MEDICATIONS FOR TREATING CANCER OR INFECTIOUS DISEASES

Abstract

The invention relates to a pharmaceutical for the treatment of cancer or infectious disease, containing as an active ingredient a peripheral blood mononuclear cell containing isolated exogenous mitochondria.

Description

[0001]

specification

[0002]

Title of Invention

[0003] Natural killer cells containing foreign mitochondria and pharmaceutical compositions containing the same

[0004] [Technology Field]

[0005] The present invention relates to a cell therapy agent, and more specifically, to natural killer cells containing foreign mitochondria, peripheral blood mononuclear cells, and a pharmaceutical composition containing the same as an active ingredient.

[0006]

Background Technology

[0007] Recently, biopharmaceuticals are being developed to treat various intractable diseases. Biopharmaceuticals are evolving from simple biological proteins to antibody drugs and cell therapies. In this context, cell therapies refer to medicines used for therapeutic, diagnostic, or preventive purposes through a series of actions, such as isolating, proliferating, or selecting autologous, allogeneic, or xenogeneic cells in vitro, or altering the biological characteristics of the cells through other methods. Depending on the type of cell used, they can be classified into somatic cell therapies and stem cell therapies.

[0008] Meanwhile, interest in cancer treatment through immunotherapy utilizing the patient's immune function is increasing. Immunotherapy utilizes the characteristic of eliminating cancer cells through the complex interactions of immune cells with various functions. Peripheral blood mononuclear cells (PBMCs) found in the human blood are blood cells that possess a round nucleus, similar to lymphocytes or monocytes; these peripheral blood mononuclear cells contain immune cells such as B cells, T cells, macrophages, dendritic cells (DCs), and natural killer cells (hereinafter abbreviated as NK cells). Immune cells that directly eliminate cancer cells include NK cells and cytotoxic T lymphocytes (CTLs), while antigen-presenting cells that present antigens to these effector cells include dendritic cells and B cells. In addition, helper T cells and regulatory T cells that secrete various cytokines participate in the immune response. Among these, NK cells are considered important as the most rapid and efficient immune cells in immunotherapy.

[0009] In particular, as the ability of NK cells to non-specifically kill cancer cells was revealed, much research has been conducted on NK cells. Based on these studies, NK cell therapy utilizing NK cells for cancer treatment is emerging. Specifically, it has been reported that NK cells play an important role in innate immune responses to combat pathogens or cancer infecting the host, as well as in adaptive immune responses through cytokine secretion.

[0010] Accordingly, the inventors of the present invention have completed the present invention by striving to find a new method to control the cytotoxicity of N cells and peripheral blood mononuclear cells, and by discovering a method to activate the cytotoxicity of NK cells and peripheral blood mononuclear cells and use this to treat cancer.

[0011]

Detailed Description of the Invention

[0012]

Technical Challenges

[0013] The object of the present invention is to provide NK cells with increased cytotoxicity and a pharmaceutical composition containing the same. Another object of the present invention is to provide peripheral blood mononuclear cells with increased cytotoxicity and a pharmaceutical composition containing the same.

[0014]

Technical Solution

[0015] To achieve the above objective, the present invention provides NK cells containing foreign mitochondria and a pharmaceutical composition containing the same for the prevention or treatment of cancer or infectious diseases.

[0016] In addition, the present invention provides peripheral blood mononuclear cells containing foreign mitochondria and a pharmaceutical composition containing the same for the prevention or treatment of cancer or infectious diseases. [Effects of the Invention]

[0017] NK cells and peripheral blood mononuclear cells into which foreign mitochondria have been introduced exhibit increased cytotoxicity, which not only increases cancer-specific killing effects but also have no side effects as immune cells present in the body. Furthermore, since the capabilities of the NK cells and peripheral blood mononuclear cells themselves are enhanced, they can be widely applied to various diseases involving NK cells and peripheral blood mononuclear cells, so pharmaceutical compositions containing NK cells and peripheral blood mononuclear cells are expected to have high commercial potential.

[0018]

Brief Description of the Drawing

[0019] Figure 1 shows the results of analyzing whether mitochondria derived from human normal liver cells (WRL68) were transferred to NK cells using the PCR analysis method.

