KR20180032366A - A medium composition for lymphocytes activating culture and the culture method using the same - Google Patents

Abstract

The present invention relates to a medium composition for lymphocyte activation culture comprising a cell culture medium, an anti-CD28 monoclonal antibody, an anti-CD56 monoclonal antibody, interleukin-2 (IL-2), aminoguanidine and self-derived plasma; and to a lymphocyte activation culture method using the same. An objective of the present invention is to provide a culture medium composition for mass-culturing activated lymphocytes having an anti-cancer high-efficiency effect on various cancers.

Description

[0001] The present invention relates to a culture medium for activating lymphocytes and a method for activating lymphocytes using the culture medium,

More particularly, the present invention relates to a culture medium for cell culture, an anti-CD28 monoclonal antibody, an anti-CD56 monoclonal antibody, interleukin-2, amino Guanidine, and autologous plasma, and a method for activating lymphocytes using the same.

A variety of methods have been developed for the treatment of cancer and malignant tumors, and the main treatment consists of cancer resection through conventional surgery, chemotherapy, and physical therapy using radiation. However, surgery can not be performed if the patient's condition is poor, or brain tumor can not be operated on, and other chemotherapy may cause severe side effects after treatment and may lead to recurrence of cancer. exist. Due to these risks and limitations, other treatment methods are required.

The fourth chemotherapeutic method is to extract the self-derived activated immune cells (natural killer cells (NK cells), T cells, etc.) from the blood of cancer patients and then to use various kinds of activators to detect and remove cancer or tumor cells , Which is a self-induced disease, has been studied extensively because of its low incidence of adverse effects, and it has been studied extensively because of its ease of use. Especially, when it is applied to treatment by chemotherapy or radiation therapy, The effect is excellent and active research is proceeding.

Korean Patent No. 10-0735081 discloses a conventional method of activating immune cells (CD4 T cells). However, since the CD4 T cell activation method selectively activates only the T cells involved in acquired immunity among the immune cells, when it is applied to the tumor treatment, the cancer cell that has been previously memorized can be effectively attacked by the massively activated T cells It is possible to kill, but there is a problem that there is a limit to the elimination of the memorized heterogeneous cancer cells. In addition, there are many difficulties in coping with the emergence of new cancer completely different from the previous one.

The conditions of the improved therapy are the cultivation of immune cells capable of recognizing and eliminating various and highly heterogeneous cancer cells, the cultivation and activation of immune cells in a stable ratio. Among them, NK cells are one of the typical innate immune cells in the human immune system. They are known to have the ability to kill cancer nonspecifically and have the ability to recognize and kill viruses, bacteria, and the like. In particular, the killing ability of NK cells in cancer patients has been reported to be closely related to the incidence of diseases such as lung cancer, liver cancer, breast cancer, uterine cancer and blood cancer. In particular, it is reported that NKT cells, one of the innate immune cells, are involved in the treatment of various diseases and autoimmune diseases, leading to a healthy life.

Currently, studies on activation and mass proliferation of NK cells, NKT cells and T cells are under way, but they are expensive to culture, difficult to mass-proliferate, and have problems in stabilizing the activation method, It is difficult to do.

Therefore, it is necessary to develop a method for stably mass-culturing high-efficiency cells in a short time at a low cost.

Korea Patent No. 10-0735081

It is an object of the present invention to provide a culture medium for mass-culturing an activated lymphocyte having an anti-cancer high-efficiency effect on various cancers.

It is another object of the present invention to provide a lymphocyte activation culture method comprising the steps of extracting lymphocytes from human peripheral blood, freezing and thawing the lymphocyte, and then culturing the lymphocyte.

In order to achieve the above object,

The present invention relates to a culture medium for mass-culturing an activated lymphocyte containing a cell culture medium and an additive added to the cell culture medium,

The additive provides a medium composition for lymphocyte activation culture comprising an anti-CD28 monoclonal antibody, an anti-CD56 monoclonal antibody, interleukin-2, aminoguanidine and an autologous plasma.

(1) extracting lymphocytes from isolated human peripheral blood;

(2) freezing the extracted lymphocytes at a temperature of -100 to -150 캜;

(3) storing and thawing the frozen lymphocytes at a temperature of -150 to -200 ° C for 1 to 24 hours;

(4) a first culturing step of culturing the thawed lymphocytes in a culture medium for cell culture and a culture medium for self-activating lymphocyte comprising an additive;

Here, the cell culture medium includes interleukin-2 and L-glutamine,

Said additive comprising interleukin-2, an anti-CD56 monoclonal antibody, aminoguanidine and an autologous plasma; And

(5) a second culturing step of culturing the cultured mixed solution in the first culturing step in a cell culture medium and a medium for lymphocyte activation culture comprising an additive;

Here, the cell culture medium includes interleukin-2 and L-glutamine,

Wherein the additive comprises an anti-CD25 monoclonal antibody, an anti-CD226 monoclonal antibody, interleukin-15, and autologous plasma.

