Methods for producing natural killer cells from pluripotent stem cells

Abstract

The present disclosure provides, inter alia, a method for efficiently producing natural killer cells from induced pluripotent cells, comprising the steps of (I) culturing pluripotent stem cells in a culture medium to produce CD56+ / CD3- immune cells.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 983,511, filed February 28, 2021, the disclosure of which is incorporated herein by reference.

[0002] [Background technology] Natural killer (NK) cells are cytotoxic lymphocytes of the immune system. NK cells are cytotoxic against cancer cells, pathogen-infected cells, and other damaged cells. NK cells are innate lymphoid cells (ILCs), specifically cytotoxic large granular lymphocytes that bridge the innate and adaptive arms of the immune response. They comprise 10–15% of circulating lymphocytes in peripheral blood. NK cells also exhibit the highest level of cytotoxic activity within the immune system. Therefore, alterations in NK cell function and numbers affect immune system function against infection and cancer.

[0003] NK cells do not contain specific cell surface antigen receptors. Therefore, NK cells can kill cancer cells and pathogen-infected cells without prior sensitization, making them part of the innate immune response. They also have a role in tumor immunosurveillance by directly influencing the adaptive immune response. These and other characteristics make NK cells a particularly attractive cell type for use in adoptive cell therapy.

[0004] Various protocols have been described for obtaining NK cells from progenitor cells, but these protocols are laborious and require multiple time-consuming steps, including cell isolation steps, which increase the manufacturing / production time and financial costs for producing NK cells. Summary of the Invention

[0005] The present application provides, among other things, an improved method for producing CD56+ / CD3− immune cells (i.e., NK cells or NK-like cells). The present application is based, at least in part, on the surprising discovery of efficient and stable production of CD56+ / CD3− immune cells from a bulk cell population derived from pluripotent stem cells (e.g., induced pluripotent stem cells (iPSCs)) without the need for a cell isolation step. Prior to the present application, methods for inducing NK cells often required the isolation of specific cell types based on known cell lineages to NK cells. As described herein, the present application unexpectedly demonstrates that NK cells can be successfully derived from a bulk cell population without the need for lineage-based isolation of any cell type. Thus, this method has many advantages over existing methods for producing NK cells, including, for example, the ability to scale up to produce large quantities of NK cells in a cost- and time-efficient manner. Thus, the present invention represents a significant advance in the field of cell therapy.

[0006] The iPSC-derived CD56+ / CD3- cells (also referred to herein as "iNK cells") produced by the methods described herein are functional and can be further genetically modified, such as through the introduction of a CAR, to target specific cell populations.

[0007] In some embodiments, a method for producing NK cells derived from pluripotent stem cells includes: (A) providing a bulk cell population (HP cell bulk) comprising hematopoietic progenitor cells (HPCs) derived from pluripotent stem cells; and (B) culturing the HP cell bulk in one or more culture media to produce CD56+ / CD3- cells, wherein the method does not include a cell isolation step.

[0008] In some embodiments, the method does not include a cell isolation step in steps (A) and (B).

[0009] In some embodiments, the HP cell bulk of (A) comprises CD34+ cells.

[0010] In some embodiments, 20% or more of the cells in the HP cell bulk are CD34+ cells.

[0011] In some embodiments, 20% to 90% of the HP cell bulk are CD34+ cells. For example, in some embodiments, about 30% of the HP cell bulk are CD34+ cells. In some embodiments, about 40% of the HP cell bulk are CD34+ cells. In some embodiments, about 50% of the HP cell bulk are CD34+ cells. In some embodiments, about 60% of the HP cell bulk are CD34+ cells. In some embodiments, about 70% of the HP cell bulk are CD34+ cells. In some embodiments, about 80% of the HP cell bulk are CD34+ cells. In some embodiments, about 90% of the HP cell bulk are CD34+ cells. In some embodiments, more than 70% of the HP cell bulk are CD34+ cells.

[0012] In some embodiments, step (B) comprises (i) culturing the HP cell bulk in a CD4 / CD8 induction medium to generate an intermediate heterogeneous cell population comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8-, and CD4+ / CD8+ cells, and (ii) culturing the intermediate heterogeneous cell population in an NK induction medium to produce CD56+ / CD3- cells.

[0013] In some embodiments, step (A) comprises culturing pluripotent stem cells in HPC induction medium to produce an HP cell bulk.

[0014] In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0015] In some embodiments, the HPC induction medium comprises at least one compound selected from bone morphogenetic protein-4 (BMP4), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), ascorbic acid, Flt3 ligand (Flt3L), thrombopoietin (TPO), and a TGFβ inhibitor, or any combination of compounds.

[0016] In some embodiments, the HPC induction medium contains BMP4 at a concentration of 5 ng / mL to 500 ng / ml.

[0017] In some embodiments, the BMP4 is at a concentration of 50 ng / ml.

[0018] In some embodiments, the HPC induction medium contains VEGF at a concentration of 5 ng / mL to 500 ng / ml.

[0019] In some embodiments, the VEGF is at a concentration of about 50 ng / ml.

[0020] In some embodiments, the HPC induction medium contains bFGF at a concentration of 5 ng / mL to 500 ng / ml.

[0021] In some embodiments, the bFGF is at a concentration of 50 ng / ml.

[0022] In some embodiments, the HPC induction medium contains ascorbic acid at a concentration of 5 μg / mL to 500 μg / ml.

[0023] In some embodiments, the ascorbic acid is at a concentration of 50 μg / ml.

[0024] In some embodiments, the HPC induction medium contains Flt3L at a concentration of 1 ng / mL to 100 ng / ml.

