KR20030085659A - Therapeutic cancer exosome and cancer vaccine using itself - Google Patents

Abstract

PURPOSE: An exosome separated from stomach cancer cell lines by a fractional centrifugal separation method to treat stomach cancer is provided. The vaccine containing the exosome and general adjuvants or carriers is widely applied to stomach cancer patients. CONSTITUTION: An exosome as a kind of an intracellular vesicle contains at least one of MHC class 1 and HSP 70 having biological activity and acts as an immunostimulating agent. The exosome can be inoculated using a conventional hypodermic needle after addition of adjuvants or carriers suitable for administration to a human body, or together with a small amount of a cytokine. The administration of the exosome vaccine to a human body causes the induction of T-cell activity and initiation of immunoactivity to tumor cells.

Description

Therapeutic cancer exosome and cancer vaccine using itself

The present invention relates to exosomes for cancer treatment and cancer vaccines using the same, and more particularly, to isolate exosomes having cancer-specific antigens from gastric cancer tumor cell lines and to administer them to gastric cancer patients to exhibit cancer treatment effects. Exosome for cancer treatment and cancer vaccine using the same.

Recent studies in the field of tumor immunology suggest that cell-mediated immunity (CMI) using T-cell mechanisms is essential to effectively extinguish tumors and suppress recurrence. In particular, dendritic cells, one of the antigen-presenting cells (APCs) that T-lymphocytes play an important role in effectively destroying tumor cells, have significantly reduced function in cancer patients. This recent research has revealed this. Using the results of these studies on dendritic cells, representative cancer treatment centers in the United States, Canada, and Japan collected blood from cancer patients, picked out a few pre-stage cells, and administered large amounts of cytokines to healthy dendritic cells. Treatment techniques for inducing an immune response by culturing and re-injecting it into cancer patients are being actively developed. The cancer vaccine therapy using dendritic cells is a new treatment that can be greatly spotted in the future because there are few side effects compared to conventional chemotherapy as a tailor-made therapy (Talor-made therapy) for each patient. However, dendritic cells are difficult to mass cultivate, which limits their benefits to many patients.

On the other hand, exosomes (exosomes) are a type of endoplasmic reticulum that can be separated from B-lymphocytes, antigen-presenting cells, etc. Although the number is somewhat small in recent years, exosomes isolated from tumor cells have tumor antigens and thus have a tumor-specific immune response. It was found to be inducible.

The exosomes of these tumor cells contain a tumor-specific antigen peptide in the MHC 1 complex and are accepted by the T lymphocyte receptor (TCR), which induces the activation of T lymphocytes by killing them in the most natural manner, thereby initiating immune action against the tumor cells.

The present invention has been made to solve the problems of the prior art described above is to provide an exosome that is isolated from several types of gastric cancer cell lines as an antigen of cancer vaccine and acts as an adjuvant.

Another object of the present invention is to provide a therapeutic cancer vaccine that can be widely applied to gastric cancer patients using the exosomes.

1 is an electron micrograph of the exosomes according to the present invention.

Figure 2 shows the result of SDS-PAGE of the exosomes according to the present invention.

Figure 3 is the result of analysis by Western blotting (exo Western blotting) and exosomes and cell lysate according to the present invention.

Figure 4 is a result of analyzing the stability of the exosomes according to the present invention using magnetic particles (magnetic bead).

5 is a result of measuring the degree of activation of T lymphocytes of exosomes according to the present invention by flow cytometry.

Exosome for cancer treatment according to the present invention for achieving the above object is separated from gastric cancer tumor cell line by fractional centrifugation method, it is preferable to include MHC class 1 or HSP 70 having biological activity.

In addition, the vaccine using exosomes for cancer treatment according to the present invention is characterized in that the exosomes and a general adjuvants or additives are added.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

Recently, exosome-related studies have shown that not only antigen-presenting cells but also tumor cells themselves can secrete exosomes, and thus, cancer vaccines can be seen through experimental animals. The present inventors isolated the exosomes from gastric cancer tumor cell lines containing a large number of gastric cancer specific antigens by using these exosomes when they are injected into the patient, so that they can be obtained even if they are phagocytic in their own antigen-presenting cells and do not conform to tissue synthesis. It was confirmed that it is possible to develop a vaccine for treating cancer that can be administered to all gastric cancer patients.

In the present invention, by separating the exosomes from about three tumor cell lines that are not too granular to fit the immune system that differs from patient to patient, the vaccine for cancer treatment can be supplied to all gastric cancer patients.

Exosome for cancer treatment according to the present invention comprises the steps of first separating the exosomes from the gastric cancer cell line, establishing a quality control system through immunobiochemical analysis of the isolated exosomes and human T lymphocyte activation of the exosomes Observing whether or not to determine the effectiveness of the cancer vaccine. Hereinafter, each step will be described.

