WO2024058421A1 - Biological cancer treatment composition of natural killer cell-derived exosomes with enhanced cytokine activity, and use thereof - Google Patents

Abstract

The present invention relates to a biological cancer treatment composition of natural killer cell-derived exosomes with enhanced cytokine activity, and a use thereof, and more specifically, to a cancer treatment composition containing natural killer cell (NK cell)-derived exosomes pretreated with cytokines, and a method for treating cancer, wherein the composition can be independently used for treating cancer or used for treating various cancers.

Description

Biological composition of natural killer cell-derived exosomes with enhanced cytokine activity for cancer treatment and use thereof

This invention was made under the support of the Ministry of Science and ICT through project identification number 1711139881 and task number 2020R1A2C1099712. The research management agency for the project is the National Research Foundation of Korea, the research project name is "Mid-career Research Support Project", and the research project name is "Hepatocellular Cancer." "Study on the mechanism of action of NK cell-derived exosomes for treatment", the host institution is the Korea Micromedical Robotics Research Institute, and the research period is 2020.09.01 ~ 2023.02.28.

This patent application claims priority to Korean Patent Application No. 10-2022-0115731 filed with the Korean Intellectual Property Office on September 14, 2022, the disclosure of which is incorporated herein by reference.

The present invention relates to a biological composition for cancer treatment of exosomes derived from natural killer cells with enhanced cytokine activity and their use, and more specifically, to exosomes derived from natural killer cells (NK cells) pretreated with cytokines. It relates to a composition and treatment method for treating cancer containing exosomes.

The first generation (chemical anticancer drugs), second generation (targeted anticancer drugs), and third generation (immunoanticancer drugs), which are existing anticancer drugs for treating cancer, each have their own side effects and problems. Specifically, first-generation chemical anti-cancer drugs attack cancer cells directly, so there are side effects in which drug toxicity also attacks normal cells, while second-generation targeted anti-cancer drugs attack cancer cell-specific target factors, so their scope of application is limited. Third-generation immunotherapy drugs have no side effects by strengthening the cancer patient-specific immune function, but have a very low immunotherapy response rate of around 20%.

In our body's immune system, natural killer cells contain various proteins such as various immune receptors, cell killing factors, and cytokines, and can directly attack cancer cells through receptor-ligand interaction. Accordingly, natural killer cells play an important role in anticancer treatment that prevents the occurrence, proliferation, metastasis, and recurrence of cancer. However, because the activation mechanisms are very diverse, it is difficult to control the activation. In particular, in treatments targeting solid cancer, it has been reported that natural killer cells have a low efficiency in accessing cancer tissue and have poor anticancer immune activity.

To overcome the above problems, research using exosomes derived from natural killer cells has been actively conducted recently. Exosomes, a type of extracellular vesicle, secrete various cell-specific components and substances into the external environment for the purpose of information exchange. They do not have the potential to self-replicate or cause cancer, and can overcome the shortcomings of existing cell therapies. It is receiving new attention in the domestic and international pharmaceutical markets.

Accordingly, recently, there has been a need for a new exosome-based cancer treatment drug that can target solid cancers and improve the low access efficiency (targeting efficiency) and low immune activity of natural killer cells to cancer tissues. There is a situation. Accordingly, the present invention proposes strengthening the high immune activity of natural killer cell-derived exosomes through cytokines IL-15 and IL-21, and these natural killer cell-derived exosomes complement the shortcomings of existing anticancer drugs and protect against cancerous tissue. It is expected that targeting performance will be improved and selective delivery to cancer tissue will be possible.

Accordingly, the present inventors developed a composition for cancer treatment containing exosomes derived from natural killer cells (NK cells) pretreated with cytokines, and confirmed that its anticancer activity was significantly superior.

Accordingly, the object of the present invention relates to a composition for treating cancer containing exosomes derived from natural killer cells.

Another object of the present invention is to provide a method for producing exosomes derived from natural killer cells.

Another object of the present invention relates to the use of exosomes derived from natural killer cells.

The present invention relates to a biological composition for the treatment of cancer using exosomes derived from natural killer cells with enhanced cytokine activity and their use. It was confirmed that the exosomes derived from natural killer cells with enhanced cytokine activity in the present invention exhibit high anticancer activity. .

