Method for producing natural killer cells

Abstract

[Problem] The present invention addresses the problem of providing a method for producing an NK cell population having a high ratio of NK cells and containing highly active NK cells. [Solution] The present invention relates to a method for producing a cell population enriched in NK cells, the method comprising a step for culturing a mononuclear cell population containing NK cells in the presence of three antibodies, namely, an anti-CD2 antibody, an anti-CD52 antibody, and an anti-CD335 antibody.

Description

Method for producing natural killer cells

[0001] The present invention relates to a method for producing natural killer cells (NK cells), and particularly to a method for producing highly pure natural killer cells having high cytotoxic activity.

[0002] NK cells, which play an important role in innate immunity, account for approximately 10% of peripheral blood lymphocytes and are responsible for the rapid elimination of pathogens and abnormal cells that invade the body. NK cells exert cytotoxic activity without undergoing differentiation after antigen stimulation. Therefore, unlike T cells, they lack antigen specificity and exhibit cytotoxic activity against cancer cells with reduced or absent MHC class 1 expression. NK cells also possess unique cytotoxic functions in addition to nonspecific cytotoxicity. The majority of NK cells express CD16 molecules (FcγRIII) on their surface. NK cells are activated by binding to the Fc portion of IgG bound to the surface of target cells via CD16, thereby exerting antibody-dependent cellular cytotoxicity (ADCC), which kills the target cells. There have been many attempts to expand NK cells, which possess this unique cytotoxic mechanism, in vitro and utilize them in adoptive immunity research. Various reagent kits for expanding NK cells are commercially available from various vendors.

[0003] These commercially available kit reagents vary significantly in performance between manufacturers, with the following problems occurring: 1) The ratio of NK cells in the harvested cells is low. There is a high level of contamination with other immune cells, such as T cells. 2) Low activity in cytotoxicity tests using cell lines (mainly K562). 3) Low activity in ADCC activity tests using cell lines (mainly SK-BR-3) and Trastuzumab (trade name Herceptin). 4) The cells do not proliferate. However, this cannot be generalized as it may be due to the condition of the original PBMCs.

[0004] For example, Patent Document 1 discloses a method for culturing blood mononuclear cells from which T cells have been removed in a serum-free medium containing ultra-high concentrations of IL-2 and IL-18 to prepare a cell suspension, and then culturing the cell suspension in a culture vessel coated with an anti-human NKp46 antibody. It is described that this method can maintain high NK cell purity while improving NK cell expansion efficiency. However, Patent Document 1 does not describe the activity of the NK cells obtained by culturing. Patent Document 2 also discloses a method for expanding NK cells, characterized by culturing NK cells in the presence of a composition containing tacrolimus or a salt thereof and dalteparin or a salt thereof. The method described in Patent Document 2 demonstrated an increase in the NK cell ratio to approximately 77.1% by culturing umbilical cord blood-derived mononuclear cells, and an increase in the NK cell ratio to 73.4% by culturing peripheral blood mononuclear cells. The NK cells expanded by the method described in Patent Document 2 showed a cytotoxic activity of approximately 55% when the effector cell to target cell ratio (E:T) was 1:1 in a cytotoxic activity test using the K562 cell line.

[0005] JP 2023-103632 A, Patent No. 5840876 Public Relations

[0006] An objective of the present invention is to provide a method for producing an NK cell population that has a high proportion of NK cells and contains highly active NK cells.

[0007] The present inventors have made intensive efforts to solve the above-mentioned problems, and as a result, have succeeded in obtaining an NK cell population containing a high proportion of NK cells and highly active NK cells by culturing the NK cell population in the presence of three antibodies, namely, anti-CD2 antibody, anti-CD52 antibody, and anti-CD335 antibody. The present invention has been completed based on the above findings and includes the following aspects: [1] One aspect of the present invention relates to a method for producing an NK cell-enriched cell population, comprising the step of culturing a mononuclear cell population containing NK cells in the presence of three antibodies, namely, anti-CD2 antibody, anti-CD52 antibody, and anti-CD335 antibody. Here, in one embodiment, the method for producing an NK cell-enriched cell population of the present invention is: [2] the production method described in [1] above, wherein the mononuclear cell population is derived from at least one cell population selected from the group consisting of peripheral blood mononuclear cells (PBMCs), bone marrow mononuclear cells (BMNCs), umbilical cord blood mononuclear cells (CBMNCs), embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells. Furthermore, one embodiment of the method for producing a cell population enriched in NK cells of the present invention is: [3] the production method described in [1] or [2] above, wherein the mononuclear cell population is derived from peripheral blood mononuclear cells (PBMCs). One embodiment of the method for producing a cell population enriched in NK cells of the present invention is: [4] the production method described in any of [1] to [3] above, wherein the step of culturing the mononuclear cell population is a step performed using a culture vessel coated with the antibody. One embodiment of the method for producing a cell population enriched in NK cells of the present invention is: [5] the production method described in any of [1] to [4] above, wherein the step of culturing the mononuclear cell population is a step of culturing the mononuclear cell population further in the presence of at least one physiologically active substance selected from the group consisting of IL-2, IL-15, IL-18, and hydrocortisone. Furthermore, in one embodiment, the method for producing a cell population enriched in NK cells of the present invention is: [6] the production method described in any one of [1] to [5] above, further comprising a step of concentrating NK cells contained in the mononuclear cell population prior to the step of culturing the mononuclear cell population.Furthermore, in one embodiment, the method for producing a cell population enriched in NK cells of the present invention is: [7] the production method described in any of [1] to [6] above, further comprising a step of recovering NK cells from the cell population enriched in NK cells or concentrating NK cells after the step of culturing the mononuclear cell population. Furthermore, in one embodiment, the method for producing a cell population enriched in NK cells of the present invention is: [8] the production method described in any of [1] to [7] above, characterized in that the recovery step is a step of recovering adherent cells from the cell population enriched in NK cells using a chelating agent. Furthermore, in one embodiment, the method for producing a cell population enriched in NK cells of the present invention is: [9] the production method described in any of [1] to [8] above, characterized in that the concentration of the antibody in the step of culturing the mononuclear cell population is in the range of 5 to 20 μg / mL.

