Composition containing cationic substance and use thereof

Abstract

According to one embodiment of the present invention, a composition containing a cationic substance as an active ingredient has the effect of increasing the activity of immune cells by improving the stability of perforin protein and inducing the accumulation of perforin protein within cells.

Description

[Technical Field]

[0001] This application claims priority to Korean Patent Application No. 10-2020-0142534, filed on October 29, 2020, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to a composition containing a cationic substance and uses thereof. [Background technology]

[0003] Natural killer cells (NK cells) are immune cells that act as the body's primary defense in the innate immune system. NK cells contain receptors that recognize abnormal cells, allowing them to instantly detect and eliminate abnormal cells such as cancer cells or viruses without specific antigens, thereby regulating the immune system and effectively suppressing the proliferation, recurrence, and metastasis of cancer cells.

[0004] Taking advantage of the above-mentioned characteristics of natural killer cells, research into anti-cancer immunotherapeutic agents using natural killer cells has recently been intensifying. Among immunotherapy methods for cancer, immune cell therapy refers to therapeutic agents that utilize immune cells within the body, genetically modify them, and then reinject them into the body. While immunotherapy aims to strengthen cellular immunity, chimeric antigen receptor T-cells (CAR-T cells) have drawbacks, such as complex genetic modification and high treatment costs. Furthermore, due to the characteristics of T cells, they can cause cytokine release syndrome and various side effects. To overcome these drawbacks, natural killer cells have recently been attracting attention in the field of immunotherapy for cancer. Summary of the Invention

[0005] [Problem to be solved by the invention] The present inventors have solved the above problems by confirming that treatment of immune cells with cationic substances improves the stabilization of perforin protein.

[0006] One embodiment provides a composition for increasing intracellular perforin protein, which comprises a cationic substance as an active ingredient.

[0007] Another embodiment provides a composition for increasing immune cell activity, which comprises a cationic substance as an active ingredient.

[0008] Yet another embodiment provides immune cells that have been pretreated with a cationic substance and have an increased expression level or content of perforin compared to normal cells.

[0009] Yet another embodiment provides a composition for preventing or treating cancer, comprising immune cells that have been pretreated with a cationic substance and have increased expression or content of perforin compared to normal cells.

[0010] In yet another embodiment, there is provided a pharmaceutical composition for preventing or treating cancer, comprising a cationic substance and an immune cell as active ingredients.

[0011] Yet another aspect provides a culture method for promoting immune cell activity, comprising culturing a cationic substance and immune cells.

[0012] Yet another embodiment provides a method for increasing perforin content or expression in immune cells, comprising culturing the immune cells with a cationic agent.

[0013] Yet another embodiment provides a method for preventing or treating cancer, comprising the step of administering to an individual a composition comprising a cationic substance and immune cells as active ingredients.

[0014] Yet another embodiment provides the use of a composition comprising a cationic substance as an active ingredient for use in increasing intracellular perforin protein.

[0015] Yet another aspect provides the use of a composition comprising a cationic substance as an active ingredient for use in increasing immune cell activity.

[0016] Yet another aspect provides the use of a composition comprising a cationic substance as an active ingredient for use in the manufacture of a medicament for the prevention or treatment of cancer.

[0017] Yet another embodiment provides the use of a cationic substance for use in increasing intracellular perforin protein.

[0018] [Means for solving the problem] One embodiment provides a composition for increasing intracellular perforin protein, which comprises a cationic substance as an active ingredient.

[0019] Another embodiment provides a composition for increasing immune cell activity, which comprises a cationic substance as an active ingredient.

[0020] As used herein, the term "cationic substance" refers to a substance that has cations on its surface, as well as a substance that has a positive charge on its surface.

[0021] In one embodiment, the cationic material is Polyethyleneimine ( P Preferably, the cationic substance is: Polyethyleneimine It is also.

[0022] As used herein, the term "polyetherimide (PEI)" refers to a polymer having a molecular weight of 1000 or more and a molecular weight of 1000 or more. 37 H 24 O6N2) n and a density of 1.27 g / cm 3 The polyetherimide is also a cationic substance. The cationic nature of the polyetherimide also allows genes to be delivered into cells.

[0023] The cell may be an immune cell, such as a T cell, a B cell, a dendritic cell or a natural killer cell.

[0024] The increased activity of the immune cells means that the immune regulatory, cytotoxic or apoptotic ability of the cells is activated compared to parent cells, such as hematopoietic cells or progenitor cells. The immune cells may also be CAR immune cells.

[0025] As used herein, the term "natural killer cell (NK cell)" refers to a type of white blood cell in the blood that plays a role in immunity and matures in the liver and bone marrow. Natural killer cells are responsible for non-specific immunity and play a role in eliminating viruses, cancer cells, etc.

