Method for culturing NK cells

Abstract

To provide an NK cell culturing method that can improve NK cell amplification efficiency while maintaining high purity.SOLUTION: An NK cell culturing method includes a vessel preparation step for preparing a culture vessel coated with an anti-human NKp46 antibody, a step for removing T-cells from hematopoietic mononuclear cells, and a culture step for suspending the hematopoietic mononuclear cells cleared of the T-cells in a serum-free medium containing very high concentrations of IL-2 and IL-18, to make a cell suspension, and adding the cell suspension to the culture vessel for cell culturing.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to a method for culturing NK cells. [Background technology]

[0002] In recent years, immunotherapy, which involves infusing peripheral blood cells amplified outside the body back into the body, has attracted attention as a method of cancer treatment. This method treats cancer by enhancing the patient's immune response to cancer cells.

[0003] As an immunotherapy utilizing the cytotoxic activity of killer cells, LAK therapy using lymphokine-activated killer cells is known (Patent Document 1). This LAK therapy involves culturing cells isolated from the patient's peripheral blood mononuclear cells (PBMCs) in the presence of interleukin-2 (IL-2), and then infusing the activated cells into the patient's body along with IL-2. Another known therapy involves culturing lymphocytes isolated from cancer tissue in the presence of IL-2, and then infusing the activated cells into the patient's body along with IL-2. Furthermore, cytotoxic T lymphocyte (CTL) therapy, which uses CD8-positive killer cells that exhibit cytotoxicity by molecularly targeting cancer-specific antigens (cancer antigens), and biologic response modifier (BRM) activated killer cell (BRM) therapy, which involves selecting cells that specifically damage cancer cells from lymphocytes isolated from the peripheral blood of cancer patients, enhancing their cytotoxicity with IL-2 and INF-α, and then returning these lymphocytes to the patient's body, are also known.

[0004] T cells possess T cell receptors (TcRs) on their surface that recognize antigens expressed on the surface of cancer cells and other cancer cells. Known T cell types include αβT cells (TcRαβ-positive cells) with TcRs composed of α and β chains, γδT cells (TcRγδ-positive cells) with TcRs composed of γ and δ chains, and NKT cells that possess properties similar to NK cells (natural killer cells). αβT cells recognize antigens in a major histocompatibility complex (MHC)-restricted manner, are activated by IL-2 stimulation, and function as killer T cells. Killer T cells act on the recognized target cancer cells, and for use in therapies such as LAK therapy and CTL therapy, cytokine stimulation and recognition of antigen-presenting cancer cells are necessary. Furthermore, αβT cells may also damage the body's own cells.

[0005] γδT cells exhibit MHC-independent cytotoxicity (natural killer activity) against cancer cells. While αβT cells recognize cancer cells expressing specific cancer-specific antigens using a single TcR and exert cytotoxicity with high specificity, γδT cells recognize cancer cells using γδTcR and activated NK receptors such as 2B4 (CD244), NKG2D (CD314), and NKp44 (CD336), and exert cytotoxicity with high diversity.

[0006] NKT cells, particularly invariant NKT cells, possess both the antigen receptor Vα14Vβ8TcR and the representative NK cell receptor CD161 on their surface. They are known to have potent cytokine production capabilities, especially IFN-γ production, and cytotoxicity mediated by Fas and perforin, due to their CD1d-restricted recognition of α-galactosylceramide. Furthermore, they possess an inhibitory receptor that binds to cells expressing MHC-class I, thus failing to recognize normal cells and instead recognizing target cancer cells that have lost their MHC molecules.

[0007] Furthermore, cancer immunotherapy using NK cells is also known. NK cells are cytotoxic cells that do not possess TcR. NK cells primarily recognize cells that do not express MHC class I and attack cancer cells.

[0008] In cancer immunotherapy, expanding the culture (amplification) of cells that exhibit cytotoxicity against cancer cells and enhancing their cytotoxic activity are important challenges. A method for amplifying NK cells using CD3 agonists, CD52 agonists, and IL-2 has been disclosed as a method for expanding MHC-unrestricted cytotoxic cells from PBMCs, which is superior to the amplification of T cells (Patent Document 2). According to this method, 1 × 10⁶ PBMCs can be cultured in 3 weeks. 8 From 1.1 × 10 11 CD16 + NK cells are cultured on a large scale.

