Method for producing natural killer cells

Abstract

This invention relates to a method for producing natural killer cells, comprising the step of culturing pluripotent stem cells in StemScale PSC complete suspension medium supplemented with CHIR 99021 to form embryoid bodies (EBs). By culturing induced pluripotent stem cells (iPSCs) in vitro under appropriate culture conditions, hematopoietic stem cells and progenitor cells are provided at low cost and with high efficiency, and further differentiation into NK cells is induced. The NK cells produced in this way have good cell proliferation ability and potent killing ability.

Description

cross reference

[0001] This invention claims priority to Chinese Patent Application No. 202311309567.2, filed with the China National Intellectual Property Office on 11 October 2023, titled "Method for Producing Natural Killer Cells," the entirety of which is incorporated herein by reference. [Technical Field]

[0002] The present invention relates to a method for producing natural killer cells, and more specifically to a method for inducing natural killer (NK) cells from pluripotent stem cells, the NK cells thus formed, and the use of said NK cells in the treatment or remission of viral infections. [Background technology]

[0003] NK cells are large granular lymphocytes that possess the intrinsic ability to kill infected and tumor cells without requiring human leukocyte antigen (HLA) restriction or prior sensitization with antigens. Human NK cells are typically characterized as CD56+CD3- lymphocytes and are broadly classified into two subsets based on CD56 and CD16 expression levels: CD56bright CD16- cells and CD56dim CD16+ cells. Within this subset, CD16 expression is associated with the maturity of NK cells.

[0004] The stimulatory and effector functions of NK cells depend on the integration of signals from two different types of receptors (activating receptors and inhibitory receptors). Normal, healthy cells express MHC class I molecules on their surface. These molecules function as ligands for inhibitory receptors, aiding in NK cell self-tolerance. However, virus-infected cells and tumor cells lose surface MHC class I expression, resulting in reduced inhibitory signaling in NK cells. Furthermore, cellular stresses associated with viral infection and tumor progression, such as DNA damage responses, cellular senescence, and tumor suppressor genes, increase ligand expression for activating receptors in these cells. Therefore, signals from activating receptors in NK cells shift the balance towards NK cell activation and target cell elimination, either directly through NK cell-mediated cytotoxicity or indirectly through the secretion of inflammatory cytokines.

[0005] Once activated, NK cells release pre-formed cytolytic granules containing granzymes and perforins to degrade infected and tumor cells. Degranulation of NK cells can also occur through CD16-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Activated NK cells further secrete various cytokines, including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), and chemokines (CCL1, CCL2, CCL3, CCL4, CCL5, and CXCL8). These cytokines can modulate the function of other innate and adaptive immune cells.

[0006] Allogeneic primary NK cells derived from peripheral blood and umbilical cord blood (PB-NK or UCB-NK, respectively) have been proven to be safe and effective cell therapies without serious toxicities such as cytokine release syndrome (CRS), neurotoxicity, or graft-versus-host disease (GVHD). However, the heterogeneous cell preparations of PB-NK and UCB-NK cells used in these studies, which vary from donor to donor, limit the development of standardized cellular immunotherapy formulations.

[0007] Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are optimal starting cells for developing various cell lineages, including NK cells. Studies have shown that hESC / iPSC-derived NK cells (iNKs) exhibit potent antitumor and antiviral activity, offering a potential approach for standardized cell therapies for these diseases. iNK cells possess potent functionality and broad cytotoxic activity against hematological malignancies and solid tumors. iNK cells are available from a homogeneous, reproducible, and sustained supply, allowing for use in multiple-dose therapies and eliminating donor variability. Furthermore, it is feasible and consistent to differentiate iNK cells with improved antitumor activity and in vivo persistence through genetic engineering techniques applied to hESCs and iPSCs. iNK cells are considered a promising new therapy for over-implantation NK cell immunotherapy and can be offered as a ready-made cell therapy or combined with antibodies targeting inhibitory checkpoint receptors to enhance antitumor effects.

[0008] Currently, improving the maturity, functionality, and in vivo persistence of iNK cells remains a challenge in this field. While NK cells have traditionally been considered a type of innate immune-mediated cell, recent studies have discovered that NK cells can acquire memory-like (ML) properties after viral infection, hapten exposure, or transient cytokine activation (such as IL-12, IL-15, and IL-18). Memory-like NK cells are highly mature, and their IFN-γ production capacity is enhanced by cytokine restimulation or re-exposure to cancer cells, which is associated with the expression of CD94, NKG2A, and NKG2C. Memory-like NK cells expressing CD19 CAR exhibit potent killing effects against NK-resistant lymphoma cell lines and major target cells. Specifically, memory-like NK cells induced by IL-12, IL-15, and IL-18 have been found to exhibit superior in vivo persistence and a more potent anti-leukemic response in mouse xenograft models. In short, these data indicate that improving the maturation of NK cells with IL-12, IL-15, and IL-18 leads to improved antitumor function. However, previous studies have mainly focused on memory-like NK cells derived from peripheral blood. Because peripheral blood has limited availability as a source of NK cells, there is an urgent need in this field for new methods that can produce mature NK cells in scalable and large quantities. [Overview of the project] [Problems that the invention aims to solve]

[0009] This invention has been made in view of the problems of the prior art described above, and aims to provide a method for scalably producing large quantities of mature NK cells that have good cell proliferation ability and strong killing ability. [Means for solving the problem]

[0010] The present invention provides a method for producing natural killer cells, comprising the following steps: Step a: Inducing differentiation from pluripotent stem cells to embryoid bodies in a culture medium to which CHIR 99021 has been added; Step b of inducing differentiation from EBs formed in Step a into hematopoietic stem cells and progenitor cells; Step c of inducing differentiation from HSPCs formed in Step b into NK cells; Step d of expanding the NK cells produced in Step c to obtain mature NK cells; Herein, the aforementioned Step a comprises: Step (a1) of performing induction on Day 0 in the presence of CHIR 99021 and Rki, and Step (a2) of performing re-induction on Day 1 in the presence of CHIR 99021.

[0011] Preferably, the aforementioned Step b comprises: Step (b1) of subjecting EBs to suspension culture in a hematopoietic differentiation medium supplemented with BMP4, FGF2, and VEGF, Step (b2) of continuing the suspension culture in a hematopoietic stem cell expansion medium supplemented with BMP4, SCF, FGF2, VEGF, and SB431542, and Step (b3) of further performing suspension culture in a hematopoietic stem cell expansion medium supplemented with SCF, FGF2, and VEGF to form HSPCs.

[0012] Preferably, the aforementioned Step (a) is performed on Days 0 to 1, the aforementioned Step (b1) is performed on Days 2 to 3, the aforementioned Step (b2) is performed on Days 4 to 5, and the aforementioned Step (b3) is performed on Days 6 to 7.

[0013] Preferably, the aforementioned HSPCs have a CD34+ phenotype.

[0014] Preferably, the aforementioned expansion in Step d comprises adding and culturing the NK cells produced in Step c in a medium containing IL12, IL15, and IL18 to obtain mature NK cells.

[0015] Preferably, the concentrations of the aforementioned IL12, IL15, and IL18 are each 5 to 50 ng / ml.

[0016] According to the present invention, there is further provided an NK cell produced by the aforementioned method, which has a CD45+ / CD56+ phenotype.

[0017] According to the present invention, there is further provided a pharmaceutical composition comprising the aforementioned NK cell and a pharmaceutically acceptable excipient.

[0018] According to the present invention, there is further provided use of the NK cell produced by the aforementioned method, the aforementioned NK cell, or the aforementioned pharmaceutical composition in the manufacture of a formulation for the treatment or prevention of an immune-related disease.

[0019] Preferably, the aforementioned immune-related disease includes one or more of tumors, viral infectious diseases, graft-versus-host disease, autoimmune diseases, or leukemia. Here, the aforementioned viral infectious disease includes one or more of human immunodeficiency virus, Epstein-Barr virus, herpes simplex virus, cytomegalovirus, varicella-zoster virus, hepatitis B virus, or hepatitis C virus.

