JP2022161046A - Method of increasing nk cell ability - Google Patents

Abstract

To provide a method of increasing the ability of NK cells, which can impart a propensity for attacking a specific pathogen to an activated NK cell population of an administration subject.SOLUTION: The method of imparting a propensity for attacking a specific pathogen to all activated NK cell population after an NK cell ability increasing step, comprises the steps of: selectively inducing NK cells from white blood cells of an administration subject; storing the induced NK cells and a prescribed NK medium in a vessel; leaving the vessel static for 6-12 hours while maintaining the vessel at a temperature of 36.5-37.5°C to activate the NK cells without allowing the NK cells to proliferate and differentiate; storing, in the vessel containing the activated NK cells, predetermined family cytokines that cause the NK cells to learn an attackability against the specific pathogen causing a disease in the administration subject; and increasing the NK cell ability without allowing the NK cells to proliferate and differentiate, by leaving the vessel static for 18-24 hours while maintaining the vessel at a temperature of 36.5-37.5°C to cause all the NK cell population in an early stage of induction to learn the attackability against the specific pathogen.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to a method for enhancing NK cell ability.

Interleukin-15 ( IL-15), and optionally one or more of stem cell factor (SCF) and interleukin-7 (IL-7), wherein IL-15 and optionally SCF and IL-7 are non-deterministic components of the medium. (a) seeding the population in a first medium not contained in and expanding the cells from step (a) in a second medium comprising interleukin-2 (IL-2) , a method for producing a population of activated natural killer (NK) cells comprising the step (b) of producing a population of activated NK cells (see Patent Document 1).

Japanese Patent Application Laid-Open No. 2020-96607

The method for producing a population of activated natural killer (NK) cells disclosed in Patent Document 1 is such that the activated natural killer (NK) cells produced by the method cannot learn aggression against a specific pathogen. and does not confer a propensity to attack specific pathogens on activated natural killer (NK) cells. Therefore, even if activated natural killer (NK) cells are administered, the activated natural killer (NK) cells do not attack specific pathogens, and the activated natural killer (NK) cells are used to identify pathogens. The symptoms of the specific disease caused by the pathogen cannot be alleviated, and the treatment aimed at curing the specific disease caused by the specific pathogen cannot be performed. In addition, the anti-disease effect (healing ability) of the activated natural killer (NK) cells produced by the method cannot be increased for specific diseases, and the activated natural killer (NK) cells are used to identify The symptoms of the disease cannot be relieved and treatment aimed at curing the specific disease cannot be given.

The object of the present invention is to provide NK cell ability capable of activating NK cells in the early stage of induction of administration subjects and imparting propensity to attack specific pathogens to the activated NK cell group of administration subjects. To provide an upgrade method. Another object of the present invention is to be able to activate the induced early NK cells of the administration subject, and to increase the anti-disease effect (healing ability) of the activated NK cell group to a specific disease of the administration subject. To provide a method for improving NK cell ability.

The premise of the present invention for solving the above problems is that a specific pathogen that increases the activity of NK cells in a subject to whom NK cells (natural killer cells) are administered and causes a specific disease in the subject It is a method for increasing the ability of NK cells to confer a propensity to attack NK cells of an administration subject, or to increase the anti-disease effect (healing ability) of the NK cells of an administration subject against a specific disease.

A feature of the present invention based on the above premise is that the method for increasing the ability of NK cells collects a predetermined amount of blood from a subject, separates leukocytes from the collected blood of the subject, and separates NK cells of the subject from the leukocytes. a first accommodation step of accommodating the NK cells induced by the NK cell induction step and a predetermined NK medium in a container having a predetermined volume; an NK cell activation step of activating NK cells without proliferating and differentiating the NK cells by leaving the container containing the medium at a temperature of 36.5 to 37.5° C. for 6 to 12 hours statically; , a container containing NK cells activated by the NK cell activation step, a predetermined family cytokine or administration subject that makes NK cells learn aggressiveness to a specific pathogen causing the disease of the administration subject a second accommodation step of accommodating a predetermined family cytokine that enhances the anti-disease effect (healing ability) against the disease of the patient; an NK cell performance improvement step in which the NK cells are left statically for 18 to 24 hours while being maintained at ~37.5°C to improve the performance of the NK cells without causing the NK cells to proliferate and differentiate, and in the NK cell performance improvement step, Aggressiveness against specific pathogens is induced by stimulating early NK cells activated by the NK cell activation process with family cytokines All early NK cell groups learn, and NK cells proliferate and differentiate All NK cell groups after the NK cell performance improvement step are given a propensity to attack a specific pathogen, or the early NK cells activated by the NK cell activation step are stimulated by family cytokines Therefore, the anti-disease effect (healing ability) of all NK cell groups against diseases after the NK cell performance improvement step is increased without the NK cells being proliferated and differentiated.

As an example of the present invention, the method for increasing the ability of NK cells includes a plasma separation step of separating autologous plasma of the administration subject from blood collected from the administration subject and inactivating the separated autologous plasma, and a first accommodation step stores the inactivated autologous plasma of the administration subject in a container together with NK cells and NK medium.

As another example of the present invention, the first accommodating step includes 2 to 5% inactivated autologous plasma relative to the total mass of NK cells, NK medium, and autologous plasma, when the total mass is 100%. Place in container.

As another example of the present invention, the second accommodation step is 0.2 to 2% of the total mass of NK cells, NK medium, autologous plasma, and family cytokines relative to the total mass of 100%. A cytokine is placed in a container.

As another example of the present invention, in the NK cell performance enhancing step, the container is replenished with a predetermined amount of family cytokines every 3-6 hours while the container is left statically for 18-24 hours.

As another example of the present invention, the specific pathogen is a tumor cell, and the family cytokine causes the early NK cell group activated by the NK cell activation process to learn aggressiveness to the tumor cell. IL-2 (interleukin-2), IL-7 (interleukin-7), IL-12 (interleukin -12), IL-15 (interleukin-15), IL-18 (interleukin-18), IL-27 (interleukin-27).

As another example of the present invention, the specific pathogen is the AIDS virus or the novel coronavirus, and the family cytokine is an NK cell group at the early stage of induction activated by the NK cell activation process to the AIDS virus or the novel coronavirus. It is IL-7 (interleukin-7) that makes all NK cell groups learn the aggressiveness of AIDS virus or new coronavirus, and increases the antiviral effect of NK cell group.

As another example of the present invention, the specific disease of the subject of administration is bronchial asthma, and the family cytokine has an anti-disease effect on bronchial asthma of the induced early NK cell group activated by the NK cell activation process. at least one of IL-4 (interleukin-4), IL-5 (interleukin-5) that increases (healing ability).

As another example of the present invention, the specific disease of the subject of administration is rheumatoid arthritis, and the family cytokine has an anti-disease effect on rheumatoid arthritis of the induced early NK cell group activated by the NK cell activation process. at least one of IL1 (interleukin 1), IL6 (interleukin 6), IL8 (interleukin 8) that increases (healing ability).

As another example of the present invention, the specific disease of the administration subject is atopic dermatitis, and the family cytokine is an induced early NK cell group activated by the NK cell activation process to atopic dermatitis. It is IL-4 (interleukin-4) that increases the anti-disease effect (healing ability) of cancer.

As another example of the present invention, the specific disease of the subject of administration is diabetes, and the family cytokine has an anti-disease effect on the diabetes of the NK cell group in the early stage of induction activated by the NK cell activation process ( It is IL-15 (interleukin-15) that increases healing ability).

