KR20190093499A - Manufacturing Method for Natural Killer Cells - Google Patents

Abstract

The present invention relates to a production method of natural killer cells. The production method of the present invention can produce high-purity natural killer cells without using various expensive cytokines, thereby enhancing the efficiency and efficacy of the prevention and treatment of cancer using natural killer cells.

Description

Manufacturing Method for Natural Killer Cells

The present invention relates to a method for producing high purity natural killer cells.

The human body is protected from pathogens by an immune response, and the immune system is composed of many immune-related cells, cytokines and the like. An important role in this immune system is leukocytes, especially lymphocytes. The cells that make up lymphocytes are typically the innate immune system and the acquired immune system.

Natural Killer Cells (NK cells) are one of the representative innate immune cells, which can kill cancer nonspecifically, kill viruses by recognizing viruses, bacteria, etc., perforin and granzyme. Is known to kill pathogens by enzymes or Fas-FasL interactions. In cancer patients, the reduction of cancer cell killing ability of NK cells may be caused by lung cancer (Carrega P, et al., Cancer, 2008: 112: 863-875), liver cancer (Jinushi M, et al., J). Hepatol., 2005: 43; 1013-1020), breast cancer (Bauernhofer T, et al., Eur J Immunol., 2003: 33: 119-124), uterine cancer (Mocchegiani E., et al., Br j Cancer., 1999: 79: 244-250), hematologic cancer (Tajima F., et al, Lekemia 1996: 10: 478-482), and has been reported to be closely associated with the development of diseases. Increase of cancer cell killing ability and activity of natural killer cells will be essential.

In order to achieve the effect of killing cancer cells, a large amount of natural killer cells with high purity are required, but it is not easy to secure a large amount of blood of cancer patients, and the rate of natural killer cells in the blood is only about 5 to 20%. It is difficult to use it as an immunotherapy.

Therefore, it is important to effectively expand and proliferate only natural killer cells, but the existing natural killer cell proliferation method requires the use of various expensive cytokines at high concentrations. In addition, other kinds of immune cells other than natural killer cells (eg, T cells, B cells, etc.) may be present together, which may cause GVHD (Graft versus host disease) when the T cells are included and administered to allogeneic cells. When administered to the same type of blood type incompatibility, there is a problem that does not maximize the anti-cancer effect, such as causing Passenger B-lymphocyte syndrome.

Domestic Publication No. 10-2013-0086820

The present inventors earnestly researched to develop a method for producing high-purity natural killer cells without using various expensive cytokines. As a result, by separating CD56 + cells from Peripheral blood mononuclear cells, and by co-culture with the irradiated feeder cells in the presence of cytokines, it was possible to produce high-purity CD56 + natural killer cells, The present invention has been completed.

It is therefore an object of the present invention to provide a method for producing natural killer cells.

The present inventors earnestly researched to develop a method for producing high-purity natural killer cells without using various expensive cytokines. As a result, after separating CD56 + cells from peripheral blood mononuclear cells (Peripheral blood mononuclear cells), it was confirmed that high-purity CD56 + natural killer cells can be produced when co-cultured with the irradiated feeder cells in the presence of cytokines.

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

One aspect of the invention relates to a method for producing a natural killer cell comprising the following steps:

A first separation step of separating peripheral blood mononuclear cells (PBMCs) from the blood sample;

A second separation step of separating any one or more selected from the group consisting of CD56 + cells and CD3- / CD56 + cells; And

A culture step of co-culturing by adding a feeder cell and a cytokine.

The present invention will be described in detail for each step.

First separation step

As used herein, the "blood sample" may be, but is not limited to, peripheral blood whole blood or leukocytes isolated from leukocytes by leukapheresis. In addition, the peripheral blood may be obtained from a normal or patient having a cancer risk or a cancer patient, but is not limited thereto.

As used herein, the term "leukapheresis" refers to the selective removal (leukocytes) of leukocytes from the collected blood and transfusion to the patient, in the case of leukocytes isolated by the method, Picol- It may be used in the present invention without a separate method such as a Ficoll-Hypaque density gradient method.

As used herein, the term "peripheral blood mononuclear cell" (Peripheral blood mononuclear cell) may be used in the same sense as "PBMC", "Mononuclear cell" or "monocytes", which is commonly used for anticancer immunotherapy Mean mononuclear cells (Mononuclear Cell) is isolated from the peripheral blood (Peripheral Blood). Peripheral blood mononuclear cells can be obtained by using a known method such as Ficoll-Hypaque density gradient method of the blood of the collected human.

The peripheral blood mononuclear cells do not necessarily need to be autologous, and allogeneic peripheral blood mononuclear cells may also be used for the production of high-purity natural killer cells for anticancer immunotherapy according to the present invention.