[0008] In another aspect, the present invention relates to:

[10] a cell population enriched in NK cells, produced by the production method according to any one of [1] to [9] above. In another aspect, the present invention relates to:

[11] an NK cell population having the following characteristics: (1) a cytotoxic activity of 50% or more when NK cells are used as effector cells (E) and K562 cells are used as target cells (T) and co-cultured at a mixing ratio (E:T) of 1:1, and (2) an ADCC activity of 5% or more when NK cells are used as effector cells (E) and SK-BR-3 cells are used as target cells (T) and co-cultured at a mixing ratio (E:T) of 0.5:1 in the presence of 0.01 μg / mL to 1 μg / mL of Herceptin. In one embodiment, the NK cell population of the present invention is:

[12] the NK cell population according to

[11] above, characterized in that (1) the cytotoxic activity is 60% or more when NK cells are used as effector cells (E) and K562 cells are used as target cells (T) at a mixing ratio (E:T) of 1:1. In one embodiment, the NK cell population of the present invention is:

[13] the NK cell population according to

[11] or

[12] above, characterized in that (2) the ADCC activity is 8% or more when NK cells are used as effector cells (E) and SK-BR-3 cells are used as target cells (T) at a mixing ratio (E:T) of 0.5:1 in the presence of 0.01 μg / mL to 1 μg / mL of Herceptin.

[0009] Another aspect of the present invention relates to

[13] a method for culturing NK cells, comprising the step of culturing a mononuclear cell population containing NK cells in the presence of three antibodies: an anti-CD2 antibody, an anti-CD52 antibody, and an anti-CD335 antibody.

[0010] The method for producing an NK cell population according to the present invention makes it possible to provide an NK cell population that has a high proportion of NK cells in the cell population and that contains highly active NK cells.

[0011] FIG. 1 is a graph showing the results of a cytotoxicity test against K562 cells for the NK cell population produced in Example 1 below, and for NK cell populations expanded and cultured using KBM NK KIT or BIN KIT. White bars represent the results of the cytotoxicity test using the NK cell population produced in the Example below. Black bars represent the results of the cytotoxicity test using the NK cell population produced using KBM NK KIT. Gray bars represent the results of the cytotoxicity test using the NK cell population produced using BIN KIT. FIG. 2 is a graph showing the results of an ADCC test against SK-BR-3 cells using Trastuzumab (Herceptin) for the NK cell population produced in Example 1 below, and for NK cell populations expanded and cultured using KBM NK KIT or BIN KIT. The left side of the graph shows the results of the cytotoxicity test using the NK cell population produced in the Example below. The center of the graph shows the results of the cytotoxicity test using the NK cell population produced using KBM NK KIT. The right side of the figure shows the results of a cytotoxicity test using an NK cell population prepared using the BIN KIT. Figure 3 is a graph showing the ratio of CD3-negative / CD56-positive cells in the NK cell population prepared in Example 1 below. Figure 4 is a graph showing the ratio of CD3-negative / CD56-positive cells in the NK cell population expanded and cultured using the KBM NK KIT. Figure 5 is a graph showing the ratio of CD3-negative / CD56-positive cells in the NK cell population expanded and cultured using the BIN KIT. Figure 6 is a graph showing the results of a cytotoxicity test against K562 for an NK cell population derived from PMBC of a cancer patient, NK cells derived from normal PMBC, and a mixed collection of NK cells derived from normal PMBC from multiple donors, all prepared in Example 2 below. White bars show the results of a cytotoxicity test at an E:T ratio of 1:1. Gray bars show the results of a cytotoxicity test at an E:T ratio of 5:1.7 is a graph showing the results of ADCC activity tests against RLEU1-7 cells (B-cell leukemia cell line) using Herceptin, Rituxan, or DARAZALEX for NK cell populations derived from PMBC of cancer patients, NK cells derived from normal PMBC, and a mixed population of NK cells derived from normal PMBC of multiple donors, all prepared in Example 2 below. White bars show the results of the cytotoxicity test in the absence of antibody. Diamond bars show the results of the ADCC activity test in the presence of Herceptin, gray bars show the results of the ADCC activity test in the presence of Rituxan, and black bars show the results of the ADCC activity test in the presence of DARAZALEX.

[0012] One aspect of the present invention relates to a method for producing a cell population enriched in NK cells, which comprises the step of culturing a mononuclear cell population containing NK cells in the presence of three antibodies: an anti-CD2 antibody, an anti-CD52 antibody, and an anti-CD335 antibody.

[0013] As used herein, "NK cells" refers to CD3-negative CD56-positive mononuclear cells. NK cells have cytotoxic activity against cells that express low or no MHC class I molecules. Furthermore, the term "NK cell population" as used herein refers to a cell population containing NK cells, and the proportion of NK cells in the cell population is not limited. The "NK cells" used in the present invention can be derived from at least one cell selected from the group consisting of peripheral blood mononuclear cells (PBMCs), bone marrow mononuclear cells (BMNCs), umbilical cord blood mononuclear cells (CBMNCs), embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells. These cells are preferably derived from humans. Furthermore, these cells are not limited to cells derived from healthy humans (normal cells) but may also be cells derived from cancer patients. When "NK cells" are derived from embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, or adult stem cells, the "NK cells" refer to cells derived from hematopoietic stem cells derived from these stem cells. In a preferred embodiment, the NK cells are derived from peripheral blood mononuclear cells (PBMCs). Blood cells collected from peripheral blood, umbilical cord blood, bone marrow, and / or lymph nodes can be used as a source of the mononuclear cell population containing NK cells. The mononuclear cell population containing NK cells may also be prepared from hematopoietic stem cells derived from any of the following cells: embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells. Methods for inducing mononuclear cells, including NK cells, from these stem cells are known, and those skilled in the art can apply them to the present invention (Domogala A. et al., Natural killer cell immunotherapy: from bench to bedside, Frontiers in Immunology, 2015;6, doi: 10.3389 / fimmu.2015.00264, and Zeng J. et al., Generation of "Off-the-Shelf" Natural Killer Cells from Peripheral Blood Cell-Derived Induced Pluripotent Stem Cells, Stem Cell Reports, 2017;9:1796-1812).