According to the method for increasing NK cell ability according to the present invention, the activated NK cells in the early stage of induction are stimulated by family cytokines, so that all of the NK cell groups in the early stages of induction of aggression against specific pathogens learn. Since NK cells do not proliferate and differentiate, all NK cell groups are endowed with a propensity to attack specific pathogens. NK cell populations can be endowed with a propensity to attack specific pathogens. The method for increasing the ability of NK cells is to administer NK cells imparted with a propensity to attack a specific pathogen by the method to a subject, and the NK cells cause a specific disease in the subject. It can intensively attack a specific pathogen, use the NK cells to alleviate the symptoms of a specific disease caused by the specific pathogen, and use the NK cells to treat the disease caused by the specific pathogen. Treatment aimed at curing a person's specific disease can be performed. In the method for increasing the ability of NK cells, activated NK cells in the early stage of induction are stimulated by family cytokines, so that the anti-disease effect (healing ability) of all NK cell groups against diseases is achieved without the proliferation and differentiation of NK cells. Therefore, it is possible to increase the anti-disease effect (healing ability) of the activated NK cell group of the administration subject against a specific disease while activating the NK cells in the initial induction period of the administration subject, and the NK cell ability By administering NK cells that have an anti-disease effect against a specific disease by the up method to a subject, it is possible to alleviate the symptoms of a specific disease using NK cells. As a result, treatment aimed at curing a specific disease of the subject of administration can be performed.

A plasma separation step of separating autologous plasma of the administration subject from blood collected from the administration subject and inactivating the separated autologous plasma is included, and the first accommodation step inactivates the administration subject together with the NK cells and the NK medium. The method for increasing the ability of NK cells to contain autologous plasma in a container uses the inactivated autologous plasma of a subject to be administered, so that NK cells in the early stage of induction can be activated by the NK medium and the autologous plasma. It is possible to give the subject's activated NK cell population a propensity to attack a specific pathogen, or to increase the anti-disease effect (healing ability) of the activated NK cell population to a specific disease. .

When the total mass of NK cells, NK medium and autologous plasma is 100%, the method of increasing the NK cell ability to accommodate 2 to 5% of the total mass of autologous plasma in a container is 2 to 2 to the total mass. By storing 5% autologous plasma in a container and using the inactivated autologous plasma, the NK cells at the early stage of induction can be activated by the NK medium and the autologous plasma, and the subject is activated. The NK cell group can be endowed with a propensity to attack a specific pathogen, or the anti-disease effect (healing ability) of the activated NK cell group against a specific disease can be increased.

When the total mass of NK cells, NK medium, autologous plasma, and family cytokines is 100%, the method for increasing the NK cell ability to accommodate 0.2 to 2% of the total mass of family cytokines in a container is based on the total mass. By using 0.2 to 2% of family cytokines, it is possible to reliably impart a propensity to attack a specific pathogen to the activated NK cell group of the administration subject, or activated The anti-disease effect (healing ability) of the NK cell group to specific diseases can be reliably increased. The method for increasing the ability of NK cells uses NK cells that have been given a propensity to attack specific pathogens by 0.2 to 2% family cytokines, and cures specific diseases of administration subjects caused by specific pathogens. Using NK cells that can perform targeted treatments and have increased anti-disease effects against specific diseases with 0.2 to 2% family cytokines, treatment aimed at curing specific diseases of the recipient. It can be carried out.

In the NK cell performance improvement step, the NK cell performance improvement method of supplying a predetermined amount of family cytokine to the container every 3 to 6 hours while the container is left statically for 18 to 24 hours is performed for 3 to 6 hours. Since the container is replenished with a predetermined amount of family cytokine every time it passes, the family cytokine will not be depleted, and by using the family cytokine, the activated NK cell group of the administration subject will have a specific pathogen. Aggressive propensity can be reliably imparted, or the anti-disease effect (healing ability) of the activated NK cell group against a specific disease can be reliably increased. The method for increasing the ability of NK cells uses NK cells that have been given a propensity to attack specific pathogens by family cytokines, and can be used to treat specific diseases caused by specific pathogens. It is possible to use NK cells whose anti-disease effects against specific diseases have been enhanced by family cytokines, and to perform treatments aimed at curing specific diseases of administration subjects.

The specific pathogen is a tumor cell, and the family cytokine is activated by the NK cell activation process. , IL-2 (interleukin-2), IL-7 (interleukin-7), IL-12 (interleukin-12), IL-15 (interleukin- 15) IL-18 (interleukin-18), IL-27 (interleukin-27) is at least one of the NK cell ability-up method activates NK cells in the early stage of induction of the administration subject. While IL-2 (interleukin-2), IL-7 (interleukin-7), IL-12 (interleukin-12), IL-15 (interleukin-15), IL-18 (interleukin-18 ), IL-27 (interleukin-27) can be used to confer a propensity to attack tumor cells in the subject's activated NK cell population. The method for increasing the ability of NK cells is to administer NK cells that have been given the propensity to attack tumor cells to the subject, and the NK cells concentrate the tumor cells that cause specific diseases in the subject. can be used to alleviate the symptoms of a specific disease caused by tumor cells, and the NK cells can be used to cure a specific disease caused by tumor cells in a subject. Treatment aimed at

The specific pathogen is the AIDS virus or the new coronavirus, and the NK cell group at the early stage of induction activated by the family cytokine by the NK cell activation process is made to learn the aggressiveness to the AIDS virus or the new coronavirus, and all NK cells The method of increasing the ability of NK cells, which is IL-7 (interleukin-7), which gives the group a tendency to attack the AIDS virus or the new coronavirus and increases the antiviral effect of the NK cell group, While activating NK cells, IL-7 (interleukin-7) can be used to confer a propensity to attack the AIDS virus or novel coronavirus to the activated NK cell group of the administration subject. The method for increasing the ability of NK cells is to administer NK cells that have been given a propensity to attack the AIDS virus or the new coronavirus to the subject, and the NK cells become the cause of AIDS or the new corona in the subject. It is possible to intensively attack the AIDS virus or the new coronavirus that is present, use the NK cells to alleviate the symptoms of AIDS or the new coronavirus in the recipient, and use the NK cells to kill the AIDS virus or the new coronavirus. Treatment aimed at curing AIDS or COVID-19 caused by a virus can be performed.

The specific disease of the administration subject is bronchial asthma, and IL-4 (intermediate Leukin-4) and IL-5 (interleukin-5), which is at least one of NK cell ability increasing methods, while activating NK cells in the initial induction period of the administration subject, IL-4 (interleukin -4) At least one of IL-5 (interleukin-5) can be used to increase the anti-disease effect (healing ability) of the activated NK cell group for bronchial asthma in the administration subject. . The method for increasing the ability of NK cells involves administering NK cells, which have an anti-disease effect on bronchial asthma by the method, to a subject, and using the NK cells to alleviate the symptoms of bronchial asthma in the subject. NK cells can be used for treatment aimed at curing bronchial asthma in the subject.

The specific disease of the administration subject is rheumatoid arthritis, and IL1 (interleukin 1) that raises the anti-disease effect (healing ability) on rheumatoid arthritis of the early NK cell group activated by the family cytokine through the NK cell activation process ), IL6 (interleukin 6), and IL8 (interleukin 8). ), IL6 (interleukin 6), and IL8 (interleukin 8) to increase the anti-disease effect (healing ability) of the activated NK cell group on rheumatoid arthritis in the administration subject. can be done. The method for increasing the ability of NK cells involves administering NK cells, which have an anti-disease effect against rheumatoid arthritis, to a subject, thereby alleviating the symptoms of rheumatoid arthritis in the subject using the NK cells. NK cells can be used to treat rheumatoid arthritis in the subject.

The specific disease of the administration subject is atopic dermatitis, and the IL that increases the anti-disease effect (healing ability) on the atopic dermatitis of the NK cell group in the early stage of induction, which is activated by the NK cell activation process of the family cytokine -4 (interleukin-4) NK cell ability increasing method, while activating the NK cells of the administration subject early induction, IL4 (interleukin 4) is used to activate the NK cells of the administration subject It is possible to increase the anti-disease effect (healing ability) of the cell group to atopic dermatitis. The method for increasing the ability of NK cells is to administer NK cells, which have an anti-disease effect against atopic dermatitis by the method, to a subject, and use the NK cells to improve the symptoms of atopic dermatitis in the subject. can be alleviated, and NK cells can be used for treatment aimed at curing atopic dermatitis in the subject.