In addition, the peripheral blood mononuclear cells are not necessarily derived from normal people, peripheral blood mononuclear cells of cancer patients can also be used for the production of high-purity natural killer cells for anticancer immune treatment according to the present invention.

As used herein, the term "CD56 + cells" can be used in the same sense as "CD56 + NK cells" or "CD56 natural killer cells," and "CD3- / CD56 + cells" are used in the same sense as "CD3- / CD56 + NK cells." Can be.

The CD56 + cells and CD3- / CD56 + cells may include cells expressing the CD56 glycoprotein on the cell surface, and cells expressing the CD56 glycoprotein and non-expressing the CD3 glycoprotein, as necessary. Even the same immune cells may have different functions depending on the type of CD attached to the cell surface and the expression rate.

Second separation step

The second separation step may be performed by separating using CD56 microbeads and / or CD3 microbeads, or separating using equipment such as CliniMACSs or Flow Cytometry Cell Sorter. Can be.

Separation using the CD56 microbeads and / or CD3 microbeads is performed by, for example, adding CD56 microbeads to PBMCs and then removing non-specific binding, or removing CD3 microbeads into PBMCs. By addition to remove specific binding, followed by addition of CD56 microbeads to remove nonspecific binding.

T cells and the like can be effectively removed through the separation of the CD56 + cells and CD3- / CD56 + cells.

Incubation stage

As used herein, the term "cytokine" refers to an immune activated cytokine that can be used to induce peripheral blood mononuclear cells into natural killer cells.

The cytokine is IL (Interleukin) -2, IL-21, IL-15, Flt3-L (FMS-like tyrosine kinase 3 ligand), SCF (Stem Cell Factor), IL-7, IL-18, IL-4 , type I interferons, GM-CSF (Granulocyte-macrophage colony-stimulating factor) and / or IGF 1 (Insulin-like growth factor 1), but is not limited thereto.

The cytokine is 50-1,000, 50-900, 50-800, 50-700, 50-600, 50-550, 100-550, 150-550, 200-550, 250-550, 300-550, 350- It can be used at a concentration of 550, 400-550 or 450-550 IU / mL, but is not limited thereto.

Meanwhile, the conventional natural killer cell proliferation method requires a high concentration of various cytokines, but the natural killer cell proliferation method according to the present invention uses two feeder cells for high-purity CD56 + cells (including CD3- / CD56 + cells). Therefore, even when the cytokine is used at a low concentration, it is possible to proliferate natural killer cells with high yield and high purity.

As used herein, the term "Feeder cell" (Feeder cell) refers to a cell that does not divide and proliferate but has metabolic activity, thereby helping to proliferate a target cell by producing various metabolites.

The feeder cells may be irradiated Jurkat cells, irradiated EBV-LCL cells (Epstein-Barr virus transformed lymphocyte continuous line), and / or PBMC, HFWT, RPMI 1866, Daudi, MM-170, TCO-2, K562 or The cells may be genetically engineered (eg, K562-mbIL-15-41BB ligand) for K562 cells, but are not limited thereto.

As used herein, the term "Jurkat cell" or "Jurkat cell line" is an immortalized acute T cell leukemia cell line, which is described by Dr. University of California at San Francisco. It is a cell line developed by Arthur Weiss. Cells expressing various chemokine receptors and capable of producing IL-2, which express MHC class I, a natural killer cell inhibitor, on the cell surface, are candidates for feeder cells for anticancer immunotherapy. It was a cell line with no potential. Jurkat cells used in the present invention can be obtained from ATCC (ATCC TIB-152).

As used herein, the term "EBV-LCL cells" or "EBV-LCL cell line" refers to lymphocyte continuous cell lines transformed with Epstein-Barr virus (EBV-LCL, Epstein-Barr virus transformed lymphocyte continuous line, DM Koelle et al., J.). Clin Invest, 1993: 91: 961-968), a B cell line produced by infecting human B cells in vitro with Epstein-Barr Virus. EBV-LCL cells used in the present invention can be prepared and used directly in a conventional laboratory through the method of adding cyclosporin A during the process of infecting EBV in PBMC.

The EBV-LCL cells can be prepared, for example, by the following method:

PXMC of ³⁰ × 10 ⁶ is placed in 9 mL of the culture medium, which is placed in a T25 culture flask, and then 9 mL of the EBV supernatant is added. Add 80 μL of cyclosporin A at a concentration of 50 μg / mL and incubate at 37 ° C. After 7 days of culture, remove the supernatant 1/2, add a new culture medium and add 40μL of cyclosporin A. Repeat the same procedure as once every 7 days until day 28 of culture. Cell lines can be used after 28 days of culture, and from this time, culture is performed without adding cyclosporin A to the culture medium.

