Patent Application
20240228958
The present disclosure relates to an immune cell culture method and, more particularly, an to immune cell proliferation/activation kit and an immune cell culture method using the proliferation/activation kit, in which the kit not only suppresses regulatory T cells (Treg) during immune cell culture to allow any one of natural killer cells (NK cells) and T cells from among immune cells to efficiently proliferate but also efficiently amplifies and activates immune cells for a long period of time.
NK cells are known to play an important role in the early biological defense mechanism and tumor immunity of the human body. In other words, NK cells can kill specific autologous cells, allogeneic cells, and even heterogeneous cancer cells without the process of acquiring immunity according to the expression of the major histocompatibility complex (MHC), and NK cells are particularly good at killing target cells that express fewer or no Class 1 MHCs. Therefore, NK cells can effectively kill most cancer cells that do not express MHCs, and can also kill some virus-infected cells and bacteria such as Salmonella typhi. However, NK cells, which have an excellent effect on killing cancer cells, make up only 5% to 15% of peripheral blood lymphocytes even in normal people, and the percentage drops to less than 1% in patients with severe cancer, so NK cells are limited in their ability to effectively attack cancer cells without further amplification through immunotherapy.
Immunotherapy is a method of extracting the most important immune cells for cancer treatment, such as natural killer cells (NK cells), dendritic cells (DCs), B cells, and T cells, from the patient's blood, cultivating them into immune cells that act strongly against cancer using various types of stimulating substance, and then injecting them back into the patient. Since immunotherapy uses the patient's own blood, immunotherapy has been actively studied in recent years because immunotherapy has fewer side effects and is easier to administer than conventional chemotherapy.
When culturing immune cells, especially when culturing of primarily NK cells is required, the role of T cells, especially help T cells, is necessary, and helper T cells (or Th cells) refer to cells that promote humoral and cellular immunity by regulating the differentiation and activation of white blood cells. They are also called CD4+ T cells because they have the CD4 protein on the cell surface. CD4+ T cells are further classified into Th1, Th2, Th17, and Treg according to their detailed functions. Th1 cells secrete interferon-gamma (IFN-γ) and tumor necrosis factor beta (TNF-β), which induce the fusion of endosomes and lysosomes inside macrophages to form endolysosomes. Meanwhile, Th2 cells secrete various types of interleukins (IL), which cause B cells to differentiate into plasma cells. Th17 cells secrete interleukin-17 (IL-17), which recruits neutrophils. Treg cells, also known as regulatory T cells, do not promote immune responses but rather suppress them, maintaining immune homeostasis and blocking autoimmune responses. Regulatory T cells (Tregs) are components of the immune system that suppress the immune response of other cells, an important “self-check” designed to prevent the immune system from overreacting. Regulatory T cells are involved in stopping the immune response after successfully killing an invading microorganism and are also involved in stopping autoimmune cell diseases. The immune system must be able to distinguish between self and non-self. When self/non-self distinction fails, the immune system destroys cells and tissues in the body, resulting in autoimmune disease. Regulatory T cells actively suppress the activity of the immune system and prevent pathological auto-reactions such as autoimmune diseases. The molecular mechanisms of how regulatory T cells exert their suppressive/regulatory activity are not yet fully understood, and the immunosuppressive cytokines TGF-beta and interleukin 10 (IL-10) have also been implicated in regulatory T cell function.
On the other hand, when different types of antibodies or cytokines are used to activate immune cells, such as regulatory T cells (Tregs) are also activated. However, Tregs take longer to activate than other cells. Therefore, it is not uncommon for cell counts to increase well in the early stages of cell culture, but when cells are cultured for more than about 8 to 9 days, cell counts are not increased as much as expected by activated Tregs. In particular, it is frequently found in cancer patients with low immunity and in patients with a large number of Treg cells and active Treg cells; this phenomenon is conspicuous because of the effect of Treg.
Therefore, there is a need to develop a technology capable of proliferating immune cells in large quantities by minimizing the effects of Tregs.
As a result of research efforts to solve the above problems, the present inventors have completed the present disclosure by developing a technology capable of inhibiting the activity of regulatory T cells (Treg) during immune cell culture.
Therefore, an objective of the present disclosure is to provide an immune cell proliferation activation kit and a method of immune cell culture using the proliferation activation kit, in which one or more immune cells, in particular NK cells and T cells, can be proliferated to a greater extent by inhibiting the activity of CD4+ T cells, in particular regulatory T cells (Treg), at a specific time in immune cell culture.
The objective of the present disclosure is not limited to the objective mentioned above, and even if not explicitly mentioned, an objective of the present disclosure that may be recognized by those skilled in the art from the detailed description of the present disclosure may be naturally included.
In order to achieve the objectives of the present disclosure as described above, the present disclosure provides an immune cell proliferation activation kit including a first unit including a T cell stimulating substance; and a second unit including a steroidal agent.
In a preferred embodiment, the first unit is formed such that the T cell stimulating substance is contained in the basic medium solution a concentration of 0.1 to 12 ug/mL when the T cell stimulating substance is an antibody and T cell stimulating substance is contained at a concentration of at least 1×105 cells/mL when the T cell stimulating substance is a feeder cell, in which the antibody is one or more selected from the group consisting of an anti-CD3 antibody, an anti-CD4 antibody, and an anti-CD28 antibody.
In a preferred embodiment, the second unit is formed by including the steroidal agent at a concentration of 0.02 μg/ml or more in a basic medium solution.
In a preferred embodiment, the steroidal agent is glucocorticoids, in which the steroidal agent is at least one selected from the group consisting of cortisol, cortisone, prednisolone, prednisone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, fludrocortisone acetate, and deoxycorticosterone acetate.
In a preferred embodiment, the steroidal agent acts to inhibit regulatory T cell (Treg) activity, thereby causing NK cell proliferation.
