Method for enhancing ability of natural killer cells to migrate to cancer cells or kill cancer cells, and pharmaceutical composition for tumor treatment comprising culture of natural killer cells

Abstract

The present invention provides a method for enhancing the ability of natural killer cells to migrate to cancer cells or to kill cancer cells, which comprises culturing natural killer cells in a medium containing a culture obtained by culturing activated natural killer cells in a serum-free medium and then removing cells and cell debris. In addition, the present invention provides a pharmaceutical composition for treating tumors through inhibition of tumor growth or tumor metastasis, which comprises the culture as an active ingredient.

Description

Method for improving the ability of natural killer cells to migrate toward cancer cells or to kill cancer cells, and pharmaceutical composition for treating tumors comprising a culture of natural killer cells

[0001] The present invention relates to a method for enhancing the ability of natural killer cells to migrate toward cancer cells or to kill cancer cells, and a pharmaceutical composition for treating tumors comprising a culture of natural killer cells. More specifically, the present invention provides a method for enhancing the ability of natural killer cells to migrate toward cancer cells or to kill cancer cells, the method comprising culturing activated natural killer cells in a serum-free medium, and then culturing the natural killer cells in a medium containing the culture obtained by removing cells and cell debris, and a pharmaceutical composition for treating tumors by inhibiting tumor growth or tumor metastasis, the method comprising the culture as an active ingredient.

[0002] Natural killer cells (NK cells) are immune cells that function as the body's first line of defense within the innate immune system. They contain receptors that recognize abnormal cells and, without specific antigens, instantly detect and eliminate abnormal cells, such as cancer cells or viruses. This helps regulate the immune system and effectively suppresses cancer cell proliferation, recurrence, and metastasis.

[0003] Taking advantage of the aforementioned characteristics of natural killer cells, research into cancer immunotherapy using natural killer cells has recently become active. Among cancer immunotherapy methods, immunocytotherapy refers to a treatment that utilizes immune cells in the body, genetically engineered, and then reintroduced. The goal of cancer immunotherapy is to enhance cellular immunity, but chimeric antigen receptor T cells (CAR-T cells) have the disadvantages of complex genetic manipulation and high treatment costs. Furthermore, the characteristics of T cells can cause cytokine release syndrome and various side effects. To overcome these issues, natural killer cells are receiving renewed attention in the field of cancer immunotherapy.

[0004] The present inventors have shown that activated natural killer cells treated with polyethylenimine (chemical priming) have high anti-tumor activity and can be useful for tumor immunotherapy (Choi SH, et al. Chemical priming of natural killer cells with branched polyethylenimine for cancer immunotherapy. Journal for ImmunoTherapy of Cancer. 2022;10:e004964 and Republic of Korea Patent Publication No. 10-2022-0057359).

[0005] Meanwhile, to apply natural killer cells to immunotherapy, it is necessary to stably expand and culture natural killer cells or activated natural killer cells (e.g., activated natural killer cells treated with polyethyleneimine) while minimizing the risk of contamination. Proliferation and culture of natural killer cells or activated natural killer cells involves the use of a serum-containing medium containing approximately 12.5% ​​fetal bovine serum (FBS). However, as a protein source of animal origin, FBS poses safety concerns, such as cross-species contamination. The international community, including the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) of the European Union, recommends avoiding the use of FBS and developing alternatives. Furthermore, not only is there a risk of contamination from other factors, such as animal diseases like mad cow disease or viruses, but the process is often complicated due to the difficulty in purifying serum components.

[0006] Moreover, even when natural killer cells or activated natural killer cells are applied to immunotherapy, their anti-tumor activity remains unsatisfactory. Therefore, the development of methods to enhance the anti-tumor activity of natural killer cells is urgently needed in the art.

[0007] The present inventors conducted various studies to develop a method for enhancing the anti-tumor activity of natural killer cells. The present inventors discovered that when activated natural killer cells treated with polyethyleneimine (chemical priming) were cultured in serum-free medium, and then cultured in a medium containing the resulting culture, which was then purified of cells and cell debris, the resulting natural killer cells exhibited enhanced cancer cell migration and / or cancer cell killing abilities. Furthermore, the present inventors discovered that the culture exhibited excellent tumor growth and / or tumor metastasis inhibition activities, making it suitable for use as a pharmaceutical composition for tumor treatment, thereby fundamentally avoiding safety issues associated with cell administration.