Effects of the Invention

[0020] In the present invention, induced pluripotent stem cells (iPSCs) are cultured in vitro under appropriate culture conditions to provide hematopoietic stem cells and progenitor cells at low cost and with high efficiency, and further induce differentiation into natural killer cells, thereby laying a solid foundation for the prevention and treatment of immune-related diseases.

[0021] According to the method for producing NK cells of the present invention, it is possible to scalably produce mature NK cells from induced pluripotent stem cells. The mature NK cells produced by the method of the present invention have favorable cell proliferation capacity and potent killing activity.

Brief Description of the Drawings

[0022] [Figure 1]This is a schematic diagram showing the differentiation and proliferation of induced NK cells (iNK cells). Human iPSCs are used to generate mesodermal embryoid bodies (EBs), then hematopoietic EBs, and subsequently differentiated into iNK cells. For iNK cell proliferation, both the conventional method (IL-2 only) and a combined activation method using IL-12, IL-15, and IL-18 are used. iNK cell proliferation is performed using K562-bmIL-21-41BBL cells in a 1:1 ratio. [Figure 2] This is a schematic diagram illustrating a differentiation method for producing HSPCs from iPSCs. HSPCs are generated from iPSCs by varying the cytokines and their addition time. [Figure 3] This diagram shows the morphological changes of cells during the differentiation process from iPSCs to HSPCs. It illustrates the timeline of the differentiation process from inducible pluripotent stem cells (iPSCs) through the embryo to hematopoietic stem cells and progenitor cells (HSPCs). The arrows in the diagram indicate the appearance of CD34+ hematopoietic stem cells in the suspension. [Figure 4] These are the results of flow cytometry analysis of HSPC markers. Figure 4A shows the gating of HSPC cells used in the flow cytometry analysis, where HSPC cells were identified as (CD235a-CD14-CD43+CD34+) on day 8 under condition #6. Figure 4B shows the gating of HSPC cells used in the flow cytometry analysis, where HSPC cells were identified as (CD235a-CD14-CD43+CD34+) on day 10 under condition #6. [Figure 5] This shows a typical bright-field image from the iPSC culture process. [Figure 6] This is a comparison of the results on day 1 and day 2 after adding CHIR99021. [Figure 7] The results of the cell stability experiment are shown. [Figure 8]Figure 8A shows a representative image of iNK cells grown in a low-adhesion 96-well plate on day 9. The combined activation method with IL-12, IL-15, and IL-18 showed superior proliferation compared to the conventional method (scale bar = 500 μm). Figure 8B shows the quantitative rate of change in the number of iNK cells in two cell lines, with the combined activation method with IL-12, IL-15, and IL-18 showing a higher rate of change than the conventional method. Figure 8C shows the quantitative results of iNK cell activity grown from the two cell lines, and these results indicate that the combined activation method with IL-12, IL-15, and IL-18 is more active than the conventional method. [Figure 9] The results of flow cytometry analysis are shown. Figure 9A shows the CD56 expression level in iNK cells on day 9 of proliferation, quantitatively analyzed by flow cytometry. The combined activation method with IL-12, IL-15, and IL-18 significantly increased the proportion of CD56+ iNK cells compared to the conventional method. Figure 9B shows the CD16 expression level in iNK cells on day 9 of proliferation, quantitatively analyzed by flow cytometry. The combined activation method with IL-12, IL-15, and IL-18 significantly increased the proportion of CD16+ iNK cells compared to the conventional method. CD16 is an effective signal for ADCC induction and can function as a marker for NK cell activation. These data suggest that CD16+ iNK cells may contribute to the enhancement of ADCC. Figure 9C shows representative flow cytometry analysis results for CD56 and CD3. Figure 9D shows representative flow cytometry analysis results for CD16 and CD56. [Figure 10] The results of measuring the cytotoxicity of caspase-3 / 7 against K562 cells or MOLM13 cells are shown. Compared to conventional methods, the combined activation method with IL-12, IL-15, and IL-18 showed similar cytotoxicity. [Modes for carrying out the invention]

[0023] To make the technical methods and beneficial effects of the present invention clearer and easier to understand, specific embodiments will be described in detail below. Note that the drawings are not necessarily drawn to a fixed scale, and local features may be enlarged or reduced to more clearly show their details. Unless otherwise defined, technical and scientific terms used herein have the same meanings as technical and scientific terms in the art to which the present invention pertains.

[0024] As used herein, the terms “one / one species (a)” or “one / one species (an)” refer to one / one species or multiple / one species, and for example, “one / one species molecule” should be understood to represent one / one species or multiple / multiple species molecules. Accordingly, the terms “one / one species (a)” or “one / one species (an),” “one / one species or multiple / multiple species,” and “at least one / at least one species” are interchangeable in this specification.

[0025] In the claims and specification of the present invention, unless otherwise indicated by context, linguistic expression, or necessary implications, the term “comprise,” or derived terms such as “comprises” or “comprising,” is used in an inclusive sense. That is, it defines the presence of the aforementioned features but does not preclude the presence or addition of other features in the various embodiments of the present invention.

[0026] As used herein, the term “approximately” encompasses values ​​within ±25% of a given number. In another embodiment, the term “approximately” refers to values ​​within ±20%, ±15%, ±10%, or ±5% of a given number. For example, in one embodiment, “approximately 3g” represents a value of 2.7 to 3.3g (i.e., 3g ± 10%).

[0027] Similarly, while methods for producing NK cells involve a sequence of events, the timing of these events may vary, for example, by at least 25%. For example, even if a specific step is disclosed to last for one day in one embodiment, the events may last longer or shorter than one day. For example, "one day" may encompass a period of approximately 18 to 30 hours. In another embodiment, the period may vary by ±20%, ±15%, ±10%, or ±5% of that period. Periods expressed as multiple days may be multiples of "one day," for example, two days may span a period of approximately 36 to 60 hours. In another embodiment, the time variation may be reduced, for example, day 2 being 48 ± 3 hours from day 0, day 4 being 96 ± 3 hours from day 0, and day 5 being 120 ± 3 hours from day 0.

[0028] As used herein, the terms “pluripotent stem cells” or “PSCs” refer to cells that can self-replicate indefinitely and have the ability to differentiate into part of a tissue or organ, or into any of the three germ layers (endoderm, mesoderm, or ectoderm). There are two main types of pluripotent stem cells: embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).

[0029] As used herein, the terms “embryonic stem cells” or “ESCs” refer to cells isolated from 5- to 7-day-old embryos donated with consent by patients who have already undergone in vitro fertilization treatment and possess remaining embryos. Ethical issues surrounding cell harvesting from human embryos hinder the use of ESCs to some extent. Appropriate human PSCs include H1 and H9 human embryonic stem cells.

[0030] As used herein, the terms “inducible pluripotent stem cells” or “iPSCs” refer to ESC-like cells derived from adult cells. iPSCs possess characteristics very similar to ESCs, but because they are not embryo-derived, they avoid the ethical issues associated with ESCs. Instead, iPSCs are typically induced by “reprogramming” fully differentiated adult cells into a pluripotent state. This reprogramming process usually involves administering reprogramming factors at specific concentrations at specific time intervals. Some exemplary methods for reprogramming adult cells into a pluripotent state include the use of RNA, proteins, or other small molecules added to adult cell cultures. Human iPSC strains are also commercially available.

[0031] Suitable human iPSCs include, but are not limited to, fibroblast-derived iPSCs 19-9-7T, MIRJT6i-mND1-4, MIRJT7i-mND2-0, and bone marrow mononuclear cell-derived iPSC BM119-9. Other suitable iPSCs are available from Cellular Dynamics International, Inc. in Madison, Wisconsin, USA.

[0032] As used herein, EB cells obtained by targeted differentiation from iPSCs are referred to as "iEB" or induced EB cells.

[0033] As used herein, HSPCs obtained by targeted differentiation from iPSCs are referred to as "iHSPCs" or induced HSPC cells.

[0034] As used herein, NK cells obtained by targeted differentiation from iPSCs are referred to as "iNK" or induced NK cells.

[0035] As used herein, the term “differentiation” refers to the process by which a cell changes from one cell type to another, particularly from a less differentiated cell type to a more differentiated cell type.

