WO2019168222A1 - Culture medium composition for culturing of anti-cancer activated lymphocytes derived from peripheral blood mononuclear cells and method for culturing anti-cancer activated lymphocytes using same - Google Patents

Abstract

The present invention relates to a culture medium composition for culturing anti-cancer activated lymphocytes derived from peripheral blood mononuclear cells and a method for culturing anti-cancer activated lymphocytes using same. More specifically, when the composition contains normal hexane fraction of Melia azedarach L., interleukin-2 (IL-2), anti-CD3 monoclonal antibody, and anti-CD16 monoclonal antibody, the effects of promoted differentiation and of mass proliferation of NK cells, NKT cells, and T cells (corresponding to anti-cancer activated lymphocytes) from peripheral blood mononuclear cells were confirmed. Accordingly, the present invention relates to a culture composition based on normal hexane fraction of Melia azedarach L. and a method for culturing peripheral blood mononuclear cells using same. Also, the anti-cancer activated lymphocytes derived from peripheral blood mononuclear cells cultured by the method according to the present invention can be used as a pharmaceutical composition which is highly effective for cancer treatment. Furthermore, the culture medium composition of the present invention may itself be used as an anti-cancer treatment, and when a chemical anticancer drug is administered in combination in a clinical use, the dosage of an existing anti-cancer drug can be lowered, thereby relieving side effects and greatly enhancing anticancer activity.

Description

Peripheral blood mononuclear cell-derived anticancer activated lymphocyte culture medium composition and anticancer activated lymphocyte culture method using the same

The present invention relates to a medium composition for culturing peripheral blood mononuclear cell-derived anti-cancer activated lymphocytes comprising a predetermined amount of interleukin-2 (IL-2), an anti-CD3 monoclonal antibody, an anti-CD16 monoclonal antibody, and a high blood normal hexane fraction, and an anticancer using the same. It relates to a method for culturing activated lymphocytes.

As a means of treating cancer or a tumor, a surgical surgical treatment to remove a cancer-generating site, a chemotherapy to administer an anticancer agent, a radiation therapy to irradiate radiation to a cancer-generating site, and the like are common.

However, these therapies have the disadvantage of showing various side effects in patients. As a result, anti-cancer drugs are used to proliferate, select, or change the biological characteristics of cells in various ways by activating autologous, allogenic, or xenogenic cells in vitro and then activating anticancer activity to patients. A fourth cancer treatment has been developed called immune cell therapy.

Representative anti-cancer immune cell therapies include self-activated lymphocyte therapy, such as lymphokine-activated killer (LAK) therapy or cytotoxic T lymphocyte (CTL) therapy.

LAK therapy is a method of treating cancer of a patient by using the immunity of the patient's own lymphocytes proliferated and activated in vitro. Specifically, after (1) blood is collected from cancer patients, peripheral blood monocytes including lymphocytes are isolated, and (2) the isolated peripheral blood monocytes are separated from IL-2 (Interleukin-2) and anti-CD3 monoclonal antibodies. cultured in a medium containing (anti-CD3 monoclonal antibody) for 2 weeks to proliferate and activate lymphocytes, and (3) wash the cell suspension containing cultured lymphocytes to treat IL-2 and anti-CD3 antibodies. The medium is removed, the lymphocytes are suspended in an appropriate solution such as saline solution containing albumin, and prepared in the form of an injection. (4) A method of treating a patient by injecting an injection containing activated lymphocytes into the patient's body. . In addition, CTL therapy is a method of proliferating and activating cytotoxic T lymphocytes, and the treatment process is similar to that of LAK cell therapy. At present, various studies have been conducted to increase the specificity and effect of anticancer immune cell therapy.

Cinnabar bark is the bark or root bark of the mulberry tree ( Meliaazedarach L. var. Japonica Makino ), and is native to Korea, China, and Japan. It is known to have antiparasitic, insecticidal, antimalarial, contraceptive, and antibacterial effects, and is used for the treatment of formal medicine, worms, and malaria fever. It has been reported that ethanol extract of Rhizobarium inhibits iNOS and that beta-carboline alkaloids exhibit anti-inflammatory activity by inhibiting iNOS in Raw264.7 cell line. In addition, the present inventors have reported the anti-cancer activity effect of the extract of Gyunpi in Korean Patent No. 10-1609935 through animal experiments, Gyunpi normal hexane fraction shows little cytotoxicity, when co-administration with anticancer agent It has been reported to show the characteristic efficacy of anticancer synergistic effect and anticancer drug side effects.