[0014] When using a peripheral blood mononuclear cell (PBMC) population as the mononuclear cell population containing NK cells for culture, a mononuclear cell population prepared from peripheral blood by density centrifugation can be used. PBMCs commercially available from LONZA, Precision BioSciences, etc. may also be used.

[0015] In the culture step of the method for producing an NK cell-enriched cell population of the present invention, NK cells are stimulated with three antibodies, anti-CD2 antibody, anti-CD52 antibody, and anti-CD335 antibody, to activate them. The NK cells may be stimulated in the presence of a physiologically active substance such as a cytokine. CD2, CD52, and CD335 are receptors present on the cell membrane surface of NK cells, and thus the anti-CD2 antibody, anti-CD52 antibody, and anti-CD335 antibody can activate NK cells by stimulating CD2, CD52, and CD335, respectively. The anti-CD2 antibody, anti-CD52 antibody, and anti-CD335 antibody used in the present invention are not limited as long as they can stimulate the respective receptors to activate NK cells; for example, commercially available antibodies can be used.

[0016] The method of stimulating NK cells using an antibody is not limited as long as it stimulates NK cells and improves their activity. A preferred embodiment includes a method of culturing a mononuclear cell population containing NK cells using a culture vessel with an antibody immobilized on its surface. The method for immobilizing an antibody on the surface of the culture vessel is not limited as long as the immobilized antibody stimulates NK cells and improves their activity, and known methods can be used. For example, an antibody solution adjusted to a desired concentration can be placed in the culture vessel and immobilized at room temperature for 3 hours. The culture vessel with the immobilized antibody can be stored, for example, at 4°C until use. When using the culture vessel on the day the antibody was immobilized, it is preferable to wash the inside of the culture vessel approximately twice with an appropriate amount of PBS. In the method for producing an NK cell-enriched cell population of the present invention, it is desirable to immobilize each antibody on a multiwell plate, flask, or the like for adherent culture. The concentrations of the anti-CD2 antibody, anti-CD52 antibody, and anti-CD335 antibody are not limited as long as they stimulate NK cells and enhance their activity, but can be immobilized at concentrations in the range of, for example, 5 to 20 μg / mL. PBS or the like can be used as a solvent for preparing the immobilized antibody solution.

[0017] Examples of media used for culture include, but are not limited to, KBM501 medium (Kohjin Bio Co., Ltd.), IMDM, MEM, DMEM, and RPMI1640.

[0018] In one embodiment, the process for culturing a mononuclear cell population containing NK cells further comprises culturing the NK cell population in the presence of at least one physiologically active substance selected from the group consisting of IL-2, IL-15, IL-18, and hydrocortisone. As described above, interleukin-2 (IL-2) may be added to the culture medium at a concentration sufficient to achieve the objectives of the present invention. The concentration of IL-2 in the culture medium may range from 2000 IU / mL to 2810 IU / mL. IL-2 preferably has a human amino acid sequence and, for safety reasons, is preferably produced using recombinant DNA technology. In this specification, the concentration of IL-2 may be expressed in Japanese National Standard Units (JRU) and International Units (IU). Adding IL-2 to the culture medium can enhance the antitumor effect of NK cells.

[0019] In the culture process of mononuclear cell populations containing NK cells, the simultaneous addition of IL-2, IL-15, and IL-18 promotes NK cell proliferation compared to stimulation with IL-2 alone. Furthermore, the addition of hydrocortisone to the medium containing these three cytokines further promotes NK cell proliferation.

[0020] In the process of culturing a mononuclear cell population containing NK cells, the concentrations of IL-15 and IL-18 in the medium are not limited as long as the desired effect is achieved, and can be, for example, in the range of 50 to 200 ng / mL. It is desirable to add them at a concentration of 100 ng / mL from the early to mid-stages of culture. Furthermore, the concentration of hydrocortisone is not limited as long as the desired effect is achieved, and can be, for example, in the range of 0.1 to 1 μM, but is desirably 1 μM from the early to mid-stages of culture. As used herein, "early stage of culture" refers to the period from approximately day 7 of culture to approximately day 14 of culture. In the process of culturing a mononuclear cell population containing NK cells, stimulation with each antibody and stimulation with at least one physiologically active substance selected from the group consisting of IL-2, IL-15, IL-18, and hydrocortisone are carried out simultaneously. When hydrocortisone is added, it is preferable to add it two or three days after the start of culture, and thereafter maintain the culture in a state where the three cytokines and hydrocortisone are added simultaneously.

[0021] The medium may be supplemented with autologous serum from the subject, human AB type serum available from Kohjin Bio Co., Ltd., or fetal bovine serum (FBS) available from Sigma-Aldrich Co., Ltd. The serum concentration in the medium is preferably 5-10%. It is preferable that the human AB type serum and FBS have been heat-inactivated.

[0022] The medium may contain other suitable proteins, cytokines, antibodies, compounds, or other components, provided that they do not impair the proliferation and activation of the resulting NK cells. The cytokines may be interleukin-12 (IL-12) or interleukin-21 (IL-21).

[0023] In the process of culturing a mononuclear cell population containing NK cells, culture vessels include, but are not limited to, commercially available dishes, flasks, plates, and culture bags. Culture conditions are not particularly limited as long as they do not impair NK cell proliferation, but culture conditions of 37°C, 5% CO2, and a saturated water vapor atmosphere are common. In the present invention, the culture period for the mononuclear cell population containing NK cells is 21 days, but the culture period is not limited as long as the desired cell number is expanded. It is preferable to change the medium appropriately during the culture period, for example, once every 2 to 3 days, but this is not limited thereto.

[0024] After culturing a mononuclear cell population containing NK cells, the resulting cell population is recovered from the culture vessel. After the culturing process, the culture vessel contains both floating cells and cells adhered to a solid surface. Because NK cells are contained in both floating and adherent cells, both floating and adherent cells are recovered. For floating cells, the culture supernatant can be collected. On the other hand, for recovering adherent cells, it is preferable to use a chelating agent to remove the cells without damaging them. That is, in one embodiment, the method for producing a cell population enriched in NK cells of the present invention includes, after culturing a mononuclear cell population, recovering adherent cells from the cell population enriched in NK cells using a chelating agent. Preferred chelating agents are those that bind to divalent metal ions such as calcium and magnesium. Examples of suitable chelating agents include, but are not limited to, EDTA. For example, the adherent cells can be detached from the culture vessel by leaving the cell population in PBS containing a chelating agent for approximately 5 to 10 minutes.