[0009] Patent Document 3 discloses a method for amplifying NK cells from PBMCs using CD56 ligands such as glycosaminoglycans. According to this method, the proportion of NK cells before culture increases from 8.5% to nearly 50%, and the culture is expanded to approximately 20 to 100 times its original size.

[0010] Non-patent document 1 describes that culturing PBMCs in the presence of IL-2 and IL-18 increased the NK cell count compared to culturing them in the presence of each individually. According to this method, when both are used in combination, the NK cell culture is expanded by approximately 14 times, and the proportion of NK cells is increased to 34%.

[0011] Patent Document 4 describes a method for expanding the culture of a cell population including γδT cells by culturing PBMCs in the presence of fibronectin, fibronectin fragments, and IL-2. According to this method, the proportion of γδT cells (CD3 and γδT cell-positive group) increased from 8.9% to approximately 30-50% of the PBMCs after 14 days from the start of culture, and the culture was expanded to approximately 5-16 times its original size.

[0012] Patent Document 5 discloses a method of culturing PBMC in the presence of bisphosphonates such as pamidronate and zoledronate and IL-2 to selectively amplify γδT cells. According to this method, the proportion of γδT cells in monocytes can be increased to nearly 90% in about 14 days of culture, and the cells are expanded and cultured about 100-fold.

[0013] In addition, Non-Patent Document 2 describes that when PBMC was cultured in the presence of zoledronate (ZOL), which is one of the bisphosphonates, or in the presence of ZOL and IL-2, the abundance ratio of γδT cells was increased. According to this method, the abundance ratio of γδT cells before culture was about 3.8%, and it was increased to about 13% in 14 days of culture. Also, γδT cells are expanded and cultured about 300 - 800-fold.

[0014] Non-Patent Document 3 and Patent Document 6 have attempted to culture PBMC in the presence of IL-2, zoledronate, and IL-18. According to this, the abundance ratio of γδT cells (Vγ9Vδ2T cells) in PBMC was increased from 1% before culture to 75% in 14 days of culture, and γδT cells were expanded and cultured about 25-fold.

[0015] Furthermore, Patent Document 7 discloses that after administering G-CSF (granulocyte colony-stimulating factor), peripheral blood mononuclear fraction (G-PBMC) is cultured in the presence of α-galactosylceramide (α-GalCer) and cytokines such as IL-2, IL-12, and IL-18 to amplify NKT cells. According to this, even without administering G-CSF, due to the presence of α-galactosylceramide and IL-2, NKT cells (Vα24+NKT cells) in PBMC increase about 4-fold, and their abundance ratio increases about 16-fold.

[0016] Incidentally, TNF-α is a substance that has the effect of causing necrosis of tumor cells. It is well known that TNF-α induces the production of INF-γ, IL-6, and IL-8. To date, the addition of TNF-α to a cell population including NK cells or NKT cells in expanded culture to enhance cytotoxicity has been disclosed, for example, in Patent Document 8. Furthermore, the use of TNF-α to expand the culture of MHC-unrestricted cytotoxic cells such as NK cells and NKT cells has been disclosed, for example, in Non-Patent Document 4, Patent Document 9, and Patent Document 10. In addition, Patent Document 10 discloses a method for promoting the amplification of activated T lymphocytes by culturing T lymphocytes that have been in contact with artificial antigen-presenting cells inactivated with a nucleic acid crosslinking agent in the presence of cytokines such as IL-2, IL-21, and TNF-α. [Prior art documents] [Patent Documents]

[0017] [Patent Document 1] Japanese Patent Publication No. 2001-314183 [Patent Document 2] Japanese Patent Publication No. 2006-340698 [Patent Document 3] Japanese Patent Publication No. 2006-115826 [Patent Document 4] International Publication No. WO2008 / 111430 [Patent Document 5] International Publication No. WO2006 / 006720 [Patent Document 6] Japanese Patent Publication No. 2010-17134 [Patent Document 7] International Publication WO01 / 94553 [Patent Document 8] Japanese Patent Publication No. 2000-245451 [Patent Document 9] Special Publication No. 2002-515756 [Patent Document 10] Special Publication No. 2010-505845 [Non-patent literature]

