CN113398258B

CN113398258B - M1 type macrophage exosome vaccine as well as preparation method and application thereof

Info

Publication number: CN113398258B
Application number: CN202110698573.6A
Authority: CN
Inventors: 李振华; 刘会芳; 吕芳芳; 张金超
Original assignee: Hebei University
Current assignee: Hebei University
Priority date: 2021-06-23
Filing date: 2021-06-23
Publication date: 2022-06-28
Anticipated expiration: 2041-06-23
Also published as: CN113398258A

Abstract

The invention provides an M1 type macrophage exosome vaccine and a preparation method and application thereof. The exosome vaccine is obtained by making M1 type macrophages absorb specific tumor antigen Ag and then extracting exosomes of M1 type macrophages carrying the tumor antigen Ag, namely the exosome vaccine M1_Ag-Exos. The invention constructs the exosome vaccine capable of regulating the tumor immune microenvironment to enhance the immunotherapy efficiency, and utilizes the M1 type macrophage exosome to realize the polarization of tumor-associated macrophages and the obvious immune activation of the tumor vaccine. The invention changes the state of tumor invasion and metastasis promotion by macrophage from immunosuppression to the state of immune support anti-tumor by polarizing tumor-related macrophage into M1 type. Meanwhile, through the immune regulation on the tumor microenvironment, the tumor vaccine can promote the proliferation and activation of T cells more efficiently and effectively inhibit the growth and metastasis of tumors.

Description

M1 type macrophage exosome vaccine as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of tumor immunotherapy drugs, in particular to an M1 type macrophage exosome vaccine and a preparation method and application thereof.

Background

The tumor immunotherapy has outstanding advantages and good therapeutic effect. But in the course of tumor development, an immunosuppressive tumor microenvironment develops. TME is mainly composed of soluble factors such as blood vessels, immune cells, fibroblasts, extracellular matrix, and cytokine growth factors. The immune suppression of TME is caused by three main factors of poor immunogenicity of tumor cells, suppression of effector T cell functions and a large number of immune suppressor cells infiltrated by a tumor microenvironment, and the phenomenon seriously restricts the effect of immunotherapy. Macrophages are a major component of immunosuppressive cells and are also an important contributory factor to immunosuppression. TAMs are divided into two distinct phenotypes in function. The M1 type macrophage promotes antigen presentation and Th1 activation, secretes proinflammatory cytokines and immune activating factors to promote antitumor function. And M2 type macrophages selectively recruit T cells to suppress anti-tumor immune responses and promote tumor invasion, growth and metastasis by secreting immunosuppressive cytokines and chemokines. Most of TAMs affected by the tumor microenvironment have M2 type functions, so that the TAM is polarized into M1 type, so that the immunosuppression of the tumor can be effectively reversed, the T cell activation efficiency is greatly improved, and the tumor immunotherapy effect is remarkably enhanced.

Macrophages are highly plastic and can be polarized to M1 form in a variety of ways. In vitro, LPS or IFN-gamma is often used to achieve efficient polarization of macrophages. In vivo, the polarization of TAMs is often achieved in a variety of ways using nanosystems. First, IL-12, a typical pro-inflammatory cytokine secreted by macrophages of the M1 type, has the ability to polarize macrophages to the M1 type, but due to the instability and systemic side effects of IL-12 administered alone, Wang et al designed and synthesized nanoparticles corresponding to the microenvironment as a cytokine delivery system to deliver IL-12 to TME for release and polarize TAM to achieve immune modulation of the microenvironment in tumor therapy; cheng et al delivered plasmid DNA encoding the IL-12 gene to TME to achieve macrophage polarization. In addition, some inorganic nanoparticles such as Fe₃O₄Macrophages are also efficiently polarized to the M1 type, which may be associated with the fenton or fenton-like reaction of these materials in the tumor microenvironment to generate ROS. While dual receptor agonists of TLR7 and TLR8, represented by R848, also have a significant effect on macrophage polarization, weissleeder et al demonstrate a phenotypic shift of macrophages towards M1 in the tumor immune microenvironment by beta cyclodextrin loading of R848 in a variety of mouse tumor models.