[0025] According to the method of the present invention for producing a cell population enriched in NK cells, the expanded and cultured NK cells do not need to be maintained only in a flask, but may be maintained in a cell culture bag with high gas permeability, such as a culture bag (product number A-350NL) manufactured by Nipro Corporation. Culture bags can be used from the middle to late stages of culture.

[0026] One embodiment of the production method of the present invention further includes a step of enriching NK cells contained in the cell population prior to the step of culturing the NK cell population. The NK cell enrichment step enriches NK cells in the cell population by separating and removing T cells, B cells, granulocytes, etc. from the cell population. Known techniques can be used to separate or enrich NK cells from a cell population containing T cells, B cells, granulocytes, etc. Preferably, NK cells are enriched by negative selection using immunomagnetic beads. Examples of immunomagnetic beads include, but are not limited to, Dynabeads (trademark) manufactured by Dynal, sold by Invitrogen, and EasySep (trademark) manufactured by STEMCELL. By culturing the cell population that has undergone the NK cell enrichment step in the presence of three antibodies: anti-CD2 antibody, anti-CD52 antibody, and anti-CD335 antibody, the proportion of NK cells in the resulting cell population can be increased to 90% or more. Preferably, it is 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more, and more preferably 99% or more.

[0027] The mononuclear cell population containing NK cells used in the present invention may contain T cells and the like in addition to NK cells. Even when the NK cell population in the present invention is adjusted by a cell separation and concentration method using immunomagnetic beads at the early stage of culture, contamination with unnecessary cells including T cells and the like cannot be completely avoided. Therefore, after the culture step of the mononuclear cell population containing NK cells using this method, expansion of cell populations other than NK cells may occur. Therefore, after expanding the NK cells, NK cells can be recovered or further concentrated from the cell population enriched in NK cells using, for example, immunomagnetic beads, FACS, or the like.

[0028] Another aspect of the present invention provides an NK cell population having the following characteristics: (1) a cytotoxic activity of 50% or more when NK cells are used as effector cells (E) and K562 cells are used as target cells (T) and co-cultured at a mixing ratio (E:T) of 1:1; and (2) an ADCC activity of 5% or more when NK cells are used as effector cells (E) and SK-BR-3 cells are used as target cells (T) and co-cultured at a mixing ratio (E:T) of 0.5:1 in the presence of 0.01 μg / mL to 1 μg / mL of Herceptin.

[0029] The NK cell populations provided by the present invention have high activity. As used herein, "activity" refers to cytotoxic activity and / or antibody-dependent cellular cytotoxicity (ADCC activity). The NK cell populations provided by the present invention have cytotoxic activity that is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, or 50-fold or more higher than that of NK cell populations expanded and cultured using existing kits (specifically, KBM NK KIT (16030210, Kohjin Bio Co., Ltd.) and BIN KIT (N501-1, Japan Biotherapy Institute). Furthermore, the NK cell populations provided by the present invention have ADCC activity that is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, or 8-fold or more higher than that of NK cell populations expanded and cultured using existing kits (specifically, KBM NK KIT (16030210, Kohjin Bio Co., Ltd.) and BIN KIT (N501-1, Japan Biotherapy Institute).

[0030] The cytotoxic activity of NK cells can be measured and calculated by methods well known to those skilled in the art. Preferably, a free LDH activity assay can be employed, using a commercially available kit (e.g., CK12 (Cytotoxicity LDH Assay Kit-WST), Dojindo Laboratories). The cytotoxic activity (%) can usually be calculated based on the viable cell count of target cells (T) after the action of effector cells (E). With the commercially available kit, the viable cell count of target cells can be measured using the absorbance of LDH in the medium as an indicator. When NK cells are used as effector cells (E) and K562 cells are used as target cells (T) and co-cultured at a 1:1 ratio (E:T) for 4 hours using the commercially available kit, the cytotoxic activity is preferably 50% or higher, more preferably 55% or higher, even more preferably 60% or higher, and even more preferably 64% or higher. In the case of NK cells prepared from cells derived from a cancer patient (e.g., PMBC), when the NK cells are used as effector cells (E) and K562 cells as target cells (T) and co-cultured at a mixing ratio (E:T) of 1:1 for 6 hours, the cytotoxic activity is preferably 15% or more, more preferably 20% or more, 25% or more, 30% or more, 35% or more, and even more preferably 40% or more.

[0031] Furthermore, when NK cells are used as effector cells (E) and K562 cells as target cells (T) and co-cultured at a mixing ratio (E:T) of 5:1 for 6 hours using the commercially available kit, the cytotoxic activity is preferably 70% or more, more preferably 75% or more, and even more preferably 80% or more. In the case of NK cells prepared from cells derived from a cancer patient (e.g., PMBC), when the NK cells are used as effector cells (E) and K562 cells as target cells (T) and co-cultured at a mixing ratio (E:T) of 5:1 for 6 hours, the cytotoxic activity is preferably 45% or more, more preferably 50% or more, 55% or more, 60% or more, 65% or more, and even more preferably 70% or more.

[0032] The ADCC activity of NK cells can be measured and calculated by methods well known to those skilled in the art. Preferably, a free LDH activity measurement method can be employed, and measurement is performed using a commercially available kit (e.g., CK12 (Cytotoxicity LDH Assay Kit-WST), Dojindo Laboratories). ADCC activity (%) can usually be calculated based on the viable cell count of target cells (T) after the action of effector cells (E). With the commercially available kit, the viable cell count of target cells can be measured using the absorbance of LDH in the medium as an indicator. Using the commercially available kit, NK cells are mixed with effector cells (E) and SK-BR-3 cells as target cells (T) at a mixing ratio (E:T) of 1:1 and co-cultured for 5 hours in the presence of Herceptin (Chugai Pharmaceutical Co., Ltd.). The ADCC activity is preferably 5% or higher, more preferably 6% or higher, even more preferably 7% or higher, even more preferably 8% or higher, and even more preferably 9% or higher.