[0025] In some embodiments, Flt3L is at a concentration of 50 ng / ml.

[0026] In some embodiments, the HPC induction medium contains TPO at a concentration of 1 ng / mL to 200 ng / mL.

[0027] In some embodiments, the TPO is at a concentration of 100 ng / ml.

[0028] In some embodiments, the CD4 / CD8 induction medium comprises at least one compound or any combination of compounds selected from the group consisting of ascorbic acid, stem cell factor (SCF), IL-7, Flt3L, thrombopoietin (TPO), a p38 inhibitor, and SDF-1. Thus, in some embodiments, the CD4 / CD8 induction medium comprises ascorbic acid. In some embodiments, the CD4 / CD8 induction medium comprises SCF. In some embodiments, the CD4 / CD8 induction medium comprises IL-7. In some embodiments, the CD4 / CD8 induction medium comprises Flt3L. In some embodiments, the CD4 / CD8 induction medium comprises TPO. In some embodiments, the CD4 / CD8 induction medium comprises a p38 inhibitor. In some embodiments, the CD4 / CD8 induction medium comprises SDF-1. In some embodiments, the CD4 / CD8 induction medium comprises a p38 inhibitor and SDF-1.

[0029] In some embodiments, the CD4 / CD8 induction medium comprises ascorbic acid at a concentration of 5 μg / ml to about 500 μg / ml.

[0030] In some embodiments, the ascorbic acid is at a concentration of 50 μg / ml.

[0031] In some embodiments, the CD4 / CD8 induction medium comprises SCF at a concentration of 5 ng / mL to 100 ng / ml.

[0032] In some embodiments, the SCF is at a concentration of 50 ng / ml.

[0033] In some embodiments, the CD4 / CD8 induction medium comprises IL-7 at a concentration of 1 ng / mL to 100 ng / ml.

[0034] In some embodiments, the IL-7 is at a concentration of 50 ng / ml.

[0035] In some embodiments, the CD4 / CD8 induction medium comprises Flt3L at a concentration of 1 ng / mL to 100 ng / ml.

[0036] In some embodiments, the Flt3L is at a concentration of 50 ng / ml Flt3L.

[0037] In some embodiments, the CD4 / CD8 induction medium comprises TPO at a concentration of 1 ng / mL to 200 ng / ml.

[0038] In some embodiments, the TPO is at a concentration of 100 ng / ml.

[0039] In some embodiments, the CD4 / CD8 induction medium comprises a p38 inhibitor at a concentration of 0.5 μM to 100 μM.

[0040] In some embodiments, the p38 inhibitor is SB203580. In some embodiments, the p38 inhibitor is BIRB796. In some embodiments, the p38 inhibitor is VX-702. In some embodiments, the p38 inhibitor is SB239063. In some embodiments, the p38 inhibitor is SB202190. In some embodiments, the p38 inhibitor is BMS582949.

[0041] In some embodiments, SB203580 is at a concentration of 15 μM.

[0042] In some embodiments, the CD4 / CD8 induction medium comprises an SDF-1 inhibitor at a concentration of 10 ng / mL to about 100 ng / ml.

[0043] In some embodiments, the SDF-1 inhibitor is at a concentration of 30 nM.

[0044] In some embodiments, the SDF-1 inhibitor is at a concentration of 30 nM and the p38 inhibitor, eg, SB203580, is at a concentration of 15 μM.

[0045] In some embodiments, the NK induction medium comprises at least one compound selected from the group consisting of a CD3 activator, IL-2, and IL7. Thus, in some embodiments, the NK induction medium comprises a CD3 activator. In some embodiments, the NK induction medium comprises IL-2. In some embodiments, the NK induction medium comprises IL-7.

[0046] In some embodiments, the NK induction medium contains IL-2 at a concentration of 1 ng / mL to 100 ng / ml.

[0047] In some embodiments, the IL-2 is at a concentration of 10 ng / ml.

[0048] In some embodiments, the NK induction medium contains IL-7 at a concentration of 1 ng / mL to 100 ng / ml.

[0049] In some embodiments, the IL-7 is at a concentration of 10 ng / ml.

[0050] In some embodiments, each of the culturing steps is carried out at about 5% oxygen.

[0051] In some embodiments, each of the culturing steps is carried out at greater than 14% oxygen.

[0052] In some embodiments, each of the culturing steps is carried out in atmospheric oxygen.

[0053] In some embodiments, each of the culturing steps is carried out at less than 5% oxygen.

[0054] In some embodiments, culturing pluripotent stem cells in bulk cell medium to obtain an HP cell bulk continues for more than 10 days.

[0055] In some embodiments, culturing pluripotent stem cells in bulk cell medium to obtain HP cell bulk continues for 11 to 15 days.

[0056] In some embodiments, culturing the pluripotent stem cells in bulk cell medium to obtain an HP cell bulk continues for 14 days.

[0057] In some embodiments, the iPSCs are obtained from peripheral blood mononuclear cells.

