CN116472344A

CN116472344A - Immune cell proliferation activating kit and immune cell culture method using same

Info

Publication number: CN116472344A
Application number: CN202180076042.1A
Authority: CN
Inventors: 申志燮; 申禹燮
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-09-15
Filing date: 2021-09-08
Publication date: 2023-07-21

Abstract

The present invention relates to an immune cell culture method, and more particularly, to an immune cell proliferation activating kit and an immune cell culture method using the proliferation activating kit, which can effectively proliferate and activate immune cells for a long period of time by not only inhibiting any one of natural killer cells (Natural Killer cell; NK cells) and T cells in regulatory T cells (Tregs) in immune cells when culturing the immune cells.

Description

Immune cell proliferation activating kit and immune cell culture method using same

Technical Field

Background

NK cells are known to play an important role in the early organism defense mechanism and tumor immunity of the human body. That is, NK cells can kill specific autologous cells, allogeneic cells, and even xenogeneic cancer cells without the need for a process of obtaining immunity based on the expression of major histocompatibility complex (Major Hist ocompatibility Complex: MHC), and particularly target cells that express little or no MHC Class 1 (Class 1 MHC) can be better killed. Therefore, NK cells can effectively kill most cancer cells that do not express MHC, but can also kill several virus-infected cells and bacteria such as salmonella typhi (salmonella typhi). However, NK cells, which exert an excellent effect on killing cancer cells, represent only 5 to 15% of peripheral blood lymphocytes even in normal humans, especially in severe cancer patients, with a reduced ratio to less than 1%, and thus have a limitation in terms of effectively attacking cancer cells without an additional amplification process by immunotherapy.

Immunotherapy is a method in which immune cells such as natural killer cells (Natural Killer cell; NK cells), dendritic Cells (DC), B cells, T cells, etc., which are most important for cancer therapy, are extracted from the blood of a patient, and then cultured into immune cells that act strongly on cancer using various stimuli, and then reinjected into the patient, and thus, compared with conventional chemotherapy, etc., research into immunotherapy has been actively conducted in recent years because of the fact that the immune cells have fewer side effects and are easier to administer by using the patient's own blood.

In culturing immune cells, particularly when it is mainly intended to culture NK cells only, the action of T cells, particularly Helper T cells (Helper T cells or Th cells)) is required, which are cells that promote humoral immunity and cellular immunity by regulating differentiation and activation of leukocytes. Also referred to as cd4+ T cells because of the characteristics of the CD4 protein on the cell surface. Cd4+ T cells are again classified into Th1, th2, th17, treg, etc. according to detailed functions. Th1 cells secrete interferon gamma (IFN-gamma) and tumor necrosis factor beta (Tumor Necrosis Factor beta, TNF-beta), thereby inducing fusion of endosomes and lysosomes inside macrophages to form endolysosomes. On the other hand, th2 cells differentiate B cells into plasma cells by secreting various Interleukins (IL). Th17 cells recruit neutrophils by secreting interleukin 17 (IL-17). Treg cells, also known as regulatory T cells, maintain immune homeostasis and block autoimmune responses, etc., by suppressing immune responses, rather than promoting immune responses. Regulatory T cells (tregs) are used as a component of the immune system to suppress immune responses of other cells, an important "self-test" for preventing immune system overreactions. Regulatory T cells are involved in stopping immune responses after successful destruction of invading microorganisms, and also in preventing autoimmune cell diseases. The immune system must be able to distinguish between self (self) and non-self. When self/non-self differentiation fails, the immune system destroys cells and tissues in the body, resulting in autoimmune disease. Regulatory T cells actively inhibit the activity of the immune system and prevent pathological autoreactions such as autoimmune diseases. It has been reported that the molecular mechanism of how regulatory T cells exert their inhibitory/regulatory activity is not clear, and that the immunosuppressive cytokine TGF-beta and interleukin 10 (IL-10) are also involved in regulatory T cell function.

On the other hand, when various antibodies or cytokines (Cyt okine) or the like are used for activating immune cells, regulatory T cells (regulatory T cells (Tregs, tregs)) are also activated. However, activation of tregs takes longer than other cells. Therefore, the increase in cell number is good at the initial stage of culturing cells, but when culturing for 8 days to 9 days or more, there are cases where the cell number does not increase as expected due to activated tregs. In particular, cancer patients with low immunity are more common, and this phenomenon is particularly prominent in patients with high and activated Treg cell numbers, simply because of the effect of Treg.

Therefore, there is a need to develop a technique that can proliferate immune cells in large amounts by minimizing the effect of tregs.

Disclosure of Invention

Technical problem

As a result of diligent studies to solve the above problems, the present inventors have developed a technique that can inhibit the activity of regulatory T cells (tregs) when immune cells are cultured, and have completed the present invention.

Accordingly, an object of the present invention is to provide an immune cell proliferation-activating kit and an immune cell culture method using the same, which can proliferate more immune cells, particularly one or more of NK cells and T cells, by inhibiting the activity of cd4+ T cells, particularly regulatory T cells (tregs), at a specific period when the immune cells are cultured.

The object of the present invention is not limited to the above-mentioned object, and it is needless to say that the object of the present invention which can be understood by those skilled in the art from the description of the detailed description of the invention described later may be included even if it is not explicitly mentioned.

Solution to the problem

In order to achieve the above object, the present invention provides an immune cell proliferation-activating kit comprising: a first unit comprising a T cell stimulating substance; and a second unit comprising a steroid.

In a preferred embodiment, in the first unit, when the T cell stimulating substance is an antibody, the antibody is contained in the basal medium solution at a concentration of 0.1 to 12. Mu.g/mL, and when the T cell stimulating substance is Feeder cells (FeederCell), the feeder cells are used in a 1X 10 ratio ⁵ The concentration of the antibody is at least one selected from the group consisting of an anti-CD 3 antibody, an anti-CD 4 antibody and an anti-CD 28 antibody, and the antibody is contained in the basal medium solution.

In a preferred embodiment, in the second unit, the steroid is contained in the basal medium solution at a concentration of 0.02 μg/mL or more.

In a preferred embodiment, the steroid is a glucocorticoid (glucocorticoids), and the steroid is at least one selected from the group consisting of Cortisol (Cortisone), cortisone (Cortisone), prednisolone (Prednisolone), prednisone (prednisone), methylprednisolone (Methylpredni solone), dexamethasone (Dexamethasone), betamethasone (Betamethasone), triamcinolone (Triamcinolone), fludrocortisone acetate (Fludrocortisone acetate), deoxycorticosterone acetate (deoxycorticosterone acetate).

In a preferred embodiment, the above-mentioned steroids act to proliferate NK cells by inhibiting regulatory T cell (Treg) activity.

In a preferred embodiment, the above kit further comprises: a third unit containing an NK cell stimulating substance, wherein in the third unit, the NK cell stimulating substance is contained in the basal medium solution at a concentration of 0.2ng/mL to 10. Mu.g/mL.

In a preferred embodiment, the NK cell stimulating substance is at least one selected from the group consisting of an anti-CD 16 antibody, an anti-CD 56 antibody, interleukin-12 (IL-12), interleukin-15 (IL-15) and interleukin-18 (IL-18).

The present invention also provides an immune cell culture method, comprising: a first culturing step of suspending Peripheral Blood Mononuclear Cells (PBMCs) isolated from blood in a basal medium solution, and then treating a first unit containing a T cell stimulating substance and culturing for 18 to 72 hours; a second culturing step of culturing for 18 to 25 hours after treating the first culture liquid subjected to the first culturing step with a second unit containing a steroid; and a third culturing step of culturing by adding one or more of the basal medium solution and fetal bovine serum (Fetal Bovine Serum, FBS, or autologous plasma, the same applies hereinafter) to the second culture medium subjected to the second culturing step at intervals of 24 hours or more from the predetermined time point, and transferring the culture medium to a larger culture vessel as the number of cells increases.

In a preferred embodiment, in the first culturing step, a third unit containing an NK cell stimulating substance is treated before or simultaneously with the treatment of the first unit.

In a preferred embodiment, in the third unit, the NK cell stimulating substance is contained in the basal medium solution at a concentration of 0.2ng/mL to 10. Mu.g/mL, and the NK cell stimulating substance is at least one selected from the group consisting of an anti-CD 16 antibody, an anti-CD 56 antibody, IL-12, IL-15 and IL-18.

In a preferred embodiment, in the first unit, when the T cell stimulating substance is an antibody, the antibody is contained in a basal medium solution at a concentration of 0.1 to 12. Mu.g/mL, and when the T cell stimulating substance is a feeder cell, the feeder cell is at 1X 10 ⁵ The concentration of at least one of the group consisting of an anti-CD 3 antibody, an anti-CD 4 antibody and an anti-CD 28 antibody is contained in the basal medium solution, the steroid is a glucocorticoid (glucosportypes) and at least one of the group consisting of Cortisone (Cortisone), prednisolone (Prednisolone), prednisone (prednisone), methylprednisolone (Methylpredni solone), dexamethasone (dexamethone), betamethasone (Betamethasone), triamcinolone (Triamcinolone), fludrocortisone acetate (Fludrocortisone acetate) and deoxycorticosterone acetate (deoxycorticosterone acetate) is contained in the basal medium solution at a concentration of at least 0.02 μg/mL.