[0033] Using the commercially available kit, NK cells were used as effector cells (E) and RLEU1-7 (a B-cell leukemia cell line; Fukushima Medical University Translational Research Institute) as target cells (T) at a mixing ratio (E:T) of 1:1. When co-cultured for 6 hours in the presence of Herceptin (Chugai Pharmaceutical Co., Ltd.), the ADCC activity was preferably equivalent to the cytotoxicity rate in the absence of antibody. This is due to the fact that the RLEU1-7 cell line does not express HER2, the target molecule of Herceptin.

[0034] When the commercially available kit is used to co-culture NK cells as effector cells (E) with RLEU1-7 as target cells (T) at a mixing ratio (E:T) of 1:1 for 6 hours in the presence of Rituxan (Zenyaku Kogyo Co., Ltd.), the ADCC activity is preferably 30% or higher, more preferably 35%, 40%, or 45% or higher, and even more preferably 50% or higher. In the case of NK cells prepared from cells derived from a cancer patient (e.g., PMBC), when the NK cells as effector cells (E) and RLEU1-7 as target cells (T) are co-cultured at a mixing ratio (E:T) of 1:1 for 6 hours in the presence of Rituxan (Zenyaku Kogyo Co., Ltd.), the ADCC activity is preferably 5% or higher, more preferably 10%, 15% or higher, and even more preferably 20% or higher.

[0035] When the commercially available kit is used, NK cells are used as effector cells (E) and RLEU1-7 as target cells (T) at a mixing ratio (E:T) of 1:1, and the cells are co-cultured for 6 hours in the presence of DARAZALEX (Janssen Pharmaceutical K.K.). The ADCC activity is preferably 55% or higher, more preferably 60%, 65%, or 70% or higher, and even more preferably 75% or higher. In the case of NK cells prepared from cells derived from a cancer patient (e.g., PMBC), when the NK cells are used as effector cells (E) and RLEU1-7 as target cells (T) at a mixing ratio (E:T) of 1:1, and the cells are co-cultured for 6 hours in the presence of DARAZALEX (Janssen Pharmaceutical K.K.), the ADCC activity is preferably 8% or higher, more preferably 10%, 15%, 20%, 25%, 30%, or 35% or higher, and even more preferably 40% or higher.

[0036] In one embodiment, the NK cell population of the present invention further has the following characteristics: (3) It contains 95% or more CD56-positive, CD3-negative NK cells, more preferably 96% or more, 97% or more, 98% or more, or 99% or more CD56-positive, CD3-negative NK cells.

[0037] (Example 1. Method for Proliferating NK Cells) 1. Materials and Methods (1) Preparation of PBMCs Normal human PBMCs were obtained from LONZA (CC2703, LONZA).

[0038] (2) Preparation of Solid-Phase Flasks. Antibody solutions were prepared by adding anti-CD2 antibody (300202, BioLegend Japan), anti-CD52 antibody (316002, BioLegend Japan), and anti-CD335 antibody (331902, BioLegend Japan) to PBS (20012027, Gibco, ThermoFisher Scientific) to a final concentration of 10 μg / mL. The prepared antibody solution was added to a T25 flask (690175, Greiner Bio-One). It is recommended to add at least 3 mL of antibody solution to cover the entire solid-phase surface. After immobilization at room temperature for 3 hours, the flasks were stored in a refrigerator at 4°C. If the immobilization flasks were to be used on the same day, the flasks could be washed twice with an appropriate amount of PBS after the 3-hour immobilization period and then used for culturing. As the culture scale expands, T75 flasks (658175, Greiner Bio-One) or T175 flasks (661175, Greiner Bio-One) can be used.

[0039] (3) Isolation and enrichment of NK cells. PBMCs were thawed in a 37°C water bath and suspended in medium or PBS. The supernatant was discarded after centrifugation. The medium consisted of IMDM (12440053, Gibco, ThermoFisher Scientific) supplemented with ITS-X supplement (094-06761, Fujifilm Wako Pure Chemical Industries, Ltd.; 100-fold dilution), monothioglycerol solution (195-15791, Fujifilm Wako Pure Chemical Industries, Ltd.; 100-fold dilution), non-essential amino acid solution (139-15651, Fujifilm Wako Pure Chemical Industries, Ltd.; 100-fold dilution), essential amino acid solution (M5550-100mL, SIGMA-Aldrich; 100-fold dilution), linoleic acid-oleic acid-albumin solution (L9655-5mL, SIGMA-Aldrich; 100-fold dilution), and GlutaMAX. TMThe pellet after centrifugation was counted, and then the cells were analyzed using Easy Separation. TM NK cells were isolated and concentrated using the method described in the Human NK Cell Isolation Kit (17955, STEMCELL Technologies).

[0040] (4) NK Cell Culture: The antibody solution in the immobilized flask prepared in step (2) was discarded and washed twice with PBS. IMDM (12440053, Gibco, ThermoFisher Scientific) was used as the basal medium for culture. This IMDM was supplemented with ITS-X supplement (094-06761, Fujifilm Wako Pure Chemical Industries, Ltd.; 100-fold dilution), monothioglycerol solution (195-15791, Fujifilm Wako Pure Chemical Industries, Ltd.; 100-fold dilution), non-essential amino acid solution (139-15651, Fujifilm Wako Pure Chemical Industries, Ltd.; 100-fold dilution), essential amino acid solution (M5550-100mL, SIGMA-Aldrich; 100-fold dilution), linoleic acid-oleic acid-albumin solution (L9655-5ML, SIGMA-Aldrich; 100-fold dilution), and GlutaMAX. TMThe medium was diluted 1:100 with 100% inactivated fetal bovine serum (35050061, Gibco, ThermoFisher Scientific). Inactivated fetal bovine serum (172012-500ML, SIGMA-Aldrich) was added to a concentration of 10%. IL-2 (202-IL-050, R&D SYSTEMS) was added to a concentration of 2000 IU / mL as cytokines. IL-15 (247-ILB-025, R&D SYSTEMS) and IL-18 (9124-IL-050, R&D SYSTEMS) were added to a final concentration of 100 ng / mL. Thus, NK cell culture medium was prepared. The NK cells isolated and enriched in step (3) were suspended in the NK cell culture medium and seeded into solid-phase flasks. The cells were cultured at 37°C under 5% CO2 and saturated water vapor for 21 days. During the culture period, the medium was replaced as needed, using color change as an indicator.