As such, the use of the bark normal hexane fraction itself as an anticancer agent is disclosed, but for the culture method in which the bark normal hexane fraction promotes the differentiation into NK cells, NKT cells, and T cells, which are immune cells that have anticancer activity, and proliferates them. It has not been disclosed at all.

Korean Patent No. 10-1039843 relates to a medium composition for culturing self-activated lymphocytes and a method for culturing self-activated lymphocytes using the same, which promotes differentiation of immune cells into NK cells, NKT cells, and T cells and lacks mass proliferation. In addition, there is a limit that the anticancer effect of killing tumor cells to inhibit proliferation is not high.

There is a need for a medium composition capable of culturing peripheral blood mononuclear cell-derived anticancer activated lymphocytes and a culture method using the same.

Prior art literature

(Patent Document 1) Korean Registered Patent No. 10-1609935

(Patent Document 2) Korean Registered Patent No. 10-1039843

Accordingly, the present inventors conducted a study to find a culture composition that is superior to a known medium for activating lymphocyte culture medium, as a result of the study, the high blood normal hexane fraction with little cytotoxicity and interleukin-2 (IL-2), anti When -CD3 monoclonal antibody and anti-CD16 monoclonal antibody are used as an additive for a predetermined amount of cell culture medium, it was found that the differentiation into NK cells, NKT cells and T cells, which are immune cells, can be greatly promoted and proliferated. The present invention has been completed. Furthermore, it was confirmed that the immune cells proliferated by the method of the present invention significantly inhibited the cell proliferation of cancer cells, and the anticancer power was greatly enhanced in comparison with the existing LAK cells in animal tests (see Experimental Example).

Therefore, an object of the present invention is to provide a medium composition for culturing natural killer cells (NK Cell), natural killer T cells (NKT Cell), and peripheral blood mononuclear cell-derived anti-cancer activated lymphocytes capable of effectively proliferating T cells.

Another object of the present invention is to provide a method for culturing peripheral blood monocyte-derived anti-cancer activated lymphocytes using the medium composition.

Another object of the present invention is to provide a pharmaceutical composition for treating cancer comprising peripheral blood mononuclear cell-derived anti-cancer activated lymphocytes cultured by the culture method.

In order to solve the above problems, the present invention is a medium composition for mass culturing anti-cancer activated lymphocytes comprising a cell culture medium and an additive added to the cell culture medium, wherein the additive is interleukin-2 (IL- 2), anti-CD3 monoclonal antibody, anti-CD16 monoclonal antibody, and provides a medium composition for culturing peripheral blood mononuclear cell derived anti-cancer activated lymphocytes, characterized in that it comprises a normal blood hexane fraction.

In addition, the present invention comprises the steps of separating peripheral blood monocytes into peripheral blood; Putting the isolated peripheral blood mononuclear cells into the cell culture medium and culturing in the presence of interleukin-2 (IL-2), anti-CD16 monoclonal antibody, and the bark normal hexane fraction; A second culture step of obtaining cells from the culture medium in which the culture was completed in the first culture step, and culturing the obtained cells in the presence of interleukin-2 (IL-2), anti-CD3 monoclonal antibody, and pulmonary normal hexane fraction; And obtaining cells from the culture medium in the second culture step, and culturing the obtained cells in the presence of interleukin-2 (IL-2), anti-CD3 monoclonal antibody, anti-CD16 monoclonal antibody, and the bark normal hexane fraction. It provides a peripheral blood mononuclear cell-derived anti-cancer activated lymphocyte culture method comprising a third culture step.

The present invention also provides a pharmaceutical composition for treating cancer comprising peripheral blood mononuclear cell-derived anticancer activated lymphocytes cultured by the above method.

The culture composition provided by the present invention comprises a fraction of high-fidelity normal hexane, thereby significantly differentiating and proliferating into NK cells, NKT cells, and T cells, which are immune cells that have anticancer activity, compared to conventional lymphokine-activated killer (LAK) cells. In this way, natural killer cells (NK Cells), natural killer T cells (NKT Cells), and T cells that can significantly inhibit the proliferation of cancer cells can be obtained.

Furthermore, the media composition of the present invention can be used as an anticancer treatment by itself, it can greatly enhance the anticancer power while alleviating side effects by lowering the dose of the existing anticancer agent when used in combination in the clinic as a chemical anticancer agent.

