Method for preparing natural killer cells

Abstract

Provided is a method for preparing natural killer cells. The method comprises culturing pluripotent stem cells in a StemScale PSC suspension complete medium supplemented with CHIR 99021 to form an embryoid body (EB). In-vitro culture of the induced pluripotent stem cells (iPSCs) under a proper culture condition provides hematopoietic stem cells and precursor cells with low cost and high efficiency, and same are further induced and differentiated into NK cells. The prepared NK cells have good cell amplification and relatively high lethality.

Description

A method for preparing natural killer cells

[0001] This disclosure claims priority to a Chinese patent application filed with the Patent Office of China on October 11, 2023, entitled “A Method for Preparing Natural Killer Cells” and having application number 202311309567.2. The entire contents of the above application are incorporated herein by reference. Technical Field

[0002] The present invention relates to a method for preparing natural killer cells, specifically a method for inducing the formation of natural killer cells from pluripotent stem cells, and also includes the NK cells formed thereby, and the use of the natural killer cells for treating or alleviating viral infections. Background Art

[0003] Natural killer (NK) cells are large granular lymphocytes with the intrinsic ability to kill infected and tumor cells without human leukocyte antigen (HLA) restriction or prior antigen sensitization. In humans, NK cells are typically characterized as CD56+CD3- lymphocytes and can be broadly divided into two subpopulations based on CD56 and CD16 expression levels: CD56brightCD16- cells and CD56dimCD16+ cells, with CD16 expression correlating with the maturation state of NK cells.

[0004] NK cell stimulation and effector function depend on the integration of the signals of two different types of receptors (activating receptors and inhibitory receptors). Normal healthy cells express MHC class I molecules on their surface, and these molecules act as ligands of inhibitory receptors and contribute to the self-tolerance of NK cells. However, virally infected cells or tumor cells have lost surface MHC class I expression, resulting in a reduction in the inhibitory signal in NK cells. Simultaneously, the cell stress associated with viral infection or tumor development, such as DNA damage response, aging program or tumor suppressor genes, will raise the ligands of the activating receptors in these cells. Therefore, the signal from the activating receptors in NK cells directly shifts the balance toward NK cell activation and target cell elimination through the cytotoxicity mediated by NK cells or indirectly through the secretion of proinflammatory cytokines.

[0005] Once activated, NK cells release preformed cytolytic granules containing granzymes and perforins to lyse infected or tumor cells. NK cell degranulation also occurs through antigen-dependent cellular cytotoxicity (ADCC) mediated by CD16. Upon activation, NK cells also secrete a variety of cytokines, such as 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 regulate the functions of other innate and adaptive immune cells.

[0006] Allogeneic primary NK cells from peripheral blood and umbilical cord blood (PB-NK or UCB-NK, respectively) have been shown to be safe and effective cell therapies without significant toxicities such as cytokine release syndrome (CRS), neurotoxicity, or graft-versus-host disease (GVHD). However, the PB-NK cells and UCB-NK cells used in these trials are heterogeneous cell products that vary from donor to donor, which limits their potential for developing standardized cell immunotherapy products.

[0007] Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) are ideal starting cell types for developing a variety of cell lineages, including NK cells. Studies have shown that hESC / iPSC-derived NK cells (iNK) exhibit potent anti-tumor and antiviral activity, providing a possible solution for standardized cell therapy for these diseases. iNK cells are powerful and have a wide range of cytotoxic activity against hematological and solid tumors. iNK cells are a uniform, reproducible, and unlimited source that can be used for multi-dose treatment, eliminating donor-to-donor variability. In addition, it is feasible and consistent to genetically engineer hESC and iPSC to derive iNK cells with improved anti-tumor activity or in vivo persistence. iNK cells represent a promising new approach to adoptive NK cell immunotherapy, and iNK cells can be provided as an off-the-shelf cell therapy or combined with antibodies against inhibitory checkpoint receptors to enhance anti-tumor responses.

[0008] Currently, improving the maturity, function, and in vivo persistence of iNK cells remains a challenge in the field. NK cells have traditionally been considered to be innate immune-mediating cells, but recent discoveries have shown that NK cells can acquire memory-like (ML) characteristics following viral infection, hapten exposure, or transient cytokine activation (e.g., IL-12, IL-15, and IL-18). Memory-like NK cells have a higher degree of maturity and enhance IFN-γ production after restimulation with cytokines or re-exposure to cancer cells, which is associated with the expression of CD94, NKG2A, and NKG2C18. Memory-like NK cells expressing the CD19 CAR have demonstrated potent killing of NK-resistant lymphoma cell lines and primary targets. Specifically, memory-like NK cells induced by IL-12, IL-15, and IL-18 were found to exhibit superior in vivo persistence and exert greater anti-leukemia responses in mouse xenograft models. Together, these data suggest that IL-12, IL-15, and IL-18 enhance NK maturation, which is associated with improved anti-tumor function. However, previous studies have mainly focused on memory-like NK cells derived from peripheral blood. Due to the limited availability of peripheral blood as a source of NK cells, a new method for scalably preparing mature NK cells is urgently needed in the field.

[0009] Summary of the Invention

[0010] Problems to be solved by the invention

[0011] Based on the above problems existing in the prior art, the purpose of the present invention is to provide a method for preparing mature NK cells on a large scale, and to enable the obtained mature NK cells to have better cell expansion and higher killing ability.

[0012] Solutions for solving problems

[0013] The present invention provides a method for preparing natural killer cells, comprising the following steps:

[0014] a. Inducing pluripotent stem cells to differentiate into embryoid bodies in culture medium supplemented with CHIR 99021;

[0015] b. inducing the EB formed in step a to differentiate into hematopoietic stem cells and progenitor cells;

[0016] c. inducing the HSPCs formed in step b to differentiate into NK cells;

[0017] d. Amplifying the NK cells prepared in step c to obtain mature NK cells;

[0018] Wherein, the step a comprises:

[0019] (a1) Induction on day 0 in the presence of CHIR 99021 and Rki;

[0020] (a2) Induction was repeated on day 1 in the presence of CHIR 99021.

[0021] Preferably, the step b comprises:

[0022] (b1) EBs were cultured in suspension in hematopoietic differentiation medium supplemented with BMP4, FGF2, and VEGF;

[0023] (b2) continuing suspension culture in hematopoietic stem cell expansion medium supplemented with BMP4, SCF, FGF2, VEGF, and SB431542;

[0024] (b3) Further suspension culture in hematopoietic stem cell expansion medium supplemented with SCF, FGF2, and VEGF to form HSPCs.

