Composition for preventing or treating cancer diseases comprising cytotoxic T cells activated by extracellular vesicles derived from helper T cells as an active ingredient

Abstract

The present invention relates to a composition for preventing or treating cancer diseases, the composition comprising CD8+T cells activated by extracellular vesicles derived from CD4+T cells as an active ingredient. It was found that the extracellular vesicles secreted from cytokine-activated CD4+T cells increased, and the extracellular vesicles enhanced the proliferation and activity of CD8+T cells to induce cancer cell death, thereby enhancing the anti-cancer effect. Therefore, the present invention provides CD8+T cells activated by extracellular vesicles derived from CD4+T cells as a medicament or immunotherapeutic agent for cancer diseases, and a method for activating CD8+T cells using extracellular vesicles derived from CD4+T cells to prepare CD8+T cells exhibiting excellent anti-cancer activity as described above.

Description

Technical Field

[0001] The present invention relates to a composition for preventing or treating cancer diseases, comprising CD8+T cells activated by extracellular vesicles derived from CD4+T cells as an active ingredient. Background Art

[0002] Small extracellular vesicles (sEV) are small membranous vesicles secreted by most cells. The diameter of extracellular vesicles is about 30nm-100nm, and it contains various types of proteins, genetic material (DNA, RNA, miRNA), lipids, etc. derived from cells. Extracellular vesicles are released and secreted outside the cell, and they originate from a specific intracellular compartment called multivesicular body (MVB), rather than being directly separated from the plasma membrane. In other words, when the fusion of multivesicular body and plasma membrane occurs, vesicles are released into the extracellular environment, which is referred to as extracellular vesicles. Although it has not been determined exactly what mechanism extracellular vesicles are produced by, it is known that they are separated and released from various cell types under normal and pathological conditions.

[0003] Cancer is the result of uncontrolled and disordered cell proliferation caused by an abnormal excess of cells. From the perspective of molecular biology, cancer is a disease caused by genetic mutations. There are dozens of types of cancer that have been identified, and they are mainly classified according to the location of the diseased tissue. Cancer is divided into benign tumors and malignant tumors. Benign tumors grow relatively slowly and do not metastasize from their primary site to other tissues, while malignant tumors leave their primary site, invade other tissues, grow rapidly, and are life-threatening due to this feature. Most cancers are asymptomatic in the early stages, and even if there are symptoms, the symptoms are mild, so most people tend to ignore them, which increases the cancer mortality rate.

[0004] Surgical therapy, chemotherapy, radiotherapy, etc. are used to treat cancer, but it is reported that despite many studies, more than 50% of all cancer patients eventually die without being cured. The reason is that cancer relapses because metastatic cancer cells that are visible under a microscope after surgical resection are not eliminated; anticancer drugs do not induce cancer cell death; or although the tumor seems to shrink in the initial stage due to the response to anticancer drugs, cancer cells that develop resistance to anticancer drugs during or after treatment increase rapidly. Today, about 60 different anticancer drugs are used, and as knowledge about cancer occurrence and cancer cell characteristics is widely understood, research on the development of new anticancer drugs is actively being carried out. However, most anticancer drugs cause serious side effects (such as nausea and vomiting, hair loss, skin and nail discoloration, and nervous system side effects), and have the disadvantage of losing their therapeutic effects because cancer cells acquire resistance to anticancer drugs when they are repeatedly administered for a long time or when cancer recurs. In addition, radiotherapy induces cancer cell death by irradiating high-energy radiation to cancer tissue, but its disadvantage is that it damages normal tissues around cancer tissue and induces side effects.

[0005] Therefore, helper T cells activated by cytokine-derived extracellular vesicles can activate cytotoxic T cells, and the development of this technology can maximize the effects of cancer prevention and treatment.