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a pharmaceutical composition for treating, preventing, alleviating or inhibiting cancer, comprising exosomes isolated from natural killer cells (NK cells) as an active ingredient.

The term “exosome” as used herein refers to a cell-derived endoplasmic reticulum that exists in the body fluids of almost all eukaryotic organisms and has a diameter of about 30-100 nm, which is larger than LDL protein but much smaller than red blood cells. It is well known that exosomes can be released from cells when multivesicular bodies fuse with the cell membrane, or can be released directly from the cell membrane, and perform important and specialized functions such as coagulation and intercellular signaling.

As used herein, the term “natural killer cell” refers to an important lymphoid cell responsible for innate immunity. It accounts for 5-10% of all lymphoid cells and, unlike T cells, matures in the liver or bone marrow. Natural killer cells are known to be able to distinguish between normal and abnormal cells by expressing various innate immune receptors on the cell surface, and are known to be able to immediately attack and eliminate target cells such as virus-infected cells or tumor cells. . Natural killer cells that recognize abnormal cells secrete perforin to puncture the cell membrane of the target cell, secrete granzymes within the cell membrane to break up the cytoplasm, causing apoptosis, or inject water and salt into the cell. This causes cell necrosis. Additionally, as an indirect method, cytotoxic T cells and B cells can be activated by secreting cytokines.

In one embodiment of the present invention, natural killer cells may be pretreated with a pretreatment material.

The term “pretreatment” in this specification refers to the process of adding a specific substance to a medium for culturing natural killer cells and culturing them. For example, it may refer to the process of culturing natural killer cells in a medium containing cytokines, and more specifically, the process of culturing natural killer cells in a medium containing added interleukin 15 and interleukin 21. It can mean.

The term “cytokine” in this specification refers to proteins secreted by immune cells. After cytokines are secreted from a cell, they can affect other cells or the secreting cell itself. In other words, it induces the proliferation of macrophages or promotes the differentiation of secretory cells themselves. Cytokines act through receptors and are particularly important in the immune system. Cytokines are important for maintaining health and disease, especially in the body's response to infection, immune response, inflammation, trauma, sepsis, cancer, and reproduction.

In one embodiment of the present invention, the pretreatment material may be a cytokine.

In one embodiment of the present invention, the pretreatment material may be Interleukin 15 and Interleukin 21.

As used herein, the term “containing as an active ingredient” refers to an amount sufficient to achieve the alleviation, inhibition, prevention or treatment activity against a specific disease of exosomes isolated from natural killer cells or pre-treated natural killer cells. it means.

In the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, silicic acid. It may contain calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It is not limited.

In the present invention, the pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

In the present invention, the pharmaceutical composition can be administered orally and parenterally, for example, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, intrathecal administration, eye. It can be administered by administration, dermal administration, transdermal administration, etc., but is not limited thereto.

In the present invention, the dosage of the pharmaceutical composition may be determined in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. It can be determined or prescribed at an effective dosage for desired treatment or prevention. For example, the daily dosage of the pharmaceutical composition of the present invention may be 0.0001-1000 mg/kg.

In the present invention, the pharmaceutical composition is formulated in unit dosage form using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art. It can be manufactured or manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer, but is limited thereto. That is not the case.

The administered dose of the pharmaceutical composition of the present invention may vary depending on the patient's age, weight, gender, dosage form, health condition, and disease level, and is administered once or several times a day at regular time intervals according to the judgment of a doctor or pharmacist. It may also be administered in divided doses. For example, the daily dosage may be 1 to 1000 μg/ml based on the active ingredient content, but this is an example of an average case and the dosage may be higher or lower depending on individual differences.

The term “cancer treatment” as used herein refers to all actions that inhibit cancer, and may specifically mean the action of regulating the cycle of cancer cells or the action of inducing apoptosis of cancer cells, but is not limited thereto. .

The term “prevention” in this specification refers to all actions that suppress or delay the onset of inflammation by administering the pharmaceutical composition according to the present invention.

As used herein, the term "improvement" means any action that results in at least reducing the degree of a parameter, such as a symptom, associated with the condition being treated by administering the composition according to the present invention.

In one embodiment of the present invention, the cancer is breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye melanoma, uterine sarcoma, ovarian cancer, and rectal cancer. , a group consisting of anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer, glioblastoma, or bone marrow cancer. It may be one or more selected from , but is not limited thereto.