[0025] Preferably, the step (a) is performed on day 0-1, the step (b1) is performed on day 2-3, the step (b2) is performed on day 4-5, and the step (b3) is performed on day 6-7.

[0026] Preferably, the HSPCs have a CD34+ phenotype.

[0027] Preferably, the amplification in step d comprises:

[0028] The NK cells prepared in step c are added to a culture medium containing IL12, IL15 and IL18 to obtain mature NK cells.

[0029] Preferably, the concentrations of IL12, IL15 and IL18 are all 5-50 ng / ml.

[0030] The present invention also provides a NK cell prepared according to the method, wherein the NK cell has a CD45+ / CD56+ phenotype.

[0031] The present invention also provides a pharmaceutical composition comprising the NK cells and a pharmaceutically acceptable excipient.

[0032] The present invention also provides a use of the NK cells prepared according to the method, the NK cells, or the pharmaceutical composition in preparing a preparation for treating or preventing immune-related diseases.

[0033] Preferably, the immune-related disease includes one or more of tumors, viral infections, graft-versus-host disease, autoimmune diseases or leukemia; wherein 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.

[0034] Effects of the Invention

[0035] The present invention provides hematopoietic stem cells and precursor cells at low cost and high efficiency by culturing induced pluripotent stem cells (iPSCs) in vitro under appropriate culture conditions, and further induces their differentiation into natural killer cells, laying a solid foundation for the prevention and treatment of immune-related diseases.

[0036] The NK cell preparation method of the present invention can prepare mature NK cells on a large scale from induced pluripotent stem cells. The mature NK cells prepared according to the method of the present invention have good cell proliferation and high lethality. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic diagram of the differentiation and expansion of induced NK cells (iNK cells). Mesodermal embryoid bodies (EBs) and hematopoietic EBs were generated from human iPSCs and then differentiated into iNK cells. iNK cells were expanded using conventional methods (IL-2 only) and a combined activation method of IL-12, IL-15, and IL-18. iNK cells were expanded at a 1:1 ratio using K562-bmIL-21-41BBL.

[0038] FIG2 is a schematic diagram of the differentiation protocol for producing HSPCs using iPSCs, utilizing variations in cytokines and addition timing to generate HSPCs from iPSCs.

[0039] Figure 3 shows cell morphological changes during iPSC-to-HSPC differentiation. It shows the timeline of hematopoietic stem and progenitor cell (HSPC) differentiation from induced pluripotent stem cells (iPSCs) through embryoid bodies, with arrows indicating the appearance of CD34+ hematopoietic stem cells in suspension.

[0040] Figure 4 is a flow cytometric analysis of HSPC markers. A is a representative of HSPC cell gating for flow cytometric analysis. HSPC cells were identified as (CD235a-CD14-CD43+CD34+) on day 8 of condition 6. B is a representative of HSPC cell gating for flow cytometric analysis; HSPC cells were identified as (CD235a-CD14-CD43+CD34+) on day 10 of condition #6.

[0041] Figure 5 shows representative bright-field images of iPSC culture stages.

[0042] Figure 6 shows the comparison results of CHIR99021 addition time of 1 day vs. 2 days.

[0043] FIG7 shows the results of the cell stability experiment.

[0044] Figure 8 (A) shows representative images of iNK cells expanded in a low-attachment 96-well plate on day 9 of iNK cell expansion. The combined IL-12, IL-15, and IL-18 activation method demonstrated superior expansion compared to the conventional method. Scale bar = 500 μm. Figure B shows quantification of the fold change in iNK cell numbers from two cell lines. The combined IL-12, IL-15, and IL-18 activation method demonstrated a higher fold change than the conventional method. Figure C shows quantification of iNK cell activity after expansion from two cell lines. The results indicate that the combined IL-12, IL-15, and IL-18 activation method demonstrated higher activity than the conventional method.

[0045] Figure 9 shows the results of flow cytometry analysis. A is a quantitative analysis of flow cytometry showing the expression of CD56 of iNK on the 9th day of expansion. Compared with the conventional method, the combined activation method of IL-12&IL-15&IL-18 significantly increased the proportion of CD56+iNK cells. B is a quantitative analysis of flow cytometry showing the expression of CD16 of iNK on the 9th day of expansion. Compared with the conventional method, the combined activation method of IL-12&IL-15&IL-18 significantly increased the proportion of CD16+iNK cells. CD16 is an effective signal for inducing ADCC and can be used as an NK activation marker. The data show that CD16+iNK may help enhance ADCC. C is a representative flow cytometry analysis of CD56 and CD3. D is a representative flow cytometry analysis of CD16 and CD56.

[0046] Figure 10 shows a caspase-3 / 7 cytotoxicity assay against K562 or MOLM13. Compared with the conventional method, the IL-12 & IL-15 & IL-18 combined activation method showed similar cytotoxicity. DETAILED DESCRIPTION

[0047] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0048] As used herein, the term "a" or "an" refers to one or more, for example, "a molecule" should be understood to represent one or more molecules. Therefore, the terms "a" or "an", "one or more", and "at least one" can be used interchangeably herein.

[0049] In the claims and description of the present invention, unless the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, that is, specifying the presence of the stated features, but not excluding the presence or addition of other features in various embodiments of the present invention.

[0050] As used herein, the term "about" encompasses a range of ±25% of a given value. In other embodiments, the term "about" encompasses a range of ±20%, ±15%, ±10%, or ±5% of a given value. For example, in one embodiment, "about 3 grams" refers to a value of 2.7-3.3 grams (i.e., 3 grams ±10%), etc.

[0051] Similarly, although the method for producing NK cells includes orderly, continuous events, the timing of the event can change, for example, by at least 25%. For example, although a specific step can be disclosed as lasting one day in one embodiment, the event can last more than or less than one day. For example, "one day" can include a time period of about 18 to about 30 hours. In other embodiments, the time period can vary by ± 20%, ± 15%, ± 10% or ± 5% of the time period. The time period indicated as multiple days can be a multiple of "one day", for example, two days can span a time period of about 36 hours to about 60 hours, etc. In another embodiment, the time variation can be reduced, for example, the 2nd day is 48 ± 3 hours from the 0th day; the 4th day is 96 ± 3 hours from the 0th day, and the 5th day is 120 hours ± 3 hours from the 0th day.

[0052] As used herein, the term "pluripotent stem cell" or "PSC" refers to a cell that is capable of replicating itself indefinitely and differentiating into all cells that form part of a tissue or organ or 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).

[0053] As used herein, the term "embryonic stem cells" or "ESCs" refers to cells isolated from 5-7 day old embryos donated by consenting patients who have completed in vitro fertilization treatment and have surplus embryos. The use of ESCs has been hampered to some extent by ethical concerns about extracting cells from human embryos. Suitable human PSCs include H1 and H9 human embryonic stem cells.