[0041] (5) Culture Process Up to Harvest: On day 2 of culture, hydrocortisone solution (H6909-10ML, SIGMA-Aldrich) was added to the NK cell culture medium to a final concentration of 1 μM. In subsequent processes involving the addition of medium or expansion of the culture scale, the serum concentration added to the medium was adjusted to 5–10%, and the cytokine concentrations added were 2000 IU / mL–2810 IU / mL for IL-2, 50–200 ng / mL for IL-15 and IL-18, and 0.1–1 μM for hydrocortisone solution. On day 5 of culture, an appropriate amount of medium was added. On day 7 of culture, a T25 flask was prepared for solid-phase cell culture, as in step (2). On day 8 of culture, approximately half of the NK cells and NK cell culture medium in the T25 flask were transferred to a newly prepared T25 flask, and the culture was expanded to two T25 flasks. On day 10 of culture, T175 flasks were prepared as in step (2). On day 11 of culture, half of the NK cells in two T25 flasks were transferred to newly prepared T175 flasks, and an appropriate amount of NK cell culture medium was added to the T25 and T175 flasks. The culture scale was expanded to two T25 flasks and one T175 flask. On day 14 of culture, half of the NK cells in two T25 flasks were transferred to a T175 flask, and an appropriate amount of NK cell culture medium was added to the T25 and T175 flasks. On day 17 of culture, an appropriate amount of NK cell culture medium was added using the same method as on day 14 of culture. On day 21 of culture, NK cells were harvested.

[0042] (6) NK Cell Recovery: NK cells suspended in the culture medium and those attached to the solid surface coexist in the flask. While cells suspended in the culture medium are easily recovered, recovering cells attached to the solid surface requires ingenuity. Repeated pipetting to detach cells from the solid surface physically damages the cells, and cell detachment using certain enzymes also damages the cells, resulting in a decrease in viability. Therefore, the culture medium in the flask was removed to recover the suspended cells, and then PBS-EDTA (13567-84, Nacalai Tesque, Inc.) was added. The NK cells attached to the solid surface were then recovered easily and without damaging the cells by leaving the flask at room temperature for approximately 5 minutes. The recovered NK cells were used in the following tests. A portion of the recovered NK cells was suspended in CELLBANKER® 1 (CB011, distributed by TAKARA Bio Co., Ltd. and manufactured by Nippon Zenyaku Kogyo Co., Ltd.), a cell cryopreservation reagent, to obtain a cell density of 1.0 × 10 NK cells per cryovial. 7 The cryovials were then slowly frozen in a programmable freezer.

[0043] (7) Culture of NK Cells for Comparison To demonstrate the superiority of the NK cells produced by the present invention, NK cells were produced using an existing NK cell culture kit and used for comparison. The existing NK cell culture kits used were the KBM NK KIT (16030210, Kohjin Bio Co., Ltd.) and the BIN KIT (N501-1, Japan Biotherapy Research Institute). For comparative studies, NK cells produced by the method of the present invention and NK cells produced using the KBM KN KIT or BIN KIT were obtained from the same donor and lot of PBMC. NK cells were produced using the KBM NK KIT and BIN KIT according to the instructions in the respective kits. NK cells cultured using the KBM NK KIT were collected according to the kit's instructions and used in the following tests. NK cells cultured using the BIN KIT were collected from the flasks used for the culture and used in the following tests.

[0044] (8) Cytotoxicity Test: Cytotoxicity tests were performed using NK cells obtained with the present and comparative kits as effector cells (E) and K562 as the target cell (T) line. Cytotoxicity was measured using a free LDH activity assay (CK12 (Cytotoxicity LDH Assay Kit-WST), Dojindo Laboratories) according to the homogeneous assay method described in the CK12 instruction manual. The E:T ratio (effector cell number:target cell number ratio) during the cytotoxicity test was 1:1, and effector and target cells were co-cultured for 4 hours. Furthermore, ADCC activity was measured using the NK cells as effector cells (E), SK-BR-3 as the target cell (T) line, and Herceptin (Chugai Pharmaceutical Co., Ltd.) as the antibody. As in the cytotoxicity assay, the ADCC activity assay was performed using the free LDH activity assay (CK12 (Cytotoxicity LDH Assay Kit-WST), Dojindo Laboratories) according to the homogeneous assay method described in the CK12 instruction manual. The E:T ratio for the ADCC activity assay was 0.5:1, and effector and target cells were co-cultured for 5 hours. The Herceptin concentrations were 0, 1, 0.1, and 0.01 μg / mL.

[0045] (9) Analysis of cell surface markers The NK cell ratio (the ratio of CD3-negative and CD56-positive cells in the NK cell population) was analyzed in the NK cell populations obtained using the kits of the present invention and comparative kits. The cells were detected by flow cytometry using an anti-CD3 antibody (344804, BioLegend Japan) and an anti-CD56 antibody (344804, BioLegend Japan).

[0046] 2. Results (1) Results of Proliferation and Culture of NK Cells from Healthy Individuals Table 1 shows the proliferation rates of NK cells obtained by steps (4) to (6) above and NK cells propagated using a comparison kit. The proliferation rate for each NK cell culture method (cell number at harvest divided by cell number seeded) was calculated based on the number of cells seeded and the number of cells at harvest after 21 days of culture. The results showed that NK cells produced using this method proliferated at a minimum of 57-fold and a maximum of 651.7-fold. NK cells produced using the KBM NK KIT proliferated at a minimum of 49.7-fold and a maximum of 152-fold. NK cells produced using the BIN KIT proliferated at a minimum of 51-fold and a maximum of 179-fold. These results compare favorably with or even better than those of commercially available kits. These results demonstrated that the method of culturing NK cells in the presence of three antibodies, anti-CD2 antibody, anti-CD52 antibody, and anti-CD335 antibody, achieved cell proliferation effects comparable to or superior to those achieved by commercially available kits.