[0058] In some embodiments, at least about 50%, 55%, 60%, 75%, 80%, 85%, 90%, 95%, 97% or more of the cells produced are CD56+ / CD3− cells without an enrichment step, e.g., without a further step of sorting / isolation / purification of CD56+ / CD3− cells. Thus, in some embodiments, at least about 50% or more of the cells produced are CD56+ / CD3− cells without an enrichment step. In some embodiments, at least about 55% or more of the cells produced are CD56+ / CD3− cells without an enrichment step. In some embodiments, at least about 60% or more of the cells produced are CD56+ / CD3− cells without an enrichment step. In some embodiments, at least about 65% or more of the cells produced are CD56+ / CD3− cells without an enrichment step. In some embodiments, at least about 70% or more of the cells produced are CD56+ / CD3− cells without an enrichment step. In some embodiments, at least about 75% or more of the cells produced are CD56+ / CD3- cells without an enrichment step. In some embodiments, at least about 80% or more of the cells produced are CD56+ / CD3- cells without an enrichment step. In some embodiments, at least about 85% or more of the cells produced are CD56+ / CD3- cells without an enrichment step. In some embodiments, at least about 90% or more of the cells produced are CD56+ / CD3- cells without an enrichment step. In some embodiments, at least about 95% or more of the cells produced are CD56+ / CD3- cells without an enrichment step. In some embodiments, at least about 97% or more of the cells produced are CD56+ / CD3- cells without an enrichment step.

[0059] In some embodiments, less than about 25%, 20%, 15%, 10%, or 5% of the cells produced are CD3+ cells.

[0060] In some embodiments, the phenotype of the produced cells can be confirmed by various means known in the art. For example, the phenotype of the produced cells can be confirmed by flow cytometry or single-cell RNA sequencing (scRNAseq).

[0061] In some embodiments, the percentage of cells produced is determined by flow cytometry. In some embodiments, the percentage of cells produced is determined by scRNAseq.

[0062] In some embodiments, the method further comprises isolating CD56+ / CD3- cells.

[0063] In some embodiments, CD56+ / CD3- cells are isolated by fluorescence-activated cell sorting (FACS) or magnetic sorting. Thus, in some embodiments, cells are isolated by FACS. In some embodiments, cells are isolated by magnetic sorting (MACS).

[0064] In some embodiments, the CD56+ / CD3- immune cells are NK cells.

[0065] In some embodiments, the CD56+ / CD3− cells are genetically modified to express one or more chimeric antigen receptors (CARs).

[0066] In some embodiments, the isolated CD56+ / CD3− cells are genetically modified to express one or more chimeric antigen receptors (CARs).

[0067] In some embodiments, the antigen is CD19.

[0068] In some embodiments, the cells are further genetically modified to express the IL-15Rα / IL-15 complex.

[0069] In some aspects, a method for producing CD56+ / CD3- immune cells from induced pluripotent stem cells (iPSCs) is provided, comprising: (1) culturing iPSCs in an HPC induction medium containing at least one compound selected from vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and ascorbic acid to obtain a heterogeneous cell population (HP cell bulk) containing hematopoietic progenitor cells (HPCs); (2) culturing the HP cell bulk obtained in (1) in a CD4 / CD8 induction medium containing one or more of ascorbic acid, a p38 inhibitor, and SDF-1 to obtain an intermediate heterogeneous cell population; and (3) culturing the intermediate heterogeneous cell population of (2) in an NK induction medium containing at least one compound selected from the group consisting of a CD3 activator, IL-2, and IL-7.

[0070] In some embodiments, a population of NK cells is provided, which is produced using the methods described herein.

[0071] In some embodiments, an unsorted cell population is provided, which comprises pluripotent stem cell-derived CD56+ / CD3- cells at a ratio of 60% or more of total pluripotent stem cell-derived CD56+ immune cells.

[0072] In some embodiments, less than 25% of the cells are CD3+ cells.

[0073] In some embodiments, less than 5% of the cells are monocytes.

[0074] In some embodiments, less than 5% of the cells are B cells.

[0075] In some aspects, a method of treating a subject in need of cell therapy is provided, the method comprising administering to the subject an NK cell described herein.

[0076] In some embodiments, the subject has cancer.

[0077] In some embodiments, the cancer is leukemia or lymphoma.

[0078] In some aspects, methods for producing CD56+ / CD3- immune cells derived from pluripotent stem cells are provided, the methods comprising: (I) culturing the pluripotent stem cells in one or more culture media to produce CD56+ / CD3- immune cells.

[0079] In some embodiments, step (I) comprises: (A) culturing pluripotent stem cells in a culture medium to produce hematopoietic progenitor cells (HPCs) (HP cell bulk); and (B) culturing the cells obtained in step (A) in a culture medium to produce CD56+ / CD3- immune cells.

[0080] In some embodiments, step (B) comprises: (a) culturing the cells obtained in step (A) in a culture medium to produce a cell population comprising CD4 / CD8 double positive cells; and (b) culturing the cells obtained in step (a) in a culture medium to produce CD56+ / CD3- immune cells.

[0081] In some embodiments, none of the previous steps include performing an isolation step of the cell population comprising CD4 / CD8 double positive cells.

[0082] In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0083] In some aspects, a method for producing CD56+ / CD3- immune cells is provided, the method comprising: (II) culturing cells, including hematopoietic progenitor cells (HPCs) (HP cell bulk), in a culture medium to produce CD56+ / CD3- immune cells.

[0084] In some embodiments, step (II) comprises: (X) culturing cells comprising hematopoietic progenitor cells (HPCs) in a culture medium to produce CD4 / CD8 double positive cells; and (Y) culturing the cells obtained in step (X) in a culture medium to produce CD56+ / CD3- immune cells.

[0085] In some embodiments, step (II) does not include performing a step of isolating CD4 / CD8 double positive cells.

[0086] In some embodiments, step (A) comprises a culture medium having at least one compound selected from bone morphogenetic protein-4 (BMP4), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), ascorbic acid, Flt3 ligand (Flt3L), thrombopoietin (TPO), and a TGFβ inhibitor.

[0087] In some embodiments, step (a) or step (X) comprises a culture medium having at least one compound selected from the group consisting of ascorbic acid, stem cell factor (SCF), IL-7, Flt3L, thrombopoietin (TPO), a p38 inhibitor, and SDF-1.