[0026] In one embodiment, treatment of natural killer cells with the composition resulted in improved immunomodulatory, cytotoxic, or cell-killing capabilities of the natural killer cells. Thus, the composition contains natural killer cells with improved immunomodulatory, cytotoxic, or cell-killing capabilities, thereby improving the ability of the natural killer cells to kill viruses or cancer cells.

[0027] As used herein, the term "perforin protein" refers to a glycoprotein that destroys cells by creating holes in the plasma membrane of the cell. The perforin protein of the cell is also present in immune cells.

[0028] The aforementioned Polyethyleneimine is also branched. The branched structure means that the chemical structure is not linear but contains branches. Polyethyleneimine may be a primary amine, a secondary amine or a tertiary amine.

[0029] The aforementioned Polyethyleneimine The molecular weight of the above may be 10,000 mM to 30,000 mM. PolyethyleneimineThe molecular weight of the cation-containing polymer may be 10,000 to 28,000, 10,000 to 27,000, 12,000 to 30,000, 12,000 to 28,000, 12,000 to 27,000, 15,000 to 30,000, 15,000 to 28,000, or 15,000 to 27,000 mM. Preferably, it is about 25,000 mM. Since gene transfer efficiency increases in proportion to the cation density, if the molecular weight range is above or below the above range, gene transfer efficiency decreases.

[0030] The aforementioned Polyethyleneimine The branched structure may be branched or linear. The branched structure may have one or more amine structures selected from the group consisting of primary amines, secondary amines, and tertiary amines in one molecule. The branched structure may have one or more amine structures in one molecule and may have a proton sponge effect, being able to convert to a cation within a wide pH range.

[0031] In one embodiment, the content of the cationic substance is 0.1 μg / ml to 10 μg / ml. For example, the content of the cationic substance is 0.1 to 9 μg / ml, 0.1 to 8 μg / ml, 0.5 to 10 μg / ml, 0.5 to 9 μg / ml, 0.5 to 8 μg / ml, 1 to 10 μg / ml, 1 to 9 μg / ml, 1 to 8 μg / ml, 2 to 10 μg / ml, 2 to 9 μg / ml, 2 to 8 μg / ml, 3 to 10 μg / ml, 3 to 9 μg / ml, or 3 to 8 μg / ml. If the content of the cationic substance is below or above the above range, immune cells may not be sufficiently activated or the level of perforin protein accumulation may be reduced.

[0032] In one embodiment, the composition is also a medium composition. The composition for increasing perforin protein or the composition for increasing immune cell activity is a medium composition, and when cultured with immune cells, it can induce stabilization or accumulation of perforin protein in the immune cells, resulting in induction of immune cell activation.

[0033] The composition may further comprise nanoparticles.

[0034] As used herein, the term "nanoparticles" refers to particles having a size of 1 to 100 nm and having a surface. The nanoparticles may also be coated.

[0035] In one embodiment, the nanoparticles are also magnetic nanoparticles. More specifically, the core of the nanoparticles also contains Zn or Fe. The nanoparticles can be made magnetic by the core layer.

[0036] The cationic substance can be bound to the nanoparticles. Specifically, the cationic substance may be present on the surface of the nanoparticles or may be chemically bound to the surface of the nanoparticles. In one embodiment, the composition can be formed by incubating the nanoparticles and the cationic substance for a certain period of time. Polyethyleneimine can be combined to obtain a composition comprising the nanoparticles and a cationic substance.

[0037] In one embodiment, the composition can induce intracellular accumulation of perforin protein by stabilizing perforin protein. The stabilization refers to resistance to proteolytic degradation, which induces an increase in the amount of perforin protein, and improved translation efficacy from perforin mRNA. Therefore, the composition can increase the number or amount of intracellular perforin protein.

[0038] The composition can be administered simultaneously with an immunological anti-cancer agent, and when administered simultaneously with the immunological anti-cancer agent, the composition can enhance the activity of the immunological anti-cancer agent.

[0039] The composition may further comprise one or more selected from the group consisting of gamma-PGA, glycol chitosan, and protamine.

[0040] Yet another embodiment provides immune cells that have been pretreated with a cationic substance and have an increased expression level or content of perforin compared to normal cells.

[0041] Yet another aspect provides a composition for preventing or treating cancer, comprising said immune cells.

[0042] The cationic substance, cells, perforin, and immune cells are as described above.

[0043] The therapeutic composition is also an immunological anti-cancer agent.

[0044] As used herein, the term "immune anticancer agent" refers to an anticancer agent that activates immune cells in the human body to kill cancer cells, and that exerts a cancer treatment effect by strengthening the patient's own immune system. In one embodiment, the immune anticancer agent is also an immune checkpoint inhibitor.

