CN111117964B

CN111117964B - Tumor-derived exosome and preparation method and application thereof

Info

Publication number: CN111117964B
Application number: CN201910696060.4A
Authority: CN
Inventors: 段海峰; 薛冰华; 于婷婷; 肖秀孝; 解晶; 庞如梦; 弓景波; 张群伟; 鲁飞
Original assignee: Beijing Shuangyin Biotechnology Co ltd
Current assignee: Beijing Huaao Xuande Biomedical Technology Co ltd
Priority date: 2019-07-30
Filing date: 2019-07-30
Publication date: 2020-12-01
Anticipated expiration: 2039-07-30
Also published as: CN111117964A

Abstract

The invention discloses a tumor-derived exosome and a preparation method and application thereof. The preparation method of the exosome is simple to operate, easy to implement and low in cost. The invention also discloses a pharmaceutical composition for vaccination and a preparation method thereof, wherein the pharmaceutical composition comprises the DC cells and the tumor-derived exosomes subjected to inactivation treatment, and is obtained by co-culturing the DC cells and the exosomes. The pharmaceutical composition for preventive vaccination provided by the invention can obviously increase the content of infiltrating lymphocytes in tumors, especially the content of killer lymphocytes.

Description

Tumor-derived exosome and preparation method and application thereof

Technical Field

The invention relates to the technical field of immunotherapy, in particular to immunotherapy by adopting exosomes, and particularly relates to a tumor-derived exosome and a preparation method and application thereof.

Background

Exosome (Exosome) is obtained by forming a multivesicular corpuscle through a series of complex mechanisms after a cell membrane is invaginated, and then fusing and releasing the multivesicular corpuscle and the cell membrane to the outside of a cell, wherein the size of the multivesicular corpuscle is 30-100 nm. The exosome contains protein, nucleic acid and lipid substances, which can be used as transportation and storage means of the above substances. Exosomes are present in various body fluids such as blood, urine, saliva, and the like, and after being endocytosed by target cells, the contents function by various mechanisms.

Tumor cells can secrete a large number of exosomes (called tumor exosomes), and tumor specific antigens contained in the exosomes can be used as tumor diagnosis markers for early clinical diagnosis, can also be used for curative effect evaluation and prognosis judgment of tumor immunotherapy, and gradually become diagnosis tools of malignant tumors. Early studies found that tumor exosomes have a promoting effect in anti-tumor immune responses, and can serve as an antigen carrier to present tumor-specific antigens to CD8+ T cells through dendritic cells, so that tumor killing effect is caused. Relevant research shows that in vitro experiments of mice, tumor rejection antigens on the surface of tumor exosomes can promote MHC I-class restricted T cell proliferation; in mouse in vivo experiments, tumor exosomes can eliminate autologous or allogeneic tumors by activating T-cell dependent cross-protection. Thus, tumor exosomes can be used as a source of tumor rejection antigens for tumor immunotherapy.

However, it has also been found in studies with patients with advanced malignant tumors that exosomes may also suppress tumor-specific immune responses. Similar to the cytokines produced by tumor cells, tumor exosomes are capable of modulating tumor development, metastasis and angiogenesis, for example: melanoma exosomes can induce myeloid stem cells to differentiate towards phenotypes that promote tumor metastasis, and can also induce vascular endothelial cells to produce TNF β, causing lymphatic endothelial cell-mediated immune tolerance responses. Meanwhile, the exosome can also transmit tumor-derived receptors, proteins and RNA of the exosome to receptor cells to generate a microenvironment beneficial to tumor growth; immunosuppressive molecules are present on the surface of tumor exosomes and these molecules can negatively regulate the immune response through a variety of pathways.

Dendritic Cells (DCs) are professional antigen-presenting cells that have the function of processing antigens, presenting antigens, and activating naive T lymphocytes. DCs are present in peripheral tissues, mostly in the immature state, where they can take up antigen and process it into small polypeptide fragments, which then activate the immature DCs to become mature DCs.

Tumor exosomes in the tumor microenvironment can reduce differentiation and maturation of DCs. In mouse models, tumor exosomes reduce differentiation of bone marrow precursor cells into DCs, are more easily induced into myeloid-derived suppressor cells (MDSCs); in vitro and in vivo experiments of mice, exosomes generated by breast cancer cells act on CD11b + bone marrow precursor cells to inhibit the differentiation of the bone marrow precursor cells into DCs; in human in vivo experiments, tumor exosomes can inhibit the differentiation and maturation of monocytes. In addition, tumor exosomes can reduce the maturation and antigen presentation functions of DCs, and related researches show that miR-212-3P in exosomes secreted by human pancreatic cancer cells can cause MHC II expression and induce immune tolerance of DCs so as to generate an immunosuppressive effect. That is, tumor exosomes can produce immunosuppressive effects by regulating differentiation, maturation and antigen-presenting functions of DCs, which greatly limits the application of tumor exosomes directly as tumor antigens.

Therefore, it is necessary to provide an exosome which can directly act as a tumor antigen for DC cells, inhibiting tumor growth.

Disclosure of Invention

In order to overcome the above problems, the present inventors have conducted intensive studies and, as a result, found that: the exosomes which are normally secreted are subjected to water bath heating treatment or chemical reagent treatment, so that the exosomes can be inactivated, the transfer promoting property of the exosomes is lost, and the inhibition effect on DC cells is relieved; the inactivated exosome and DC cells are prepared into a pharmaceutical composition for preventive inoculation, so that the growth of tumors can be obviously inhibited, the content of infiltrating lymphocytes in the tumors, particularly the content of killer lymphocytes, is increased, and the effect of the pharmaceutical composition is improved, thereby completing the invention.