The specific disease of the subject of administration is diabetes, and IL-15 (interleukin- 15), a method for increasing the ability of NK cells, activates NK cells in the early stage of induction of the subject, and uses IL5 (interleukin 5) to promote the activation of the NK cell group in the subject to diabetes. Anti-disease effect (healing ability) can be increased. The method for increasing the ability of NK cells is capable of alleviating the symptoms of diabetes in the subject by using the NK cells by administering the NK cells whose anti-disease effect on diabetes has been enhanced by the method to the subject. , NK cells can be used for treatment aimed at curing diabetes in the subject.

Schematic diagram of NK cell separation process shown as an example. Schematic which shows an example of a 1st accommodation process. Schematic diagram showing an example of the NK cell activation process. Schematic which shows an example of a 2nd accommodation process. The figure which shows the administration to the administration subject of the NK cell (NK cell group) after the NK cell performance improvement process. Schematic which shows another example of a 2nd accommodation process. Schematic which shows another example of a 2nd accommodation process. Schematic which shows another example of a 2nd accommodation process. Schematic which shows another example of a 2nd accommodation process. Schematic which shows another example of a 2nd accommodation process.

The details of the NK cell capacity enhancing method according to the present invention will be described below with reference to the accompanying drawings such as FIG. 1, which is a schematic diagram of the NK cell separation process shown as an example. FIG. 2 is a schematic diagram showing an example of the first accommodation step, and FIG. 3 is a schematic diagram showing an example of the NK cell activation step.

The NK cell ability increasing method increases the activity of the NK cells of the administration subject 11 to whom NK cells 10 (natural killer cells) are administered, and increases the aggressiveness against the specific pathogen causing the specific disease of the administration subject 11. is given to the NK cells 10 of the administration subject 11, or the anti-disease effect (healing ability) of the NK cells 10 of the administration subject 11 against a specific disease is increased.

In the NK cell capacity increasing method, a predetermined amount (15 to 16 cc) of blood is collected from an administration subject 11 (blood collector), leukocytes 12 are separated from the collected blood of the administration subject 11, and leukocytes 12 are administered. NK cells 10 of a subject 11 are selectively induced (NK cell induction (extraction) step). Further, the autologous plasma 13 of the administration subject 11 is separated from the blood collected from the administration subject 11, and the separated autologous plasma 13 is inactivated (plasma separation step).

As shown in FIG. 1, the blood collected from the administration subject 11 is injected (contained) in a vertically elongated glass test tube 14 (separation container). A glass test tube 14 is placed in a centrifuge, and the blood in the glass test tube 14 is centrifuged into autologous plasma 13, white blood cells 12, and red blood cells 15 (three layers) by the centrifuge. After the blood is separated into layers of autologous plasma 13, leukocytes 12, and erythrocytes 15, leukocytes 12 are extracted (aspirated) using a syringe (not shown) or a pipette (not shown), and extracted from the leukocytes 12. Selectively induce (extract) NK cells 10 of administration subject 11 (blood collector), extract (separate) autologous plasma 13 using a syringe or pipette, and extract (separate) autologous plasma 13 is inactivated.

The NK cells 10 can be extracted (collected) by a process of separating mononuclear cells from blood (white blood cells 12) collected from the subject 11 of administration. For example, NK cells 10 can be harvested using immunomagnetic beads, or immunofluorescently stained with specific antibodies against cell surface markers and isolated using a FACS (Fluorescence activated cell sorter) or flow cytometer.・Can be identified.

After inducing (extracting) NK cells 10 from white blood cells 12 by the NK cell separation step and separating autologous plasma 13 by the plasma separation step, the NK cells 10 of the administration subject 11, the inactivated autologous plasma 13, and a predetermined NK medium 16 are accommodated in a flat container 17 (container) having a predetermined volume (first accommodation step). As shown in FIG. 2, the flat container 17 is a flat container that is made of transparent glass or transparent plastic, has a small capacity, has a bottom surface with a predetermined area, and has a substantially square planar shape. The injection port of the flat container 17 is watertightly (airtightly) closed with a lid. The flat container 17 has a capacity of about 20-30 cc (preferably 25 cc), a bottom surface area of about 25-36 mm ² and a side length of 5-6 mm. As the flat container 17, a flat container having a small capacity and a bottom surface with a predetermined area and having a circular or elliptical planar shape can also be used.

In the first accommodation step, when the total mass of the NK cells 10, the NK medium 16 and the autologous plasma 13 is 100%, the inactivated autologous plasma 13 having a mass of 2 to 5% with respect to the total mass is flattened. Store (add) to container 17 . If the mass of the autologous plasma 13 to the total mass is less than 2%, the inactivated autologous plasma 13 cannot be used sufficiently, and the NK medium 16 and the autologous plasma 13 are used to activate the NK cells 10 at the early stage of induction. and cannot confer NK cells 10 (NK cell group) with a propensity to attack specific pathogens, or anti-disease effects of NK cells 10 (NK cell group) on specific diseases (Healing ability) cannot be increased.

In the NK cell ability increasing method, autologous plasma 13 with a mass of 2 to 5% with respect to the total mass (100%) is stored in a flat container 17, and the inactivated autologous plasma 13 is used to create an NK medium 16. and autologous plasma 13 can activate NK cells 10 in the early stage of induction, and can impart a propensity to attack specific pathogens to the activated NK cells 10 (NK cell group) of the administration subject, Alternatively, the anti-disease effect (healing ability) of the activated NK cells 10 (NK cell group) to a specific disease can be increased.

NK medium 16 includes KBM501 medium (Kohjin Bio Co., Ltd., containing IL-2 at 1,750 JRU/ml. For human NK cell Primary Culture), CellGro SCGM medium (Cellgenics, Iwai Chemicals Co., Ltd.), X- VIVO15 medium (Lonza, Takara Bio Inc.), Cosmedium 008 (Cosmo Bio. Contains 1,750 JRU/ml IL-2. For human NK cell Primary Culture), CTS AIM V Medium Gibco ^™ CTS ^™ AIM V ^™ Medium ( CTS OpTmizer T Cell Expansion Basal Medium (Thermo Fisher Scientific. For the growth and expansion of human T lymphocytes), CTS OpTmizer T Cell Expansion Basal Medium. ), IMDM, MEM, DMEM, RPMI-1640, etc., but not limited thereto, other NK media can also be used.

The NK medium 16 may be supplemented with subject's autologous serum, human type AB serum available from BioWhittaker, and donated human serum albumin available from the Japanese Red Cross Society. Autologous serum and human type AB serum are preferably added at a concentration of 1-10%, more preferably at a concentration of 5-7%. Donated human serum albumin is preferably added at a concentration of 1-10%, more preferably at a concentration of 5-7%. A serum-free medium can also be used as the NK medium 16 . The serum-free medium preferably contains serum albumin, transferrin and insulin.

After accommodating the NK cells 10, the NK medium 16, and the autologous plasma 13 in the flat container 17 by the first accommodation step, as shown in FIG. While maintaining the flat container 17 at a temperature of 36.5 to 37.5° C. in the humidifier 18, the flat container 17 is left statically for 6 to 12 hours to activate the NK cells 10 (NK cell activation step). . In the NK cell activation step, the NK cells 10 are activated by the NK medium 16 and the autologous plasma 13 without proliferating and differentiating the NK cells 10 housed in the flat container 17, and all NK cells 10 (NK cell group) are activated. activity increases.