In a preferred embodiment, the basal medium solution in which the Peripheral Blood Mononuclear Cells (PBMCs) are suspended is treated with the first unit at any one of the time points of culture day 0, culture day 1, and culture day 2, and the second unit is treated at a time point of 20 hours to 25 hours after the first culture solution is treated with the first unit.

In a preferred embodiment, the culture is carried out in the second culture solution obtained in the second culture step for 5 days and then treated with the second unit more than once.

The invention also provides a pet immune cell culture method, which comprises the following steps: a first culturing step of suspending Peripheral Blood Mononuclear Cells (PBMCs) isolated from blood of dogs in a basal medium solution, and then simultaneously or sequentially treating a third unit including an NK cell stimulating substance and a first unit including a T cell stimulating substance and culturing for 4 days; a second culturing step of culturing for 18 to 25 hours after simultaneously or sequentially treating the first culture solution having undergone the first culturing step with a second unit containing a steroid and the third unit; and a third culturing step of culturing by adding one or more of the basal medium solution and fetal bovine serum (Fetal Bovine Serum, FBS) to the second culture medium subjected to the second culturing step at intervals of 24 hours or more from a predetermined time point, and transferring the culture medium to a larger culture vessel for culturing as the number of cells increases.

In a preferred embodiment, in the first unit, when the T cell stimulating substance is an antibody, the antibody is contained in a basal medium solution at a concentration of 0.1 to 12. Mu.g/mL, and when the T cell stimulating substance is a feeder cell, the feeder cell is at 1X 10 ⁵ The concentration of the steroid is greater than or equal to 0.02 μg/mL, the steroid glucocorticoid (glucocorticoids) is contained in the basal medium solution, and the steroid is selected from Cortisol (Cortisone), prednisolone (Prednisolone), prednisone (prednisone), methylprednisolone (Methylpredni solone), dexamethasone (De-oxametalone), betamethasone, triamcinolone (Triamcinolone), and Fludrocortisone acetate (Fludrocortisone)acetate), deoxycorticosterone acetate (deoxyco rticosterone acetate), wherein in the third unit, the NK cell stimulating substance is contained in the basal medium solution at a concentration of 0.2ng/mL to 10. Mu.g/mL, and the NK cell stimulating substance is at least one selected from the group consisting of an anti-CD 16 antibody, an anti-CD 56 antibody, IL-12, IL-15 and IL-18.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the immune cell culture method using the immune cell proliferation activating kit of the present invention, the immunosuppressive activity of CD 4+T cells, particularly regulatory T cells (tregs), is suppressed by the proliferation activating kit at a specific period when culturing immune cells, so that not only immune cells, particularly one or more of NK cells and T cells, but also large-scale proliferation can be efficiently proliferated by at least 2-fold or more, as compared with known immune cell culture methods.

Such technical effects of the present invention are not limited to the above-mentioned effects, but include, of course, effects of the invention that can be understood by those of ordinary skill in the art from the descriptions of specific matters for carrying out the invention described later, even if they are not explicitly mentioned.

Drawings

Fig. 1a is a graph showing the change of the number of cells cultured by the immune cell culture method according to an embodiment of the present invention and the culture method according to a comparative example thereof with respect to the culture time, and fig. 1b is a graph showing FACS data (FACS data) obtained by analyzing each culture medium by flow cytometry.

Fig. 2a is a graph showing the change of the number of cells cultured by the immune cell culture method according to still another embodiment of the present invention and the culture method according to the comparative example thereof with respect to the culture time, and fig. 2b is a graph showing FACS data (FACS data) obtained by analyzing each culture medium by flow cytometry.

Fig. 3a is a graph showing the change of the number of cells cultured by the immune cell culture method according to another embodiment of the present invention and the culture method according to the comparative example thereof with respect to the culture time, and fig. 3b is a graph showing FACS data (FACS data) obtained by analyzing each culture medium by flow cytometry.

Fig. 4a is a graph showing the change of the number of cells cultured by the immune cell culture method according to still another embodiment of the present invention and the culture method according to the comparative example thereof with respect to the culture time, and fig. 4b is a graph showing FACS data (FACS data) obtained by analyzing each culture medium by flow cytometry.

FIG. 5 is a graph showing the change in the number of cells cultured by the immune cell culture method according to still another embodiment of the present invention and the culture method according to the comparative example thereof with the time of the culture.

Fig. 6a is a graph showing FACS data (FACS data) of a medium cultured by an immune cell culture method according to still another embodiment of the present invention, and fig. 6b is a graph showing FACS data (FACS data) of a medium cultured according to a comparative example thereof.

FIG. 7 is a graph showing the change in the number of cells cultured by the immune cell culture method according to still another embodiment of the present invention and the culture method according to the comparative example thereof with the time of the culture.

Fig. 8a and 8b are graphs showing FACS data (FACS data) of treated and untreated cells, respectively, using cells having NK cell ratio of about 50%, increasing steroid concentration contained in the epidemic cell proliferation activation kit about 2-fold, treating and untreated cells on days 3, 4, 8, and culturing to obtain FACS data (FACS data).

Detailed Description

The terminology used in the present invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular forms include the plural unless the context clearly indicates otherwise. In the present invention, the terms "comprises" and "comprising" are to be interpreted as referring to the features, numbers, steps, operations, structural elements, components, or combinations thereof recited in the specification, without precluding the presence or addition of more than one other features or numbers, steps, operations, structural elements, components, or combinations thereof.

The terms "first", "second", etc. may be used to describe various structural elements, but the above structural elements should not be limited by the above terms. The above terms are used only to distinguish one structure from another. For example, a first structural element may be termed a second structural element, and, similarly, a second structural element may be termed a first structural element, without departing from the scope of the present claims.

Unless defined otherwise, all terms including technical or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The meaning of terms defined in commonly used dictionaries should be interpreted as having the same meaning as in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

The term "immune cell" as used in the present invention is used in the sense of including only NK cells as well as T cells. "NK cells" are used as meaning including NK cells (NK cells) and NKT cells (NKT cells) each, but may be used as meaning including only NK cells (NK cells) according to circumstances.

The term "Feeder Cell" as used in the present invention is used in the sense of including cancer Cell lines as allogeneic cells belonging to the same species.

The term "basal medium" used in the present invention means a medium containing the most basic components which are added and mixed with nutrients required for the culture in order to culture tissues of microorganisms or animals and plants, and known common basal media include basal Eagle medium (Basal medi um Eagle's, BME), minimum basal medium (minimum essential medium, MEM), dulbecco Modified Eagle Medium (DMEM), RPMI 1640, etc., and "basal medium for suspension cell culture" used in the present invention means a basal medium suitable for culturing suspension cells among known basal media.

In the case of the description of the time relationship, for example, in the case of the description of the time sequence relationship with, for example, "immediately" or "directly", unless "immediately" or "directly" is used, the discontinuous case is also included.

Hereinafter, the technical structure of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein, but may be embodied in other forms. The same reference numerals used to describe the present invention denote the same structural elements throughout the specification.

The technical features of the present invention relate to a novel use of a drug for inhibiting regulatory T cell (Treg) activity, particularly a steroid, and particularly to an immune cell proliferation activating kit and an immune cell culture method using the same, which use a first unit comprising a T cell stimulating substance and a second unit comprising a steroid, and optionally a third unit comprising a NK cell stimulating substance, in a specific order and a specific period of time when culturing immune cells, thereby inhibiting the immunosuppressive activity of cd4+ T cells, particularly regulatory T cells (Treg), so that more NK cells and one or more T cells can be proliferated for a long period of time.

That is, when the NK cells are cultured, not only the NK cells need to be stimulated at the beginning but also the NK cells need to be stimulated during the culture, but the T cells are sufficient only by the initial stimulation, and therefore, the NK cells are first stimulated with the NK cell stimulating substance, that is, the third unit, and then the T cells are stimulated and activated with the T cell stimulating substance, that is, the first unit, so that when the activated T cells and the NK cell stimulating substance further stimulate the NK cells together, cd4+ T cells, particularly the immunosuppressive activity of regulatory T cells (tregs), in the activated T cells can be suppressed immediately, and more NK cells can be activated or proliferated, because it is demonstrated that even in the case where more T cells are to be activated or proliferated, when the first unit and the second unit are used in sequence without using the third unit, the T cells can be proliferated and activated more effectively.

In other words, steroids are known as agents for inhibiting regulatory T cell (Treg) activity that inhibit cd4+ T cells and further inhibit Treg cells therein, and the present invention proves that when the steroids are used in a specific culturing step for culturing immune cells, they can be more effectively proliferated and activated, thereby developing a novel use of the steroids as agents for inhibiting regulatory T cell (Treg) activity.