The Jurkat cells and EBV-LCL cells can be used as feeder cells through irradiation.

The irradiated Jurkat cells and irradiated EBV-LCL cells were 1: 0.1-5, 1: 0.1-4, 1: 0.1-3, 1: 0.1-2, 1: 0.1-1.5, 1: 0.5-1.5 Or it may be included in the content ratio of 1: 0.75-1.25, but is not limited thereto.

The irradiated Jurkat cells and irradiated EBV-LCL cells treated 50-500, 50-400, 50-300, 50-200, 50-150, 70-130, 80-120 or 90-110 Gy radiation, respectively It can be obtained by, but is not limited thereto.

The culture step is 0-45, 0-42, 0-40, 0-35, 0-30, 0-20, 0-19, 0-18, 0-17, 0-16, 0-15 or 0- It may be performed for 14 days, but is not limited thereto.

The culturing step may further comprise the following steps.

A first culture step of co-culture by adding the feeder cells and the first cytokine; And

A second culture step of co-culture by adding a second cytokine.

The second culture step may be performed by adding the second cytokine one or more times during culture for 0-6 days.

Specifically, the second culture step may be performed by adding a second cytokine once every 0 days and 3 days of culture.

In addition, the second culture step may be performed by adding the second cytokine and feeder cells one or more times for six days in a 14-day cycle of culture.

Specifically, the second culture step may be performed by adding the feeder cells every 14 days of culture, and adding the second cytokine once every 3 and 6 days of each cycle.

The first cytokine may be IL-2, but is not limited thereto.

The second cytokine may be IL-21, but is not limited thereto.

The second cytokine may be used at a concentration of 10-1,000, 10-500, 10-100, 20-100, 30-100, 40-100, 50-100 or 10-50ng / mL, but is not limited thereto. no.

When the second cytokine is cultured by adding at least one time during culture for 0-6 days, it may exhibit better proliferative capacity and anticancer activity.

When the second cytokine and feeder cells are added at least once for 6 days at 14 days of cultivation, the second cytokine and the feeder cells may exhibit better proliferative capacity.

The coculture cultures peripheral blood mononuclear cells and feeder cells (eg, Jurkat cells and EBV-LCL cells) in 1: 1-100, 1: 1-90, 1: 1-80, 1: 1-70, 1 It may be performed by including a mixing ratio of: 10-65, 1: 20-65, 1: 30-65, 1: 40-65, 1: 50-65, or 1: 55-65, but is not limited thereto.

The co-culture can be carried out in a medium, wherein the medium can be used without limitation any conventional medium used for induction and proliferation of peripheral blood mononuclear cells into natural killer cells. As such a medium, for example, RPMI-1640, DMEM, x-vivo 10, x-vivo 20 or cellgro SCGM medium can be used. In addition, the culture conditions such as temperature may follow the culture conditions of ordinary peripheral blood mononuclear cells.

The natural killer cells produced by the production method of the present invention may have a ratio (purity) of CD56 + (and / or CD3- / CD56 +) natural killer cells in total cells of 85%, 90%, 95% or 98% or more.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a method for producing natural killer cells.

(b) Since the production method of the present invention can produce high-purity natural killer cells without T cells and the like without the use of various expensive cytokines, the prevention and treatment of cancer using natural killer cells, in particular allopathic Can enhance the effect.

1 is a result confirming the proliferation ability of NK cells prepared according to an embodiment of the present invention.
Figure 2 is a result confirming the purity of the NK cells prepared according to an embodiment of the present invention.
Figure 3 is a plate designed to confirm the anticancer activity of the NK cells prepared according to an embodiment of the present invention for hematological cancer cells.
Figure 4a is a result of confirming the anticancer activity of the blood cancer cells of the NK cells prepared according to an embodiment of the present invention.
Figure 4b is a result of confirming the anticancer activity of the NK cells prepared according to an embodiment of the solid cancer cells.
Figure 5a is a result confirming the proliferation capacity of the cells according to the cytokine treatment time of the NK cells prepared according to an embodiment of the present invention.
Figure 5b is a result confirming the proliferation capacity of the cells according to the number of cytokine treatment of NK cells prepared according to an embodiment of the present invention.
Figure 6 is a result confirming the proliferation capacity of the cells according to the cytokine treatment concentration of NK cells prepared according to an embodiment of the present invention
Figure 7a is a result confirming the anticancer activity for hematological cancer cells according to the cytokine treatment time of NK cells prepared according to an embodiment of the present invention.
Figure 7b is the result of confirming the anticancer activity for solid cancer cells according to the cytokine treatment time of NK cells prepared according to an embodiment of the present invention.
Figure 7c is the result of confirming the anticancer activity for solid cancer cells according to the number of cytokine treatment of NK cells prepared according to an embodiment of the present invention.
Figure 8a is a result confirming the anticancer activity for hematological cancer cells according to the cytokine treatment concentration of NK cells prepared according to an embodiment of the present invention.
Figure 8b is a result confirming the anticancer activity for solid cancer cells according to the cytokine treatment concentration of NK cells prepared according to an embodiment of the present invention.
Figure 9 is a result confirming the proliferation capacity of the cells according to the number of feeder cell treatment of NK cells prepared according to an embodiment of the present invention.
Figure 10a is a result confirming the proliferative capacity of NK cells prepared using the blood of a cancer patient according to an embodiment of the present invention.
Figure 10b is the result of confirming the purity of NK cells prepared using the blood of cancer patients according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1. Preparation of CD56 + Natural Killer (NK) Cells (IL-2 Treatment)