In one embodiment of the present invention, the cancer may be liver cancer, but is not limited thereto.

Another aspect of the present invention is a food composition for alleviating, suppressing or improving cancer, comprising exosomes isolated from natural killer cells (NK cells) as an active ingredient.

Since the food composition according to the present invention contains exosomes derived from natural killer cells according to the present invention in the same way as the pharmaceutical composition described above, the description of common information between the two is omitted to avoid excessive complexity of the present specification. .

The food composition according to the present invention may include ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavorings, but is not limited thereto. .

Carbohydrates that can be included in the food composition according to the present invention include monosaccharides such as glucose and fructose, disaccharides such as maltose, sucrose, and oligosaccharides, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol, and erythritol. Sugar alcohols such as sugar alcohols may be included, but are not limited thereto.

Flavoring agents that may be included in the food composition according to the present invention may include natural flavoring agents such as thaumatin and stevia extract and synthetic flavoring agents such as saccharin and aspartame, but are not limited thereto.

The present invention relates to a biological composition for cancer treatment of exosomes derived from natural killer cells with enhanced cytokine activity and their use, and more specifically, to exosomes derived from natural killer cells (NK cells) pretreated with cytokines. It relates to a composition and treatment method for cancer treatment containing exosomes, and can be used independently for the treatment of cancer or for the treatment of various cancers.

Figure 1 is a diagram showing the results of screening for cytokine activity of natural killer cells (NK cells) prepared according to an embodiment of the present invention.

Figure 2 is a diagram showing the results of quantifying cytokine activity of natural killer cells prepared according to an embodiment of the present invention through immunoblotting.

Figure 3 is a diagram showing the results of evaluating the activity of cytokines IL-15 and IL-21 against natural killer cells according to an embodiment of the present invention.

Figure 4 is a diagram showing the results of quantifying the activity of cytokines IL-15 and IL-21 against natural killer cells according to an embodiment of the present invention.

Figure 5 is a TEM image of exosomes derived from natural killer cells activated with cytokines IL-15 and IL-21 prepared according to an embodiment of the present invention.

Figure 6 is a graph of the NTA size distribution of exosomes derived from natural killer cells activated with cytokines IL-15 and IL-21 prepared according to an embodiment of the present invention.

Figure 7 is a DLS size distribution graph of exosomes derived from natural killer cells activated with cytokines IL-15 and IL-21 prepared according to an embodiment of the present invention.

Figure 8 is a diagram showing the results of exosome marker confirmation through immunoblotting of exosomes derived from natural killer cells activated with cytokines IL-15 and IL-21 prepared according to an embodiment of the present invention.

Figure 9 is a diagram showing the results of confirmation of natural killer cell-specific markers through immunoblotting of exosomes derived from natural killer cells activated with cytokines IL-15 and IL-21 prepared according to an embodiment of the present invention.

Figure 10 shows quantification of natural killer cell-specific marker, perforin, through immunoblotting of exosomes derived from natural killer cells activated with cytokines IL-15 and IL-21 prepared according to an embodiment of the present invention. This is a drawing showing the results.

Figure 11 shows granzyme B, a natural killer cell-specific marker, through immunoblotting of exosomes derived from cytokines IL-15 and IL-21 activated natural killer cells prepared according to an embodiment of the present invention. This is a diagram showing the quantification results.

Figure 12 is a diagram showing the results of confirming the cytotoxicity of exosomes derived from natural killer cells activated with cytokines IL-15 and IL-21 prepared according to an embodiment of the present invention against hepatocellular carcinoma cell line (Hep3B).

Figure 13 is a diagram showing the results of a cell survival experiment on the hepatocellular carcinoma cell line (Hep3B) of exosomes derived from natural killer cells activated with cytokines IL-15 and IL-21 prepared according to an embodiment of the present invention.

Figure 14 is a diagram showing the results of a cell viability test on the hepatocellular carcinoma cell line (Hep3B) of exosomes derived from natural killer cells activated with cytokines IL-15 and IL-21 prepared according to an embodiment of the present invention.