1) Isolation of Exosomes

35 ml of fresh medium is added per 5 × 10 ⁶ cells removed from about 3 gastric cancer cell lines and incubated for 48 hours and separated by differential centrifugation. Separation First, the supernatant of the cultured medium is centrifuged at 300 g for 5 minutes, the supernatant is taken, centrifuged at 1,200 g for 20 minutes, and the supernatant is separated from 10,000 g for 30 minutes. The exosome pellet obtained by centrifuging the separated supernatant again at 95,000g for 60 minutes was washed with PBS (phosphate buffered saline) and finally centrifuged at 95,000g for 1 hour to remove residual impurities. Can be obtained. As a result of quantifying the exosome protein by the BCA solution method, the obtained exosomes can obtain 0.2-0.5 µg of exosomes per 1 × 10 ⁶ cell, and an electron micrograph of the isolated exosomes is shown in FIG. 1. As can be seen in Figure 1, the exosomes have a specific shape of 40 to 90nm diameter.

2) Immunobiochemical Analysis of Isolated Exosomes

The exosomes isolated in 1) were subjected to SDS-PAGE and Western blotting to analyze protein expression in exosomes and gastric cancer tumor cells. Figure 2 shows the result of SDS-PAGE of the separated exosomes. As can be seen in Figure 2, the exosomes have a different protein profile (cell profile) than the cell lysates, these specific bands can be confirmed that the common band that all the gastric cancer cell line exosomes have.

In addition, in order to confirm whether the exosome having the specific band is a substance capable of effectively presenting the antigen, the expression pattern of MHC class 1, which is a specific molecule of antigen presenting cells, can be confirmed by Western blotting using an antibody. .

In the present invention, in addition to the above-described SDS-PAGE and Western blotting, the method for immunobiochemical analysis of the exosomes measures the amount of protein expressed in the exosomes and further analyzes using magnetic particles to test their stability. Can be done. That is, exosomes were measured by measuring the amount of proteins including MHC class 1 and 2 expressed in exosomes isolated from gastric cancer cell lines and exosomes refrigerated for a certain period of time after separation using beads without exosomes as a control. It is possible to determine whether this ability is capable of presenting the antigen effectively and how stable the isolated exosomes are.

3) Activation of Exosomal Specific T Cells

The following method can be used to confirm whether the exosomes which have been separated and immunobiochemically analyzed by the above-described method can effectively activate human T lymphocytes.

In general, T lymphocytes cause clonal expansion when a specific antigen is stimulated, and the stimulated T lymphocytes first form blasts that grow in size. Therefore, by confirming whether the exosomes isolated and immunobiochemically analyzed according to the above method can stimulate T lymphocytes to form blasts, it is possible to know whether T lymphocytes are activated on the isolated exosomes. Subcellular formation of can be observed by flow cytometry by staining with CD4 ⁺ , CD8 ⁺ antibody. In this case, it is preferable to use autologous serum in the present invention so as to avoid nonspecific stimulation against serum in T lymphocyte culture.

When stimulating T lymphocytes with the exosomes according to the present invention it can be seen that the formation of CD4 ⁺ T lymphocytes is much more than when stimulated with cell lysates or media (media), whereby the exosomes according to the present invention is human Activation of T lymphocytes can have a therapeutic effect on tumor cells.

Exosome according to the present invention can be inoculated by subcutaneous injection or a small amount of cytokine at the same time by adding a suitable adjuants or additives (carrier) suitable for administration to the human body. Adjuvant may include complete Freund's commonly used in microbial vaccines or KHL, QS-21 or alum, which is currently being clinically tested for cancer vaccines in the United States. When the exosome vaccine is administered to a living body, tumor-specific antigen peptides of exosomes are effectively presented to MHC class 1 antigen, which is an antigen presenting cell, and T lymphocyte receptors are accepted to induce activation of killing T lymphocytes and to immunize tumor cells. The action is initiated, thereby treating the tumor.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are for illustration only and should not be understood as limiting the scope of the invention.

EXAMPLE

1.Isolation of Exosomes from Gastric Cancer Cell Lines

35 ml of fresh medium per 5 × 10 ⁶ cells from gastric cancer tumor cell lines were incubated for 48 hours in a T75 flask, and then the supernatant of the medium was taken into a conical tube and centrifuged at 300 g for 5 minutes. Discard the resulting cell pellet, take only the supernatant, transfer to a new conical tube, centrifuge at 1,200 g for 20 minutes, centrifuge the separated supernatant again at 10,000 g for 30 minutes, and then centrifuge the separated supernatant at 95,000 g for 60 minutes. To obtain an exosome pellet. The obtained exosome pellet was washed with PBS to remove impurities, and centrifuged again at 95,000 g for 60 minutes to finally obtain an exosome pellet.