[0054] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to ESC-like cells derived from adult cells. iPSC has very similar characteristics to ESC, but avoids the ethical issues associated with ESC because iPSC is not derived from embryos. Instead, iPSC is typically derived from fully differentiated adult cells that have been "reprogrammed" back to a pluripotent state. This reprogramming step typically involves the use of reprogramming factors at specific time intervals and at certain concentrations. Some exemplary methods for reprogramming adult cells back to a pluripotent state involve the use of RNA, proteins or other small molecules applied to adult cell cultures. Alternatively, human iPSC lines are also commercially available.

[0055] Suitable human iPSCs include, but are not limited to, iPSCs 19-9-7T, MIRJT6i-mND1-4, and MIRJT7i-mND2-0 derived from fibroblasts and iPSC BM119-9 derived from bone marrow mononuclear cells. Other suitable iPSCs can be obtained from Cellular Dynamics International in Madison, Wisconsin, USA.

[0056] As used herein, EBs obtained by directed differentiation of iPSCs are referred to as "iEBs" or induced EB cells.

[0057] As used herein, HSPCs obtained by directed differentiation of iPSCs are referred to as "iHSPCs" or induced HSPC cells.

[0058] As used herein, NK cells obtained by directed differentiation of iPSCs are referred to as "iNK" or induced NK cells.

[0059] As used herein, the term "differentiation" refers to the process by which a cell changes from one cell type to another, particularly a less specialized type of cell to a more specialized type of cell.

[0060] As used herein, the term "medium" or its plural "media" refers to a liquid or gel designed to support the growth of cells.

[0061] The differentiation of PSCs into NK cells is typically carried out under controlled conditions, particularly when the generated NK cells are intended to be administered to human subjects in accordance with good manufacturing practices (GMP). The initial steps of the process involve culturing PSCs in culture, for example, on tissue culture plates or culture dishes or in bioreactors. The use of bioreactors is particularly attractive in view of the ability to scale up NK cell production at the clinical level for adoptive transfer. However, the scheme utilizing tissue culture plates or culture dishes can also be appropriately scaled up for adoptive NK cell transfer.

[0062] PSCs are cultured in a specific culture medium. Suitable basal culture media include, but are not limited to, Iscove's Modified Dulbecco's Medium / F12 (IMDM / F12), TeSR1 basal medium without FGF2 and TGF-β (mTeSR1 TM Basal medium, Stem Cell Technologies); DF4S basal medium, i.e. Essential 8 TM Culture medium (Life Technologies; also known as "E8" medium). The cell culture medium can be supplemented with other growth factors to improve the cloning efficiency and single cell survival of PSCs. An exemplary supplement that can be used is CloneR (Stem Cell Technologies). Once the PSCs reach the desired confluence, the cells can be harvested and plated to form embryoid bodies.

[0063] Although the culture media disclosed herein may include specific components (e.g., morphogens, small molecules, and hematopoietic cytokines), it is contemplated that other components having the same, equivalent, or similar properties may be used in addition to or in place of those disclosed, as are known in the art.

[0064] As used herein, the term "embryoid body" (EB) refers to a floating three-dimensional aggregate composed of PSC. EB includes hematopoietic EB, which is an EB capable of forming endothelial progenitor cells and blood progenitor cells. Various methods for generating EB are known in the art. For example, traditional methods generally involve generating a single cell suspension of PSC. The PSC can then be cultured in an uncoated non-tissue culture treated culture dish or microwell to prevent PSC attachment, thereby promoting cell aggregation while maintaining suspension. Alternatively, PSC can be cultured in a low-attachment culture dish or microwell. Newer methods for forming EB involve the use of spin aggregation or bioreactors to improve efficiency and process control. ROCK inhibitors, such as Y-27632, have also been shown to promote PSC aggregation, leading to the formation of EB. EB has a teratoma-like structure similar to that of a developing embryo, and the formation of EB is a common platform for establishing differentiation into cells (such as NK cells) from any of the three germ layers.

[0065] As used herein, the term "hematopoietic stem and progenitor cells" (HSPC) refers to cells that are categorized as hematopoietic lineages, but are capable of further hematopoietic differentiation, and include multipotent hematopoietic stem cells (blood embryos), bone marrow progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells, and lymphoid progenitor cells. Hematopoietic stem and progenitor cells are multipotent stem cells that produce all blood cell types, including bone marrow (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells).

[0066] In certain embodiments, at about 10,000 cells / cm 2 Up to 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 / cm 2 or 40,000 cells / cm 2 , PSCs were plated to generate EBs.

[0067] In case EB is formed, EB is cultured in a differentiation medium comprising NK differentiation factors to promote differentiation into NK cells. The EB quantity used depends on the type of tissue culture plate, tissue culture dish or bioreactor used. For example, if 6-well plates are used, about 10-30 EB plates can be inoculated. However, it will be understood by those skilled in the art that, according to the surface area of ​​the tissue culture plate, tissue culture dish or bioreactor used, the method for determining the optimal number of EBs to be plated is known in the art.

[0068] In certain embodiments, differentiation medium or expansion medium can be xeno-free and incorporate human proteins that have been isolated from natural sources, such as from the placenta or other human tissues, or that can be produced using recombinant technology.

[0069] In certain embodiments, all proteins described herein are human.

[0070] In certain embodiments, all proteins used in the differentiation medium or expansion medium are human proteins.

[0071] In certain embodiments, all proteins used in the differentiation medium or expansion medium are human proteins.

[0072] In certain embodiments, all proteins described herein are human recombinant proteins.

[0073] In certain embodiments, all proteins used in the differentiation medium or expansion medium are recombinant human proteins.

[0074] All of the proteins described herein are known to those skilled in the art, and most, if not all, of the proteins described herein are commercially available.

[0075] Those skilled in the art will appreciate that the cell culture medium can be replaced at fixed or varying intervals, depending on the specific circumstances. As cells remain in culture, toxic metabolites may be produced, and the cultured cells continuously utilize nutrients, their numbers steadily increasing during the expansion phase. Therefore, old cell culture medium can be replaced with fresh cell culture medium.

[0076] In certain embodiments, the differentiation medium or expansion medium can be changed about every 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0077] Once the NK cell population becomes more mature, and / or a sufficient number of PSCs have differentiated into NK cells, more medium changes are needed to replenish the growing and maturing NK cell population. In some embodiments, this period is about 10-20 days. After this stage, the differentiation medium or expansion medium can be changed about every 1 day, about 2 days, or about 3 days.