[0045] As used herein, the term "immune checkpoint inhibitor" refers to an immunological anticancer agent that activates T cells to attack cancer cells by blocking the activation of immune checkpoint proteins involved in T cell suppression, such as proteins such as PD-L1 expressed in tumor cells.

[0046] In one embodiment, the immune checkpoint inhibitor is any one or more selected from the group consisting of an NK cell therapeutic agent, a T cell therapeutic agent, a CAR immune cell therapeutic agent, a DC vaccine, a CTL therapeutic agent, anti-PD-L1, anti-PD-1, and anti-CTLA-4, for example, an NK cell therapeutic agent.

[0047] In one embodiment, the CAR immunotherapy agent refers to an immune checkpoint inhibitor including CAR-T (Chimeric antigen receptor-T) cells or CAR-NK (Chimeric antigen receptor-NK) cells.

[0048] Yet another aspect provides a method of treating cancer in an individual comprising administering to the individual the pharmaceutical composition.

[0049] Yet another embodiment provides a method for preventing or treating cancer, comprising administering to an individual a composition comprising a cationic substance as an active ingredient.

[0050] the cationic substance, Polyethyleneimine The specific details of the immune cells are as described above.

[0051] As used herein, the term "individual" refers to a subject in need of cancer treatment, and more specifically refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cows. The cancer may be one or more selected from the group consisting of breast cancer, thyroid cancer, gastric cancer, colon cancer, lung cancer, liver cancer, prostate cancer, pancreatic cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, oral cancer, lip cancer, testicular cancer, acute myeloid leukemia, basal cell carcinoma, ovarian epithelial cancer, brain tumor, multiple myeloma, blood cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, bladder cancer, peritoneal cancer, tongue cancer, non-small cell lung cancer, small cell lung cancer, small intestine cancer, esophageal cancer, kidney cancer, heart cancer, malignant lymphoma, urethral cancer, cervical cancer, rectal cancer, tonsillar cancer, and laryngeal cancer.

[0052] As used herein, the term "prevention" refers to any action that suppresses or delays the onset of cancer by administering the composition according to the present invention.

[0053] As used herein, the term "treatment" means or includes the alleviation, slowing down of progression, or prevention of a disease, disorder, condition, or one or more symptoms thereof, and the term "active ingredient" or the term "pharmaceutically effective amount" means any amount of a composition utilized in the course of practicing the inventions provided herein sufficient to alleviate, slow down or prevent a disease, disorder, condition, or one or more symptoms thereof.

[0054] The pharmaceutical composition contains immune cells with increased perforin content due to the cationic substance, and thus contains immune cells with activated immune responses as active ingredients, thereby enabling effective treatment of cancer.

[0055] The composition may further contain other known immunoadjuvants, preferably one of monophosphoryl lipid A (MPL) and GLA-SE (Glucopyranosyl Lipid Adjuvant, formulated in a stable nano-emulsion of squalene oil in water).

[0056] The method of administration of the pharmaceutical composition is not particularly limited, and may be parenteral administration such as intravenous, subcutaneous, intraperitoneal, inhalation, or topical application, or oral administration, depending on the intended method. The dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. A daily dosage refers to the amount of a therapeutic substance according to one embodiment that is sufficient to treat a disease state when administered to an individual in need of treatment. The effective amount of a therapeutic substance varies depending on the specific compound, the disease state and its severity, and the individual in need of treatment, and can be routinely determined by one of ordinary skill in the art. As a non-limiting example, the dosage of a composition according to one embodiment administered to a human body may also vary depending on the patient's age, weight, sex, administration method, health condition, and disease severity. Based on an adult patient weighing 70 kg, the dosage is, for example, about 1,000 to 10,000 cells / dose, 1,000 to 100,000 cells / dose, 1,000 to 1,000,000 cells / dose, 1,000 to 10,000,000 cells / dose, 1,000 to 100,000,000 cells / dose, 1,000 to 1,000,000,000 cells / dose, or 1,000 to 10,000,000,000 cells / dose, and can be administered once or several times a day at regular intervals, or multiple times at regular intervals.

[0057] The pharmaceutical composition may also contain pharmaceutically acceptable carriers and / or additives. For example, it may contain sterilized water, physiological saline, conventional buffers (such as phosphate, citric acid, and other organic acids), stabilizers, salts, antioxidants (such as ascorbic acid), surfactants, suspending agents, isotonicity agents, or preservatives. For local administration, it may also contain organic materials such as biopolymers, or inorganic materials such as hydroxyapatite, specifically, collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. When the pharmaceutical composition according to one embodiment is formulated into a dosage form suitable for injection, the substance that increases immune cells or their activity is dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution.