The present invention also provides a composition for treating or preventing cancer comprising an activated lymphocyte cultured by the above culture method.

The culture medium and the culture method for mass-culturing the activated lymphocytes of the present invention can stably activate lymphocytes such as NK cells, NKT cells and T cells.

In addition, the cultured activated lymphocytes are capable of removing various cancer cells, have little side effects during chemotherapy, and can be used in combination with conventional chemotherapy, so that an elevated anticancer effect can be expected. Thus, cancer patients can be effectively treated have.

1 is a distribution diagram of NK cells, NKT cells and T cells obtained by the culture method of Example 1. Fig.
2 is a distribution diagram of NK cells, NKT cells and T cells obtained by the culture method of Comparative Example 1. Fig.
FIG. 3 is a graph showing cytotoxicity against K562 blood cancer cells according to the activation time of lymphocytes obtained by the culture method of Example 1 and Comparative Example 1. FIG.
4 is a graph showing cytotoxicity of lymphocytes obtained by the culture methods of Example 1, Comparative Example 2 and Comparative Example 3 to various cancer cells.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a culture medium for mass-culturing an activated lymphocyte containing a cell culture medium and an additive added to the cell culture medium,

The additive relates to a medium composition for lymphocyte activation culture comprising an anti-CD28 monoclonal antibody, an anti-CD56 monoclonal antibody, interleukin-2, aminoguanidine and an autologous plasma.

The term "medium" used in the present invention refers to a cell such as adult stem cells in vitro which contains essential elements for growth and proliferation of cells such as sugar, amino acid, various nutrients, serum, For growth and proliferation. In particular, the medium of the present invention is a medium for culturing lymphocyte activation.

The above-mentioned "lymphocyte activation" means that the lymphocyte is present in an inactive state in the absence of a heterogeneous antigen in the blood, and activates the cell circuit when the antigen is introduced or newly emerged. It means to change.

The term "cultivation" as used above includes activation and proliferation of lymphocytes.

A lymphocyte is a type of leukocyte in which hematopoietic stem cells, ancestral cells, are differentiated and matured into lymphocytic hematopoietic stem cells through hematopoiesis, a process of making blood. These mature lymphocytes can be largely divided into B cells, T cells and NK cells (natural killer cells) and play a pivotal role in the immune response in the body. Lymphocytes can be found in bone marrow, lymph nodes, lymphoid tissue, and peripheral blood. Lymphocytes are usually made from yolk sacs (yolk sacs), liver and spleen (spleen) during birth, but they are produced in the bone marrow and thymus (thymus), the primary lymphogenic organ, after birth. Subsequently, mature lymphocytes are distributed in the form of circulating lymphocytes of peripheral blood and lymphatic tissues such as lymph nodes, spleen, digestive tract and respiratory tract called secondary or reactive lymphoid organs.

The activated lymphocyte of the present invention includes NK cells (natural killer cells), T cells (T cells) and NKT (natural killer T cell) cells.

The NK cell (natural killer cell) is a cell which is present in 10-20% of the peripheral blood lymphocytes, and is an important cell responsible for innate immunity. There are about 100 million NK cells in the body, and unlike T cells, they mature in the liver and bone marrow. It is known to attack virus-infected cells or tumor cells. In this method, if abnormal cells are first recognized, perforin is sprayed on the cell membrane to dissolve the cell membrane to puncture the cell membrane, and the granzyme is sprayed into the cell membrane to disrupt the cytoplasm to cause apoptosis (cell apoptosis) It is a way to inject necrosis (necrosis) by injecting saline. Many cancer patients are found to have defects in the number of NK cells or in their anticancer activities, and the abnormalities of NK cells are known to be closely related to the occurrence of these cancers. NK cells also play a key role in regulating inflammation and immune responses through the production of cytokines such as IFN-γ (interferon-γ) and TNF-α (tumor necrosis factor-α). Many studies have shown that NK cells play a key role in the removal of cancer cells and virus-infected cells, and they also play an important role in bone marrow transplantation, embryo implantation and reproduction regulation. In addition, NK cells play a key role in regulating immune responses through direct interaction with dendritic cells, macrophages, T cells and indirect cytokines. It has been found that NK cells play an important role in the development of inflammatory diseases, autoimmune diseases and various intractable diseases such as asthma.