More specifically, it is known that steroids have an effect of eliminating inflammation and pain by inhibiting cd4+ T cells such as Th1, th2, th17, treg, etc., but serious related side effects such as a decrease in immunity are generated when used for a long period of time, because it is confirmed that proliferation and activation of NK cells are not inhibited when the Treg cells are inhibited with steroids after the NK cells are activated, and NK cell activation and cell proliferation are not hindered even if the immune cells are dense to some extent in culture, and proliferation and activation of T cells but not NK cells are not inhibited even if the immune cells are dense to some extent in culture when the NK cells are not stimulated and the Treg cells are inhibited with steroids.

Thus, the immune cell proliferation-activating kit of the present invention comprises: a first unit comprising a T cell stimulating substance; and a second unit comprising a steroid. Optionally, the composition may further comprise a third unit comprising an NK cell stimulating substance.

The first unit contains a T Cell stimulating substance, and when the T Cell stimulating substance is an antibody, the antibody can be contained in a basal medium solution at a concentration of 0.1 to 12. Mu.g/mL, and when the T Cell stimulating substance is a Feeder Cell, the Feeder Cell can be 1X 10 ⁵ The concentration of the antibody used as the T cell stimulating substance may be at least one selected from the group consisting of an anti-CD 3 antibody, an anti-CD 4 antibody, and an anti-CD 28 antibody, which is contained in the basal medium solution at a concentration of at least one. Further, feeder cells (Feeder cells) are not limited as long as they are allogeneic cells of the same species (species) as the immune cells to be cultured, but basically, 5% or more of the number of immune cells to be cultured is required to be used as the stimulating cells, and up to 100% of the number may be used. Wherein the basic medium solution is formed by incorporating interleukin-2 (IL-2) at a concentration of 300-4000 IU/mL into a basic medium for culturing suspension cells, and the suspension cell culture is used for culturing immune cellsBasal medium was used.

In general, when immune cells are cultured, th1 is stimulated and activated by IL-2 contained in a basal medium solution while NK cells are stimulated to some extent, and when Th cells are stimulated with T cell stimulating substances such as anti-CD 3 antibodies and NK cells and Tc cells are stimulated with activated Th1 cells in this state, NK cells proliferate to some extent as NK cells and Tc cells are activated. In particular, as described below, when NK cells are further stimulated with more than one NK cell stimulating substance selected from the group consisting of IL-12, IL-15, IL-18, anti-CD 16 antibodies, anti-CD 56 antibodies and the like contained in the third unit before the first unit is treated, NK cells are further activated and more effectively grown.

The second unit comprises a steroid which may be included in the basal medium solution at a concentration of 0.02 μg/mL or more. The steroid contained in the second unit may use all known steroid drugs, but may be corticosteroids (glucocorticoids). As an example, one or more of the group consisting of Cortisol (Cortisone), cortisone (Cortisone), prednisolone (Prednisolone), prednisone (prednisone), methylprednisolone (Methylpredni solone), dexamethasone (Dexamethasone), betamethasone (Betamethasone), triamcinolone (Triamcinolone), fludrocortisone acetate (Fludrocortisone acetate), deoxycorticosterone acetate (deoxycorticosterone acetate) may be used.

As described above, when immune cells are cultured, when the first unit is treated, i.e., immune cells are stimulated with T cell stimulating substances such as anti-CD 3 antibodies, all T cells, particularly, humoral immune cells such as Th2 and tregs are stimulated, but these cells do not help NK cells activate and proliferate. In particular, treg cells interfere with activation and proliferation of NK cells, so that T cells can be proliferated better than NK cells in immune cells when treating the second unit in a first culture solution treated with the first unit to inactivate Treg cells, and NK cells can be proliferated better than T cells when treating the second unit in a first culture solution treated with the first unit after treating the third unit.

Wherein the steroid contained in the second unit acts on the proliferation of immune cells, in particular NK cells, by inhibiting the immunosuppressive activity of cd4+ T cells, in particular regulatory T cells (tregs), experiments have shown that the steroid has the property to suppress the overall growth of T cells to some extent by eliminating the immunosuppressive activity of regulatory T cells.

That is, as described in the following experimental examples, it was confirmed that the steroid used as a drug for inhibiting the activity of regulatory T cells (tregs) did not affect the proliferation of activated NK cells, tc cells and other cellular immune cells at all, because the proportion of NK cells was further increased with the lapse of the culture time. In particular, when the concentration of the steroid increases, the proliferation rate of T cells is suppressed to some extent, and thus the NK cell ratio increases in proportion to the concentration of the steroid.

When the content of the steroid contained in the second unit is less than 0.02 μg/mL, the characteristic of suppressing the immunosuppressive activity of cd4+ T cells, especially regulatory T cells (tregs), is not sufficiently exerted, and even if a high concentration of the steroid is treated, when the concentration is more than 9 μg/mL, it is predicted that the influence on immune cell culture is not greatly changed. In view of the fact that even if the culture medium is contained at a concentration of 20. Mu.g/mL in the experiment, there is no greater progress or adverse effect on the proliferation effect of immune cells, and therefore, the upper limit of the steroid content can be determined in consideration of economic feasibility. Therefore, in the second unit of the immune cell proliferation activation kit of the present invention, the steroid used as a drug for inhibiting regulatory T cell (Treg) activity can be contained at a concentration of 0.02 μg/mL or more to obtain a desired effect.

The third unit contains an NK cell stimulating substance, which may be present in the basal medium solution at a concentration of 0.2ng/mL to 10. Mu.g/mL, and which may be one or more selected from the group consisting of an anti-CD 16 antibody, an anti-CD 56 antibody, IL-12, IL-15 and IL-18. Wherein the concentration of the NK cell stimulating substance contained in the third unit varies depending on the kind of the NK cell stimulating substance, as a first example, the third unit may be prepared by adding and dissolving IL-122 and IL-18 in the basic medium solution at a concentration of 0.5 to 15ng/mL and 2 to 50ng/mL, respectively, or as a second example, the third unit may be prepared by adding and dissolving only IL-15 in the basic medium solution at a concentration of 1 to 100 ng/mL. As a third example of another example, the third unit may be prepared by including one or more of an anti-CD 16 antibody at a concentration of 0.1 to 10 μg/mL and an anti-CD 56 antibody at a concentration of 0.1 to 10 μg/mL in the basal medium solution. According to circumstances, the third unit may also be realized by further including one or more of an anti-CD 16 antibody and an anti-CD 56 antibody in the first example.

As described above, when the NK cells are further stimulated with the NK cell stimulating substance such as the anti-CD 16 antibody or the anti-CD 56 antibody in a state where Th1 is stimulated and activated with IL-2, the NK cells can be proliferated and activated more effectively than the T cells and cultured for a long time, and when the third unit is not used, the basal medium solution contains IL-2, and therefore the T cells can be proliferated and activated more effectively than the NK cells and cultured for a long time.

The immune cell proliferation-activating kit of the present invention having such a structure is individually packaged so as to be added to a culture medium in each step of forming the immune cell culture method of the present invention described later, and may contain different contents, i.e., be composed of a first unit, a second unit, and/or a third unit of different compositions. Also, in culturing immune cells, since NK cells need to be assisted by stimulated T cells to be fully activated after initial stimulation, the proliferation activation kit of the present invention must use a second unit after stimulating T cells in immune cell culture medium with the first unit. Thus, when the immune cell proliferation activation kit of the present invention is used in order, that is, when the T cells in the immune cell medium are stimulated by the treatment of the first unit, the proliferation rate of the T cells in the immune cells can be very effectively increased without using the third unit and the proliferation rate of NK cells in the immune cells can be increased with using the third unit before using the first unit when the second unit is treated in an appropriate period when the immune cells are cultured.

Second, the immune cell culture method of the present invention may include: a first culturing step of suspending Peripheral Blood Mononuclear Cells (PBMCs) isolated from blood in a basal medium solution, and then treating a first unit containing a T cell stimulating substance and culturing for 18 to 72 hours; a second culturing step of culturing for 18 to 25 hours after treating the first culture liquid subjected to the first culturing step with a second unit containing a steroid; and a third culturing step of culturing by adding one or more of the basal medium solution and fetal bovine serum (Fetal Bovine Serum, FBS) to the second culture medium subjected to the second culturing step at intervals of 18 to 25 hours, and transferring the culture medium to a larger culture vessel as the number of cells increases.

In the first culturing step, the third unit containing the NK cell stimulating substance may be treated before or simultaneously with the treatment of the first unit. That is, as described above, NK cells are stimulated to some extent by the inclusion of IL-2 in the basal medium solution, but when the second unit is post-treated in a state of further treatment with one or more NK cell stimulating substances selected from the group consisting of anti-CD 16 antibody, anti-CD 56 antibody, IL-12, IL-15, IL-18, NK cells in immune cells can grow better, and therefore, when it is desired to especially increase the proportion of NK cells in immune cells and proliferate NK cells, it is preferable to treat the third unit before treating the first unit.

The second culturing step is performed by culturing for a prescribed time after the second unit is treated in the first culture liquid passing through the first culturing step, and thus may be performed after 18 to 48 hours after the treatment of the first unit.