[0058] The pharmaceutical composition may contain, as needed, suspending agents, solubilizers, stabilizers, isotonicity agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, soothing agents, buffers, reducing agents, antioxidants, etc., depending on the administration method and dosage form. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those mentioned above, are described in detail in [Remington's Pharmaceutical Sciences, 19th ed., 1995]. The pharmaceutical composition may be formulated using pharmaceutically acceptable carriers and / or excipients by a method easily performed by those skilled in the art to which this invention pertains, and prepared in a unit-dose form or in a large-volume container. In this case, the dosage form may be a solution, suspension, or emulsion in an oily or aqueous medium, or may be in the form of a powder, granules, tablet, or capsule.

[0059] Yet another aspect provides a culture method for promoting immune cell activity, comprising culturing a cationic substance and immune cells.

[0060] Yet another embodiment provides a method for increasing perforin content or expression in immune cells, comprising culturing the immune cells with a cationic agent.

[0061] the cationic substance, Polyethyleneimine The specific details regarding immune cells and the promotion of their activity are as described above.

[0062] The culturing step may involve culturing the cationic substance and immune cells for 5 to 60 hours, e.g., 5 to 60 hours, 12 to 60 hours, 6 to 58 hours, 6 to 55 hours, 6 to 53 hours, 6 to 50 hours, 8 to 60 hours, 8 to 58 hours, 8 to 55 hours, 8 to 53 hours, 8 to 50 hours, 10 to 60 hours, 10 to 58 hours, 10 to 55 hours, 10 to 53 hours, or 10 to 50 hours. If the culturing time exceeds or is shorter than the above values, perforin may not be sufficiently stabilized.

[0063] The method improves the stabilization of perforin protein by culturing immune cells with a cationic substance, and induces its intracellular accumulation, thereby resulting in enhanced cell killing ability.

[0064] Yet another embodiment provides the use of a composition comprising a cationic substance as an active ingredient for use in increasing intracellular perforin protein.

[0065] Yet another aspect provides the use of a composition comprising a cationic substance as an active ingredient for use in increasing immune cell activity.

[0066] Yet another aspect provides the use of a composition comprising a cationic substance as an active ingredient for use in the manufacture of a medicament for the prevention or treatment of cancer.

[0067] Yet another embodiment provides the use of a cationic substance for use in increasing intracellular perforin protein.

[0068] the cationic substance, Polyethyleneimine The meanings of terms such as immune cells, activity promotion, individual, administration, prevention or treatment are also within the scope mentioned above. [Effects of the Invention]

[0069] According to one embodiment of the present invention, a composition containing a cationic substance as an active ingredient has the effect of increasing the activity of immune cells by improving the stability of perforin protein and inducing the accumulation of perforin protein within cells. [Brief explanation of the drawings]