The T cell (T cell) refers to a cell having a T cell antigen receptor (TCR) on the cell surface. TCR forms a heterodimer with the CD3 antigen, which is a duplex of a-chain and -chain. Some of the T cells (around 5% of peripheral blood T cells) are not α, β, but a dimer of γ and δ chains. TCR complexes with the CD3 antigen (γ, δ, ε, ζ, η), and the CD3 antigen is responsible for transferring the signal into the cell when the TCR recognizes the antigen.

The NKT (natural killer T cell) cell belongs to the innate immune system and is known as a kind of NK cell or T cell. NKT cells have not been known in the world for less than 10 years. The NKT cells express T cell receptors and NK cell specific surface markers. T cells expressing NK cell markers are often collectively referred to as NKT cells. NKT cells are characterized by the activation of interleukin-4, interleukin-10, interleukin-13, interferon-γ, and TNF-a tumor necrosis factor -α), and other cytokines in a very short time. This feature implies that NKT cells can have a large impact on the adaptive immune response. Overall, it is very similar to NKT cells, and both NK cells and NKT cells have NK cell receptors, which are larger in size than other T cells. However, NKT cells are mature in the thymus and NK cells in the liver and bone marrow, and NKT cells are T cells, which express T cell receptor (TCR), but NK cells lack TCR.

The culture medium for cell culture is a mixture containing essential saccharide, amino acid, water, etc. required for cell survival, and is a mixture excluding serum, nutrients and various self-activating lymphocyte growth factors. The culture medium for cell culture of the present invention may be artificially synthesized and used, or a commercially produced medium may be used. Commercially produced media include, for example, RPMI 1640, KBM 540, but are not limited thereto.

In the present invention, the cell culture medium includes L-glutamine and interleukin-2.

The L-glutamine is contained in an amount of 1 to 5 mM, and activation and proliferation of the lymphocyte occurs within the above range at a high rate.

In addition, the amount of interleukin-2 is included in the range of 100 to 3000 IU / mL. When the amount of interleukin-2 is less than 100 IU / mL, the lymphocyte cell cycling rate is slowed, and when it exceeds 3000 IU / mL, the content of NK cells and NKT cells is decreased.

In the present invention, the lymphocyte is firstly cultured in a medium composition comprising an anti-CD28 monoclonal antibody, an interleukin-2, an anti-CD56 monoclonal antibody, an aminoguanidine and an additive containing autologous plasma and a medium composition for cell culture.

After the primary culture, the cultured mixed liquor is secondarily cultured in a cell culture medium and a self-activating lymphocyte medium composition containing an additive added to the cell culture medium, wherein the additive is an anti-CD25 monoclonal antibody, an anti-CD226 single antibody Antibodies, interleukin-15 and autologous plasma. The self-activating lymphocyte can be mass-cultured through the secondary culture.

The lymphocytes used in the primary culture are frozen at a temperature of -100 to -150 캜, and then stored at -150 to -200 캜 for 1 to 24 hours and thawed.

When the lymphocytes are frozen, the function of the cells deteriorates and the inherent functions of the cells are lost. It is effective in restoring the deteriorated growth state stored in these cells to the original state, and the cells are more actively activated and proliferated after thawing .

Therefore, when the lymphocyte is frozen and thawed and cultured, a large amount of self-activating lymphocyte can be proliferated. In the present invention, the proliferation can be about 200 times or more.

The anti-CD28 monoclonal antibody is a co-stimulatory protein required for the survival and activation of T cells. Stimulation of T cells with the anti-CD28 monoclonal antibody produces a variety of cytokines. The anti-CD28 monoclonal antibody is solidified at 25 to 45 캜 for 1 to 48 hours, and is included at 1 to 50 μg, preferably at 5 to 30 μg.

The solidification of the anti-CD28 monoclonal antibody can be accomplished by conventional methods used in the art. In the present invention, the anti-CD28 monoclonal antibody is solidified using a method of adhering to the bottom of the culture container.

Although the kind of the anti-CD28 monoclonal antibody is not particularly limited, a product (product name: Purified NA / LE Mouse Anti-Human CD28, product number: 555725) of BD Bioscience Inc. was used in the present invention.

The interleukin-2 is a glycoprotein having a molecular weight of 14 to 17 kDa which is produced when a T cell recognizes and activates an antigen. The interleukin-2 is secreted outside the T cell, and the T cell producing the interleukin- It reacts with itself and promotes the growth of the T cell. Interleukin-2 acts on NK cells to promote growth, enhances the killing capacity of NK cells, and acts on B cells to promote its growth. In the present invention, interleukin-2 is added to a culture solution to activate killer T cells, NK cells, and NKT cells.

