | 2022-04-15 | 深圳先进技术研究院 | Exosome for promoting tumor infiltration of T lymphocytes and preparation method thereof |
| CN114478772A (en) * | 2021-12-31 | 2022-05-13 | 苏州大学 | Engineered immune cells, nanogels, and preparation methods and applications thereof |
| CN114478772B (en) * | 2021-12-31 | 2023-08-22 | 苏州怡然生物科技有限公司 | Engineered immune cells, nanogels, methods of making and uses thereof |
| CN114732905A (en) * | 2022-04-24 | 2022-07-12 | 中山大学·深圳 | Engineered exosome nano material and preparation method and application thereof |
| CN114887075A (en) * | 2022-06-08 | 2022-08-12 | 南京涵台余生物科技有限公司 | Pharmaceutical composition containing immune cell exosomes and application thereof |
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Application publication date: 20201030 |