As an example, the basal medium solution in which PBMCs are suspended is treated with the first unit and cultured for 20 to 25 hours at any time point of the culture day 0, the culture day 1 or the culture day 2 in the first culturing step to form a first culture solution, and the second culturing step may be performed by treating the second unit in the above-described first culture solution. That is, when NK cells are stimulated with IL-2 contained in a basal medium solution to a certain extent on day 0 of culturing immune cells, and then T cells are stimulated with T cell stimulating substances such as anti-CD 3 antibody, anti-CD 4 antibody or anti-CD 28 contained in the first unit to activate Tc, NK cells, etc. on day 0, 1 or 2 of culturing, cd4+ T cells, especially regulatory T cells (Treg) are inhibited in treating the second unit containing steroid on any specific day of culturing on days 1 to 4, thus immune cells including NK cells, etc. are proliferated and/or activated because the inhibitory activity caused by regulatory T cells (Treg) cannot be exhibited.

The third culturing step is a process of culturing a large number of immune cells and collecting immune cells, and culturing is performed by adding one or more of a basal medium solution and fetal bovine serum (Fetal Bovine Serum, FBS) to the second culture medium subjected to the second culturing step at intervals of 24 hours or more from a predetermined time point, but may be performed by adding at intervals of 48 hours or more as in the later-described examples, and transferring to a larger culture vessel for culturing as the number of cells increases due to proliferation of immune cells. According to circumstances, the second cell may be further treated in the third culturing step, and as an example, the second culture solution subjected to the second culturing step may be treated with the second cell 1 or more times after culturing for 5 days. It is known that the proliferation rate is reduced and the growth is poor at the early stage of the culture of PBMCs, that is, at the young stage of the immune cells (initial stage of the culture), but the proliferation rate is reduced and the growth is poor after about 8 to 10 days of the culture, but in the present invention, the third culture step from 11 days to 21 days can be performed, so that a large amount of NK cells can be collected as compared with the conventional immune cell culture method which must be generally performed within 8 days.

As described above, the third step, which is based on the experimental results of the present invention, can be performed for a long period of time by stimulating immune cells with a basal medium solution at the initial stage of culturing immune cells, that is, in the first culturing step, and further stimulating T cells with the first unit to post-treat the second unit containing steroid to inhibit Treg, in which state immune cells can be activated to proliferate relatively well by increasing the concentration of cytokines (cytokine) including IL-2 even after culturing for 8 to 10 days, and therefore, as in the examples described later, one or more of basal medium solution and FBS can be added to the second culture solution passing through the second culturing step at desired times and contents at intervals of 18 to 25 hours from the prescribed time point in consideration of the number of proliferated cells. The predetermined time point may be a time point when about 24 hours or more passes from the time point when the second unit is added in the second step.

Secondly, the pet immune cell culture method of the invention comprises the following steps: a first culturing step of suspending Peripheral Blood Mononuclear Cells (PBMCs) isolated from blood of dogs in a basal medium solution, and then simultaneously or sequentially treating a first unit including a T cell stimulating substance and a third unit including an NK cell stimulating substance and culturing for 4 days; a second culturing step of culturing for 18 to 25 hours after simultaneously or sequentially treating the first culture solution having undergone the first culturing step with a second unit containing a steroid and the third unit; and a third culturing step of culturing by adding one or more of the basal medium solution and fetal bovine serum (Fetal Bovine Serum, FBS) to the second culture medium subjected to the second culturing step at intervals of 24 hours or more from a predetermined time point, and culturing by transferring the number of cells to a larger culture vessel.

The pet immune cell culture method of the present invention is a method for proliferating immune cells including NK cells in a large amount in animals PBMC but not in human PBMC, and is similar to the above-described immune cell culture method in its entirety in the point of using the immune cell proliferation activating kit of the present invention, but in order to proliferate immune cells more effectively using PBMCs isolated from pets, particularly dogs, it is not described in detail since the first to third culture steps are performed only if two or more of the first to third units are used at the same time and the time points of treatment are different, and the remaining structures are the same.

However, it was confirmed through experiments that the effect was more excellent only when IL-15 was contained in the third unit in the pet immune cell culture method.

Example 1

The first unit to the third unit were prepared as follows, thereby preparing the immune cell proliferation-activating kit 1.

1. Preparation of basal Medium solution

A basal medium solution was prepared by adding and dissolving IL-2 at a concentration of 2000IU/mL in a basal medium for suspension cell culture (product name: RPMI 1640).

2. Preparation of the first Unit

The first unit-1 was prepared by adding and dissolving an anti-CD 3 antibody in a basal medium solution at a concentration of 1. Mu.g/mL.

3. Preparation of the second Unit

The second unit-1 was prepared by dissolving dexamethasone sodium phosphate (Dexamethasone sodium phosphate) in the basal medium solution at a concentration of 0.02 μg/mL.

Example 2

An immune cell proliferation-activating kit 2 was prepared by the same method as example 1, except that, in preparing the second unit, dexamethasone sodium phosphate (Dexamethasone sod ium phosphate) was dissolved in a basal medium solution at a concentration of 20. Mu.g/mL to prepare the second unit.

Example 3

An immune cell proliferation-activating kit 3 was prepared by the same method as example 1, except that, in preparing the second unit, dexamethasone sodium phosphate (Dexamethasone sod ium phosphate) was dissolved in a basal medium solution at a concentration of 1. Mu.g/mL to prepare the second unit-3.

Example 4

An immune cell proliferation-activating kit 4 was prepared by the same method as in example 1, except that, in the preparation of the second unit, prednisolone (solondo tablet, 5 mg/tablet, yuhan Corporation) was dissolved in the basal medium solution at a concentration of 167. Mu.g/mL, and then the second unit-4 was prepared by diluting 20-fold with the basal medium solution to a concentration of 8.3. Mu.g/mL.

Example 5

An immune cell proliferation-activating kit 5 was prepared by the same method as in example 1, except that methylprednisolone (PD tablet, 4 mg/tablet, middle and outside pharmacy) was dissolved in the basal medium solution at a concentration of 80. Mu.g/mL at the time of preparation of the second unit, and then the second unit-5 was prepared by diluting 10-fold to a concentration of 8. Mu.g/mL with the basal medium solution.

Example 6

An immune cell proliferation-activating kit 6 was prepared by the same method as in example 1, except that 1. Mu.L of betamethasone sodium phosphate (betamethasone sodium phosphate) (Hanall betamethasone injection, 5.2mg/1mL ampoule) was dissolved in 10mL of the basal medium solution to a concentration of 0.4. Mu.g/mL to prepare the second unit-6.

Example 7

Except that the K562 cell line (cell line) was used instead of the anti-CD 3 antibody at 5X 10 in the preparation of the first cell ⁶ An immunocyte proliferation-activating kit 7 was prepared in the same manner as in example 1, except that the first unit-2 was prepared by dissolving the concentration of each/mL in the basal medium solution.

Example 8

An immune cell proliferation-activating kit 8 was prepared by the same method as example 1, except that the third unit was also prepared as follows.

IL-12 and IL-18 were added and dissolved in the basal medium solution at concentrations of 10ng/mL and 50ng/mL, respectively, to prepare a third unit-1.

Example 9

An immune cell proliferation-activating kit 9 was prepared by the same method as in example 7, except that in preparing the third unit, the anti-CD 16 antibody and the anti-CD 56 antibody were added and dissolved in the basal medium solution at a concentration of 1. Mu.g/mL, respectively.

Example 10

An immune cell proliferation-activating kit 10 was prepared by the same method as example 7, except that IL-15 was added at a concentration of 20ng/mL and dissolved in a basal medium solution to prepare a third unit-3.

Experimental method

1. Cell count (Cell count)

The first Day of PBMC isolation from blood and culture was taken as Day0 (Day 0) (1X 10) ⁷ Individual cells (cells)) were cultured for a total of 14 days or 19 days and the cell number was determined on a specific culture day. All cells and media were collected in 500mL conical tubes (medical tube) and centrifuged at 1800rpm for 5 minutes at 4℃and the supernatant removed. Cells were suspended by adding 10mL of DPBS to Cell pellet (Cell pellet) and diluted by removing 10 μl therefrom, then mixed with an equal amount of 0.4% trypan blue staining solution (Trypan blue staining solution, gibco, new York (NY), USA (USA)), then placed on C-chip (encyto, georgia (GA), USA) and counted under a culture microscope CKX53 (Culture microscope CKX, olympus, tokyo, japan).