The interleukin-2 is contained in an amount of 50 to 100 μL of 17 × 10 ⁶ to 19 × 10 ⁶ IU / mL based on 20 mL of the cell culture medium.

The anti-CD56 monoclonal antibody is a glycoprotein having a molecular weight of 180 to 200 kDa and is expressed in all of NK cells and a part of T cells. The CD56 antigen is the same molecule as NCAM-1 (neural cell adhesion molecule-1), which is widely distributed in human nerve tissue. NCAM-1 is known as a molecule involved in intercellular adhesion in nerves or tissues. Therefore, it can be concluded that the anti-CD56 monoclonal antibody expressed in NK cells also functions as intercellular adhesion.

Although the kind of the anti-CD56 monoclonal antibody is not particularly limited, a product (product name: Purified NA / LE Mouse Anti-Human CD56, product number: 555513) of BD Bioscience Inc. was used in the present invention.

The anti-CD56 monoclonal antibody is contained in an amount of 2 to 20 μg at a concentration of 0.1 to 2 mg / mL based on 20 mL of the cell culture medium.

The aminoguanidine acts to inhibit or prevent the cross-linking and complications of the product of the condensation reaction and the binding protein and the sugar. The aminoguanidine is contained in an amount of 2 to 20 μg at a concentration of 0.1 to 2 mg / mL based on 20 mL of the cell culture medium.

In addition, the self-derived plasma is required for culturing and is contained in an amount of 0.1 to 15% by volume based on 20 ml of the cell culture medium.

The self-derived plasma refers to a supernatant centrifuged after artificially coagulated by adding calcium ion (Ca ^{2 +} ) or the like to plasma, and since it does not undergo a blood clotting reaction depending on platelets unlike normal serum, It does not contain the substance of the origin, and contains the nutrients from which erythrocytes have been removed.

The anti-CD25 monoclonal antibody is an antibody to CD25 which is an alpha chain of interleukin-2 receptor on T cells, which is used as a therapeutic agent for rejection of xenograft transplantation. The CD25 monoclonal antibody is contained at a concentration of 0.1 to 2 mg / mL in an amount of 50 to 100 μg based on 500 mL of the cell culture medium.

Although the kind of the anti-CD25 monoclonal antibody is not particularly limited, a product (product name: Purified NA / LE Mouse Anti-Human CD25, product number: 555429) of BD Bioscience Inc. was used in the present invention.

The CD226 antigen is expressed on the surface of NK cells, platelets, and monocytes and regulates the function of adhering to other cells. The CD226 monoclonal antibody is contained at a concentration of 0.1 to 2 mg / mL in an amount of 50 to 100 μg based on 500 mL of the cell culture medium.

Although the kind of the anti-CD226 monoclonal antibody is not particularly limited, a product of BD Bioscience (product name: Purified NA / LE Mouse Anti-Human CD226, product number: 559786) was used in the present invention.

The interleukin-15 promotes the proliferation of cytotoxic T cells, the activation of specific cytotoxic T cells and the nonspecific NK cells of the antigen. The interleukin-15 is contained in an amount of 50 to 100 μL at a concentration of 50 to 150 μg / mL based on 500 mL of the cell culture medium.

In addition, the self-derived plasma is required for culturing and is contained in an amount of 0.1 to 8% by volume based on 20 ml of the cell culture medium.

In addition,

(1) extracting lymphocytes from isolated human peripheral blood;

(2) freezing the extracted lymphocytes at a temperature of -100 to -150 캜;

(3) storing and thawing the frozen lymphocytes at a temperature of -150 to -200 ° C for 1 to 24 hours;

(4) a first culturing step of culturing the thawed lymphocytes in a culture medium for cell culture and a culture medium for self-activating lymphocyte comprising an additive;

Here, the cell culture medium includes interleukin-2 and L-glutamine,

Said additive comprising interleukin-2, an anti-CD56 monoclonal antibody, aminoguanidine and an autologous plasma; And

(5) a second culturing step of culturing the cultured mixed solution in the first culturing step in a cell culture medium and a medium for lymphocyte activation culture comprising an additive;

Here, the cell culture medium includes interleukin-2 and L-glutamine,

Wherein the additive comprises an anti-CD25 monoclonal antibody, an anti-CD226 monoclonal antibody, interleukin-15 and an autologous plasma.

Hereinafter, each step will be described in more detail.

The step (1) is a step of extracting lymphocytes from the separated human peripheral blood.

The lymphocyte is extracted from a peripheral blood of a human, which is extracted by a method known in the art, and the method is not particularly limited in the present invention.