[0006] [Prior art literature]

[0007] [Patent Document]

[0008] Patent Document 1: Korean Patent Application Publication No. 10-2018-0063841 (published on June 12, 2018) Summary of the invention

[0009] Technical issues

[0010] The present invention relates to a composition for preventing or treating cancer diseases, the composition comprising cytotoxic T cells (CD8+T cells) activated by extracellular vesicles derived from helper T cells (CD4+T cells) as an active ingredient. It has been found that the extracellular vesicles secreted from cytokine-activated CD4+T cells increase, and the extracellular vesicles enhance the proliferation and activity of CD8+T cells to induce cancer cell death, thereby enhancing the anti-cancer effect. CD8+T cells activated by extracellular vesicles derived from CD4+T cells can be usefully used as a composition for preventing or treating cancer diseases, a composition used as an immune anticancer drug, etc. Therefore, the present invention provides CD8+T cells activated by extracellular vesicles derived from CD4+T cells as a medicament or immunotherapeutic agent for cancer diseases, and a method for activating CD8+T cells using extracellular vesicles derived from CD4+T cells, to prepare CD8+T cells showing excellent anti-cancer activity as described above.

[0011] Technical Solution

[0012] The present invention provides a pharmaceutical composition for preventing or treating cancer diseases, the composition comprising CD8+T cells activated by extracellular vesicles derived from CD4+T cells as an active ingredient.

[0013] Furthermore, the present invention provides a composition for an immunological anticancer agent, comprising, as an active ingredient, CD8+ T cells activated by extracellular vesicles derived from CD4+ T cells.

[0014] In addition, the present invention provides a method for activating CD8+T cells, the method comprising: activating CD4+T cells by treating with cytokines; and treating the CD8+T cells with extracellular vesicles derived from the activated CD4+T cells.

[0015] Beneficial Effects

[0016] According to the present invention, it was found that the secretion of extracellular vesicles from cytokine-activated CD4+T cells increased, and the extracellular vesicles enhanced the proliferation and activity of CD8+T cells to induce cancer cell death, thereby enhancing the anti-cancer effect. Therefore, CD8+T cells activated by extracellular vesicles derived from CD4+T cells can be usefully used as a composition for preventing or treating cancer diseases, a composition of immune anticancer drugs, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The graph shows the increased secretion of extracellular vesicles from CD4+ T cells activated by IL-2 and the anti-cancer effect of CD8+ T cells activated by the extracellular vesicles.

[0018] Figure 2 Shown are the results of the validation of the increased secretion and extracellular vesicle size induced by IL-2 in CD4+ T cells and primary CD4+ T cells determined by nanoparticle tracking analysis.

[0019] Figure 3 Shown are the results of Western blot, real-time polymerase chain reaction and flow cytometry analysis to determine the increased expression of Rab5, Rab7, and Rab27a in CD4+ T cells and primary CD4+ T cells induced by IL-2.

[0020] Figure 4 and Figure 5 Shown are the results of determining the increase in proliferation and activity of CD8+ T cells and primary CD8+ T cells using extracellular vesicles derived from CD4+ T cells and primary CD4+ T cells activated by IL-2.

[0021] Figure 6Shown are results of determining the increase in anti-cancer activity of CD8+ T cells and primary CD8+ T cells using extracellular vesicles derived from CD4+ T cells and primary CD4+ T cells activated by IL-2. DETAILED DESCRIPTION

[0022] The terms used herein are selected as currently widely used general terms as much as possible in consideration of the functions of the present invention, but may change according to the intention of those skilled in the art or precedents, the emergence of new technologies, etc. In addition, in specific cases, there are terms arbitrarily selected by the applicant, and in this case, their meanings will be described in detail in the description of the corresponding invention. Therefore, the terms used herein should be defined according to the meaning of the terms and the overall content of the present invention, rather than just according to the names of the terms.

[0023] Unless otherwise defined, all terms (including technical or scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Common terms (such as those defined in dictionaries) should be interpreted as having a meaning consistent with that in the context of the relevant art, and should not be interpreted as an ideal or overly formal meaning unless explicitly defined in this application.