A preferred embodiment further includes a third unit, including an NK cell stimulating substance, in which the third unit is formed such that the NK cell stimulating substance is contained in the basic medium solution at a concentration in a range of 0.2 ng/ml to 10 μg/mL.
In a preferred embodiment, NK cells stimulating substance is at least one selected from the group consisting of an anti-CD16 antibody, anti-CD56 antibody, IL-12, IL-15, and IL-18.
In addition, the present disclosure provides a method of culturing immune cells, the method including: a first culturing step in which peripheral blood mononuclear cells (PBMC) isolated from blood are suspended in a basic medium solution and is then cultured for 18 to 72 hours while being treated with the first unit including a T cell stimulating substance; a second culturing step in which the first culture solution having undergone the first culturing step is treated with the second unit including a steroidal agent and incubated for 18 to 25 hours; and a third culturing step in which at least one of the basic medium solution and FBS (FBS, or autologous plasma, the same hereinafter) is added to the second culture solution having undergone the second culturing step, and the second culture solution is cultured at intervals of 24 hours or more from a certain point in time and transferred to a larger culture container as the number of cells increases.
In a preferred embodiment, in the first culturing step, before or simultaneously with the treatment of the first unit, a third unit including an NK cell stimulating substance is treated.
In a preferred embodiment, the third unit is formed such that the NK cell stimulating substance is contained at a concentration of 0.2 ng/ml to 10 μg/mL in a basic medium solution, and the NK cell stimulating substance is at least one selected from the group consisting of anti-CD16 antibodies, anti-CD56 antibodies, IL-12, IL-15, and IL-18.
In a preferred embodiment, the first unit is formed such that T cell stimulating substance is contained in the basic medium solution at a concentration of 0.1 to 12 ug/mL when the T cell stimulating substance is an antibody and T cell stimulating substance is contained at a concentration of at least 1×105 cells/mL when the T cell stimulating substance is a feeder cell, and the antibody is at least one selected from the group consisting of an anti-CD3 antibody, anti-CD4 antibody, and anti-CD28 antibody. The second unit is formed such that a steroidal agent is contained in the basic medium solution at a concentration of 0. 02 μg/ml or more, in which the steroidal agent is a glucocorticoid and is at least one selected from the group consisting of cortisol, cortisone, prednisolone, prednisone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, fludrocortisone acetate, and deoxycorticosterone acetate.
In a preferred embodiment, the first unit is used for the treatment of the PBMC suspended in the basic medium solution on the 0th day of culture, the 1st day of culture, or the 2nd day of culture, and the second unit is used for treatment 20 to 25 hours after the first culture solution is treated with the first unit.
In a preferred embodiment, the second unit is further treated once or more after 5 days of culture in the second culture solution in which the second culturing was performed.
In addition, the disclosure provides a method of culturing immune cells for pets, the method including: a first culturing step in which peripheral blood mononuclear cells (PBMCs) isolated from the blood of a dog are suspended in a basic medium solution and is then cultured for 4 days while sequentially or simultaneously being treated with a third unit including a NK cell stimulating substance and a first unit including a T cell stimulating substance; a second culturing step in which the first culture solution having undergone the first culturing step is simultaneously or sequentially treated with a second unit including a steroidal agent and the third unit and then cultured for 18 to 25 hours; and a third culturing step in which at least one of the basic medium solution and FBS (FBS) is added to the second culture solution having undergone the second culturing, the second culture solution is cultured at intervals of 24 hours or more from a certain point in time and transferred to a larger culture container as the number of cells increases.
In a preferred embodiment, the first unit is formed such that T cell stimulating substance is contained in the basic medium solution at a concentration of 0.1 to 12 ug/mL when the T cell stimulating substance is an antibody and T cell stimulating substance is contained at a concentration of at least 1×105 cells/mL when the T cell stimulating substance is a feeder cell, and the antibody is at least one selected from the group consisting of an anti-CD3 antibody, anti-CD4 antibody, and anti-CD28 antibody. The second unit is formed such that a steroidal agent is contained in the basic medium solution at a concentration of 0.02 μg/ml or more, in which the steroidal agent is a glucocorticoid and is at least one selected from the group consisting of cortisol, cortisone, prednisolone, prednisone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, fludrocortisone acetate, and deoxycorticosterone acetate. The third unit is formed such that the NK cell stimulating substance is contained in the basic medium solution at a concentration of 0.2 ng/ml to 10 μg/mL, in which the NK cell stimulating substance is one or more selected from the group consisting of an anti-CD16 antibody, an anti-CD56 antibody, IL-12, IL-15, and IL-18.
According to the immune cell culture method using the immune cell proliferation activation kit of the above-described disclosure, the immunosuppressive activity of CD4+ T cells, especially regulatory T cells (Treg), is suppressed by the proliferation activation kit at a certain time during immune cell culture, so that immune cells, especially one or more of NK cells and T cells, can be proliferated with high efficiency of at least two times or more compared to the known immune cell culture method, and also mass proliferation is possible.
These technical effects of the present disclosure are not limited to the above-mentioned scope and may be recognized by those skilled in the art from specific descriptions for implementing the present disclosure, even if not explicitly mentioned.
The terms used herein are used only to describe specific embodiments and are not intended to limit the present disclosure. As used herein, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well unless the context clearly indicates otherwise. In this specification, the term “include” or “have” should be understood to designate that one or more of the described features, numbers, steps, operations, components, or a combination thereof exist, and the possibility of addition of one or more other features or numbers, operations, components, or combinations thereof should not be excluded in advance.
Terms such as first, second, and the like may be used to describe various components, but the components should not be limited by such terms. The above terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present disclosure, a first component may be referred to as a second component, and similarly, the second component may also be referred to as a first component.
Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by those of ordinary skilled in the art to which this disclosure belongs. Terms such as those defined in a generally used dictionary should be interpreted as having meanings consistent with those of the context of related technologies and are not interpreted as ideal or excessively formal meanings unless clearly defined in the present disclosure.
As used herein, the term “immune cell” is used to include only NK cells and T cells. “NK cell” is used to mean both NK cells and NKT cells, but may also be used to mean only NK cells in some cases.
The term “feeder cell” used in the present disclosure is used as an allogeneic cell belonging to a cancer cell line.
As used in the present disclosure, the term “basic medium” refers to a medium including the most basic components of a medium mixed with the nutrients required by the cultured organism to cultivate microorganisms or animal or plant tissues, and commercialized basic media include Basal medium eagle's (BME), minimum essential medium (MEM), Dulbecco's modified eagle's medium (DMEM), and RPMI 440. As used in the present disclosure, “basic medium for culturing suspension cells” refers to a basic medium suitable for culturing suspension cells among the known basic media.
In the case of description of the time relationship, for example, when the time-post relationship is described as “after”, “and then”, “thereafter”, “before”, etc., it includes cases where “right” or “direct” is not continuous unless used.
Hereinafter, the technical configuration of the present disclosure will be described in detail with reference to the accompanying drawings and preferred embodiments.
However, the present disclosure is not limited to the embodiments described herein and may be embodied in other forms. Like reference numbers used to describe the disclosure throughout the specification refer to like elements.
The technical features of the present disclosure relate to a novel use of drugs, in particular steroidal agents, for suppressing regulatory T cell (Treg) activity, and in particular to an immune cell proliferation activation kit and a method of immune cell culture using the kit, in which a first unit including a T cell stimulating substance, a second unit including a steroidal agent, and, if necessary, a third unit including an NK cell stimulating substance, are used in a specific sequence and at a specific time in immune cell culture to suppress the immunosuppressive activity of CD4+ T cells, in particular regulatory T cells (Tregs), resulting in a greater and longer proliferation of one or more of NK cells and T cells.
In other words, when culturing immune cells, NK cells need to be stimulated not only at the beginning but also during the culture, while the stimulation of T cells is sufficient for the initial stimulation. Therefore, it is possible to activate more NK cells by stimulating NK cells first with an NK cell stimulating substance, i.e., third unit, and then stimulating T cells with a T cell stimulating substance, i.e., the first unit, so that the activated T cells and the NK cell stimulating substance together stimulate NK cells further, and if the immunosuppressive activity of CD4+ T cells, especially regulatory T cells (Treg), among the activated T cells can be suppressed, NK cells can be activated or proliferated more. This is because it proved the hypothesis that if the first and second units are used sequentially without using the third unit, T cells can be more efficiently proliferated and activated even if more T cells are to be activated or proliferated.
In other words, the present disclosure has developed a new use for steroidal drugs for inhibiting regulatory T cell (Treg) activity by demonstrating that steroidal agents, drugs for inhibiting regulatory T cell (Treg) activity, which is known to suppress CD4+ T cells and, in particular, Treg cells, can be utilized in immune cell cultures at certain stages of culture, immune cells can proliferate and activate more efficiently.
More specifically, steroidal agents are known to inhibit CD4+ T cells such as Th1, Th2, Th17, and Treg from removing inflammation and pain, but they have serious side effects such as immunosuppression when used for a long period of time. In immune cell culture, it was found that inhibition of Treg cells by steroidal agents after NK cell activation did not inhibit NK cell proliferation and activation and that a certain degree of overcrowding of immune cells in culture did not interfere with NK cell activation and cell proliferation. This is because it was found that when Treg cells are inhibited by steroidal agents without stimulating NK cells, steroidal agents proliferate and activate T cells rather than NK cells and that a certain degree of overcrowding of immune cells in culture does not interfere with T cell activation and cell proliferation.
Accordingly, the immune cell proliferation activation kit of the present disclosure includes a first unit including a T cell stimulating substance; and a second unit including a steroidal agent. If necessary, a third unit including a NK cell stimulating substance may be further included.
The first unit includes a T cell stimulating substance, which, when the T cell stimulating substance is an antibody, may be formed by causing the basic medium solution to include the antibody at a concentration of 0.1 to 12 ug/mL, or, when the T cell stimulating substance is a feeder cell, may be formed by causing the basic medium solution to contain at least 1×105 feeder cells/mL, and the antibody used as the T cell stimulating substance may be one or more selected from the group consisting of an anti-CD3 antibody, an anti-CD4 antibody, and an anti-CD28 antibody. In addition, feeder cells are not limited as long as they are of the same species as the immune cells to be cultured; basically, at least 5% of the number of immune cells to be cultured should be used as stimulatory cells, and in many cases up to 100%. Here, the basic medium solution is formed such that interleukin-2 (IL-2) is contained at a concentration of 300 to 4000 IU/mL in the basic medium for suspension cell culture, in which the basic medium for suspension cell culture is used for immune cell culture.
In general, in immune cell culture, NK cells are stimulated to a certain extent by stimulating and activating Th1 by IL-2 included in the basic medium solution, and in this state, if Th cells are stimulated by T cell stimulating substance such as anti-CD3 antibodies to stimulate NK cells and Tc cells by activated Th1 cells, NK cells, and Tc cells are activated, and NK cells are also proliferated to a certain extent. In particular, further stimulation with one or more NK cell stimulating substance selected from the group consisting of IL-12, IL-15, IL-18, anti-CD16 antibodies, and anti-CD56 antibodies included in the third unit prior to treatment with the first unit, as described herein, causes the NK cells to become more activated and grow efficiently.