[0078] The number of NK cells in the culture can be determined by various methods known in the art. For example, flow cytometry or microscopy can be used. If flow cytometry is used, the cell sample can be stained with markers commonly associated with NK cells, such as CD3, CD56, CD45, CD94, CD122, CD127, CD16, KIR, NKG2A, NKG2D, NKp30, NKp44, NKp46, and NKp80.

[0079] In certain embodiments, supplementation of the cell culture medium with specific cytokines, chemokines, proteins, signaling factors, and growth factors occurs within defined time intervals, after which supplementation of these factors is ceased.

[0080] In certain embodiments, supplementation of the cell culture medium with specific cytokines, chemokines, proteins, signaling factors, and growth factors is stopped after about 5-20 days. For example, supplementation with specific factors can be stopped after about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days.

[0081] In certain embodiments, supplementation of one or more of the following factors: IL-3, SCF, IL-7, IL-15, FLT3L, and IL-2 is discontinued after a defined interval.

[0082] After a period of time, expanded, floating NK cells with a specific spindle-shaped or elongated morphology begin to appear in the culture. Typically, NK cells with a spindle-shaped or elongated morphology are observed between about 15 and 50 days in culture. In certain embodiments, NK cells with a spindle-shaped or elongated morphology are observed after about 15 days, about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 45 days, or about 50 days in culture.

[0083] The verification of NK cell markers and functional properties can be carried out using many tests known in the art. The cell surface markers commonly associated with NK cells include CD3, CD56, CD45, CD94, CD122, CD127, CD16, KIR, NKG2A, NKG2D, NKp30, NKp44, NKp46 and NKp80. Mature NK cells act by the release of granule exocytosis and cytotoxic proteins, cytokines and chemokines to induce targeted cell death. An exemplary mechanism of NK cell-mediated cell death is through the interaction with caspase enzymatic cascade, apoptosis signaling and inflammatory signaling. Therefore, the maturity and functional properties of NK cells, including cytotoxicity and tumor killing ability, can be measured by measuring the expression level of proteins such as Fas ligand (FasL), tumor necrosis factor (TNF)-α, TNF-related apoptosis-inducing ligand (TRAIL) or other cytokines and chemokines. In addition, the cell surface expression of certain markers is also a sign that NK cell homing properties improve. Changes in NK cell functional properties can be indicated by increases or decreases in the expression levels of certain markers or proteins. In addition, changes in NK cell functional properties can be indicated by increases in certain markers or proteins and concomitant decreases in other markers or proteins.

[0084] The suitable method for measuring the cytotoxicity of the NK cells produced by the method of the present invention is to co-cultivate NK cells and tumor cell lines as target cells, such as K562, LN-18, U937, WERI-RB-1, U-118MG, HT-29, HCC2218, KG-1 or U266 tumor cells, etc. The tumor cells can then be, for example, labeled with fluorescent markers specific for tumor cells. The cytotoxicity in the co-cultivation process can then be assessed based on the reduction of fluorescent marker expression, which will indicate tumor cell apoptosis or cell death. Examples of labels suitable for labeling tumor cells include carboxyfluorescein succinimidyl ester (CFSE), mCherry or any other suitable fluorescent marker known in the art. Alternatively, flow cytometry can be used, such as by staining with Annexin-V (annexin V) or any other fixable viability dyes, to further analyze apoptosis and dead cell populations. Co-cultivation of NK cells and tumor cells is generally carried out with a specific effector: target ratio. In certain embodiments, the ratio is about 1: 1 to 10: 1. In certain embodiments, the ratio is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. Methods and assays for measuring ADCC are also known in the art, for example, by incubating NK cells with target cells and a suitable effector antibody, such as NKp46. ADCC assays and kits and reagents therefor are also commercially available, for example, the Promega ADCC Bioassay.

[0085] As used herein, the term "pharmaceutical composition" refers to a composition comprising NK cells or NK cell populations described herein that has been formulated for administration to an individual. Preferably, the pharmaceutical composition is sterile. In certain embodiments, the pharmaceutical composition is pyrogen-free.

[0086] NK cells or NK cell groups will be prepared, dosed and used in a manner consistent with good medical practice. Factors considered in this context include the type of the specific disease being treated, the specific individual being treated, the clinical condition of the individual, the site of administration, the method of administration, the time of administration, possible side effects and other factors known to the physician. Other considerations include maximizing the cytotoxicity and persistence of NK cells in vivo. The therapeutically effective amount of NK cells or NK cell groups to be administered will be determined by these considerations.

[0087] NK cells or NK cell groups can be administered to an individual by any suitable method, including intravenous (IV) injection and subcutaneous injection. NK cells can also be administered together with other therapeutic agents such as antibodies or modifiers intended to enhance innate NK cell properties or NK cell activity in vivo. NK cells can also be pretreated or initiated with a modifier before NK cells are administered to an individual to enhance innate NK cell properties or NK cell activity in vivo. The example of a suitable initiator or modifier is IL-2 or IL-15. Other suitable protein constructs, agonists, antagonists, regulators or inflammatory factors are also contemplated.

[0088] NK cells, due to their effector properties in inducing ADCC, can also be used in antibody cancer therapy, which uses antibodies such as monoclonal antibodies to target tumor cells. Indeed, NK cells can mediate antibody-dependent tumor killing through cytotoxic granule exocytosis, TNF death receptor signaling, and the release of cytokines such as interferon-γ.

[0089] As used herein, the term "effective amount" refers to an amount of NK cells or a population of NK cells effective to treat a condition, disease, or disorder in a subject.

[0090] As used herein, the terms "treat," "treating," or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or ameliorate a condition, disease, or disorder, or to slow (lessen) the progression of a condition, disease, or disorder in a subject. Subjects in need of treatment include those already suffering from a condition, disease, or disorder as well as those in which the condition, disease, or disorder is to be prevented.

[0091] As used herein, the terms "prevent," "preventation," "preventative," or "prophylactic" refer to preventing the occurrence of, or hindering, guarding against, or resisting the occurrence of, a disease condition, illness, or disorder, including abnormalities or symptoms. The subject in need of prevention may be susceptible to developing the disease condition, disease, or disorder.

[0092] As used herein, the terms "ameliorate" or "amelioration" refer to the reduction, alleviation, or elimination of a condition, disease, or disorder, including an abnormality or symptom. A subject in need of treatment may already have the condition, disease, or disorder, may be susceptible to the condition, disease, or disorder, or may be in whom the condition, disease, or disorder is to be prevented.