Preferably, however, lymphocytes can be extracted using the point that the specific gravity of mononuclear cells such as human lymphocytes or mononuclear cells is lower than 1.077. The blood of the patient to be treated was superimposed on a Ficoll-Paque Plus solution having a specific gravity of 1.077 and centrifuged at a constant centrifugal force. By the specific gravity difference, red blood cells and granulocyte layers having specific gravity larger than 1.077 were downward, Can be separated to obtain a mononuclear cell layer containing the lymphocyte, and only the lymphocyte can be extracted from the separated mononuclear cell layer.

Wherein the step (2) is a step of freezing the extracted lymphocyte at a temperature of -100 to -150 ° C., and the step (3) is a step of storing the frozen lymphocyte at a temperature of -150 to -200 ° C. for 1 to 24 hours And then thawing.

The freezing method is not particularly limited, but in the present invention, the extracted lymphocytes are frozen using a controlled temperature freezer (CRF).

The frozen lymphocytes are stored at -150 to -200 ° C for 1 to 24 hours, thawed, and the lymphocytes are frozen and thawed to allow the self-activating lymphocytes to proliferate in a large amount.

The step (4) is a first culturing step of culturing the thawed lymphocytes in a culture medium for cell culture and a culture medium for self-activating lymphocyte comprising an additive.

The cell culture medium includes L-glutamine and interleukin-2.

The L-glutamine is contained in an amount of 1 to 5 mM, and activation and proliferation of the lymphocyte occurs within the above range at a high rate.

In addition, the amount of interleukin-2 is included in the range of 100 to 3000 IU / mL. When the amount of interleukin-2 is less than 100 IU / mL, the lymphocyte cell cycling rate is slowed, and when it exceeds 3000 IU / mL, the content of NK cells and NKT cells is decreased.

Such additives include interleukin-2, anti-CD56 monoclonal antibodies, aminoguanidine and autologous plasma.

More specifically, it is preferable that 50 to 100 μL of interleukin-2 at 17 × ^{10 6} to 19 × 10 ⁶ IU / mL, 2 to 20 μg of anti-CD56 monoclonal antibody at 0.1 to 2 mg / mL, and 0.1 to 2 mg / mL < / RTI > of aminoguanidine. Also, the self-derived plasma is contained in an amount of 0.1 to 15% by volume.

The first culture is to culture the thawed lymphocytes, the cell culture medium and the additives in a flask in which the anti-CD28 monoclonal antibody is solidified.

The anti-CD28 monoclonal antibody is contained in a flask in an amount of 1 to 50 μg, and is solidified at 25 to 45 ° C. for 1 to 48 hours.

The first culture is incubated for 3 to 5 days at a temperature of 25 to 45 DEG C in a 1 to 10% carbon dioxide atmosphere.

NK cells, NKT cells and T cells of the lymphocytes can be activated through the first culture.

The step (5) is a second culturing step of culturing the cultured mixed liquid in the first culturing step in a culture medium for cell culture and a medium for self-activating lymphocyte culture comprising the additives.

The cell culture medium includes L-glutamine and interleukin-2.

The L-glutamine is contained in an amount of 1 to 5 mM, and activation and proliferation of the lymphocyte occurs within the above range at a high rate.

In addition, the amount of interleukin-2 is included in the range of 100 to 3000 IU / mL. When the amount of interleukin-2 is less than 100 IU / mL, the lymphocyte cell cycling rate is slowed, and when it exceeds 3000 IU / mL, the content of NK cells and NKT cells is decreased.

Such additives include anti-CD25 monoclonal antibodies, anti-CD226 monoclonal antibodies, interleukin-15 and autologous plasma.

More preferably 50 to 150 mg / mL of interleukin-15 50 to 100 μL, 0.1 to 2 mg / mL of anti-CD25 monoclonal antibody 50 to 100 μg and 0.1 to 2 mg / mL of anti-CD25 monoclonal antibody Lt; / RTI > monoclonal antibody. In addition, the self-derived plasma is contained in an amount of 0.1 to 8% by volume.

The second culture is cultured at a temperature of 25 to 45 DEG C for 7 to 10 days in a carbon dioxide atmosphere at a concentration of 1 to 10%.

NK cells, NKT cells and T cells of lymphocytes can be mass-grown through the second culture.

The present invention also relates to a composition for treating or preventing cancer comprising an activated lymphocyte cultured by the above-mentioned culturing method.

"Prevention" means any act that inhibits cancer or delays onset by the administration of activated lymphocytes.

"Treatment" means any action that improves or alleviates the symptoms of cancer by the administration of activated lymphocytes.

The route of administration of the self-activating lymphocyte cultured by the method of the present invention can be administered through any conventional route, preferably intravenously, as long as it can reach the target tissue.