2. Flow cytometry analysis (Flow cytometric analysis)

The cultured cells were centrifuged at 2000rpm at 4℃for 3 minutes and the supernatant was removed, and 200. Mu.l of FCS buffer (buffer) (eBioscience ^TM Flow cell staining buffer (Flow cytometry staining buffer), invitrogen) was suspended and centrifuged at 2000rpm for 3 minutes at 4 ℃. After removing the supernatant, 5. Mu.l of an antibody (anti-ibody) (anti-human CD 56-PE), anti-human CD16 PE-Cy-7 (anti-human CD16 PE-Cy-7), anti-human CD3-FITC (anti-human CD 3-FITC)) and isotype control antibody (isotype control antibody) (Mouse IgG1 kappa isotype control PE (Mouse IgG1 kappa isotype control-PE), mouse IgG1 kappa isotype control FITC (Mouse IgG1 kappa isotype control-FITC), mouse IgG1 kappa isotype control PE-Cy-7 (Mouse IgG1 kap pa isotype control PE-Cy-7)) were added, respectively, and then the reaction was performed under dark conditions and at 2-4 ℃. After 30 minutes, the supernatant was removed after centrifugation at 2000rpm for 3 minutes at a temperature of 4 ℃. After suspending with 500. Mu.l of FCS buffer (FCS buffer), the supernatant was removed by centrifugation at 2000rpm for 3 minutes at 4 ℃. After washing the total cells 2 times, the cells were suspended in 500. Mu.l of FC S buffer (FCS buffer) and used as FACSCanto II @ The analysis was performed by Bidi medical company (Becton Dickinson).

Example 11

The immune cells were cultured using the immune cell proliferation activation kit 1 as follows. In the course of culturing immune cells, cells were counted and analyzed by flow cytometry using the above experimental method, and the results thereof are shown in table 1, fig. 1a and fig. 1 b.

1. Isolation of human peripheral blood mononuclear cells (peripheral blood mononuclear cell s:PBMC)

(1) To a 50mL Leukosep separation tube (Leukosep tube) (Greiner Bio-One) was added 15mL Ficoll-Paque ^TM PLUS (GE healthcare, sweden) and centrifuged at 1200rpm for 3 minutes at a temperature of 4 ℃.

(2) Based on 30cc of blood collected in a vacuum blood collection tube (BD vacutainer, N.J., U.S.), 30cc of blood was added to one (1) of the separation tubes (tube) and centrifuged at 2000rpm at a temperature of 4℃for 20 minutes.

(3) After centrifugation, the plasma (plasma) corresponding to the uppermost portion was transferred to a new 50mL conical tube (fresh tube) and centrifuged at 1500rpm for 5 minutes at a temperature of 4℃after Heating (Heating) 3 times in a water bath (water bath) at 56℃for a total of 10 minutes, and then the supernatant was labeled as Heat-inactivated plasma (Heat-Inactivated plasma, HIP) and stored at a temperature of 4 ℃.

(4) The Peripheral Blood Mononuclear Cell (PBMC) layer remaining after plasma separation was transferred to a new 50mL conical tube (medical tube) and centrifuged at 1500rpm for 5 minutes at a temperature of 4 ℃.

(5) After removing the supernatant, the cells were washed (washing) with 20mL of Dulbecco's phosphate-Buffered Saline (DPBS, welgene, korea) and centrifuged at 1200rpm at 4℃for 5 minutes.

(6) After removal of the supernatant, cells were suspended (suspension) iN 5mL of DPBS, from which 10. Mu.L of the mixture was mixed with the same amount of 0.4% trypan blue staining solution (Trypan blue staining solution, gibco, new York, USA) and diluted, and then 10. Mu.L of the diluted cell solution was inserted into C-Chip (iN)CYTO, georgia, U.S.) and counting cells under a culture microscope CKX53 (Culture microscope CKX, olympus, japan (JAPAN)), confirming 1X 10 cells per 1mL of blood ⁷ Individual cells (cells).

2. Culturing immune cells

(1) Day0 (Day 0): the day of PBMC isolation from blood

PBMC (1X 10) ⁷ Individual cells (cells)) were suspended in 5mL of the basal medium solution and placed in T25 cell culture flasks (cell culture flask, SPL, korea: example flask) and adding the first unit-1 to give an anti-CD 3 antibody concentration of 1 μg/mL, then 5% co at 37 °c ₂ Cells were cultured under culture conditions for 24 hours. For use as a control group, cultures were performed in other flasks under the same conditions as in comparative example 1.

(2) Day1 (Day 1): adding a second unit

Only the second unit-1 was added to the example flask so as to contain 0.2. Mu.g/mL dexamethasone sodium phosphate (Dexamethasone sodium phosphate), and then the culture was continued under the same conditions.

(3) Day4 (Day 4) to Day14 (Day 14): addition of basal medium solution and FBS

25mL of basal medium solution and 2.5mL of FBS (or autologous plasma) were added to each of the example flask and the control flask and the flask was replaced with a T75 flask. Thereafter, under the conditions shown in table 1 below, the cells were transferred to a larger flask as the number of cells increased and cultured for a total of 2 weeks.

TABLE 1

As shown in table 1 and fig. 1a, it was confirmed from the culture results that the cell proliferation of example 11, in which the immune cells were stimulated with the anti-CD 3 antibody contained in the first unit-1 and then cultured by adding the steroid contained in the second unit-1 to the cell culture medium, was more superior to that of comparative example 1, in which the second unit-1 was not treated, and, in particular, increased significantly from the time of 8 days after the culture, by at most 5-fold or at least 4-fold. As shown in fig. 1b, it was confirmed from the results of flow cytometry analysis (Flow cytometric analysis) of the cultured immune cells that, before the first unit was treated, the NK cells were not stimulated in large amounts, and thus, example 11 and comparative example 1 each showed that the T cells proliferated more than the NK cells. It was found that example 11 in which the second unit was treated proliferated relatively more NK cells than comparative example 1 in which the second unit was not treated at all, and therefore it was confirmed that the second unit further increased the NK cell proliferation rate under the same conditions.

Example 12

After preparing human PBMCs by the same method as in example 11, immune cells were cultured using the immune cell proliferation activation kit 7 as follows. In the course of culturing immune cells, cells were counted and analyzed by flow cytometry using the above experimental method, and the results thereof are shown in table 2, fig. 2a and fig. 2 b.

(1) Day0 (Day 0): the day of PBMC isolation from blood

PBMC (1X 10) ⁷ Individual cells (cells)) were suspended in 5mL of the basal medium solution and placed in T25 cell culture flasks (cell culture flask, SPL, korea: example flask) and at 37℃5% CO ₂ Cells were cultured under culture conditions for 48 hours. For use as a control group, cultures were performed in other flasks under the same conditions as in comparative example 2.

(2) Day2 (Day 2): adding the first unit

The first unit-2 was added to both the example flask and the control flask so as to contain 5X 10 ⁶ K562 cells (cells) were used as Feeder cells (Feeder cells) and then cultured under the same conditions.

(3) Day4 (Day 4): adding a second unit, basal medium solution and FBS

25mL of basal medium solution and 2.5mL of FBS were added to each of the example flask and the control flask and the flask was replaced with a T75 flask. In particular, unlike the control flask, the second unit-2 was added to the example flask so as to contain 20. Mu.g/mL dexamethasone sodium phosphate (Dexamethasone sodium phosphate). The culture was continued under the same conditions with the example flask and the control flask.

(4) Day5 (Day 5) to Day14 (Day 14): addition of basal medium solution and FBS

Under the conditions shown in Table 2 below, the cells were transferred to a larger flask and cultured for a total of 2 weeks as the number of cells increased.

TABLE 2

As shown in table 2 and fig. 2a, it is understood from the culture results that the Cell proliferation of example 12, which was cultured by adding the steroid contained in the second unit-2 to the Cell culture medium after one day after stimulation of immune cells with the K562 Cell line (Cell line) as Feeder cells (Feeder cells) contained in the first unit-2, was more superior to that of comparative example 2, which was not treated with the second unit-2, and, in particular, increased significantly from 8 days after the culture, at least increased by a factor of 2 or more. Further, as shown in fig. 2b, it was confirmed from the results of flow cytometry analysis (Flow cytometric analysis) of the cultured immune cells that, although the third cell was not treated before the first cell was treated, example 12 and comparative example 2 each showed that NK cells proliferated more than T cells. In particular, when the ratio of NK cells to T cells in example 12 in which the second unit was treated and comparative example 2 in which the second unit was not treated at all was examined, it was found that NK cells of example 12 were proliferated more than 2 times more than T cells, but the proliferation ratio of NK cells to T cells of comparative example 2 was similar, so that it was confirmed that the second unit was more elevated in proliferation rate of NK cells than T cells under the same conditions. In particular, in the case of using Feeder cells (Feeder cells) as T Cell stimulating substances, it was confirmed that the proliferation rate of NK cells was increased even without using the third Cell before the first Cell was treated.

Example 13

After preparing human PBMCs by the same method as in example 11, immune cells were cultured using the immune cell proliferation activation kit 8 as follows. In the course of culturing immune cells, cells were counted and analyzed by flow cytometry using the above experimental method, and the results are shown in table 3, fig. 3a and fig. 3 b.

(1) Day0 (Day 0): the day of PBMC isolation from blood

PBMC (1X 10) ⁷ Individual cells (cells)) were suspended in 5mL of the basal medium solution and placed in T25 cell culture flasks (cell culture flask, SPL, korea: example flask) and adding the third unit-1 such that the IL-12 concentration is 10ng/mL and the IL-18 concentration is 50ng/mL, then at 37℃with 5% CO ₂ Cells were cultured under culture conditions for 24 hours. For use as a control group, cultures were performed in other flasks under the same conditions as in comparative example 3.