[0018] [Non-Patent Document 1] Young-Ik Son, et al., Cancer Research 61, 884-888 [Non-Patent Document 2] Kiyoshi Sato et al., Int. J. Cancer, 116, p.94-99(2005) [Non-Patent Document 3] Wen Li et al., J. Immunother. 33(3), P287-206 2010 [Non-Patent Document 4] Jpn J Cancer Chemother 30(11), p.1776-1779 [Overview of the project] [Problems that the invention aims to solve]

[0019] In cancer immunotherapy, it is necessary to infuse large amounts of NK cells into cancer patients. Furthermore, it is well known that the frequency of NK cells in peripheral blood varies greatly from person to person; some patients have a high proportion of NK cells, while others have a low proportion.

[0020] This invention has been made in view of the above problems, and aims to provide a method for culturing NK cells that are highly pure and have improved amplification efficiency. [Means for solving the problem]

[0021] The present invention relates to a method for culturing NK cells, comprising: a container preparation step of preparing a culture vessel coated with an anti-human NKp46 antibody; a removal step of removing T cells from hematopoietic mononuclear cells; and a culture step of preparing a cell suspension by suspending the hematopoietic mononuclear cells, from which the T cells have been removed, in a serum-free medium containing ultra-high concentrations of IL-2 and IL-18, and then adding the cell suspension to the culture vessel to perform cell culture. [Effects of the Invention]

[0022] According to the NK cell culture method of the present invention, it is possible to maintain high purity while improving the amplification efficiency of NK cells. [Brief explanation of the drawing]

[0023] [Figure 1] This figure shows the results of analyzing NK cells using a flow cytometer. [Modes for carrying out the invention]

[0024] The embodiments of the present invention will be described below with reference to the attached drawings. However, these embodiments are provided to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the embodiments described below. Other embodiments in which those skilled in the art appropriately substitute the configurations of the embodiments below are also included in the scope of the present invention.

[0025] The method for culturing NK cells according to the present invention is: A container preparation step involves preparing culture vessels coated with anti-human NKp46 antibody, A removal process to remove T cells from hematopoietic mononuclear cells, The method includes a culture step of preparing a cell suspension by suspending hematopoietic mononuclear cells, from which the T cells have been removed, in a serum-free medium containing ultra-high concentrations of IL-2 and IL-18, and then adding the cell suspension to the culture vessel to perform cell culture.

[0026] (i) Container preparation process In the container preparation step, culture vessels coated with anti-human NKp46 antibody are prepared.

[0027] The culture vessel is made of plastic, although it is not particularly limited. Examples of plastics include polystyrene, polypropylene, polyvinyl chloride, polyethylene, cyclic polyolefin, polyethylene terephthalate, polycarbonate, silicone resin, or acrylic resin. From the viewpoint of versatility, polymethyl methacrylate (PMMA) is preferred as the acrylic resin.

[0028] NKp46 is an activating receptor for NK cells, and therefore, anti-human NKp46 antibodies can stimulate NKp46 and activate NK cells.

[0029] Add 0.1% (w / v%) human serum albumin (HSA)-containing phosphate buffer to an antibody solution of 4-6 μg / ml using anti-human NKp46 antibody (antibody clone name: 195314). Dispense this antibody solution at a rate of 0.15-0.2 ml / cm³. 2 Place the culture vessel in such a manner. For example, if the culture vessel is a 6-well plate (culture area 9 cm²) 2 If so, add it to a culture vessel so that the total volume is 1.5 ml, and use a T25 flask (culture area 25 cm²). 2 If necessary, add the mixture to a culture vessel to a total volume of 4 ml. Then, leave it to stand overnight at 4°C to allow the anti-human NKp46 antibody to immobilize and coat the surface of the culture vessel.

[0030] Furthermore, it is preferable that the surface of the culture vessel be coated not only with anti-human NKp46 antibody but also with anti-human CD16 antibody. Anti-human CD16 antibody is an antibody against the CD16 antigen. The CD16 antigen is a marker for NK cells and granulocytes and is known as FcγRIII, a constituent protein of the Fc receptor present on the cell surface of most NK cells in the quiescent phase. Anti-human CD16 antibody has NK cell proliferation-inducing activity. Although the mechanism by which anti-human CD16 antibody induces NK cell proliferation has not been clarified, co-adding anti-human CD16 antibody with cytokines such as IL-2 can dramatically increase the rate of NK cell proliferation compared to when cytokines are added alone.