Specifically, the present invention aims to provide the following:

in a first aspect, a tumor-derived exosome is provided, wherein the exosome is obtained by inactivating a normally secreted exosome.

In a second aspect, there is provided a method for preparing the tumor-derived exosome of the first aspect, wherein the method comprises the following steps:

step 1, obtaining normally secreted tumor-derived exosomes;

step 2, detecting the tumor source exosomes obtained in the step 1;

and 3, inactivating the tumor source exosomes detected in the step 2.

In a third aspect, there is provided a use of the tumor-derived exosome of the first aspect or the tumor-derived exosome prepared by the method of the second aspect in preparing a medicament for treating tumor.

In a fourth aspect, a pharmaceutical composition for vaccination is provided, wherein the pharmaceutical composition comprises DC cells and the tumor-derived exosomes of the first aspect or the tumor-derived exosomes prepared by the method of the second aspect.

In a fifth aspect, a method for preparing the pharmaceutical composition of the fourth aspect is provided.

The invention has the advantages that:

(1) the tumor source exosome provided by the invention loses the transfer promoting property of the exosome, removes the inhibiting effect of the exosome on DC cells, can be directly used as a tumor antigen for DC treatment, activates an immune system and inhibits the growth of tumors;

(2) the treatment method of the tumor-derived exosome provided by the invention is simple, easy to realize, low in cost and suitable for large-scale application;

(3) the pharmaceutical composition for preventive vaccination provided by the invention can limit and inhibit the growth of tumors and can inhibit the metastasis of the tumors in the lung;

(4) the pharmaceutical composition for preventive vaccination provided by the invention can obviously increase the content of infiltrating lymphocytes in tumors, especially the content of killer lymphocytes.

Drawings

FIG. 1 is a graph showing the amount of each cytokine secreted after treatment of DC cells with different exosomes according to a preferred embodiment of the present invention;

FIG. 2 shows the effect of different temperature-treated exosomes in conjunction with DC cell application on tumor size according to a preferred embodiment of the present invention;

FIG. 3 shows the effect of different temperature treated exosomes in combination with DC cell application on tumor weight according to a preferred embodiment of the present invention;

FIG. 4 shows the effect of different temperature-treated exosomes in combination with DC cell application on tumor lung metastases for a preferred embodiment of the present invention;

FIG. 5 shows the effect of different pharmaceutical compositions of a preferred embodiment of the present invention on tumor size;

FIG. 6 shows a tumor anatomy map treated with different pharmaceutical compositions according to a preferred embodiment of the present invention;

FIG. 7 shows the effect of different pharmaceutical compositions of a preferred embodiment of the present invention on tumor weight;

FIG. 8 shows the proportion of non-tumorous mice during treatment with different pharmaceutical compositions according to a preferred embodiment of the invention;

FIG. 9 shows the effect of different pharmaceutical compositions of a preferred embodiment of the present invention on tumor lung metastases;

FIG. 10 shows the survival curves of mice during treatment with different pharmaceutical compositions according to a preferred embodiment of the present invention;

FIG. 11 is a graphical representation of a metaphase spleen lymphocyte content statistics for different pharmaceutical compositions according to a preferred embodiment of the present invention;

FIG. 12 is a flow chart showing spleen lymphocyte content after termination of treatment with various pharmaceutical compositions in accordance with a preferred embodiment of the present invention;

FIG. 13 is a statistical graph showing spleen lymphocyte content after treatment with various pharmaceutical compositions according to a preferred embodiment of the present invention is completed;

FIG. 14 shows a flow chart of tumor infiltrating lymphocyte content after treatment is over for various pharmaceutical compositions according to a preferred embodiment of the present invention;

FIG. 15 shows a statistical graph of the tumor infiltrating lymphocyte content after treatment is concluded with various pharmaceutical compositions according to a preferred embodiment of the present invention.

Detailed Description

The present invention will be described in further detail below with reference to preferred embodiments and examples. The features and advantages of the present invention will become more apparent from the description.

The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

In a first aspect of the present invention, an exosome is provided, which is obtained by inactivating a normally secreted exosome.

The inventor finds that tumor cells can help escape to transfer to other parts of the body through the exocrine bodies secreted by the tumor cells, therefore, the exocrine bodies are preferably inactivated so that the exocrine bodies lose the metastasis promoting characteristics of the exocrine bodies, thereby being better called tumor antigens in the tumor immunotherapy process.

According to a preferred embodiment of the invention, the normally secreted exosomes are tumor-derived exosomes.

Wherein, the tumor-derived exosome can be prepared by separating from body fluids such as ascites, urine or serum of a patient and is easy to obtain; the exosome obtained by separation can keep activity for a long time and is not easy to degrade, and the exosome is convenient to transport; carrying abundant tumor specific antigens, RNAs and proteins.

In a further preferred embodiment, the tumor is a solid tumor, including but not limited to melanoma, liver tumor, lung tumor, and kidney tumor.

According to a preferred embodiment of the invention, the inactivation treatment comprises a water bath heating treatment or a chemical agent treatment.

In the present invention, the inactivation treatment refers to the loss of the exosome's own pro-metastatic property.

In a further preferred embodiment, the temperature of the water bath treatment is 37 ℃ to 70 ℃, preferably 42 ℃ to 65 ℃;

the treatment time is 0.25-3 h, preferably 0.5-2 h.

The inventor researches and discovers that the inactivation effect on exosomes is best when the temperature of water bath treatment is 37-70 ℃, preferably 42-65 ℃, and the inactivation of exosomes cannot be realized when the temperature is lower than 37 ℃; when the temperature is higher than 70 ℃, the envelope structure of the exosome is damaged.