[0020] Figure 2 shows the results of FACS analysis on whether human normal liver cell-derived mitochondria were delivered to NK cells.

[0021] Figure 3 shows the results of analyzing whether human normal liver cell-derived mitochondria were delivered to NK cells using a fluorescence microscope.

[0022] Figures 4a and 4b show the results of analyzing the change in anticancer activity of NK cells delivered with foreign mitochondria using the CD107a degranulation assay. Figure 5 shows the results of analyzing the change in anticancer activity of NK cells delivered with foreign mitochondria using the cytotoxicity assay against K562.

[0023] Figure 6 shows the results of FACS analysis on whether rat-derived mesenchymal stem cell mitochondria were transferred to NK cells.

[0024] Figure 7 shows the results of analyzing the change in anticancer activity of NK cells delivered with umbilical cord-derived mesenchymal stem cell mitochondria using a cytotoxicity assay for K562.

[0025] Figures 8a to 8c show the results of analyzing the therapeutic effect of K cells delivered with rat-derived mesenchymal stem cell mitochondria in an animal model of acute myeloid leukemia, based on mouse body weight and survival rate.

[0026] Figure 9 shows the results of the expression distribution of tumor markers in the blood of an animal model of acute myeloid leukemia administered with NK cells delivered with umbilical cord-derived mesenchymal stem cell-derived mitochondria.

[0027] FIG. 10 shows the results of analyzing changes in anticancer activity of superblood mononuclear cells (hereinafter referred to as PBMCs) delivered with foreign mitochondria using the K562 cytotoxicity assay.

Best Mode for Carrying Out the Invention

[0028] The present invention will be described in detail below.

[0029] One aspect of the present invention provides NK cells comprising foreign mitochondria. As used in the present invention, the term "foreign mitochondria" refers to mitochondria introduced from outside, which are not mitochondria present within the NK cells. In this case, the foreign mitochondria may be obtained from the same individual as the individual from which the NK cells were obtained, or may be obtained from a different individual. In this case, the foreign mitochondria may be obtained from mammals, and preferably from humans. For example, the foreign mitochondria may be obtained from muscle cells, liver cells, fibroblasts, epithelial cells, ᅵ It is obtained from nerve cells, adipocytes, osteocytes, leukocytes, lymphocytes, or mucosal cells, and preferably from muscle cells with excellent mitochondrial activity. Additionally, the mitochondria may be obtained from cells cultured in vitro.

[0030] Meanwhile, foreign mitochondria can be obtained by lysing the cell and centrifuging the mitochondria, or by culturing the cell and then lysing the cell and centrifuging the mitochondria. The method for obtaining mitochondria may utilize conventional methods used for collecting cell organelles.

[0031] At this time, NK cells containing foreign mitochondria are 10 5It may be obtained by introducing 0.01 to 500 fig, 0.1 to 450 β, 0.5 to 300 g, 1 to 100 µg, or 2 to 10 mitochondria per NK cell. In this case, the NK cells are 1 to 10 3 Pieces, 10 to 10 2 It may contain foreign mitochondria. Specifically, the number of foreign mitochondria contained within a single NK cell may be approximately 1, 10, 100, or 500. In this case, the number of foreign mitochondria contained in the NK can be controlled by adjusting the amount of mitochondria introduced when introducing foreign mitochondria into the NK cell. The number of foreign mitochondria contained in each individual NK cell may vary. Furthermore, the NK cell may be derived from mammals or humans. Preferably, it may be obtained from an individual who wishes to receive NK cell therapy. In this case, the NK cell may be used by directly isolating it from the individual's blood, or by differentiating immature N cells or stem cells obtained from the individual.

[0032] ' Meanwhile, in order to introduce the above-mentioned foreign mitochondria into NK cells, the foreign mitochondria and NK cells can be mixed and then centrifuged to deliver the mitochondria to the NK cells. The conditions during centrifugation can be appropriately controlled to efficiently introduce mitochondria without damaging the cells. At this time, the centrifugation can be performed in a room, and the conditions can also be appropriately selected for cell stability. At this time, when introducing foreign mitochondria, the NK cells and foreign mitochondria can be mixed in the presence of a surfactant to increase the permeability of the NK cell membrane, thereby increasing the efficiency of introducing foreign mitochondria.