[0031] In such cases, an antibody solution is prepared by adding 0.1% (w / v) human serum albumin (HSA)-containing phosphate buffer to each of the anti-human NKp46 antibody (antibody clone name: 195314) and anti-human CD16 antibody (antibody clone name: 3G8), resulting in an antibody solution with a concentration of 4-6 μg / ml for both the anti-human NKp46 antibody and the anti-human CD16 antibody. This antibody solution is then dispensed at a rate of 0.15-0.2 ml / cm³. 2 Place the mixture into a culture vessel in this manner. Then, leave it to stand overnight at 4°C to allow the anti-human NKp46 antibody and anti-human CD16 antibody to immobilize and coat the surface of the culture vessel.

[0032] (ii) Removal process In the removal process, T cells are removed from hematopoietic mononuclear cells.

[0033] Hematopoietic mononuclear cells are not limited to any specific type, but include, for example, peripheral blood mononuclear cells (PBMCs), bone marrow-derived mononuclear cells (BMNCs), or umbilical cord blood mononuclear cells (CBMNCs).

[0034] In the removal process, it is preferable to remove only T cells from hematopoietic mononuclear cells and not isolate NK cells. In other words, in the removal process, T cells are removed from hematopoietic mononuclear cells while ensuring that antigen-presenting cells such as B cells and monocytes are included. It is even more desirable in the removal process to remove not only T cells but also red blood cells, which are unwanted components.

[0035] Hematopoietic mononuclear cells include T cells (CD3+CD56-), NK cells (CD3-CD56+), NKT cells (CD3+CD56+), B cells (CD3-CD56-CD20+), and monocytes (CD3-CD56-CD11b+). If only T cells are removed and NK cells are not isolated, NK cells, NKT cells, B cells, monocytes, etc., will remain. Culture is more efficient when only T cells are removed from hematopoietic mononuclear cells than when only NK cells are isolated and cultured. B cells and monocytes are called antigen-presenting cells and express various costimulatory molecules, and it is thought that these cells stimulate NK cells, thereby enhancing amplification efficiency.

[0036] T cell removal can be performed, for example, by adding an antibody that recognizes CD3 expressed on T cells to remove only T cells from hematopoietic mononuclear cells, without isolating NK cells.

[0037] Specifically, blood is collected intravenously into a vacuum blood collection tube to which heparin has been added to a final concentration of 10 μg / ml to 100 μg / ml; 1.2 U / ml to 12 U / ml. Using RosetteSep Human CD3 Depletion Cocktail (Stem Cell Technologies), antibodies that recognize Glycophorin A expressed on red blood cells and CD3 expressed on T cells (a tetramer in which CD3 antibody and Glycophorin A antibody are linked) are added to the collected blood and allowed to react at room temperature for 20 minutes. Alternatively, CD3-positive cells can also be removed using antibody-conjugated magnetic beads (MACS) from Miltenyi biotech. In this case, PBMCs are separated using lymphocyte separatory before the CD3-positive cell removal procedure is performed.

[0038] Subsequently, add the same volume of cell culture medium (e.g., RPMI, DMEM, AIM-V, X-VIVO15, NK MACS Medium, AlyS 505NK, etc.) as the blood and mix. This mixture is then overlaid on lymphocyte separator Lymphoprep (Stem Cell Technologies) and centrifuged at 1,200G for 30 minutes. Alternatively, Ficoll-PAQUE or similar lymphocyte separators can be used. After centrifugation, carefully remove the hematopoietic mononuclear cells, which have been separated at the boundary between the lymphocyte separator and plasma, using a measuring pipette, and transfer them to another centrifuge tube. Add the cell culture medium to the transferred cell solution, wash by centrifugation at 620G for 7 minutes, remove the supernatant, and then use for cell culture.

[0039] (iii) Cultivation process In the culture process, a cell suspension is prepared by suspending hematopoietic mononuclear cells, from which T cells have been removed, in serum-free medium containing ultra-high concentrations of IL-2 and IL-18. This cell suspension is then added to a culture vessel for cell culture.