[0024] Numerical ranges include the values ​​defined within the ranges. Each maximum numerical limitation given throughout this specification includes any lower numerical limitations, as if the lower numerical limitations were explicitly written. Each minimum numerical limitation given throughout this specification includes any higher numerical limitations, as if the higher numerical limitations were explicitly written. Each numerical limitation given throughout this specification will include any narrower numerical range within the broader numerical range, as if the narrower numerical limitations were explicitly written.

[0025] Hereinafter, the present invention will be described in more detail.

[0026] like Figure 1 As shown, the inventors of the present invention determined that when CD4+T cells are treated with cytokines, the secretion of extracellular vesicles increases, and when CD8+T cells are treated with extracellular vesicles derived from CD4+T cells, CD8+T cells exhibit a stronger effect than their anticancer effect in a normal state. In other words, the present invention is accomplished by determining that extracellular vesicles increase the proliferation ability and cytotoxic ability of CD8+T cells to induce cancer cell death, thereby enhancing the anticancer effect.

[0027] The present invention provides a pharmaceutical composition for preventing or treating cancer diseases, the composition comprising CD8+T cells activated by extracellular vesicles derived from CD4+T cells as an active ingredient.

[0028] CD4+T cells are activated by cytokines, and the cytokines may be one or more selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, and IL-2 is preferred, but not limited thereto.

[0029] Extracellular vesicles can increase the proliferation of CD8+ T cells and increase the expression of IFN-γ, perforin, and granzyme B, which are markers of cytotoxicity, thereby enhancing the anti-cancer effect.

[0030] The extracellular vesicle is a small extracellular vesicle (sEV) having a diameter of 50-100 nm, and may include exosomes and microvesicles, but it should be noted that the present invention is not limited thereto.

[0031] The cancer disease may be selected from the group consisting of, but not limited to, melanoma, skin cancer, lung cancer, liver cancer, stomach cancer, pancreatic cancer, bone cancer, head and neck cancer, uterine cancer, ovarian cancer, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hawkins disease, esophageal cancer, small intestine cancer, colorectal cancer, colon cancer, rectal cancer, perianal cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic leukemia or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumors, brain stem gliomas and pituitary adenomas.

[0032] In the case where the composition of the present invention is a pharmaceutical composition, in addition to the above-mentioned active ingredient, it can also include a pharmaceutically acceptable carrier, excipient or diluent for administration. Carrier, excipient and diluent can include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate / ester, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0033] The pharmaceutical composition of the present invention can be prepared into oral dosage forms (such as powders, granules, tablets, capsules, suspensions, emulsions, syrups or aerosols), or external preparations, suppositories or sterile injections and used according to conventional methods. Specifically, during preparation, diluents or excipients, such as commonly used fillers, loosening agents, adhesives, wetting agents, disintegrants and surfactants can be used. Solid dosage forms for oral administration include, but are not limited to, tablets, pills, powders, granules and capsules. In addition to active ingredients, such solid dosage forms can also be prepared by mixing one or more excipients (such as starch, calcium carbonate, sucrose, lactose and gelatin). In addition to simple excipients, lubricants (such as magnesium stearate and talc) can also be used. In addition to liquids and liquid paraffin for oral use, various excipients (such as wetting agents, sweeteners, aromatics, preservatives) can also be added to prepare. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils (eg olive oil), and injectable esters (eg ethyl oleate) can be used. As bases for suppositories, Witepsol, Macrosol, Tween 61, cocoa butter, laurel fat, and glycerin gelatin can be used.

[0034] The suitable dosage of the pharmaceutical composition of the present invention varies according to the patient's condition and body weight, disease extent, drug form and time, but can be appropriately selected by those skilled in the art. The daily dose of the composition is preferably 0.001 mg / kg-50 mg / kg, and can be administered once a day or in divided doses as needed.