FIG. 4 is a schematic diagram showing an example of the second accommodation step, and FIG. 5 is a diagram showing administration of NK cells 10 (NK cell group) to administration subject 11 after the NK cell performance improvement step. After activating the NK cells 10 (NK cell group) by the NK cell activation step, the flat container 17 is removed from the constant temperature and humidity chamber 18, and the activated NK cells 10 (NK cell group) are placed in the flat container 17. A predetermined family cytokine is accommodated (second accommodation step). The family cytokine imparts the NK cell 10 with a propensity to attack a specific pathogen by making the NK cell 10 learn the aggressiveness to a specific pathogen that causes the disease of the administration subject 11, or NK cells The anti-disease effect (healing ability) of 10 administration subjects 11 is increased (enhanced).

When the specific pathogen is a tumor cell (malignant tumor cell), an example of the family cytokine IL2, IL7, IL12, IL15, IL18, IL27 (stimulatory substance) accommodated in the flat container 17 in the second accommodation step is IL- 2 (interleukin-2), IL-7 (interleukin-7), IL-12 (interleukin-12), IL-15 (interleukin-15), IL-18 (interleukin-18), IL- 27 (interleukin-27) and utilizes one or more of these interleukins.

IL-2 (interleukin-2), IL-7 (interleukin-7), IL-12 (interleukin-12), IL-15 (interleukin-15), IL-18 (interleukin-18), IL-27 (interleukin-27) stimulates early separation NK cells 10 (NK cell group) activated by the NK cell activation process, and activates early separation NK cells 10 (NK cell group). Aggressiveness to tumor cells (malignant tumor cells) is learned to give all NK cells 10 (NK cell group) aggressiveness to tumor cells (malignant tumor cells), and NK cells 10 (NK cell group) Increases cell death induction of tumor cells (malignant tumor cells).

In the second accommodation step, NK cells 10, NK medium 16, autologous plasma 13, family cytokines IL2, IL7, IL12, IL15, IL18, IL27 (IL-2 (interleukin-2) and IL-7 (interleukin-7 ), IL-12 (interleukin-12), IL-15 (interleukin-15), IL-18 (interleukin-18), IL-27 (interleukin-27)) When the total mass is 100%, 0.2 to 2% of the mass of the family cytokines IL2, IL7, IL12, IL15, IL18, and IL27 (these interleukins) is contained in the flat container 17 with respect to the total mass. (add). The family cytokines IL2, IL7, IL12, IL15, IL18, IL27 (these interleukins) are present in NK medium 16 at concentrations of 20-200 ng/mL.

Family cytokines IL2, IL7, IL12, IL15, IL18, IL27 (IL-2 (interleukin-2), IL-7 (interleukin-7), IL-12 (interleukin-12), IL -15 (interleukin-15), IL-18 (interleukin-18), at least one of IL-27 (interleukin-27)) is less than 0.2% of the administration subject 11 Activated NK cells 10 (NK cell group) cannot be given a propensity to attack tumor cells (malignant tumor cells) (specific pathogens), and tumor cells (malignant tumors) in NK cells 10 (NK cell group) cell) death-inducibility.

Family cytokines IL2, IL7, IL12, IL15, IL18, IL27 (IL-2 (interleukin-2), IL-7 (interleukin-7), IL-12 (interleukin-12), IL -15 (interleukin-15), IL-18 (interleukin-18), IL-27 (interleukin-27)) by mass more than 2% by the NK cell activation step The activity of activated NK cells 10 (NK cell population) may be reduced by family cytokines IL2, IL7, IL12, IL15, IL18, IL27 (their interleukins).

The method for increasing the ability of NK cells is to use 0.2 to 2% mass of family cytokines IL2, IL7, IL12, IL15, IL18, IL27 (IL-2 (interleukin-2) and IL-7 (interleukin-7), IL-12 (interleukin-12), IL-15 (interleukin-15), IL-18 (interleukin-18), IL-27 (interleukin-27) 1), the concentration of family cytokines IL2, IL7, IL12, IL15, IL18, IL27 (their interleukins) in NK medium 16 was maintained at 20-200 ng/mL, and NK cells 10 of administration subject 11 ( While maintaining the activity of the NK cell group), the activated NK cells 10 (NK cell group) of the administration subject 11 are surely endowed with the propensity to attack tumor cells (malignant tumor cells) (specific pathogens). can be done.

Family cytokines IL2, IL7, IL12, IL15, IL18, IL27 (IL-2 (interleukin-2), IL-7 (interleukin-7), IL-12 (interleukin-12), IL -15 (interleukin-15), IL-18 (interleukin-18), at least one of IL-27 (interleukin-27)) in a flat container 17, NK cells 10 and autologous plasma The flat container 17 containing 13, NK medium 16, and family cytokines IL2, IL7, IL12, IL15, IL18, and IL27 (their interleukins) was placed again in the thermo-hygrostat 18, and in the thermo-hygrostat 18 While maintaining the flat container 17 at a temperature of 36.5 to 37.5 ° C., the flat container 17 is left statically for 18 to 24 hours, and the activity of the NK cells 10 is maintained while the NK cells 10 (NK cell group) (NK cell performance improvement step).

In the NK cell performance improvement step, family cytokines IL2, IL7, IL12, IL15, IL18, IL27 (IL-2 (interleukin-2), IL-7 (interleukin-7), IL-12 (interleukin-12) , IL-15 (interleukin-15), IL-18 (interleukin-18), IL-27 (interleukin-27)) at a concentration of 20-200 ng/mL in NK medium 16 While the flat container 17 is statically left for 18 to 24 hours, a predetermined amount of family cytokines IL2, IL7, IL12, IL15 is added to the flat container 17 every 3 to 6 hours from the initial stage of standing. , IL18, IL27 (IL-2 (interleukin-2), IL-7 (interleukin-7), IL-12 (interleukin-12), IL-15 (interleukin-15), IL-18 (inter at least one of leukin-18), IL-27 (interleukin-27)), and NK cells 10 by these family cytokines IL2, IL7, IL12, IL15, IL18, IL27 (these interleukins) Keep stimulating at all times.

In the NK cell performance improvement step, isolated early NK cells 10 activated by the NK cell activation step are family cytokines IL2, IL7, IL12, IL15, IL18, IL27 (IL-2 (interleukin-2) and IL-7 (interleukin-7), IL-12 (interleukin-12), IL-15 (interleukin-15), IL-18 (interleukin-18), IL-27 (interleukin-27) 1), all of the early NK cells 10 (NK cell group) learn to aggressively attack tumor cells (malignant tumor cells) (specific pathogens), and NK cells 10 proliferate and differentiate All the NK cells 10 (NK cell group) after the NK cell performance improvement step are endowed with a propensity to attack tumor cells (malignant tumor cells).

NK cells 10 (NK cell group) imparted with a propensity to attack tumor cells (malignant tumor cells) by the NK cell performance improvement process are administered to a subject 11 by an infusion 19 as shown in FIG. It is used for treatment of cancer of the administration subject 11 caused by cells (malignant tumor cells). NK cells 10 (NK cell group) that have been given the propensity to attack tumor cells (malignant tumor cells) (specific pathogens) by the NK cell potency-enhancing method induce cell death of tumor cells (malignant tumor cells).

The method for increasing the ability of NK cells activates the NK cells in the early stage of induction of the subject, while the family cytokines IL2, IL7, IL12, IL15, IL18, IL27 (IL-2 (interleukin-2), IL-7 ( at least one of interleukin-7), IL-12 (interleukin-12), IL-15 (interleukin-15), IL-18 (interleukin-18), IL-27 (interleukin-27) ) can be used to give the activated NK cells 10 (NK cell group) of the administration subject 11 the propensity to attack tumor cells (malignant tumor cells) (specific pathogens), and the NK cells 10 ( NK cell group) can increase the cell death inducibility of tumor cells (malignant tumor cells).