1 is a graph comparing the number of immune cells proliferated using a conventional culture method (Comparative Example 1) and a culture method using the activated lymphocyte culture medium composition according to the present invention (Example 1).

2A-2C are diagrams illustrating the differentiation and phenotypic changes of lymphocytes using flow cytometry.

Figures 3a-3c is a result of measuring the viability of the cells using the ⁵¹ Cr release assay.

Figure 4 is a result confirming the cancer cell proliferation inhibitory effect using a lung cancer model animal.

The present invention will be described in more detail as follows.

The present invention relates to a medium composition for mass culturing anti-cancer activated lymphocytes comprising a cell culture medium and an additive added to the cell culture medium. Specifically, the additive is interleukin-2 (IL-2), anti -CD3 monoclonal antibody, anti-CD16 monoclonal antibody, and cinnabar normal hexane fraction. Herein, the anti-cancer activated lymphocytes in the present invention mean natural killer cells (NK Cell), natural killer T cells (NKT Cell), and T cells.

Prior to the detailed description of the present invention, each of the additives used for amplifying and activating the facet cells together with the characteristics of each immune cell will be described.

The NK cells are large granular lymphocytes (LGL), a characteristic type of lymphocytes, and their antitumor activity occurs in necrosis, apoptosis, or both mechanisms. It is done by NK cells respond to cytokines such as IL-2, IL-12, Interferon, and the like, thereby raising cytotoxicity, secretory, and proliferative functions. Phenotypes of NK cells are CD16 (FcγRIII) and CD56 in humans, and CD16 and CD56 do not have a T-cell receptor complex (TRC) on the cell surface and thus are used as markers for NK cells.

T cells refer to cells having an T cell receptor (TCR) on the cell surface. TCR forms a heterodimer with the CD3 antigen of the di- and β-chain dimers. Some of the T cells (around 5% of peripheral blood T cells) are dimers of γ and δ chains, not αβ. TCR forms a complex with the CD3 antigen (γ, δ, ε, ζ, ζ or η), which plays a role in delivering the signal into the cell when TCR recognizes the antigen.

NKT cells are a type of T cell whose function is being revealed relatively recently as an attendant of endogenous immunity. As the name suggests, it expresses T-cell receptors and NK cell-specific surface markers. One surprising feature of NKT cells is the rapid release of several cytokines such as IL-4, IL-10, IL-13, IFN-γ, TNF-α after activation. This feature suggests that NKT cells can have a significant effect on the adaptive immune response.

On the other hand, one of the additives, interleukin-2 (IL-2) is a glycoprotein (glycoprotein) having a molecular weight of 14 to 17 kDa that is produced when T cells recognize and activate an antigen, is secreted out of T cells, and then IL-2. It reacts with the T cells themselves, which promotes the growth of the corresponding T cells. IL-2 also acts on NK cells to promote growth, enhances NK cell killing capacity, and acts on B cells to promote their growth.

When lymphocytes are cultured in the presence of anti-CD3 monoclonal antibody, anti-CD16 monoclonal antibody or antigen antibody complex, CD16 antigen is added to NK cells and signaling occurs. Transferrin receptors such as α chains, or tumor necrosis factor (TNF) or IFN-γ can be produced.

Therefore, in the present invention, IL-2, anti-CD3 monoclonal antibody and anti-CD16 monoclonal antibody were used as a medium additive in culture. Among the additives, IL-2 was used for proliferation of T cells and NK cells, and each monoantibody was used. To play the role of the antigen to express CD3, CD16 in immune cells, in the present invention further comprises a high blood normal hexane fraction, NK cells, T cells and NKT cells to proliferate and activate significantly.

Specifically, the effect on goryeon blood normal hexane fraction cells to avoid an extract extracted goryeon blood with C _{1 ~ 4} alcohol and the use in that the fraction obtained by the fractionation as normal hexane, which cytotoxicity is substantially not grown in the present invention In the present invention, the bark normal hexane fraction greatly promotes differentiation and proliferation into NK cells, T cells and NKT cells.

Specifically, the medium composition of the present invention is a cell culture medium that can be used in conventional cell culture, and interleukin-2 (IL-2) 100 ~ 200000 IU / ㎖ as an additive; Anti-CD3 monoclonal antibody 10-100000 ng / ml; Anti-CD16 monoclonal antibody 10-100000 ng / ml; And 25-250 μg / ml of the bark normal hexane fraction.