When the water bath treatment time is less than 0.25h, the exosome is not completely inactivated; when the water bath treatment time is more than 3 hours, part of protein in the exosome is degraded.

In a still further preferred embodiment, the temperature of the water bath treatment is a combination of one or more of 42 ℃, 56 ℃ and 65 ℃.

According to a preferred embodiment of the invention, the temperature of the water bath treatment is 42 ℃, and the time of the water bath treatment is 1.5-2.5 h, preferably 2 h; or

The temperature of the water bath treatment is 56 ℃, and the time of the water bath treatment is 0.5-1.5 h, preferably 1 h; or

The temperature of the water bath treatment is 65 ℃, and the time of the water bath treatment is 0.25-0.75 h, preferably 0.5 h.

According to a preferred embodiment of the invention, the water bath treatment is performed for 0.5-1.5 h, preferably 1h, at 42 ℃ and then for 0.5-1.5 h, preferably 1h, at 56 ℃; or

The water bath treatment is carried out for 0.75-2 h, preferably 1.5h, at 42 ℃ and then for 0.25-1 h, preferably 0.5h, at 65 ℃; or

The water bath treatment is carried out for 0.25-1 h, preferably 0.5h, at 56 ℃, and then for 0.25-1 h, preferably 0.5h, at 65 ℃.

The inventor researches and discovers that the tumor exosome can remarkably reverse the transfer promoting property of the tumor exosome through the treatment of the single temperature or the combination of the temperatures, relieves the inhibition effect of the tumor exosome on DC cells, and can be directly used as a tumor antigen of DC treatment to activate an immune system and inhibit the growth of tumors.

According to a preferred embodiment of the invention, the chemical agent comprises one or more of beta-propiolactone, formaldehyde, glutaraldehyde and formalin.

In a further preferred embodiment, the chemical agent is one or more of beta-propiolactone, formaldehyde, glutaraldehyde, and formalin, preferably beta-propiolactone.

In a further preferred embodiment, the final concentration of the beta-propiolactone is from 1:800 to 1:4500, preferably 1:1000 to 1: 4000.

In the invention, the final concentration of the beta-propiolactone is 1: 800-1: 4500, preferably 1: 1000-1: 4000, which has the following specific meanings: diluting beta-propiolactone by 800-4500 times, preferably 1000-4000 times.

The inventor researches and discovers that the inactivation effect on exosomes is optimal when the final concentration is selected, and the exosomes are not inactivated thoroughly when the final concentration is lower than 1: 800; when the final concentration is higher than 1:4500, there is a subsequent residue of too high a concentration of the reagent, further affecting the application of exosomes.

According to a preferred embodiment of the present invention, the treatment time of the chemical agent is 6 to 15 hours, preferably 8 to 12 hours.

Wherein when the treatment time is less than 6h, the exosomes are not completely inactivated; when the treatment time is more than 15 hours, the degradation of partial protein in the exosome is caused, and the envelope structure of the exosome is damaged.

In a further preferred embodiment, the chemical treatment is carried out at a temperature of 4 ℃.

In a second aspect of the present invention, there is provided a method for preparing the tumor-derived exosome of the first aspect, comprising the steps of:

step 1, obtaining normally secreted tumor-derived exosomes.

Wherein the tumor-derived exosomes are obtained according to the following steps:

step 1-1, tumor cells are pre-cultured.

In the present invention, the tumor cell is preferably a solid tumor cell, such as a melanoma cell, and the tumor cell needs to be cultured in advance to adjust the cell state before collecting normally secreted exosomes, and in the present invention, the tumor cell is preferably cultured for 3-5 generations.

And step 1-2, adding a DMEM medium into the cells during normal subculture, culturing, and collecting cell supernatant.

Wherein, the cells are normally passaged into a culture dish, preferably a culture dish with the diameter of 150mm, so that the confluence degree of the attached cells is not more than 30%.

According to a preferred embodiment of the present invention, DMEM medium is added to the cells, and the cells are cultured, and the cell supernatant is collected when the confluency of the cells reaches 90%.

The DMEM culture medium is a culture medium containing various amino acids and glucose, and is widely applied to vaccine production, cell culture of various primary virus host cells and single cell culture.

In the invention, cell supernatants can be repeatedly collected by passage until the required dosage is met, and the collected cell supernatants are stored at 4 ℃ for later use.

In a further preferred embodiment, the DMEM is a medium containing 10% fetal bovine serum.

And 1-3, centrifuging the collected cell supernatant for multiple times to obtain a precipitate, rinsing the precipitate, and resuspending.

According to a preferred embodiment of the present invention, the multi-step centrifugation comprises three times of centrifugation, wherein,

the rotating speed of the first centrifugation is 3000-4000 rpm, the centrifugation time is 6-12 min to remove residual cells and large particles, and preferably, the rotating speed of the first centrifugation is 3500rpm, and the centrifugation time is 10 min;

the second centrifugation is to carry out second centrifugation on the supernatant obtained by the first centrifugation to remove cell debris, wherein the rotation speed of the second centrifugation is 8000-12000 g, the centrifugation time is 25-35 min, preferably the rotation speed of the second centrifugation is 10000g, and the centrifugation time is 30 min;

the third centrifugation is to carry out ultracentrifugation on the supernatant obtained by the second centrifugation, the rotating speed of the third centrifugation is 100000-150000 g, the centrifugation time is 1.5-2.5 h, preferably the rotating speed of the third centrifugation is 120000g, and the centrifugation time is 2 h.

Wherein, after centrifugation many times, can appear circular scar and deposit at the bottom of centrifuging tube, abandon the supernatant carefully, rinse.

According to a preferred embodiment of the invention, the rinsing is carried out with a PBS solution, preferably 2 times.