[0093] As used herein, the term "subject" refers to a mammal. The mammal can be a primate, particularly a human, or can be a domestic animal, a zoo animal, or a companion animal. Although the methods disclosed herein and the resulting NK cells or NK cell populations are particularly contemplated as suitable for medical treatment of humans, they are also suitable for veterinary treatment, including treatment of domestic animals such as horses, cattle, and sheep, companion animals such as dogs and cats, or zoo animals such as felines, canines, bovines, and ungulates.

[0094] Unless otherwise stated, the experimental methods in the following examples without specific conditions are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight. Unless otherwise stated, the room temperature described in the present invention is 20°C to 30°C. Unless otherwise stated, the various materials and reagents used in the present invention can be obtained using conventional methods in the art or through commercial channels, as shown below:

[0095] StemScale PSC Suspension Medium: ThermoFisher, #A4965001

[0096] Y27632:Tocris, #1254

[0097] CHIR99021:Sigma,SML1046

[0098] Stemline II Hematopoietic Stem Cell Medium: Sigma, #S0192

[0099] CTS TM NK-Xpander TM Culture medium: ThermoFisher, #A5019001

[0100] Penicillin-Streptomycin (P / S): ThermoFisher, #15140122

[0101] GlutaMAX Supplement: ThermoFisher, #35050061

[0102] L-Ascorbic acid: Sigma-Aldrich, #A4544

[0103] ITS-G: ThermoFisher, #41400045

[0104] BMP4: Miltenyi Biotec, #130-111-167

[0105] FGF2: Miltenyi Biotec, #130-093-564

[0106] VEGF: Miltenyi Biotec, #130-109-396

[0107] SB431542: Tocris, #1614 / TB1614-GMP

[0108] SCF: Miltenyi Biotec, #130-096-695

[0109] DMEM / F-12GlutaMAX: ThermoFisher, #10565018

[0110] F12+GlutaMAX: ThermoFisher, #31765-035

[0111] Heat-inactivated human Ab serum: Sigma, #H3667

[0112] L-Glutamine: ThermoFisher, #25030081

[0113] B-Mercaptoethanol: ThermoFisher, #21985023

[0114] Sodium selenite: Sigma, #214485

[0115] Ethanolamine: MP, #193845

[0116] IL-3: Miltenyi Biotec, #130-095-070

[0117] IL-7: Miltenyi Biotec, #130-095-362

[0118] IL-2: Miltenyi Biotec, #130-097-748

[0119] IL-15: Miltenyi Biotec, #130-095-764

[0120] IL-18: MBL, #B001-5

[0121] IL-12: Miltenyi Biotec, #130-096-705

[0122] FLT3L: Miltenyi Biotec, #130-096-477

[0123] DPBS: ThermoFisher, #14190-144

[0124] Accutase: Stem Cell Technologies, #07922

[0125] CellTraceTM Violet: Thermo-Fisher Scientific, #C34557

[0126] Caspase-3 / 7 Green Detection Reagent: Thermal Fisher Scientific, #C10423

[0127] The present invention provides a method for preparing natural killer (NK) cells, comprising the following steps:

[0128] a. Inducing pluripotent stem cells to differentiate into embryoid bodies (EBs) in a medium supplemented with CHIR 99021;

[0129] b. inducing the EB formed in step a to differentiate into hematopoietic stem and progenitor cells (HSPC);

[0130] c. inducing the HSPCs formed in step b to differentiate into NK cells;

[0131] d. Amplifying the NK cells prepared in step c to obtain mature NK cells;

[0132] Wherein, the step a comprises:

[0133] (a1) Induction on day 0 in the presence of CHIR 99021 and Rki;

[0134] (a2) Induction was repeated on day 1 in the presence of CHIR 99021.

[0135] In certain embodiments, step b comprises:

[0136] (b1) EBs were cultured in suspension in hematopoietic differentiation medium supplemented with BMP4, FGF2, and VEGF;

[0137] (b2) continuing suspension culture in hematopoietic stem cell expansion medium supplemented with BMP4, SCF, FGF2, VEGF, and SB431542;

[0138] (b3) Further suspension culture in hematopoietic stem cell expansion medium supplemented with SCF, FGF2, and VEGF to form HSPCs.

[0139] In certain embodiments, the methods do not require a feeder layer during preparation.

[0140] In certain embodiments, the culture medium in step (a) is selected from one or more of StemScale PSC, Essential 8, KSR / FGF2, mTeSR, AKIT, or B8.

[0141] In certain embodiments, the culture medium in step (a) is StemScale PSC.

[0142] In certain embodiments, the concentration of CHIR 99021 in step (a) is 0.1-100 μM.

[0143] In certain embodiments, the concentration of CHIR 99021 in step (a) is 0.2-50 μM.

[0144] In certain embodiments, the concentration of CHIR 99021 in step (a) is 0.5-25 μM.

[0145] In certain embodiments, the concentration of CHIR 99021 in step (a) is 1-20 μM.

[0146] In certain embodiments, the concentration of CHIR 99021 in step (a) is about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM or about 20 μM.

[0147] In certain embodiments, the culture medium in step (b1) further contains one or more of P / S, GlutaMax, ascorbic acid or ITS-G.

[0148] In certain embodiments, the culture medium in step (b1) contains 1% P / S, 2mM GlutaMaX, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / mL BMP4, 1-100ng / mL FGF2 and 1-100ng / mL VEGF.

[0149] In certain embodiments, the concentration of BMP4 in the culture medium in step (b1) 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.

[0150] In certain embodiments, the concentration of FGF2 in the culture medium in step (b1) 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.

[0151] In certain embodiments, the concentration of VEGF in the culture medium in step (b1) 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.

[0152] In certain embodiments, the culture medium in step (b2) further contains one or more of P / S, GlutaMaX, ascorbic acid and ITS-G.

[0153] In certain embodiments, the culture medium in step (b2) contains 1% P / S, 2mM GlutaMax, 50μg / ml ascorbic acid, 1% ITS-G, 1-100ng / mL BMP4, 1-100ng / mL SCF, 1-100ng / mL FGF2, 1-100ng / mL VEGF and 1-20μM SB431542.

[0154] In certain embodiments, the concentration of BMP4 in the culture medium in step (b2) 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.

[0155] In certain embodiments, the concentration of SCF in the culture medium in step (b2) 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.

[0156] In certain embodiments, the concentration of FGF2 in the culture medium in step (b2) 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.

[0157] In certain embodiments, the concentration of VEGF in the culture medium in step (b2) 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.

[0158] In certain embodiments, the concentration of SB431542 in the culture medium in step (b2) is about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM or about 20 μM.

[0159] In certain embodiments, the culture medium in step (b3) further contains one or more of P / S, GlutaMax, ascorbic acid or ITS-G.

[0160] In certain 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.