[0088] In some embodiments, step (b) or step (Y) comprises a culture medium having at least one compound selected from the group consisting of a CD3 activator, IL-2, and IL7.

[0089] In some embodiments, the method further comprises culturing the cells produced after step (b) or step (Y) in a culture medium comprising IL-7 and / or IL-15.

[0090] In some embodiments, the culturing is carried out at about 5% oxygen. In some embodiments, step (A) lasts for about 10 days, hi other embodiments, step (A) lasts for about 10-18 days.

[0091] In some embodiments, iPSCs are produced from peripheral blood monocytic cells.

[0092] In some embodiments, at least about 50%, 55%, 60%, 75%, 80%, 85%, or 90% of the cells produced are CD56+ / CD3- cells.

[0093] In some embodiments, less than about 25% of the cells are CD3+ cells.

[0094] In some embodiments, the method further comprises isolating CD56+ / CD3− cells.

[0095] In some embodiments, CD56+ / CD3- cells are isolated by fluorescence-activated cell sorting (FACS).

[0096] In some embodiments, the CD56+ immune cells are CD56+ / CD3-.

[0097] In some embodiments, any pluripotent, multipotent, or patient-derived HPCs can be used in the methods described herein. For example, in some embodiments, the cells are embryonic stem cells. In some embodiments, the cells are adult stem cells. Various adult stem cells are known in the art, including, for example, mesenchymal stem cells, hematopoietic stem cells, umbilical cord-derived cells, bone marrow stem cells, adipose stem cells, and the like. In some embodiments, the cells are induced pluripotent stem cells (iPSCs). Thus, NK cells produced according to the methods described herein can be generated from any pluripotent, multipotent, or patient-derived HPCs, such as primary HPCs derived directly from a donor.

[0098] In some embodiments, the cells used to produce the NK cells described herein are genetically modified at any stage of cell differentiation. In some embodiments, the cells used to produce the NK cells described herein are genetically modified at the pluripotent, multipotent, or unipotent stage. For example, in some embodiments, the cells used to produce the NK cells described herein are genetically modified at the pluripotent stage. For example, the cells can be genetically modified at the embryonic stem cell stage or the iPSC stem cell stage. In some embodiments, the cells used to produce the NK cells described herein are genetically modified at the multipotent stage. For example, the cells can be genetically modified at the HSC stage.

[0099] In some embodiments, the cells are genetically modified to express one or more chimeric antigen receptors (CARs).

[0100] In some embodiments, the isolated CD56+ / CD3− cells are genetically modified to express one or more chimeric antigen receptors (CARs).

[0101] In some embodiments, the antigen is CD19.

[0102] In some embodiments, the cells are further genetically modified to express the IL-15Rα / IL-15 complex.

[0103] In some embodiments, an unsorted cell population is provided, which comprises pluripotent stem cell-derived CD56+ / CD3- cells at a ratio of 60% or more of total pluripotent stem cell-derived CD56+ immune cells.

[0104] In some embodiments, less than 25% of the cells are CD3+ cells.

[0105] In some embodiments, less than 5% of the cells are monocytes.

[0106] In some embodiments, less than 5% of the cells are B cells.

[0107] In some embodiments, a method of treating a subject in need of cell therapy is provided, the method comprising administering to the subject the pluripotent stem cell-derived CD56+ / CD3- immune cells of any one of the preceding claims.

[0108] In some embodiments, the subject has cancer.

[0109] In some embodiments, the cancer is leukemia or lymphoma.

[0110] The methods described herein use a variety of culture media, including, for example, HPC induction medium, CD4 / CD8 induction medium, and NK induction medium.

[0111] In some aspects, induced pluripotent cells (iPSCs) are cultured in HPC induction medium for a period of time to generate a cell population comprising hematopoietic progenitor cells; this cell population is referred to herein as an HP cell bulk. In some embodiments, this period is about 10-14 days. In some embodiments, this period is about 13 days. In some embodiments, the HPC induction medium comprises one or more of BMP4, VEGF, bFGF, ascorbic acid, a TGFβ inhibitor, stem cell factor (SCF), thrombopoietin (TPO), and Flt3L. In some embodiments, the iPSCs are cultured in HPC induction medium comprising VEGF, bFGF, and ascorbic acid from day 1 to about day 10. In some embodiments, the VEGF is at a concentration of about 50 ng / mL. In some embodiments, the bFGF is at a concentration of about 50 ng / mL. In some embodiments, the ascorbic acid is at a concentration of about 50 μg / mL. In some embodiments, iPSCs are cultured in HPC induction medium containing BMP4 from day 1 to about day 3. In some embodiments, BMP4 is at a concentration of about 50 ng / mL. In some embodiments, iPSCs are cultured with a TGFβ inhibitor, such as SB431542, at a concentration of about 6 μM from about day 2 to about day 3. In some embodiments, one or more of stem cell factor (SCF), thrombopoietin (TPO), and Flt3L are added to the culture between days 7 and 13 of culture. In some embodiments, SCF is added at a concentration of about 50 ng / mL. In some embodiments, TPO is added at a concentration of about 30 ng / mL. In some embodiments, Flt3L is added at a concentration of about 10 ng / mL.