In a further preferred embodiment, the rinsed pellet is resuspended in physiological saline.

Among them, 1ml of physiological saline is preferably used for resuspension.

And 2, detecting the tumor-derived exosomes obtained in the step 1.

Wherein, the collected precipitate needs to be identified to determine whether the precipitate is an exosome, in the invention, Western Blot is preferably used for detecting the expression of an exosome marker protein CD 63.

According to a preferred embodiment of the present invention, the total protein of the precipitate identified as exosome is quantified, and according to the quantification, exosome is diluted to 1 μ g/ml with physiological saline.

In the present invention, total protein quantification is performed on exosomes by using a BCA protein quantification method or a BCA protein quantification kit (e.g., a BCA protein quantification kit in cloudy days) commonly used in the prior art.

In a further preferred embodiment, the diluted exosomes are subjected to filter sterilization using a 0.22 μm filter.

And 3, inactivating the tumor source exosomes detected in the step 2.

the treatment time is 0.25-3 h, preferably 0.5-2 h.

In the present invention, the exosomes after the water bath treatment are all rapidly placed on ice for cooling, and are filtered and sterilized again by using a 0.22 mu m filter membrane for standby.

In a further preferred embodiment, the final concentration of the beta-propiolactone is from 1:800 to 1:4500, preferably 1:1000 to 1: 4000. According to a preferred embodiment of the present invention, the treatment time of the chemical agent is 6 to 15 hours, preferably 8 to 12 hours.

In a third aspect of the present invention, there is provided a use of the tumor-derived exosome of the first aspect or the tumor-derived exosome prepared by the method of the second aspect in preparing a medicament for treating tumor.

In the present invention, the tumor is preferably a solid tumor, such as melanoma, liver tumor, lung tumor and kidney tumor.

In a fourth aspect of the present invention, there is provided a pharmaceutical composition for vaccination, for activating the immune system and inhibiting tumor growth, comprising a DC cell and a tumor-derived exosome according to the first aspect or a tumor-derived exosome prepared by the method according to the second aspect, the pharmaceutical composition being obtained by co-culturing the DC cell and exosome.

Among them, Dendritic Cells (DCs) are the most powerful Antigen Presenting Cells (APCs) found at present, and are the only APCs capable of activating naive T cells. The DC absorbs tumor antigens, high expression of MHC I and II molecules is carried out on the membrane after the development and maturation, a large amount of tumor antigens are presented to a T cell receptor, meanwhile, the DC improves the expression of co-stimulatory molecules B7-1, B7-2, CD40 and the like, the T cells are activated, and the DC is combined with the T cells to secrete a large amount of IL-12, and the IL-12 can strongly induce the T cells, NK cells and LAK cells to generate a large amount of TNF-gamma, perforin and granzyme, so that the dissolving effect of CTL cells and NK cells on target cells is enhanced.

As the exosome derived from the tumor almost contains all information of the tumor, mainly comprising protein, RNA and DNA of the tumor cell, the exosome derived from the tumor has better effect as the antigen of the tumor vaccine than the antigen of the polypeptide antigen hRNA which is cracked by the tumor cell or simply synthesized, and can present more tumor information to the DC cell.

Therefore, in the present invention, it is preferable that the inactivated tumor-derived exosomes are loaded on the DC cells, and the suppression effect of the tumor-derived normal secreted exosomes on the DC cells is released, so as to significantly increase the DC vaccine effect.

According to a preferred embodiment of the present invention, the DC cells are bone marrow cell-derived DC cells and/or lymphocyte-derived DC cells, preferably bone marrow cell-derived DC cells.

In a fifth aspect of the present invention, there is provided a process for preparing a pharmaceutical composition according to the fourth aspect, the process comprising the steps of:

and step I, obtaining DC cells.

In the present invention, the DC cells are preferably bone marrow cell-derived DC cells, wherein the step 1 comprises the following substeps:

step I-1, bone marrow cells are isolated.

In the present invention, it is preferable to isolate bone marrow cells of a mouse according to the following procedure: killing 6-8 week old mice by dislocation of cervical vertebrae, removing femur and tibia by operation, removing muscle tissue around bone with scissors and forceps, soaking in sterile culture dish containing 70% alcohol for 2-5min for sterilization, and washing with sterile PBS for 2 times. And (3) moving the bone into another new culture dish containing PBS, shearing two ends of the bone by using scissors, extracting the PBS by using a syringe, respectively inserting the needle heads into the marrow cavity from two ends of the bone, and repeatedly washing out the bone marrow into the culture dish until the bone is completely whitened. Collecting bone marrow suspension, and filtering with 200 mesh nylon net to remove small pieces and muscle tissue; the filtrate was centrifuged at 1200rpm for 5min and the supernatant was discarded.

And step I-2, performing induction culture on the bone marrow cells to obtain the DC cells.

Wherein, the step I-2 comprises the following substeps:

step I-2-1, counting the obtained bone marrow cells, then adjusting the cell concentration, adding an induction factor I, and carrying out primary culture.

According to a preferred embodiment of the invention, the cell concentration is adjusted using 10% FBS in RPMI1640 complete medium.

In a further preferred embodiment, the adjusted cell concentration is 0.5X 10⁶～1.5×10⁶Preferably 1 × 10⁶And (4) respectively.

According to a preferred embodiment of the invention, the first inducing factors are GM-CSF (granulocyte-macrophage colony stimulating factor) and IL-4 (interleukin-4),

the concentration of the GM-CSF is 20-60 mug/ml, preferably 30-50 mug/ml, such as 40 mug/ml;

the concentration of the IL-4 is 5-15 mu g/ml, preferably 8-12 mu g/ml, such as 10 mu g/ml.