[0040] Serum-free media are, for example, serum-free media containing 2-10% autologous plasma or serum (e.g., AIM-V, X-VIVO15, NK MACS Medium, AlyS 505NK, etc.).

[0041] The serum-free medium contains ultra-high concentrations of IL-2 and IL-18. While not particularly limited, the serum-free medium may, for example, contain IL-2 at 3,000 IU / ml or more and IL-18 at 50 IU / ml or more, preferably IL-2 at 3,000 IU / ml to 6,000 IU / ml and IL-18 at 50 IU / ml to 100 IU / ml. It is also possible for the serum-free medium to contain only either ultra-high concentrations of IL-2 or ultra-high concentrations of IL-18. For example, it may contain IL-2 at 3,000 IU / ml or more, or IL-18 at 50 IU / ml or more. Preferably, it may contain IL-2 at 3,000 IU / ml to 6,000 IU / ml, or IL-18 at 50 IU / ml to 100 IU / ml.

[0042] Although the concentration of the cell suspension is not particularly limited, for example, in a serum-free medium, blood mononuclear cells from which T cells have been removed are suspended at a concentration of 0.5 to 1.5 x 10 6 / ml.

[0043] The amount of the solution added to the culture container for the cell suspension is not particularly limited, but for example, it can be a cell solution amount of 0.22 to 0.40 ml / cm 2 . For example, if the culture container is a 6-well plate (culture area 9 cm 2 ), it is placed in the culture container so that it becomes 2 ml, and if it is a T25 flask (culture area 25 cm 2 ), it is placed in the culture container so that it becomes 10 ml.

[0044] The number of days of cell culture is not particularly limited, but for example, it is a culture period of 7 days or more and 21 days or less. The culture conditions are, for example, an environment of 37°C, 5% CO2, and 100% humidity.

[0045] According to cell growth, a serum-free medium (plasma may not be included) containing a high concentration of IL-2 is added in a volume 2 to 3 times the original, and transferred to an appropriate culture container according to the volume. When the culture solution exceeds 300 ml, it is also possible to transfer it to a gas-permeable cell culture bag. It can be cultured for up to 21 days, but after that, the cells hardly grow.

[0046] In this way, NK cells can be cultured while maintaining a high purity and improving the amplification efficiency (2 to 6 times that of the prior art).

Example

[0047] 1. Example 1 (1) Preparation process of a plastic culture container coated with an anti-human NKp46 antibody Anti-human NKp46 antibody (antibody clone name: 195314) was prepared in phosphate buffer saline (PBS) containing 0.1% (w / v) human serum albumin (HSA) to a concentration of 5 μg / ml. Specifically, the concentration of anti-human NKp46 antibody in the phosphate buffer was 5 μg / ml.

[0048] The plastic culture vessel is a 6-well plate (culture area 9 cm²). 2 A 1.5 ml prepared antibody solution was transferred to the plastic culture vessel and allowed to stand overnight at 4°C to solidify. This created a culture vessel coated with anti-human NKp46 antibody.

[0049] (2) T cell removal process from PBMCs Blood was collected intravenously from subjects using vacuum blood collection tubes to which heparin was added to a final concentration of 10 μg / ml to 100 μg / ml; 1.2 U / ml to 12 U / ml. CD3-positive cells were removed using RosetteSep® Human CD3 Depletion Cocktail. Specifically, the collected blood was mixed with Glycophorin A (expressed abundantly and specifically on the red blood cell membrane and playing a role in preventing cell aggregation) and an antibody that recognizes CD3 expressed on T cells, and reacted at room temperature for 20 minutes (a tetramer in which the CD3 antibody and the glycophorin A antibody were linked was added). Then, the same amount of cell culture medium (RPMI medium) was added and mixed, and the mixture was overlaid on lymphocyte separator Lymphoprep (Stem Cell Technologies), and centrifugation was performed at 1,200 G for 30 minutes. After centrifugation, peripheral blood mononuclear cells (PBMCs), from which CD3-expressing cells had been removed, were carefully removed using a volumetric pipette and transferred to another centrifuge tube. Cell culture medium was added to the transferred cell solution, and the mixture was washed by centrifugation at 620G for 7 minutes, after which the supernatant was removed.