[0036] As used herein, “medium” or its plural form “media” refers to a liquid or gel designed to support cell growth.

[0037] Differentiation from PSCs to NK cells is typically carried out under controlled conditions, especially when the resulting NK cells are intended to be administered to human organisms according to good GMP (Good Manufacturing Practice). The initial stages of this process include culturing PSCs, for example, on tissue culture plates or in culture dishes, or in a bioreactor. The use of bioreactors is particularly attractive because it allows for scaling up NK cell production to a clinical level for adoptive transplantation. However, protocols using tissue culture plates and culture dishes have also been appropriately extended and are used for NK cell adoptive transplantation.

[0038] PSCs are cultured in specific media. Suitable basal media include Iscove's Modified Dulbecco's Medium / F12 (IMDM / F12) and TeSR1 basal medium (mTeSR1) which does not contain FGF2 and TGF-β. TM Basal culture medium, manufactured by Stem Cell Technologies; DF4S basal medium, i.e., Essential 8 TM Examples of suitable culture media include, but are not limited to, Life Technologies' "E8" medium. Other growth factors may be added to the cell medium to improve the clonal efficiency and single-cell viability of PSCs. One example of a usable additive is CloneR (Stem Cell Technologies). Once the PSCs reach the desired confluence, the cells can be harvested and seeded or cultured on plates to form embryoid bodies.

[0039] The culture media disclosed herein may contain certain components (e.g., morphogenetic factors, small molecule compounds, and hematopoietic cytokines), but as is well known in the art, other components having the same, equivalent, or similar properties may be used in addition to or instead of the disclosed components.

[0040] As used herein, the term "embryoid body" (EB) refers to a three-dimensional floating aggregate composed of PSCs. EBs include hematopoietic EBs that are capable of forming endothelial progenitor cells and blood progenitor cells. Various methods for generating EBs are known in the art. For example, conventional methods typically generally involve generating a single-cell suspension of PSCs. The PSCs are then cultured in uncoated, non-tissue culture-treated culture dishes or microwells to prevent PSC attachment, thereby promoting cell aggregation while maintaining a floating state. Alternatively, PSCs may be cultured in low-adhesion culture dishes or microwells. New methods for forming EBs include improving efficiency and process control by using spin aggregation or bioreactors. ROCK inhibitors such as Y-27632 have also been shown to promote the aggregation of pluripotent stem cells (PSCs), leading to EB formation. EBs have teratoma-like structures resembling developing embryos, and EB formation is a common platform for establishing differentiation into cells (e.g., NK cells) from any of the three germ layers.

[0041] As used herein, the term "hematopoietic stem and progenitor cells" (HSPCs) refers to cells that are classified in the hematopoietic lineage and further have hematopoietic differentiation potential, including pluripotent hematopoietic stem cells (hemocytoblasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem and progenitor cells are pluripotent stem cells that produce all types of blood cells, including the myeloid lineage (monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and the lymphoid lineage (T cells, B cells, NK cells).

[0042] In some embodiments, about 10,000 cells / cm 2 to about 40,000 cells / cm 2 initial density, for example 10,000 cells / cm 2 , 15,000 cells / cm 2 , 20,000 cells / cm 2 , 25,000 cells / cm 2 , 30,000 cells / cm 2 , 35,000 cells / cm2 or 40,000 cells / cm 2 Then, PSCs are cultured on a plate to produce EBs.

[0043] Once EBs are formed, they are cultured in a differentiation medium containing NK differentiation factors to promote differentiation into NK cells. The amount of EBs used varies depending on the type of tissue culture plate, tissue culture dish, or bioreactor used. For example, when using a 6-well plate, approximately 10 to 30 EBs can be cultured on the plate. However, those skilled in the art will understand that methods for determining the optimal number of EBs to be cultured on the plate, depending on the surface area of ​​the tissue culture plate, tissue culture dish, or bioreactor used, are well known in the art.

[0044] In some embodiments, the differentiation medium or growth medium may be free of heterologous substances and may contain naturally occurring human proteins isolated from the placenta or other human tissues, or human proteins produced using recombinant technology.

[0045] In some embodiments, all proteins used herein are of human origin.

[0046] In some embodiments, all proteins used in the differentiation medium or growth medium are human proteins.

[0047] In some embodiments, all proteins as used herein are recombinant human proteins.

[0048] In some embodiments, all proteins used in the differentiation medium or growth medium are recombinant human proteins.

[0049] All proteins described herein are well known to those skilled in the art, and most (but not all) of the proteins described herein are available through commercial channels.

[0050] Those skilled in the art will understand that, depending on the specific circumstances, cell culture media can be replaced at fixed or variable intervals. When cells are retained in culture, toxic metabolites may be produced, and the cultured cells continuously utilize nutrients, leading to a steady increase in cell number during the growth phase. Therefore, new cell media can be used in place of old cell media.

[0051] In some embodiments, the differentiation medium or growth medium can be replaced every 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days.

[0052] As the NK cell population matures and / or a sufficient number of PSCs differentiate into NK cells, more medium changes are required to replenish the subsequently proliferating and maturing NK cell population. In some embodiments, this period is approximately 10–20 days. From this stage onward, the differentiation medium or growth medium can be changed approximately every day, every two days, or every three days.

[0053] The number of NK cells in a culture can be determined by various methods known in this field. For example, flow cytometry or microscopy can be used. When using flow cytometry, cell samples can be stained with markers typically associated with NK cells, such as CD3, CD56, CD45, CD94, CD122, CD127, CD16, KIR, NKG2A, NKG2D, NKp30, NKp44, NKp46, and NKp80.

[0054] In some embodiments, the addition of specific cytokines, chemokines, proteins, signaling factors, and growth factors to the cell culture medium occurs within a limited time interval, after which the addition of these factors is stopped.

[0055] In some embodiments, the addition of specific cytokines, chemokines, proteins, signaling factors, and growth factors to the cell culture medium is stopped after approximately 5 to 20 days. For example, the addition of specific factors can be stopped after approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0056] In some embodiments, after a defined interval has elapsed, the addition of one or more factors selected from the group consisting of IL-3, SCF, IL-7, IL-15, FLT3L, and IL-2 is stopped.

[0057] After a certain period, proliferative suspension NK cells with a specific spindle-shaped or elongated morphology begin to appear in the culture medium. Typically, NK cells with a spindle-shaped or elongated morphology are observed approximately 15 to 50 days after the start of culture. In some embodiments, NK cells with a spindle-shaped or elongated morphology are observed approximately 15, 20, 25, 30, 35, 40, 45, or 50 days after the start of culture.

[0058] The validation of NK cell markers and functional properties can be performed using many tests known in this field. Typically, cell surface markers associated with NK cells include CD3, CD56, CD45, CD94, CD122, CD127, CD16, KIR, NKG2A, NKG2D, NKp30, NKp44, NKp46, and NKp80. Mature NK cells induce targeted cell death through granular exocytosis and the release of cytotoxic proteins, cytokines, and chemokines. Exemplary mechanisms include NK cells mediating cell death through interactions with caspase enzyme cascades, apoptotic signaling, and inflammatory signaling. Therefore, the maturity and functional properties of NK cells (such as cytotoxicity and tumor-killing ability) can be measured by measuring the expression levels of proteins such as Fas ligand (FasL), tumor necrosis factor (TNF)-α, TNF-related apoptosis-inducing ligand (TRAIL), or other cytokines and chemokines. Furthermore, the expression of specific markers on the cell surface is also an indicator of improved NK cell homing ability. Changes in the functional properties of NK cells can be indicated by an increase or decrease in the expression levels of specific markers or proteins. Changes in the functional properties of NK cells can also be indicated by an increase in specific markers or proteins and a corresponding decrease in other markers or proteins.