From a mechanistic point of view, activation or inhibition of some signaling pathways can have an effect on the phenotype of macrophages. For example, when the antimalarial chloroquine which clinically shows an anti-tumor effect recently is researched, the chloroquine shows the anti-tumor activity by activating an NF-kB signal pathway to polarize TAM into M1 type macrophages. Chen et al and Zhang et al, respectively, by MnO₂The NF-kB signal channel is activated by the construction of the nano particles and the knockout of the Ndrg2 gene, and the polarization of macrophages to M1 type is also realized. Inhibition of the CSF1R, MAPK and STAT3 signaling pathways, in turn, polarized macrophages to M1. Besides, the microRNA also has an influence on the phenotype of the macrophage. Mutant p53 cancers can reprogram macrophages to a state that supports tumors through miR-1246 in exosomes. miR155 can be used for up-regulating the expression of IL-12, iNOS and MHC II in TAM,and miR-125b repolarizes macrophages to an immune-activated phenotype to enhance the anti-tumor effect of paclitaxel. Furthermore, Kim et al also noted in their studies of macrophage vesicles that miR-155, miR-125 and miR-21 polarize macrophages to the M1 phenotype, while miR-34a, let-7c and let-7f polarize macrophages to the M2 phenotype.

Polarization of TAM in vivo is usually completed by means of nano-carriers, and exogenous materials as carriers usually have the defects of high immunogenicity, potential toxic and side effects and the like. Therefore, a vaccine with small toxic and side effects and good effect is urgently needed to be developed, and tumor-related macrophages are polarized into M1 type, so that the macrophages are changed from a state of promoting tumor invasion and metastasis through immunosuppression into a state of supporting and resisting tumors in an immune manner.

Disclosure of Invention

The invention aims to provide an M1 type macrophage exosome vaccine and a preparation method and application thereof, and aims to solve the problems that immune response cannot be effectively activated only by using a conventional immunotherapy means under an immunosuppressive tumor microenvironment, and the existing drugs for changing the tumor microenvironment have high immunogenicity, potential toxic and side effects and the like.

The purpose of the invention is realized by the following steps: an M1 type macrophage exosome vaccine is prepared by making M1 type macrophage take up specific tumor antigen Ag, and then extracting exosome of M1 type macrophage carrying tumor antigen Ag to obtain an exosome vaccine M1_Ag-Exos。

The M1 type macrophage is formed by polarizing primary macrophage into M1 type macrophage by using lipopolysaccharide or IFN-gamma.

The primary macrophages are human or mouse derived macrophages.

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.

The preparation method of the exosome vaccine comprises the following steps:

(a) culture of M1 type macrophages carrying antigen: incubating the cultured primary macrophage cells for 12-24 h by 50-200ng/mL lipopolysaccharide to polarize the primary macrophage cells into M1 type macrophage cells; then co-incubating the M1 type macrophage with 0.05-0.15mg/mL tumor antigen Ag to obtain M1 type macrophage carrying tumor antigen Ag for 24-48 h;

(b) extracting an exosome vaccine: culturing M1 type macrophage carrying tumor antigen Ag for 2-5 days, collecting cell culture supernatant, concentrating by using ultrafiltration centrifugal tube to remove impurities, filtering the supernatant, centrifuging again, removing supernatant, and resuspending to obtain exosome vaccine M1_Ag-Exos。

In step (b), the treatment of the cell culture supernatant comprises the steps of: firstly, centrifuging at 4500 g rotation speed and 4 ℃ by using a 100 KD ultrafiltration centrifugal tube, centrifuging the concentrated cell supernatant at 2000 g rotation speed and 4 ℃ for 20-40 min, collecting the supernatant, and centrifuging again at 10000 g rotation speed and 4 ℃ for 40-50 min; collecting supernatant, filtering with 0.45 μ M filter membrane, collecting filtrate, transferring the filtrate to new centrifuge tube, centrifuging at 100000 g rotation speed and 4 deg.C for 60-80min, removing supernatant, re-suspending with precooled PBS, centrifuging at 100000 g rotation speed and 4 deg.C for 60-80min, removing supernatant, and re-suspending with precooled PBS to obtain exosome vaccine M1 _Ag-Exos。

The M1 type macrophage exosome vaccine is applied to an immunomodulatory drug aiming at a tumor microenvironment.