2. Immunohistochemical Analysis of Isolated Exosomes

The expression of MHC class 1 of the exo-some according to the present invention was examined by Western blotting of the exo-some isolated in step 1 using an antibody against gastric cancer specific antigen.

20 ug each of the exosomes and cell lysates separated in Example 1 were subjected to Western blotting, and the separated proteins were transferred to a nylon membrane. Each antibody that recognizes MHC, HSP70, LMP1, and EBNA2 proteins was treated to identify proteins that were recognized by chemophotometry.

The result of western blotting is shown in FIG. Exosomes obtained in Example 1 can be seen that the expression of MHC class 1 more than the cell lysate, it can be seen that through the MHC class 1 can present antigen to T lymphocytes specifically MHC. In addition, it can be seen that the exosome contains a large amount of HSP70 having the function of entering the antigen presenting cells and effectively presenting the antigens, and activating the antigen presenting cells.

3. Confirmation step of antigen presentation ability and stability of exo

In order to confirm the antigen presenting ability and stability of exosomes, exosomes isolated from human B cell lines and exosomes refrigerated for 20 days after separation were used. Each of these two types of exosomes were mixed with DALAL beads (M-450) with MHC class 1 antibodies, and then incubated at room temperature for 24 hours to remove bead-free exosomes. The exosome-bead complex thus obtained was stained with anti-HLA-DR, anti-HLA-ABC, and anti-CD80, respectively, and the results were analyzed by flow cytometry. In this case, beads without addition of exosomes were used as controls. The results are shown in FIG. Exosome immediately after separation contains a large amount of both MHC class 1, 2, and even about 20 days after separation, about half of the degradation (degradation) occurred over time, but beads without exo It was confirmed that much more MHC class 1, 2 than expressed in case of. As a result, the exosome of the present invention has the ability to present the antigen, was confirmed to be stable for a certain period after separation.

4. Measurement of T Lymphocyte Activity by Exosomes

To measure the activity of T lymphocytes by exosomes, exosomes were extracted from human B cell lines, and lymphocytes were isolated from peripheral blood of normal humans with almost identical HLA types. The isolated lymphocytes were placed in 6 well plates at 8 × 10 ⁶ cells / well. In order to suppress cell proliferation, the lymphocytes were cultured with the addition of irradiated B cell line at 2 × 10 ⁵ cells / well as a control and the addition of exosomes from the B cell line as a treatment group. On the 6th day of culture, 3 IU / ml rhIL-2 was added and the amount was increased from 5 days to 5 IU / ml rhIL-2 until 20 days. All cultures used normal human serum obtained when separating lymphocytes instead of animal serum. Activated lymphocytes induced stronger activity by adding SNU9 cell line or exosomes once more in the same manner as above at day 10. The degree of activation was observed by increasing the size of T lymphocytes with CD4 ⁺ , CD8 ⁺ antibodies at intervals of 0, 10, and 20 days with flow cytometry, and the results are shown in FIG. 5.

In Figure 1 it can be seen that the blast formation of CD4 + T lymphocytes by exosomes is 72% much more than when stimulated with cell lysate (48%) or culture (28%).

In the step of confirming the antigen-presenting ability of the exosomes and measuring the activity of T lymphocytes by the exosomes, the expression of antigen-presenting cells and the degree of activation of T lymphocytes were confirmed using exosomes isolated from B cell lines of normal individuals. Exosomes isolated from tumor cell lines show the same function or effect except that MHC class 2, a specific molecule of antigen presenting cells, is absent.

According to the above embodiment, it was confirmed that the exosomes isolated from gastric cancer tumor cell lines had specific bands of proteins, and the exosomes having the specific bands effectively expressed MHC class 1 antigen-presenting cells. In addition, the exosomes according to the present invention showed a therapeutic effect on tumor cells by effectively activating human T lymphocytes, and confirmed the effectiveness as a vaccine using the same.

According to the present invention, exosomes isolated from gastric cancer tumor cell lines were identified by immunobiochemical analysis and activation of T lymphocytes.

In addition, the cancer vaccine using the exosomes according to the present invention has the effect that can be widely administered to patients with gastric cancer, rather than individuals that are too segmented to meet the immune system that differs from patient to patient by eliminating the side effects of existing anticancer drugs.

Although the present invention has been described in detail only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the present invention, and such modifications and variations are included in the appended claims. Belonging is natural.

Claims (4)

Exosome for cancer treatment isolated from gastric cancer tumor cell line. The method of claim 1, The exosomes for cancer treatment, characterized in that the exosomes comprise MHC class 1 or HSP70 having biological activity. The method according to claim 1 or 2, The exosomes for cancer treatment, characterized in that the exosomes are separated from the gastric cancer cell line by a fractional centrifugal separation method. The vaccine for cancer treatment which added the usual adjuvants or an additive to the exosome of Claim 1.