WO2022092784A1 - 양이온성 물질을 포함하는 조성물 및 이의 용도 - Google Patents
양이온성 물질을 포함하는 조성물 및 이의 용도 Download PDFInfo
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- WO2022092784A1 WO2022092784A1 PCT/KR2021/015147 KR2021015147W WO2022092784A1 WO 2022092784 A1 WO2022092784 A1 WO 2022092784A1 KR 2021015147 W KR2021015147 W KR 2021015147W WO 2022092784 A1 WO2022092784 A1 WO 2022092784A1
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- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
Definitions
- the present invention relates to compositions comprising cationic materials and uses thereof.
- Natural killer cells are immune cells that perform the primary defense action in the body in the innate immune system. Natural killer cells contain receptors that recognize abnormal cells, and they regulate the immune system by immediately detecting and removing abnormal cells such as cancer cells or viruses without specific antigens, and effectively inhibits the proliferation, recurrence, and metastasis of cancer cells. do
- immune cell therapy refers to a therapeutic agent that is genetically modified using immune cells in the body and then put back into the body.
- CAR-T cells chimeric antigen receptor T-cells
- the present inventors solved the above problems by confirming that the stabilization of the perforin protein is increased when the immune cells are treated with a cationic substance.
- compositions for increasing intracellular perforin protein comprising a cationic material as an active ingredient.
- compositions for enhancing immune cell activity comprising a cationic material as an active ingredient.
- the cationic material is pre-treated to provide immune cells with increased perforin expression level or content compared to normal cells.
- compositions for preventing or treating cancer including immune cells that have been pretreated with a cationic material and have increased perforin expression or content compared to normal cells.
- Another aspect provides a pharmaceutical composition for preventing or treating cancer comprising a cationic material and immune cells as active ingredients.
- Another aspect provides a culturing method for promoting the activity of immune cells comprising the step of culturing the cationic material and immune cells.
- Another aspect provides a method for increasing the content or expression of perforin in immune cells comprising the step of culturing the cationic material and immune cells.
- Another aspect provides a method for preventing or treating cancer, comprising administering to a subject a composition comprising a cationic material and immune cells as active ingredients.
- compositions comprising a cationic material as an active ingredient for use in increasing intracellular perforin protein.
- compositions comprising a cationic material as an active ingredient for use in increasing immune cell activity.
- compositions comprising a cationic material as an active ingredient for use in the manufacture of a medicament for the prevention or treatment of cancer.
- Another aspect provides the use of a cationic material for use in increasing the intracellular perforin protein.
- compositions for increasing intracellular perforin protein comprising a cationic material as an active ingredient.
- compositions for enhancing immune cell activity comprising a cationic material as an active ingredient.
- cationic substance may be a material having a cation on the surface, or a material having a positive charge on the surface.
- the cationic material may be polyetherimide (PEI) or chitosan (chitosan).
- PEI polyetherimide
- chitosan chitosan
- the cationic material may be a polyetheramide.
- polyetherimide is a polymer having a chemical formula of (C37H24O6N2)n, and has a density of 1.27 g/cm3.
- the polyetheramide may be a cationic material.
- the cationic property of the polyetheramide may be to deliver a gene into the cell.
- the cells may be immune cells, T cells, B cells, dendritic cells or natural killer cells.
- the enhancement of immune cell activity means that the immunomodulatory ability, cytotoxicity or apoptosis ability of the cell is activated compared to parental cells, for example, hematopoietic cells, or progenitor cells.
- the immune cells may be CAR-immune cells.
- NK cell Natural Killer cell
- the natural killer cells may be responsible for non-specific immunity, and may serve to remove viruses, cancer cells, and the like.
- the composition includes natural killer cells with increased immunomodulatory ability, cytotoxicity or apoptosis, and the virus or cancer cell killing ability of the natural killer cells is increased.