The invention constructs the exosome vaccine capable of regulating the tumor immune microenvironment to enhance the immunotherapy efficiency, and realizes the polarization of tumor-related macrophages and the obvious immune activation of the tumor vaccine by using M1 type macrophage exosomes as a carrier.

The invention changes the state of tumor invasion and metastasis promotion by immunosuppression into the state of immune support anti-tumor by polarizing tumor-related macrophages to M1 type. Meanwhile, through the immune regulation on the tumor microenvironment, the tumor vaccine can promote the proliferation and activation of T cells more efficiently and effectively inhibit the growth and metastasis of tumors.

Proved by experimentsMixing M1_OVAExos (OVA is a widely used antigen model) can target tumor sites and polarize tumor-associated macrophages into M1-type macrophages after tail vein injection into tumor-bearing mice. M1 type macrophage can kill tumor cells through ways of secreting cell factor, phagocytosis and the like, and meanwhile, the change of tumor-related macrophage phenotype can relieve the immune suppression of a tumor microenvironment and enhance the treatment efficiency of the tumor vaccine. Cytotoxic T lymphocytes are activated by cross-presentation of antigen, triggering the production of anti-OVA specific antibodies by melanoma cells (B16-OVA), activating the CD8+ T cell response.

The invention organically combines the M1 type macrophage exosome with the tumor vaccine, realizes the conversion of the tumor-related macrophage function from promoting the tumor invasion and metastasis to killing tumor cells, relieves the immune suppression phenomenon of the tumor microenvironment, improves the immune response efficiency of the tumor vaccine, and effectively inhibits the growth and metastasis of the tumor. The preparation method is simple, the preparation cost is reduced, and the toxic and side effects are reduced.

Drawings

FIG. 1 is a schematic diagram of the preparation steps and therapeutic principles of the exosome vaccine of the present invention.

Figure 2 is a western blot analysis of exosome vaccines.

Figure 3 is a TEM image of an exosome vaccine.

Figure 4 is a particle size distribution plot for an exosome vaccine.

FIG. 5 is confocal laser analysis M1_OVAExos macrophage polarizing ability in vitro.

FIG. 6 flow cytometry analysis M1_OVAExos in vitro immune activation capacity.

FIG. 7 is M1_OVA-inhibition of tumor growth by Exos.

FIG. 8 is M1_OVASurvival of tumor-bearing mice after Exos treatment.

FIG. 9 is a pathological analysis of organs and tumor tissues.

FIG. 10 is M1_OVA-flow cytometry analysis of local macrophage polarization of tumor after Exos treatment.

FIG. 11 is M1_OVA-flow cytometry analysis of tumor local T cell activation efficiency after Exos treatment.

FIG. 12 is an assay of relevant immunocytokines in mouse serum after treatment.

FIG. 13 is a tumor-bearing mouse lung tissue metastasis nodule assay.

Detailed Description

The technical solution of the present invention will be described in detail with reference to specific examples. The experimental 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 manufacturers.

Example 1:

the following description will discuss M1 in detail by taking an example of a widely used antigen model OVA_OVA-Exos preparation method, as shown in FIG. 1, comprising the following steps:

(1) preparation of OVA-bearing M1-type macrophages

Female C57BL/6 mice, 6-10 weeks old, were sacrificed on an empty stomach one day prior to sacrifice and starved for at least 8 h. After being soaked and disinfected by 75 percent alcohol, the cleaning agent is operated on a super clean bench, and all the used articles are required to be clean and sterile. Mice were fixed to foam plates and tweezers lifted the abdominal fur of the mice and cut open with scissors without cutting the endothelium. Lifting endothelium, cutting a small opening, lifting with forceps, pouring cold DMEM complete culture medium into abdominal cavity, sucking several times in abdominal cavity, gently touching mouse abdomen with fingers, collecting lavage liquid in abdominal cavity, repeating several times, centrifuging (1000 rpm, 5 min), resuspending with DMEM complete culture medium, placing in incubator, and washing non-adherent cells with PBS after adherence. The cells extracted were at 37 ℃ and 5% CO ₂And culturing under the condition of saturated humidity to obtain primary macrophages.