[0070] [Figure 1] FIG. 1 is a schematic diagram showing the results of treating natural killer cells with polyethyleneimine. [Figure 2A] 1 is a graph showing the activity of natural killer cells as a result of treating the cells with branched or linear polyethyleneimine. [Figure 2B] 1 is a graph showing the activity of natural killer cells as a result of treating the cells with branched or linear polyethyleneimine. [Figure 2C] 1 is a graph showing the activity of natural killer cells as a result of treating the cells with branched or linear polyethyleneimine. [Figure 2D] 1 is a graph showing the activity of natural killer cells as a result of treating the cells with branched or linear polyethyleneimine. [Figure 2E] 1 is a graph showing the activity of natural killer cells as a result of treating the cells with branched or linear polyethyleneimine. [Figure 2F] 1 is a graph showing the activity of natural killer cells as a result of treating the cells with branched or linear polyethyleneimine. [Figure 2G] 1 is a graph showing the activity of natural killer cells as a result of treating the cells with branched or linear polyethyleneimine. [Figure 2H] 1 is a graph showing the activity of natural killer cells as a result of treating the cells with branched or linear polyethyleneimine. [Figure 2I] 1 is a graph showing the activity of natural killer cells as a result of treating the cells with branched or linear polyethyleneimine. [Figure 3A] 1 is a graph showing the activity of natural killer cells as a result of treating breast cancer cells with polyethyleneimine having a molecular weight of 25K at different concentrations. [Figure 3B] 1 is a graph showing the activity of natural killer cells as a result of treating breast cancer cells with polyethyleneimine having a molecular weight of 25K at different concentrations. [Figure 3C] 1 is a graph showing the activity of natural killer cells as a result of treating breast cancer cells with polyethyleneimine having a molecular weight of 25K at different concentrations. [Figure 3D] 1 is a graph showing the activity of natural killer cells as a result of treating breast cancer cells with polyethyleneimine having a molecular weight of 25K at different concentrations. [Figure 3E] 1 is a graph showing the activity of natural killer cells as a result of treating breast cancer cells with polyethyleneimine having a molecular weight of 25K at different concentrations. [Figure 3F] 1 is a graph showing the activity of natural killer cells as a result of treating breast cancer cells with polyethyleneimine having a molecular weight of 25K at different concentrations. [Figure 4] 1 is a graph showing the quantification of natural killer cell activity as a result of treating breast cancer cells with various concentrations of polyethyleneimine having a molecular weight of 25K. [Figure 5A] 1 is a graph showing the degree of cell death of breast cancer cells when breast cancer cells are cultured with 5 μg / ml of 25K molecular weight polyethyleneimine at different E:T ratios. [Figure 5B] 1 is a graph showing the degree of cell death of breast cancer cells when breast cancer cells are cultured with 5 μg / ml of 25K molecular weight polyethyleneimine at different E:T ratios. [Figure 5C] 1 is a graph showing the degree of cell death of breast cancer cells when breast cancer cells are cultured with 5 μg / ml of 25K molecular weight polyethyleneimine at different E:T ratios. [Figure 6] 1 is a graph quantitatively showing the degree of cell death of breast cancer cells when breast cancer cells are cultured with 5 μg / ml of 25K molecular weight polyethyleneimine at different E:T ratios. [Figure 7A]1 is a graph showing the activity of natural killer cells as a result of treating natural killer cells with polyethyleneimine after inhibiting its cationicity. [Figure 7B] 1 is a graph showing the activity of natural killer cells as a result of treating natural killer cells with polyethyleneimine after inhibiting its cationicity. [Figure 7C] 1 is a graph showing the activity of natural killer cells as a result of treating natural killer cells with polyethyleneimine after inhibiting its cationicity. [Figure 7D] 1 is a graph showing the activity of natural killer cells as a result of treating natural killer cells with polyethyleneimine after inhibiting its cationicity. [Figure 8] 1 is a graph showing the quantification of the activity of natural killer cells as a result of treating natural killer cells with polyethyleneimine after inhibiting its cationicity. [Figure 9] 1 is a graph showing changes in granzyme protein expression as a result of culturing natural killer cells with 5 μg / ml of polyethyleneimine over time. [Figure 10] 1 is a graph showing changes in perforin protein expression as a result of culturing natural killer cells with 5 μg / ml of polyethyleneimine over time. [Figure 11] 10 is an image showing changes in expression of granzyme proteins and perforin proteins as a result of culturing natural killer cells with 5 μg / ml of polyethyleneimine over time. [Figure 12] 1 is a graph showing changes in the expression of granzyme proteins and perforin proteins as a result of culturing natural killer cells with 5 μg / ml of polyethyleneimine for 48 hours. [Figure 13] 10 is an image showing changes in perforin protein expression as a result of treating polyethyleneimine and natural killer cells with MG132 and culturing them. DETAILED DESCRIPTION OF THE INVENTION

[0071] The present invention encompasses, for example, the following embodiments: [Embodiment 1] A composition for increasing intracellular perforin protein, comprising a cationic substance as an active ingredient. [Embodiment 2] A composition for increasing intracellular perforin protein described in embodiment 1, wherein the cells are immune cells. [Embodiment 3] The cationic substance is Polyethyleneimine ( P 10. The composition for increasing intracellular perforin protein according to embodiment 1, wherein the composition is EI). [Embodiment 4] Polyethyleneimine The composition for increasing intracellular perforin protein according to embodiment 1, wherein the molecular weight of the compound is 10,000 mM to 30,000 mM. [Embodiment 5] Polyethyleneimine A composition for increasing intracellular perforin protein according to embodiment 1, wherein the compound is branched. [Embodiment 6] Polyethyleneimine The composition for increasing intracellular perforin protein according to embodiment 1, wherein the content of [Embodiment 7] A composition for increasing immune cell activity, comprising a cationic substance as an active ingredient. [Embodiment 8] A culture method for promoting the activity of natural killer cells, comprising culturing a cationic substance and immune cells. [Embodiment 9] A method for preventing or treating cancer, comprising administering the composition described in embodiment 1 to an individual. [Embodiment 10] Use of the composition described in embodiment 1 for increasing intracellular perforin protein. [Embodiment 11] Use of the composition described in embodiment 1 for increasing immune cell activity. [Embodiment 12] Use of a cationic substance for increasing intracellular perforin protein. The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0072] Example 1. Preparation of materials and animal models 1.1 Preparation or supplier of the compound branched Polyethyleneimine (Sigma-Aldrich, Sigma 408727) was diluted to 5 mg / ml in distilled water and used to make up the volume.