CD56 + cells and CD3- / CD56 + cells were isolated from PBMC in the following manner. First, PBMCs were separated from blood using the Ficoll-Hypaque density gradient method and the cells were counted.

1-1. Preparation Steps for the Preparation of CD56 + Cells

After adding MACS buffer (1x PBS + 0.5% HSA) to the counted PBMCs, the cells were suspended, and after adding CD56 microbeads (Miltenyi Biotec) to 1-20 μL per PBMC 1.0x10 ⁷ cells, 5- Incubate at 2-8 ° C. for 30 minutes. After incubation, the MACS buffer was added and mixed, followed by centrifugation (600xg) to precipitate the cells.

After centrifugation, the supernatant was removed and the MACS buffer was added to suspend the cells and placed in a column bound to a MACS separator. MACS buffer was passed through the column to remove non-specific binding. The column was separated in a MACS separator, transferred to a 15 mL conical tube, and MACS buffer was added to separate CD56 + cells attached to the column.

1-2. Preparation steps for the preparation of CD3- / CD56 + cells

MACS buffer (1x PBS + 0.5% HSA) was added to the counted PBMCs to suspend the cells, and for 5-30 minutes after the addition of CD3 microbeads (Miltenyi Biotec) to 1-20 μL per PBMC 1.0x10 ⁷ cells. Incubate at 2-8 ° C. After incubation, the MACS buffer was added and mixed, followed by centrifugation (600xg) to precipitate the cells.

After centrifugation, the supernatant was removed and the cells suspended in a MACS buffer and placed in a column bound to the MACS separator. CD3-cells were recovered by passing the MACS buffer through the column.

The cells were suspended by adding MACS buffer (1 × PBS + 0.5% HSA) to the recovered CD3-cells, and after adding CD56 microbeads (Miltenyi Biotec) to 1-20 μL per 1.0 × 10 ⁷ cells of CD3- cells, 5- Incubate at 2-8 ° C. for 30 minutes. After incubation, the MACS buffer was added and mixed, followed by centrifugation (600xg) to precipitate the cells.

After centrifugation, the supernatant was removed and the cells suspended in a MACS buffer and placed in a column bound to the MACS separator. MACS buffer was passed through the column to remove nonspecific binding. The column was separated in a MACS separator, transferred to a 15 mL conical tube, and MACS buffer was added to separate CD3- / CD56 + cells attached to the column.

1-3. Preparation of Natural Killer Cells Using CD56 + Cells and CD3- / CD56 + Cells

CD56 + cells or CD3- / CD56 + cells isolated in Examples 1-1 and 1-2 above were pre-prepared with 100 Gy irradiated feeder cells (Jurkat cells and EBV-LCL cells) at a concentration of 500 IU / mL. It was placed in RPMI-1640 medium containing 10% of added FBS and co-cultured in a 37 ° C., 5% CO ₂ incubator. At this time, (CD56 + cells or CD3- / CD56 + cells) :( Jurkat cells) :( EBV-LCL cells) were cultured at a ratio of 1:30:30.

Example 2. Preparation of CD56 + Natural Killer (NK) Cells (Treatment of IL-2 and IL-21)

In the same manner as in Example 1, except that IL-2 and IL-21 were added at concentrations of 500IU / mL and 50ng / mL, respectively, instead of IL-2 at 500IU / mL. Natural killer cells were prepared.

Comparative Example 1. Preparation of natural killer (NK) cells excluding CD56 + cell separation step (IL-2 treatment)

PBMCs were isolated from blood using the Ficoll-Hypack density gradient separation method and 10% FBS with IL-2 added at 500 IU / mL concentration, together with 100 Gy irradiated feeder cells (Jurkat cells and EBV-LCL cells) prepared in advance It was placed in RPMI-1640 medium containing and co-cultured in 37 ℃, 5% CO ₂ incubator.