The inventor researches and discovers that the separated mononuclear cells can be obviously reduced to be differentiated into macrophages by adopting the induction factors with the concentration, and the quantity of immature DC cells can be increased.

In a further preferred embodiment, the ratio by volume of GM-CSF and IL-4 added is 1: (0.7 to 1.4), preferably 1: (0.8-1.2), such as 1: 1.

In the present invention, it is preferable that the preliminary culture is carried out at 37 ℃ with 5% CO₂Is carried out in an incubator.

In a further preferred embodiment, the ratio of the volume of the first induction factor to the volume of the cells is (8-15): 10000, preferably 10: 10000.

In the present invention, it is preferable to spread the cells adjusted in concentration into 100mm dishes of bacteria, 10ml of cells per dish, and then add 10. mu.l of the first inducer.

And step I-2-2, adding the induction factor I again, culturing for a period of time, and then carrying out liquid changing treatment to obtain the DC cell.

According to a preferred embodiment of the present invention, on the 3 rd day after the addition of the first induction factor, a complete culture solution containing the first induction factor is added again, and the volume ratio of the complete culture solution to the first induction factor added is 10000: 10.

wherein, the complete culture solution is to add some natural substances rich in amino acids, vitamins and bases in the basic culture solution to meet the requirement of cell growth. In the present invention, the concentration and volume of the first induction factor added to the complete culture medium are the same as those of the first induction factor added for the first time.

In a further preferred embodiment, half-volume change treatments are performed from the time of initial induction factor addition to the 6 th and 8 th days of culture, respectively.

Wherein, the half amount of the replacement fluid is used for collecting old culture fluid, the complete culture fluid containing the first induction factor is used for resuspending cell precipitation after centrifugation, and then the cell suspension is put back to the original container.

And II, loading exosomes to obtain the pharmaceutical composition.

In the present invention, the exosome is the exosome according to the first aspect or the exosome prepared by the method according to the second aspect, namely the exosome subjected to inactivation treatment.

Wherein the step II comprises the following substeps:

step II-1, exosomes are added to DC cells, and the cells are collected after co-culture for a period of time.

According to a preferred embodiment of the present invention, exosomes are added to DC cells at the 8 th day of culture from the start of the initial addition of induction factors,

wherein the exosome is added in an amount of 1 × 10⁷The amount of the cell is 0.8 to 1.2. mu.g, preferably 1. mu.g.

In a further preferred embodiment, the co-cultivation time is 1 to 3 days, preferably 2 days.

The co-culture time is 1-3 days, preferably 2 days, namely, the cells can be collected from the beginning of the initial addition of the induction factor to the 10 th day of the culture. The collection is to gently blow and collect the suspension cells by using a pipette.

In the present invention, it is preferable that 9.2X 10 in average per culture dish can be obtained⁶And (4) cells.

And step II-2, centrifuging the collected cells, re-suspending the cell sediment, adding a second induction factor, and continuing to culture.

According to a preferred embodiment of the present invention, the centrifugation is performed at 1000 to 1500rpm for 3 to 8min at room temperature, preferably at 1200rpm for 5min at room temperature.

In a further preferred embodiment, the cell pellet is resuspended in complete culture medium RPMI 1640.

According to a preferred embodiment of the invention, the second inducing factor comprises GM-CSF, IL-4 and LPS (lipopolysaccharide), wherein,

the concentration of the IL-4 is 5-15 mu g/ml, preferably 8-12 mu g/ml, such as 10 mu g/ml;

the concentration of LPS is 0.5-1.5 mg/ml, preferably 0.8-1.2 mg/ml, such as 1 mg/ml.

The inventor researches and discovers that the induction factor with the concentration can remarkably reduce the differentiation of the separated mononuclear cells to macrophages and increase the number of induced mature DC cells.

In a further preferred embodiment, the ratio by volume of GM-CSF, IL-4 and LPS is (2-3): (2-3): 10, preferably 2.5:2.5: 10.

In a further preferred embodiment, the ratio of the volume of the second induction factor to the volume of the complete RPMI1640 culture medium is (10-20): 10000, preferably 15: 10000.

Preferably, the induction factor II is added followed by 5% CO at 37 deg.C₂The culture is continued for 1 to 2 days under the condition (1).

And step II-3, collecting cells, washing and precipitating after centrifuging, re-suspending after centrifuging, and adjusting the concentration.

Collecting cells by using a cell scraper, and centrifuging the collected cell suspension at the speed of 1300-1800 rpm, preferably 1600 rpm; the centrifugation time is 7-15 min, preferably 10 min.

In the present invention, the washing is performed with physiological saline, preferably 2 times, and then centrifugation is performed according to the above-mentioned centrifugation conditions, and the obtained cell pellet is resuspended.

Finally, cell counting was performed and the concentration was adjusted to 2X 10⁶One/ml for standby.

Examples

The present invention is further described below by way of specific examples, which are merely exemplary and do not limit the scope of the present invention in any way.

Example 1

Preparing an inactivated tumor-derived exosome:

(1) mouse-derived melanoma B16F10 cells (purchased from ATCC company) were cultured in DMEM medium containing 10% fetal bovine serum (FBS, purchased from Bioind company); before collecting exosomes, B16F10 cells need to be cultured for 3-5 additional generations to adjust the cell state; when exosome is to be collected, cells are normally passaged to a culture dish of 150mm (the confluence degree of the cells after being attached to the wall can not exceed 30%), 20ml of DMEM culture medium containing 10% fetal bovine serum (FBS, purchased from Bioind company) is added for culture, when the confluence degree of the cells reaches 90%, cell supernatant is collected and stored at 4 ℃ for standby, and the cell supernatant is repeatedly passaged and collected until the required dosage is met;

the collected cell supernatant is firstly centrifuged at 3500rpm for 10 minutes to remove residual cells and large particles, then the centrifugally collected supernatant is centrifuged at 10000g for 30 minutes to remove cell debris, finally the centrifuged supernatant is ultracentrifuged at 120000g for 2 hours, a round scar precipitate appears at the bottom of an ultracentrifuge tube, the supernatant is carefully discarded, then the cell supernatant is softly rinsed with PBS for 2 times, finally the precipitate is resuspended with 1ml of physiological saline, and the cell supernatant is stored at 4 ℃ for standby.