[0050] (3) Cell culture using ultra-high concentrations of IL-2 and IL-18 PBMCs (with 10 CD3-positive cells) were mixed in 2 ml of serum-free medium (AIM-V) containing 3,000 IU / ml of interleukin (IL)-2, 50 IU / ml of IL-18, and 10% autologous plasma. 6 (pieces) 0.5 x 10 6 The cells were suspended at a concentration of / ml to obtain a cell suspension.

[0051] Plastic culture vessels coated with anti-human NKp46 antibody were washed with physiological saline, and the cell suspension described above was transferred to the washed plastic culture vessels.

[0052] 6-well plate (culture area 9 cm²) 2 Transferred 2 ml of the above cell suspension to a plastic culture vessel. The initial cell solution volume was approximately 0.22 ml / cm³. 2 That was the case.

[0053] Cell suspension was transferred to plastic culture vessels and culture was started at 37°C, 5% CO2, and 100% humidity. As cell growth progressed, serum-free medium containing a high concentration of IL-2 (plasma-free was not required) was added in 2 to 3 volumes, and the culture was transferred to appropriate culture vessels according to the volume. This process was continued for 14 days.

[0054] 2. Example 2 (1) Process for preparing plastic culture vessels coated with anti-human NKp46 antibody and anti-human CD16 antibody. Anti-human NKp46 antibody (antibody clone name: 195314) and anti-human CD16 antibody (antibody clone name: 3G8) were each prepared in phosphate buffer saline (PBS) containing 0.1% (w / v) human serum albumin (HSA) to a concentration of 5 μg / ml. Specifically, the concentrations of the anti-human NKp46 antibody and anti-human CD16 antibody in the phosphate buffer were both 5 μg / ml.

[0055] The plastic culture vessel is a 6-well plate (culture area 9 cm²). 2 A 1.5 ml prepared antibody solution was transferred to the plastic culture vessel and allowed to stand overnight at 4°C to solidify. This created culture vessels coated with anti-human NKp46 antibody and anti-human CD16 antibody.

[0056] (2) T cell removal process from PBMCs Blood was collected intravenously from subjects using vacuum blood collection tubes to which heparin was added to a final concentration of 10 μg / ml to 100 μg / ml; 1.2 U / ml to 12 U / ml. CD3-positive cells were removed using RosetteSep® Human CD3 Depletion Cocktail. Specifically, the collected blood was mixed with Glycophorin A, which is expressed on red blood cells, and an antibody that recognizes CD3, which is expressed on T cells, and reacted at room temperature for 20 minutes (a tetramer in which the CD3 antibody and the glycophorin A antibody were linked was added). Then, the same amount of cell culture medium (RPMI medium, etc.) was added and mixed, and the mixture was overlaid on lymphocyte separator Lymphoprep (Stem Cell Technologies), and centrifuged at 1,200 G for 30 minutes. After centrifugation, peripheral blood mononuclear cells (PBMCs), from which CD3-expressing cells had been removed, were carefully removed using a volumetric pipette and transferred to another centrifuge tube. Cell culture medium was added to the transferred cell solution, and the mixture was washed by centrifugation at 620G for 7 minutes, after which the supernatant was removed.

[0057] (3) Cell culture using ultra-high concentrations of IL-2 and IL-18 PBMCs (with 10 CD3-positive cells) were mixed in 2 ml of serum-free medium (AIM-V) containing 3,000 IU / ml of interleukin (IL)-2, 50 IU / ml of IL-18, and 10% autologous plasma. 6 (pieces) 0.5 x 10 6 The cells were suspended at a concentration of / ml to obtain a cell suspension.

[0058] Plastic culture vessels coated with anti-human NKp46 antibody and anti-human CD16 agonist antibody were washed with physiological saline, and the cell suspension was transferred to the washed plastic culture vessels.

[0059] 6-well plate (culture area 9 cm²) 2 Transferred 2 ml of the above cell suspension to a plastic culture vessel. The initial cell solution volume was approximately 0.22 ml / cm³. 2 The cell suspension was transferred to plastic culture vessels, and cultivation was started at 37°C, 5% CO2, and 100% humidity. As the cells proliferated, serum-free medium containing a high concentration of IL-2 (plasma was not required) was added in 2 to 3 times the volume, and the culture was transferred to an appropriate culture vessel according to the volume. The cells were cultured in this manner for 14 days.