(2) Day1 (Day 1): adding the first unit

The first unit-1 was added to both the example flask and the control flask to give an anti-CD 3 antibody concentration of 1. Mu.g/mL, followed by 5% CO at 37 ℃ ₂ Cells were cultured under culture conditions for 24 hours.

(3) Day2 (Day 2): adding a second unit

(4) Day4 (Day 4) to Day14 (Day 14): addition of basal medium solution and FBS

25mL of basal medium solution and 2.5mL of FBS were added to each of the example flask and the control flask and the flask was replaced with a T75 flask. Thereafter, under the conditions shown in Table 3 below, the cells were transferred to a larger flask as the number of cells increased and cultured for a total of 2 weeks.

As shown in Table 3 and FIG. 3a, it is understood from the results of the culture that the proliferation of cells of example 13, in which the NK cell stimulating substances IL-12 and IL-18 contained in the third unit-1 were treated first, and then immune cells were stimulated with the anti-CD 3 antibody contained in the first unit-1 after one day, and then the steroid contained in the second unit-1 was added to the cell culture medium for culture, was more superior to that of comparative example 3, in which the second unit-1 was not treated, and in particular, increased significantly from the time of 8 days later, by at least 2-fold or more.

TABLE 3 Table 3

As shown in fig. 3b, it was confirmed from the results of flow cytometry analysis (Flow cytometric analysis) of the cultured immune cells that NK cells were stimulated by the treatment of the third cell before the treatment of the first cell, and that NK cells were proliferated more than T cells in each of example 13 and comparative example 3. In particular, when the ratio of NK cells to T cells proliferated in example 13 in which the second unit was treated and comparative example 3 in which the second unit was not treated at all was examined, it was found that NK cells of example 13 proliferated almost 30 times or more than T cells, but NK cells of comparative example 3 proliferated about 4.5 times more than T cells, so that it was confirmed that the second unit significantly increased the proliferation rate of NK cells than T cells under the same conditions. In particular, when the third unit-1 comprising IL-12 and IL-18 is used before the first unit is treated, it is known that the proportion of T cells in very efficiently cultured immune cells is very small and NK cells can proliferate to almost a large proportion of proliferation.

Example 14

After preparing human PBMCs by the same method as in example 11, immune cells were cultured using immune cell proliferation activation kit 9 as follows. In the course of culturing immune cells, cells were counted and analyzed by flow cytometry using the above experimental method, and the results thereof are shown in table 4 and fig. 4a to 4 b.

(1) Day0 (Day 0): the day of PBMC isolation from blood

PBMC (1X 10) ⁷ Individual cells (cells)) were suspended in 5mL of the basal medium solution and placed in T25 cell culture flasks (cell culture flask, SPL, korea: example flask) and adding the third unit-2 to give anti-CD 16 antibody and anti-CD 56 antibody concentrations of 1. Mu.g/mL, respectively, then at 37℃with 5% CO ₂ Cells were cultured under culture conditions for 24 hours. For use as a control group, cultures were performed in other flasks under the same conditions as in comparative example 4.

(2) Day1 (Day 1): adding the first unit

(3) Day2 (Day 2): adding a second unit

Only the second unit-2 was added to the example flask so as to contain 20. Mu.g/mL dexamethasone sodium phosphate (Dexamethasone sodium phosphate), and then the culture was continued under the same conditions.

(4) Day4 (Day 4) to Day14 (Day 14): addition of basal medium solution and FBS

25mL of basal medium solution and 2.5mL of FBS were added to each of the example flask and the control flask and the flask was replaced with a T75 flask. Thereafter, under the conditions shown in Table 4 below, the cells were transferred to a larger flask as the number of cells increased and cultured for a total of 2 weeks.

As shown in Table 4 and FIG. 4a, it is understood from the results of the culture that the cell proliferation of example 14, in which the anti-CD 16 antibody and the anti-CD 56 antibody contained in the third unit-2 as NK cell stimulating substances were treated first, and then immune cells were stimulated with the anti-CD 3 antibody contained in the first unit-1, after one day, and then the steroid contained in the second unit-1 was added to the cell culture medium for culture, was more superior to that of comparative example 4, in which the second unit-1 was not treated, and in particular, increased significantly from 8 days after the culture, by at least 2-fold or more.

TABLE 4 Table 4

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As shown in fig. 4b, it was confirmed from the results of flow cytometry analysis (Flow cytometric analysis) of the cultured immune cells that NK cells were stimulated by the treatment of the third cell before the treatment of the first cell, and that NK cells were proliferated more than T cells in each of example 14 and comparative example 4. In particular, when the ratio of NK cells to T cells in example 14 in which the second unit was treated and comparative example 4 in which the second unit was not treated at all was examined, it was found that NK cells of example 14 were proliferated almost 2.5 times or more than T cells, but NK cells of comparative example 3 were proliferated about 1.5 times more than T cells, so that it was confirmed that the second unit was more enhanced in proliferation rate of NK cells than T cells under the same conditions. However, compared to example 13 in which the third unit-1 including IL-12 and IL-18 was used before the first unit was treated, the proportion of NK cells to T cells that proliferated was not high, and thus it was found that IL-12 and IL-18 included in the third unit-1 exert more effective actions as NK cell stimulating substances than the anti-CD 16 antibody and anti-CD 56 antibody included in the third unit-2.

Example 15

The immune cell culture process was performed as follows using the medium addition kit (NKT M) for immune cell culture disclosed in the prior patent and the immune cell proliferation activation kits 3 to 6 of the present invention instead of the basal medium solution.

On the other hand, the medium addition kit (NKTM) for immune cell culture is packaged separately for each, so that immune cells can be added to the medium in each step of culturing, and is composed of different contents, i.e., different components, including B units (base solution, B solution), C1-1 units (cytokine 1-1 solution), C1-2 units, C2 units, A1 units, A2 units, and D units. Details of each unit and specific conditions of a culture process (NKTM culture process) for culturing immune cells using the above units are disclosed in patent publication No. 10-2018-0057359 (hereinafter, "prior patent"), and thus a part different from that described in the prior patent is emphasized. However, the components contained in the first and third units forming the immune cell proliferation activation kit of the present invention are contained in each unit forming NKTM, and therefore only the second unit-3 to the second unit-6 are used without using the first and third units in addition.

1. Preparation of Medium addition kit (NKTM) for immune cell culture

(1) Preparation of the base solution (solution B)

The base solution (solution B) was prepared by adding 2.2mg of IL-2 and 100mL of 500mM L-glutamine (L-glutamine) solution to and dissolving in a base medium for suspension cell culture (the addition amount was adjusted to make the final concentration when IL-2 or L-glutamine (L-glutamine) was already present in the base medium) and finally making 10L.

(2) Preparation of cytokine 1-1 solution

A cytokine solution (solution C) was prepared by dissolving 20. Mu.g of IL-12 and 125. Mu.g of IL-18 in distilled water to prepare 10 mL. Cytokine 1-1 solution (C1-1 solution) was prepared by dissolving 1mL of the C solution in 1000mL of the base solution (B1 solution).

(3) Solution A: solutions prepared by dissolving anti-CD 16 antibody and anti-CD 56 antibody in B1 solution at a concentration of 0.01-1.5. Mu.g/mL, respectively

(4) A1 solution: solutions prepared by dissolving anti-CD 16 in a C1-1 solution at a concentration of 0.1 to 15. Mu.g/mL

(5) A2 solution: solutions prepared by dissolving anti-CD 56 antibodies in a C1-1 solution at a concentration of 0.1-15. Mu.g/mL

(6) B1 solution: solution with concentration of IL-2 in suspension cell culture basic culture medium solution of 1000-4000 IU/mL

(7) B2 solution: IL-2 concentration 2 times B1 solution

(8) C1-1 solution: solutions prepared by dissolving IL-12 and IL-18 in B1 solution at concentrations of 0.5 to 5ng/mL and 2 to 50ng/mL, respectively

(9) C5 solution: adding 5 times concentration of IL-2 and IL-18 solution into C1-1 solution

A solution D: solutions prepared by dissolving anti-CD 3 antibodies in a C1-1 solution at a concentration of 0.5 to 12. Mu.g/mL

R solution: basal medium solution for suspension cell culture containing L-glutamine (L-glutamine) at a concentration of 3 to 12mM, without Cytokine (Cytokine) or antibody

2. Culture process

After the lymphocyte extraction step and the autologous plasma preparation step were performed in the same manner as in the conventional patent, the culture was performed by the same method except that some of the conditions in the culture method described in example 3 of the conventional patent described below were changed as follows.

Day0 (Day 0): 1.0X10 to be separated from blood ⁷ The culture was started after the PBMC were dissolved in the C1-1 solution. That is, after treatment of isolated PBMC with 1 bottle (4.5 mL) of C1-1 solution (C1-1 solution) and 0.5mL of serum, the PBMC were placed in a T25 Flask (T25 flash) and in CO ₂ Culturing was performed in an Incubator (Incubator). Since the components contained in the third unit are contained in the C1-1 solution, it is equivalent to treating the third unit.