[0059] A suitable method for determining the cytotoxicity of NK cells produced by the method of the present invention is to co-culture NK cells with tumor cell lines such as K562, LN-18, U937, WERI-RB-1, U-118MG, HT-29, HCC2218, KG-1, and U266 as target cells. Next, the tumor cells can be labeled with a fluorescent label specific to tumor cells, for example. Then, the cytotoxicity in co-culture, indicating apoptosis or cell death of tumor cells, is evaluated based on a decrease in the expression level of the fluorescent label. Examples of suitable labels for labeling tumor cells include carboxyfluorescein succinimide (CFSE), mCherry, or any other suitable fluorescent label known in the art. Alternatively, apoptosis and dead cell populations can be further analyzed, for example, by staining with annexin-V (membrane-bound protein V) or any other fixable viability stain using flow cytometry. Co-culture of NK cells and tumor cells is usually performed in a specific effector:target ratio. In some embodiments, this ratio is approximately 1:1 to 10:1. In some embodiments, this ratio is approximately 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. Methods and tests for measuring ADCC are also known in the art, such as methods for incubating NK cells with target cells and appropriate effector antibodies such as NKp46. ADCC tests, as well as their kits and reagents, are also available through commercial channels, such as the Promega ADCC Bioassay.

[0060] As used herein, the term “pharmaceutical composition” refers to a composition formulated for administration to an individual, comprising NK cells or a population of NK cells as described herein. Preferably, the pharmaceutical composition is sterile. In some embodiments, the pharmaceutical composition is nonpyrogenic.

[0061] NK cells or NK cell populations are formulated, dosage-determined, and administered in accordance with appropriate medical practices. Factors considered in this context include the specific type of disease being treated, the specific individual being treated, the individual's clinical condition, the administration site, the method of administration, the administration schedule, possible side effects, and other factors known to the physician. Furthermore, maximizing the cytotoxicity and persistence of NK cells in vivo is also considered. The therapeutically effective dose of NK cells or NK cell populations to be administered is determined based on these considerations.

[0062] NK cells or populations of NK cells can be administered to an individual by any suitable method, including intravenous (IV) and subcutaneous injection. NK cells may be administered in combination with additional therapeutic agents, such as antibodies, or modifiers designed to enhance the intrinsic properties or activity of NK cells in vivo. Furthermore, NK cells may be pre-treated or induced with modifiers before administration to an individual to enhance their intrinsic properties or activity in vivo. Examples of suitable initiators or modifiers include IL-2 or IL-15. Other suitable protein constructs, agonists, antagonists, modulators, or inflammatory factors are also conceivable.

[0063] NK cells possess effector properties in inducing ADCC, making them useful in antibody-based cancer therapy. Antibody-based cancer therapy targets tumor cells using antibodies such as monoclonal antibodies. In fact, NK cells can mediate antibody-dependent tumor killing through the release of cytotoxic granules, TNF death receptor signaling, and cytokines such as interferon-gamma.

[0064] As used herein, the term “effective dose” refers to the amount of NK cells or NK cell population that is effective in treating an individual’s disease, illness, or condition.

[0065] As used herein, the terms “treat,” “treating,” and “treatment” refer to therapeutic treatments and preventive or preventive measures aimed at preventing or improving an individual’s disease, illness, or condition, or slowing (mitigating) the progression of such a condition. Individuals requiring treatment include those already suffering from a disease, illness, or condition, as well as those for whom prevention of disease, illness, or condition is necessary.

[0066] As used herein, the terms “prevent,” “preventation,” “preventative,” and “prevention” refer to preventing the onset of a disease, illness, or condition, or preventing, defending against, or resisting its onset, including any abnormalities or symptoms. Subjects requiring prevention may have predisposing factors that make them susceptible to developing such disease, illness, or condition.

[0067] As used herein, the terms “ameliorate” or “amelioration” refer to a reduction, mitigation, or elimination of a disease, illness, or condition, including an abnormality or symptom. Individuals requiring treatment are either already suffering from such disease, illness, or condition, are susceptible to such disease, illness, or condition, or require prevention of such disease, illness, or condition.

[0068] As used herein, the term “individual” refers to a mammal. A mammal may be a primate, particularly a human, or a livestock, zoo animal, or pet animal. The methods disclosed herein, and the NK cells or NK cell populations obtained thereby, are considered particularly suitable for human medical treatment, but are also applicable to veterinary treatment, including the treatment of livestock such as cattle, horses, and sheep, pet animals such as dogs and cats, or zoo animals such as felines, canines, bovines, and ungulates.

[0069] Unless otherwise specified, experimental methods in the following examples that do not specify concrete conditions are typically carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all percentages, ratios, proportions, or parts are on a weight basis. Unless otherwise specified, room temperature in this invention is 20°C to 30°C. Unless otherwise specified, the various materials and reagents used in this invention are available through conventional methods in the art or through commercial channels. Specifically, these are as follows:

[0070] StemScale PSC suspension medium: Thermo Fisher, #A4965001 Y27632: Tocris, #1254 CHIR99021: Sigma SML1046 Stemline II hematopoietic stem cell medium: Sigma-Ace, #S0192 CTS TM NK-Xpander TM Culture medium: Thermo Fisher, #A5019001 Penicillin-streptomycin (P / S): Thermo Fisher, #15140122 GlutaMAX Supplement: Manufactured by Thermo Fisher, #35050061 L-ascorbic acid: Sigma-Aldrich, #A4544 ITS-G: Manufactured by Thermo Fisher, #41400045 BMP4: Manufactured by Milteny Biotech, #130-111-167 FGF2: Milteny Biotech, #130-093-564 VEGF: Milteny Biotech, #130-109-396 SB431542: Manufactured by Tocris, #1614 / TB1614-GMP SCF: Milteny Biotech, #130-096-695 DMEM / F-12GlutaMAX: Manufactured by Thermo Fisher, #10565018 F12+GlutaMAX: Thermo Fisher, #31765-035 Thermally inactivated human ab serum: Sigma-Ace, #H3667 L-Glutamine: Thermo Fisher, #25030081 β-mercaptoethanol: Thermo Fisher, #21985023 Sodium selenite: Sigma-Ace, #214485 Ethanolamine: MP Corporation, #193845 IL-3: Manufactured by Milteny Biotech, #130-095-070 IL-7: Manufactured by Milteny Biotech, #130-095-362 IL-2: Manufactured by Milteny Biotech, #130-097-748 IL-15: Manufactured by Milteny Biotech, #130-095-764 IL-18: Manufactured by MBL, #B001-5 IL-12: Manufactured by Milteny Biotech, #130-096-705 FLT3L: Manufactured by Milteny Biotech, #130-096-477 DPBS: Thermo Fisher, #14190-144 Accutase: Stem Cell Technologies, #07922 CellTrace TM Violet: Manufactured by Thermo Fisher Scientific, #C34557 Caspase-3 / 7 Green Assay Reagent: Thermal Fisher Scientific, #C10423

[0071] The present invention provides a method for producing natural killer cells (NK) comprising the following steps: Step a: Inducing differentiation from pluripotent stem cells to embryoid bodies (EBs) in a culture medium to which CHIR 99021 has been added; Step b involves inducing differentiation of EBs formed in step a into hematopoietic stem cells and progenitor cells (HSPCs); Step c involves inducing differentiation of HSPCs formed in step b into NK cells; Step d involves proliferating the NK cells produced in step c to obtain mature NK cells; Here, step a is, The process (a1) involves induction on day 0 in the presence of CHIR 99021 and Rki, The process includes (a2) a step of re-induction on day 1 in the presence of CHIR 99021.

[0072] In some embodiments, step b is, (b1) A step in which EB is cultured in suspension in hematopoietic differentiation medium supplemented with BMP4, FGF2, and VEGF, (b2) A step in which suspension culture is continued in hematopoietic stem cell proliferation medium to which BMP4, SCF, FGF2, VEGF, and SB431542 have been added, The process includes (b3) further suspension culture in hematopoietic stem cell proliferation medium supplemented with SCF, FGF2, and VEGF to form HSPCs.

[0073] In some embodiments, the method does not require a feeder layer during the preparation process.

[0074] In some embodiments, the culture medium in step (a) is one or more selected from the group consisting of StemScale PSC, Essential 8, KSR / FGF2, mTeSR, AKIT, and B8.

[0075] In some embodiments, the culture medium in step (a) is StemScale PSC.

[0076] In some embodiments, the concentration of CHIR 99021 in step (a) is 0.1 to 100 μM.