[0035] In addition, the present invention provides a method for activating CD8+T cells, the method comprising: activating CD4+T cells by treating with cytokines; and treating the CD8+T cells with extracellular vesicles derived from the activated CD4+T cells.

[0036] The cytokines in the step of activating CD4+ T cells may be selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, but are not limited thereto.

[0037] Example

[0038] Hereinafter, the present invention will be described in more detail using examples. These examples are only used to more specifically illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples.

[0039] Example 1: Cell culture

[0040] B16F10 cells (mouse melanoma) were cultured in DMEM medium (Hyclone) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (Hyclone), and CD4+T cells (human T lymphocytes) were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin. CD8+T cells (mouse cytotoxic T lymphocytes) were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin and streptomycin and 20 IU / mL recombinant IL-2 (R&D system). Primary CD4+T cells and primary CD8+T cells (isolated from PBMC) were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin.

[0041] Example 2: Isolation of primary CD4+ T cells and primary CD8+ T cells from human peripheral blood mononuclear cells

[0042] After collecting 50 mL of blood using a 50 mL syringe and transferring it to a vacuum tube (BD Vacutainer) containing heparin, the supernatant was removed by centrifugation at 550 × g for 10 minutes. After that, it was mixed with PBS at 1:1. Then, 5 mL of ficoll (GE Healthcare) was added to every 8 mL of blood mixed with PBS and centrifuged at 550 × g for 30 minutes. Then, the supernatant was removed and the buffy coat was transferred to a new 50 mL conical tube. After adding PBS to 30 mL, centrifuge at 550 × g for 10 minutes. Thereafter, the precipitate was mixed with RPMI 1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin and cultured on a 10 cm culture dish (Cat. 430167; Corning) for 16 hours.

[0043] After culturing human peripheral blood mononuclear cells for 16 hours, primary CD4+ T cells and primary CD8+ T cells were isolated using a human CD4+ T cell isolation kit (Cat. 130-096-533; MACS) and a human CD8+ T cell isolation kit (Cat. 130-096-495; MACS).

[0044] The number of human peripheral blood mononuclear cells cultured on 10 cm culture dishes (Cat. 430167; Corning) was counted by trypan blue staining, and then they were centrifuged at 550 × g for 10 minutes. 7 40 μL of Macs buffer was added every 10 7 Add 10 μL of biotin-Ab mixture to each well and mix, and incubate at 4°C for 5 min.7 20 μL of the bead mixture was added and mixed, and incubated at 4°C for 10 minutes. After that, the LS column was placed in the magnetic field of the MACS separator. After that, the column was washed 3 times with 1 mL of Macs buffer each time. Thereafter, the cell supernatant incubated for 10 minutes was placed in the column and the flow-through was received in a 5 mL tube. The above process was repeated 3 times to obtain a total of 3 mL of cell supernatant, which was centrifuged at 550 × g for 10 minutes. The precipitate was then mixed with RPMI 1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin and cultured on a 10 cm culture dish (Cat. 430167; Corning).

[0045] Example 3: Analysis of extracellular vesicles (sEVs) derived from CD4+ T cells activated by IL-2 and primary CD4+ T cells

[0046] 3-1. Nanoparticle tracking analysis

[0047] 2×10 6 CD4+T cells were added at a density of 100 μg / 10 cm culture dishes (Cat. 430167; Corning) and treated with various concentrations of IL-2 (0 IU / mL, 1 IU / mL, 10 IU / mL, 100 IU / mL, 500 IU / mL), and then cultured for 48 hours. Then, in order to collect extracellular vesicles, the culture medium was replaced with a medium not supplemented with FBS, and the cells were further cultured for 24 hours. The cell culture supernatant was recovered and centrifuged to remove cells and debris. Then, in order to isolate extracellular vesicles (CE) derived from CD4+T cells or extracellular vesicles (IL-2E) derived from IL-2-activated T cells, each supernatant obtained from the cells was centrifuged continuously at 300 × g, 2500 × g, and 10,000 × g. The supernatant was then filtered through a 0.2 μm syringe filter and centrifuged at 120,000 × g. The extracellular vesicle pellet was resuspended in PBS and centrifuged again at 120,000 × g. The purified pellet was resuspended in PBS or 1× cell lysis buffer for the next experiment. Thereafter, the size and concentration of extracellular vesicles were measured using Nanosight LM10 (Malvern Instruments) equipped with fast video capture and particle tracking software. The process was performed in the same manner in primary CD4+ T cells.