Day1 (Day 1): 1 bottle (1 mL) of A2 solution was added to the T25 flask during cultivation and the flask was placed in an incubator with slight shaking for continuous cultivation. Since the component contained in the third unit is contained in the A2 solution, it is equivalent to treating the third unit.

Day2 (Day 2): 1 bottle (1 mL) of the D solution was added to the T25 flask during cultivation and the flask was placed in an incubator with slight shaking to continue cultivation. Since the components contained in the first unit are contained in the D solution, it is equivalent to treating the first unit.

Day3 (Day 3): after preparing 5T 25 flasks of cells that had been cultured through the previous step, 5mL of the second unit-3 (example 15-1), 4.5mL of the second unit-4 (example 15-2), 4.5mL of the second unit-5 (example 15-3), and 4.5mL of the second unit-6 (example 15-4) were added, respectively. The remaining 1 was used as a control group without adding the second unit as comparative example 5. Thereafter, the culturing step was performed in the same manner.

Day4 (Day 4): the bottom of the T25 flask in culture was scraped with a scraper (wiper) and all cells were transferred to a T75 flask. 3mL of R solution was added to the cells in the cell culture, or the medium was changed to 10mL of C1-1 solution and the mixture was gently shaken and then placed in an incubator for continuous culture.

Day5 (Day 5): 20mL of A1 solution was added to the T75 flask during cultivation and the flask was gently shaken and then placed in an incubator to continue cultivation.

Day6 (Day 6): the bottom of the T25 flask in culture was scraped with a scraper (wiper) and all cells were transferred to a T150 flask. Wherein 20mL of solution a and 3mL of autologous plasma or FBS (or the like) were added and gently shaken and then placed in an incubator for continuous culture.

Day7 (Day 7): cells in all cell cultures were transferred to a T150 flask and cultured after 40mL of B1 solution was added.

Day8 (Day 8): after clamping half of the 1L culture bag (bag) with a clamp, the culture solution containing the cells under culture in the T150 flask was added to the culture bag (bag), and 300mL of R solution was added.

Day10 (Day 10): from clamping half of 1L CO with a clamp ₂ The clamps were removed from the air-permeable culture bag (bag) and the culture was continued after 300mL of R solution was added.

Day11 to Day14 (Day 11 to Day 14): 300mL of B2 solution and 50mL of C5 solution were added to the cells in cell culture. The cells in culture were thoroughly rubbed to grow well, then incubated at 37℃with 5% CO ₂ Is continuously cultured in the incubator of (a).

Day15 (Day 15): the immune cells were collected by centrifugation (400 Xg) to separate them into immune cells and culture broth. The separated culture solution was stored in cold storage as an immune cell culture solution.

If necessary, the immunocytes isolated on Day15 (Day 15) were dissolved in 100mL of B solution. After sandwiching half of the 1L culture bag (bag) containing the cell culture medium with a clamp, the culture solution containing the cells and 50mL of the C1-2 unit cytokine 1-2 solution were added to the above culture bag (bag), and further cultured as follows.

Day16 (Day 16): the cells in culture were thoroughly rubbed to grow well, then incubated at 37℃with 5% CO ₂ Is continuously cultured in the incubator of (a).

Day17 (Day 17): from clamping half of 1L CO with a clamp ₂ The culture was continued after the clip was removed from the air-permeable culture bag (bag).

Day18 (Day 18): the cells in culture were thoroughly rubbed to grow well, then incubated at 37℃with 5% CO ₂ Is continuously cultured in the incubator of (a).

The procedure described above on days 15 to 18 (Day 15 to Day 18) was repeated 1 to 2 times or more depending on the cell state, whereby a total of 4 liters or more of cells could be obtained in one batch.

3. Cell count

In order to confirm the effect of the immune cell proliferation activation kits 3 to 6 of the present invention containing one or more drugs for inhibiting regulatory T cells (Treg) as an active ingredient on immune cell culture, the cell proliferation results obtained in examples 15-1 to 15-4 and comparative example 4 are shown in Table 5 (unit: 1X 10 ⁷ And so on) and in fig. 5.

TABLE 5

Days (Day)	0	4	6	7	8	11	13	14	15	17	19
												Example 15-1	1.0	0.89	4.6	7.0	13.5	178	352	384	447	512	570
Example 15-2	1.0	0.7	3.1	6.6	13.5	168	312	365	395	462	505
												Example 15-3	1.0	0.6	3.2	6.5	13.2	153	325	374	420	480	520
Examples 15 to 4	1.0	0.7	3.2	6.9	14.2	172	330	385	452	515	530
												Comparative example 5	2.0	1.45	6.4	10.0	19.1	78	175	180	182	175	120

As shown in Table 5 and FIG. 5, it was confirmed that, in the course of culturing immune cells, after stimulating Th cells by treatment with an anti-CD 3 antibody on day2 (day 2), immune cell proliferation-activating kit 3 containing 1. Mu.L of dexamethasone as a steroid series injection was added to the medium on day3 (day 3) for culturing as in example 15-1, the original medium was removed on day4 (day 4) to remove cytokines (cytokine) produced by Th2, treg and the like, and then culturing was continued after changing to C1 solution, and when comparing the number of cells obtained after continuing culturing with the number of cells obtained in comparative example 5 without using steroid, the proliferation rate was increased by about 4.3 times or more on day14 (day 14) and about 5.9 times or more on day17 (day 17) by the method of example 15-1, in view of the difference in the number of initial cells as compared with comparative example 5. As shown in table 5, it was confirmed that the proliferation of examples 15-2 to 15-4, in which the immunocyte proliferation-activating kit 4 comprising prednisolone, the immunocyte proliferation-activating kit 5 comprising methylprednisolone, and the immunocyte proliferation-activating kit 6 comprising betamethasone sodium phosphate were each added, was also very good, unlike the immunocyte proliferation-activating kit 3 comprising dexamethasone.

4. Flow cytometry analysis

The flow cytometry analysis was performed on the cultured cells obtained in example 15-1 and comparative example 5 according to the flow cytometry analysis method described in the experimental method, and the results are shown in Table 6, FIG. 6a and FIG. 6 b.

TABLE 6

As shown in Table 6, FIG. 6a and FIG. 6b, it was found that the NK cell content and the activity of the proliferation medium of example 15-1 were higher than those of comparative example 5. On the other hand, it was found in example 15-1 that the effect was probably better if the steroid was also added after the medium was changed on day4 (day 4), but the effect was very good even when treated only once on day3 (day 3). Also, it is known to use activated lymphocytes obtained by culturing lymphocytes as effector cells, use a blood cancer cell line (K562) as target cells, and set the ratio of activated lymphocytes to cancer cells to 10:1, whereby when a titer analysis for measuring the killing ability of activated lymphocytes against a blood cancer cell line is performed, the higher the ratio of NK cells in activated lymphocytes, the higher the value thereof, and therefore, it can be predicted that the therapeutic effect of activated lymphocytes having a very high specific gravity of NK cells cultured by the present invention is excellent.

Example 16

PBMC from dogs (Canine) were prepared from blood collected from veins of dogs by the same method as in example 11, and then the immune cells were cultured using the immune cell proliferation activation kit 10 according to the culture conditions of Table 7 by the following method. The cells were counted during the period of culturing the immune cells by the above-described experimental method, and the results are shown in table 7 and fig. 7.

(1) Day0 (Day 0): the day of PBMC isolation from canine blood

Separation of lymphocytes from blood of healthy dogs yields 2.24X10 ⁷ Each cell was divided into two parts and each of the two T25 flasks was then filled with 4.5mL of third unit-3 and 0.5mL of plasma and each of the two parts was divided into an example flask (example 16) and a control flask (comparative example 6), and then placed in an incubator for culturing according to the conventional method described above.

(2) Day5 (Day 5)

In the example flask and the control flask, 4.5mL of the third unit-3 and 0.5mL of plasma were further added, and only the second unit-1 was added to the example flask so as to be contained in the medium at a concentration of 1. Mu.g/mL, and the culture was continued.

(3) Day6 (Day 6)

9mL of the basal medium solution and 1mL of plasma were added to each of the example flask and the control flask, and the mixture was transferred to a T75 flask and further cultured.

(4) Day9 (Day 9)

18mL of the basal medium solution and 2mL of plasma were added to the example flask and the control flask, and the mixture was transferred to a T150 flask and further cultured.

(5) Day12 (Day 12)

All the culture solutions including the cells under culture in the example flask and the control flask were each placed in a gas-permeable bag (air per meable bag) containing 100mL of the basal medium solution, and then cultured. In this case, the culture bag (bag) was clamped with a clamp at about 1:3 for use and the clamp was released appropriately as the number of days of culture increased.

(6) Day14 (Day 14)

The cultivation was continued after moving the clamp of the cultivation bag (bag) in cultivation to about 2 parts of 3.

(7) Day16 (Day 16)

The clamps of the culture bags (bag) in culture were completely released and the culture was continued.

(8) Day18 to Day21 (Day 18 to Day 21)

Cells were obtained by conventional methods.