[0077] In some embodiments, the concentration of CHIR 99021 in step (a) is 0.2 to 50 μM.

[0078] In some embodiments, the concentration of CHIR 99021 in step (a) is 0.5 to 25 μM.

[0079] In some embodiments, the concentration of CHIR 99021 in step (a) is 1 to 20 μM.

[0080] In some embodiments, the concentration of CHIR 99021 in step (a) is approximately 1 μM, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 11 μM, approximately 12 μM, approximately 13 μM, approximately 14 μM, approximately 15 μM, approximately 16 μM, approximately 17 μM, approximately 18 μM, approximately 19 μM, or approximately 20 μM.

[0081] In some embodiments, the culture medium in step (b1) further contains one or more of P / S, GlutaMaX, ascorbic acid, or ITS-G.

[0082] In some embodiments, the culture medium in step (b1) contains 1% P / S, 2 mM GlutaMaX, 50 μg / mL ascorbic acid, 1% ITS-G, 1-100 ng / mL BMP4, 1-100 ng / mL FGF2, and 1-100 ng / mL VEGF.

[0083] In some embodiments, the concentration of BMP4 in the culture medium in step (b1) is approximately 10 ng / mL, approximately 20 ng / mL, approximately 30 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, or approximately 100 ng / mL.

[0084] In some embodiments, the concentration of FGF2 in the culture medium in step (b1) is approximately 10 ng / mL, approximately 20 ng / mL, approximately 30 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, or approximately 100 ng / mL.

[0085] In some embodiments, the concentration of VEGF in the culture medium in step (b1) is approximately 10 ng / mL, approximately 20 ng / mL, approximately 30 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, or approximately 100 ng / mL.

[0086] In some embodiments, the culture medium in step (b2) further contains one or more of P / S, GlutaMaX, ascorbic acid, and ITS-G.

[0087] In some embodiments, the culture medium in step (b2) contains 1% P / S, 2 mM GlutaMaX, 50 μg / mL ascorbic acid, 1% ITS-G, 1-100 ng / mL BMP4, 1-100 ng / mL SCF, 1-100 ng / mL FGF2, 1-100 ng / mL VEGF, and 1-20 μM SB431542.

[0088] In some embodiments, the concentration of BMP4 in the culture medium in step (b2) is approximately 10 ng / mL, approximately 20 ng / mL, approximately 30 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, or approximately 100 ng / mL.

[0089] In some embodiments, the concentration of SCF in the culture medium in step (b2) is approximately 10 ng / mL, approximately 20 ng / mL, approximately 30 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, or approximately 100 ng / mL.

[0090] In some embodiments, the concentration of FGF2 in the culture medium in step (b2) is approximately 10 ng / mL, approximately 20 ng / mL, approximately 30 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, or approximately 100 ng / mL.

[0091] In some embodiments, the concentration of VEGF in the culture medium in step (b2) is approximately 10 ng / mL, approximately 20 ng / mL, approximately 30 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, or approximately 100 ng / mL.

[0092] In some embodiments, the concentration of SB431542 in the culture medium in step (b2) is approximately 1 μM, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 11 μM, approximately 12 μM, approximately 13 μM, approximately 14 μM, approximately 15 μM, approximately 16 μM, approximately 17 μM, approximately 18 μM, approximately 19 μM, or approximately 20 μM.

[0093] In some embodiments, the culture medium in step (b3) further contains one or more of P / S, GlutaMaX, ascorbic acid, or ITS-G.

[0094] In some embodiments, the culture medium in step (b3) contains 1% P / S, 2 mM GlutaMaX, 50 μg / mL ascorbic acid, 1% ITS-G, 1-100 ng / mL SCF, 1-100 ng / mL FGF2, and 1-100 ng / mL VEGF.

[0095] In some embodiments, the SCF concentration in the culture medium in step (b3) is approximately 10 ng / mL, approximately 20 ng / mL, approximately 30 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, or approximately 100 ng / mL.

[0096] In some embodiments, the concentration of FGF2 in the culture medium in step (b3) is approximately 10 ng / mL, approximately 20 ng / mL, approximately 30 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, or approximately 100 ng / mL.

[0097] In some embodiments, the concentration of VEGF in the culture medium in step (b3) is approximately 10 ng / mL, approximately 20 ng / mL, approximately 30 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, or approximately 100 ng / mL.

[0098] In some embodiments, step (a) is performed on day 0-1, step (b1) on day 2-3, step (b2) on day 4-5, and step (b3) on day 6-7.

[0099] In some embodiments, the EB is an iEB.

[0100] In some embodiments, the HSPC is an iHSPC.

[0101] In some embodiments, the NK cells are iNK cells.

[0102] In some embodiments, the HSPC has a CD34+ phenotype. In some embodiments, step c includes (c1) adding the HSPC formed in step b to a first culture medium and performing cell culture, and (c2) adding the cells obtained in step (c1) to a second culture medium and performing cell culture to obtain NK cells.

[0103] In some embodiments, the first culture medium contains heat-inactivated human type AB serum, a colony-stimulating factor, and an interleukin. Here, the colony-stimulating factor is one or more selected from the group consisting of G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF, and FLT3L, and the interleukin is one or more selected from the group consisting of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27.

[0104] In some embodiments, the second culture medium contains heat-inactivated human type AB serum, a colony-stimulating factor, and an interleukin. Here, the colony-stimulating factor is one or more selected from the group consisting of G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF, and FLT3L, and the interleukin is one or more selected from the group consisting of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27.

[0105] In some embodiments, the colony-stimulating factors in the first culture medium are SCF and FLT3L.

[0106] In some embodiments, the interleukins in the first culture medium are IL-3, IL-7, and IL-15.

[0107] In some embodiments, the first culture medium further contains one or more of the following: DMEM / F12+GlutaMAX, F12+GlutaMAX, P / S, L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, and retrotronectin.

[0108] In some embodiments, the first medium contains 56.6% DMEM / F12+GlutaMAX, 28.3% F12+GlutaMAX, 1-20% heat-inactivated human AB serum, 1% P / S, 2 mM L-glutamine, 1 μM β-mercaptoethanol, 5-50 ng / mL sodium selenite, 50-200 μM ethanolamine, 20-100 mg / L ascorbic acid, 1-50 ng / mL IL-3, 1-100 ng / mL SCF, 1-100 ng / mL IL-7, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, 1-100 μg / mL DLL4, and 1-100 μg / mL retronectin.

[0109] In some embodiments, the concentration of IL-3 in the first culture medium is approximately 1 ng / mL, approximately 2 ng / mL, approximately 3 ng / mL, approximately 4 ng / mL, approximately 5 ng / mL, approximately 6 ng / mL, approximately 7 ng / mL, approximately 8 ng / mL, approximately 9 ng / mL, approximately 10 ng / mL, approximately 11 ng / mL, approximately 12 ng / mL, approximately 13 ng / mL, approximately 14 ng / mL, approximately 15 ng / mL, approximately 16 ng / mL, approximately 17 ng / mL, approximately 18 ng / mL, approximately 19 ng / mL, approximately 20 ng / mL, approximately 21 ng / mL, approximately 22 ng / mL, approximately 23 ng / mL, approximately 24 ng / mL, The levels are approximately 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, or 50 ng / mL.

[0110] In some embodiments, the concentration of SCF in the first culture medium is about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL.

[0111] In some embodiments, the concentration of IL-7 in the first culture medium is about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL.

[0112] In some embodiments, the concentration of IL-15 in the first culture medium is about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL.

[0113] In some embodiments, the concentration of FLT3L in the first culture medium is about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL.

[0114] In some embodiments, the concentration of DLL4 in the first culture medium is approximately 1 μg / mL, approximately 5 μg / mL, approximately 10 μg / mL, approximately 15 μg / mL, approximately 20 μg / mL, approximately 25 μg / mL, approximately 30 μg / mL, approximately 35 μg / mL, approximately 40 μg / mL, approximately 45 μg / mL, approximately 50 μg / mL, approximately 55 μg / mL, approximately 60 μg / mL, approximately 65 μg / mL, approximately 70 μg / mL, approximately 75 μg / mL, approximately 80 μg / mL, approximately 85 μg / mL, approximately 90 μg / mL, approximately 95 μg / mL, or approximately 100 μg / mL.