[0112] In some aspects, the HP cell bulk population is cultured in CD4 / CD8 induction medium for a period of time to generate a population of cells comprising an intermediate heterogeneous cell population comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8-, and CD4+ / CD8+ cells. In some embodiments, the HP cell bulk population is cultured in CD4 / CD8 induction medium for 19-22 days. In some embodiments, the HP cell bulk population is cultured in CD4 / CD8 induction medium for about 21 days. In some embodiments, the CD4 / CD8 induction medium comprises one or more of ascorbic acid, SCF, TPO, Flt3L, IL7, a p38 MAPK inhibitor such as SB203580, SDF1a, etc. In some embodiments, the ascorbic acid is at a concentration of about 50 μg. In some embodiments, the SCF is at a concentration of 50 ng / mL. In some embodiments, the TPO is at a concentration of about 100 ng / mL. In some embodiments, Flt3L is at a concentration of about 50 ng / mL. In some embodiments, IL7 is at a concentration of about 50 ng / mL. In some embodiments, SB203580 is at a concentration of about 15 μM. In some embodiments, SDF1a is at a concentration of about 30 nM. In some embodiments, HP cell bulk is cultured on a culture dish coated with hDLL4 / RetroNectin.

[0113] In some embodiments, the CD4 / CD8 induction medium comprises at least one compound selected from the group consisting of ascorbic acid, stem cell factor (SCF), IL-7, Flt3L, thrombopoietin (TPO), a p38 inhibitor, and SDF-1. In some embodiments, the CD4 / CD8 induction medium comprises a p38 inhibitor. In some embodiments, the CD4 / CD8 induction medium comprises SDF-1. In some embodiments, the CD4 / CD8 induction medium comprises a p38 inhibitor and SDF-1.

[0114] In some aspects, a heterogeneous cell population comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8-, and CD4+ / CD8+ cells is cultured in NK induction medium for a period of time to generate a cell population comprising CD56+ / CD3- NK cells. In some embodiments, the heterogeneous cell population is cultured in NK induction medium for a period of about 5-9 days. In some embodiments, the cell population is cultured for about 7 days. In some embodiments, the NK cell induction medium comprises one or more of IL-7, IL-2, and an anti-CD3 antibody. In some embodiments, the heterogeneous cell population is cultured in NK cell induction medium comprising IL-7 and IL-2 for about 7 days. In some embodiments, the IL-7 is at a concentration of about 10 ng / mL. In some embodiments, the IL-2 is at a concentration of about 10 ng / mL. In some embodiments, the heterogeneous population is cultured in NK cell induction medium comprising anti-CD3 for about 3 days. In some embodiments, the method generates greater than 50%, 60%, 70%, 80%, 90%, or 95% CD56+ / CD3- cells. Thus, in some embodiments, the method generates greater than 50% CD56+ / CD3- cells. In some embodiments, the method generates greater than 60% CD56+ / CD3- cells. In some embodiments, the method generates greater than 70% CD56+ / CD3- cells. In some embodiments, the method generates greater than 80% CD56+ / CD3- cells. In some embodiments, the method generates greater than 90% CD56+ / CD3- cells. In some embodiments, the method generates greater than 95% CD56+ / CD3- cells. [Brief explanation of the drawings]

[0115] [Figure 1]

[0023] Figure 1 is a series of flow cytometry plots showing the results of flow cytometry analysis of NK cell bulk populations obtained using the methods described herein. Briefly, iPS cells were cultured as described herein without an intermediate isolation step. The flow cytometry graphs show the presence of a population of CD3-negative cells that are CD56high (approximately 63.5% of the NK cell bulk population) and a population of CD3-negative cells that are CD56dim (approximately 26.7% of the NK cell bulk population). Both of these populations are NK cells. Thus, the total CD56-positive, CD3-negative NK cell population obtained represents more than 90% of the total cell population. [Figure 2]

[0023] Figure 1 shows the results of scRNA analysis of NK cell bulk populations obtained using the methods described herein. Briefly, iPS cells were cultured as described herein without an intermediate isolation step. Cells were sorted into four cell populations: monocytes, B cells, NK cells, and T cells. Approximately 75% of the cells in the NK cell bulk population were identified as NK cells (CD56+ / CD3- cells), and approximately 25% of the cells in the bulk cell population were identified as T cells. T cells as described herein may include NKT cells (CD56+ / CD3+ cells). [Figure 3] This is a series of photographs showing the results of an antitumor activity assay in NSG (NOD / Shi-scid, IL-2R gamma null) mice injected with luciferase-expressing Nalm6 cells followed by iNK-CAR19 cells. NSG mice implanted with Nalm6 cells were treated with (a) PBS buffer or (b) iNK-CAR cells, and the antitumor effects of these treatments were observed. The data show that iNK-CAR cells reduced the proliferation of Nalm6 cells.

[0116] [Detailed Description of the Invention] definition Administration: As used herein, the terms "administer," "administering," "administration," "introducing," or "introduction" are used interchangeably in the context of delivering therapeutic cells, e.g., iPSC- or HPC-derived CD56+ / CD3- immune cells, to a subject by a method or route that results in delivery of such cells. Various methods for administering cells are known in the art, e.g., intravenously, topically, orally, intramuscularly, intraperitoneally, intrathecally, subcutaneously, or transdermally. Cells may be administered with or without a carrier.

[0117] Adoptive cell therapy: As used interchangeably herein, the terms "adoptive cell therapy" or "adoptive cell transfer" or "cell therapy" or "ACT" refer to the transplantation of cells, e.g., a population of CD56+ / CD3- cells, generated using the methods described herein and administered to a subject or patient in need. In some embodiments, the cells are CD56+ / CD3- immune cells generated using the methods described herein and further expressing a CAR.