Figure 15 is a diagram confirming the apoptosis effect of exosomes derived from natural killer cells activated with cytokines IL-15 and IL-21 prepared according to an embodiment of the present invention on hepatocellular carcinoma cell line (Hep3B).

Figure 16 is a diagram showing the results of quantification of apoptosis for hepatocellular carcinoma cell line (Hep3B) of exosomes derived from natural killer cells activated with cytokines IL-15 and IL-21 prepared according to an embodiment of the present invention.

Figure 17 is a diagram showing the results of confirming apoptosis-specific markers through immunoblotting of exosomes derived from natural killer cells activated with cytokines IL-15 and IL-21 prepared according to an embodiment of the present invention.

A pharmaceutical composition for treating, preventing, alleviating or suppressing cancer, comprising exosomes isolated from natural killer cells (NK cells) as an active ingredient.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Isolation of exosomes derived from natural killer cells activated with cytokines IL-15 and IL-21

Natural killer cells (NK-92) were purchased from ATCC (American Type Culture Collection) and mixed with 12.5% fetal bovine serum (FBS; Corning) and 12.5% horse serum (based on the total volume of medium). HS; Sigma-Aldrich) and 1% antibiotics, 0.2mM inositol (Sigma-Aldrich), 0.1mM 2-mercaptoethaol (Sigma-Aldrich), 0.02mM folic acid (Sigma) -Aldrich) and 100 IU/mL interleukin-2 (IL-2; Miltenyi Biotec) were cultured in MEM alpha (Thermofisher Scientific) medium for 3 days. All cells were cultured under 5% CO ₂ and 37°C incubator conditions.

Afterwards, the cytokines IL-15 and IL-21 were pretreated at a concentration of 10 ng/ml in the culture medium of NK-92 cells. After 48 hours, the cell culture supernatant was recovered and centrifuged to remove cells and debris.

To remove extracellular vesicles basically contained in each serum (fetal bovine serum and horse serum), they were centrifuged at 4°C and 100,000 g for 18 hours. Fetal bovine serum and horse serum from which extracellular vesicles were removed were cultured in natural killer cell culture medium at 37°C and 5% CO ₂ for 3 to 5 days.

After incubation, the supernatant was collected and centrifuged at 300 g for 10 minutes to remove cells, and the supernatant was centrifuged again at 2,000 g for 10 minutes to filter out cell destruction and dead cells. The supernatant was then centrifuged at 10,000 g for 30 minutes to obtain a medium containing extracellular vesicles and no apoptotic bodies.

The medium containing extracellular vesicles was concentrated to a volume ratio of 1/5 to 1/10 while removing molecules smaller than the size of the extracellular vesicles using tangential flow filtration (TFF). The culture medium containing concentrated extracellular vesicles was centrifuged at 100,000 g and 4°C for 90 minutes using an ultra-high-speed centrifuge to obtain an extracellular vesicle pellet. After washing the extracellular vesicle pellet once using PBS (phosphate buffer saline), the extracellular vesicles were separated by dispersing them in about 1 mL of PBS.

Example 2: Evaluation of the activity of cytokine-activated natural killer cells

To find the combination of various types of cytokines that most effectively activate natural killer cells, the expression of related proteins that induce cytotoxicity was screened using immunoblotting. β-actin was used as the standard protein.

The cytoplasm of the natural killer cell group pretreated with various types of cytokines was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and then incubated with each primary antibody (Perforin, Granzyme B, β-actin (Santa Cruz Biotechnology, Dallas). , TX, USA) and re-cultured with HRP-conjugated secondary antibody. Expression of cytotoxicity-related proteins was visualized using ECL detection reagent and quantified using ImageJ (Ver.1.53e, NIH, Bethesda, MD, USA) software.

As a result, as can be seen in Figures 1 to 4, the expression of natural killer cell-specific proteins (Perforin, Granzyme B) showing cytotoxic ability in natural killer cells simultaneously pretreated with the cytokines IL-15 and IL-21. appeared highest.