[0115] In some embodiments, the concentration of retrolectin in the first culture medium is approximately 1 μg / mL, approximately 5 μg / mL, approximately 10 μg / mL, approximately 15 μg / mL, approximately 20 μg / mL, approximately 25 μg / mL, approximately 30 μg / mL, approximately 35 μg / mL, approximately 40 μg / mL, approximately 45 μg / mL, approximately 50 μg / mL, approximately 55 μg / mL, approximately 60 μg / mL, approximately 65 μg / mL, approximately 70 μg / mL, approximately 75 μg / mL, approximately 80 μg / mL, approximately 85 μg / mL, approximately 90 μg / mL, approximately 95 μg / mL, or approximately 100 μg / mL.

[0116] In some embodiments, the colony-stimulating factors in the second culture medium are SCF and FLT3L.

[0117] In some embodiments, the interleukins in the second culture medium are IL-7 and IL-15.

[0118] In some embodiments, the second culture medium further contains one or more of the following: DMEM / F12+GlutaMAX, F12+GlutaMAX, P / S, L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, and retrotronectin.

[0119] In some embodiments, the second medium contains 56.6% DMEM / F12+GlutaMAX, 28.3% F12+GlutaMAX, 1-20% heat-inactivated human AB serum, 1% P / S, 2 mM L-glutamine, 1 μM β-mercaptoethanol, 5-50 ng / mL sodium selenite, 50-200 μM ethanolamine, 20-100 mg / L ascorbic acid, 1-100 ng / mL SCF, 1-100 ng / mL IL-7, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, 1-100 μg / mL DLL4, and 1-100 μg / mL retronectin.

[0120] In some embodiments, the concentration of SCF in the second culture medium is about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL.

[0121] In some embodiments, the concentration of IL-7 in the second culture medium is about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL.

[0122] In some embodiments, the concentration of IL-15 in the second culture medium is about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL.

[0123] In some embodiments, the concentration of FLT3L in the second culture medium is approximately 10 ng / mL, approximately 20 ng / mL, approximately 30 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, or approximately 100 ng / mL.

[0124] In some embodiments, the concentration of DLL4 in the second culture medium is approximately 1 μg / mL, approximately 5 μg / mL, approximately 10 μg / mL, approximately 15 μg / mL, approximately 20 μg / mL, approximately 25 μg / mL, approximately 30 μg / mL, approximately 35 μg / mL, approximately 40 μg / mL, approximately 45 μg / mL, approximately 50 μg / mL, approximately 55 μg / mL, approximately 60 μg / mL, approximately 65 μg / mL, approximately 70 μg / mL, approximately 75 μg / mL, approximately 80 μg / mL, approximately 85 μg / mL, approximately 90 μg / mL, approximately 95 μg / mL, or approximately 100 μg / mL.

[0125] In some embodiments, the concentration of retrolectin in the second culture medium is approximately 1 μg / mL, approximately 5 μg / mL, approximately 10 μg / mL, approximately 15 μg / mL, approximately 20 μg / mL, approximately 25 μg / mL, approximately 30 μg / mL, approximately 35 μg / mL, approximately 40 μg / mL, approximately 45 μg / mL, approximately 50 μg / mL, approximately 55 μg / mL, approximately 60 μg / mL, approximately 65 μg / mL, approximately 70 μg / mL, approximately 75 μg / mL, approximately 80 μg / mL, approximately 85 μg / mL, approximately 90 μg / mL, approximately 95 μg / mL, or approximately 100 μg / mL.

[0126] In some embodiments, the cell culture in step (c1) is carried out for 6 to 8 days.

[0127] In some embodiments, the cell culture in step (c1) is carried out for approximately 6 days, 7 days, or 8 days.

[0128] In some embodiments, the cell culture in step (c2) is carried out for 8-10 days, after which the cells are cultured for a further 8-10 days in fresh second culture medium.

[0129] In some embodiments, the cell culture in step (c2) is carried out for approximately 8, 9, or 10 days, after which the cells are cultured for a further 8, 9, or 10 days in a fresh second medium.

[0130] In some embodiments, the growth in step (d) is The process includes adding the NK cells produced in step c to a culture medium containing IL12, IL15, and IL18 to obtain mature NK cells.

[0131] In some embodiments, the concentrations of IL12, IL15, and IL18 are 5 to 50 ng / ml, respectively.

[0132] In some embodiments, step d includes adding the NK cells prepared in step c to an NK cell growth medium and performing cell culture.

[0133] In some embodiments, step d includes adding the NK cells prepared in step c to an activation medium and performing cell culture, and replacing the activation medium with maintenance medium on the third day of NK cell proliferation.

[0134] In some embodiments, step d further includes replacing half of the culture medium with maintenance medium on day 6 of NK cell proliferation.

[0135] In some embodiments, step d further includes transferring the NK cells to 24-well Grex cells on day 9 of NK cell proliferation.

[0136] In some embodiments, step d further includes replacing half of the culture medium with maintenance medium on day 12 of NK cell proliferation.

[0137] In some embodiments, step d further includes harvesting iNK cells on day 15 of NK cell proliferation.

[0138] In some embodiments, the activation medium is NK Xpander medium supplemented with human type AB serum, P / S, IL-15, IL-12, IL-18, and IL-2.

[0139] In some embodiments, the activated medium is a medium supplemented with 1-20% human type AB serum, 1% P / S, 5-50 ng / ml IL-15, 5-50 ng / ml IL-12, 5-50 ng / ml IL-18, and 50 U / ml IL-2.

[0140] In some embodiments, the concentration of IL-12 is approximately 5 ng / mL, approximately 6 ng / mL, approximately 7 ng / mL, approximately 8 ng / mL, approximately 9 ng / mL, approximately 10 ng / mL, approximately 11 ng / mL, approximately 12 ng / mL, approximately 13 ng / mL, approximately 14 ng / mL, approximately 15 ng / mL, approximately 16 ng / mL, approximately 17 ng / mL, approximately 18 ng / mL, approximately 19 ng / mL, approximately 20 ng / mL, approximately 21 ng / mL, approximately 22 ng / mL, approximately 23 ng / mL, approximately 24 ng / mL, approximately 25 ng / mL, approximately 26 ng / mL, approximately The levels are approximately 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, or 50 ng / mL.

[0141] In some embodiments, the concentration of IL-15 is approximately 5 ng / mL, approximately 6 ng / mL, approximately 7 ng / mL, approximately 8 ng / mL, approximately 9 ng / mL, approximately 10 ng / mL, approximately 11 ng / mL, approximately 12 ng / mL, approximately 13 ng / mL, approximately 14 ng / mL, approximately 15 ng / mL, approximately 16 ng / mL, approximately 17 ng / mL, approximately 18 ng / mL, approximately 19 ng / mL, approximately 20 ng / mL, approximately 21 ng / mL, approximately 22 ng / mL, approximately 23 ng / mL, approximately 24 ng / mL, approximately 25 ng / mL, approximately 26 ng / mL, approximately The levels are approximately 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, or 50 ng / mL.

[0142] In some embodiments, the concentration of IL-18 is approximately 5 ng / mL, approximately 6 ng / mL, approximately 7 ng / mL, approximately 8 ng / mL, approximately 9 ng / mL, approximately 10 ng / mL, approximately 11 ng / mL, approximately 12 ng / mL, approximately 13 ng / mL, approximately 14 ng / mL, approximately 15 ng / mL, approximately 16 ng / mL, approximately 17 ng / mL, approximately 18 ng / mL, approximately 19 ng / mL, approximately 20 ng / mL, approximately 21 ng / mL, approximately 22 ng / mL, approximately 23 ng / mL, approximately 24 ng / mL, approximately 25 ng / mL, approximately 26 ng / mL, approximately The levels are approximately 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, or 50 ng / mL.

[0143] In some embodiments, the activated medium is a medium supplemented with 1-20% human type AB serum, 1% P / S, 10 ng / mL IL-15, 50 ng / mL IL-12, 50 ng / mL IL-18, and 50 U / mL IL-2.