[0118] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human, at any stage of development. In some embodiments, "animal" refers to a non-human animal, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0119] Antigen-specific targeting domain: An "antigen-specific targeting domain" confers on a CAR the ability to bind to a target antigen of interest. In some embodiments, the antigen-specific targeting domain targets an antigen of clinical interest, where it would be desirable to elicit an effector immune response that results in tumor killing. An antigen-specific targeting domain can be any protein or peptide that possesses the ability to specifically recognize and bind to a biomolecule (e.g., a cell surface receptor or tumor protein, or component thereof). Antigen-specific targeting domains include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biomolecule of interest.

[0120] Exemplary antigen-specific targeting domains include, for example, antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands of cell surface molecules / receptors or receptor-binding domains thereof, and tumor-binding proteins.

[0121] In some embodiments, the antigen-specific targeting domain is an antibody or is derived from an antibody. An antibody-derived targeting domain can be an antibody fragment or a genetically engineered product of one or more fragments of an antibody, which fragment is responsible for binding to the antigen. Examples include a variable region (Fv), a complementarity-determining region (CDR), a Fab, a single-chain antibody (scFv), a heavy chain variable region (VH), a light chain variable region (VL), and a camelid antibody (VHH).

[0122] In some embodiments, the binding domain is a single chain antibody (scFv). The scFv can be murine, human, or humanized scFv.

[0123] Allogeneic: As used herein, "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to one another if the genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently genetically different to interact antigenically.

[0124] Approximately or about: As used herein, the term "approximately" or "about," as applied to one or more values ​​of interest, refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about," unless otherwise specified or otherwise clear from the context, refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of any of the indicated reference values ​​(above or below), except where this number exceeds 100% of possible values. When the term "about" or "approximately" is used to modify a stated reference value, it is understood that the stated reference value itself is encompassed, along with values ​​close to the stated reference value on either side of the stated reference value.

[0125] Ascorbic Acid or Vitamin C: As used herein, "ascorbic acid" or "vitamin C" refers to L-ascorbic acid and its derivatives, and "L-ascorbic acid derivatives" refers to derivatives that undergo enzymatic reaction in vivo to become vitamin C. Examples of L-ascorbic acid derivatives include vitamin C phosphate, ascorbic acid glucoside, ascorbyl ethyl, vitamin C ester, ascorbyl tetrahexyldecanoate, ascorbyl stearate, and ascorbyl 2-phosphate 6-palmitate. Examples of vitamin C phosphate include salts of L-ascorbic acid phosphate, such as L-ascorbic acid phosphate Na and L-ascorbic acid phosphate Mg. In one embodiment, vitamin C can be ascorbic acid 2-phosphate.

[0126] Bulk cell population: The terms "bulk cell population," "bulk cells," and the like refer to a heterogeneous cell population. In some embodiments, the bulk cell population comprises hematopoietic cells. In some embodiments, the bulk cell population can be obtained from pluripotent cells, e.g., induced pluripotent stem cells. In some embodiments, the bulk cell population is obtained from donor tissue, e.g., blood, etc.

[0127] CD4 / CD8 induction medium: As used herein, the term CD4 / CD8 induction medium refers to a cell culture medium used to obtain a population of cells including CD4- / CD8- cells, CD4+ / CD8+ cells, CD4+ / CD8- cells, and CD4- / CD8+ cells. In some embodiments, the CD4 / CD8 induction medium is used to differentiate HP cell bulk into cell populations including CD4- / CD8- cells, CD4+ / CD8+ cells, CD4+ / CD8- cells, and CD4- / CD8+ cells. In some embodiments, the CD4 / CD8 induction medium includes one or more or all of ascorbic acid, SCF, TPO, Flt3L, IL7, a p38MAPKi inhibitor such as SB203580, SDF1a, etc.

[0128] Chimeric Antigen Receptor (CAR): As used herein, the term "chimeric antigen receptor" or "CAR" refers to a receptor that can confer antigen specificity to cells (e.g., NK cells, T cells such as naive T cells, central memory T cells, effector memory T cells, or a combination thereof). CARs are also known as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some embodiments, the CARs of the present invention comprise an antigen-specific targeting domain, an extracellular domain, a transmembrane domain, optionally one or more costimulatory domains, and an intracellular signaling domain. In some embodiments described herein, the CAR is introduced into CD56+ / CD3- immune cells (e.g., NK or NK-like cells) generated using the methods described herein to redirect their specificity to a desired cell surface antigen or MHC-peptide complex. These synthetic receptors typically comprise a target-binding domain linked to one or more signaling domains via a flexible linker within a single fusion molecule. The target-binding domain is used to direct immune cells (e.g., CD56+ / CD3+ immune cells) to specific targets on the surface of diseased cells (e.g., cancer cells), while the signaling domain contains the molecular machinery for activating and expanding immune cells (e.g., CD56+ / CD3- immune cells). Typically, a flexible linker that passes through the immune cell (e.g., CD56+ / CD3- cell) membrane (i.e., forming a transmembrane domain) allows the target-binding domain of the CAR to be displayed on the cell membrane. CARs have been successfully used to redirect immune cells to antigens expressed on the surface of tumor cells in various malignancies, such as lymphomas and solid tumors (Gross et al., (1989) Transplant Proc., 21(1 Pt 1):127-30; Jena et al., (2010) Blood, 116(7):1035-44). The extracellular binding domain of a CAR may be composed of a single-chain variable fragment (scFv) derived from a fusion of the variable heavy and light chain regions of a murine or humanized monoclonal antibody. In some embodiments, the extracellular binding domain comprises a single-domain antibody.Alternatively, scFv derived from Fabs (e.g., rather than from antibodies obtained from a Fab library) may be used. In various embodiments, the scFv is fused to a transmembrane domain and then to an intracellular signaling domain. At least three generations of CARs have been developed. First-generation CARs consisted of a target-binding domain attached to a signaling domain derived from the cytoplasmic region of CD3 zeta or the gamma chain of the Fc receptor. First-generation CARs were shown to successfully redirect immune cells to selected targets, but failed to confer long-term expansion and anti-tumor activity in vivo. Second- and third-generation CARs have focused on enhancing the survival and proliferation of engineered cells by including costimulatory molecules such as CD28, OX-40 (CD134), and 4-1BB (CD137).