The method for increasing the ability of NK cells is to administer NK cells 10 (NK cell group) imparted with a propensity to attack tumor cells (malignant tumor cells) (specific pathogens) to an administration subject 11, thereby increasing the NK cells 10 (NK cell group) intensively attacks tumor cells (malignant tumor cells) that cause cancer (specific disease) in administration subject 11, and uses the NK cell 10 (NK cell group) The symptoms of cancer (specific disease) caused by tumor cells (malignant tumor cells) can be alleviated (malignant tumor cells are reduced), and the NK cells 10 (NK cell group) can be used to utilize tumor cells (malignant tumors). It is possible to perform treatment aimed at curing cancer (a specific disease) of the administration subject 11 caused by cells).

FIG. 6 is a schematic diagram showing another example of the second accommodation step. When the specific pathogen is the AIDS virus or novel coronavirus, the family cytokine IL7 (stimulatory substance) contained in the flat container 17 in the second containment step utilizes IL-7 (interleukin-7). IL-7 (interleukin-7) stimulates early separation NK cells 10 (NK cell group) activated by the NK cell activation process, and activates early separation NK cells 10 (NK cell group). Learning aggressiveness to AIDS virus or new coronavirus to give all NK cells 10 (NK cell group) aggressiveness to AIDS virus or new coronavirus, AIDS virus in NK cell 10 (NK cell group) Or increase the cell death inducibility of the novel coronavirus.

In the second accommodation step, when the total mass of NK cells 10, NK medium 16, autologous plasma 13 and family cytokine IL7 (IL-7 (interleukin-7)) is 100%, the total mass 0.2-2% by mass of family cytokine IL7 (IL-7 (interleukin-7)) is contained (added) to flat container 17 . The family cytokine IL7 (IL-7 (interleukin-7)) is present in NK medium 16 at a concentration of 20-200 ng/mL.

If the mass of the family cytokine IL7 (IL-7 (interleukin-7)) relative to 100% of the total mass is less than 0.2%, the AIDS virus (specific pathogen) or new coronavirus (specific pathogen), and increase the cell (virus) death induction of AIDS virus or new coronavirus in NK cells 10 (NK cell group) can't When the mass of the family cytokine IL7 (IL-7 (interleukin-7)) exceeds 2% relative to the total mass of 100%, the activity of NK cells 10 (NK cell group) activated by the NK cell activation process increases with the family cytokine It may be decreased by IL7 (IL-7 (interleukin-7)).

The NK cell potency enhancement method utilizes 0.2-2% mass of family cytokine IL7 (IL-7 (interleukin-7)) relative to 100% total mass, and family cytokine IL7 (IL -7 (interleukin-7)) at a concentration of 20 to 200 ng / mL, while maintaining the activity of NK cells 10 (NK cell group) of administration subject 11, activated NK of administration subject 11 Cells 10 (NK cell group) can be reliably endowed with a propensity to attack the AIDS virus (specific pathogen) or novel coronavirus (specific pathogen).

After housing family cytokine IL7 (IL-7 (interleukin-7)) in flat container 17 by the second housing step, flat container 17 housing NK cells 10, autologous plasma 13, NK medium 16, and family cytokine IL7 is housed again in the constant temperature and humidity chamber 18 (see FIG. 3), and the flat container 17 is held at a temperature of 36.5 to 37.5° C. in the constant temperature and humidity chamber 18 for 18 to 24 hours. The performance of the NK cells 10 (NK cell group) is increased while the activity of the NK cells 10 is maintained by being left statically (NK cell performance improvement step).

In the NK cell performance improvement step, the flat container 17 was statically placed for 18-24 hours so that the family cytokine IL7 (IL-7 (interleukin-7)) was present in the NK medium 16 at a concentration of 20-200 ng/mL. During the period of leaving to stand, a predetermined amount of family cytokine IL7 (IL-7 (interleukin-7)) is appropriately replenished to the flat container 17 every 3 to 6 hours from the initial stage of leaving, and family cytokine IL7 (IL- 7 (interleukin-7)) keeps NK cells 10 in a state of being able to be stimulated at all times.

In the NK cell performance improvement step, the isolated early NK cells 10 activated by the NK cell activation step are stimulated by the family cytokine IL7 (IL-7 (interleukin-7)), resulting in AIDS virus (specific pathogen ) or the aggressiveness to the new coronavirus (specific pathogen) All of the NK cells 10 (NK cell group) in the early stage of isolation learn, and all of the NK cells after the NK cell performance improvement process do not proliferate and differentiate NK cells 10 (NK cell group) are endowed with a propensity to attack AIDS virus or novel coronavirus.

NK cells 10 (NK cell group) imparted with a propensity to attack AIDS virus or novel coronavirus by the NK cell performance improvement step are administered to an administration subject 11 by intravenous drip 19 (see FIG. 5), and the AIDS virus or novel coronavirus It is used to treat diseases of the administration subject 11 caused by coronavirus. NK cells 10 (NK cell group), which have been given the propensity to attack AIDS virus (specific pathogen) or novel coronavirus (specific pathogen) by the NK cell ability-up method, are cells of AIDS virus or novel coronavirus (virus ) induce death.

The method for increasing the ability of NK cells activates the NK cells 10 in the initial induction period of the subject 11, and activates the subject 11 using the family cytokine IL7 (IL-7 (interleukin-7)). NK cells 10 (NK cell group) can be given an aggressive propensity to AIDS virus (specific pathogen) or new coronavirus (specific pathogen), and AIDS virus or new corona in NK cells 10 (NK cell group) It can increase the cell (virus) death inducibility of the virus.

The NK cell ability-up method is to administer NK cells 10 (NK cell group) that have been given a propensity to attack the AIDS virus (specific pathogen) or the new coronavirus (specific pathogen) to the administration subject 11. The NK cells 10 (NK cell group) intensively attack the AIDS virus or novel coronavirus that causes the disease of the administration subject 11, and the NK cells 10 (NK cell group) are used to attack the AIDS virus. Or the symptoms of the disease caused by the new coronavirus can be alleviated (reduced virus), and the NK cells 10 (NK cell group) can be used to treat the disease of the administration subject 11 caused by the AIDS virus or the new coronavirus Treatment aimed at curing the disease can be performed.

FIG. 7 is a schematic diagram showing another example of the second accommodation step. When the specific disease of the administration subject 11 is bronchial asthma, an example of the family cytokines IL4 and IL5 (stimulating substances) housed in the flat container 17 in the second housing step is IL-4 (interleukin-4), IL-5 (interleukin-5), which utilizes one of these interleukins or two of them. IL-4 (interleukin-4) and IL-5 (interleukin-5) stimulate early separation NK cells 10 (NK cell group) activated by the NK cell activation process, and activated early separation to increase (enhance) the anti-disease effect (healing ability) of NK cells 10 (NK cell group) in bronchial asthma (specific disease).

In the second accommodation step, a total of NK cells, NK medium, autologous plasma, and family cytokines IL4 and IL5 (at least one of IL-4 (interleukin-4) and IL-5 (interleukin-5)) Family cytokines IL4, IL5 (IL-4 (interleukin-4), IL-5 (interleukin-5)) with a mass of 0.2-2% relative to the total mass when the mass is 100% is housed in (added to) the flat container 17 . Family cytokines IL4, IL5 (IL-4 (interleukin-4), IL-5 (interleukin-5)) are present in NK medium 16 at concentrations of 20-200 ng/mL.

If the mass of family cytokines IL4 and IL5 (at least one of IL-4 (interleukin-4) and IL-5 (interleukin-5)) relative to 100% of the total mass is less than 0.2%, administration subjects A treatment aimed at curing bronchial asthma in subject 11, unable to increase the anti-disease effect (healing ability) of 11 activated NK cells 10 (NK cell group) on bronchial asthma (specific disease) The NK cells 10 cannot be used for the first time. When the mass of family cytokines IL4, IL5 (at least one of IL-4 (interleukin-4) and IL-5 (interleukin-5)) exceeds 2% relative to 100% of the total mass, NK cell activation The activity of NK cells 10 (NK cell group) activated by the process may be reduced by the family cytokines IL4, IL5 (their interleukins).