- Perforin protein refers to a glycoprotein that destroys cells by making holes in the plasma membrane of cells.
- the perforin protein of the cell may be present in the immune cell.
- the polyetheramide may be branched.
- the branching means that the chemical structure is not linear and includes branches.
- the polyetheramine may be a primary, secondary or tertiary amine.
- the molecular weight of the polyetheramide may be 10,000 mM to 30,000 mM.
- the molecular weight of the polyetheramide 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.
- it may be about 25,000 mM. Since the gene transfer efficiency increases in proportion to the density of the cations, when the molecular weight range is greater than or less than the numerical value, the gene transfer efficiency may be decreased.
- the polyetheramide may be branched or linear.
- the branched type may have one or more selected from the group consisting of primary, secondary and tertiary amines in one molecule.
- the branched type may have one or more amine structures in one molecule and thus may have a cation sponge effect that can be changed to a cation within a wide pH range.
- the content of the cationic material may be 0.1 ⁇ g/ml to 10 ⁇ g/ml.
- the content of the cationic material is 0.1 to 9, 0.1 to 8, 0.5 to 10, 0.5 to 9, 0.5 to 8, 1 to 10, 1 to 9, 1 to 8, 2 to 10, 2 to 9 , 2 to 8, 3 to 10, 3 to 9 or 3 to 8 ⁇ g/ml.
- the content of the cationic material is less than or more than the above range, there is a problem in that immune cells are not sufficiently activated or the scale of the perforin protein is reduced.
- the composition may be a medium composition.
- the composition for increasing perforin protein or the composition for enhancing immune cell activity when cultured with immune cells as a medium composition, induces stabilization or accumulation of perforin protein in immune cells, and consequently induces activation of immune cells it could be
- the composition may additionally include nanoparticles.
- nanoparticles may refer to particles having a size of 1 to 100 nm having a surface.
- the nanoparticles may be coated.
- the nanoparticles may be magnetic nanoparticles. More specifically, the core of the nanoparticles may include Zn or Fe. The nanoparticles may have magnetism due to the core layer.
- the cationic material may be bound to nanoparticles.
- the cationic material may be present on the surface of the nanoparticles, and may be chemically bonded to the surface of the nanoparticles.
- the composition according to an embodiment may be obtained by culturing the nanoparticles and the cationic material for a predetermined time, thereby binding polyetheramine to the surface of the nanoparticles, and obtaining a composition including the nanoparticles and the cationic material.
- the composition may induce the accumulation of the perforin protein in the cell by stabilizing the perforin protein.
- the stabilization may mean resistance to proteolytic degradation that induces an increase in the amount of perforin protein and improvement in translation efficacy from perforin mRNA. Accordingly, the composition may increase the number or amount of perforin protein in the cell.
- the composition may be administered simultaneously with the immuno-cancer agent.
- the composition When the composition is administered simultaneously with the immuno-oncology agent, it may increase the activity of the immuno-cancer agent.
- the composition may additionally include one or more selected from the group consisting of gamma-PGA (Gamma-PGA), glycol chitosan, and protamine.
- gamma-PGA Gamma-PGA
- glycol chitosan glycol chitosan
- protamine protamine
- the cationic material is pre-treated to provide immune cells with increased perforin expression level or content compared to normal cells.
- Another aspect provides a composition for preventing or treating cancer comprising the immune cells.
- the cationic material, cells, perforin, and immune cells are the same as described above.
- the therapeutic composition may be an immune anticancer agent.
- the term “immune anticancer agent” is an anticancer agent that kills cancer cells by activating immune cells in the body, and may refer to a drug that exhibits a therapeutic effect on cancer by enhancing the patient's own immunity.
- the immune anticancer agent may be an immune cell therapeutic agent.
- immune checkpoint inhibitor refers to T cells by blocking the activation of immune checkpoint proteins involved in T cell suppression, for example, PD-L1 expressed in tumor cells. It may refer to an immune anticancer agent that activates and attacks cancer cells.