(2) Detecting the expression condition of an exosome marker CD63 by using Western Blot on the collected exosomes, and identifying and collecting precipitates as exosomes; then, the total protein was quantified using BCA protein quantification kit (BCA protein quantification kit from Biyuntian), and according to the quantification result, the exosome was diluted to 1ug/ml with physiological saline, and then filtered and sterilized with 0.22um filter membrane for use.

(3) The prepared exosomes are placed in a water bath at 65 ℃ for continuous treatment for 0.5h, then quickly placed on ice for cooling, and filtered and sterilized by a 0.22 mu m filter membrane for later use.

Example 2

The procedure used in this example is similar to that of example 1, except that in step (3) the exosomes are placed in a water bath at 42 ℃ for a continuous treatment of 2 h.

Example 3

The procedure used in this example is similar to that of example 1, except that in step (3) the exosomes are placed in a 56 ℃ water bath for a duration of 1 h.

Example 4

The procedure used in this example is similar to that of example 1, except that in step (3) the exosomes are first treated in a 42 ℃ water bath for 1h and then in a 56 ℃ water bath for 1 h.

Example 5

The procedure used in this example is similar to that of example 1, except that in step (3) the exosomes are first treated in a 42 ℃ water bath for 1.5h and then in a 65 ℃ water bath for 0.5 h.

Example 6

The procedure used in this example is similar to that of example 1, except that in step (3) the exosomes are first treated in a 56 ℃ water bath for 0.5h and then in a 65 ℃ water bath for 0.5 h.

Example 7

The procedure used in this example is similar to that of example 1, except that in step (3) the exosomes are treated with beta-propiolactone at a final concentration of 1:2000 for 10h at 4 ℃.

Example 8

The procedure used in this example is similar to that of example 1, except that in step (3) the exosomes are treated with beta-propiolactone at a final concentration of 1:1000 for 12h at 4 ℃.

Example 9

The procedure used in this example is similar to that of example 1, except that in step (3) the exosomes are treated with beta-propiolactone at a final concentration of 1:4000 for 8h at 4 ℃.

Example 10

The procedure used in this example is similar to that of example 1, except that in step (3) the exosomes are treated with formaldehyde at a final concentration of 1:2000 for 10h at 4 ℃.

Example 11

Preparation of a pharmaceutical composition for vaccination:

(1) killing 6-8 week old mice by cervical dislocation, taking out all thighbones and shinbones by operation, removing muscle tissues around the bones as clean as possible by scissors and tweezers, soaking in a sterile culture dish containing 70% alcohol for 2-5min for disinfection and sterilization, and washing with sterile PBS for 2 times; moving the bone into another new culture dish containing PBS, shearing off two ends of the bone by using scissors, extracting the PBS by using an injector, respectively inserting the needle heads into marrow cavities from two ends of the bone, and repeatedly washing out the bone marrow to the culture dish until the bone is completely whitened; collecting bone marrow suspension, and filtering with 200 mesh nylon net to remove small pieces and muscle tissue; the filtrate was centrifuged at 1200rpm for 5min and the supernatant was discarded.

(2) The bone marrow cells of the obtained mice were counted and then adjusted to a cell concentration of 1X 10 by using 10% FBS-containing RPMI1640 complete medium (purchased from Gibco Co., Ltd.)⁶One/ml, spread into 100mm bacterial culture dishes, 10ml of cells per dish, and simultaneously add recombinant murine factor GM-CSF 5ul (40ug/ml) and IL-45 ul (10ug/ml) (both from Peprotech Corp.), 37 ℃ and 5% CO₂Culturing in an incubator; on day 3, 10ml of complete culture medium containing recombinant murine factor GM-CSF 5ul (40ug/ml) and IL-45 ul (10ug/ml) was added to the culture dish; half of the culture medium was replaced on day 6 and day 8, respectively, by collecting the old culture medium, resuspending the cell pellet with a complete culture medium containing recombinant murine factor GM-CSF 5ul (40ug/ml) and IL-45 ul (10ug/ml) after centrifugation, and then returning the cell suspension to the original dish.

(3) On day 8, the tumor-derived exosomes prepared in example 1 were administered per 1 × 10⁷Adding 1ug of exosome into each cell, directly adding the exosome into a culture dish, and then continuously culturing; on day 10, cells, i.e., BMDCs, were harvested at 9.2X 10 cells/dish⁶(ii) individual cells;

the DCs cultured on day 10 were gently pipetted to collect suspension cells, centrifuged at 1200rpm for 5min at room temperature, the supernatant was discarded, the cells were resuspended in 10ml of RPMI1640 complete medium and plated on 100mm cell culture plates, and recombinant murine factors GM-CSF 2.5ul (40ug/ml), IL-42.5 ul (10ug/ml) and LPS 10ul (1mg/ml) (purchased from Sigma) were added at 37 ℃, 5% CO₂Continuously culturing for 1-2 days in the incubator; finally, collecting cells by using a cell scraper, centrifuging the collected cell suspension for 10 minutes at 1600rpm, discarding the supernatant, washing the cells for 2 times by using physiological saline, resuspending the obtained cell precipitate under the same centrifugation conditions, counting the cells, and readjusting the concentration to be 2 x 10⁶One/ml is ready for use.

