阅读说明：本技术 外泌体联合免疫检查点阻断剂的肿瘤疫苗及其制备方法 (Tumor vaccine of exosome combined immune checkpoint blocking agent and preparation method thereof ) 是由 李振华 戴欣悦 张金超 于 2020-06-17 设计创作，主要内容包括：本发明提供了一种外泌体联合免疫检查点阻断剂的肿瘤疫苗及其制备方法。所述肿瘤疫苗是以特定的肿瘤抗原Ag激活的树突状细胞的外泌体Exo-Ag为载体,利用磷脂-聚乙二醇-琥珀酰亚胺酯DSPE-PEG-NHS为交联剂,将PD-L1抗体修饰到外泌体Exo-Ag表面而得到的aPD-L1修饰的肿瘤疫苗Exo-Ag-aPD-L1。本发明利用树突状细胞外泌体为载体实现了肿瘤疫苗高效的淋巴结富集及显著的免疫激活作用,同时通过阻断免疫检查点通路,解除其对免疫应答的“刹车”效应,达到一加一大于二的效果。本发明以树突状细胞外泌体为载体与免疫检查点阻断剂有效联合,协同清除肿瘤,并防止肿瘤转移及复发。(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.)

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.

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.