[0060] 3. Example 3 (1) Confirmation of the amplification efficiency and purity of NK cells Blood was collected from three healthy donors and amplified culture was performed using the method described above. For each of the three donors, the amplified culture was performed using either a plastic incubator coated with anti-human NKp46 antibody or a plastic culture vessel coated with both anti-human NKp46 antibody and anti-human CD16 antibody.

[0061] PBMCs from which CD3-positive cells were removed at the start of the study, and cultured cells after 14 days of culture were stained with trypan blue, and the number of cells was counted using a cell counting chamber. Furthermore, the purity of CD3-negative CD56-positive NK cells present in these cell populations was analyzed using a flow cytometer (FACSCalibur: BD). Antibody-based cell staining was performed. 6Cells were suspended in 100 μl of Stain Buffer (BD), and 2 μl each of PE / Cy5-labeled anti-CD3 antibody (antibody clone name: HIT3a) and AlexaFluoro488-labeled anti-human CD56 antibody (antibody clone name: NCAM1) were added. The cells were reacted at 4°C for 30 minutes. After the reaction, the cells were washed with 1 ml of Stain Buffer and suspended in 500 μl of 1% paraformaldehyde / PBS solution. The cells were reacted at 4°C for 30 minutes, and analysis was performed using a flow cytometer. The results are shown in Table 1 and Figure 1.

[0062] [Table 1]

[0063] As shown in Table 1 and Figure 1, in samples 1, 2, and 3, the cell population at the start of culture contained 48.2%, 52.1%, and 60.7% CD3-negative CD56-positive NK cells, respectively, and the number of CD3-negative CD56-positive NK cells was 0.48 x 10⁶. 6 cells, 0.52 x 10 6 cells, 0.61 x 10 6 It was a cell.

[0064] On day 14 of culture, in Sample 1, the control, anti-human CD16 antibody-conjugated plate, anti-human NKp46 antibody-conjugated plate, and anti-CD16 and anti-human NKp46 antibody-conjugated plate each measured 214.0 x 10⁶. 6 cells, 243.7 x 10 6 cells, 353.3 x 10 6 cells, 403.3 x 10 6 The cells were amplified down to the cellular level, and the purity of CD3-negative CD56-positive NK cells was 90.1%, 80.8%, 96.2%, and 96.9%, respectively, with the number of CD3-negative CD56-positive NK cells being 192.8 x 10⁶. 6 cells, 196.9 x 10 6 cells, 339.9 x 10 6 cells, 390.8 x 10 6The amplification factors for cells and CD3-negative CD56-positive NK cells were 400.0x, 408.5x, 705.1x, and 810.8x, respectively.

[0065] On day 14 of culture, in sample 2, the control, anti-human CD16 antibody-conjugated plate, anti-human NKp46 antibody-conjugated plate, and anti-human CD16 and anti-human NKp46 antibody-conjugated plate each measured 88.8 x 10⁶. 6 cells, 130.0 x 10 6 cells, 382.2 x 10 6 cells, 412.2 x 10 6 The cells were amplified down to the cellular level, and the purity of CD3-negative CD56-positive NK cells was 95.2%, 97.4%, 97.6%, and 98.6%, respectively, with the number of CD3-negative CD56-positive NK cells being 84.5 x 10⁶. 6 cells, 126.6 x 10 6 cells, 373.0 x 10 6 cells, 406.4 x 10 6 The amplification factors for cells and CD3-negative CD56-positive NK cells were 162.3x, 243.0x, 716.0x, and 780.1x, respectively.

[0066] On day 14 of culture, in sample 3, the control, anti-human CD16 antibody-conjugated plate, anti-human NKp46 antibody-conjugated plate, and anti-human CD16 and anti-human NKp46 antibody-conjugated plate each measured 186.0 x 10⁶. 6 cells, 348.8 x 10 6 cells, 447.2 x 10 6 cells, 478.4 x 10 6 The cells were amplified down to the cellular level, and the purity of CD3-negative CD56-positive NK cells was 45.9%, 35.0%, 83.2%, and 86.7%, respectively, with the number of CD3-negative CD56-positive NK cells being 85.4 x 10⁶. 6 cells, 122.1 x 10 6 cells, 372.1 x 10 6 cells, 414.8 x 10 6 The amplification factors for cells and CD3-negative CD56-positive NK cells were 140.6x, 201.1x, 613.0x, and 683.3x, respectively.