[0008] Accordingly, the present invention aims to provide a method for improving the ability of natural killer cells to migrate toward cancer cells or to kill cancer cells, which comprises culturing natural killer cells in a medium containing the culture.

[0009] In addition, the present invention aims to provide a pharmaceutical composition for treating tumors by suppressing tumor growth or tumor metastasis, which comprises the culture as an effective ingredient.

[0010] According to one aspect of the present invention, a method is provided for improving the ability of natural killer cells to migrate toward cancer cells or to kill cancer cells, comprising the steps of: (a) culturing natural killer cells in a first medium containing serum and polyethyleneimine, and then culturing them in a second medium that is serum-free; and (b) culturing natural killer cells in a third medium containing a culture obtained by removing cells and cell debris from the culture solution obtained in step (a).

[0011] According to another aspect of the present invention, a pharmaceutical composition for treating a tumor by inhibiting tumor growth or tumor metastasis is provided, comprising a culture obtained by a manufacturing method including the steps of (a) culturing natural killer cells in a first medium containing serum and polyethyleneimine and then culturing the cells in a second serum-free medium; and (b') removing cells and cell debris from the culture solution obtained in step (a) to obtain a culture, as an active ingredient.

[0012] According to the present invention, it has been discovered that when activated natural killer cells treated with polyethyleneimine (chemical priming) are cultured in a serum-free medium, and then cultured in a medium containing the resulting culture by removing cells and cell debris, the resulting natural killer cells exhibit enhanced ability to migrate toward cancer cells and / or to kill cancer cells. Therefore, the natural killer cells obtained by the method according to the present invention can be usefully used as a cell therapy agent for immunotherapy of cancer.

[0013] Furthermore, the present invention has revealed that the cultured product exhibits excellent tumor growth and / or tumor metastasis-inhibiting activity, and thus can be used as a pharmaceutical composition for tumor treatment. Therefore, the pharmaceutical composition according to the present invention can be effectively used as a tumor treatment agent, fundamentally avoiding safety issues associated with cell administration.

[0014] Figure 1 is a schematic diagram showing the manufacturing process of a culture of natural killer cells (secretome), including a culture of natural killer cells (Chem_NK culture) according to the present invention.

[0015] Figure 2 shows the results obtained by evaluating the ability of natural killer cells to migrate toward cancer cells and to kill cancer cells in an in vitro test in the presence of a culture of natural killer cells (C_NK culture or Chem_NK culture).

[0016] Figure 3 shows the results obtained by evaluating the effect on endogenous natural killer cells in an in vivo test when tumor cells were injected after repeated administration of a culture of natural killer cells (C_NK culture or Chem_NK culture).

[0017] Figure 4 shows the results obtained by evaluating the effects on cancer cell growth and immune activation through in vivo testing when a culture of natural killer cells (C_NK culture or Chem_NK culture) was repeatedly administered to tumor-implanted mice.

[0018] Figure 5 shows the results of cytokine array analysis for cultures of natural killer cells (C_NK culture or Chem_NK culture).

[0019] The present invention provides a method for improving the ability of natural killer cells to migrate toward cancer cells or to kill cancer cells, comprising the steps of: (a) culturing natural killer cells in a first medium containing serum and polyethyleneimine, and then culturing them in a second serum-free medium; and (b) culturing natural killer cells in a third medium containing a culture obtained by removing cells and cell debris from the culture solution obtained in step (a).