On the other hand, the present invention provides a peripheral blood mononuclear cell-derived anti-cancer activated lymphocyte culture method using the culture composition.

Specifically, separating peripheral blood monocytes with peripheral blood; Putting the isolated peripheral blood mononuclear cells into the cell culture medium and culturing in the presence of interleukin-2 (IL-2), anti-CD16 monoclonal antibody, and the bark normal hexane fraction; A second culture step of obtaining cells from the culture medium in which the culture was completed in the first culture step, and culturing the obtained cells in the presence of interleukin-2 (IL-2), anti-CD3 monoclonal antibody, and pulmonary normal hexane fraction; And obtaining cells from the culture medium in the second culture step, and culturing the obtained cells in the presence of interleukin-2 (IL-2), anti-CD3 monoclonal antibody, anti-CD16 monoclonal antibody, and the bark normal hexane fraction. To provide a third culture step. This will be described in detail step by step as follows.

(Isolation of Peripheral Blood Monocytes)

In the present invention, peripheral blood mononuclear cells refer to monocytes (Mononuclear Cell) isolated from peripheral blood (Peripheral Blood) commonly used for anticancer immunotherapy. Peripheral blood monocytes can be separated from blood by centrifugation using specific gravity, and the degree and composition of immune cells is confirmed by flow cytometry. In addition, as an embodiment of the present invention, the peripheral blood monocytes may be obtained from a patient or cancer patient having a normal risk of cancer. In addition, the peripheral blood mononuclear cells used in the present invention are not necessarily self-derived, and if the peripheral blood mononuclear cells derived from the same species can be used for the induction and proliferation of natural killer cells for anticancer immune treatment according to the present invention.

(First stage of culture)

In the first culture step, the peripheral blood mononuclear cells isolated in the cell culture medium (culture medium) are put and cultured in the presence of Interleukin-2 (IL-2), anti-CD16 monoclonal antibody, and the pulmonary blood normal hexane fraction.

In this case, the cell culture medium may further include a nutrient component or a pH adjuster for culturing peripheral blood mononuclear cells. As a medium containing such a component, RPMI-1640, DMEM, serum-free medium, and the like may be used, and reagents commonly used in cell culture may be included. Examples of such reagents include serum (human or fetal), albumin, antibiotics and antifungal agents, L-glutamine, sodium pyruvate, transferrin, insulin and the like.

In addition, the culture medium containing the additive IL-2, anti-CD3 monoclonal antibody, anti-CD16 monoclonal antibody and the hexane fraction can be prepared by the following method. For example, the culture medium is prepared by adding IL-2, anti-CD3 monoclonal antibody, anti-CD16 monoclonal antibody, and bark hexane fraction to the cell culture medium, or a medium containing anti-CD3 monoclonal antibody, anti-CD16 monoclonal antibody. It can also be prepared by coating each antibody in a flask in advance, and adding the remaining culture component to the flask.

In addition, for the differentiation and proliferation of peripheral blood monocytes to NK cells, NKT cells and T cells, the cell culture medium during the first to the third culture step, the concentration of IL-2 100 ~ 200000 IU / ㎖, anti-CD3 Concentrations of monoclonal antibodies of 10 to 100000 ng / ml and concentrations of anti-CD16 monoclonal antibodies of 10 to 100000 ng / ml, and bark normal hexane fractions can be used within the range of 25 to 250 μg / ml. The amount of these may be adjusted and used within the above range through each culture step, but is not necessarily limited.

Preferably, in the first culture step, interleukin-2 (IL-2) 200-1000 IU / ml, anti-CD16 monoclonal antibody 1000-30000 ng / ml, and the bark normal hexane fraction 30-75 ㎍ / ml are used. .

In addition, the culture conditions of the first culture step are not particularly limited, and conditions used in normal cell culture can be used. In one embodiment, it is preferable to incubate at a temperature of 30 ~ 38 ℃ and a condition of CO ₂ concentration of 5 to 10%. The culture period is 3 to 5 days in order to activate and proliferate peripheral blood monocytes, and to fully differentiate and proliferate into NK cells, NKT cells and the like, and may be longer. In addition, culture medium exchange can be appropriately carried out during the culture.