[0144] In some embodiments, the maintenance medium is NK Xpander medium supplemented with human type AB serum, P / S, IL-2, and IL-15.

[0145] In some embodiments, the maintenance medium is a medium supplemented with 1-20% human type AB serum, 1% P / S, 5-500 U / ml of IL-2, and 1-100 ng / ml of IL-15.

[0146] According to the present invention, NK cells produced by the method described above are further provided. The NK cells exhibit increased expression of one or more of IL-12, IL-15, and IL-18.

[0147] In some embodiments, the NK cells show an increase of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more in the NK cell marker or protein.

[0148] In some embodiments, the NK cells show an increase of approximately 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or more in the NK cell marker or protein.

[0149] In some embodiments, the NK cells have a CD45+ / CD56+ phenotype.

[0150] In some embodiments, the proportion of cells having the CD45+ / CD56+ phenotype exceeds 80%.

[0151] The present invention further provides a pharmaceutical composition comprising the NK cells and a pharmaceutically acceptable excipient.

[0152] In some embodiments, the pharmaceutical composition further comprises additional therapeutic agents.

[0153] In some embodiments, the additional therapeutic agent is an antibody.

[0154] The present invention further provides for the use of NK cells produced by the method, the NK cells, or the pharmaceutical composition in the manufacture of a formulation for the treatment or prevention of an immune-related disease.

[0155] In some embodiments, the immune-related disease includes one or more of the following: tumors, viral infections, graft-versus-host diseases, autoimmune diseases, or leukemias.

[0156] In some embodiments, the viral infection includes one or more of the following: human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), cytomegalovirus (CMV), varicella-zoster virus (VZV), hepatitis B virus (HBV), or hepatitis C virus (HCV).

[0157] (Example 1) Fabrication of HSPC from iPSC (Condition #8) Human iPSCs are typically maintained in commercially available Essential 8 medium. When the iPSCs reached 70% confluence, their healthy morphology and lack of spontaneous differentiation were confirmed. The iPSCs were dissociated into single cells using Accutase. The collected single cells were counted and seeded in StemScale PSC complete suspension medium supplemented with 5–50 μM Y27632.

[0158] Cells were cultured in a 37°C, 5% CO2 incubator while continuously rotating at a speed of 30-100 rpm using a CO2-tolerant orbital shaker. Within 24 hours (day 0), half of the medium was changed using StemScale PSC suspension complete medium, and the ROCK inhibitor Rki and the GSK-3 inhibitor CHIR 99021 were added.

[0159] 24 hours later (Day 1), CHIR 99021 was added.

[0160] After 24 hours (day 2), the obtained embryoid bodies (EBs) were collected and resuspended in Stemline II hematopoietic stem cell proliferation medium (as hematopoietic differentiation medium 1) supplemented with 1% P / S, 2 mM GlutaMaX, 50 μg / ml ascorbic acid, 1% ITS-G, 1-100 ng / ml BMP4, 1-100 ng / ml FGF2, and 1-100 ng / ml VEGF.

[0161] After 24-48 hours (days 3-4), EB was collected and resuspended in Stemline II hematopoietic stem cell proliferation medium (as hematopoietic differentiation medium 2) containing an additional 1% P / S, 2 mM GlutaMaX, 50 μg / ml ascorbic acid, 1% ITS-G, 1-100 ng / ml BMP4, 1-100 ng / ml FGF2, 1-100 ng / ml VEGF, 1-100 ng / ml SCF, and 1-20 μM SB431542.

[0162] After 24-72 hours (days 5-7), EB was collected and resuspended in Stemline II hematopoietic stem cell proliferation medium (as hematopoietic differentiation medium 3) supplemented with 1% P / S, 2 mM GlutaMaX, 50 μg / ml ascorbic acid, 1% ITS-G, 1-100 ng / ml SCF, 1-100 ng / ml FGF2, and 1-100 ng / ml VEGF. HSPC was collected on days 8-10.

[0163] (Example 2) Fabrication of HSPC from iPSC (Condition #4) Human iPSCs are typically maintained in commercially available Essential 8 medium. When the iPSCs reached 70% confluence, their healthy morphology and lack of spontaneous differentiation were confirmed. The iPSCs were dissociated into single cells using Accutase. The collected single cells were counted and seeded in StemScale PSC complete suspension medium supplemented with 5–50 μM Y27632.

[0164] Cells were cultured in a 37°C, 5% CO2 incubator while continuously rotating at a speed of 30-100 rpm using a CO2-tolerant orbital shaker. Within 24 hours (day 0), half of the medium was changed using StemScale PSC suspension complete medium, and the ROCK inhibitor Rki was added.

[0165] 24 hours later (Day 1), CHIR 99021 (double concentration) was added.

[0166] After 24 hours (day 2), the obtained embryoid bodies (EBs) were collected and resuspended in Stemline II hematopoietic stem cell proliferation medium (as hematopoietic differentiation medium 1) supplemented with 1% P / S, 2 mM GlutaMaX, 50 μg / ml ascorbic acid, 1% ITS-G, 1-100 ng / ml BMP4, 1-100 ng / ml FGF2, and 1-100 ng / ml VEGF.

[0167] After 24-48 hours (days 3-4), EB was collected and resuspended in Stemline II hematopoietic stem cell proliferation medium (as hematopoietic differentiation medium 2) containing an additional 1% P / S, 2 mM GlutaMaX, 50 μg / ml ascorbic acid, 1% ITS-G, 1-100 ng / ml BMP4, 1-100 ng / ml FGF2, 1-100 ng / ml VEGF, 1-100 ng / ml SCF, and 1-20 μM SB431542.

[0168] After 24-72 hours (days 5-7), EB was collected and resuspended in Stemline II hematopoietic stem cell proliferation medium (as hematopoietic differentiation medium 3) containing 1% P / S, 2 mM GlutaMaX, 50 μg / ml ascorbic acid, 1% ITS-G, 1-100 ng / ml SCF, 1-100 ng / ml FGF2, and 1-100 ng / ml VEGF. HSPC was collected on days 8-10.

[0169] (Example 3) Fabrication of HSPC from iPSC (Condition #6) Human iPSCs are typically maintained in commercially available Essential 8 medium. When the iPSCs reached 70% confluence, their healthy morphology and lack of spontaneous differentiation were confirmed. The iPSCs were dissociated into single cells using Accutase. The collected single cells were counted and seeded in StemScale PSC complete suspension medium supplemented with 5–50 μM Y27632.

[0170] Cells were cultured in a 37°C, 5% CO2 incubator while continuously rotating at a speed of 30-100 rpm using a CO2-tolerant orbital shaker. Within 24 hours (day 0), half of the medium was changed using StemScale PSC suspension complete medium, and the ROCK inhibitor Rki and the GSK-3 inhibitor CHIR 99021 were added.

[0171] 24 hours later (day 1), CHIR 99021 was added. After 24-48 hours (days 2-3), the obtained embryoid bodies (EBs) were collected and resuspended in Stemline II hematopoietic stem cell proliferation medium (as hematopoietic differentiation medium 1) supplemented with 1% P / S, 2 mM GlutaMaX, 50 μg / ml ascorbic acid, 1% ITS-G, 1-100 ng / ml BMP4, 1-100 ng / ml FGF2, and 1-100 ng / ml VEGF.

[0172] After 24-48 hours (days 4-5), EB was collected and resuspended in Stemline II hematopoietic stem cell proliferation medium (as hematopoietic differentiation medium 2) supplemented with 1% P / S, 2 mM GlutaMaX, 50 μg / ml ascorbic acid, 1% ITS-G, 1-100 ng / ml BMP4, 1-100 ng / ml FGF2, 1-100 ng / ml VEGF, 1-100 ng / ml SCF, and 1-20 μM SB431542.

[0173] After 24-48 hours (days 6-7), EB cells were collected and resuspended in Stemline II hematopoietic stem cell proliferation medium (as hematopoietic differentiation medium 3) supplemented with 1% P / S, 2 mM GlutaMaX, 50 μg / ml ascorbic acid, 1% ITS-G, 1-100 ng / ml SCF, 1-100 ng / ml FGF2, and 1-100 ng / ml VEGF. HSPC cells were collected on days 8-10, and cell surface markers were measured.