[0033] At this time, centrifugation may be performed at 100Xg, 300Xg, 500Xg, 800Xg, 100Xg, 1200Xg, 1500Xg, 1800Xg, 2000Xg, 2400Xg, 3000Xg, 5000Xg, or 10000Xg. In addition, the centrifugation time may be from 0.1 min to 60 min, but is not limited thereto. Specifically, 1 min, 2 min, 3 min, 5 min, 10 min ᅵ , it may be 20 minutes or 30 minutes. In addition, the centrifugation is 0 to 40 ° C, 20 to 38 ° C or 30 to 37 ° It can be performed at a temperature of C.

[0034] In this way, by applying centrifugal force to both NK cells and foreign mitochondria, mitochondria can be delivered to NK cells with high efficiency while minimizing damage to the NK cells. Additionally, a surfactant may be used to enhance the cell membrane permeability of NK cells. The surfactant may be added before, during, or after mixing the NK cells and foreign mitochondria. Furthermore, after adding the surfactant to the NK cells, the NK cells may be incubated for a certain period of time to increase the cell membrane permeability of the NK cells. The incubation time may be 0.1 to 60 minutes. Specifically, it may be 1 minute, 5 minutes, 10 minutes, 20 minutes, or 30 minutes, but is not limited thereto.

[0035] Specifically, the surfactant is preferably a nonionic surfactant and may be a poloxamer. In this case, the poloxamer is a triblock copolymer composed of a central hydrophobic chain of polyoxypropylene arranged laterally by two hydrophilic chains of polyoxyethylene. Additionally, the concentration of the surfactant in the mixture may be 1 to 100 mg / ml, 3 to 80 mg / ml, or 5 to 40 mg / ml, and preferably 10 to 30 mg / ml.

[0036] In addition, the method may further include a step of incubating the mixture under predetermined time and temperature conditions. The incubation is 0 to 40 ° C or 20 to 38 ° C or 30 to 37 ° It can be performed at a temperature of C. Additionally, it can be performed for 0.1 to 4 hours, 0.5 to 3.8 hours, or 0.8 to 3.5 hours. Furthermore, incubation can be performed for a predetermined time after foreign mitochondria are delivered to K cells by performing centrifugation. Additionally, the incubation time can be appropriately selected depending on the type of cell and the amount of mitochondria.

[0037] Another aspect of the present invention provides a pharmaceutical composition for treating cancer or infectious diseases comprising NK cells containing foreign mitochondria as an active ingredient. In this case, the cancer may be any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. Additionally, the infectious disease may be any one selected from the group consisting of hepatitis B, hepatitis C, human papillomavirus (HPV) infection, cytomegalovirus infection, viral respiratory disease, and influenza.

[0038] In addition, the above pharmaceutical composition can be prepared as a liquid or frozen formulation. Even when thawed after freezing, cell function is not impaired, and high cell viability and cytotoxicity can be maintained. Therefore, storage and supply in a liquid or frozen form can be easily facilitated without additional processing.

[0039] Another aspect provides a method for preventing or treating a disease comprising the step of administering to an individual a pharmaceutical composition containing NK cells containing foreign mitochondria as an active ingredient.

[0040] This method comprises the step of administering an effective amount of the NK cells of the present invention to an individual with a disease or an individual suspected of having a disease. For example, cells into which foreign mitochondria have been introduced may be administered as a therapeutic agent to an individual, preferably a mammal. The cells may be administered via intravenous or subcutaneous routes. When the composition of the present invention is provided parenterally, such as intravenously, subcutaneously, ophthalmically, intraperitoneally, or intramuscularly, the composition is preferably aqueous or comprises a physiologically applicable body fluid, suspension, or solution. Accordingly, since the carrier or vehicle is physiologically acceptable, it can be added to the composition and delivered to the patient without adversely affecting the patient's electrolytes. Therefore, physiological saline may generally be used as a carrier for the formulation.