(2nd culture stage)

In the second culture step, the culture is performed under conditions that selectively differentiate peripheral blood monocytes into cytotoxic T cells, and the peripheral blood monocytes, which have not been stimulated in the first culture step, are selectively differentiated and expanded into T cells. NK cells and NKT cells differentiated, activated and proliferated in the first culture stage can be stimulated, directly or indirectly, by T cells activated in the second culture stage to significantly activate and proliferate NK cells and NKT cells. .

Specifically, in the second culture step, in order to selectively differentiate peripheral blood monocytes into cytotoxic T cells, cells are obtained from the culture medium cultured in the first culture step, and the obtained cells are interleukin-2 (IL-2), Incubated in the presence of anti-CD3 monoclonal antibody, and a barley normal hexane fraction.

In addition, as described above, the cell culture medium during the first to third culture stages has a concentration of 100-20000 IU / ml of IL-2, a concentration of 10-100000 ng / ml of anti-CD3 monoclonal antibody, and an anti-CD16 monoclonal antibody. The concentration of 10 to 100000 ng / ml, the concentration of the bark normal hexane fraction can be used within the range of 25 ~ 250 ㎍ / ㎖.

Preferably, the second culture step uses 200-1000 IU / ml of interleukin-2 (IL-2), 1000-30000 ng / ml of anti-CD3 monoclonal antibody, and 30-75 [mu] g / ml of the bark normal hexane fraction.

In addition, the culture solution is exchanged at the start of the second culture step to perform the culture. In addition, the culture conditions of the second culture step is not particularly limited, and may be cultured under the same conditions as specified in the first culture step, and the culture period is sufficient for the differentiation and proliferation of peripheral blood monocytes into cytotoxic T cells. It can be 3 to 5 days or more. In addition, culture medium exchange can be appropriately carried out during the culture.

(3rd culture stage)

The third culture step is a step of additionally culturing the cells obtained in the culture medium of the second culture step in a culture medium containing IL-2, anti-CD16 monoclonal antibody, anti-CD3 monoclonal antibody and the bark hexane fraction. The third culture step may further activate and proliferate the immune cells differentiated and expanded in the first and second culture steps.

Specifically, in the third culture step, cells are obtained from the culture medium in the second culture step, and the obtained cells are interleukin-2 (IL-2), anti-CD3 monoclonal antibody, anti-CD16 monoclonal antibody, and cinnabar rind. Incubate in the presence of normal hexane fractions.

In addition, as described above, the cell culture medium undergoes a concentration of 100-20000 IU / ml of IL-2, a concentration of 10-100000 ng / ml of anti-CD3 monoclonal antibody, and an anti-CD16 monoclonal antibody during the first to third culture steps. The concentration of 10 to 100000 ng / ml, the concentration of the bark normal hexane fraction can be used within the range of 25 ~ 250 ㎍ / ㎖.

Preferably, in the third culture step, interleukin-2 (IL-2) 200 to 1000 IU / ml, anti-CD3 monoclonal antibody 1000 to 30000 ng / ml, anti-CD16 monoclonal antibody 1000 to 30000 ng / ml, and high blood normal Hexane fractions 30-75 μg / ml are used.

In addition, the culture medium is exchanged at the start of the third culture step to perform the culture. In addition, the culture conditions of the third culture step is not particularly limited, can be cultured under the same conditions as specified in the first and second culture step, the culture period is 3 to 5 in order for the immune cells to fully differentiate and proliferate I can do it more than a day. In addition, culture medium exchange can be appropriately carried out during the culture.

The present invention as described above will be described in more detail based on the following examples, but the present invention is not limited thereto.

Preparation Example 1 Preparation of High-velocity Normal Hexane Fraction

10L of 80% ethanol solution was added to 1kg of dried barley bark, extracted and filtered for 8 hours between 70 and 80 ° C, concentrated with reduced pressure concentrator until it became 500ml, and 500ml of normal hexane solution. After transferring to the fractionation rod and fractionated, the supernatant was taken, concentrated again by a vacuum condenser and the solvent was removed. Approximately 8 g of oil extract obtained by removing the solvent was obtained in an oil phase.

Comparative Example 1. Proliferation of immune cells by LAK cell culture (control)

First, peripheral blood of 30 ml was collected, and peripheral blood mononuclear cells were separated from blood by centrifugation and selected as experimental groups (peripheral blood mononuclear cells). The peripheral blood mononuclear cells thus separated were added to 50 ml of a medium containing IL-2 (Interleukin-2) 200 IU / ml and anti-CD3 monoclonal antibody 10000 ng / ml, and cultured for 2 weeks, thereby proliferating and activating lymphocyte cells.