[0020] Culturing in the first medium can be performed according to a known method in a natural killer cell culture medium containing serum and polyethylenimine. For example, culturing in the first medium can be performed according to the method disclosed in Choi SH, et al. Chemical priming of natural killer cells with branched polyethylenimine for cancer immunotherapy. Journal for ImmunoTherapy of Cancer . 2022;10:e004964 and / or Korean Patent Publication No. 10-2022-0057359. In one embodiment, culturing in the first medium can be performed in a-MEM medium supplemented with L-glutamine, inositol, 2-mercaptoethanol, folic acid, fetal bovine serum, and 1% penicillin / streptomycin. Polyethylenimine can have a molecular weight of 10 kDa to 30 kDa, for example, can have a molecular weight of about 25 kDa. Polyethyleneimine may be present in the first medium in an amount of 1 μg / ml to 10 μg / ml, for example, 5 μg / ml. Fetal bovine serum may be present in an amount of 5 to 20 w / v%, for example, about 12.5 w / v%. Other supplements, such as L-glutamine, may be present in amounts commonly used in the field of natural killer cell culture. Culturing in the first medium may be performed, for example, at 37°C for 12 hours, but is not limited thereto.

[0021] The culture in the above serum-free second medium can be performed in a conventional cell culture medium that does not contain serum, and for example, can be performed in a conventional cell culture medium such as RPMI 1640 medium. The culture in the above serum-free second medium can be performed for 24 hours at 37°C, for example, but is not limited thereto.

[0022] In the method of the present invention, the culture can be obtained by removing cells and cell debris from the culture solution obtained in step (a). The removal of the cells and cell debris can be performed by centrifuging to obtain a supernatant according to a conventional method. In addition, if necessary, the obtained supernatant can be filtered through a conventional membrane filter (i.e., a 0.22 μm membrane filter). In addition, if necessary, the supernatant or filtrate can be concentrated or lyophilized by a conventional method. Therefore, in the present invention, the culture can be a supernatant obtained by centrifuging the culture solution obtained in step (a); a filtrate obtained by filtering the supernatant; a concentrate of the supernatant or filtrate; or a lyophilized product of the supernatant or filtrate.

[0023] In the cultivation of natural killer cells in the third medium, the natural killer cells may be the same natural killer cells as the natural killer cells used in step (a) or different natural killer cells (e.g., human-derived natural killer cells used clinically for immune cell therapy). The cultivation of natural killer cells in the third medium may be performed in a conventional cell culture medium that includes the culture and does not include serum, and may be performed in a conventional cell culture medium such as RPMI 1640 medium including the culture. The amount of the culture may be appropriately determined depending on the amount of natural killer cells inoculated into the third medium and the form of the culture (supernatant, supernatant filtrate, or concentrate thereof, etc.). For example, 5 x 10 5 For natural killer cells inoculated at a cell / ml concentration, the culture may be present in the form of a 10-fold concentrate at an amount of about 700 to 900 μg / mL. Culturing of natural killer cells in the third medium may be performed, for example, at 37°C for 24 hours, but is not limited thereto.

[0024] The present invention also provides a pharmaceutical composition for treating a tumor by inhibiting tumor growth or tumor metastasis, comprising a culture obtained by a manufacturing method including the steps of (a) culturing natural killer cells in a first medium containing serum and polyethyleneimine and then culturing the cells in a second serum-free medium; and (b') removing cells and cell debris from the culture solution obtained in step (a) to obtain a culture, as an active ingredient.

[0025] In the pharmaceutical composition of the present invention, steps (a) and (b') can be performed in substantially the same manner as steps (a) and (b) in the method for improving the ability of natural killer cells to migrate toward cancer cells or to kill cancer cells.

[0026] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, and may be formulated into a parenteral dosage form such as a solution, suspension, emulsion, or lyophilisate according to a conventional method. The pharmaceutically acceptable carrier includes an aqueous diluent or solvent such as phosphate buffered saline, purified water, or sterile water, and a non-aqueous diluent or solvent such as propylene glycol, polyethylene glycol, or olive oil. In addition, the composition may include a wetting agent, a fragrance, a preservative, or the like, as needed. The culture contained in the pharmaceutical composition may vary depending on the patient's condition and weight, the degree of disease, the route of administration, and the period of administration, but may be appropriately selected by a person skilled in the art. For example, the culture may be administered at a dose of 0.5 to 5.0 mg / kg per day, and the administration may be administered once a day or in several divided doses.