The NK cell ability enhancement method is to use 0.2 to 2% mass of family cytokines IL4, IL5 (IL-4 (interleukin-4), IL-5 (interleukin-5) with respect to 100% of the total mass). at least one of), maintaining the concentration of family cytokines IL4 and IL5 (their interleukins) in NK medium 16 at 20 to 200 ng / mL, and the activity of NK cells 10 (NK cell group) of administration subject 11 while maintaining the anti-disease effect (healing ability) of the activated NK cells 10 (NK cell group) of the administration subject 11 against bronchial asthma (specific disease).

After housing the family cytokines IL4 and IL5 (at least one of IL-4 (interleukin-4) and IL-5 (interleukin-5)) in the flat container 17 by the second housing step, NK cells 10 and The flat container 17 containing the autologous plasma 13, the NK medium 16, and the family cytokines IL4 and IL5 (their interleukins) was placed in the thermo-hygrostat 18 again (see FIG. 3). While maintaining the container 17 at a temperature of 36.5 to 37.5 ° C., the flat container 17 is left statically for 18 to 24 hours, and while maintaining the activity of the NK cells 10, NK cells 10 (NK cell group) Improve performance (NK cell performance improvement step).

In the NK cell performance improvement step, family cytokine IL4, IL5 (at least one of IL-4 (interleukin-4) and IL-5 (interleukin-5)) is 20 to 200 ng / mL in NK medium 16 A predetermined amount of family cytokines IL4 and IL5 (IL -4 (interleukin-4), at least one of IL-5 (interleukin-5)), and can constantly stimulate NK cells 10 by those family cytokines IL4, IL5 (these interleukins) hold in state.

In the NK cell performance improvement step, the isolated early NK cells 10 activated by the NK cell activation step are among the family cytokines IL4, IL5 (IL-4 (interleukin-4), IL-5 (interleukin-5) At least one of), the anti-disease effect (healing ability) of NK cells 10 (NK cell group) activated without proliferation and differentiation of NK cells 10 against bronchial asthma (specific disease) rise (improve) The NK cells 10 (NK cell group) whose anti-disease effect (healing ability) against bronchial asthma has been increased by the NK cell performance improvement process are administered to the administration subject 11 by infusion 19 (see FIG. 5), and the administration subject 11 used in the treatment of bronchial asthma.

The method for increasing the ability of NK cells activates the NK cells 10 in the early stage of induction of the administration subject 11, and the family cytokines IL4, IL5 (IL-4 (interleukin-4), IL-5 (interleukin-5) at least one of them) can be used to increase (enhance) the anti-disease effect (healing ability) of the activated NK cells 10 (NK cell group) of the administration subject 11 against bronchial asthma. The method for increasing the ability of NK cells utilizes NK cells 10 (NK cell group) by administering NK cells 10 (NK cell group) whose anti-disease effect against bronchial asthma has been enhanced by the method to administration subject 11. The NK cells 10 (NK cell group) can be used to treat the subject 11 with the aim of curing the bronchial asthma.

Drawing 8 is a schematic diagram showing other examples of the 2nd accommodation process. When the specific disease of the administration subject 11 is rheumatoid arthritis, an example of the family cytokines IL1, IL6, and IL8 (stimulating substances) housed in the flat container 17 in the second housing step is IL-1 (interleukin-1 ), IL-6 (interleukin-6), IL-8 (interleukin-8), which utilize one or more of these interleukins. IL-1 (interleukin-1), IL-6 (interleukin-6), and IL-8 (interleukin-8) are isolated early NK cells 10 (NK cell group) activated by the NK cell activation process. ) to increase (enhance) the anti-disease effect (healing ability) of the activated isolated early NK cells 10 (NK cell group) against rheumatoid arthritis (specific disease).

In the second accommodation step, NK cells, NK medium, autologous plasma and family cytokines IL1, IL6, IL8 (IL-1 (interleukin-1), IL-6 (interleukin-6), IL-8 (interleukin- 8) at least one of)) and family cytokines IL1, IL6, IL8 (IL-1 (interleukin -1), IL-6 (interleukin-6), and IL-8 (interleukin-8)) are accommodated (added) to the flat container 17 . Family cytokines IL1, IL6, IL8 (IL-1 (interleukin-1), IL-6 (interleukin-6), IL-8 (interleukin-8)) are 20-200 ng/mL in NK medium 16 present at a concentration of

family cytokines IL1, IL6, IL8 (at least one of IL-1 (interleukin-1), IL-6 (interleukin-6), IL-8 (interleukin-8)) relative to 100% total mass If the mass is less than 0.2%, the anti-disease effect (healing ability) of the activated NK cells 10 (NK cell group) of the administration subject 11 against rheumatoid arthritis (a specific disease) cannot be increased. The NK cells 10 cannot be used for treatment aimed at curing the subject's 11 rheumatoid arthritis.

family cytokines IL1, IL6, IL8 (at least one of IL-1 (interleukin-1), IL-6 (interleukin-6), IL-8 (interleukin-8)) relative to 100% total mass If the mass exceeds 2%, the activity of NK cells 10 (NK cell group) activated by the NK cell activation process may be reduced by the family cytokines IL1, IL6, IL8 (their interleukins).

The method for increasing the ability of NK cells is to use 0.2 to 2% mass of family cytokines IL1, IL6, IL8 (IL-1 (interleukin-1), IL-6 (interleukin-6) relative to 100% of the total mass). , at least one of IL-8 (interleukin-8)), maintaining the concentration of family cytokines IL1, IL6, IL8 (these interleukins) in NK medium 16 at 20-200 ng / mL, and administering While maintaining the activity of the NK cells 10 (NK cell group) of the subject 11, the anti-disease effect (healing ability) can be increased.

family cytokines IL1, IL6, IL8 (at least one of IL-1 (interleukin-1), IL-6 (interleukin-6), IL-8 (interleukin-8)) by the second accommodation step; After housing in the flat container 17, the flat container 17 housing the NK cells 10, the autologous plasma 13, the NK medium 16, and the family cytokines IL1, IL6, and IL8 (their interleukins) is housed again in the thermo-hygrostat 18. (see FIG. 3), while maintaining the flat container 17 at a temperature of 36.5 to 37.5 ° C. in the constant temperature and humidity chamber 18, the flat container 17 is left statically for 18 to 24 hours, and the activity of the NK cells 10 while maintaining the performance of NK cells 10 (NK cell group) (NK cell performance improvement step).

In the NK cell performance improvement step, at least one of the family cytokines IL1, IL6, IL8 (IL-1 (interleukin-1), IL-6 (interleukin-6), IL-8 (interleukin-8) ) is present in the NK medium 16 at a concentration of 20 to 200 ng/mL. 17 with a predetermined amount of family cytokines IL1, IL6, IL8 (at least one of IL-1 (interleukin-1), IL-6 (interleukin-6), IL-8 (interleukin-8)) NK cells 10 are maintained in a state capable of being constantly stimulated by these family cytokines IL1, IL6 and IL8 (these interleukins) by appropriate supplementation.

In the NK cell performance improvement step, the isolated early NK cells 10 activated by the NK cell activation step are family cytokines IL1, IL6, IL8 (IL-1 (interleukin-1), IL-6 (interleukin-6) , at least one of IL-8 (interleukin-8)), NK cells 10 (NK cell group) activated without proliferating and differentiating from rheumatoid arthritis (specific The anti-disease effect (healing ability) against disease) is increased (improved). NK cells 10 (NK cell group) whose anti-disease effect (healing ability) against rheumatoid arthritis has been increased by the NK cell performance improvement process are administered to the administration subject 11 by infusion 19 (see FIG. 5), and the administration subject 11 used in the treatment of rheumatoid arthritis.