[0073] 1.2 NK cell preparation process NK cells were cultured in a T75 flask at a concentration of 3 × 10 cells / ml. The cells were homogenously diluted with α-MEM medium (Gibco 12561-056), 12.5% ​​FBS (Gibco 16000-044), 1% P / S (Gibco 15140-122), 2 mM L-glutamine (Gibco 25030-081), 0.2 mM inositol (Sigma I7508), 0.1 mM 2-mercaptoethanol, and 0.02 mM folic acid (Sigma F8785). The cells were then sterilized using a filter (Corning 430758). 5 The cells were cultured at 1000 x g / ml in a 37°C, 5% CO2 incubator.

[0074] 1.3 Method for analyzing the ability to induce natural killer cell activity in vitro Green fluorescently labeled MDA MB 231 cells and natural killer cells were mixed in a 1.5 ml Eppendorf tube and incubated for 4 hours in a 37°C CO2 5% incubator. After the incubation, the cells were stained with 7AAD (Invitrogen A1310) for 20 minutes and fixed. The mixed cells were separated into two cell populations based on the presence or absence of green fluorescence using a flow cytometer, and the percentage of dead cells was measured. The groups were compared and analyzed for activity.

[0075] 1.4 Western blot analysis method Protein extracts from natural killer cells were separated using 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Amersham Biosciences) at 120 V for 90 minutes. The membrane was blocked for 1 hour in Tris-buffered saline-Tween (TBST; 0.2 M NaCl, 0.1% Tween-20, and 10 mM Tris (pH 7.4)) containing 3% bovine serum albumin (BSA). The blocked membrane was then incubated with rabbit polyclonal anti-perforin antibody (1:1000; ab180773, Abcam) or rabbit monoclonal anti-GAPDH antibody (1:1000; 3683S, Cell Signaling). After incubation, the membrane was incubated with anti-rabbit polyclonal IgG (1:5000; #7074, Cell Signaling Technology) for 1 h at room temperature. After each step, the membrane was washed several times with TBST, and bound antibody was detected using an enhanced chemiluminescence detection system (Thermo Fisher Scientific Biosciences) according to the manufacturer's instructions.

[0076] Example 2. Confirmation of the ability of polyethyleneimine structures to induce natural killer cell activity Polyethyleneimine In order to confirm the ability of the natural killer cells to be activated according to the molecular weight and morphology of the natural killer cells of Example 1, Polyethyleneimine Or the control group was mixed and cultured for 48 hours.

[0077] Molecular weight and form of the compounds used in the experiment Polyethyleneimine The molecular weight, charge and structure of the comparative compounds are shown in Table 1 below.

[0078] Specifically, as shown in Table 1 below, a branched copolymer with a molecular weight of 1.8K was used. Polyethyleneimine , branched with a molecular weight of 10K Polyethyleneimine , linear with a molecular weight of 25K Polyethyleneimine , branched with a molecular weight of 25K Polyethyleneimine Natural killer cells were mixed with γ-PGA (gamma-PGA) having a molecular weight of 750K, glycol chitosan having a molecular weight of 5K, and protamine having a molecular weight of 4.5K, and the mixture was cultured for 48 hours. Polyethyleneimine uses primary amines, secondary amines and tertiary amines, and the linear Polyethyleneimine used a secondary amine.

[0079] [Table 1]

[0080] The cultured natural killer cells were then washed and resuspended in fresh culture medium. The natural killer cells were then mixed with the triple-negative breast cancer cell line MDA_MB231 at an E:T ratio of 10:1 and cultured for 4 hours. Finally, the cultured cells were quantitatively analyzed for the degree of target cell death using CFSE-7AAD assay.

[0081] Figure 2 shows the branched or linear structure of natural killer cells. Polyethyleneimine 1 is a graph showing the activity of natural killer cells as a result of treatment with .

[0082] As a result, as shown in Figure 2, when only the target (1.32) was present, when only NK cells were present (21.36), and when a branched chain with a molecular weight of 1.8K was present. Polyethyleneimine When mixed with (22.99), a branched chain with a molecular weight of 10K Polyethyleneimine When mixed with (24.07), a linear molecule with a molecular weight of 25K PolyethyleneimineThe degree of cell death was low when mixed with γ-PGA (gamma-PGA) with a molecular weight of 750K (26.12), when mixed with γ-PGA (gamma-PGA) with a molecular weight of 750K (23.08), when mixed with glycol chitosan with a molecular weight of 5K (32.14), and when mixed with protamine with a molecular weight of 4.5K (25.31). On the other hand, natural killer cells and branched chain PGA with a molecular weight of 25K were Polyethyleneimine When the triple-negative cancer cells were cultured (Branched PET (25K)) (59.28), the degree of cell death was higher than that of the control group. Polyethyleneimine This means that when used in combination with other compounds, it increases the activity of natural killer cells.