Example 1 Proliferation and Identification of Immune Cells According to the Culture Method of the Present Invention

First, peripheral blood of 30 ml was collected, and peripheral blood mononuclear cells were separated from blood by centrifugation and selected as experimental groups (peripheral blood mononuclear cells).

The peripheral blood monocytes were suspended in 50 ml of medium to which 200 IU / ml of IL-2 and 50 µg / ml of the bark normal hexane fraction obtained in Preparation Example 1 were added, and 10000 ng / ml of anti-CD16 monoclonal antibody. In a 75 cm 2 flask coated with a 37 ° C, 5% CO ₂ conditions were incubated for 5 days using a cell incubator (first culture step).

Next, the cells were recovered from the flask at the end of the first culture step (Day 5), and the recovered cells were again added with 200 IU / mL of IL-2 and 50 μg / mL of the bark normal hexane fraction. Suspended in ㎖ medium (primary culture medium), and then placed in a sterile gas permeable cell culture medium coated with anti-CD3 monoclonal antibody 10000 ng / ㎖ for 4 days at 37 ℃, 5% CO ₂ conditions using a cell incubator The culture was carried out (second culture step).

Next, the cells were recovered from the flask at the end of the second culture step (Day 9), and the recovered cells were again subjected to 200 IU / ml of IL-2, 10000 ng / ml anti-CD3 monoclonal antibody, 10000 ng / ml. Into a gas-permeable cell culture medium containing an anti-CD16 monoclonal antibody and 50 μg / ml of the high-fidelity normal hexane fraction, the cells were incubated at 37 ° C. and 5% CO ₂ for 4 days using a cell culture medium. 3 culture steps).

The cells in Comparative Example 1 and Example 1 obtained through the above culture process were compared with the total lymphocyte cell numbers including NK cells, NKT cells, and T cells, and are shown in FIG. 1.

As shown in Figure 1, the number of cells before the culture was 2.0x10 ⁶ ~ 1.3x10 ^7, but by the conventional LAK cell culture method (Comparative Example 1) was 2.3x10 ⁹ ~ 3.70x10 ⁹ or more, according to the present invention By culture method (Example 1), it was confirmed that the growth was 3.1x10 ⁹ ~ 4.8x10 ⁹ or more.

Therefore, it was confirmed that the lymphocytes prepared by the culturing method according to the present invention had an increase in cell number of about 35% or more compared with the lymphocytes prepared by the conventional LAK cell culture method.

Experimental Example 1. Confirmation of differentiation and activity of immune cells

The activated lymphocytes cultured in Comparative Example 1 and Example 1 were analyzed for surface antigen of each immune cell using flow cytometry, and the results are shown in FIG. In FIG. 2A, the H1 region shows T cells, the H2 region shows NKT cells, the H4 region shows NK cells, and the H3 region shows the distribution of other immune cells.

Specifically, referring to the result of FIG. 2A, the surface antigen distribution is most distributed in the H4 region before the cultivation, whereas the distribution of the H1 and H2 regions is obtained after the culturing by the culturing method (Example 1) according to the present invention. Was found to be increased.

As a result of calculating the ratios of actual NK cells, NKT cells and T cells, 40.3%: 4.6%: 27.5% by the conventional LAK cell culture method, whereas 51.6%: 7.7%: 38.2% by the culture method according to the present invention. It can be seen that the lymphocytes produced by the culture method according to the present invention is increased in differentiation and activity compared to the lymphocytes produced by the conventional LAK cell culture method.

Also, the activated lymphocytes cultured in Comparative Examples 1 and 1 were confirmed the secretion level of IFN-γ by ELISPOT assay, and CD69 expression, which is an active surface antigen of lymphocytes, was measured by flow cytometry. b) and (c).

Referring to the results of (b) and (c) of Figure 2, the lymphocytes prepared by the culture method according to the present invention (Example 1) is IFN-γ compared to lymphocytes prepared by the conventional LAK cell culture method (Comparative Example 1) Increased secretion and expression of CD69 was found to increase the activity was confirmed.

From the above results, it was confirmed that the ratio of NK cells, NKT cells, and T cells in the lymphocyte cells prepared by the culture method according to the present invention was significantly increased, and the effect of the composition change of the final culture on the killing ability of various cancer cells The following analysis was performed to evaluate.