[0129] Culture: The terms "culture" or "cell culture" or "culturing" refer to the maintenance, growth, and / or differentiation of cells in an in vitro environment. In various methods described herein, cells are cultured in a particular cell culture medium(s) that facilitates or promotes the growth or differentiation of one type of cell into a different type of cell. For example, in certain embodiments described herein, culturing iPSCs in cell culture medium results in at least 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the cells in the total cell population becoming CD34+ cells (i.e., HPCs). In some embodiments described herein, at least 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the 30% HPCs result in a cell population that includes CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8-, and CD4+ / CD8+ cells. In yet another embodiment, culturing a cell population comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8-, and CD4+ / CD8+ cells results in an enriched CD56+ / CD3- cell population (i.e., at least 50% of the cells in the total cell population are CD56+ / CD3-). The cell culture medium serves as a source of nutrients, hormones, and / or other factors that aid in the propagation and / or maintenance of the cells.

[0130] Differentiating: The terms "differentiating," "inducing," "converting," "deriving," and the like refer to the process by which cells of one phenotype change into cells of another phenotype.

[0131] Engineered: As used herein, the term "engineered" refers to a polynucleotide, polypeptide, or cell that has been designed or altered and whose presence and production requires intervention and / or activity. For example, an engineered cell is purposefully designed to elicit a particular effect and that is different from the effect of a naturally occurring cell of the same species. In some embodiments, the engineered cell is a CD56+ / CD3- cell derived from iPSCs or HPCs using the methods described herein and further expresses a chimeric antigen receptor.

[0132] Enriched: As used herein, the term "enriched" with respect to a particular cell type refers to a cell population having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% of a particular cell type within the cell population as determined by flow cytometry or other analytical methods.

[0133] Ex vivo: As used herein, the term "ex vivo" refers to a process in which cells are removed from an organism and grown outside of the organism (e.g., in a test tube, in a culture bag, in a bioreactor).

[0134] Functional equivalent or derivative: As used herein, the term "functional equivalent" or "functional derivative," in the context of a functional derivative of an amino acid sequence, refers to a molecule that retains substantially similar biological activity (either function or structure) as the original sequence. Functional derivatives or equivalents can be natural derivatives or are synthetically prepared. Exemplary functional derivatives include amino acid sequences with one or more amino acid substitutions, deletions, or additions, provided that the biological activity of the protein is preserved. The substituted amino acids desirably have similar chemical and physical properties to the substituted amino acids. Desirable similar chemical and physical properties include charge similarity, bulkiness, hydrophobicity, hydrophilicity, etc.

[0135] Hematopoietic progenitor cells: The term "hematopoietic progenitor cell(s)" or "HPC(s)" refers to CD34+ cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation and include hematopoietic stem cells, multipotent hematopoietic stem cells, common myeloid progenitors, megakaryocytic progenitors, erythroid progenitors, and lymphoid progenitors.

[0136] HPC bulk: The terms "HPC bulk," "hematopoietic progenitor cell bulk," or "HP cell bulk" refer to a heterogeneous cell population comprising hematopoietic progenitor cells. In some embodiments, the HPC bulk is derived from iPSCs. In some embodiments, the HPC bulk is derived from blood.

[0137] HPC-derived NK cells: The term "HPC-derived NK cells" refers to CD56+ / CD3- immune cells (e.g., NK or NK-like cells) obtained from an HPC bulk population after culture in cell culture medium.

[0138] HPC induction medium: As used herein, the term HPC induction medium refers to a culture medium used to produce a cell population comprising hematopoietic cells from a starting cell population. In some embodiments, the starting cell population is iPSCs. In some embodiments, the HPC induction medium comprises one or more of BMP4, VEGF, bFGF, ascorbic acid, a TGFβ inhibitor, stem cell factor (SCF), thrombopoietin (TPO), and Flt3L.

[0139] Immune cell: As used herein, the term "immune cell(s)" refers to cells of the immune system, such as T cells, NK cells, T / NK cells, dendritic cells, macrophages, B cells, neutrophils, erythrocytes, monocytes, basophils, neutrophils, mast cells, neutrophils, and any combination thereof. In various embodiments, the immune cells produced using the methods described herein are NK cells and are characterized as CD56+ / CD3- cells.

[0140] Induced pluripotent stem cells (iPSCs): As used herein, the term "induced pluripotent stem cells" or "iPSCs" refers to pluripotent stem cells that are artificially derived (e.g., induced) from non-pluripotent cells, typically adult cells, by inducing expression of a combination of one or more genes, such as POU4F1 / OCT4 (Gene ID; 5460) and, but not limited to, SOX2 (Gene ID; 6657), KLF4 (Gene ID; 9314), cMYC (Gene ID; 4609), NANOG (Gene ID; 79923), and LIN28 / LIN28A (Gene ID; 79727). Stem cells may be genetically modified at any stage with a marker or gene such that the marker or gene is retained through any stage of culture. Markers may also be used to purify or enrich differentiated or undifferentiated stem cell populations at any stage of culture.