The method for increasing the ability of NK cells activates the NK cells 10 in the early stage of induction of the administration subject 11, and the family cytokines IL1, IL6, IL8 (IL-1 (interleukin-1), IL-6 (interleukin-6 ), at least one of IL-8 (interleukin-8)), the anti-disease effect (healing ability) of the activated NK cells 10 (NK cell group) of the administration subject 11 to rheumatoid arthritis can raise (increase) The method for increasing the ability of NK cells utilizes NK cells 10 (NK cell group) by administering NK cells 10 (NK cell group) whose anti-disease effect on rheumatoid arthritis has been enhanced by the method to administration subject 11. can alleviate the symptoms of rheumatoid arthritis of the subject 11, and the NK cells 10 (NK cell group) can be used to treat rheumatoid arthritis of the subject 11.

FIG. 9 is a schematic diagram showing another example of the second housing step. When the specific disease of the administration subject 11 is atopic dermatitis, an example of the family cytokine IL4 (stimulatory substance) accommodated in the flat container 17 in the second accommodation step is IL-4 (interleukin-4). use. IL-4 (interleukin-4) stimulates early separation NK cells 10 (NK cell group) activated by the NK cell activation process, and activates early separation NK cells 1 (NK cell group). Raise (enhance) the anti-disease effect (healing ability) for atopic dermatitis (specific disease).

In the second housing step, when the total mass of NK cells 10, NK medium 16, autologous plasma 13 and family cytokine IL4 (IL-4 (interleukin-4)) is 100%, the total mass is 100% 0.2-2% mass of family cytokine IL4 (IL-4 (interleukin-4)) is accommodated (added) to flat container 17 . The family cytokine IL4 (IL-4 (interleukin-4)) is present in NK medium 16 at concentrations of 20-200 ng/mL.

When the mass of the family cytokine IL4 (IL-4 (interleukin-4)) relative to 100% of the total mass is less than 0.2%, atopic dermatitis of the activated NK cells 10 (NK cell group) of the administration subject 11 The anti-disease effect (healing ability) against (specific disease) cannot be increased, and the NK cells 10 cannot be used for treatment aimed at curing the atopic dermatitis of the administration subject 11. When the mass of family cytokine IL4 (IL-4 (interleukin-4)) exceeds 2% relative to the total mass of 100%, the activity of NK cells 10 (NK cell group) activated by the NK cell activation process increases It may be decreased by IL4 (IL-4 (interleukin-4)).

The NK cell potency enhancement method utilizes 0.2-2% mass of family cytokine IL4 (IL-4 (interleukin-4)) relative to 100% total mass, and family cytokine IL4 (IL -4 (interleukin-4)) at a concentration of 20 to 200 ng / mL, while maintaining the activity of NK cells 10 (NK cell group) of administration subject 11, activated NK of administration subject 11 The anti-disease effect (healing ability) of cells 10 (NK cell group) to atopic dermatitis (specific disease) can be reliably increased.

After housing the family cytokine IL4 (IL-4 (interleukin-4)) in the flat container 17 by the second housing step, NK cells 10, autologous plasma 13, NK medium 16 and family cytokine IL4 (IL-4 (interleukin-4) -4)) is housed again in the constant temperature and humidity chamber 18 (see FIG. 3), and the flat container 17 is maintained at a temperature of 36.5 to 37.5° C. in the constant temperature and humidity chamber 18. The flat container 17 is left statically for 18 to 24 hours while maintaining the activity of the NK cells 10 while increasing the performance of the NK cells 10 (NK cell group) (NK cell performance improving step).

In the NK cell performance improvement step, the flat container 17 was statically placed for 18-24 hours so that the family cytokine IL4 (IL-4 (interleukin-4)) was present in the NK medium 16 at a concentration of 20-200 ng/mL. During the period of leaving to stand, a predetermined amount of family cytokine IL4 (IL-4 (interleukin-4)) is appropriately replenished to the flat container 17 every 3 to 6 hours from the initial stage of leaving, and family cytokine IL4 (IL- 4 (interleukin-4)) maintains the NK cells 10 in a constantly stimulable state.

In the NK cell performance improvement step, the NK cells 10 in the early stage of separation activated by the NK cell activation step are stimulated by the family cytokine IL4 (IL-4 (interleukin-4)), so that the NK cells 10 proliferate and differentiate. The anti-disease effect (healing ability) of the NK cells 10 (NK cell group) activated without treatment for atopic dermatitis (specific disease) is increased (improved). NK cells 10 (NK cell group) whose anti-disease effect (healing ability) on atopic dermatitis has been increased by the NK cell performance improvement step are administered to the administration subject 11 by infusion 19 (see FIG. 5), and the administration subject It is used to treat the atopic dermatitis of Person 11.

The NK cell ability-up method uses the family cytokine IL4 (IL-4 (interleukin-4)) while activating the NK cells 10 (NK cell group) in the initial induction period of the administration subject 11. It is possible to increase (enhance) the anti-disease effect (healing ability) of 11 activated NK cells 10 (NK cell group) to atopic dermatitis. The method for increasing the ability of NK cells is to administer NK cells 10 (NK cell group), whose anti-disease effect on atopic dermatitis has been enhanced by the method, to administration subject 11, thereby increasing NK cells 10 (NK cell group). The NK cells 10 (NK cell group) can be used to alleviate the symptoms of atopic dermatitis in the administration subject 11, and treatment aimed at curing the atopic dermatitis in the administration subject 11 can be performed. be able to.

FIG. 10 is a schematic diagram showing another example of the second housing step. When the specific disease of the administration subject 11 is diabetes, IL-15 (interleukin-15) is used as an example of the family cytokine IL15 (stimulatory substance) contained in the flat container 17 in the second containing step. IL-15 (interleukin-15) stimulates early separation NK cells 10 (NK cell group) activated by the NK cell activation process, and activates early separation NK cells 10 (NK cell group). Raise (enhance) the anti-disease effect (healing ability) for diabetes (a specific disease).

In the second accommodation step, when the total mass of NK cells 10, NK medium 16, autologous plasma 13 and family cytokine IL15 (IL-15 (interleukin-15)) is 100%, the total mass is 100% 0.2-2% mass of family cytokine IL15 (IL-15 (interleukin-15)) is accommodated (added) to flat container 17 . The family cytokine IL15 (IL-15 (interleukin-15)) is present in NK medium 16 at concentrations of 20-200 ng/mL.

If the mass of family cytokine IL15 (IL-15 (interleukin-15)) relative to 100% of the total mass is less than 0.2%, the activated NK cells 19 (NK cell group) of administration subject 11 diabetes (specific disease), and the NK cells 10 cannot be used for treatment aimed at curing diabetes (blood sugar control) of the administration subject 11. When the mass of the family cytokine IL15 (IL-15 (interleukin-15)) exceeds 2% relative to the total mass of 100%, the activity of NK cells 10 (NK cell group) activated by the NK cell activation process increases with the family cytokine It may be decreased by IL15 (IL-15 (interleukin-15)).

The NK cell potency enhancement method utilizes 0.2-2% mass of family cytokine IL15 (IL-15 (interleukin-15)) relative to 100% total mass, and family cytokine IL15 (IL -15 (interleukin-15)) at a concentration of 20 to 200 ng / mL, while maintaining the activity of NK cells 10 (NK cell group) of administration subject 11, activated NK of administration subject 11 The anti-disease effect (healing ability) of the cells 10 (NK cell group) to diabetes (a specific disease) can be reliably increased.

After housing the family cytokine IL15 (IL-15 (interleukin-15)) in the flat container 17 by the second housing step, NK cells 10, autologous plasma 13, NK medium 16 and family cytokine IL15 (IL-15 (interleukin-15) -15)) is housed again in the constant temperature and humidity chamber 18 (see FIG. 3), and the flat container 17 is held at a temperature of 36.5 to 37.5° C. in the constant temperature and humidity chamber 18. The flat container 17 is left statically for 18 to 24 hours while maintaining the activity of the NK cells 10 while increasing the performance of the NK cells 10 (NK cell group) (NK cell performance improving step).