[0067] In Sample 1, the purity and amplification factor of NK cells in the control group were approximately as shown in Table 1. Compared to the control group, the anti-human CD16 antibody resulted in a 9.3% lower purity and a 1.02-fold higher amplification factor. The anti-human NKp46 antibody resulted in a 6.1% higher purity and a 1.76-fold higher amplification factor. The anti-human CD16 antibody and anti-human NKp46 antibody both resulted in a 6.8% higher purity and a 2.02-fold higher amplification factor. These results indicate that the anti-human NKp46 antibody significantly enhanced the amplification factor of NK cells, and the anti-human CD16 antibody and anti-human NKp46 antibody further enhanced this efficiency.

[0068] In Sample 2, the control group showed NK cell purity similar to that described in Table 1, but the amplification efficiency tended to be significantly lower. Compared to the control group, the anti-human CD16 antibody showed a 2.2% higher purity and a 1.49-fold higher amplification factor. The anti-human NKp46 antibody showed a 2.4% higher purity and a 4.41-fold higher amplification factor. The anti-human CD16 antibody and anti-human NKp46 antibody showed a 3.4% higher purity and a 4.80-fold higher amplification factor. From these results, the anti-human NKp46 antibody significantly enhanced the amplification factor of NK cells, and the anti-human CD16 antibody and anti-human NKp46 antibody further enhanced it.

[0069] In Sample 3, the purity and amplification efficiency of NK cells tended to be significantly lower in the control group, as shown in Table 1. Compared to the control group, the anti-human CD16 antibody resulted in a 10.9% lower purity and a 1.43-fold higher amplification factor. The anti-human NKp46 antibody resulted in a 37.3% higher purity and a 4.35-fold higher amplification factor. The anti-human CD16 antibody and anti-human NKp46 antibody both resulted in a 40.8% higher purity and a 4.86-fold higher amplification factor. These results indicate that the anti-human NKp46 antibody significantly enhanced the amplification factor of NK cells, and this enhancement was further enhanced by the anti-human CD16 antibody and anti-human NKp46 antibody. [Industrial applicability]

[0070] It can be used for cancer prevention and treatment.

Claims

1. A container preparation step involves preparing culture vessels coated with anti-human NKp46 antibody and anti-human CD16 antibody, A removal process that removes only T cells from peripheral blood mononuclear cells (PBMCs) without isolating NK cells, A cell culture step is to prepare a cell suspension by suspending hematopoietic mononuclear cells, from which the T cells have been removed, in a serum-free medium containing 3,000 IU / ml or more of IL-2 and 50 IU / ml or more of IL-18, and then add the cell suspension to the culture vessel to perform cell culture. A method for culturing NK cells, characterized by having the following characteristics.

2. The method for culturing NK cells according to claim 1, characterized in that the removal step involves adding an antibody that recognizes CD3 expressed on T cells to remove only T cells from hematopoietic mononuclear cells, without isolating NK cells.

3. In the container preparation step, the antibody is added to phosphate buffer to prepare an antibody solution of 4-6 μg / ml, and this antibody solution is dispensed at a rate of 0.15-0.2 ml / cm³. 2 A method for culturing NK cells according to claim 1 or 2, characterized in that the cells are placed in the culture vessel in such a manner and allowed to stand, thereby immobilizing and coating the surface of the culture vessel with the antibody.

4. The method for culturing NK cells according to any one of claims 1 to 3, characterized in that the culture vessel is made of plastic.

5. In the aforementioned culture step, 0.5 to 1.5 x 10⁶ hematopoietic mononuclear cells, from which T cells have been removed, are placed in the serum-free medium. 6 The method for culturing NK cells according to claim 1, characterized in that a cell suspension is prepared by suspending the cells at a concentration of / ml.

6. In the culture step, the cell suspension is added to the culture vessel at a rate of 0.22 to 0.40 ml / cm³. 2 A method for culturing NK cells according to any one of claims 1 to 5, characterized in that the cell solution volume is as follows.

7. The method for culturing NK cells according to any one of claims 1 to 6, characterized in that the cell culture in the culture step is performed for a period of 7 days or more and 21 days or less.

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