- the immune cell therapeutic agent in the immune cell therapeutic agent, is 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-PD-L1 It may be any one or more selected from the group consisting of -CTLA-4, for example, it may be an NK cell therapeutic agent.
- the CAR-immunocyte therapeutic agent may refer to an immune cell therapeutic agent comprising CAR T (Chimeric Antigen Receptor-T) or CAR-NK (Chimeric Antigen Receptor-NK) cells.
- Another aspect provides a method of treating cancer in a subject, comprising administering the pharmaceutical composition to the subject.
- Another aspect provides a method for preventing or treating cancer, comprising administering to an individual a composition comprising a cationic material as an active ingredient.
- the term “individual” refers to a subject in need of treatment for cancer, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses and cattle. means mammals.
- the cancer is breast cancer, thyroid cancer, stomach cancer, colon cancer, lung cancer, liver cancer, prostate cancer, pancreatic cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, oral cancer, oral cancer, testicular cancer, acute myeloid leukemia, basal cell cancer, ovarian epithelial cancer, brain tumor, Multiple myeloma, hematologic cancer, chronic myelogenous leukemia, chronic lymphocytic leukemia, bladder cancer, peritoneal cancer, tongue cancer, non-small cell lung cancer, small cell lung cancer, small-cell lung cancer, small-cell cancer, esophageal cancer, kidney cancer, heart cancer, malignant lymphoma, urethral cancer, cervical cancer, rectal cancer, It may be at least one selected from the group
- prevention refers to any action that inhibits or delays the occurrence of cancer by administration of the composition according to the present invention.
- treatment refers to or includes the alleviation, progression inhibition or prevention of a disease, disorder or condition, or one or more symptoms thereof
- active ingredient or the term “pharmaceutically effective amount” refers to a disease, disorder or may mean any amount of a composition employed in the practice of the invention provided herein sufficient to alleviate, inhibit the progression or prevent the condition, or one or more symptoms thereof.
- the pharmaceutical composition may include immune cells in which the content of perforin is increased due to a cationic substance, thereby effectively treating cancer by including immune cells in which an immune response is activated as an active ingredient.
- composition may additionally contain other known adjuvants, preferably monophosphoryl lipid A (MPL) and Glucopyranosyl Lipid Adjuvant (GLA-SE), formulated as other adjuvants. in a stable nano-emulsion of squalene oil in water).
- MPL monophosphoryl lipid A
- GLA-SE Glucopyranosyl Lipid Adjuvant
- the method of administering the pharmaceutical composition is not particularly limited, but may be administered parenterally or orally, such as intravenously, subcutaneously, intraperitoneally, inhalation or topical application, depending on the desired method.
- the dosage varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease.
- a daily dose refers to an amount of a therapeutic agent according to one aspect sufficient to treat a disease state ameliorated by administration to a subject in need thereof.
- An effective amount of a therapeutic agent depends on the particular compound, the disease state and its severity, and the individual in need of treatment, which can be routinely determined by one of ordinary skill in the art.
- the dosage for the human body of the composition according to one aspect may vary depending on the patient's age, weight, sex, dosage form, health status, and disease degree. Based on an adult patient weighing 70 kg, for example, about 1,000-10,000 cells/time, 1,000-100,000 cells/time, 1,000-1000,000 cells/time, 1,000-10,000,000, 1,000-100,000,000 cells/time, 1,000 to 1,000,000,000 cells/time, 1,000 to 10,000,000,000 cells/time, may be administered in divided doses once or several times a day at regular time intervals, or may be administered several times at regular time intervals.
- the pharmaceutical composition may include a pharmaceutically acceptable carrier and/or additive.
- a pharmaceutically acceptable carrier and/or additive for example, sterile water, physiological saline, conventional buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic acid, or preservatives, etc. can do.