Comparative Example 2. Preparation of Natural Killer (NK) Cells Except for CD56 + Cell Separation (Treatment of IL-2 and IL-21)

Natural killing in the same manner as in Comparative Example 1, except that IL-2 and IL-21 were added at a concentration of 500IU / mL and 50ng / mL, respectively, instead of IL-2 at 500IU / mL. Cells were prepared.

Comparative Example 3-4. Preparation of Natural Killer (NK) Cells Except for CD56 + Cell Separation

In Comparative Examples 1 and 2, PBMC: (Jurkat cells) :( EBV-LCL cells) were cultured in the same manner as in Comparative Examples 1 and 2, except that they were cultured at a ratio of 1: 0.5: 0.5. Prepared.

Experimental Example 1. Confirmation of proliferative capacity of NK cells

For each NK cell cultured in a CO ₂ incubator, inoculated into 350 mL bag on the basis of the number of cells 1.0x10 ⁵ -2.0x10 ⁶ / mL on the ^6th day of culture and further incubated for 4 days Further incubation for 4 days after inoculation into 1 L bag based on ⁵ -2.0x10 ⁶ / mL. Finally, on the 14th day of culture, the cells were further cultured for 3-6 days after inoculation into 1L bag based on the cell number of 1.0x10 ⁵ -2.0x10 ⁶ / mL.

As can be seen in Figure 1, the cell number of Comparative Example 1 (PBMC of W / O IL-21) and 2 (PBMC of W / IL-21) is 243 times and 1,248 times, respectively, compared to the number of cells on day 0 culture In contrast, the number of cells in Example 1 (CD56 of W / O IL-21) and 2 (CD56 of W / IL-21) was increased by 2,990 and 20,434 times, respectively, compared to the number of cells on day 0 of culture. there was.

Experimental Example 2. Confirmation of Purity of NK Cells

Each NK cell was washed once with FACS staining buffer and suspended in 100 μL, and then fluorescence-bound CD56 / CD3 / CD20 / CD14 monoclonal antibody was mixed and stored at 2-8 ° C. for 20-30 minutes in dark conditions. . After one additional wash, cells were suspended in 300-500 μL FACS staining buffer, and then 10,000-100,000 cells were obtained per tube using the CD56-FITC / CD3-PE / CD20-PerCP5 / CD14-APC panel of the flow cytometer. The analysis was carried out.

At this time, the purity of CD56 + NK cells was defined as the ratio of cells entering the CD3- / CD56 + region after FSC / SSC gating, and further confirmed that CD20 and CD14 were not expressed in the cells of the CD3- / CD56 + region. .

As can be seen in Figure 2, the purity of NK cells of Comparative Example 1 (PBMC of W / O IL-21) and 2 (PBMC of W / IL-21) is only 84.2% and 84.7%, respectively, Example 1 The purity of the NK cells of (CD56 of W / O IL-21) and 2 (CD56 of W / IL-21) reached 98.6% and 99.2%, respectively.

Experimental Example 3. Confirmation of cancer cell killing ability of NK cells

First, the killing ability against K562 cells (blood cancer, ATCC® CCL-243?), A chronic myeloid leukemia cell line, was confirmed.

K562 cells were suspended in RPMI 1640 medium containing 10% of FBS before being used in experiments and passaged at 3 ± 3 days at 37 ± 1 ° C., followed by incubation for at least 7 days.

The prepared K562 cells were suspended in RPMI-1640 medium at a concentration of 1.0 × 10 ⁶ cells / mL and fluorescent material (Calcein-AM) was added at a concentration of 4 μM. Inverting was performed at 10 minute intervals while staining at 37 ± 1 ° C. for 30 minutes. K562 cells stained with fluorescent material were centrifuged at 3,300 rpm for 3 minutes and washed three times and then suspended in SNK medium containing 10% FBS at a rate of 1.0x10 ⁶ cells / mL. K562 cells were seeded in round bottom microwell plates (96-wells) at 1.0 × 10 ⁴ cells per well.

The Experimental Example 1 14-20 day culture of NK cells (Effector cell) of each 1.0x10 ⁶ cells / mL, 3.0x10 ⁵ cells / mL, 1.0x10 ⁵ cells / mL and 0.5x10 FBS at a rate of ⁵ cells / mL It was suspended and diluted in RPMI-1640 medium containing 10%.

Diluted cells were inoculated with each well for 3 wells (Triplication) at a concentration of 100 μL per well on the plate inoculated with the K562 cells (Target cells). At this time, the ratio of the effector cells and the target cells is shown in Table 1 below.