[0161] In certain embodiments, the concentration of SCF in the culture medium in step (b3) 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.

[0162] In certain embodiments, the concentration of FGF2 in the culture medium in step (b3) 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.

[0163] In certain embodiments, the concentration of VEGF in the culture medium in step (b3) 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.

[0164] In certain embodiments, step (a) is performed on day 0-1, step (b1) is performed on day 2-3, step (b2) is performed on day 4-5, and step (b3) is performed on day 6-7.

[0165] In certain embodiments, the EB is an iEB.

[0166] In certain embodiments, the HSPCs are iHSPCs.

[0167] In certain embodiments, the NK cells are iNK cells.

[0168] In some embodiments, the HSPC has a CD34+ phenotype. In some embodiments, step c comprises:

[0169] (c1) adding the HSPCs formed in step b to a first culture medium for cell culture;

[0170] (c2) adding the cells cultured in step (c1) to a second culture medium for cell culture to obtain NK cells.

[0171] In certain embodiments, the first culture medium contains heat-inactivated human AB serum, colony stimulating factors, and interleukins, wherein the colony stimulating factors are selected from one or more of G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF, and FLT3L, and the interleukins are selected from one or more of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27;

[0172] In certain embodiments, the second culture medium contains heat-inactivated human AB serum, colony stimulating factors, and interleukins, wherein the colony stimulating factors are selected from one or more of G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF, and FLT3L, and the interleukins are selected from one or more of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27.

[0173] In certain embodiments, the colony stimulating factors in the first culture medium are SCF and FLT3L.

[0174] In certain embodiments, the interleukins in the first culture medium are IL-3, IL-7 and IL-15.

[0175] In certain embodiments, the first culture medium further contains one or more of DMEM / F12+GlutaMAX, F12+GlutaMAX, P / S, L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, and Retronectin.

[0176] In certain embodiments, the first culture 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.

[0177] In certain embodiments, the concentration of IL-3 in the first culture medium is about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 31 ng / mL, about 32 ng / mL, about 33 ng / mL, about 34 ng / mL, about 35 ng / mL, about 36 ng / mL, about 37 ng / mL, about 38 ng / mL, about 39 ng / mL, about 40 ng / mL, about 41 ng / mL, about 42 ng / mL, about 43 ng / mL, about 44 ng / mL, about 45 ng / mL, about 46 ng / mL, about 47 ng / mL, about 48 ng / mL, about 49 ng / mL, or about 50 ng / mL.

[0178] In certain 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.

[0179] In certain 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.

[0180] In certain 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.

[0181] In certain 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.

[0182] In certain embodiments, the concentration of DLL4 in the first culture medium is about 1 μg / mL, about 5 μg / mL, about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL, about 50 μg / mL, about 55 μg / mL, about 60 μg / mL, about 65 μg / mL, about 70 μg / mL, about 75 μg / mL, about 80 μg / mL, about 85 μg / mL, about 90 μg / mL, about 95 μg / mL, or about 100 μg / mL.

[0183] In certain embodiments, the concentration of Retronectin in the first culture medium is about 1 μg / mL, about 5 μg / mL, about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL, about 50 μg / mL, about 55 μg / mL, about 60 μg / mL, about 65 μg / mL, about 70 μg / mL, about 75 μg / mL, about 80 μg / mL, about 85 μg / mL, about 90 μg / mL, about 95 μg / mL, or about 100 μg / mL.

[0184] In certain embodiments, the colony stimulating factors in the second culture medium are SCF and FLT3L.

[0185] In certain embodiments, the interleukins in the second culture medium are IL-7 and IL-15.

[0186] In certain embodiments, the second culture medium further contains one or more of DMEM / F12+GlutaMAX, F12+GlutaMAX, P / S, L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, and Retronectin.

[0187] In certain embodiments, the second culture 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.

[0188] In certain 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.

[0189] In certain 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.

[0190] In certain 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.

[0191] In certain embodiments, the concentration of FLT3L 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.

[0192] In certain embodiments, the concentration of DLL4 in the second culture medium is about 1 μg / mL, about 5 μg / mL, about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL, about 50 μg / mL, about 55 μg / mL, about 60 μg / mL, about 65 μg / mL, about 70 μg / mL, about 75 μg / mL, about 80 μg / mL, about 85 μg / mL, about 90 μg / mL, about 95 μg / mL, or about 100 μg / mL.

[0193] In certain embodiments, the concentration of Retronectin in the second culture medium is about 1 μg / mL, about 5 μg / mL, about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL, about 50 μg / mL, about 55 μg / mL, about 60 μg / mL, about 65 μg / mL, about 70 μg / mL, about 75 μg / mL, about 80 μg / mL, about 85 μg / mL, about 90 μg / mL, about 95 μg / mL, or about 100 μg / mL.

[0194] In certain embodiments, the cell culture in step (c1) is carried out for 6-8 days.

[0195] In certain embodiments, the cells in step (c1) are cultured for about 6 days, about 7 days, or about 8 days.

[0196] In certain embodiments, the cells in step (c2) are cultured for 8-10 days and then cultured for another 8-10 days using a fresh second culture medium.

[0197] In certain embodiments, the cells in step (c2) are cultured for about 8 days, about 9 days, or about 10 days, and then cultured with fresh second culture medium for another about 8 days, about 9 days, or about 10 days.

[0198] In certain embodiments, the amplifying in step d comprises:

[0199] The NK cells prepared in step c are added to a culture medium containing IL12, IL15 and IL18 to obtain mature NK cells.

[0200] In certain embodiments, the concentrations of IL12, IL15, and IL18 are all 5-50 ng / ml.

[0201] In certain embodiments, step d comprises adding the NK cells prepared in step c to NK expansion medium for cell culture.

[0202] In certain embodiments, step d comprises adding the NK cells prepared in step c to an activation medium for cell culture, and replacing the activation medium with a maintenance medium on day 3 of NK cell expansion.

[0203] In certain embodiments, step d further comprises performing a half-medium replacement using maintenance medium on day 6 of NK cell expansion.

[0204] In certain embodiments, step d further comprises transferring the NK cells into 24-well Grex on day 9 of NK cell expansion.

[0205] In certain embodiments, step d further comprises performing a half-medium replacement using maintenance medium on day 12 of NK cell expansion.

[0206] In certain embodiments, step d further comprises harvesting iNK cells on day 15 of NK cell expansion.

[0207] In certain embodiments, the activation medium is NK Xpander medium supplemented with human AB serum, P / S, IL-15, IL-12, IL-18, and IL-2.

[0208] In certain embodiments, the activation medium is supplemented with 1-20% human 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.