[0027] Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0028] Example 1: Culture of natural killer cells (C_NK culture and Chem_NK culture)

[0029] Human natural killer cell NK92MI cells (ATCC) were cultured at 5 x 10 in a-MEM medium supplemented with 2 mM L-glutamine (Gibco / Life Technologies), 0.2 mM inositol (Sigma-Aldrich, St. Louis, USA), 0.1 mM 2-mercaptoethanol (Gibco / Life Technologies), 0.02 mM folic acid (Sigma-Aldrich), 12.5% ​​fetal bovine serum, and 1% penicillin / streptomycin (Gibco / Life Technologies). 5 Cells were inoculated at a concentration of 10 cells / ml. Control NK (C_NK) was not treated additionally, and Chem_NK was additionally treated with 25 kb polyethyleneimine at a concentration of 5 μg / ml, and cultured at 37°C for 12 hours, respectively. After harvesting C_NK and Chem_NK, 5 x 10 5 The cultures were inoculated at a concentration of 10 cells / ml and cultured at 37°C for 24 hours. Each culture of C_NK and Chem_NK was centrifuged at 1300 rpm for 10 minutes, and the supernatants were filtered through a 0.22 μm filter (#SLGPR33RS) to remove cells and cell debris, and then concentrated 2-fold or 10-fold by volume using Pierce™Protein Concentrator PES, 3K MWCO (#88526) (4°C, 8000 g) to obtain 2X or 10X C_NK cultures and 2X or 10X Chem_NK cultures, respectively. (Fig. 1)

[0030] Example 2: Evaluation of the ability of natural killer cells to migrate toward cancer cells and kill cancer cells treated with C_NK culture or Chem_NK culture (in vitro test)

[0031] (1) Natural killer cells treated with C_NK culture or Chem_NK culture

[0032] NK92MI cells (ATCC), which are human natural killer cells, were seeded at 5 x 10 in serum-free medium (RPMI 1640 Medium, no phenol red, #11835030) containing the C_NK culture obtained in Example 1. 5 After inoculating at a concentration of 1 cell / ml and culturing at 37°C for 24 hours, natural killer cells (hereinafter referred to as ‘C-NK CM cells’) were harvested.

[0033] In addition, NK92MI cells (ATCC), which are human natural killer cells, were seeded at 5 x 10 in serum-free medium (RPMI 1640 Medium, no phenol red, #11835030) containing the Chem_NK culture obtained in Example 1. 5 After inoculating at a concentration of 1 cell / ml and culturing at 37°C for 24 hours, natural killer cells (hereinafter referred to as ‘Chem_NK CM cells’) were harvested.

[0034] For comparison, 5 x 10 human natural killer cells, NK92MI cells (ATCC), were seeded in serum-free medium (RPMI 1640 Medium, no phenol red, #11835030) of Example 1. 5 After inoculating at a concentration of 10 cells / ml and culturing at 37°C for 24 hours, the harvested natural killer cells were referred to as ‘Media cells.’

[0035] (2) Evaluation of migration ability toward cancer cells

[0036] To evaluate the mobility toward cancer cells, a transwell assay was performed using a 24-well insert transwell chamber (Corning, #353097) with a pore size of 8.0 μm. MDA-MB231 cells (ATCC), a human breast cancer cell line, were used as the cancer cells, and 5 x 10 5 Cells were seeded and cultured at 37°C for 24 h. Media cells, C-NK CM cells, and Chem_NK CM cells were labeled with 1 μM carboxy fluorescein succinimidyl ester (CFSE, #C34554) and seeded at 5 x 10 in the upper chamber (insert). 5 Each cell was inoculated, and all media (upper and lower chambers) used at this time were serum-free DMEM (#11995073). After incubation at 37℃ for 18 hours, CFSE-labeled natural killer cells that migrated to the lower chamber were harvested and counted using a Luna cell counter. In addition, the migrated CFSE-labeled natural killer cells were photographed using an EVOS microscope (M5000). As can be confirmed from the results in A and B of Fig. 2, natural killer cells cultured in the presence of Chem_NK culture medium (Chem_NK CM cells) showed significantly enhanced migration ability toward cancer cells.