[0041] A method for preventing or treating a disease using the cell of the present invention may also include administering another drug or physiologically active substance having a preventive or therapeutic effect on the disease in combination with the cell of the present invention, and the route, timing, and dosage of the combined administration may be determined according to the type of disease, the patient's disease state, the purpose of treatment or prevention, and the other drug or physiologically active substance used in combination.

[0042] In addition, the above disease may be a cancer or an infectious disease, wherein the cancer may be selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma, as described above. In addition, the infectious disease may be any one selected from the group consisting of hepatitis B, hepatitis C, human papillomavirus (HPV) infection, cytomegalovirus infection, viral respiratory disease, and influenza.

[0043] Another aspect of the present invention is to provide a peripheral blood mononuclear cell containing foreign mitochondria.

[0044] The term "peripheral blood mononuclear cell" used in the present invention refers to peripheral blood mononuclear cells or PBMCs, and means cells having a spherical nucleus present in peripheral blood. Such PBMCs may include immune cells such as B cells, T cells, macrophages, dendritic cells, and N cells. The PBMCs may be obtained through the blood of an individual. In this case, the foreign mitochondria may be obtained from the tissues or cells of an individual as described above. In this case, the PBMC cells containing foreign mitochondria are 10 5 It may be obtained by introducing 0.01 to 500 g, 0.1 to 450 μ, 0.5 to 300 μ, 1 to 100 μ, or 2 to 10 g of mitochondria per PBMC cell. In this case, the foreign mitochondria are 1 to 10 per monocyte cell 3A number of individuals may be included, or 10 to 100 individuals may be included. Additionally, the method of introducing foreign mitochondria into PBMC can be performed through centrifugation as described above. Another aspect of the present invention is to provide a pharmaceutical composition for treating cancer or infectious diseases comprising PBMC containing the foreign mitochondria as an active ingredient.

[0045] At this time, the above cancer may be selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma, as described above. Additionally, the above infectious disease may be any one selected from the group consisting of hepatitis B, hepatitis C, human papillomavirus (HPV) infection, cytomegalovirus infection, viral respiratory disease, and influenza.

[0046] The present invention will be explained in more detail below through the following examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto.

[0047]

Form for carrying out the invention

[0048] I. Example of Production and Function Verification of NK Cells with Introduced Foreign Mitochondria i. Production of K Cells with Introduced Foreign Mitochondria

[0049] Human normal hepatocytes (WRL-68) (CRL1458, ATCC) were inoculated into DMEM (Dulbecco Modified Eagle Medium) containing 10% fetal bovine serum (FBS, Gibco), 100 g / ml streptomycin, and 100 U / ml ampicillin, and cultured for 72 hours. After incubation, the cells were washed twice with DPBS (Dulbecco Phosphate Buffered Saline, Gibco). The washed cells were treated with 0.25% Trypsin-EDTA (TE, Gibco) to obtain the cells. To extract mitochondria from the obtained cells, the cell count was measured using a hemocytometer and 3 x 10⁶ cells were collected. 6 Cells of approximately cells / ml were recovered.

[0050] Subsequently, the above cell line is approximately 4 ° A first centrifugation was performed at a speed of 350Xg for 10 minutes at a temperature of C, and the obtained pellet was recovered, resuspended in a buffer solution, and homogenized for 10 to 15 minutes. A composition containing the pellet was approximately 4 ° The supernatant was obtained by centrifuging a second time at a speed of 1,100Xg for 3 minutes at a temperature of C. Subsequently, the supernatant was centrifuged a third time at a speed of 12,000Xg for 15 minutes at a temperature of approximately 4°C to isolate mitochondria from the cell line.

[0051] Isolated mitochondria were transferred to separate human NK cells (NK92mi) (CRL2408, ATCC) at a rate of 1 x 10⁶ 5 Inject into a test tube in the amount of, and about 4 ° Centrifuged at a rate of 2,500 x g for 15 minutes at a temperature of C. After removing the supernatant, washed with PBS and approximately 4 ° Centrifugation was performed for 5 minutes at a temperature of C. Washing was performed twice under the same conditions. At this time, the isolated mitochondria were 1 x 10⁶ target cells. 5It was delivered at weights of 0.05, 0.05, 0.5, and 5 / g per piece.