Example 12

The procedure used in this example is similar to that of example 11, except that the exosome prepared in example 2 was used in step (3).

Example 13

The procedure used in this example is similar to that of example 11, except that the exosome prepared in example 3 was used in step (3).

Example 14

The procedure used in this example is similar to that of example 11, except that the exosome prepared in example 4 was used in step (3).

Example 15

The procedure used in this example is similar to that of example 11, except that the exosome prepared in example 5 was used in step (3).

Example 16

The procedure used in this example is similar to that of example 11, except that the exosome prepared in example 6 was used in step (3).

Comparative example

Comparative example 1

The procedure used in this comparative example is similar to that of example 1, except that normally collected tumor-derived exosomes were not subjected to inactivation treatment.

Comparative example 2

This comparative example uses a method similar to example 11, except that the DC cells in step (3) are loaded with tumor-derived exosomes as described in comparative example 1, which have not been subjected to inactivation treatment.

Comparative example 3

This comparative example used a procedure similar to that of example 11, except that the DC cells were not loaded with exosomes.

Examples of the experiments

Experimental example 1

Mouse bone marrow-derived DC cells (as described in example 11) were incubated with the exosomes prepared in example 1 and comparative example 1, respectively, every 1X 10 at day 8 of mouse DC cell culture⁶0.1ug of exosome was added to each DC cell, and incubated at 37 ℃ for 24 hours, and then cell supernatants were collected and centrifuged at 12000g for 10 minutes to remove the cells.

The supernatant obtained by centrifugation was used to detect the expression of TNF- α, IL-6, IFN- γ and IL-12 secreted by DC cells, and the specific procedures were performed according to the ELISA kits for the corresponding murine factors (all purchased from Xinbo biosciences, Inc.), and the results are shown in FIG. 1.

As can be seen from FIG. 1, the exosome prepared in example 1 can significantly increase the secretion of TNF-alpha and IL-6 by mouse DC cells, but has no influence on the secretion of IFN-gamma and IL-12, which indicates that the processing and extracting capacity of the DC cells to antigen is increased.

Experimental example 2

Selecting 80 male C57BL/6 mice (purchased from Scibefu (Beijing) Biotechnology limited) with age of 6-8 weeks, recovering melanoma cells of the mice B16F10, washing twice with PBS, injecting subcutaneously into the mice, killing the mice after 3 weeks of culture, stripping off tumors, cutting under aseptic condition, and using 70 mesh sterile mouseGently grinding the tissue with a bacterial cell screen, washing the collected cells with PBS, counting, centrifuging, and processing at 5 × 10⁵Each 100. mu.L of the mice were resuspended, injected subcutaneously into the right axilla, and the mice were randomly divided into 8 groups, designated as groups 1-8, each group consisting of 10 mice.

On day 5 after tumor inoculation, mice in each group were given different cell treatments, wherein group 1 was a control group and was given a saline treatment; group 2 treatment with the pharmaceutical composition prepared in comparative example 2; group 3 treatment with the pharmaceutical composition prepared in example 12; group 4 treatment with the pharmaceutical composition prepared in example 13; group 5 treatment with the pharmaceutical composition prepared in example 11; group 6 treatment with the pharmaceutical composition prepared in example 14; group 7 treatment with the pharmaceutical composition prepared in example 15; group 8 was treated with the pharmaceutical composition prepared in example 16.

The administration mode of each group is tail vein injection, and all groups require administration in the morning.

The administration time is as follows: groups 2-8 required a total of 3 injections of cells every 7 days.

Control group (group 1): each mouse was injected with 100. mu.l of physiological saline each time, and tail vein injection was performed.

Group 2 to group 8: the injection amount per mouse is 2X 10⁶Cells/100 ul, once every 7 days, tail vein injection.

The model was made and the mice were weighed every other day. After tumor growth, mice tumors were measured twice a week, and the length and width of the tumors were measured with a vernier caliper, and the tumor volume was calculated as length × width/2. On day 32 of molding, mice were sacrificed by decapitation, immediately dissected, and the body weight and tumor weight of the mice were weighed, and the number of mice that died naturally before the mice were sacrificed was counted, and the results are shown in fig. 2, 3, and table 1.

TABLE 1

As can be understood from fig. 2, fig. 3 and table 1, after the exosomes were treated with 65 ℃ for 0.5h in group 5 (treated with the pharmaceutical composition prepared in example 11), the application of the combination DC cells was able to suppress the growth of tumor in mice more, and the number of naturally dead mice before the sacrifice by 32 days was relatively smaller than that in the other groups.

The lungs were dissected and fixed with 4% paraformaldehyde, and then metastasis was detected, with the results shown in fig. 4.

As can be seen from fig. 4, after the exosomes were treated with 65 ℃ for 0.5h in group 5 (treated with the pharmaceutical composition prepared in example 11), the application of the combination of DC cells could significantly inhibit the metastasis of mouse tumors in the lung, with a significantly reduced number of metastases.

Experimental example 3

Selecting 120 male C57BL/6 (purchased from Beijing Biotechnology limited) with age of 6-8 weeks, recovering melanoma cells of mouse B16F10, washing twice with PBS, injecting into mouse subcutaneously, killing mouse after culturing for 3 weeks, stripping tumor, shearing under aseptic condition, lightly grinding tissue with 70 mesh aseptic cell screen, washing collected cells with PBS, counting, centrifuging, and processing according to 5 × 10⁵Each 100. mu.L of the mice were resuspended, injected subcutaneously into the right axilla, and the mice were randomly divided into 4 groups, designated groups A-D, of 30 mice each.