The second unit includes a steroidal agent and may be formed such that the steroidal agent is contained at a concentration of 0.02 μg/ml or more in the basic medium solution. The steroidal agent included in the second unit may be of the glucocorticoid class, although any steroidal drug known in the art may be used. In one embodiment, the steroidal agent may be at least one selected from the group consisting of cortisol, cortisone, prednisolone, prednisone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, fludrocortisone acetate, and deoxycorticosterone acetate.
As mentioned above, if the first unit is processed in immune cell culture, i.e., if the immune cells are stimulated with T cell stimulating substance such as anti-CD3 antibodies, all T cells are stimulated, especially humoral immune cells such as Th2 and Treg, which are not conducive to NK cell activation and proliferation. In particular, since Treg cells interfere with the activation and proliferation of NK cells, T cells can be proliferated better than NK cells among immune cells if the second unit is treated with the first culture solution. NK cells can be proliferated better than T cells by neutralizing Treg cells by treating the third unit first and then treating the second unit in the first culture solution treated with the first unit.
Here, the steroidal agent included in the second unit acts on the proliferation of immune cells, especially NK cells, by inhibiting the immunosuppressive activity of CD4+ T cells, especially regulatory T cells (Treg), and experimentally, the steroidal agent has been shown to eliminate the immune response inhibitory activity of regulatory T cells, thereby inhibiting the growth of T cells in general to some extent.
In other words, it was confirmed that steroidal agents, which are drugs for suppressing the activity of regulatory T cells (Treg), do not affect the proliferation of cellular immune cells, such as once activated NK cells and Tc cells, at all, as shown in the experimental examples described later. This is because it was confirmed that the NK cell ratio increased as the culture time increased. In particular, when the concentration of the steroidal agent is increased, the proliferation rate of T cells is suppressed to some extent, and the NK cell ratio is relatively increased in proportion to the steroidal agent concentration.
When the content of the steroidal agent included in the second unit is less than 0.02 μg/ml, the properties of suppressing the immunosuppressive activity of CD4+ T cells, especially regulatory T cells (Treg), are not sufficiently exhibited, and even if the steroidal agents are treated at a high concentration, it was predicted that the effect on immune cell culture would not change significantly when the concentration exceeded 9 μg/ml. Experimentally, considering that even if the culture contains more than 20 μg/ml, there is no greater progress or adverse effect on the immune cell proliferation effect, the upper limit of the steroid content can be determined in consideration of economic feasibility. Therefore, the steroidal agents used as a drug for suppressing Treg activity in the second unit of the kit for activating immune cell proliferation of the present disclosure can obtain desired effects as long as it is included at a concentration of 0.02 μg/ml or more.
The third unit includes an NK cell stimulating substance, which may be formed such that the NK cell stimulating substance is contained in the basic medium solution at a concentration of 0.2 ng/ml to 10 μg/mL, in which the NK cell stimulating substance may be one or more selected from the group consisting of an anti-CD16 antibody, an anti-CD56 antibody, IL-12, IL-15, and IL-18. Here, the concentration of the NK cell stimulating substance included in the third unit varies depending on the type of NK cell stimulating substance, in which the third unit may be prepared by adding IL-12 and IL-18 to a basic medium solution and then dissolving the mixed solution, such that IL-12 is included at a concentration of 0.5-15 ng/ml and IL-18 is included at a concentration of 2-50 ng/ml, in a first embodiment, or by adding IL-15 alone to a basic medium solution and then dissolving the mixed solution, such that IL-15 is included at a concentration of 1-100 ng/ml, in a second embodiment. As another embodiment, the third embodiment, the third unit can be prepared to include at least one of an anti-CD16 antibody at a concentration of 0.1 to 10 g/mL and an anti-CD56 antibody at a concentration of 0.1 to 10 μg/mL in the basic medium solution. It will be appreciated that the first embodiment may further include one or more anti-CD16 antibody and an anti-CD56 antibody to implement the third unit if desired.
As described above, when Th1 is stimulated and activated by IL-2 in immune cell culture using the immune cell proliferation activation kit of the present disclosure, and NK cells are further stimulated by NK cell stimulating substance such as anti-CD16 antibody and anti-CD56 antibody, NK cells can be proliferated and activated more efficiently than T cells for long-term culture, and T cells can be proliferated and activated more efficiently than NK cells for long-term culture when the third unit is not used because the basic medium solution includes IL-2.
The immune cell proliferation activation kit of the present disclosure having such a configuration may include a first unit, a second unit, and/or a third unit, each individually packaged and having different contents, i.e., different components, to be added to the medium at each step of the immune cell culture method of the present disclosure described herein. In addition, since NK cells in immune cell culture require the help of stimulated T cells to be fully activated after initial stimulation, the proliferation activation kit of the present disclosure requires the first unit to be used and the second unit to be used after the T cells in the immune cell culture medium are stimulated. In this way, when the immune cell proliferation activation kit of the present disclosure is used sequentially in immune cell culture, that is, when the first unit is treated to stimulate T cells in the immune cell culture medium and the second unit is treated at the appropriate time, the proliferation rate of T cells among immune cells can be increased very effectively when the third unit is not used, and the proliferation rate of NK cells among immune cells can be increased when the third unit is used before the first unit is used.
In addition, the immune cell culture method of the present disclosure includes: a first culturing step in which peripheral blood mononuclear cells (PBMC) isolated from blood are suspended in a basic medium solution and is then cultured for 18 to 72 hours while being treated with a first unit including a T cell stimulating substance; a second culturing step in which the first culture solution having undergone the first culturing step is treated with a second unit including a steroidal agent and incubated for 18 to 25 hours; and a third culturing step in which at least one of the basic medium solution and FBS (FBS) is added to the second culture solution having undergone the second culturing step, the second culture solution is cultured at intervals of 18 to 25 hours and transferred to a larger culture container as the number of cells increases.