In the NK cell performance improvement step, the flat container 17 was statically placed for 18-24 hours so that the family cytokine IL15 (IL-15 (interleukin-15)) was present in the NK medium 16 at a concentration of 20-200 ng/mL. During the standing period, a predetermined amount of family cytokine IL15 (IL-15 (interleukin-15)) is appropriately supplied to the flat container 17 every 3 to 6 hours from the initial stage of standing, and family cytokine IL15 (IL- 15 (interleukin-15)) keeps NK cells 10 in a state of being able to be stimulated at all times.

In the NK cell performance improvement step, the NK cells 10 at the initial stage of separation activated by the NK cell activation step are stimulated by the family cytokine IL15 (IL-15 (interleukin-15)), so that the NK cells 10 proliferate and differentiate. The anti-disease effect (healing ability) of diabetes (specific disease) of NK cells 10 (NK cell group) activated without treatment is increased (improved). The NK cells 10 (NK cell group) whose anti-disease effect (healing ability) against diabetes has been increased by the NK cell performance improvement process are administered to the administration subject 11 by infusion 19 (see FIG. 5), and the administration subject 11 Used in the treatment of diabetes.

The NK cell ability-up method uses the family cytokine IL15 (IL-15 (interleukin-15)) while activating the NK cells 10 (NK cell group) in the initial induction period of the administration subject. It is possible to increase (enhance) the anti-disease effect (healing ability) of the activated NK cells 10 (NK cell group) to diabetes. The NK cell capacity increasing method uses NK cells 10 (NK cell group) by administering NK cells 10 (NK cell group) whose anti-disease effect to diabetes has been improved by the method to the administration subject 11. It is possible to alleviate the symptoms of diabetes (hyperglycemia) in the subject 11, and to provide treatment aimed at curing diabetes (control of blood sugar level) in the subject 11 using NK cells 10 (NK cell group). It can be carried out.

10 NK cells (natural killer cells)
11 administration subject 12 leukocyte 13 autologous plasma 14 glass test tube (separation container)
15 erythrocyte 16 NK medium 17 flat container 18 thermohygrostat 19 drip IL1, IL2, IL4, IL5, IL6, IL7, IL8, IL12, IL15, IL18, IL27 family cytokine

Claims (11)

NK cells (natural killer cells) of a subject to be administered increase the activity of the NK cells, and the NK cells of the subject have a propensity to attack a specific pathogen causing a specific disease of the subject. Or, in the method for increasing the ability of NK cells to increase the anti-disease effect (healing ability) of the NK cells of the administration subject to the specific disease,
The method for increasing the ability of NK cells collects a predetermined amount of blood from the subject, separates leukocytes from the collected blood of the subject, and selectively removes NK cells of the subject from the leukocytes. Inducing NK cell induction step,
a first accommodation step of accommodating the NK cells induced by the NK cell induction step and a predetermined NK medium in a container having a predetermined volume;
The container containing the NK cells and the NK medium in the first containing step is kept at a temperature of 36.5 to 37.5° C. and allowed to stand statically for 6 to 12 hours to prevent the NK cells from proliferating and differentiating. an NK cell activation step of activating NK cells;
A predetermined family cytokine that causes the NK cells to learn aggression against a specific pathogen causing the disease of the subject, or a second accommodation step of accommodating a predetermined family cytokine that enhances the anti-disease effect (healing ability) against the disease of the administration subject;
The container containing the NK cells, the NK medium, and the family cytokine in the second housing step is maintained at a temperature of 36.5 to 37.5° C. and left statically for 18 to 24 hours to proliferate and differentiate the NK cells. and an NK cell performance improvement step of improving the performance of the NK cells without
In the NK cell performance improving step, the NK cells in the early stage of induction activated by the NK cell activation step are stimulated by the family cytokine, thereby increasing the aggressiveness to the specific pathogen. is learned, and all NK cell groups after the NK cell performance improvement step are given a propensity to attack the specific pathogen without proliferation and differentiation of the NK cells, or by the NK cell activation step By stimulating the activated NK cells in the early stage of induction with the family cytokine, the anti-disease effect of all NK cell groups after the NK cell performance improvement step on the disease without the NK cells being proliferated and differentiated ( A method for increasing the ability of NK cells, characterized by increasing healing ability). The method for increasing NK cell capacity includes a plasma separation step of separating the autologous plasma of the administration subject from the blood collected from the administration subject and inactivating the separated autologous plasma, and the first containing step includes the 2. The method for increasing the ability of NK cells according to claim 1, wherein the inactivated autologous plasma of the administration subject is accommodated in the container together with the NK cells and the NK medium. In the first containing step, when the total mass of the NK cells, the NK medium, and the autologous plasma is 100%, 2 to 5% of the inactivated autologous plasma is placed in the container. 3. The method for increasing the ability of NK cells according to claim 2, wherein the cells are housed. In the second housing step, when the total mass of the NK cells, the NK medium, the autologous plasma and the family cytokine is 100%, 0.2 to 2% of the family cytokine is added to the total mass. 4. The method for increasing the ability of NK cells according to claim 3, wherein the NK cells are housed in the container. Any one of claims 1 to 4, wherein in the NK cell performance improvement step, the container is replenished with a predetermined amount of family cytokine every 3 to 6 hours while the container is left statically for 18 to 24 hours. 2. The method for enhancing NK cell ability according to . The specific pathogen is a tumor cell, and the family cytokine causes the early NK cell group activated by the NK cell activation step to learn aggression against the tumor cell, and all the NK cell groups IL-2 (interleukin-2), IL-7 (interleukin-7), IL-12 (interleukin-12) that imparts aggressiveness to the tumor cells and enhances the antitumor effect of the NK cell group ), IL-15 (interleukin-15), IL-18 (interleukin-18), IL-27 (interleukin-27) according to any one of claims 1 to 5 method for improving NK cell ability. The specific pathogen is the AIDS virus or the novel coronavirus, and the family cytokine causes the NK cell group at the early stage of induction activated by the NK cell activation process to be aggressive to the AIDS virus or the novel coronavirus. Claim 1, which is IL-7 (interleukin-7) that makes all of the NK cell groups learn to attack the AIDS virus or the novel coronavirus and increases the antiviral effect of the NK cell group 6. The method for increasing the ability of NK cells according to any one of claims 1 to 5. The specific disease of the administration subject is bronchial asthma, and the family cytokine suppresses the anti-disease effect (healing ability) of the early NK cell group activated by the NK cell activation process on the bronchial asthma. 6. The method for increasing the ability of NK cells according to any one of claims 1 to 5, wherein at least one of IL-4 (interleukin-4) and IL-5 (interleukin-5) is used. The specific disease of the administration subject is rheumatoid arthritis, and the family cytokine exhibits an anti-disease effect (healing ability) on the rheumatoid arthritis of the early NK cell group activated by the NK cell activation process. 6. The method for increasing the ability of NK cells according to any one of claims 1 to 5, wherein at least one of IL1 (interleukin 1), IL6 (interleukin 6), and IL8 (interleukin 8) is increased. The specific disease of the administration subject is atopic dermatitis, and the family cytokine has an anti-disease effect on the atopic dermatitis of the initial NK cell group activated by the NK cell activation process ( 6. The method for enhancing the ability of NK cells according to any one of claims 1 to 5, wherein IL-4 (interleukin-4) is used to increase healing ability. The specific disease of the administration subject is diabetes, and the family cytokine increases the anti-disease effect (healing ability) of the NK cell group in the early stage of induction activated by the NK cell activation process to the diabetes. IL -15 (interleukin-15).

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