- organic materials such as biopolymers, inorganic materials such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers and chemical derivatives thereof, etc. may also be combined for topical administration.
- the pharmaceutical composition according to one embodiment is formulated in a dosage form suitable for injection, the immune cells, or substances that increase their activity, are dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution.
- the pharmaceutical composition can be used as a suspending agent, solubilizer, stabilizer, isotonic agent, preservative, adsorption inhibitor, surfactant, diluent, excipient, pH adjuster, analgesic agent, buffer, reducing agent, Antioxidants and the like may be included as appropriate.
- Pharmaceutically acceptable carriers and agents suitable for the present invention including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.
- the pharmaceutical composition is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person skilled in the art to which the present invention pertains, or It can be prepared by introducing into a multi-dose container.
- the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of a powder, granules, tablets or capsules.
- Another aspect provides a culturing method for promoting the activity of immune cells comprising the step of culturing the cationic material and immune cells.
- Another aspect provides a method for increasing the content or expression of perforin in immune cells comprising the step of culturing the cationic material and immune cells.
- the culturing may include culturing the cationic material and immune cells for 5 to 60 hours. For example, 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, 10 to 50 hours may be cultured. If the incubation time is greater than or less than the above value, the stabilization of Perforin may not be sufficiently achieved.
- the method may be to increase the stabilization of the perforin protein and induce intracellular accumulation by culturing the cationic material and immune cells, and consequently enhance apoptosis.
- compositions comprising a cationic material as an active ingredient for use in increasing intracellular perforin protein.
- compositions comprising a cationic material as an active ingredient for use in increasing immune cell activity.
- compositions comprising a cationic material as an active ingredient for use in the manufacture of a medicament for the prevention or treatment of cancer.
- Another aspect provides the use of a cationic material for use in increasing the intracellular perforin protein.
- composition comprising a cationic material according to an aspect as an active ingredient, there is an effect of increasing the activity of immune cells by increasing the stability of the perforin protein and inducing the accumulation of the perforin protein in the cell.
- 1 is a schematic diagram showing the results of polyetheramide treatment on natural killer cells.
- FIG. 2 is a diagram showing the activity of natural killer cells as a result of treating branched or linear polyetheramides in the natural killer cells.
- FIG. 3 is a diagram showing the activity of natural killer cells as a result of treating breast cancer cells with polyethylenamine having a molecular weight of 25K for each concentration.
- FIG. 4 is a graph showing the quantification of the activity of natural killer cells as a result of treating breast cancer cells with polyethylenamine having a molecular weight of 25K by concentration.
- FIG. 5 is a diagram showing the degree of apoptosis of breast cancer cells as a result of culturing breast cancer cells and 5 ⁇ g/ml of polyethylenamine having a molecular weight of 25K at each E:T ratio.
- FIG. 6 is a graph quantitatively showing the degree of apoptosis of breast cancer cells as a result of culturing breast cancer cells and 5 ⁇ g/ml of polyethylenamine having a molecular weight of 25K at each E:T ratio.
- FIG. 7 is a diagram showing the activity of natural killer cells as a result of treatment with natural killer cells by inhibiting the cationicity of polyetheramide.
- FIG. 9 is a graph showing the change in expression of granzyme protein as a result of culturing polyetheramide 5 ⁇ g/ml and natural killer cells by time.
- 10 is a graph showing the change in expression of perforin protein as a result of culturing polyetheramide 5 ⁇ g/ml and natural killer cells for each hour.
- 11 is an image showing changes in expression of granzyme protein and perforin protein as a result of culturing polyetheramide 5 ⁇ g/ml and natural killer cells by time.
- FIG. 12 is a diagram showing changes in expression of granzyme and perforin proteins as a result of culturing polyetheramide 5 ⁇ g/ml and natural killer cells for 48 hours.
- FIG. 13 is an image showing the change in expression of perforin protein as a result of culturing polyetheramide and natural killer cells treated with MG132.