A third unit including an NK cell stimulating substance may be processed before or simultaneously as the first unit in the first culturing step. In other words, as described above, NK cells are stimulated to some extent by the inclusion of IL-2 in the basic medium solution, but when the first unit is treated and then the second unit is treated with one or more NK cell stimulating substance selected from the group consisting of an anti-CD16 antibody, an anti-CD56 antibody, IL-12, IL-15, and IL-18, the NK cells among the immune cells may grow better, so it may be desirable to treat the third unit before treating the first unit when it is desired to increase the proportion of NK cells among the immune cells.
The second culturing step is performed by culturing the second unit for a period of time after treating the first unit in the first culture solution having undergone the first culturing step, so that the second culturing step may be performed 18 to 48 hours after the treatment of the first unit.
In one embodiment, the second culturing can be performed by treating the second unit with the first culture solution formed by treating the first unit at any point during day 0, day 1, or day 2 of culture with the PBMC-suspended basic medium solution and culturing for 20 to 25 hours. In other words, on day 0 of immune cell culture, NK cells are stimulated to some extent by IL-2 included in the basic medium solution, and then on day 0, 1, or 2 of culture, T cells are stimulated with T cell stimulating substance such as an anti-CD3 antibody, anti-CD4 antibody, or anti-CD28 included in the first unit to activate Tc, NK cells, etc. Subsequent treatment with the second unit including a steroidal agent on any one of days 1 to 4 of the culture, inhibits the immunosuppressive activity of CD4+ T cells, in particular regulatory T cells (Treg), so that immune cells, including NK cells, proliferate and/or are activated because the suppressive activity of regulatory T cells (Treg) is prevented.
The third culturing is a process for harvesting immune cells by culturing immune cells in large quantities, and the second culture solution in which the second culturing was performed is cultured by adding one or more of the basic medium solution and fetal bovine serum (FBS) at intervals of 24 hours or more from a predetermined time and may be transferred to a larger culture container as the immune cells proliferate, and the number of cells increases while adding at intervals of 48 hours or more, as in the embodiment described below. If necessary, the second unit may be further processed in the third culturing, and as one embodiment, the second culture solution in which the second culturing has been performed may be further processed once or more after 5 days of culture. As is known, PBMCs proliferate well in the early stage of culture, that is, when the immune cells are young (early stage of culture), but after about 8 to 10 days of culture, the proliferation rate decreases, and they do not grow well, but in the present disclosure, the third stage of culture can be performed for as short as 11 days and as long as 21 days, so a large amount of NK cells can be harvested compared to the conventional immune cell culture method, which must be performed for as long as 8 days.
This prolongation of the third culturing is based on the experimental results of the present disclosure, which show that when immune cells are stimulated with the basic medium solution at the beginning of immune cell culture, i.e., the first culturing step, and when T cells are further stimulated with the first unit and then treated with the second unit including a steroidal agent to suppress Treg, immune cells are activated and proliferate relatively well by increasing the concentration of cytokines including IL-2 even after 8 to 10 days of culture. Considering the number of cells proliferated in the second culture solution in which the second culturing is performed, the process can be performed by adding one or more of the basic medium solutions and FBS in the required number and content at intervals of 18 to 25 hours from a predetermined time point, as in the embodiment described later. Here, the predetermined time point may be a time point after about 24 hours or more from the time point at which the second unit is added in the second culturing.
Next, the pet immune cell culture method of the present disclosure includes: a first culturing step in which peripheral blood mononuclear cells (PBMCs) isolated from the blood of a dog are suspended in a basic medium solution and is then cultured for 4 days while simultaneously or sequentially being treated with the first unit including a T cell stimulating substance and a third unit including a NK-cell stimulating substance; a second culturing step in which the first culture solution having undergone the first culturing step is treated with a second unit including a steroidal agent and the third unit simultaneously or sequentially and is then cultured for 18 to 25 hours; and a third culturing step in which at least one of the basic medium solution and FBS (FBS) is added to the second culture solution having undergone the second culturing step at intervals of 24 hours or more from a certain point in time and the second culture solution is transferred to a larger culture container as the number of cells increases.
The pet immune cell culture method of the present disclosure is a method for massively proliferating immune cells, including NK cells, from PBMCs of animals rather than PBMCs of humans and is broadly similar to the immune cell culture method described above in that the culturing utilizes the immune cell proliferation activation kit of the present disclosure. However, in order to more efficiently proliferate immune cells with PBMCs isolated from pets, especially dogs, rather than human blood, when performing the first culturing step to the third culturing step, it differs only in whether two or more units of the first unit or the third unit are used simultaneously and the time of processing, and the rest of the composition is the same, so a detailed description will be omitted.
However, it was experimentally confirmed that in the pet immune cell culture method, the effect is more excellent only when IL-15 is included in the third unit.
An immune cell proliferation activation kit 1 was prepared by preparing the first to third units as follows.
Basic medium for suspension cell culture (trade name: RPMI 1640) to which IL-2 was added to a concentration of 2000 IU/mL and then dissolved to prepare the basic medium solution.
First unit-1 was prepared by adding and dissolving anti-CD3 antibody to the basic medium solution to a concentration of 1 ug/mL.
Second unit-1 was prepared by dissolving dexamethasone sodium phosphate in a basic medium solution at a concentration of 0.02 ug/mL.
An immune cell proliferation activation kit 2 was prepared by performing the same method as in Example 1, except that in the preparation of the second unit, dexamethasone sodium phosphate was dissolved to include a concentration of 20 ug/mL in the basic medium solution to prepare the second unit-2.
An immune cell proliferation activation kit 3 was prepared by performing the same method as in Example 1, except that in the preparation of the second unit, dexamethasone sodium phosphate was dissolved to include a concentration of 1 ug/mL in the basic medium solution to prepare the second unit-3.