[0037] (3) Evaluation of cancer cell apoptotic ability

[0038] To evaluate the cancer cell killing ability, Caspase-3 / 7 activity assay was performed using CellEvent caspase-3 / 7 green detection kit (#C10723). 1 x 10 human breast cancer cells (ATCC) MDA-MB231 cells were seeded in a 96-well plate. 4Dog, Media cells, C-NK CM cells, and Chem_NK CM cells were labeled with CellTrace far-red reagent (#C34564) and 2 x 10 4 Dogs were co-cultured. The co-culture medium used was DMEM (#11995073) containing 10% FBS. Afterwards, 5 μM CellEvent caspase-3 / 7 green detection reagent was added to the co-culture medium, and the fluorescence of caspase activity was captured using a confocal microscope for 12 hours at 37°C and quantified. As can be confirmed from the results in Figures 2A and B, natural killer cells (Chem_NK CM) cultured in the presence of Chem_NK culture medium showed significantly enhanced cancer cell killing ability.

[0039] Example 3: Evaluation of the effect on endogenous natural killer cells (in vivo test)

[0040] To determine whether NK cell cultures could affect endogenous NK cells in an in vivo setting, tumor cells were injected and compared with endogenous NK cells infiltrated into tumor cells. Balb / c nude mice (5 weeks old) with functional T cell deficiency were intraperitoneally administered once daily for 3 days with 200 μl of DPBS, 200 μl of 2X C_NK culture, or 200 μl of 2X Chem_NK culture. One day after the 3-day intraperitoneal administration, 2 x 10 mouse breast cancer cells EO771 (ATCC) were injected. 5 The dog was mixed with Matrigel (#354230) (DPBS:Matrigel ratio = 1) and transplanted into the fourth mammary fat pad of the mouse (orthotopic modeling). (Fig. 3A)

[0041] Body weight was measured twice a week and tumor volume was measured using a vernier caliper. Tumor volume (mm) 3 ) is (short for 2 x long axis) x 0.52 was calculated as follows. The tumor size was approximately 700 mm 3 Seventeen days later, mice were sacrificed and tumors were removed. As a result, there was no change in mouse body weight during the experimental period, and the weight of the formed tumors also did not differ between treatment groups. (Figures 3B to E)

[0042] Tumor cells isolated from tumor tissue through a single-cell suspension process were resuspended in FACS buffer (0.09% sodium azide and 2% FBS in DPBS). 1 x 10 6 After staining the cells with appropriate antibodies, endogenous NK cells within the tumors were quantified by flow cytometry. As antibodies, the Zombie Aqua™ Fixable Viability Kit (#423102; BioLegend, San Diego, CA) was used to distinguish Live / Dead cells, and anti-mouse CD45 antibody (#103114, BioLegend) and anti-mouse CD335 (NKp46) antibody (#137608, Biolegend) were used to distinguish immune cells and NK cells. As can be confirmed from Fig. 3F, the amount of mouse NK cells present within the tumors was significantly higher in the group treated with Chem_NK culture.

[0043] From the above results, it can be confirmed that administration of Chem_NK culture improves the homing ability to tumors by affecting endogenous natural killer cells.

[0044] Example 4: Evaluation of the effect on lung metastasis and immune activation of cancer cells (in vivo test)

[0045] To determine whether natural killer cell cultures could influence the immune system in an intact in vivo environment, tumors were implanted in C57BL / 6 mice (6 weeks old), and tumorigenicity and immune activation in the lungs were evaluated after repeated administration of natural killer cell cultures. 5 x 10 of mouse melanoma cells, B16F10_Luc (ATCC), were injected. 5 Mice were intravenously injected. One day after tumor modeling, 300 μl of DPBS, 300 μl of 2X C_NK culture, or 300 μl of 2X Chem_NK culture were intraperitoneally administered three times a week. Body weights were measured twice a week, and mice were sacrificed 14 days after cancer cell injection. (Fig. 4A)

[0046] No change in mouse body weight was observed during the test period (Fig. 4B).