- Polyethylenimine in branched form (Sigma-Aldrich, Sigma 408727) was diluted to 5 mg/ml in distilled water and used according to the dose.
- Alpha-MEM media (gibco12561-056) media base with 12.5% FBS (gibco 16000-044), 1% P/S (gibco15140-122), 2 mM L-glutamine (gibco25030-081), 0.2 mM inositol (Sigma I7508) ); 0.1 mM 2-mercaptoethanol; After uniformly diluting 0.02 mM folic acid (Sigma F8785), it was used after sterilization using a filter (corning 430758). NK cells were cultured in a 37°C CO 2 5% incubator at 3X105/ml in a T75 flask.
- Green-type fluorescence-labeled MDA MB 231 cells and natural killer cells were put in 1.5 ml eppendorf tube at respective ratios, mixed, and reacted in a 37°C CO 2 5% incubator for 4 hours. After the reaction was completed, it was stained with 7AAD (Invitrogen A1310) for 20 minutes and fixed. The presence or absence of green fluorescence among the mixed cells through flow cytometry was also analyzed for activity by dividing the two cell populations, measuring the percentage of dead cells, and comparing them by group.
- the protein extracts of natural killer cells were separated using 10% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (polyvinylidene difluoride amersham Biosciences) under 120V conditions for 90 minutes.
- Tris-buffered saline-Tween containing 3% bovine serum albumin (BSA) [TBST; 0.2 M NaCl, 0.1% Tween-20, and 10 mM Tris (pH 7.4)] to block the membrane for 1 hour.
- the blocking membrane was incubated with rabbit polyclonal anti-Perforin antibody (1:1000; ab180773, abcam) or rabbit monoclonal anti-GAPDH antibody (1:1000; 3683S, Cell signaling).
- membranes were incubated with anti-rabbit polyclonal IgG (1:5000; #7074, Cell Signaling Technology) for 1 hour 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.
- the natural killer cells of Example 1 were mixed with polyetheramide or a comparative group for each molecular weight and type and cultured for 48 hours.
- Table 1 below shows the molecular weight, charge, and structure of the polyetheramide or comparative compound by molecular weight and form used in the experiment.
- branched polyetheramides having a molecular weight of 1.8K branched polyetheramides having a molecular weight of 10K, linear polyetheramides having a molecular weight of 25K, branched polyetheramides having a molecular weight of 25K, branched polyetheramides having a molecular weight of 750K
- Gamma-PGA gamma-PGA
- glycol chitosan with a molecular weight of 5K and protamine with a molecular weight of 4.5K were mixed with natural killer cells and cultured for 48 hours.
- the branched polyetheramine primary, secondary and tertiary amines were used, and the prior polyetheramine used secondary amines.
- the cultured natural killer cells were washed, and repositioned in a fresh culture medium (resuspension). Then, the natural killer cells and the triple-negative breast cancer cell line MDA_MB231 were mixed at an E:T ratio of 10:1 and cultured for 4 hours. Finally, the degree of apoptosis of the target cells was quantitatively analyzed for the cultured cells using the CFSE-7AAD assay.
- FIG. 2 is a diagram showing the activity of natural killer cells as a result of treating branched or linear polyetheramides in the natural killer cells.
- polyetheramide having a molecular weight of 25K was added to the natural killer cells of Example 1 by concentration (0 ⁇ g/ml, 0.63 ⁇ g/ml, 1.25 ⁇ g/ml). , 2.5 ⁇ g/ml, or 5 ⁇ g/ml) and incubated for 48 hours.
- the cultured natural killer cells were washed, and repositioned in a fresh culture medium (resuspension). Then, the natural killer cells and the triple-negative breast cancer cell line MDA_MB231 were mixed at an E:T ratio of 10:1 and cultured for 4 hours. Finally, the degree of apoptosis of the target cells was quantitatively analyzed for the cultured cells using the CFSE-7AAD assay.