[0083] Example 3: Confirmation of cancer cell killing ability at different concentrations of polyethyleneimine Polyethyleneimine In order to confirm the ability of each concentration to induce natural killer cell activity, the natural killer cells of Example 1 were treated with a 25K molecular weight IgG. Polyethyleneimine were treated with different concentrations (0 μg / ml, 0.63 μg / ml, 1.25 μg / ml, 2.5 μg / ml, or 5 μg / ml) and cultured for 48 hours.

[0084] The cultured natural killer cells were then washed and resuspended in fresh culture medium. The natural killer cells were then mixed with the triple-negative breast cancer cell line MDA_MB231 at an E:T ratio of 10:1 and cultured for 4 hours. Finally, the cultured cells were quantitatively analyzed for the degree of target cell death using CFSE-7AAD assay.

[0085] Figure 3 shows that breast cancer cells are Polyethyleneimine1 shows the activity of natural killer cells after treatment with various concentrations of IL-16. The activity is shown for the following conditions: when only the target (comparison target) is present (5.0), when there is no target (N, 9.1), when treated with 0.63 μg / ml (14.6), when treated with 1.25 μg / ml (24.2), when treated with 2.5 μg / ml (32.8), and when treated with 5 μg / ml (55.1).

[0086] Figure 4 shows that breast cancer cells are Polyethyleneimine 1 is a graph showing the quantification of natural killer cell activity as a result of treatment with various concentrations of CI 16101-16101.

[0087] As a result, as shown in Figs. 3 and 4, Polyethyleneimine The higher the concentration, the better the cell killing ability. At a concentration of 5 μg / ml, NK cell activity was maintained at 90% or more, while the highest immune activity was observed. At concentrations of 10 μg / ml or higher, the cell killing ability was significantly reduced.

[0088] Example 4. Confirmation of cell killing ability according to the ratio of polyethyleneimine and breast cancer cells Polyethyleneimine In order to confirm the cancer cell killing ability depending on the mixture ratio of cancer cells, the natural killer cells of Example 1 were added with 5 μg / ml of 25K molecular weight IgG. Polyethyleneimine The treated natural killer cells were cultured with triple-negative breast cancer cells MDA_MB231 at different E:T ratios (1.25:1, 2.5:1, 5:1, or 10:1) for 48 hours.

[0089] The cultured natural killer cells were then washed and resuspended in fresh culture medium. The natural killer cells were then mixed with the triple-negative breast cancer cell line MDA_MB231 at an E:T ratio of 10:1 and cultured for 4 hours. Finally, the cultured cells were quantitatively analyzed for the degree of target cell death using CFSE-7AAD assay.

[0090] Figure 5 shows breast cancer cells and 25K molecular weight Polyethyleneimine This graph shows the degree of cell death of breast cancer cells cultured at various E:T ratios with 5 μg / ml PEI. When only the target was present, the value was 2.58. The non-treated group showed 9.80 at an E:T ratio of 1.25, 9.53 at an E:T ratio of 2.5, 11.92 at an E:T ratio of 5, and 17.56 at an E:T ratio of 10. The group treated with PEI at a concentration of 5 μg / ml showed 36.55 at an E:T ratio of 1.25, 39.90 at an E:T ratio of 2.5, 43.82 at an E:T ratio of 5, and 49.55 at an E:T ratio of 10.

[0091] Figure 6 shows breast cancer cells and 25K molecular weight Polyethyleneimine 1 is a graph quantitatively showing the degree of cell death of breast cancer cells after culturing at 5 μg / ml at different E:T ratios.

[0092] As a result, as shown in Figures 5 and 6, the activity of natural killer cells at all E:T ratios was higher than that of the untreated group, and the E:T ratio of 10:1 was the most excellent.

[0093] Example 5. Cation-dependent analysis of polyethyleneimine Polyethyleneimine In order to confirm the ability of the nanoparticles of Example 1 to induce natural killer cell activation in response to the charge, the nanoparticles were coated with anionic hyaluronic acid.

[0094] Specifically, cationic Zn / Fe nanoparticles were added by an electrical interaction method. Polyethyleneimine First, anionic hyaluronic acid is bonded to the surface of the Polyethyleneimine The zeta potential of the nanoparticles was measured, confirming their anionic nature.

[0095] The cultured natural killer cells were then washed and resuspended in fresh culture medium. The natural killer cells were then mixed with the triple-negative breast cancer cell line MDA_MB231 at an E:T ratio of 10:1 and cultured for 4 hours. Finally, the cultured cells were quantitatively analyzed for the degree of target cell death using CFSE-7AAD assay.