In other words, simultaneous treatment with cytokines IL-15 and IL-21 can increase the activity of natural killer cells to the highest degree. From the above results, when inducing apoptosis of cancer cells, the above combination is most suitable and has the highest effect. It was assumed that it would be induced

Example 3: Size and distribution of exosomes derived from cytokine-activated natural killer cells

Exosomes derived from cytokine-activated natural killer cells To confirm proper separation, 1 to 10 uL of exosome solution was placed on a Formvar-coated TEM grid (TMA), dried, and then analyzed using a transmission electron microscope (TEM). The shape was visualized. As can be seen in Figure 5, exosomes derived from cytokine-activated natural killer cells (NK-exos, NK-exos ^IL-15 , NK-exos ^IL-21 , NK-exos ^IL-15/21 ) are well separated in a spherical shape. It has been done.

The size of exosomes was confirmed through nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) methods.

First, the size and number of exosomes (10 uL, diluted 1:100 in PBS) through nanoparticle tracking analysis (NTA) was determined using a Nanosight LM10 system equipped with a 405 nm laser (Malvern Ins., Malvern, UK) was used for analysis. Data were collected and analyzed using NTA Software (Nanosight version 3.1; Malvern), and each experiment was repeated three times. As can be seen in Figure 6, respectively (NK-exos: 148.3 ± 64.1 nm, NK-exos ^IL-15 : 148.0 ± 67.2 nm, NK-exos ^IL-21 : 141.3 ± 56.5 nm, NK-exos ^IL-15/21 : 157.0 ± 53.2 nm).

Next, Zetasizer Nano ZS (Malvern Ins., Malvern, UK) equipped with a 633 nm laser was used to analyze exosome particle size through dynamic light scattering (DLS). Samples were placed in a square cuvette (1 mL, diluted 1:100 with PBS) before analysis, and each measurement was repeated three times. As can be seen in Figure 7, respectively (NK-exos: 103.4 ± 69.6 nm, NK-exos ^IL-15 : 107.0 ± 87.2 nm, NK-exos ^IL-21 : 104.2 ± 65.3 nm, NK-exos ^IL-15/21 : 102.1 ± 89.4 nm).

Compared to the control group (NK-exos), there was no difference in exosome size between the cytokine activation groups (NK-exos ^IL-15 , NK-exos ^IL-21 , NK-exos ^IL-15/21 ).

Example 4: Confirmation of characteristics of exosomes derived from cytokine-activated natural killer cells

To confirm whether exosomes derived from isolated cytokine-activated natural killer cells express specific markers of extracellular vesicles and the same markers as parent natural killer cells, protein immunoblotting was performed using GAPDH as a standard protein. carried out.

Cytosolic or exosomal proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. Afterwards, the cells were incubated with each primary antibody (CD9, CD63, Perforin, Granzyme B, GAPDH), and incubated once again with the HRP-conjugated secondary antibody. Protein marker expression was visualized using ECL detection reagent and quantified using ImageJ software.

As a result of the experiment, as can be seen in Figures 8 and 9, the specific markers of extracellular vesicles (CD9, CD63) were hardly expressed in natural killer cells, and the level of expression was high only in exosomes derived from cytokine-activated natural killer cells. was confirmed. In addition, specific markers (perforin, granzyme B) of natural killer cells, which are parent cells, were observed to be expressed equally in exosomes derived from parent cells and cytokine-activated natural killer cells, confirming that the extracellular vesicles were derived from natural killer cells. .

In addition, as can be seen in Figures 10 and 11, the control group (NK-exos) and the cytokine activation group (NK-exos ^IL-15 , NK-exos ^IL-21 , NK-exos ^IL-15/21 ) were compared. It was confirmed that natural killer cell-specific proteins (perforin, Granzyme B) were expressed at the highest level in the group pretreated simultaneously with IL-15 and IL-21 (NK-exos ^IL-15/21 ). This activates cytokines This suggests the possibility of improving the cytotoxic ability of exosomes derived from natural killer cells (NK-exos ^IL-15/21 ).

Example 5: Confirmation of toxicity and proliferation inhibitory effects of exosomes derived from cytokine-activated natural killer cells on cancer cells

To confirm the cell viability of exosomes derived from cytokine-activated natural killer cells against the liver cancer cell line (Hep3B), live cell activity was first measured using a Live/Dead staining kit ( Thermo Fisher Scientific ). Hep3B was cultured at 1 x 10 ⁴ each in a confocal dish and a 96-well plate for one day to induce adhesion.