Effector: Target Effector cell Target cell 10: 1 1.0 x 10 ⁵ 1.0 x 10 ⁴ 3: 1 3.0 x 10 ⁴ 1.0 x 10 ⁴ 1: 1 1.0 x 10 ⁴ 1.0 x 10 ⁴ 0.5: 1 0.5 x 10 ⁴ 1.0 x 10 ⁴

On the other hand, the plate design used in this experiment is shown in Figure 3, the negative control (Spontaneous) added fluorescent stained live K562 cells, the positive control (Maximum release) completely killed K562 cells using TX-100 The maximum fluorescence was shown.

The plates inoculated with the target cells and the effector cells were centrifuged at 1,000 rpm for 5 minutes, incubated for 3-4 hours at 37 ± 1 ° C., and then centrifuged at 1,000 rpm for 5 minutes. After centrifugation, 80 μL of the supernatant was transferred to a black plate (96-well), and the amount of fluorescence was measured by using a fluorescence microplate reader. Calculated by

[Calculation 1]

Cytotoxicity =

As can be seen in Figure 4a and Table 2, Example 1 (CD56 + of W / O IL-21) compared to Comparative Example 3 (PBMC of W / O IL-21) and 4 (PBMC of W / IL-21) And 2 (CD56 + of W / IL-21) showed higher anticancer activity.

E: T ratio 10: 1 3: 1 1: 1 0.5: 1 PBMC (W / O IL-21) 97.5 94.6 75.3 60.0 PBMC (W / IL-21) 102.3 97.1 84.8 66.9 CD56 + (W / O IL-21) 103.3 99.9 83.8 67.4 CD56 + (W / IL-21) 102.9 100.7 87.7 80.0

Next, the killing ability was also confirmed for solid cancer cells reported to have stronger resistance to NK cells. AGS (gastric cancer, ATCC® CRL-1739?), A549 (lung cancer, ATCC® CCL-185?) And MDA-MB-231 (breast cancer, ATCC® HTB-26?) Cells were used as solid cancer cells.

Each solid cancer cell was fluorescently labeled with a CYTO-ID® Green long-term cell tracer kit (Enzo Life Science Inc.), inoculated on a plate and incubated for 24 hours. The next day, the ratio of NK cells to cancer cells was 0.5: 1 and reacted for 48 hours. After 48 hours, the killing ability was confirmed by measuring the number of green fluorescence expressing cells using flow cytometry.

As can be seen in Figure 4b, in solid cancer cells compared to Comparative Example 1 (PBMC of W / O IL-21) and 2 (PBMC of W / IL-21) Example 1 (CD56 of W / O IL-21) and When cultured by the method of 2 (CD56 of W / IL-21) it can be seen that the higher anticancer activity.

Experimental Example 4. Confirmation of proliferative capacity of NK cells according to the time and number of treatment of IL-21

First, in order to evaluate the proliferative capacity of NK cells according to the treatment time of IL-21, further experiments were conducted as follows.

Preparation of CD56 + NK cells by the method of Example 1, the culture 0-6 days (D0-6), 6-10 days (D6-10), 10-14 days (D10-14) and 14-17 days ( D14-17) was compared to the proliferative capacity of NK cells prepared by treating IL-21 at a concentration of 50ng / mL using the method of Experimental Example 1 above.

The D0-6 group was treated twice a day for each 0 and 3 days of culture, the D6-10 group once every 6 days, the D10-14 group once every 10 days, and the D14-17 group was cultured 14 Treatment was once a day. At this time, the group not treated with IL-21 was used as a control.

As can be seen in Figure 5a and Table 3, the D10-14 and D14-17 group showed no difference in proliferative capacity compared to the control group, while the D0-6 and D6-10 group showed higher proliferative capacity than the control group, In particular, the highest proliferation fold was shown in the group treated with IL-21 (D0-6) at 0-6 days of incubation.

Hyperplasia control D0-6 D6-10 D10-14 D14-17 Donor 1 2996 21859 6388 2894 2330

Next, based on the above results, further experiments were carried out as follows to evaluate the proliferative capacity of NK cells according to the number of treatment of IL-21 in culture 0-6 days.

Preparation of CD56 + NK cells by the method of Example 1, the concentration of 50ng / mL in culture 0-3 days (D0-3), 3-6 days (D3-6) and 0-6 days (D0-6) The proliferative capacity of NK cells prepared by treatment with IL-21 was compared using the method of Experimental Example 1 above.

The D0-3 group was treated once on day 0 of cultivation, the D3-6 group was treated once on day 3 of cultivation, and the D0-6 group was treated twice on day 0 and day of cultivation each. At this time, the group not treated with IL-21 was used as a control.

As can be seen in Figure 5b and Table 4, the initial cultivation was increased in fold growth compared to the control group in all groups treated with IL-21 irrespective of the timing and frequency, especially IL-21 at 0-6 days 2 The highest proliferation fold was shown in the group treated twice (D0-6).