In addition, the composition may be administered together with all therapeutic medicines or procedures that are generally used in chemotherapy, and the composition for treating or preventing cancer of the present invention may be administered orally or parenterally, And a preferable dosage is about 10 ⁶ to 10 ¹⁰ cells.

The term "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective amount will vary depending on factors such as the severity of the disease, Route and rate of excretion, duration of treatment, factors including co-administered drugs, and other well known factors in the medical field. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example  1. Production of self-activated lymphocytes

5x10 ⁶ cells of lymphocytes harvested from 60 cc of the patient's peripheral blood were slowly frozen at -130 ° C using a Controlled Rate Freezer (CRF). Thereafter, it was stored at -196 DEG C for 24 hours and thawed.

The thawed lymphocytes were mixed in 20 mL of cell culture medium containing 1200 IU / mL of interleukin-2 and 2 mM of L-glutamine.

To the cell culture medium, 80 μL of interleukin-2 at 18 × 10 ⁶ IU / mL, 1 mg / mL of anti-CD56 monoclonal antibody (BD Bioscience, product name: Purified NA / LE Mouse Anti-Human CD259. Product number: 555513), 10 mg of 1 mg / mL aminoguanidine and 4 vol% of autogenous plasma.

Then, the mixed cell culture medium was transferred to a flask solidified with an anti-CD28 monoclonal antibody (BD Bioscience, product name: Purified NA / LE Mouse Anti-Human CD28, product number: 555725), and 5% Lt; RTI ID = 0.0 > 37 C < / RTI > for 4 hours.

The anti-CD28 monoclonal antibody was included in the flask at 20 μg and was solidified at 37 ° C. for 24 hours.

The culture mixture obtained in the primary culture was mixed with 500 mL of cell culture medium containing 1200 IU / mL of interleukin-2 and 2 mM of L-glutamine. To the mixed cell culture medium, 80 μL of interleukin-15 at 100 mg / mL and 1 mg / mL of anti-CD25 monoclonal antibody (BD Bioscience, product name: Purified NA / LE Mouse Anti-Human CD25, 80 μg of anti-CD226 monoclonal antibody (BD Bioscience, product name: Purified NA / LE Mouse Anti-Human CD226, product number: 559786) and 4 vol% of autologous plasma were added.

Thereafter, the culture mixture was transferred to a vinyl bag for CO2 permeation culture, and secondary culture was carried out for 9 days at 37 DEG C in a quartet of 5% carbon dioxide to obtain a large amount of lymphocyte activation.

Comparative Example  One.

We isolated 5 x 10 ⁶ cells of lymphocytes harvested from 60 cc of isolated peripheral blood of the patient. The lymphocytes were mixed with 20 mL of cell culture medium containing 1200 IU / mL of interleukin-2 and 2 mM of L-glutamine. 10 μg of an anti-CD3 monoclonal antibody (BD Bioscience, product name: Purified NA / LE Mouse Anti-Human CD3, product number: 555329) of 1 mg / mL was added to the cell culture solution in an amount of 1 mg / 10 μg of a CD16 monoclonal antibody (BD Bioscience, product name: Purified NA / LE Mouse Anti-Human CD16, product number: 555403), 1 mg / ml of anti-CD56 monoclonal antibody (BD Bioscience, -Human CD56, product number: 555513) and 5% by volume of autogenous plasma. Then, the mixed cell culture was transferred to a flask and cultured in a 5% carbon dioxide incubator at a temperature of 37 DEG C for 7 days.

The culture mixture obtained in the primary culture was mixed with 500 mL of cell culture medium containing 1200 IU / mL of interleukin-2 and 2 mM of L-glutamine. 10 μg of 1 mg / ml anti-CD3 monoclonal antibody (BD Bioscience, product name: Purified NA / LE Mouse Anti-Human CD3, product number: 555329), 1 mg / ml of the cell culture medium 10 μg of anti-CD16 monoclonal antibody (BD Bioscience, product name: Purified NA / LE Mouse Anti-Human CD16, product number: 555403), 1 mg / ml of anti-CD56 monoclonal antibody (BD Bioscience, Mouse Anti-Human CD56, Product Number: 555513) and 5% by volume of autologous plasma. Thereafter, the culture mixture was transferred to a vinyl bag for permeation of carbon dioxide, and secondary culture was carried out in a 5% carbon dioxide incubator at 37 DEG C for 7 days to obtain lymphocyte activation.