[0048] Results, such as Figure 2 As shown, in the case of IL-2 treatment of CD4+ T cells and primary CD4+ T cells, it was determined that the secretion of extracellular vesicles increased in a concentration-dependent manner.

[0049] 3-2. Flow cytometry

[0050] CD4+T cells and primary CD4+T cells were washed with PBS and then fixed with 4% paraformaldehyde for 15 minutes at room temperature. Then, PBS (Cat.SH30028.02; Hyclone) containing 0.1% Triton-X (CAS#.9002-93-1; Biosang) was added and reacted at room temperature for 10 minutes to penetrate the cells. After penetration, the samples were reacted with the primary antibody at 4°C for 1 hour, washed with PBS, reacted with the secondary antibody (1:2000 dilution) at 4°C for 1 hour, washed with PBS, and subjected to flow cytometry (FACSAria III; Becton Dickinson). Data were analyzed using FlowJo software (Flowjo, Ashland, OR, USA).

[0051] The primary antibodies used in this experiment were: Rab5 (ab18211, 1:100; Abcam), Rab7 (ab50533, 1:100; Abcam), and Rab27a (ab55667, 1:100; Abcam).

[0052] Results, such as Figure 3 As shown, in the case where CD4+ T cells and primary CD4+ T cells were treated with 500 IU / mL IL-2, the expression of Rab enzymes (Rab5, Rab7, Rab27a) involved in the production and secretion of extracellular vesicles was determined to be increased.

[0053] Example 4: Purification of extracellular vesicles (sEVs) from IL-2 activated CD4+ T cells and primary CD4+ T cells

[0054] CD4+ T cells or CD4+ T cells activated with IL-2 (500 IU / mL) were washed with phosphate-buffered saline (PBS) and further cultured in RPMI 1640 medium without FBS for 24 hours.

[0055] For extracellular vesicle purification, the cell culture supernatant was recovered by centrifugation at 1,200 rpm for 5 minutes at room temperature, and the supernatant was then centrifuged at 2000 × g for 30 minutes to remove cells and debris. Then, in order to separate extracellular vesicles (CE) derived from CD4+T cells or extracellular vesicles (IL-2E) derived from IL-2 activated T cells, the respective supernatants obtained from the cells were centrifuged continuously at 300 × g, 2500 × g and 10,000 × g. The supernatant was then filtered through a 0.2 μm syringe filter and centrifuged at 120,000 × g. The extracellular vesicle pellet was resuspended in PBS and centrifuged again at 120,000 × g. The purified pellet was resuspended in PBS or 1 × cell lysis buffer for the next experiment.

[0056] Example 5: Analysis of the anti-cancer effects of CD8+ T cells and primary CD8+ T cells activated by extracellular vesicles of CD4+ T cells and primary CD4+ T cells

[0057] 5-1. Flow cytometry

[0058] Next, CD8+ T cells were treated with extracellular vesicles purified from CD4+ T cells activated with IL-2 to evaluate the effects of extracellular vesicles. 5 CD8+T cells were seeded into 6-well plates at a density of 1 cell / well and cultured with CE (extracellular vesicles derived from CD4+T cells) or IL-2E (extracellular vesicles derived from IL-2-activated CD4+T cells) (25 μg / mL) for 24 hours. Then, flow cytometry was performed for Ki-67, IFN-γ, perforin, and granzyme B. In addition, primary CD8+T cells were treated with extracellular vesicles purified from primary CD4+T cells and flow cytometry was performed in the same manner as above.