[0174] (Example 4-8) Fabrication of HSPC from iPSC (Conditions #1-3, 5, 7) The culture methods for Examples 4-8 (conditions #1-3, 5, and 7) are almost the same as those for Example 1. The specific conditions are shown in Figure 2.

[0175] (Example 9) Comparison of the effects of the differentiation protocol From a morphological standpoint, the addition of CHIR (condition 6) during the iPSC culture stage (i.e., day 0 in Figure 2) was observed. The results are shown in Figure 5. The addition of CHIR initiated differentiation during the iPSC culture stage and after iPSC spheroid formation. As a result, it became possible to use a larger number of more uniform spheroids in the subsequent differentiation process.

[0176] Furthermore, the experiment was conducted by extending the induction period of CHIR99021 to two days during the induction stage of mesoderm formation from iPSCs (i.e., days 1-3 of condition 6). The results are shown in Figure 6. It was found that adding CHIR99021 for one day promoted the release of HPC cells from HE EB and was more advantageous for overall cell activity / proliferation than adding it for two days.

[0177] Stability tests were conducted on four batches under condition 6. The results are shown in Figure 7. While most CD34 cells in hematopoietic EB cells were earlier progenitor cells (CD43-negative), more than 50% of the suspension cells released from EB spheroids were CD34-positive and more than 90% were CD43-positive, suggesting that most of the suspension cells released from EB spheroids are hematopoietic progenitor cells.

[0178] (Example 10) Production of iNK cells from HSPC The HSPCs and suspension cell cultures recovered in Examples 1-8 were mixed with 56.6% DMEM / F12+GlutaMAX. TM -I, 28.3% F12+GlutaMAX TM -I was resuspended in NK differentiation medium 1 containing 1-20% heat-inactivated human AB serum, 1% P / S, 2 mM L-glutamine, 1 μM β-mercaptoethanol, 5-50 ng / mL sodium selenite, 50-200 μM ethanolamine, 20-100 mg / L ascorbic acid, 1-50 ng / mL IL-3, 1-100 ng / mL SCF, 1-100 ng / mL IL-7, 1-100 ng / mL IL-15, and 1-100 ng / mL FLT3L.

[0179] On day 8 of iNK cell differentiation, the culture was converted to 56.6% DMEM / F12+GlutaMAX. TM -I, 28.3% F12+GlutaMAX TM -I was resuspended in NK differentiation medium 2 containing 1-20% heat-inactivated human AB serum, 1% P / S, 2 mM L-glutamine, 1 μM β-mercaptoethanol, 5-50 ng / mL sodium selenite, 50-200 μM ethanolamine, 20-100 mg / L ascorbic acid, 1-100 ng / mL SCF, 1-100 ng / mL IL-7, 1-100 ng / mL IL-15, and 1-100 ng / mL FLT3L.

[0180] Ten days later (day 18 of iNK cell differentiation), the suspension cells were collected and resuspended in fresh NK differentiation medium 2.

[0181] Ten days later (day 28 of iNK cell differentiation), we monitored whether or not spindle-shaped iNK-like cells appeared in the culture and evaluated their CD45 / CD56 expression. As a result, the proportion of CD45+ / CD56+ cells exceeded 80%.

[0182] (Example 11) Measurement of iNK cell proliferation, maturation, and activity iNK cells were proliferated on day 28. 3 x 10 4 Individual iNK cells and 3 × 10 4 K562-mbII21-41BBL cells (iNK cells:feeder layer = 1:1) were cultured in 200 μl of NK growth medium on a 96-well low-adhesion plate. The composition of the NK cell growth and maturation medium is shown in Table 1. The cells were incubated at 37°C under 5% CO2 conditions.

[0183] [Table 1]

[0184] On day 3 of iNK cell proliferation, the activation medium was replaced with maintenance medium. On day 6 of iNK cell proliferation, half of the medium was replaced with maintenance medium. On day 9 of iNK cell proliferation, the iNK cells were transferred to Grex cells in a 24-well plate, and on day 12, half of the culture medium was replaced. The iNK cells were harvested on day 15 of iNK cell proliferation.

[0185] To evaluate the cytotoxicity of iNK cells, a caspase assay was performed. Simply put, target cells (K562 or MOLM13) were subjected to a caspase assay in PBS at a final concentration of 5 μM. TM The cells were pre-stained with Violet at 37°C for 20 minutes. After staining, the cells were washed in complete culture medium and then mixed with NK cell culture at a predetermined effector cell-to-target cell (E:T) ratio. After short-term centrifugation, the co-culture was incubated at 37°C for 3.5 hours. The cells were then baited. Next, the CellEvent® caspase-3 / 7 green detection kit (Thermo Fisher, C10423) was added and the cells were re-cultured for another 30 minutes. The total incubation time was 4 hours. Next, the cells were analyzed by flow cytometry.

[0186] As a result, as indicated by the multiplicative changes in cell number and CD16 surface marker expression, transient treatment with IL-12, IL-15, and IL-18 improved the proliferation and maturation state of NK cells, thereby enhancing their antitumor function in vivo.

[0187] It should be understood that all the embodiments described above are illustrative and do not encompass all possible embodiments included in the claims. Various modifications and changes can be made based on the embodiments described above without departing from the scope of the invention. Similarly, it is possible to combine any technical features of the embodiments described above to form other embodiments of the invention not expressly described. Therefore, the embodiments described above are merely examples of some embodiments of the invention and do not limit the scope of protection of the invention.

Claims

1. Step a involves inducing differentiation from pluripotent stem cells to embryoid bodies in a culture medium to which CHIR 99021 has been added, Step b involves inducing differentiation of EB formed in step a into hematopoietic stem cells and progenitor cells, Step c is a step in which the HSPC formed in step b is differentiated into NK cells, The process includes step d, which involves proliferating the NK cells produced in step c to obtain mature NK cells, However, step a is, The process (a1) involves induction on day 0 in the presence of CHIR 99021 and Rki, A method for producing natural killer cells, characterized by comprising the step (a2) of re-inducing the cells on day 1 in the presence of CHIR 99021.

2. The aforementioned step b is, (b1) A step in which EB is cultured in suspension in a hematopoietic differentiation medium to which BMP4, FGF2, and VEGF have been added, (b2) A step in which suspension culture is continued in hematopoietic stem cell proliferation medium to which BMP4, SCF, FGF2, VEGF, and SB431542 have been added, The method according to claim 1, characterized by comprising the step (b3) of further suspension culture in hematopoietic stem cell proliferation medium to which SCF, FGF2, and VEGF have been added to form HSPCs.

3. The method according to the 2, characterized in that step (a) is performed on day 0 to 1, step (b1) is performed on day 2 to 3, step (b2) is performed on day 4 to 5, and step (b3) is performed on day 6 to 7.

4. The method according to claim 1, characterized in that the HSPC has a CD34+ phenotype.

5. The method according to claim 1, characterized in that the proliferation in step d includes adding the NK cells produced in step c to a culture medium containing IL12, IL15, and IL18 and culturing them to obtain mature NK cells.

6. The method according to claim 5, characterized in that the concentrations of IL12, IL15, and IL18 are each 5 to 50 ng / ml.

7. NK cells produced by the method described in 1, characterized in that they have a CD45+ / CD56+ phenotype.

8. A pharmaceutical composition comprising NK cells as described in claim 7 and a pharmaceutically acceptable excipient.

9. Use of NK cells produced by the method of any one of claims 1 to 6, NK cells according to claim 7, or the pharmaceutical composition according to claim 8 in the manufacture of a preparation for the treatment or prevention of an immune-related disease.

10. The use according to claim 9, wherein the immune-related disease includes one or more of tumors, viral infections, graft-versus-host diseases, autoimmune diseases, or leukemia, and the viral infection includes one or more of human immunodeficiency virus, Epstein-Barr virus, herpes simplex virus, cytomegalovirus, varicella-zoster virus, hepatitis B virus, or hepatitis C virus.

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