Comparative Example  2. NK  Mass culture

We isolated 5 × 10 ⁶ cells of lymphocytes harvested from 60 cc of isolated peripheral blood of the patient. The isolated lymphocytes were isolated using NK cell magnetic beads. The separated lymphocytes were mixed with 20 mL of cell culture medium containing 1200 IU / mL of interleukin-2 and 2 mM of L-glutamine. 10 μg of 1 mg / ml anti-CD16 monoclonal antibody (BD Bioscience, product name: Purified NA / LE Mouse Anti-Human CD16, product number: 555403), 1 mg / ml of anti-CD16 monoclonal antibody 10 μg of a CD56 monoclonal antibody (BD Bioscience, product name: Purified NA / LE Mouse Anti-Human CD56, product number: 555513) and 5% by volume of autologous plasma were added. Then, the mixed cell culture was transferred to a flask and cultured in a 5% carbon dioxide incubator at a temperature of 37 DEG C for 7 days.

The culture mixture obtained in the primary culture was mixed with 500 mL of cell culture medium containing 1200 IU / mL of interleukin-2 and 2 mM of L-glutamine. 10 μg of 1 mg / ml anti-CD16 monoclonal antibody (BD Bioscience, product name: Purified NA / LE Mouse Anti-Human CD16, product number: 555403) and 1 mg / ml of the cell culture medium 10 μg of anti-CD56 monoclonal antibody (BD Bioscience, product name: Purified NA / LE Mouse Anti-Human CD56, product number: 555513) and 5% by volume of autologous plasma were added. Thereafter, the culture mixture was transferred to a vinyl bag for permeation of carbon dioxide, and secondary culture was carried out in a 5% carbon dioxide incubator at 37 DEG C for 7 days to obtain lymphocyte activation.

Comparative Example  3. CAT Culture

We isolated 5 × 10 ⁶ cells of lymphocytes harvested from 60 cc of isolated peripheral blood of the patient. The lymphocytes were mixed with 20 mL of cell culture medium containing 1200 IU / mL of interleukin-2 and 2 mM of L-glutamine. To the cell culture medium, 5% by volume of autologous plasma was added based on 20 mL of the cell culture solution. Then, the mixed cell culture medium was transferred to a flask solidified with an anti-CD3 monoclonal antibody (BD Bioscience, product name: Purified NA / LE Mouse Anti-Human CD3, product number: 555329) and incubated in a 5% carbon dioxide incubator Followed by incubation at 37 DEG C for 7 days. The anti-CD3 monoclonal antibody was included in the flask at 20 μg and solidified at 37 ° C. for 24 hours.

The culture mixture obtained in the primary culture was mixed with 500 mL of cell culture medium containing 1200 IU / mL of interleukin-2 and 2 mM of L-glutamine. 5% by volume of autologous plasma was added to the mixed cell culture medium based on 500 mL of the cell culture. Thereafter, the culture mixture was transferred to a vinyl bag for permeation of carbon dioxide, and secondary culture was carried out in a 5% carbon dioxide incubator at 37 DEG C for 7 days to obtain lymphocyte activation.

Experimental Example  One. NK  cell, NKT  Measure the distribution of cells and T cells

The distribution of NK cells, NKT cells and T cells of the lymphocytes cultured in Example 1 and Comparative Example 1 was measured.

The NK cells, NKT cells and T cells were analyzed for surface antigens using flow cytometry.

As a result, in Example 1 which is a culture method of the present invention, the ratio of NK cells was remarkably higher than that of Comparative Example 1.

Experimental Example  2. Cytotoxicity against K562 blood cancer cells ( cytotoxicity ) Measure

The activated lymphocytes cultured by the methods of Example 1 and Comparative Example 1 were used as effector cells and blood cancer cell lines (K562) as target cells, and the ratio of activated lymphocytes to cancer cells was 10 : 1 to analyze the killing ability of activated lymphocytes against blood cancer cell lines.

As a result, the cytotoxicity of the activated lymphocytes cultured by the conventional culture method of Comparative Example 1 was less than 40%, whereas the cytotoxicity of the activated lymphocytes cultured by the culture method of the present invention was about 75% (FIG. 3) . That is, it was confirmed that the activated lymphocyte cultured by the present invention is superior to the activated lymphocyte cultured by the conventional culture method, in terms of cancer cell killing ability.

Experimental Example  3. Cytotoxicity against various cancer cells ( cytotoxicity ) Measure

The activated lymphocytes cultured by the methods of Examples 1 and 2 and 3 were used as effector cells and MKN74, NCI-N87, MDA-MB-231, SNU354, NCI-H40, Raji, SW620 And K562 were used as target cells and the ratio of activated lymphocytes to cancer cells was set to 10: 1. The results are shown in Table 1 below. And FIG. 4, respectively.