On day 5 after tumor inoculation, mice in each group were given different cell treatments, wherein group a was a control group and given a normal saline treatment; group B was administered exosome-unloaded DC cell therapy described in comparative example 3; group C treatment with the pharmaceutical composition loaded with exosomes not subjected to inactivation treatment as described in comparative example 2; group D was treated with the pharmaceutical composition prepared in example 11.

The administration time is as follows: groups B-D required a total of 3 injections of cells every 7 days.

Control group (group a): each mouse was injected with 100. mu.l of physiological saline each time, and tail vein injection was performed.

Cell: the injection amount per mouse is 2X 10⁶Cells/100 ul, once every 7 days, tail vein injectionAnd (4) shooting.

The model was made and the mice were weighed every other day. After the tumor outgrowth, the length and width of the tumor were measured daily with a vernier caliper, and the tumor volume was calculated as length × width/2. On the 20 th day and the 30 th day of model building, 10 mice are respectively taken out of necks and killed, immediately dissected, the weight and the tumor weight of the mice are weighed, the tumor size of the mice and the proportion of mice without tumors in the treatment process are counted, and the results are shown in fig. 5-8.

As can be seen from fig. 5 to 7, the tumor growth of the mice treated with the pharmaceutical composition prepared in example 11 in group D was significantly inhibited, and as can be seen from fig. 8, the proportion of the mice without tumor growth in the treatment process was higher in the experimental group treated with the pharmaceutical composition prepared in example 11 in group D.

Dissecting lung, fixing with 4% paraformaldehyde, and detecting metastasis, the result is shown in FIG. 9; then, 10 mice remained, and the survival curve of the mice was normally counted for 6 weeks, and the results are shown in fig. 10.

As can be seen from fig. 9 and 10, the inactivated exosome can significantly inhibit the metastasis of melanoma in the lung, and prolong the survival time of tumor mice.

Experimental example 4

The mice treated in Experimental example 3 were dissected, about 1g of tumor tissue and the whole spleen were weighed, ground with a 10ml syringe stopper and passed through a 70 μm mesh, and the cell suspension was centrifuged at 800g for 5 minutes, and the supernatant was removed, followed by lymphocyte separation according to a mouse organ lymphocyte separation kit (purchased from Tianjin ocean Biotechnology Ltd.) in the following manner: resuspending the centrifuged cells with a sample diluent, separating the cell suspension with a sample separating medium, centrifuging at 800g for 20 min, aspirating the buffy coat, and separating the resulting 2X 10⁶Washing the tumor infiltrating lymphocytes twice by PBS, transferring the tumor infiltrating lymphocytes into a flow tube, respectively adding anti-CD 3, CD4, CD8 and NK1.1 mouse flow antibodies according to requirements, giving the antibody dosage according to an antibody specification, adjusting the system to 100ul by PBS, incubating for 30min at room temperature in a dark place, washing twice by PBS after the incubation is finished, and finally resuspending the cells by 400ul PBS and detecting on a machine. Treg detection was labelled according to the mouse Treg cell staining kit (purchased from eBioscience).

The spleen lymphocyte content and the tumor infiltrating lymphocyte content were counted in the middle stage of treatment and after the end of treatment, respectively, by flow cytometry (model: Beckman AS28118), and the results are shown in FIGS. 11 to 15.

As can be seen from fig. 11 to 15, the pharmaceutical composition prepared in example 11 (inactivated exosome-loaded DC cells) significantly increased the amount of infiltrating lymphocytes, particularly killer lymphocytes, in tumors compared to the amount of infiltrating lymphocytes in tumors and the amount of spleen lymphocytes.

The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to be construed in a limiting sense. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention.

Claims

1. A method for preparing a pharmaceutical composition for vaccination comprising the steps of:

step I, obtaining DC cells;

step II, loading exosomes to obtain the pharmaceutical composition; step II comprises the following substeps:

step II-1, adding exosomes into DC cells, co-culturing for a period of time, and collecting the cells;

the exosome is prepared according to a method comprising the following steps:

step 1, obtaining normally secreted tumor-derived exosomes;

the method comprises the following steps:

step 1-1, pre-culturing tumor cells; the tumor cell is a melanoma cell, and the tumor cell is a tumor cell,

step 1-2, adding a DMEM medium into the cells during normal subculture, culturing, collecting cell supernatant,

step 1-3, centrifuging the collected cell supernatant for multiple times to obtain a precipitate, rinsing and resuspending the precipitate;

step 2, detecting the tumor source exosomes obtained in the step 1;

step 3, inactivating the tumor source exosomes detected in the step 2;

the exosome is heated by adopting water bath to process,

the temperature of the water bath treatment is one or a combination of two of 56 ℃ and 65 ℃; the treatment time is 0.25-3 h;

step II-2, centrifuging the collected cells, resuspending the cell sediment, adding an induction factor II, and continuing to culture;

the second induction factor comprises GM-CSF, IL-4 and LPS;

2. Use of a pharmaceutical composition prepared according to the method of claim 1 for the preparation of a medicament for the treatment of tumors.

3. An application of inactivated tumor-derived exosome in preparing a tumor treatment drug is characterized in that,

the inactivated tumor-derived exosome is prepared according to a method comprising the following steps:

step 1, obtaining normally secreted tumor-derived exosomes;

the method comprises the following steps:

step 2, detecting the tumor source exosomes obtained in the step 1;

step 3, inactivating the tumor source exosomes detected in the step 2;

the exosome is heated by adopting water bath to process,

the preparation of the tumor treatment drug comprises the step of loading the inactivated tumor-derived exosomes prepared by the method on DC cells.