Natural killer cell-derived exosomes (NK-exos), IL-15 treated exosomes (NK-exos ^IL-15 ), IL-21 treated exosomes (NK-exos ^IL-21 ), IL-15 and IL- 21 Add 50 μg of simultaneously processed exosomes (NK-exos ^IL-15/21 ) into each well, and use Calcein-AM, which selectively shows strong green fluorescence only in living cells, to measure live cell activity after 24 hours. It was detected. The number of surviving cells was visualized with a fluorescence image, and the fluorescence value was quantified using ImageJ software, and the results are shown in Figures 12 to 13 and Table 1.

As can be seen in Figures 12 to 13 and Table 1, compared to natural killer cell-derived exosomes (NK-exos), cytokine-activated natural killer cell-derived exosomes (NK-exos ^IL-15 , NK-exos ^IL-21 , NK-exos ^IL-15/21 ) decreased Hep3B cell survival rate, especially when compared with the group treated with IL-15 and IL-21 alone (NK-exos ^IL-15 , NK-exos ^IL-21 ). When exosomes co-treated with IL-15 and IL-21 (NK-exos ^IL-15/21 ) showed the greatest decrease in cell viability.

experimental group	control	NK-exos	NK-exos IL-15	NK-exos IL-21	NK-exos IL-15/21
Surviving cells (%)	97.4	62.5	33.0	37.2	20.0

In addition, natural killer cell-derived exosomes (NK-exos), IL-15 treated exosomes (NK-exos ^IL-15 ), IL-21 treated exosomes (NK-exos ^IL-21 ), IL-15 and 50 μg of exosomes (NK-exos ^IL-15/21 ) co-treated with IL-21 were added to each well, and after 24 hours, the medium was removed, dead cells were removed with phosphate buffer solution, and MTT (3-(4) , 10 μL of 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was applied to each well and MTT assay was performed. After 3 hours, the solution was removed, 100 μL of DMSO was added to each well, and the absorbance at 570 nm was measured using a microplate reader. The results are shown in Figure 14 and Table 2.

As a result of the experiment, as can be seen in Figure 14 and Table 2, the survival rate of the liver cancer cell line (Hep3B) treated alone with natural killer cell-derived exosomes (NK-exos) decreased to 91.6%, and the survival rate of the exosomes treated with IL-15 ( The survival rate of NK-exos ^IL-15 ) was 65.8%, the survival rate of exosomes treated with IL-21 (NK-exos ^IL-21 ) was 62.5%, and the survival rate of exosomes simultaneously treated with IL-15 and IL-21 (NK- The survival rate of exos ^IL-15/21 ) was significantly reduced to 47.0%.

In other words, natural killer cell-derived exosomes co-treated with IL-15 and IL-21 had improved cytotoxic ability and showed the highest proliferation inhibitory effect on liver cancer cell line (Hep3B), confirming that the anticancer effect was enhanced.

experimental group	control	NK-exos	NK-exos IL-15	NK-exos IL-21	NK-exos IL-15/21
Cell viability (%)	100	91.6	65.8	62.5	47.0

Example 6: Confirmation of apoptosis effect of exosomes derived from cytokine-activated natural killer cells on cancer cells

To confirm the apoptotic effect of exosomes derived from cytokine-activated natural killer cells on liver cancer cell line (Hep3B), flow cytometry and immunoblotting were used.

First, the level of cell death was measured by flow cytometry. Hep3B (1x106) was grown in a ⁶ -well plate with 50 μg each of natural killer cell-derived exosomes (NK-exos), IL-15-treated exosomes (NK-exos ^IL-15 ), and IL-21-treated exosomes ( NK-exos ^IL-21 ), exosomes co-treated with IL-15 and IL-21 (NK-exos ^IL-15/21 ). After 24 hours, each cell group was resuspended in fluorescein isothiocyanate (FITC)-conjugated Annexin V (Annexin V-FITC) binding buffer and stained with Annexin V and propidium iodide (PI) for 15 minutes in the dark. Flow cytometry was performed on each stained cell group within 2 hours, and the results are shown in Figures 15 and 16.