[0047] As a result of photographing the lungs extracted from the sacrificed mice, the number of cancer nodules formed in the lungs, i.e., melanoma nodules formed as a result of cancer cell metastasis, was significantly lower in the Chem_NK CM treatment group (C in Fig. 4).

[0048] In addition, cells isolated from spleen tissue through a single-cell suspension process were resuspended in FACS buffer (0.09% sodium azide and 2% FBS in DPBS). 1 x 10 6After staining the cells with appropriate antibodies, immune cells were analyzed by flow cytometry. The Zombie Aqua™ Fixable Viability Kit (#423102; BioLegend, San Diego, CA) was used to distinguish live / dead cells, anti-mouse CD45 antibody (#103114, BioLegend) was used to distinguish immune cells, and anti-mouse NK-1.1 antibody (#108724, BioLegend) and anti-mouse CD335 (NKp46) antibody (#137608, Biolegend) were used to distinguish NK cell populations. As a result, in the spleen tissue of the Chem_NK culture treatment group, the amount of mouse NK cell group expressed as NK1.1(+) among the immune cells expressed as CD45(+), and the amount of activated NK cells expressed as NKp46(+) among the NK1.1(+) NK cells were significantly higher (Fig. 4D).

[0049] From the above results, it can be confirmed that when Chem_NK culture is administered, natural killer cells in spleen tissue are activated and lung metastasis of tumors is suppressed.

[0050] Example 5: Cytokine Array Analysis of C_NK and Chem_NK Cultures

[0051] To determine the composition of C_NK and Chem_NK cultures, cytokines in each culture were analyzed using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems, Minneapolis, MN, USA), which can analyze 105 cytokines and chemokines. The analysis was performed according to the manufacturer's protocol. Briefly, the membrane blot was blocked for 1 hour, and then 500 μl of C_NK and Chem_NK cultures were incubated overnight at 4°C. After washing away any remaining material on the membrane, Streptavidin-HRP was added. Finally, after reacting with a chemiluminescent substrate, the intensity of each spot was detected and compared. Spot intensities were quantified using ImageJ, and 27 cytokines and chemokines were confirmed to be increased in Chem_NK cultures based on spots with an intensity value of 3000 or higher (Fig. 5A). Gene Ontology (GO) enrichment analysis performed using DAVID suggests that these increased proteins may be associated with various signaling pathways, including the ERK pathway and the JAK / STAT pathway (Fig. 5B and C).

Claims

1. (a) a step of culturing natural killer cells in a first medium containing serum and polyethyleneimine, and then culturing them in a second serum-free medium; and (b) A step of culturing natural killer cells in a third medium containing a culture obtained by removing cells and cell debris from the culture solution obtained in step (a). A method for improving the ability of natural killer cells to migrate toward cancer cells or to kill cancer cells, including:

2. A method according to claim 1, characterized in that the polyethyleneimine is present in the first medium in an amount of 1 ㎍ / ml to 10 ㎍ / ml.

3. A method according to claim 1, characterized in that the culture is a supernatant obtained by centrifuging the culture solution obtained in step (a); a filtrate obtained by filtering the supernatant; a concentrate of the supernatant or filtrate; or a lyophilized product of the supernatant or filtrate. 4.(a) A step of culturing natural killer cells in a first medium containing serum and polyethyleneimine, and then culturing them in a second serum-free medium; and (b') A step of obtaining a culture by removing cells and cell debris from the culture solution obtained in step (a). A pharmaceutical composition for treating a tumor by inhibiting tumor growth or tumor metastasis, comprising a culture obtained by a manufacturing method including the above as an active ingredient.

5. A pharmaceutical composition according to claim 4, characterized in that the polyethyleneimine is present in the first medium in an amount of 1 ㎍ / ml to 10 ㎍ / ml.

6. A pharmaceutical composition characterized in that, in the fourth paragraph, the culture is a supernatant obtained by centrifuging the culture solution obtained in step (a); a filtrate obtained by filtering the supernatant; a concentrate of the supernatant or filtrate; or a lyophilized product of the supernatant or filtrate.

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