[0209] In certain embodiments, the concentration of IL-12 is about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about or about 50 ng / mL.

[0210] In certain embodiments, the concentration of IL-15 is about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about or about 50 ng / mL.

[0211] In certain embodiments, the concentration of IL-18 is about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 31 ng / mL, about 32 or about 50 ng / mL.

[0212] In certain embodiments, the activation medium is supplemented with 1-20% human 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.

[0213] In certain embodiments, the maintenance medium is NK Xpander medium supplemented with human AB serum, P / S, IL-2, and IL-15.

[0214] In certain embodiments, the maintenance medium is supplemented with 1-20% human AB serum, 1% P / S, 5-500 U / ml IL-2, and 1-100 ng / ml IL-15.

[0215] The present invention also provides a NK cell prepared according to the method, wherein the NK cell shows increased expression of one or more of IL-12, IL-15 and IL-18.

[0216] In certain embodiments, the NK cells exhibit about a 1% increase, about a 2% increase, about a 3% increase, about a 4% increase, about a 5% increase, about a 6% increase, about a 7% increase, about an 8% increase, about a 9% increase, about a 10% increase, about a 20% increase, about a 30% increase, about a 40% increase, about a 50% increase, about a 60% increase, about a 70% increase, about an 80% increase, about a 90% increase, about a 100% increase, or more, in an NK cell marker or protein.

[0217] In certain embodiments, the NK cells exhibit an increase of about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold or more in an NK cell marker or protein.

[0218] In certain embodiments, the NK cells have a CD45+ / CD56+ phenotype.

[0219] In certain embodiments, the proportion of cells having a CD45+ / CD56+ phenotype is >80%.

[0220] The present invention also provides a pharmaceutical composition comprising the NK cells and a pharmaceutically acceptable excipient.

[0221] In certain embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.

[0222] In certain embodiments, the additional therapeutic agent is an antibody.

[0223] The present invention also provides a use of the NK cells prepared according to the method, the NK cells, or the pharmaceutical composition in preparing a preparation for treating or preventing immune-related diseases.

[0224] In certain embodiments, the immune-related disease comprises one or more of a tumor, a viral infection, a graft-versus-host disease, an autoimmune disease, or a leukemia.

[0225] In certain embodiments, the viral infection comprises one or more of 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).

[0226] Example 1: Generation of HSPCs from iPSCs (Condition #8)

[0227] Human iPSCs are typically maintained in commercially available Essential 8 Medium. When iPSCs reach 70% confluence, they are checked for healthy morphology and absence of spontaneous differentiation. iPSCs are dissociated into single cells using Accutase. Collected single cells are counted and plated in StemScale PSC Suspension Complete Medium supplemented with 5-50 μM Y27632.

[0228] The cells were incubated in a 37°C, 5% CO2 incubator on a CO2-resistant orbital shaker with continuous rotation at 30-100 rpm. Within 24 hours (day 0), a half-fluid change was performed using StemScale PSC suspension complete medium with the addition of the ROCK inhibitor Rki and the GSK-3 inhibitor CHIR 99021.

[0229] After 24 hours (day 1), CHIR 99021 was added.

[0230] After 24 hours (day 2), the formed embryoid bodies (EBs) were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml BMP4, 1-100ng / ml FGF2, and 1-100ng / ml VEGF as hematopoietic differentiation medium 1.

[0231] After 24-48 hours (days 3-4), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml BMP4, 1-100ng / ml FGF2, 1-100ng / ml VEGF, 1-100ng / ml SCF and 1-20uM SB431542 as hematopoietic differentiation medium 2.

[0232] After 24-72 hours (days 5-7), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml SCF, 1-100ng / ml FGF2, and 1-100ng / ml VEGF as hematopoietic differentiation medium 3. On days 8-10, HSPCs were harvested.

[0233] Example 2: Generation of HSPCs from iPSCs (Condition #4)

[0234] Human iPSCs are typically maintained in commercially available Essential 8 Medium. When iPSCs reach 70% confluence, they are checked for healthy morphology and absence of spontaneous differentiation. iPSCs are dissociated into single cells using Accutase. Collected single cells are counted and plated in StemScale PSC Suspension Complete Medium supplemented with 5-50 μM Y27632.

[0235] The cells were incubated in a 37°C, 5% CO2 incubator on a CO2-resistant orbital shaker with continuous rotation at 30-100 rpm. Within 24 hours (day 0), a half-fluid change was performed with StemScale PSC suspension complete medium supplemented with the ROCK inhibitor Rki.

[0236] After 24 hours (day 1), CHIR 99021 (2X) was added.

[0237] After 24 hours (day 2), the formed embryoid bodies (EBs) were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml BMP4, 1-100ng / ml FGF2, and 1-100ng / ml VEGF as hematopoietic differentiation medium 1.

[0238] After 24-48 hours (days 3-4), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml BMP4, 1-100ng / ml FGF2, 1-100ng / ml VEGF, 1-100ng / ml SCF and 1-20uM SB431542 as hematopoietic differentiation medium 2.

[0239] After 24-72 hours (days 5-7), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml SCF, 1-100ng / ml FGF2, and 1-100ng / ml VEGF as hematopoietic differentiation medium 3. On days 8-10, HSPCs were harvested.

[0240] Example 3: Generation of HSPCs from iPSCs (Condition #6)

[0241] Human iPSCs are typically maintained in commercially available Essential 8 Medium. When iPSCs reach 70% confluence, they are checked for healthy morphology and absence of spontaneous differentiation. iPSCs are dissociated into single cells using Accutase. Collected single cells are counted and plated in StemScale PSC Suspension Complete Medium supplemented with 5-50 μM Y27632.

[0242] The cells were incubated in a 37°C, 5% CO2 incubator on a CO2-resistant orbital shaker with continuous rotation at 30-100 rpm. Within 24 hours (day 0), a half-fluid change was performed using StemScale PSC suspension complete medium with the addition of the ROCK inhibitor Rki and the GSK-3 inhibitor CHIR 99021.

[0243] After 24 hours (day 1), CHIR 99021 was added.

[0244] After 24-48 hours (day 2-3), the formed embryoid bodies (EBs) were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml BMP4, 1-100ng / ml FGF2, and 1-100ng / ml VEGF as hematopoietic differentiation medium 1.

[0245] After 24-48 hours (days 4-5), EBs were harvested and resuspended in Stemline II Hematopoietic Stem Cell Expansion Medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml BMP4, 1-100ng / ml FGF2, 1-100ng / ml VEGF, 1-100ng / ml SCF and 1-20uM SB431542 as hematopoietic differentiation medium 2.