[0096] Figure 7 shows Polyethyleneimine 1 shows the activity of natural killer cells after treatment with cation-blocking agents, with values ​​of 1.02 for MDA-MB-231, 18.47 for MDA-MB-231 and NK-92MI, and 18.21 for MDA-MB-231, NK-92MI, and aNP.

[0097] Figure 8 shows Polyethyleneimine 1 is a graph showing the quantification of the activity of natural killer cells after treatment with natural killer cells in which the cation property of the compound was inhibited.

[0098] As a result, as shown in Figures 7 and 8, it can be seen that when the cations are reduced by anionic coating, the activation ability of natural killer cells is inhibited. Polyethyleneimine This means that the natural killer cell activation ability of α-glucan depends on cations.

[0099] Example 6. Analysis of the ability of polyethyleneimine to stabilize perforin protein 6.1 Analysis of stabilization ability by incubation time Polyethyleneimine In order to confirm whether or not the amount of intracellular perforin increases, 5 μg / ml of the natural killer cells of Example 1 was added. Polyethyleneimine After treatment with α-glucan and culturing for different time periods (0, 3, 6, 12, 24, or 48 hours), the cultured natural killer cells were subjected to Western blotting to determine the amount of granzyme B and perforin protein.

[0100] Figure 9 shows Polyethyleneimine 1 is a graph showing the change in expression of granzyme proteins as a result of culturing 5 μg / ml and natural killer cells over time.

[0101] Figure 10 shows Polyethyleneimine 1 is a graph showing changes in perforin protein expression as a result of culturing 5 μg / ml and natural killer cells over time.

[0102] Figure 11 shows Polyethyleneimine 10 shows images showing changes in the expression of granzyme proteins and perforin proteins as a result of culturing 5 μg / ml and natural killer cells over time.

[0103] Figure 12 shows Polyethyleneimine 1 is a graph showing changes in the expression of granzyme proteins and perforin proteins as a result of culturing 5 μg / ml and natural killer cells for 48 hours.

[0104] As a result, as shown in Figure 12, it was confirmed that the amount of granzyme B protein in the cultured natural killer cells was unchanged, but the amount of perforin protein was significantly increased. In particular, as shown in Figures 9 to 11, it was confirmed that the amount of perforin protein increased significantly when the cells were cultured for 48 hours compared to cells cultured for less than 48 hours. These results suggest that Polyethyleneimine This means that it induces the stabilization of perforin protein and increases its amount, thereby inducing the activation of natural killer cells.

[0105] 6.2 Confirmation of the dependence of intracellular perforin amount on perforin stability To determine whether the increase in intracellular perforin levels in Example 6.2 correlates with the degree of intracellular perforin stabilization, the natural killer cells of Example 1 were treated with MG132, a proteolysis inhibitor, to inhibit intracellular proteolysis and then cultured. Western blotting was then performed on the cultured natural killer cells to determine the levels of granzyme B and perforin protein.

[0106] Figure 13 shows Polyethyleneimine 10 is an image showing changes in perforin protein expression as a result of treating natural killer cells with MG132 and culturing them.

[0107] As a result, as shown in Figure 13, the amount of perforin protein was analyzed after blocking intracellular proteolysis, and it was confirmed that the amount of perforin protein increased with MG132 treatment. Polyethyleneimine This study confirmed that MG132 treatment did not further increase perforin protein levels, because treatment improved protein stability, resulting in increased intracellular perforin levels. This suggests that perforin levels in natural killer cells are regulated by protein stability.

Claims

1. A composition for increasing perforin protein in natural killer cells, comprising a cationic substance as an active ingredient, wherein the cationic substance is branched polyethyleneimine (PEI), and the molecular weight of the polyethyleneimine is 15,000 Da to 27,000 Da.

2. The composition according to claim 1, wherein the content of the polyethyleneimine is 1 μg / ml to 10 μg / ml.

3. A composition described in claim 1 for use in increasing perforin protein in natural killer cells.

4. A composition described in claim 1 for use in increasing the activity of natural killer cells.

5. A composition for increasing the activity of natural killer cells, comprising a cationic substance as an active ingredient, wherein the cationic substance is branched polyethyleneimine (PEI), and the molecular weight of the polyethyleneimine is 15,000 Da to 27,000 Da.

6. A culture method for promoting the activity of natural killer cells, comprising the step of culturing a cationic substance and natural killer cells, wherein the cationic substance is branched polyethyleneimine (PEI), and the molecular weight of the polyethyleneimine is 15,000 Da to 27,000 Da.

7. 10. A method for preventing or treating cancer, comprising administering the composition of claim 1 to an individual, wherein the individual is a non-human primate or mammal.

8. 8. The method of claim 7, wherein the non-human mammal comprises a mouse, rat, dog, cat, horse, or cow.

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