[0059] The primary antibodies used in this experiment were: anti-Ki-67 (ab16667, 1:500; Abcam), perforin (ab16074, 1:500; Abcam), IFN-g (ab9657, 1:500; Abcam), and granzyme B (ab4059, 1:500; Abcam).

[0060] Results, such as Figure 4 As shown, when CD8+T cells were treated with extracellular vesicles (IL-2E) derived from IL-2 activated CD4+T cells, the expression of the cell proliferation marker Ki-67 protein was determined to be increased by about 7% compared with the control group. In addition, when primary CD8+T cells were treated with extracellular vesicles (IL-2E) derived from primary CD4+T cells activated by IL-2, the expression of the cell proliferation marker Ki-67 protein was determined to be increased by about 40% compared with the control group.

[0061] In addition, if Figure 5 As shown, when CD8+T cells were treated with extracellular vesicles (IL-2E) derived from IL-2 activated CD4+T cells and primary CD8+T cells were treated with extracellular vesicles (IL-2E) derived from primary CD4+T cells activated by IL-2, it was determined that the expression of IFN-γ, perforin, and granzyme B proteins, which are cytotoxicity markers in both groups, was also increased compared to the control group or the CE treatment group. In particular, perforin and granzyme B showed significant increases compared to the control group or the CE treatment group.

[0062] Therefore, it was determined that extracellular vesicles derived from IL-2-activated CD4+ T cells and extracellular vesicles derived from IL-2-activated primary CD4+ T cells (IL-2E) enhanced the cytotoxic capacity of CD8+ T cells, thereby enhancing the anti-cancer effect.

[0063] 5-2. Real-time polymerase chain reaction

[0064] After extracting mRNA from CD8+T cells (MiniBEST Universal RNA Extraction Kit, #9767; TaKaRa), the concentration of mRNA was measured using nanodrop (DS-11 series spectrophotometer; DeNovix). After synthesizing cDNA with 100 ng mRNA (PrimeScriptTM 1st strand cDNA Synthesis Kit, #6110A; TaKaRa), gene expression was detected using TBGreenTM Premix Ex TaqTM (Tli RNaseH Plus) Kit (#RR420A; TaKaRa).

[0065] The primers used in this experiment are shown in Table 1 below.

[0066] Table 1

[0067]

[0068] Real-time polymerase chain reaction was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). The relative mRNA levels of the samples were calculated by Ct (comparative threshold cycle) analysis after normalization to the amount of GAPDH in the same sample and expressed as 2 modified from the initial Ct value. -△△Ct value.

[0069] Results, such as Figure 4 As shown, when CD8+ T cells were treated with extracellular vesicles from primary CD4+ T cells, it was determined that the mRNA expression level of Ki-67, a T cell proliferation marker, was increased. Figure 5 As shown, when CD8+ T cells were treated with extracellular vesicles from primary CD4+ T cells, the mRNA expression levels of IFN-γ, granzyme B, and perforin, which are markers of T cell activation, increased. This means that the ability of CD8+ T cells to attack cancer cells is enhanced, thereby enhancing the anti-cancer effect.

[0070] Example 6: Analysis of co-culture of CD8+ T cells pretreated with extracellular vesicles and melanoma cells

[0071] In 1×10 5After CD8+T cells were seeded into 6-well plates at a density of cells / well, they were pretreated with CE (extracellular vesicles derived from CD4+T cells) or IL-2E (extracellular vesicles derived from CD4+T cells activated by IL-2) (25μg / mL). After 24 hours of culture, the cells were cultured in 96-well plates with melanoma cells at different ratios (1:1 to 1:10) for 72 hours. At the end of the culture, MTS (#G3582; Promega) reagent was added to each well and cultured at 37°C for 2 hours. Afterwards, the MTS reagent containing the supernatant of each well was removed, and the absorbance was measured at 490nm using a microplate reader. In addition, in the same manner, CD8+T cells isolated from human PBMCs were pretreated with CE (extracellular vesicles derived from primary CD4+T cells) or IL-2E (extracellular vesicles derived from primary CD4+T cells activated by IL-2) (25μg / mL), and the same procedure was performed.