[0246] After 24-48 hours (days 6-7), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml SCF, 1-100ng / ml FGF2, and 1-100ng / ml VEGF as hematopoietic differentiation medium 3. On days 8-10, HSPCs were harvested and assayed for cell surface markers.

[0247] Example 4-8: Preparation of HSPCs from iPSCs (Conditions #1-3, 5, 7)

[0248] The culture methods of Examples 4-8 (Conditions #1-3, 5, 7) are basically the same as those of Example 1. For specific conditions, see Figure 2.

[0249] Example 9: Comparison of Differentiation Scheme Effects

[0250] From a morphological perspective, we observed the addition of CHIR (condition 6) during the iPSC culture stage (i.e., day 0 in Figure 2). The results are shown in Figure 5. It can be seen that the addition of CHIR allows the formation of iPSC spheres during the iPSC culture stage before differentiation begins, thereby achieving a larger number of spheres with better uniformity for downstream differentiation.

[0251] We further attempted to extend the induction time of CHIR99021 to 2 days during the stage of iPSC induction to form mesoderm (i.e., days 1-3 of condition 6). The results are shown in Figure 6. It can be seen that adding CHIR99021 for only 1 day is more conducive to the release of HPC cells from HE EBs than adding it for 2 days, and is also more beneficial to the overall cell activity / growth.

[0252] Four batches of stability experiments were conducted under condition 6. The results are shown in Figure 7. The CD34 cells in the hematopoietic EBs are mostly early progenitor cells (CD43-), while the suspended cells released from the EB spheres can reach >50% CD34 positivity and >90% CD43 positivity, indicating that the suspended cells released from the EB spheres are mostly hematopoietic lineage progenitor cells.

[0253] Example 10: Preparation of iNK cells from HSPCs

[0254] The HSPCs and suspension cell cultures harvested in Examples 1-8 were resuspended in NK differentiation medium 1 containing 56.6% DMEM / F12+GlutaMAX TM -I, 28.3% F12+GlutaMAX TM -I, 1-20% heat-inactivated human AB serum, 1% P / S, 2mM L-glutamine, 1μM β-mercaptoethanol, 5-50ng / mL sodium selenite, 50-200μM ethanolamine, 20-100mg / L ascorbic acid, 1-50ng / mL IL-3, 1-100ng / mL SCF, 1-100ng / mL IL-7, 1-100ng / mL IL-15, 1-100ng / mL FLT3L.

[0255] On day 8 of iNK cell differentiation, resuspend the culture in NK differentiation medium 2 containing 56.6% DMEM / F12 + GlutaMAX. TM -I, 28.3% F12+GlutaMAX TM -I, 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.

[0256] After 10 days (iNK cell differentiation day 18), the suspended cells were collected and resuspended in fresh NK differentiation medium 2.

[0257] After 10 days (day 28 of iNK cell differentiation), the cultures were monitored for the emergence of spindle-shaped iNK-like cells and their CD45 / CD56 expression was assessed. The results showed that the ratio of CD45+ / CD56+ cells was >80%.

[0258] Example 11: iNK cell expansion, maturation and activity testing

[0259] iNK cells were expanded on day 28. 3x10 4 iNK cells and 3x10 4K562-mbII21-41BBL cells (iNK cell:feeder layer ratio of 1:1) were cultured in 96-well low attachment plates in 200 μl of NK expansion medium. The composition of the NK expansion and maturation medium is shown in Table 1. The cells were incubated at 37°C in 5% CO2.

[0260] Table 1: Composition of NK cell expansion medium

[0261] On day 3 of iNK cell expansion, the activation medium was replaced with maintenance medium. On day 6 of iNK cell expansion, a half-medium change with maintenance medium was performed. On day 9 of iNK cell expansion, iNK cells were transferred to 24-well Grex plates, and a half-medium change was performed on day 12. iNK cells were harvested on day 15 of iNK cell expansion.

[0262] Caspase assay was performed to test iNK cell cytotoxicity. Briefly, target cells (K562 or MOLM13) were incubated at 37°C with CellTrace ELISA in PBS at a final concentration of 5 μM. TM Violet pre-stained for 20 minutes. After staining, cells were washed in complete medium and then mixed with NK cell cultures at the indicated effector to target (E:T) ratios. After brief centrifugation, the co-cultures were incubated at 37°C for 3.5 hours. Caspase-3 / 7 Green Detection Reagent (Thermal Fisher Scientific, C10423) was added for an additional 30 minutes, for a total incubation time of 4 hours, and the cells were then analyzed by flow cytometry.

[0263] The results showed that transient treatment with IL-12, IL-15, and IL-18 improved NK expansion and maturation status, as indicated by fold change in cell number and CD16 surface marker expression, which may lead to enhanced anti-tumor function in vivo.

[0264] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A method for preparing natural killer cells, characterized in that: The method comprises the following steps: a. Inducing pluripotent stem cells to differentiate into embryoid bodies in a medium supplemented with CHIR 99021; b. inducing the EB formed in step a to differentiate into hematopoietic stem cells and progenitor cells; c. inducing the HSPCs formed in step b to differentiate into NK cells; d. amplifying the NK cells prepared in step c to obtain mature NK cells; Wherein, the step a comprises: (a1) Induction on day 0 in the presence of CHIR 99021 and Rki; (a2) Induction was repeated on day 1 in the presence of CHIR 99021.

2. The method according to claim 1, characterized in that The step b comprises: (b1) EBs were cultured in suspension in hematopoietic differentiation medium supplemented with BMP4, FGF2, and VEGF; (b2) continuing suspension culture in hematopoietic stem cell expansion medium supplemented with BMP4, SCF, FGF2, VEGF and SB431542; (b3) Further suspension culture in hematopoietic stem cell expansion medium supplemented with SCF, FGF2 and VEGF to form HSPCs.

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

4. The method according to claim 1, characterized in that The HSPCs have a CD34+ phenotype.

5. The method according to claim 1, characterized in that The amplification in step d comprises: The NK cells prepared in step c are added to a culture medium containing IL12, IL15 and IL18 to obtain mature NK cells.

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

7. A NK cell prepared according to the method of claim 1, characterized in that: The NK cells have a CD45+ / CD56+ phenotype.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the NK cells according to claim 7 and a pharmaceutically acceptable excipient.

9. Use of the NK cells prepared according to the method of any one of claims 1 to 6, the NK cells according to claim 7, or the pharmaceutical composition according to claim 8 in the preparation of a preparation for treating or preventing immune-related diseases.

10. The use according to claim 9, characterized in that The immune-related diseases include one or more of tumors, viral infections, graft-versus-host disease, autoimmune diseases or leukemia; wherein the viral infections include 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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