As can be seen in Figures 15 to 16 and Table 3, natural killer cell-derived exosomes (NK-exos IL ^{-15/21) pretreated simultaneously with IL-15 and IL-21} induce apoptosis (Hep3B) in hepatocellular carcinoma cell line (Hep3B). It showed the highest effect by inducing about 60% of apoptosis.

experimental group	control	NK-exos	NK-exos IL-15	NK-exos IL-21	NK-exos IL-15/21
Cell necrosis rate (%) (Necrosis)	0.00	0.70	0.70	0.66	0.24
Cell death rate (%) (Apoptosis)	0.00	22.74	38.07	38.84	60.36
Cell viability (%) (Living cells)	100	76.56	61.23	60.50	39.40

Additionally, the expression levels of specific proteins involved in apoptosis were measured by immunoblotting. Hep3B (1x106) was grown in a ⁶ -well plate with 50 μg each of natural killer cell-derived exosomes (NK-exos), IL-15-treated exosomes (NK-exos ^IL-15 ), and IL-21-treated exosomes ( NK-exos ^IL-21 ), exosomes co-treated with IL-15 and IL-21 (NK-exos ^IL-15/21 ) were cultured together. After 24 hours, the cytoplasmic proteins of each cell group recovered were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Then, each primary antibody (Bax, Bcl-2, Clv-PARP (Cell Signaling Technology, Danvers, MA, USA), Perforin, Granzyme B, β-actin, CD63 (Santa Cruz Biotechnology, Dallas, TX, USA)) and incubated again with HRP-conjugated secondary antibody. Protein marker expression was detected using an ECL detection reagent, and the results are shown in Figure 17.

As can be seen in Figure 17, exosomes derived from natural killer cells (NK-exos ^IL-15/21 ) simultaneously pretreated with IL-15 and IL-21 most effectively kill apoptotic proteins (Bax, Clv-PARP) in Hep3B cells. , perforin, and granzyme B), and suppressed anti-apoptotic protein (Bcl-2).

In other words, from the above results, natural killer cell-derived exosomes (NK-exos ^IL-15/21 ) pretreated simultaneously with IL-15 and IL-21 induced an improvement in the activity of important cytotoxic factors, and thus hepatocellular carcinoma cell lines ( It was confirmed that the anti-cancer efficacy of Hep3B) was increased.

The present invention relates to a biological composition for cancer treatment of exosomes derived from natural killer cells with enhanced cytokine activity and their use, and more specifically, to exosomes derived from natural killer cells (NK cells) pretreated with cytokines. It relates to a composition and treatment method for treating cancer containing exosomes.

Claims (11)

1. A pharmaceutical composition for treating, preventing, alleviating or suppressing cancer, comprising exosomes isolated from natural killer cells (NK cells) as an active ingredient.
2. The pharmaceutical composition for treating, preventing, alleviating or inhibiting cancer according to claim 1, wherein the natural killer cells are pretreated with a pretreatment material.
3. The pharmaceutical composition for treating, preventing, alleviating or inhibiting cancer according to claim 2, wherein the pretreatment material is a cytokine.
4. The pharmaceutical composition for treating, preventing, alleviating or inhibiting cancer according to claim 3, wherein the pretreatment material is Interleukin 15 and Interleukin 21.
5. The method of claim 1, wherein the cancer is breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye melanoma, uterine sarcoma, ovarian cancer, rectal cancer, In the group consisting of anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer, glioblastoma, or bone marrow cancer. A pharmaceutical composition for the treatment, prevention, alleviation or inhibition of cancer, which is one or more selected.
6. The pharmaceutical composition for treating, preventing, alleviating or suppressing cancer according to claim 5, wherein the cancer is liver cancer.
7. A food composition for alleviating, suppressing or improving cancer, comprising exosomes isolated from natural killer cells (NK cells) as an active ingredient.
8. The food composition for alleviating, suppressing or improving cancer according to claim 7, wherein the natural killer cells are pretreated with a pretreatment material.
9. The food composition for alleviating, suppressing or improving cancer according to claim 8, wherein the pretreatment material is a cytokine.
10. The food composition for alleviating, suppressing or improving cancer according to claim 9, wherein the pretreatment material is Interleukin 15 and Interleukin 21.
11. The method of claim 7, wherein the cancer is breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye melanoma, uterine sarcoma, ovarian cancer, rectal cancer, In the group consisting of anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer, glioblastoma, or bone marrow cancer. A food composition for alleviating, suppressing or ameliorating cancer, which is one or more selected ones.

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