Hyperplasia control D0-3 D3-6 D0-6 Donor 1 2996 16420 4360 21859

Experimental Example 5. Confirmation of proliferation ability of NK cells according to the concentration of IL-21 treatment

Preparation of CD56 + NK cells by the method of Example 1, IL-21 at a concentration of 0ng / mL, 10ng / mL, 30ng / mL, 50ng / mL and 100ng / mL, respectively on the 0 and 3 days of the initial culture The proliferative ability of NK cells prepared by two treatments in total was compared using the method of Experimental Example 1.

As can be seen in FIG. 6, even if only 10ng / ml of IL-21 was treated, the proliferation fold of NK cells was increased compared to the case of not treating IL-21 (0ng / ml), and the concentration of 10ng / mL-50ng / mL was increased. It was found that the proliferation ratio of NK cells also increased with the concentration of IL-21. However, it can be seen that there is no significant difference in the proliferation rate when IL-21 is treated at a concentration of 50ng / mL when treated with 100ng / mL.

Experimental Example 6. Confirmation of cancer cell killing ability of NK cells according to the treatment time and frequency of IL-21

First, in order to evaluate the cancer cell killing ability of NK cells according to the treatment time of IL-21, further experiments were conducted as follows.

Preparation of CD56 + NK cells by the method of Example 1, the culture 0-6 days (D0-6), 6-10 days (D6-10), 10-14 days (D10-14) and 14-17 days ( D14-17) NK cells prepared by treatment with IL-21 at a concentration of 50 ng / mL, the killing ability of blood cancer cells (K562 cells, CCL-243?) Was compared using the method of Experimental Example 3 .

The D0-6 group was treated twice a day for each 0 and 3 days of culture, the D6-10 group once every 6 days, the D10-14 group once every 10 days, and the D14-17 group was cultured 14 Treatment was once a day. At this time, the group not treated with IL-21 was used as a control.

As can be seen in Figure 7a and Table 5, it was found that most of the NK cells treated with IL-21 except for the D10-14 group shows a higher anticancer activity than the control.

E: T ratio 10: 1 3: 1 1: 1 0.5: 1 Control (No treat) 98.8 96.9 73.1 55.1 D0-6 98.6 97.0 77.8 68.4 D6-10 96.78 99.1 80.3 69.8 D10-14 98.4 96.6 68.5 52.4 D14-17 104.5 98.8 79.1 68.8

In addition, the NK cells of each group prepared above, the killing ability against solid cancer cells were compared using the method of Experimental Example 3. AGS (CRL-1739?), A549 (CCL-185?), And MDA-MB-231 (HTB-26?) Cells were used as solid cancer cells.

As shown in FIG. 7b, it was found that NK cells of the group treated with IL-21 (D0-6) showed the highest anticancer activity in all carcinomas at the initial stage of culture.

Next, based on the above results, further experiments were carried out as follows to evaluate the cancer cell killing ability of NK cells according to the number of treatment of IL-21 in culture 0-6 days.

Preparation of CD56 + NK cells by the method of Example 1, the concentration of 50ng / mL in culture 0-3 days (D0-3), 3-6 days (D3-6) and 0-6 days (D0-6) The killing ability of NK cells prepared by treating IL-21 with respect to solid cancer cells was compared using the method of Experimental Example 3. AGS (CRL-1739?), A549 (CCL-185?), And MDA-MB-231 (HTB-26?) Cells were used as solid cancer cells.

The D0-3 group was treated once on day 0 of cultivation, the D3-6 group was treated once on day 3 of cultivation, and the D0-6 group was treated twice on day 0 and day of cultivation each. At this time, the group not treated with IL-21 was used as a control.

As can be seen in Figure 7c, regardless of the timing and frequency of the initial culture was found to show a high anti-cancer activity in all groups treated with IL-21 compared to the control.

Experimental Example 7. Confirmation of cancer cell killing ability of NK cells according to the concentration of IL-21 treatment

First, CD56 + NK cells were prepared by the method of Example 1, but IL- at concentrations of 0 ng / mL, 10 ng / mL, 30 ng / mL, 50 ng / mL and 100 ng / mL, respectively, on the 0 and 3 days of culture. Killing ability of blood cancer cells (K562 cells, CCL-243?) Of NK cells prepared by treating a total of 21 two times was confirmed using the method of Experimental Example 3.

As can be seen in Figure 8a, most of the NK cells treated with IL-21 showed high cytotoxicity compared to NK cells not treated with IL-21, but in the conditions treated with IL-21 at 100ng / ml There was no difference.