Primary macrophages were polarized into M1-type macrophages by incubation with 100 ng/mL LPS for 12 h. Working concentrations of OVA 0.1 mg/mL were used to incubate macrophages for 24 h. M1 type macrophages carrying OVA antigen were obtained.

(2) Extraction of exosome vaccines

Collecting cell culture supernatant after the cells are normally cultured for 2 days, and firstly using a 100 KD ultrafiltration centrifugal tubeThe suspension was centrifuged at 4500 g at 4 ℃ to concentrate the exosomes and remove small particle impurities from the culture supernatant. The concentrated cell supernatant was centrifuged at 2000 g, 4 ℃ for 30 min and the supernatant was collected and then centrifuged again at 10000 g, 4 ℃ for 45 min to remove larger vesicles. Collecting supernatant, filtering with 0.45 μm filter membrane, and collecting filtrate. Transferring the filtrate to a new centrifuge tube, selecting an overspeed rotor, centrifuging at 4 ℃ and 100000 g for 70 min. The supernatant was removed, resuspended in 10 mL of pre-cooled PBS, and the ultracentrifuge selected for 70 min at 4 ℃ and 100000 g. The supernatant was removed and resuspended in 100. mu.L of pre-cooled PBS to yield M1_OVA-Exos. The obtained exosome can be stored for a long time at the temperature of 80 ℃ below zero. For the obtained M1 _OVAExos, results are shown in FIGS. 2-4.

Example 2

（1）M1_OVAEvaluation of Exos in vitro Immunity activation Capacity

A large number of TAMs are present in the tumor microenvironment and are affected by the tumor microenvironment, mostly manifested as the immunosuppressive M2 phenotype, a major obstacle to immunotherapy. We then used M1-Exos to polarize macrophages to the M1 phenotype. As shown in FIG. 5, the results of laser confocal measurements showed that M1-Exos and M1_OVAExos can increase the expression of CD80 and decrease the expression of CD206 on the surface of RAW 264.7. This indicates M1-Exos and M1_OVAExos can efficiently polarize macrophages to the M1 phenotype. Figure 6 materials were analyzed by flow cytometry for in vitro T cell activation capacity. T cell Jurkat (clone E6-1) was mixed with PBS, OVA, M0-Exos, M1-Exos and M1, respectively_OVAAfter Exos co-incubation, PBS was centrifuged to resuspend the cells and CD4+ T cells and CD8+ T cells were labeled with fluorescent antibodies, respectively. The results, which were examined by flow cytometry, showed that OVA and M0-Exos were substantially the same as the control group and had no ability to activate the immune system. And M1_OVAExos is effective in promoting the differentiation of T cells into CD4+ T cells and CD8+ T cells.

（2）M1_OVAEvaluation of Exos tumor suppressor levels in vivo

To verify whether TAM polarization-regulated tumor microenvironment in combination with OVA-specific tumor vaccine treatment could enhance tumor growth inhibition Different materials, PBS, OVA, M0-Exos, M1-Exos, M1 were used_OVA-Exos（Exos 10¹⁰ OVA 100 μ g/mouse) tail vein administration of B16-OVA tumor-bearing mice, and the PBS group was used as a blank control group for the experiment. Tumor growth in tumor-bearing mice is shown. The survival of tumor bearing mice was recorded, and the tumor size of each group of mice was measured every two days to detect the tumor growth. As shown in FIG. 7, the PBS, OVA and M0-Exos group tumors exhibited extremely fast growth rates, while the M1-Exos treatment group showed limited inhibition of tumor growth, indicating that TAM polarization to the M1 phenotype also had some inhibition of tumor growth. M1_OVAThe Exos treatment group showed the most significant inhibition of tumor growth with a significant difference compared to the PBS control group. Furthermore, until day 40 post-treatment, M1_OVAThe tumor volume growth rate in the Exos-treated group was still slow, showing a significant antitumor effect.