[0047]

[0048] (2) Quantification of Cytotoxic Activity Figure 1 is a graph showing the results of a cytotoxicity test against K562 cells using NK cells obtained by steps (4) to (6) above (NK cells produced by this method) and NK cells expanded and cultured using KBM NK KIT or BIN KIT (KBM NK KIT-produced NK or BIN KIT-produced NK). The vertical axis represents cytotoxic activity (unit: %). In Figure 1, the white bars represent the results of the cytotoxicity test using NK cells obtained by steps (4) to (6) above. The black bars represent the results of the cytotoxicity test using NK cells produced using KBM NK KIT. The gray bars represent the results of the cytotoxicity test using NK cells produced using BIN KIT. As shown in Figure 1, the NK cells obtained by steps (4) to (6) above exhibited 64% cytotoxicity against K562 cells. On the other hand, NK cells obtained using the KBM NK KIT showed 0.43% cytotoxicity, and NK cells obtained using the BIN KIT showed 15% cytotoxicity. These results indicate that NK cells obtained by culturing NK cells in the presence of anti-CD2, anti-CD52, and anti-CD335 antibodies have higher cytotoxicity against K562 than NK cells obtained using the existing kits.

[0049] (3) Quantification of ADCC Activity Figure 2 shows the results of an ADCC activity assay using NK cells obtained by steps (4) to (6) above (NK cells produced by this method) and NK cells produced by existing kits (NK cells produced by KBM NK KIT or NK cells produced by BIN KIT). The vertical axis represents cytotoxic activity (unit: %). The left graph in the figure shows the results of ADCC activity assay using NK cells produced by steps (4) to (6) above. The center graph shows the results of ADCC activity assay using NK cells produced by KBM NK KIT. The right graph shows the results of ADCC activity assay using NK cells produced by BIN KIT. From the results shown in Figure 2, first, in the absence of Herceptin, only NK cells produced by this method exhibited cytotoxic activity, while NK cells produced by either KBM NK KIT or BIN KIT did not exhibit cytotoxic activity. Under Herceptin-containing conditions, all NK cells exhibited ADCC activity, but the ADCC activity of NK cells produced by culturing NK cells in the presence of three antibodies, anti-CD2 antibody, anti-CD52 antibody, and anti-CD335 antibody, was higher than that of NK cells produced with each kit.

[0050] (4) Analysis of NK Cell Surface Markers Figures 3, 4, and 5 show the ratios of CD3-negative / CD56-positive cells in the NK cell population obtained by steps (4) to (6) above (NK cells produced by this method) and in the NK cell population obtained by each of the existing kits (KBM NK KIT-produced NK or BIN KIT-produced NK). Figure 3 shows the results for the NK cell population obtained by steps (4) to (6) above (NK cells produced by this method). As shown in Figure 3, the ratio of CD3-negative / CD56-positive cells in the NK cell population obtained by steps (4) to (6) above was a very high 98.65%. This indicates that the NK cell population obtained by culturing NK cells in the presence of three antibodies, anti-CD2 antibody, anti-CD52 antibody, and anti-CD335 antibody, has a high NK cell purity. Meanwhile, Figure 4 shows the results for NK cell populations expanded and cultured using KBM NK KIT (KBM NK KIT-produced NK), and Figure 5 shows the results for NK cell populations expanded and cultured using BIN KIT (BIN KIT-produced NK). As shown in Figures 4 and 5, the CD3-negative / CD56-positive cell ratios were 18.43% and 7.72%, respectively. The cell populations obtained using KBM NK KIT and BIN KIT showed a significant expansion of immune cells other than NK cells.

[0051] The above experimental results demonstrate that, compared to existing NK cell culture kits, it is possible to produce highly pure NK cells with high cytotoxic activity and / or ADCC activity without the need for complicated procedures.

[0052] (Example 2. Method for expanding NK cells using PBMCs derived from cancer patients) It is said that the immune function of cancer patients is often weakened due to various factors such as medication and radiation therapy in cancer treatment. In this example, NK cells were actually produced from PBMCs derived from cancer patients and normal human PBMCs, and it was confirmed whether there was a difference in their proliferation ability and inhibitory ability.

[0053] 1. Materials and Methods PBMCs from cancer patients were obtained from 13 sources: 10 from ProteoGenex and 2 from EWBio. The cancer types obtained were: 3 gastric cancer, 2 melanoma, 2 ovarian cancer, 2 bladder cancer, 1 cervical cancer, 1 prostate cancer, 1 head and neck cancer, and 1 renal cancer. Normal human PBMCs were also obtained from LONZA for comparison (CC2703, LONZA).

[0054] (1) Method for Proliferating NK Cells NK cells were obtained by the same procedure and conditions as those described in "1. Materials and Methods" in Example 1.

[0055] (2) Cytotoxicity Test: Cytotoxicity tests were performed using NK cells isolated from cancer patient-derived PBMCs and normal PBMCs as effector cells (E) and K562 as the target cell (T) line. Cytotoxicity was measured using the free LDH activity assay (CK (Cytotoxicity Assay Kit-WST), Dojindo Laboratories) according to the homogeneous assay method described in the CK12 instruction manual. The E:T ratio (effector cell number:target cell number) used in the cytotoxicity test was 1:1 or 5:1, and the effector and target cells were co-cultured for 6 hours. Only samples containing the required number of cells were tested.

[0056] Furthermore, ADCC activity was measured using the NK cells as effector cells (E) and RLEU1-7 (a B-cell leukemia cell line; Fukushima Medical University Translational Research Institute) as target cells (T). The antibodies used were Herceptin (Chugai Pharmaceutical Co., Ltd.), Rituxan (Zenyaku Kogyo Co., Ltd.), and DARAZALEX (Janssen Pharmaceuticals K.K.). Similar to the cytotoxicity assay, the ADCC assay employed a free LDH activity assay (CK12 (Cytotoxicity LDH Assay Kit-WST), Dojindo Laboratories) according to the homogeneous assay method described in the CK12 instruction manual. The E:T ratio for the ADCC assay was 1:1, and the effector and target cells were co-cultured for 6 hours. The concentration of each antibody was 1 μg / mL.