An immune cell proliferation activation kit 4 was prepared by performing the same method as in Example 1, except that in the preparation of the second unit, prednisolone (Sorondo, 5 mg/tablet, Yuhan Yanghaeng) was dissolved in the basic medium solution to a concentration of 167 ug/mL, and then diluted 20 times with the basic medium solution to a concentration of 8.3 ug/mL to prepare the second unit-4.
An immune cell proliferation activation kit 5 was prepared by performing the same method as in Example 1, except that in the preparation of the second unit, methylprednisolone (PD tablet, 4 mg/tablet, Jungwoo Pharmaceutical) was dissolved in the basic medium solution to a concentration of 80 ug/mL, and then diluted 10 times with the basic medium solution to a concentration of 8 μg/mL to prepare the second unit-5.
An immune cell proliferation activation kit 6 was prepared by performing the same method as in Example 1, except that in the preparation of the second unit, 1 uL of betamethasone sodium phosphate (Hanol Betamethasone Ju, 5.2 mg/1 mL ampule, Hanol Biopharma) was dissolved in 10 mL of the basic medium solution to have a concentration of 0.4 ug/mL to prepare the second unit-6.
An immune cell proliferation activation kit 7 was prepared by performing the same method as in Example 1, except that in the preparation of first unit, 5×106 K562 cell lines were dissolved per 1 ml of the basic medium solution instead of anti-CD3 antibody to prepare the first unit-2.
An immune cell proliferation activation kit 8 was prepared in the same manner as in Example 1, except that the third unit was further prepared as follows.
IL-12 and IL-18 were added to the basic medium solution to include IL-12 at a concentration of 10 ng/ml and IL-18 at a concentration of 50 ng/mL and then dissolved to prepare the third unit-1.
An immune cell proliferation activation kit 9 was prepared by performing the same method as in Example 7, except that in the preparation of the third unit, anti-CD16 and anti-CD56 were added to the basic medium solution to include a concentration of 1 μg/mL each, and then dissolved to prepare the third unit-2.
An immune cell proliferation activation kit 10 was prepared by performing the same method as in Example 7, except that in the preparation of the third unit, IL-15 was added to the basic medium solution to include IL-15 at a concentration of 20 ng/ml and then dissolved to prepare the third unit-3.
Cell counts were measured on specific culture days, with the first day PBMCs were isolated from blood and cultured being Day0 (1×107 cells) and cultured for a total of 14 or 19 days. Collect both cells and medium in a 500 ml conical tube and centrifuge at 1,800 rpm for 5 minutes at 4° C., then remove the supernatant. Cells were suspended by adding 10 ml of DPBS to the cell pellet, 10 μg of which was taken and diluted, mixed with an equal amount of 0.4% Trypan blue staining solution (Gibco, NY, USA), and placed on a C-Chip (iNCYTO, GA, USA) to count the number of cells under a culture microscope CKX53 (Olympus, Tokyo, Japan).
The cultured cells were centrifuged at 2,000 rpm for 3 min at 4° C. to remove the supernatant, resuspended in 200 μl of FCS buffer (eBioscienceTMFlow cytometry staining buffer, Invitrogen), and centrifuged at 2,000 rpm for 3 min at 4° C. The supernatant was removed, and 5 μl of each antibody (anti-human CD56-PE, anti-human CD16 PE-Cy-7, anti-human CD3-FITC) and isotype control antibody (Mouse IgG1 kappa isotype control-PE, Mouse IgG1 kappa isotype control-FITC, Mouse IgG1 kappa isotype control PE-Cy-7) were added and reacted under dark conditions and at 2° C. to 4° C. After 30 minutes, centrifugation was performed at 2,000 rpm at 4° C. for 3 minutes, and the supernatant was removed. After resuspension with 500 μl FCS buffer, centrifugation was performed at 2,000 rpm at 4° C. for 3 minutes, and the supernatant was removed. After washing the total cells twice, the cells were suspended in 500 μl of FCS buffer and analyzed by FACSCanto II (Becton Dickinson).
Immune cells were cultured using the immune cell proliferation activation kit 1 in the following manner. During immune cell culture, cells were counted, and flow cytometric analysis was performed using the experimental methods described above, and the results are shown in Table 1 and
PBMCs (1×107 cells) were suspended in 5 mL of basic medium solution and placed in T25 cell culture flasks (SPL, KOREA: example flask), the anti-CD3 antibody was added to a concentration of 1 ug/mL, and the cells were cultured for 24 hours at 37° C. and 5% CO2 culture conditions. In order to use as a control group, cells were cultured in the same conditions in another flask as in Comparative Example 1.
After adding the second unit-1 so that only the example flask contained 0.2 ug/mL of dexamethasone sodium phosphate, the culture was continued under the same conditions.
While adding 25 ml of basic medium solution and 2.5 ml of FBS (or autologous plasma) to both the example flask and the control flask, the flask was transferred to T75. Thereafter, as the number of cells increased under the conditions shown in Table 1 below, the cells were transferred to a large flask and cultured for a total of 2 weeks.
As shown in Table 1 and
Human PBMCs were prepared in the same manner as in Example 11 and immune cells were cultured using the immune cell proliferation activation kit 7 as described below. During immune cell culture, cells were counted, and flow cytometric analysis was performed using the experimental methods described above, and the results are shown in Table 2 and
PBMCs (1×107 cells) were suspended in 5 mL of basic medium solution and placed in T25 cell culture flasks (SPL, KOREA: example flask), and cultured the cells for 48 hours at 37° C. and 5% CO2 culture conditions. In order to use as a control group, cells were cultured in the same conditions in another flask as in Comparative Example 2.
Both the example flask and the control flask contained 5×106 of the feeder cell, K562 cells, and continued to be cultured under the same conditions after the addition of the first unit-2.