[0052] Example 2 · Confirmation of delivery of human normal hepatocyte (WRL68)-derived mitochondria into NK cells (PCR analysis method)

[0053] A DNA Purification Kit (NucleoSpin, Macherey-Nagel) was used to extract the whole gene from the NK cells recovered in Example 1 above. WRL-68 mitochondrial-specific identification primers (Sequence No. 1) and R: 5' -AAG TAT ΉA TGG TAC CGT ACG-3' (Sequence No. 2)) were mixed with the extracted DNA, respectively, then 2XPCR Master Mix (Applied Biosystems, Foster City, CA, USA) and triple distilled water were added to make the total volume 10^, and the desired DNA portion was amplified using a Veriti 96-well Thermal Cycler (Applied Biosystems).

[0054] Amplified DNA was obtained by performing a PCR reaction, and to confirm this, electrophoresis was performed on a 1.5% agarose gel, stained with a loading star (DYNE Bio, Seongnam, Korea), and the amplified DNA bands were confirmed using a UV-spectrometer (Chemi-Doc XRS, Bio-Rad, Hercules, CA, USA). GAPDH was selected as the housekeeping gene, and for this purpose, primers capable of amplifying GAPDH (F- 5'- GGA AGG TGA AGG TCG GAG-3' (Sequence No. 3) and R- 5'-GGC AAC AAT ATC CAC TTT ACC-3' (Sequence No. 4) were used. This is shown in Figure 1.

[0055] Through Figure 1, it was confirmed that as the amount of mitochondria mixed with NK cells (0.005, 0.05, 0.5, and 5 g) increased, the amount of foreign mitochondria delivered to NK cells increased.

[0056] Example 3. Confirmation of delivery of human normal hepatocyte (WRL68)-derived mitochondria into NK cells (FACS analysis method)

[0057] Fluorescence-activated cell sorter (FACS) analysis was performed to confirm the delivery of hepatocyte-derived mitochondria into NK cells. Mitochondria isolated from hepatocytes were treated with 500 nM Green Mitotracker (Thermo Fisher Scientific, Waltham USA) and 37 ° The cells were incubated in a 5% CO2 incubator for 10 minutes and washed. Fluorescence-labeled hepatocyte mitochondria were transferred to immune cells using centrifugation, and the cells were resuspended in 1 mL of PBS. Subsequently, the transfer of mitochondria was confirmed and analyzed using a FACS Calibur flow cytometer (BDBiosciences, San Jose, CA, USA). The results are shown in Figure 2.

[0058] Through Figure 2, it was confirmed that NK cells can be distinguished according to the amount of mitochondria delivered to NR cells (0.005, 0.05, 0.5, and 5 g).

[0059] Example 4. Confirmation of delivery of human normal hepatocyte (WRL68)-derived mitochondria into K cells (fluorescence microscopy)

[0060] To confirm the delivery of mitochondria derived from normal hepatocytes (WRL-68) into human NK cells (NK92mi), NK cell mitochondria were treated with 500 nM Green mitochondria nitrate (Thermo Fisher Scientific, Waltham, USA) and 37 ° C, incubated for 10 minutes in a 5% CO2 incubator. Isolated hepatocyte mitochondria were treated with 500 nM red mitotracker and 37 ° After reacting for 10 minutes in a 5% CO2 incubator, they were delivered to immune cells. After delivering 5 lig of mitochondria, they were seeded into a 24-well plate and 37 ° After culturing in a 5% CO2 incubator, the presence or absence of intracellular delivery was confirmed using fluorescence microscopy within 24 hours. DAPI reagent for nuclear staining was used as a counterstain. The results are shown in Figure 3.

[0061] Through Fig. 3, it was confirmed that foreign mitochondria were delivered into K cells. Example 5. Analysis of changes in anticancer activity of K cells delivered with mitochondria (CD107a degranulation analysis)

[0062] In order to confirm the presence or absence of CD107a degranulation expression, an indicator of NK cell activity, in human NK cells to which foreign mitochondria recovered in Example 1 were introduced, human NK cells (NK92mi) and target cells (K562) were mixed in a ratio of 10:1, treated with fluorescently conjugated anti-CD107a, and co-cultured for 4 hours. After co-culture, anti-CD56 was treated for surface staining and reacted for 30 minutes, after which FACS (Fluorescence-activated cell sorter) analysis was performed. The results are shown in Figures 4a and 4b.