[0057] 2. Results (1) Results of proliferation and culture of NK cells obtained from PMBCs derived from cancer patients Table 2 shows the proliferation rates of NK cells obtained from PMBCs derived from cancer patients or normal human PMBCs obtained by the above process. The proliferation rate of each NK cell type (number of cells at harvest ÷ number of cells seeded) was calculated from the number of cells seeded and the number of cells after 21 days of culture, i.e., at harvest.

[0058] The results in Table 2 show that for some samples, even NK cells generated from PBMCs derived from cancer patients were significantly proliferated by culturing NK cells in the presence of three antibodies: anti-CD2, anti-CD52, and anti-CD335. However, the proliferation rate of NK cells generated from PBMCs derived from cancer patients tended to be lower than that of NK cells generated from PBMCs of normal donors, and some samples showed poor proliferation, forcing the culture to be discontinued midway through.

[0059]

[0060] (2) Quantification of Cytotoxic Activity Figure 6 is a graph showing the results of a cytotoxicity test of NK cells obtained by the above process against K562. In this example, the results of the cytotoxicity test of NK cells derived from donors 1, 4, 7, 8, 11, and normal PBMCs are shown, which satisfied the cell numbers required for the experiment. For comparison, Figure 6 also shows the results of a cytotoxicity test using normal donor-derived mixed NK cells, which were prepared from normal PBMCs by the same culture process, but were combined into one lot by mixing NK cells from multiple donors. In Figure 6, the vertical axis represents cytotoxic activity (unit: %), and white bars represent the cytotoxicity test results at an E:T ratio of 1:1. Gray bars represent the cytotoxicity test results at an E:T ratio of 5:1.

[0061] As shown in Figure 6, NK cells generated from PBMCs derived from cancer patients showed improved cytotoxicity when cultured in the presence of three antibodies: anti-CD2, anti-CD52, and anti-CD335. NK cells derived from cancer patients exhibited lower cytotoxicity than NK cells derived from normal PBMCs. However, mixed NK cells derived from normal donors also exhibited stable and high cytotoxicity.

[0062] (3) Quantification of ADCC Activity Figure 7 is a graph showing the results of an ADCC activity test of NK cells obtained by the above process. In this example, the results of the cytotoxicity test of NK cells derived from donors 1, 3, 4, 5, 7, 8, and 11, and normal PBMCs, which satisfied the conditions for the number of cells required for the experiment, are shown. For comparison, Figure 7 also shows the results of the cytotoxicity test using normal donor-derived mixed NK cells, which were prepared from normal PBMCs by the same culture process, but were combined into one lot of NK cells from multiple donors.

[0063] In Figure 7, the vertical axis represents cytotoxic activity (unit: %), and the white bars represent the results of the ADCC assay without antibody. The diamond bars represent the results of the ADCC assay with Herceptin, the gray bars represent the results of the ADCC assay with Rituxan, and the black bars represent the results of the ADCC assay with Darazalex. The results in Figure 7 demonstrate that NK cells generated from PBMCs derived from cancer patients exhibited improved cytotoxic activity in the ADCC assay when cultured in the presence of anti-CD2, anti-CD52, and anti-CD335 antibodies. Furthermore, NK cells derived from cancer patients exhibited lower cytotoxicity than NK cells derived from normal PBMCs. However, stable and high cytotoxicity was also demonstrated when mixed NK cells derived from normal donors were used.

Claims

1. A method for producing a cell population enriched in NK cells, comprising the step of culturing a mononuclear cell population containing NK cells in the presence of three antibodies: an anti-CD2 antibody, an anti-CD52 antibody, and an anti-CD335 antibody.

2. The method of claim 1, wherein the mononuclear cell population is derived from at least one cell population selected from the group consisting of peripheral blood mononuclear cells (PBMC), bone marrow mononuclear cells (BMNC), umbilical cord blood mononuclear cells (CBMNC), embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells.

3. The method of claim 1, wherein the mononuclear cell population is derived from peripheral blood mononuclear cells (PBMCs).

4. The method for producing a cell population enriched in NK cells according to claim 1, wherein the step of culturing the mononuclear cell population is carried out using a culture vessel coated with the antibody.

5. The method for producing a cell population enriched in NK cells according to claim 1, wherein the step of culturing the mononuclear cell population further comprises culturing the mononuclear cell population in the presence of at least one physiologically active substance selected from the group consisting of IL-2, IL-15, IL-18, and hydrocortisone.

6. The method for producing a cell population enriched in NK cells according to claim 1, further comprising a step of concentrating NK cells contained in the mononuclear cell population prior to the step of culturing the mononuclear cell population.

7. The method for producing a cell population enriched in NK cells according to claim 1, further comprising a step of recovering NK cells from the cell population enriched in NK cells or concentrating NK cells after the step of culturing the mononuclear cell population.

8. The method for producing a cell population enriched in NK cells according to claim 1, wherein the recovery step is a step of recovering adherent cells from the cell population enriched in NK cells using a chelating agent.

9. The method for producing a cell population enriched in NK cells according to claim 1, wherein the concentration of each of the antibodies in the mononuclear cell population culture step is in the range of 5 to 20 μg / mL.

10. A cell population enriched in NK cells, produced by the production method described in claim 1.

11. A NK cell population having the following characteristics: (1) a cytotoxic activity of 50% or more when NK cells are used as effector cells (E) and K562 cells are used as target cells (T) and co-cultured at a mixture ratio (E:T) of 1:1, and (2) an ADCC activity of 5% or more when NK cells are used as effector cells (E) and K562 cells are used as target cells (T) and co-cultured at a mixture ratio (E:T) of 0.5:1 in the presence of 0.01 μg / mL to 1 μg / mL of Herceptin.

12. The NK cell population according to claim 11, (1) having a cytotoxic activity of 60% or more when co-cultured at a mixture ratio (E:T) of 1:1 using NK cells as effector cells (E) and K562 cells as target cells (T).

13. The NK cell population according to claim 11, (2) having an ADCC activity of 8% or more when NK cells are used as effector cells (E) and SK-BR-3 cells are used as target cells (T) and co-cultured at a mixture ratio (E:T) of 0.5:1 in the presence of 0.01 μg / mL to 1 μg / mL of Herceptin.

Images