Types of Cancer Cells site of origin Cytotoxicity (%) Example 1 Comparative Example 2 Comparative Example 3 MKN74 Stomach 67 50 47 NCI-N87 Stomach 46 21 13 MDA-MB-231 Breast 60 28 22 SNU-354 Liver 74 60 36 NCI-H40 Lung 47 34 8.5 Raji Lymphocyte 65 50 38 SW620 Colon 50 39 12 K562 WBC 75 69 43 Cell distribution (content) NK 37 70 0 NKT 19.2 3 0 T 43.4 20 98

As a result, contents of NK cells, NKT cells and T cells were different according to the culture method of lymphocyte activation, and in the result of Example 1 according to the culturing method of the present invention, NK cells, NKT cells and T cells were uniformly distributed .

In addition, it was confirmed that the cytotoxicity of the activated lymphocytes cultured in Example 1, which is a culturing method of the present invention, is superior to that of the activated lymphocytes cultured by the culture methods of Comparative Examples 2 and 3, in all of the cancer cell killing ability.

Claims (12)

A culture medium for mass-culturing an activated lymphocyte comprising a cell culture medium and an additive added to the cell culture medium,
Wherein the additive comprises an anti-CD28 monoclonal antibody, an anti-CD56 monoclonal antibody, interleukin-2, aminoguanidine and an autologous plasma. The culture medium for lymphocyte activation culture according to claim 1, wherein the cell culture medium comprises 1 to 5 mM L-glutamine and 100 to 1200 IU / mL of interleukin-2. . The method according to claim 1, wherein, when 20 mL of the cell culture medium is used as a reference,
The additive may comprise 50-100 μL of interleukin-2 at 17 × ^{10 6} to 19 × 10 ⁶ IU / mL, 2 to 20 μg of anti-CD56 monoclonal antibody at 0.1 to 2 mg / mL, 0.1 to 2 mg / mL of aminoguanidine, 2 to 20 μg, and 0.1 to 15% by volume of autogenous plasma. The culture medium for lymphocyte activation culture according to claim 1, wherein the anti-CD28 monoclonal antibody is contained in the flask at 1 to 50 μg and is solidified at 25 to 45 ° C. for 1 to 48 hours. The lymphocyte activation culture according to claim 1, wherein the lymphocyte is frozen at a temperature of -100 to -150 ° C and then stored at -150 to -200 ° C for 1 to 24 hours and thawed, Lt; / RTI > The composition of claim 1, wherein the additive further comprises an anti-CD25 monoclonal antibody, an anti-CD226 monoclonal antibody, interleukin-15 and an autologous plasma. [Claim 6] The method according to claim 6, wherein, when 500 mL of the cell culture medium is used as a reference,
Wherein said additive is selected from the group consisting of 50-100 μL of 50-150 mg / mL of interleukin-15, 50-100 μg of anti-CD25 monoclonal antibody of 0.1-2 mg / mL, 50-100 μg of anti-CD226 monoclonal antibody of 0.1-2 mg / And 0.1 to 8% by volume of self-derived plasma. (1) extracting lymphocytes from isolated human peripheral blood;
(2) freezing the extracted lymphocytes at a temperature of -100 to -150 캜;
(3) storing and thawing the frozen lymphocytes at a temperature of -150 to -200 ° C for 1 to 24 hours;
(4) a first culturing step of culturing the thawed lymphocytes in a culture medium for cell culture and a culture medium for self-activating lymphocyte comprising an additive;
Here, the cell culture medium includes interleukin-2 and L-glutamine,
Said additive comprising interleukin-2, an anti-CD56 monoclonal antibody, aminoguanidine and an autologous plasma; And
(5) a second culturing step of culturing the cultured mixed solution in the first culturing step in a culture medium for cell culture and a culture medium for self-activating lymphocyte comprising the additives;
Here, the cell culture medium includes interleukin-2 and L-glutamine,
Wherein the additive comprises an anti-CD25 monoclonal antibody, an anti-CD226 monoclonal antibody, interleukin-15, and autologous plasma. 9. The method of claim 8, wherein said first incubation step is incubated in a flask in which the anti-CD28 monoclonal antibody is solidified and incubated for 3-5 days at a concentration of 1 to 10% Lymphocyte activated culturing method. The method of claim 9, wherein the anti-CD28 monoclonal antibody is contained at 1 to 50 μg and is solidified at 25 to 45 ° C for 1 to 48 hours. The method according to claim 8, wherein the second culturing step is carried out at a temperature of 25 to 45 ° C for 7 to 10 days at a concentration of 1 to 10% of carbon dioxide. A composition for treating or preventing cancer comprising an activated lymphocyte cultured by the culturing method of claim 8.

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