[0063] Through Figures 4a and 4b, it was confirmed that the anticancer activity of NK cells increased with the amount of foreign mitochondria (0.05, 0.5, and 5 g).

[0064] Example 6. Confirmation of changes in anticancer activity of NK cells delivered with mitochondria (K562 cytotoxicity assay)

[0065] To confirm the anticancer activity of the NK cells recovered in Example 1 above, the recovered NK cells were mixed with target cells (K562) labeled with green fluorescent staining (CFSE, Invitrogen) at a ratio of 10:1, and then 37t, 5%, CO 2 Co-culture was performed for 4 hours in an incubator under certain conditions. After co-culture, to analyze target cells killed by NK cells, the cells were treated with red fluorescent staining (7-AAD, Invitrogen) and reacted for 10 minutes, and the fluorescence concentration of the killed target cells was analyzed using FACS (Fluorescence-activated cell sorter). The results are shown in Figure 5. Through Figure 5, it was confirmed that the cytotoxicity of K562 increased with the amount of delivered foreign mitochondria (0.05, 0.5, and 5 / g).

[0066] Example 7. Mesenchymal stem cells derived from a placenta (provided by Bundang CHA Hospital, IRBNo, 1044308— 201511-BR-022-02) into which mitochondria of umbilical cord-derived mesenchymal stem cells were inoculated into Alpha-MEM (Alpha-Minimum Essential Medium) medium containing 10% fetal bovine serum (FBS, Gibco), 100 g / ml streptomycin, and 100 U / ml ampicillin and cultured for 72 hours.

[0067] After the culture was completed, the cells were washed twice with DPBS (Dulbecco Phosphate Buffered Saline, Gibco). The washed cells were treated with 0.2 Trypsin-EDTA (TE, Gibco) to obtain the cells. To extract mitochondria from the obtained cells, the cell count was measured using a hemocytometer and found to be 2 x 10⁶. 7 Cells of approximately cells / ml were recovered.

[0068] Subsequently, the above cell line is approximately 4 ° A first centrifugation was performed at a speed of 350Xg for 10 minutes at a temperature of C, and the obtained pellet was recovered, resuspended in a buffer solution, and homogenized for 10 to 15 minutes. A composition containing the pellet was approximately 4 ° The supernatant was obtained by centrifuging a second time at a speed of 1,100Xg for 3 minutes at a temperature of C. Subsequently, the supernatant was centrifuged a third time at a speed of 12,000Xg for 15 minutes at a temperature of approximately 4°C to isolate mitochondria from the cell line.

[0069] Isolated mitochondria were transferred to separate human NK cells (NK92mi) (CRL2408, ATCC) at a rate of 1 x 10⁶ 5 Inject into a test tube in the amount of, and about 4 ° Centrifuged at a rate of 2,500 x g for 15 minutes at a temperature of C. Removed the supernatant, washed with PBS, and approximately 4 ° Centrifugation was performed for 5 minutes at a temperature of C. Washing was performed twice under the same conditions. At this time, the isolated mitochondria were 1 x 10⁶ target cells. 5 They were delivered with weights of 0.3, 1, 3, 5, and 10 per unit.

[0070] Example 8. Confirmation of delivery of umbilical cord-derived mesenchymal stem cell (UC-MSC) mitochondria into NK cells (FACS analysis method)

[0071] FACS (Fluorescence-activated cell sorter) analysis was performed to confirm the delivery of mitochondria derived from umbilical cord-derived mesenchymal stem cells into NK cells. Mitochondria isolated from umbilical cord-derived mesenchymal stem cells were treated with 500 nM Red Mitochondria Sorter (Thermo Fisher Scientist, Waltham, USA), incubated in a 37 5% CO2 incubator for 30 minutes, and then washed. After delivering the fluorescence-labeled mitochondria from umbilical cord-derived mesenchymal stem cells to immune cells using centrifugation, the cells were resuspended in 1 mL of PBS. Subsequently, the delivery of mitochondria was confirmed and analyzed using a FACS Caliber Flow Cytometer (BDB Biosciences, San Jose, CA, USA). The results are shown in Figure 6.