Experimental Example 2: Confirmation of apoptosis effect on cancer cells

The immune cell-containing compositions prepared in Comparative Example 1 and Example 1 were placed in a 15 ml centrifuge tube, centrifuged (1400 rpm, 5 minutes), and the supernatant was removed to separate cells. Using the isolated cells, apoptosis effects on the human lung cancer cell line A549, the human liver cancer cell line HepG2, and the human cervical cancer cell line SiHa were confirmed using a ⁵¹ Cr release assay, and the results are shown in FIG. 3. For reference, the cell death effect was calculated based on Equation 1 below.

[Equation 1]

% Apoptosis = ((experimental release-spontaneous release) / (maximal release-spontaneous release)) × 100

As shown in Figure 3, it was confirmed that the lymphocytes prepared by the culture method according to the present invention (Example 1) has a higher killing effect on cancer cells than lymphocytes prepared by the conventional LAK cell culture method (Comparative Example 1). .

Experimental Example 3: Confirmation of cancer cell proliferation inhibitory effect in lung cancer model animals

The immune cell-containing compositions prepared in Comparative Example 1 and Example 1 were placed in a 15 ml centrifuge tube, centrifuged (1400 rpm, 5 minutes), and the supernatant was removed to separate cells. The isolated immune cells were administered intravenously to BALB / c nude mice in which tumors were formed by administering A549, a non-small cell lung cancer line, to confirm the effect of inhibiting tumor growth.

As shown in Figure 4, it was confirmed that the lymphocytes prepared by the culture method according to the present invention (Example 1) showed a higher tumor growth inhibitory effect than lymphocytes prepared by the conventional LAK cell culture method (Comparative Example 1).

Claims (5)

In the medium composition for mass culturing anti-cancer activated lymphocytes comprising a cell culture medium and an additive added to the cell culture medium, The additives include interleukin-2 (IL-2), anti-CD3 monoclonal antibodies, anti-CD16 monoclonal antibodies, and hardened normalhexane fractions, The cell culture medium is Interleukin-2 (IL-2) 100-200000 IU / ml; Anti-CD3 monoclonal antibody 10--100000 ng / ml; Anti-CD16 monoclonal antibody 10-100000 ng / ml; And Pulp normal hexane fraction 25-250 μg / ml; Peripheral blood mononuclear cell-derived anticancer activated lymphocyte culture medium composition comprising a. The method of claim 1, The blood goryeon normal hexane fraction goryeon avoid the C _{1 ~ 4} peripheral blood mononuclear cells derived from anti-cancer active lymphocyte culture medium composition of the extract extracted with an alcohol characterized in that the fractions obtained by fractionation with normal hexane. The method of claim 1, The anticancer activated lymphocytes are natural killer cells (NK Cells), natural killer T cells (NKT Cells), and the peripheral blood mononuclear cell-derived anti-cancer activated lymphocyte culture medium composition, characterized in that T cells. Separating peripheral blood monocytes into peripheral blood; Putting the isolated peripheral blood mononuclear cells into the cell culture medium and culturing in the presence of interleukin-2 (IL-2), anti-CD16 monoclonal antibody, and the bark normal hexane fraction; A second culture step of obtaining cells from the culture medium in which the culture was completed in the first culture step, and culturing the obtained cells in the presence of interleukin-2 (IL-2), anti-CD3 monoclonal antibody, and pulmonary normal hexane fraction; And Obtaining cells from the culture medium in the second culture step, and culturing the obtained cells in the presence of interleukin-2 (IL-2), anti-CD3 monoclonal antibody, anti-CD16 monoclonal antibody, and the bark normal hexane fraction Third culture step; Containing, the culture medium used through the first to the third culture step, the concentration of interleukin-2 (IL-2) 100 ~ 200000 IU / ㎖, the concentration of the anti-CD3 monoclonal antibody 10 ~ 100000 ng / ㎖, anti- A method for culturing peripheral blood mononuclear cell-derived lymphocytes, characterized in that it comprises a concentration of CD10 monoclonal antibody of 10 ~ 100000 ng / ㎖, and 25 ~ 250 ㎍ / ㎖ of the high blood normal hexane fraction. The method of claim 4, wherein Said first to third culture step is a peripheral blood mononuclear cell-derived anti-cancer activated lymphocyte culture method characterized in that the culture for 3 to 5 days at 30 ~ 80 ℃ temperature and 5 ~ 10% CO ₂ conditions.

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