[0072] Results, such as Figure 6 As shown, it was determined that in the co-culture group of CD8+ T cells treated with extracellular vesicles and melanoma cells, the viability of melanoma cells was significantly reduced.

[0073] The specific parts of the present invention are described in detail above. It is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the essential scope of the present invention will be defined by the appended claims and their equivalents.

[0074] The scope of the present invention is indicated by the following claims, and all modifications or substitutions derived from the spirit and scope of the claims and their equivalents should be construed to be included in the scope of the present invention. <110> Kyungpook National University Daegu Gyeongbuk Institute of Science and Technology <120> Contains cytotoxicity activated by extracellular vesicles derived from helper T cells Composition for preventing or treating cancer diseases using T cells as active ingredients <130> AOP-2020-0048PCT <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> PD-1-f <400> 1 agaatcctgg agacctcaac 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> PD-1-r <400> 2 atacccacta gggcactcat 20 <210> 3 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Ki-67-f <400> 3 catcaaggaa cagcctcaac c 21 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Ki-67-r <400> 4 gacctacggc gttgatcact 20 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> IFN-rf <400> 5 gacaatgaac gctacacact 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> IFN-rr <400> 6 taggctttca atgactgtgc 20 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Granzyme Bf <400> 7 ctactgctga ccttgtctct 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Granzyme Br <400> 8 aaagtaaggc catgtagggt 20 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Perforin-f <400> 9 tttcgcctgg tacaaaaacc 20 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Perforin-r <400> 10 agggctgtaa ggaccgagat 20

Claims

1. A pharmaceutical composition for preventing or treating cancer diseases, comprising CD8+T cells activated by extracellular vesicles derived from CD4+T cells as an active ingredient, wherein: The CD4+ T cells were activated only by IL-2 at a concentration of 500 IU / mL to produce the extracellular vesicles.

2. The pharmaceutical composition according to claim 1, wherein The cancer disease is one or more selected from the group consisting of: melanoma, skin cancer, lung cancer, liver cancer, stomach cancer, pancreatic cancer, bone cancer, head and neck cancer, uterine cancer, ovarian cancer, breast cancer, fallopian tube cancer, vaginal cancer, vulvar cancer, Hawkins disease, esophageal cancer, small intestine cancer, colorectal cancer, perianal cancer, endocrine gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic leukemia or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer or ureteral cancer, renal pelvis cancer, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumors.

3. The pharmaceutical composition according to claim 1, wherein The cancer disease is one or more selected from the group consisting of: endometrial cancer, cervical cancer, colon cancer, rectal cancer, thyroid cancer, parathyroid cancer, adrenal cancer, renal cell carcinoma, brain stem glioma and pituitary adenoma.

4. A composition for an immune anticancer agent, comprising CD8+T cells activated by extracellular vesicles derived from CD4+T cells as an active ingredient, wherein: The CD4+ T cells were activated only by IL-2 at a concentration of 500 IU / mL to produce the extracellular vesicles.

5. Use of extracellular vesicles derived from CD4+T cells activated only by IL-2 at a concentration of 500 IU / mL in the preparation of CD8+T cells, wherein: The CD8+T cells are CD8+T cells activated by extracellular vesicles derived from CD4+T cells, and the CD4+T cells are activated only by IL-2 at a concentration of 500 IU / mL to produce the extracellular vesicles.

6. A method for preparing CD8+T cells, the method comprising: CD4 + T cells were activated by treatment with IL-2 alone at a concentration of 500 IU / mL; And, CD8+ T cells were treated with extracellular vesicles derived from activated CD4+ T cells.

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