- FIG. 3 is a diagram showing the activity of natural killer cells as a result of treating breast cancer cells with polyethylenamine having a molecular weight of 25K for each concentration.
- FIG. 4 is a graph showing the quantification of the activity of natural killer cells as a result of treating breast cancer cells with polyethylenamine having a molecular weight of 25K by concentration.
- the natural killer cells of Example 1 were treated with polyetheramide having a molecular weight of 25K and 5 ⁇ g/ml by concentration, and the treated natural killer cells were tripled.
- Negative breast cancer cells MDA-MB231 and E:T ratios (1.25:1, 2.5:1, 5:1 or 10:1) were cultured for 48 hours.
- the cultured natural killer cells were washed, and repositioned in a fresh culture medium (resuspension). Then, the natural killer cells and the triple-negative breast cancer cell line MDA_MB231 were mixed at an E:T ratio of 10:1 and cultured for 4 hours. Finally, the degree of apoptosis of the target cells was quantitatively analyzed for the cultured cells using the CFSE-7AAD assay.
- FIG. 5 is a diagram showing the degree of apoptosis of breast cancer cells as a result of culturing breast cancer cells and 5 ⁇ g/ml of polyethylenamine having a molecular weight of 25K at each E:T ratio.
- the non-treated group showed 9.80 for E:T ratio 1.25, 9.53 for E:T ratio 2.5, 11.92 for E:T ratio 5, and 17.56 for E:T ratio 10.
- the group treated with PEI at a concentration of 5 ⁇ g/ml showed 36.55 for E:T Ratio 1.25, 39.90 for E:T Ratio 2.5, 43.82 for E:T Ratio 5, and 49.55 for E:T Ratio 10. .
- FIG. 6 is a graph quantitatively showing the degree of apoptosis of breast cancer cells as a result of culturing breast cancer cells and 5 ⁇ g/ml of polyethylenamine having a molecular weight of 25K at each E:T ratio.
- Example 1 In order to confirm the ability to induce natural killer cell activity in response to the charge of polyetheramine, the nanoparticles of Example 1 were coated with anionic hyalunic acid.
- cationic polyetheramide was first bound to Zn/Fe nanoparticles by electric interaction, and anionic hyalunic acid was bound to cationic polyetheramide thereon, and the zeta potential of the nanoparticles was By measuring, anionicity was confirmed.
- the cultured natural killer cells were washed, and repositioned in a fresh culture medium (resuspension). Then, the natural killer cells and the triple-negative breast cancer cell line MDA_MB231 were mixed at an E:T ratio of 10:1 and cultured for 4 hours. Finally, the degree of apoptosis of the target cells was quantitatively analyzed for the cultured cells using the CFSE-7AAD assay.
- FIG. 7 is a diagram showing the activity of natural killer cells as a result of treatment with natural killer cells by inhibiting the cationicity of polyetheramide.
- the values were 1.02 for MDA-MB-231, 18.47 for MDA-MB-231 and NK-92MI, and 18.21 for MDA-MB-231 and NK-92MI and aNP.
- Example 1 In order to determine whether polyetheramine increases the amount of intracellular perforin, the natural killer cells of Example 1 were treated with 5 ⁇ g/ml of polyetheramide, and timed (0 hours, 3 hours, 6 hours) , 12 hours, 24 hours or 48 hours), and then Western blot was performed on the cultured natural killer cells to confirm the amount of granzyme B and perforin protein.
- FIG. 9 is a graph showing the change in expression of granzyme protein as a result of culturing polyetheramide 5 ⁇ g/ml and natural killer cells by time.
- 10 is a graph showing the change in expression of perforin protein as a result of culturing polyetheramide 5 ⁇ g/ml and natural killer cells for each hour.
- 11 is an image showing changes in expression of granzyme protein and perforin protein as a result of culturing polyetheramide 5 ⁇ g/ml and natural killer cells by time.