[0072] Through Figure 6, it was confirmed that NK cells can be distinguished based on the amount of UC-MSCs-derived mitochondria delivered into the NK cells (0.3, 1, 3, 5, and 10 / g).

[0073] Example 9. Confirmation of changes in anticancer activity of NK cells delivered with mitochondria derived from umbilical cord-derived mesenchymal stem cells (UC-MSC) (K562 cytotoxicity assay)

[0074] To confirm the anticancer activity of the NK cells recovered in Example 7 above, the recovered NK cells were mixed with target cells (K562) labeled with green fluorescent staining (CFSE, Invitrogen) in a ratio of 10:1, and then 37 °Cultured together for 4 hours in an incubator under conditions of 5% CO₂. After co-culture, to analyze the target cells killed by NK cells, red fluorescence staining (7-AAD, Invitrogen) was treated and then incubated for 10 minutes, and the fluorescence concentration of the killed target cells was analyzed by FACS (Fluorescence-activated cell sorter). The results are shown in Fig. 7. Through Fig. 7, it was confirmed that the cytotoxicity against K562 increased according to the amount of exogenous mitochondria delivered (0.5, 1, 3, 5, and 10 μg).

[0075] Example 10. Evaluation of acute myeloid leukemia treatment through body weight change and survival rate Male NOD.cg-Prkdcscid Il2rgtm1Sug / JicKoat mice aged 6 to 8 weeks were purchased from Checkmate (Koatech Co., Ltd., Gyeonggi-do, Korea). The purchased mice were acclimated in the clean area of the Experimental Animal Center of Cha University of Medicine and then the experiment was conducted. The environment where the mice stayed during the acclimation period consisted of day and night at 12-hour intervals, and the indoor temperature was maintained at 23 ± 2 ° °C and the humidity at 40 to 60%. After undergoing such an acclimation period for 7 days, they were introduced into the experiment. The mice thus prepared were administered K562 cells at 2×10 5 cells / 100 μl via the tail vein (i.v., Intravenous injection) to establish an acute myeloid leukemia model.

[0076] At this time, the K cells (NK92mi) prepared according to Example 7 were administered at 2×10 6 cells / 100 μl via the tail vein (i.v., Intravenous injection) of the mice with induced acute myeloid leukemia to prepare an experimental group. In the same way, normal NK cells (NK92mi) without mitochondrial delivery were administered at 2×10 6A control group was prepared by administering cel ls / 100^. Changes in body weight and survival of the experimental group and the control group were analyzed for 24 days from the time of administration of the acute myeloid leukemia cell line (K562). The results are shown in Figures 8a to 8c.

[0077] Through Figures 8a to 8c, it was confirmed that the group administered NK cells delivered with umbilical cord-derived mesenchymal stem cell mitochondria showed a body weight increase of about 5% and a survival rate increase of about 40% or more compared to the group administered with normal NK cells without delivered mitochondria.

[0078] Example 11. Confirmation of Acute Myeloid Leukemia Treatment Evaluation through Analysis of Tumor-Related Markers. To confirm the expression distribution of the tumor-related markers p53 and c-Myc, blood samples were collected from the experimental group and the control group according to Example 10, and serum was separated by centrifugation at 12,000 x g for 15 minutes. The separated serum was analyzed for the expression distribution of p53 and c-Myc using a Western Blot Kit (WB Bio-rad). The results are shown in Figure 9.

[0079] Figure 9 confirms that the expression of the tumor marker c-Myc in the blood decreased and the expression of p53 increased in the group administered NK cells delivered with umbilical cord-derived mesenchymal stem cell-derived mitochondria.