TABLE 7

As shown in Table 7 and FIG. 7, it is understood from the results of the culture that the proliferation of example 16, in which the NK cell stimulating substance IL-15 contained in the third unit-3 was first treated and then the immune cells were stimulated with the anti-CD 3 antibody contained in the first unit-1, and then the steroid contained in the second unit-1 was added to the cell culture medium at day 5 for culture, was more superior to that of comparative example 6, in which the second unit-1 was not treated, and in particular, increased significantly from the time of 8 days later, at least by a factor of 2 or more. Although not specifically shown, it was confirmed that it was excellent to culture even when PBMC (or T cells) or anti-h-CD 3 (or ca-CD 3) of other dogs were used instead of the first unit-1 on day 1.

Experimental example 1

Results were obtained by the same method as in example 15-1 except that cells having an NK cell proportion of about 50% were used, the concentration of the steroid contained in the immune cell proliferation-activating kit was increased about 2-fold and added on days 3, 4, and 8 for culture, and the results were obtained by the same method as in comparative example 5 except that cells having an NK cell proportion of about 50% were used, and the two results were compared and are shown in fig. 8a and 8b, respectively.

Fig. 8a and 8b show that when the immune cell proliferation-activating kit of the present invention is used, the NK cell ratio is 81.35%, but when not used, the NK cell ratio is 51.76%, and therefore it was confirmed that even if the culture is started in a state where the NK cell ratio is high from the beginning, the proliferation rate of NK cells is further improved during the culture when the immune cell proliferation-activating kit of the present invention is treated.

Experimental example 2

To test the effect of the immune cell proliferation activation kit of the present invention on reactivation of cells with reduced activity, treg cells were suppressed as in example 15-1 using old cells with reduced activity cultured for 25 days, while reactivation was performed as follows. The supernatant was removed by centrifugation to remove the original medium, and then placed in a conventional medium (RPMI) for about 2 hours, then changed to a C1 solution and cultured for 3 days.

Thereafter, the activity of the reactivated NK cells was measured, and the results thereof are shown in Table 8.

As shown in table 8 below, an increase in activity was observed without Treg inhibition activity, and a dramatic decrease in cell number was not observed. However, a decrease in the degree to which the number of cells naturally decreases due to aging of cells is observed, which can also be regarded as a measurement error. This method is expected to activate NK cells in a short period of time (1 to 3 days), and is very effective for reactivation of NK cells having decreased activity due to thawing after freezing, as well as activating old cells and NK cells in PBMC isolated directly from peripheral blood.

TABLE 8

Experimental example 3

After NK cells were cultured by the same method as in example 15-1 except that PBMC were obtained from blood of a human which appeared to cause high activation of Treg cells due to high concentration of IL-10 in blood, cells were taken out and the original culture solution was removed on day 12, and then after 2 days of culture with C1 solution, a medium composition remaining after isolation of NK cells was obtained.

The components of the resulting culture medium composition were confirmed, and the results are shown in table 9.

TABLE 9

As shown in Table 9, when cultured for 2 days after stimulation with the C1 solution on day 12 of the culture, the cell number increased well and IFN-. Gamma.as well as IL-10 increased significantly with little IL-8 production. In contrast, it was found that when isolated directly without activation with C1 solution, a small amount of cytokine was increased overall. Thus, it was confirmed that when the immune cells of a human having a large number of Treg cells were stimulated with the C1 solution on day 12 of culture after treatment with the immune cell proliferation activation kit of the present invention, the cell number of immune cells did not increase in an anti-reflection manner and a large amount of IFN- γ and IL-10 were produced.

On the other hand, IL-8 is known to be an inflammatory-related cytokine (cytokine) that recruits neutrophils, and IL-10 induces macrophages into M2b type to exert an effect of eliminating inflammation, so that the resulting culture medium composition is effective in improving skin problems and treating skin diseases caused by inflammation, etc.

While the present invention has been illustrated and described by the above-described preferred embodiments, the present invention is not limited to the above-described embodiments, and various changes and modifications may be made by one of ordinary skill in the art to which the present invention pertains without departing from the spirit of the present invention.

Claims

1. An immune cell proliferation activation kit comprising:

a first unit comprising a T cell stimulating substance; and

a second unit comprising a steroid.

2. The kit for activating immune cell proliferation according to claim 1, wherein in the first unit, when the T-cell stimulating substance is an antibody, the antibody is contained in a basic medium solution at a concentration of 0.1 to 12. Mu.g/mL, and when the T-cell stimulating substance is a feeder cell, the feeder cell is a cellThe concentration of the antibody is at least one selected from the group consisting of an anti-CD 3 antibody, an anti-CD 4 antibody and an anti-CD 28 antibody, and the antibody is contained in the basal medium solution.

3. The immune cell proliferation-activating kit according to claim 1, wherein in the second unit, the steroid is contained in the basal medium solution at a concentration of 0.02 μg/mL or more.

4. The immune cell proliferation activating kit according to claim 3, wherein the steroid is a glucocorticoid and the steroid is at least one selected from the group consisting of cortisol, cortisone, prednisolone, prednisone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, fludrocortisone acetate, deoxycorticosterone acetate.

5. The immune cell proliferation activating kit according to claim 1, wherein the steroid acts to proliferate NK cells by inhibiting regulatory T cell activity.

6. The immune cell proliferation-activating kit according to claim 1,

also comprises: a third unit containing an NK cell stimulating substance,

in the third unit, the NK cell stimulating substance is contained in the basal medium solution at a concentration of 0.2ng/mL to 10. Mu.g/mL.

7. The kit for activating immune cell proliferation according to claim 6, wherein the NK cell stimulating substance is at least one selected from the group consisting of an anti-CD 16 antibody, an anti-CD 56 antibody, interleukin-12, interleukin-15 and interleukin-18.

8. An immune cell culture method, comprising:

a first culturing step of suspending peripheral blood mononuclear cells isolated from blood in a basal medium solution, and then treating a first unit containing a T cell stimulating substance and culturing for 18 to 72 hours;

a second culturing step of culturing for 18 to 25 hours after treating the first culture liquid subjected to the first culturing step with a second unit containing a steroid; and

and a third culturing step of culturing by adding one or more of the basal medium solution and the fetal bovine serum to the second culture medium subjected to the second culturing step at intervals of 24 hours or more from the predetermined time point, and transferring the culture medium to a larger culturing container for culturing as the number of cells increases.

9. The method according to claim 8, wherein in the first culturing step, a third unit containing an NK cell stimulating substance is treated before or simultaneously with the treatment of the first unit.

10. The method according to claim 9, wherein the NK cell stimulating substance is contained in the basal medium solution at a concentration of 0.2ng/mL to 10. Mu.g/mL in the third unit, and the NK cell stimulating substance is at least one selected from the group consisting of an anti-CD 16 antibody, an anti-CD 56 antibody, interleukin-12, interleukin-15 and interleukin-18.

11. The method for culturing immune cells according to claim 8,

in the first unit, when the T cell stimulating substance is an antibody, the antibody is contained in a basal medium solution at a concentration of 0.1 to 12. Mu.g/mL, and when the T cell stimulating substance is a feeder cell, the feeder cell is a cellThe concentration of at least one antibody is at least one selected from the group consisting of an anti-CD 3 antibody, an anti-CD 4 antibody and an anti-CD 28 antibody,

in the second unit, a steroid is contained in the basal medium solution at a concentration of 0.02 μg/mL or more, the steroid is a glucocorticoid, and the steroid is one or more selected from the group consisting of cortisol, cortisone, prednisolone, prednisone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, fludrocortisone acetate, deoxycorticosterone acetate.

12. The method according to claim 11, wherein the basal medium solution in which the peripheral blood mononuclear cells are suspended is treated with the first unit at any one of the time points of culture day 0, culture day 1, and culture day 2, and the second unit is treated at a time point of 20 hours to 25 hours after the first culture solution is treated with the first unit.

13. The method according to claim 12, wherein the cells are cultured in the second culture medium obtained in the second culturing step for 5 days and then treated with the second unit more than once.

14. A method of pet immune cell culture comprising:

a first culturing step of suspending peripheral blood mononuclear cells isolated from blood of a canine in a basal medium solution, and then simultaneously or sequentially treating a third unit containing an NK cell stimulating substance and a first unit containing a T cell stimulating substance and culturing for 4 days;

a second culturing step of culturing for 18 to 25 hours after simultaneously or sequentially treating the first culture solution having undergone the first culturing step with a second unit containing a steroid and the third unit; and

15. The method for culturing pet immune cells of claim 14,

in the second unit, a steroid is contained in the basal medium solution at a concentration of 0.02. Mu.g/mL or more, the steroid is a glucocorticoid, and the steroid is one or more selected from the group consisting of cortisol, cortisone, prednisolone, prednisone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, fludrocortisone acetate, deoxycorticosterone acetate,

in the third means, the NK cell stimulating substance is contained in the basal medium solution at a concentration of 0.2ng/mL to 10. Mu.g/mL, and the NK cell stimulating substance is at least one selected from the group consisting of an anti-CD 16 antibody, an anti-CD 56 antibody, interleukin-12, interleukin-15 and interleukin-18.