While adding 25 ml of the basic medium solution and 2.5 ml of FBS to both the example flask and the control flask, the flask was transferred to T75. In particular, the second unit-2 was added to the example flask to contain 20 ug/mL of dexamethasone sodium phosphate, unlike the control flask. Example flasks and control flasks were continuously cultured under the same conditions.
As the number of cells increased under the conditions shown in Table 2 below, they were transferred to a large flask and cultured for a total of 2 weeks.
As shown in Table 2 and
Human PBMCs were prepared in the same manner as in Example 11, and immune cells were cultured using the immune cell proliferation activation kit 8 as described below. During immune cell culture, cells were counted, and flow cytometric analysis was performed using the experimental methods described above, and the results are shown in Table 3 and
PBMCs (0.5×107 cells) were suspended in 5 mL of basic medium solution and placed in a T25 cell culture flask (SPL, KOREA: example flask), IL-12 was present at a concentration of 10 ng/ml and IL-18 was present at 50 ng/ml, and then the third unit-1 was added, and the cells were cultured for 24 hours at 37° C. under 5% CO2 culture conditions. In order to use as a control group, cells were cultured in the same conditions in another flask as in Comparative Example 3.
The cells were cultured for 24 hours at 37° C. under 5% CO2 culture conditions after the first unit-1 was added so that anti-CD3 antibodies were present in both the example flask and the control flask at a concentration of 1 ug/mL.
After adding the second unit-1 so that only the example flask contained 0.2 ug/mL of dexamethasone sodium phosphate, the culture was continued under the same conditions.
While adding 25 ml of basic medium solution and 2.5 ml of FBS to both the example flask and the control flask, the flask was transferred to T75. Thereafter, as the number of cells increased under the conditions shown in Table 3 below, the cells were transferred to a large flask and cultured for a total of 2 weeks.
As shown in Table 3 and
Furthermore, flow cytometric analysis of cultured immune cells showed that both Example 13 and Comparative Example 3 proliferated more NK cells than T cells as a result of stimulation of NK cells by treatment with the third unit before treatment with the first unit, as shown in
Human PBMCs were prepared in the same manner as in Example 11, and immune cells were cultured using the immune cell proliferation activation kit 9 as described below. During immune cell culture, cells were counted, and flow cytometric analysis was performed using the experimental methods described above, and the results are shown in Table 4 and
PBMCs (0.5×107 cells) were suspended in 5 mL of basic medium and placed in T25 cell culture flasks (SPL, KOREA: example flasks), anti-CD16 and anti-CD56 antibodies were added at a concentration of 1 μg/mL each, and the cells were cultured for 24 hours at 37° C. under 5% CO2 culture conditions. In order to use as a control group, cells were cultured in the same conditions in another flask as in Comparative Example 4.
The cells were cultured for 24 hours at 37° C. under 5% CO2 culture conditions after the first unit-1 was added so that anti-CD3 antibodies were present in both the example flask and the control flask at a concentration of 1 ug/mL.
After adding the second unit-2 so that only the example flask contained 20 ug/mL of dexamethasone sodium phosphate, the culture was continued under the same conditions.
While adding 25 ml of basic medium solution and 2.5 ml of FBS to both the example flask and the control flask, the flask was transferred to T75. Thereafter, as the number of cells increased under the conditions shown in Table 4, the cells were transferred to a large flask and cultured for a total of 2 weeks.
As shown in Table 4 and
Furthermore, flow cytometric analysis of cultured immune cells showed that both Example 14 and Comparative Example 4 proliferated more NK cells than T cells as a result of stimulation of NK cells by treatment with the third unit before treatment with the first unit, as shown in
The immune cell culture process was performed as follows, using not the basic medium solution but the medium addition kit for immune cell culture (NKTM) disclosed in the related patent and the immune cell proliferation activation kits 3 to 6 of the present disclosure.
In the other hand, a medium addition kit for immune cell culture (NKTM) is composed of B unit (basic solution, B solution), C1-1 unit (cytokine 1-1 solution), C1-2 unit, C2 unit, A1 unit, A2 unit, and D unit, each individually packaged and including different components to be added to the medium at each stage of immune cell culture. Since a detailed description of each unit and a detailed description of the specific conditions for the culture process of culturing immune cells using the unit (NKTM culture process) are disclosed in Patent Publication No. 10-2018-0057359 (hereinafter referred to as the “prior patent”), only on the parts that differ from the parts described in the prior patent will be focused. However, since the components included in the first unit and third unit including the immune cell proliferation activation kit of the present disclosure are included in each unit including the NKTM, the test was performed using only the second unit-3 to second unit-6 without using the first unit and third unit separately.
The basic solution (B-solution) was prepared by dissolving 2.2 mg of IL-2 and 100 mL of 500 mM L-glutamine solution in the basic medium for suspension cell culture (when IL-2 or L-glutamine is already present in the basic medium, adjust the amount of addition to achieve the final concentration) to make a final 10 L.
A cytokine solution (C solution) was prepared by dissolving 20 ug of IL-12 and 125 ug of IL-18 in distilled water to make 10 mL. Cytokine 1-1 solution (C1-1 solution) was prepared by dissolving 1 mL of C solution in 1000 mL of basic solution (B1 solution).
The lymphocyte extraction and autologous plasma preparation steps were performed as in the prior patent and then cultured using the same method as described in Example 3 of the prior patent, except that some conditions were different, as described below.
Day 0: Culture was started by dissolving 1.0×107 of PBMCs isolated from the blood into C1-1 solution. That is, the isolated PBMC was treated with 1 vial (4.5 mL) of C1-1 solution and 0.5 mL of serum, put into a T25 flask, and cultured in a CO2 incubator. Since the C1-1 solution contains the components included in the third unit, it is the same as the third unit treated.