- FIG. 12 is a diagram showing changes in expression of granzyme and perforin proteins as a result of culturing polyetheramide 5 ⁇ g/ml and natural killer cells for 48 hours.
- Example 6.2 In order to check whether the increase in the amount of intracellular perforin in Example 6.2 is related to the degree of intracellular perforin stabilization, the natural killer cells of Example 1 were treated with MG132, a proteolysis inhibitor, to prevent intracellular proteolysis. After that, it was cultured. Then, Western blot was performed on the cultured natural killer cells to confirm the amount of granzyme B and perforin protein.
- FIG. 13 is an image showing the change in expression of perforin protein as a result of culturing polyetheramide and natural killer cells treated with MG132.
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Abstract
Description
번호 | 화합물명 (Mw) | 분자량 | 전하 | 아민의 구조 |
1 | Branched PET (25K) | 25,000 | (+ + +) | 1차, 2차 및 3차 |
2 | Linear PEL (25K) | 25,000 | (+) | 2차 |
3 | Branched PET (10K) | 10,000 | (+) | 1차, 2차 및 3차 |
4 | Branched PET (1.8K) | 1,800 | (+) | 1차, 2차 및 3차 |
5 | Gamma-PGA | 750,000 | (-) | - |
6 | Glycol Chitosan | 5,000 | (+) | 1차 및 2차 |
7 | Protamine | 4,500 | (+) | 1차 및 2차 |
Claims (12)
- 양이온성 물질을 유효성분으로 포함하는 세포 내 퍼포린 단백질 증가용 조성물.
- 청구항 1에 있어서, 상기 세포는 면역 세포인 것인, 세포 내 퍼포린 단백질 증가용 조성물.
- 청구항 1에 있어서, 상기 양이온성 물질은 폴리에테르아미드(Polyetherimide: PEI)인 것인, 세포 내 퍼포린 단백질 증가용 조성물.
- 청구항 1에 있어서, 상기 폴리에테르아미드의 분자량은 10,000 mM 내지 30,000 mM 인 것인, 세포 내 퍼포린 단백질 증가용 조성물.
- 청구항 1에 있어서, 상기 폴리에테르아미드는 분지된 것인, 세포 내 퍼포린 단백질 증가용 조성물.
- 청구항 1에 있어서, 상기 폴리에테르아미드의 함량은 1 ㎍/㎖ 내지 10 ㎍/㎖ 인 것인, 세포 내 퍼포린 단백질 증가용 조성물.
- 양이온성 물질을 유효성분으로 포함하는 면역세포 활성 증진용 조성물.
- 양이온성 물질 및 면역세포를 배양하는 단계를 포함하는 자연살해세포의 활성을 촉진하는 배양 방법.
- 청구항 1의 조성물을 개체에 투여하는 단계를 포함하는 암의 예방 또는 치료하는 방법.
- 세포 내 퍼포린 단백질 증가에 사용하기 위한 청구항 1의 조성물의 용도.
- 면역세포 활성 증가에 사용하기 위한 청구항 1의 조성물의 용도.
- 세포 내 퍼포린 단백질 증가에 사용하기 위한 양이온성 물질의 용도.