[0080] Π. Fabrication and functional verification of PB with introduced foreign mitochondria

[0081] Example 12. Production of PBMC with introduced foreign mitochondria

[0082] Peripheral blood from the inventor collected by a clinician (CHA Hospital) was treated with Fi col l-Paque (Amersham Biosciences) at a 1:1 ratio, and the PBMC pellet was recovered by centrifugation at 400 xg for 35 minutes. The recovered PBMC was washed twice with PBS, and then human hepatocyte (WRL-68)-derived mitochondria isolated according to Example 1 were added to target cells IX 10 5 It was delivered in weights of 0.05, 0.05, 0.5, and 5 g per piece.

[0083] Example 13. Confirmation of changes in anticancer activity of PBMCs delivered with mitochondria (K562 cytotoxicity assay)

[0084] Confirm the anticancer activity of the PBMC recovered in Example 12 above. ' To do so, the recovered PBMC cells were mixed with target cells (K562) labeled with green fluorescent staining (CFSE, Invitrogen) at a ratio of 10:1, and then 37 ° Co-culture was performed for 4 hours in an incubator under 5% CO2 conditions at a temperature of C. After co-culture, to analyze target cells killed by NK cells, the cells were treated with red fluorescent staining (the AAD, Invitrogen) and reacted for 10 minutes, and the fluorescence concentration of the killed target cells was analyzed using FACS (Fluorescence-activated cell sorter). The results are shown in Figure 10.

[0085] Through Figure 10, it was confirmed that cytotoxicity against K562 increased with the amount of delivered foreign mitochondria (0.005, 0.05, and 0.5; g).

Claims

【Scope of Claim】

1. Natural killer cells containing foreign mitochondria.

2. In Paragraph 1, Natural killer cells in which the above-mentioned foreign mitochondria are obtained from muscle cells, liver cells, fibroblasts, epithelial cells, neurons, adipocytes, osteocytes, leukocytes, lymphocytes, or mucosal cells.

3. In Paragraph 1, The above foreign mitochondria are 1 to 10 per natural killer cell 3 Natural killer cells, including dogs.

4. In that U clause, A natural killer cell in which the above composition of mixed foreign mitochondria and natural killer cells is centrifuged to transfer the foreign mitochondria into the natural killer cell.

5. A pharmaceutical composition for treating cancer or infectious diseases comprising natural killer cells of claim 1 as an active ingredient.

6. In Paragraph 5, The above cancers are stomach cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, A pharmaceutical composition selected from the group consisting of cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

7. In Paragraph 5, A pharmaceutical composition in which the above infectious disease is selected from the group consisting of hepatitis B, hepatitis C, human papillomavirus (HPV) infection, cytomegalovirus infection, viral respiratory disease, and influenza.

8. In that 15th paragraph, A pharmaceutical composition in which the above composition is in a liquid or frozen form.

9. A method for preventing or treating a disease comprising the step of administering to an individual a pharmaceutical composition containing the natural killer cells of claim 1 as an active ingredient.

10. In Paragraph 9, A method for preventing or treating a disease, wherein the route of administration of the above composition is selected from the group consisting of intravenous, subcutaneous, ocular, intraperitoneal, and intramuscular. 【Claim 111 In Article 9, A method for preventing or treating a disease, comprising administering another drug or physiologically active substance having a preventive or therapeutic effect on the disease in combination with the cell.

12. Peripheral blood mononuclear cells containing foreign mitochondria.

13. In Paragraph 12, Peripheral blood mononuclear cells in which the above-mentioned foreign mitochondria are obtained from muscle cells, liver cells, fibroblasts, epithelial cells, neurons, adipocytes, osteocytes, leukocytes, lymphocytes, or mucosal cells. Claim 14 In Paragraph 12, The above foreign mitochondria are 1 to 10 per natural killer cell 3 Individual, peripheral blood mononuclear cell.

15. A pharmaceutical composition for treating cancer or infectious diseases comprising peripheral blood mononuclear cells of claim 12.

16. In Paragraph 15, Pharmaceutical that the above cancer is selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma Composition.

17. In Paragraph 15, A pharmaceutical composition wherein the above infectious disease is selected from the group consisting of hepatitis B, hepatitis C, human papillomavirus (HPV) infection, cytomegalovirus infection, viral respiratory disease, and influenza.

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