If necessary, dissolve the isolated immune cells on Day 15 into 100 ml of B solution. After picking up half of the 1 L culture bag containing the cell culture medium with forceps, add the culture medium containing the cells to this bag and add 50 mL of cytokine1-2 solution of C1-2 units to the resultant and place the resultant culture medium in a 37° C., 5% CO2 incubator for further incubation as follows.
The process described above for Days 15 through 18 can be repeated one or two more times, depending on the condition of the cells, to obtain a total of 4 liters or more of culture in a single batch.
To determine the effect of the immune cell proliferation kits 3 to 6 of the present disclosure, which includes at least one of the 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: 1×107 cells) and
As can be seen from Table 5 and
Flow cytometric analysis was performed on the cultured cells obtained in Example 15-1 and Comparative Example 5 according to the flow cytometry method described in experimental methods, and the results are shown in Table 6,
As can be seen from Table 6,
Canine PBMCs were prepared by performing the same method as in Example 11 from blood collected from a vein of a canine, and then immune cells were cultured using the immune cell activation kit 10 according to the culture proliferation conditions in Table 7 as described below. In addition, while culturing the immune cells, the number of cells was counted using the experimental method described above, and the results are shown in Table 7 and
Lymphocytes were isolated from the blood of a healthy dog to obtain 2.24×107 cells, which were divided into two, put into two T25 flasks, and further added 4.5 mL of the third unit-3 and 0.5 mL of plasma was to form an example flask (Example 16) and a control flask (Comparison 6), respectively, and incubated in an incubator in the conventional manner described above.
4.5 mL of the third unit-3 and 0.5 mL of plasma were further added to both the example flask and the control flask, and the second unit-1 was added only to the example flask at a concentration of 1 ug/mL in the medium, and continued culturing.
9 mL of basic medium solution and 1 mL of plasma were added to both the example flask and the control flask and transferred to a T75 flask and continued culturing.
After adding 18 mL of basic medium solution and 2 mL of plasma to both the example flask and the control flask, transferred to a T150 flask, and continued culturing.
Put all the culture mediums containing the cells being cultured in the example flask and the control flask into an air permeable bag containing 100 mL basic medium solution, respectively, and continue culturing. At this time, the bag is used by picking up about ⅓ of the bag with forceps, and as the culture days pass, the forceps should be released appropriately.
Move the forceps of the bag being cultured to about two-thirds of the way and continue culturing.
Release the forceps of the bag in culture and continue culturing.
Cells are harvested in a conventional manner.
As shown in Table 7 and
The results were obtained by performing the same method as Example 15-1, except that cells with a ratio of about 50% NK cells were used, and the concentration of the steroidal agent included in the immune cell proliferation activation kit was increased by about two times and added on day 3, day 4, and day 8. The results obtained by performing the same method as Comparative Example 5, except that cells with a ratio of about 50% NK cells were used, are compared, and the results are shown in
To test the effect of the immune cell proliferation activation kit of the present disclosure on the reactivation of cells with decreased vitality, old cells that had been cultured for 25 days and had decreased in vitality while inhibiting Treg cells according to the method of Example 15-1 were used for reactivation as follows. The supernatant was removed by centrifugation to remove the old medium and placed in a regular medium (RPMI) for culturing for about 2 hours, after which the medium was replaced with C1 solution and cultured for 3 days.
Then, the activity of the reactivated NK cells was measured, and the results are shown in Table 8.
As shown in Table 8 below, an uninhibited increase in activity was observed by Tregs, and no dramatic decrease in cell number was observed. However, a slight decrease in the number of cells, which can be thought of as a measurement error, was observed to the extent that cells naturally decrease due to aging. This method can activate NK cells in a very short period of time (1 to 3 days) and not only activates old cells but also in PBMCs immediately isolated from peripheral blood, as well as for reactivation of NK cells that have been frozen and thawed and have lost their vitality.
Experimental Example 3, NK cells were cultured in the same manner as in Example 15-1, except that PBMCs were obtained from a person who had a high concentration of IL-10 in his blood, indicating that Treg cells were likely to be highly activated. The cells were then taken out on day 12, the existing culture medium was removed, and the cells were cultured for 2 days using C1 solution, after which the NK cells were isolated, and the remaining culture medium composition was obtained.
The components of the obtained culture medium composition were confirmed, and the results are shown in Table 9.
Table 9 shows that when cultured for 2 days after stimulation with C1 solution on day 12 of culture, the number of cells increased well, and IFN-γ and IL-10 increased significantly, as well as only a very small amount of IL-8 was produced. On the other hand, it can be seen that the total cytokine increases less when it is separated without activating with the C1 solution. As such, even if Treg cells are human immune cells that have developed, it can be seen that the number of cells does not decrease but increases, and a large amount of IFN-γ and IL-10 is produced when stimulated with C1 solution on the 12th day of culture after treatment with the immune cell proliferation activation kit of the present disclosure.
On the other hand, IL-8 is a cytokine that recruits neutrophils and is often associated with inflammation, and IL-10 is known to induce macrophages into M2b type and play a role in removing inflammation, so the culture medium composition obtained is effective in improving skin problems as well as treating skin diseases caused by inflammation.
Although the present disclosure is described with reference to the preferred embodiment as described above, the present disclosure is not limited thereto, and various changes and modifications may be made by those skilled in the art without departing from the spirit of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0118665 | Sep 2020 | KR | national |
| 10-2021-0099651 | Jul 2021 | KR | national |
This application claims benefit under 35 U.S.C. 119, 120, 121, or 365 (c), and is a National Stage entry from International Application No. PCT/KR2021/012211, filed Sep. 8, 2021, which claims priority to the benefit of Korean Patent Application Nos. 10-2020-0118665 filed on Sep. 15, 2020 and 10-2021-0099651 filed on Jul. 29, 2021 in the Korean Intellectual Property Office, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/012211 | 9/8/2021 | WO |