Priority Applications (3)
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US18/250,876 US20230390326A1 (en) | 2020-10-29 | 2021-10-27 | Composition comprising cationic substance, and use for same |
JP2023526242A JP2023549078A (ja) | 2020-10-29 | 2021-10-27 | 陽イオン性物質を含む組成物及びその用途 |
CN202180088281.9A CN117255853A (zh) | 2020-10-29 | 2021-10-27 | 包括阳离子物质的组合物以及该组合物的用途 |
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KR10-2020-0142534 | 2020-10-29 | ||
KR1020200142534A KR20220057359A (ko) | 2020-10-29 | 2020-10-29 | 양이온성 물질을 포함하는 조성물 및 이의 용도 |
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WO2022092784A1 true WO2022092784A1 (ko) | 2022-05-05 |
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US (1) | US20230390326A1 (ko) |
JP (1) | JP2023549078A (ko) |
KR (3) | KR20220057359A (ko) |
CN (1) | CN117255853A (ko) |
WO (1) | WO2022092784A1 (ko) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2003146887A (ja) * | 2001-11-07 | 2003-05-21 | Gotoo Corporation:Kk | Nk細胞活性化作用を有する製剤および飲食品 |
JP2007509040A (ja) * | 2003-10-11 | 2007-04-12 | イネックス ファーマシューティカルズ コーポレイション | 先天性免疫及び抗体依存性細胞傷害を強化するための方法及び組成物 |
WO2010088927A1 (en) * | 2009-02-09 | 2010-08-12 | Curevac Gmbh | Use of pei for the improvement of endosomal release and expression of transfected nucleic acids, complexed with cationic or polycationic compounds |
-
2020
- 2020-10-29 KR KR1020200142534A patent/KR20220057359A/ko active Application Filing
-
2021
- 2021-10-27 CN CN202180088281.9A patent/CN117255853A/zh active Pending
- 2021-10-27 JP JP2023526242A patent/JP2023549078A/ja active Pending
- 2021-10-27 US US18/250,876 patent/US20230390326A1/en active Pending
- 2021-10-27 WO PCT/KR2021/015147 patent/WO2022092784A1/ko active Application Filing
-
2023
- 2023-06-19 KR KR1020230078496A patent/KR20230093226A/ko not_active Application Discontinuation
- 2023-08-04 KR KR1020230102231A patent/KR20230119094A/ko not_active Application Discontinuation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2003146887A (ja) * | 2001-11-07 | 2003-05-21 | Gotoo Corporation:Kk | Nk細胞活性化作用を有する製剤および飲食品 |
JP2007509040A (ja) * | 2003-10-11 | 2007-04-12 | イネックス ファーマシューティカルズ コーポレイション | 先天性免疫及び抗体依存性細胞傷害を強化するための方法及び組成物 |
WO2010088927A1 (en) * | 2009-02-09 | 2010-08-12 | Curevac Gmbh | Use of pei for the improvement of endosomal release and expression of transfected nucleic acids, complexed with cationic or polycationic compounds |
Non-Patent Citations (2)
Title |
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CHUNG YANG SOOK, ET AL.: " Effects of Chitosan on Anti-tumor Cellular Activity in Mice", JOURNAL OF LIFE SCIENCE, KOREAN SOCIETY OF LIFE SCIENCE, KR, vol. 14, no. 2, 1 January 2004 (2004-01-01), KR , pages 209 - 214, XP055926733, ISSN: 1225-9918, DOI: 10.5352/JLS.2004.14.2.209 * |
LIN LI, HE JUNYI, LI JIAWEI, XU YAN, LI JINGXIA, WU YANGZHE: "Chitosan Nanoparticles Strengthen Vγ9Vδ2 T-Cell Cytotoxicity Through Upregulation Of Killing Molecules And Cytoskeleton Polarization", INTERNATIONAL JOURNAL OF NANOMEDICINE, DOVE MEDICAL PRESS LTD., AUCKLAND, NZ, vol. Volume 14, 1 November 2019 (2019-11-01), AUCKLAND, NZ , pages 9325 - 9336, XP055923051, ISSN: 1176-9114, DOI: 10.2147/IJN.S212898 * |
Also Published As
Publication number | Publication date |
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KR20230093226A (ko) | 2023-06-27 |
JP2023549078A (ja) | 2023-11-22 |
CN117255853A (zh) | 2023-12-19 |
KR20230119094A (ko) | 2023-08-16 |
KR20220057359A (ko) | 2022-05-09 |
US20230390326A1 (en) | 2023-12-07 |
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