Next, CD56 + NK cells were prepared by the method of Example 1, but IL-21 was totally added at concentrations of 0 ng / mL, 10 ng / mL, 30 ng / mL, and 50 ng / mL, respectively, on the 0 and 3 days of culture. The killing ability of solid tumor cells of NK cells prepared by two treatments was confirmed using the method of Experimental Example 3. AGS (CRL-1739?), A549 (CCL-185?), And MDA-MB-231 (HTB-26?) Cells were used as solid cancer cells.

As can be seen in Figure 8b, NK cells cultured by treating 50-ng / mL IL-21 in solid cancer cells also showed the highest anti-cancer activity.

Experimental Example 8. Confirmation of NK cell proliferation capacity according to the number of feeder cell treatment

In order to see if the growth of NK cells can be continued by treating the feeder cells several times, the feeder cells were treated at intervals of 14 days for NK cells in culture to observe NK cell growth rate for 42 days.

At this time, in order to check whether the growth rate of NK cells increased further according to the treatment of IL-21, 3 days for 6 days (each 0-6 days, 14-20 days and 28-34 days) at the time of treating the feeder cells IL-21 was treated at a concentration of 50 ng / mL twice at intervals.

As can be seen in Figure 9, when treated with two or more feeder cells and IL-21 together, the proliferation fold of NK cells significantly increased, the number of cells at 42 days of culture compared to the number of cells at day 0 of culture The group treated with -21 (approximately 3.4x10 ¹⁰ fold) was increased compared to the group not treated with IL-21 (approximately 5.3x10 ⁸ fold).

Experimental Example 9. Confirmation of NK cell culture effect using blood of some cancer patients

After culturing for 17 days in the method of Example 1 except for using PBMC of colorectal cancer patients to prepare CD56 + NK cells, the proliferation capacity and purity of the prepared NK cells using the methods of Experimental Examples 1 and 2 Measured.

At this time, in order to confirm the effects of the treatment of IL-21, IL-21 was treated at a concentration of 50ng / mL for some experimental groups (two times on the 0 and 3 days of culture).

As can be seen in Figure 10a, the number of NK cells not treated with IL-21 increased 8-fold compared to the number of cells on day 0, whereas the number of NK cells when treated with IL-21 and the number of cells on day 0 It can be seen that the increase is 1,461 times.

In addition, as can be seen in Figure 10b, the purity of NK cells not treated with IL-21 is only 84.2%, the purity of NK cells when treated with IL-21 was found to reach 99.19%.

In summary, it is expected that NK cells can be produced even in some cancer patients in which the growth of NK cells is poor when the method of the present invention is used.

Claims (15)

Natural killer cell production method comprising the following steps:
A first separation step of separating peripheral blood mononuclear cells (PBMCs) from the blood sample;
A second separation step of separating any one or more selected from the group consisting of CD56 + cells and CD3- / CD56 + cells; And
A culture step of co-culturing by adding a feeder cell and a cytokine. The method of claim 1, wherein the second separation step is performed using at least one selected from the group consisting of CD56 microbeads and CD3 microbeads. The method of claim 1, wherein the cytokine is IL-2, IL-21, IL-15, Flt3-L, SCF, IL-7, IL-18, IL-4, type I interferons, GM-CSF and IGF 1 At least one selected from the group consisting of: The method of claim 1, wherein the cytokine is added at a concentration of 50-1,000 IU / mL. The method of claim 1, wherein the feeder cells comprise irradiated Jurkat cells and irradiated EBV-LCL cells (Epstein-Barr virus transformed lymphocyte continuous line). The method of claim 5, wherein the irradiated Jurkat cells and irradiated EBV-LCL cells are included in a content ratio of 1: 0.1-5. The method of claim 5, wherein the irradiated Jurkat cells and irradiated EBV-LCL cells are each obtained by treating 50-500 Gy radiation. The method of claim 1, wherein the culturing step is performed for 0-45 days. The method of claim 1, wherein the culturing step comprises the following steps:
A first culture step of co-culture by adding the feeder cells and the first cytokine; And
A second culture step of co-culture by adding a second cytokine. 10. The method of claim 9, wherein the second culturing step is adding at least one second cytokine for 0-6 days of culture. The method of claim 10, wherein the second culturing step is to add the second cytokine and feeder cells one or more times for six days every 14 days of culture. 12. The method of any one of claims 9-11, wherein the first cytokine is IL-2. The method of any one of claims 9-11, wherein the second cytokine is IL-21. The method of claim 9, wherein the second cytokine is added at a concentration of 10-50 ng / mL. The method according to claim 1, wherein the co-culture is carried out in a mixing ratio of 1: 1-100 with any one or more cells selected from the group consisting of CD56 + cells and CD3- / CD56 + cells.

Images