The results in FIG. 8 show that M1_OVATreatment with Exos effectively extended the mean survival time of mice, with 5 mice surviving until the end of the 40 day experiment.

FIG. 9 shows H&E, the heart, the liver, the spleen, the lung and the kidney are stained, and no obvious damage is caused to the organs of each group when the organs are observed. The result of tumor tissue staining was shown to be M1 _OVAEvidence of necrosis of Exos-treated tumor cells.

After tumor-bearing mice were treated, groups of tumor tissues were collected and analyzed by flow cytometry. As shown in fig. 10, M1_OVAExos and M1 cells highly expressing CD80 were significantly increased in the Exos and M206 cells were significantly decreased compared to the control group, indicating that tumor-associated macrophages are reduced by M1_OVAExos and M1 Exos polarization to the M1 phenotype. As shown in FIG. 11, M1 compared to untreated group_OVAInfiltration of Exos groups CD4 + T cells and CD8+ T cells was significantly increased. 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.

Serum was analyzed for relevant immunocytokines during treatment. As shown in FIG. 11, interferon-gamma, TNF-alpha, interleukin-6 and interleukinThe level of interleukin-6 was measured by M1_OVAThe increase in IL-10 levels and the decrease in IL-10 levels after Exos treatment further confirmed M1_OVAExos efficiently polarizes macrophages to M1 type.

（3）M1_OVAEvaluation of Exos Effect on inhibiting tumor metastasis

To test M1_OVAExos therapeutic potential, tested by establishing a metastatic mouse model. Collecting metastatic lung tissue, analyzing each group of lung tissue to count the number of lung nodules, and finding that the blank group has obvious metastatic points after M1 _OVAIn Exos-treated groups, the mean number of lung metastases was significantly reduced, with very significant differences from the blank group, as shown in fig. 13.

Claims

1. An M1 type macrophage exosome vaccine, which is prepared by the following method:

(a) culture of M1 type macrophages carrying antigen: incubating the cultured primary macrophages with 50-200ng/mL lipopolysaccharide for 12-24 h to polarize the primary macrophages into M1-type macrophages; then co-incubating the M1 type macrophage with 0.05-0.15mg/mL tumor antigen OVA for 24-48 h to obtain M1 type macrophage carrying tumor antigen OVA;

(b) extracting an exosome vaccine: normally culturing the cells for 2 days, collecting cell culture supernatant, centrifuging at 4500 g rotation speed 4 ℃ by using a 100 KD ultrafiltration centrifugal tube, concentrating exosome and removing small particle impurities in the culture supernatant; centrifuging the concentrated cell supernatant at 2000 g, 4 deg.C for 30 min, collecting supernatant, and centrifuging again at 10000 g, 4 deg.C for 45 min to remove larger vesicles; collecting supernatant, filtering with 0.45 μm filter membrane, and collecting filtrate; transferring the filtrate into a new centrifuge tube, selecting an overspeed rotor, centrifuging at 4 ℃ for 70 min at 100000 g; removing supernatant, re-suspending with 10 mL precooled PBS, selecting an overspeed rotor, and ultracentrifuging again at 4 ℃ and 100000 g for 70 min; the supernatant was removed and resuspended in 100. mu.L of pre-cooled PBS to yield M1 _OVA-Exos exosome vaccines.

2. An exosome vaccine according to claim 1, characterised in that the primary macrophages are macrophages of human or mouse origin.

3. Use of the M1-type macrophage exosome vaccine of claim 1 in the preparation of an immunomodulatory medicament directed against a tumor microenvironment.