[0141] Induction Medium: The term "induction medium" generally refers to a cell culture medium used to differentiate a population of cells from a first cell phenotype to a second cell phenotype. In some embodiments, the first cell phenotype and the second cell phenotype comprise a heterogeneous cell population. In some embodiments, the first cell phenotype comprises a heterogeneous cell population. In some embodiments, the second cell phenotype comprises a heterogeneous cell population. In some embodiments, the induction medium is used to differentiate a heterogeneous cell population into a population of cells that is substantially homogeneous.

[0142] In vitro: As used herein, the term "in vitro" refers to events that take place in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than inside a multicellular organism.

[0143] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (e.g., as opposed to in vitro systems).

[0144] Isolation step: As used herein, the term "isolation step" or "cell isolation step" refers to a step of separating a specific cell type from a mixture of cells. Various methods for separating a specific cell type from a mixture of cells are known in the art, including, for example, magnetic bead-based sorting strategies such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS).

[0145] Natural Killer (NK) Cells: As used herein, "natural killer cells" or NK cells are lymphoid cells defined by their marker expression and function / activity. For example, in humans, NK cells express CD56. In further embodiments, such NK cells may express CD56 and CD16. In another example, such NK cells may be CD56+ / CD3- cells. NK cells may express varying levels of CD56. For example, NK cells may be "CD56 high ", meaning that the NK cells express high levels of CD56 when assessed by methods in the art, e.g., when assessed by flow cytometry. As another example, NK cells can be "CD56 dim ", which means that the NK cells express low but detectable levels of CD56 when assessed by methods in the art, for example, when assessed by flow cytometry.

[0146] NK induction medium: In some embodiments, the term "NK induction medium" refers to a cell culture medium used to generate a population of cells comprising CD56+ / CD3- cells. In some embodiments, the NK cell induction medium comprises one or more of IL-7, IL-2, and an anti-CD3 antibody.

[0147] iPS NK cells: As used herein, "iPS NK cells" are iPSC-derived NK cells, e.g., NK cells derived from iPSCs as starting material. Such iPS NK cells express CD56. In a further embodiment, such iPS NK cells may express CD56 and CD16. In another example, such iPS NK cells may express CD56 or may be CD3- (CD56+ / CD3-). iPS NK cells are also referred to herein as "iNK cells."

[0148] T cell: As used herein, a "T cell" is a lymphoid cell defined by its marker expression and function / activity. For example, in humans, T cells express CD3. CD56+ / CD3+ cells are known as NKT cells.

[0149] A "single chain Fv antibody" or "scFv" refers to an engineered antibody comprising a light chain variable region and a heavy chain variable region connected to each other either directly or via a peptide linker sequence.

[0150] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, a subject is a human. A subject can be a patient, which refers to a human who visits a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject is afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0151] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or condition. The disease can include cancer, such as lymphoma and leukemia.

[0152] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent (e.g., cell therapy) refers to an amount (e.g., a specific number of cells or a population of cells enriched for a specific percentage of cell(s) of a particular species or species) sufficient to treat, diagnose, prevent, and / or delay the onset of a symptom(s) of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to such a disease, disorder, and / or condition. Those skilled in the art will appreciate that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one dose. In some embodiments, the CD56+ / CD3- cells described herein are modified to express one or more transgenes. In some embodiments, the CD56+ / CD3- cells described herein are modified to express a chimeric antigen receptor, such as CD19. In some embodiments, approximately 100 million to 900 million CD56+ / CD3- cells described herein are administered to a subject in need thereof. In some embodiments, about 100 to 700 million CD56+ / CD3- cells described herein are administered to a subject in need thereof. In some embodiments, about 100 to 500 million CD56+ / CD3- cells described herein are administered to a subject in need thereof. In some embodiments, about 200 to 900 million CD56+ / CD3- cells described herein are administered to a subject in need thereof. In some embodiments, about 200 to 700 million CD56+ / CD3- cells described herein are administered to a subject in need thereof. In some embodiments, about 200 to 500 million CD56+ / CD3- cells described herein are administered to a subject in need thereof.

[0153] Treating: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, or prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of, one or more symptoms or characteristics of a particular disease, injury, and / or condition. Treatment may also be administered to subjects who do not show signs of disease and / or who show only early signs of disease, with the intent of reducing the risk of developing pathology associated with the disease.

[0154] The recitation herein of numerical ranges by endpoints includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4, and 5). It is also understood that all such numbers and fractions are presumed to be modified by the term "about."

[0155] Various aspects of the present invention are described in detail in the following sections. The use of sections is not intended to limit the present invention. Each section may be applicable to any aspect of the present invention. In this application, the use of "or" means "and / or" unless stated otherwise. As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0156] Various aspects of the present invention are described in detail in the following clauses. The use of clauses is not intended to limit the invention. Each clause may be applicable to any aspect of the present invention. In this application, the use of "or" means "and / or" unless stated otherwise. DETAILED DESCRIPTION OF THE INVENTION

[0157] The present disclosure provides a method for producing NK cells from pluripotent cells, such as iPS cells or hematopoietic progenitor cells (HPCs). NK cells produced from iPSCs are referred to herein as iPS-derived NK cells, iPS NK cells, or iNK cells. The present disclosure provides a cell culture method that can differentiate progenitor cells, such as iPSCs or HPCs, into NK cells or iPS NK cells with high efficiency without the need for an isolation step. The present disclosure also demonstrates that the resulting iPS NK cells are functional and can be further genetically modified, for example, through the introduction of a chimeric antigen receptor (CAR), useful for the treatment of various diseases or disorders, such as cancer.

[0158] Various methods for generating CD56+ / CD3- cells (NK cells) are described in further detail below.

[0159] A method for producing NK or iPS-derived NK cells from pluripotent cells without an isolation step In some embodiments, methods of producing NK cells or iPS-derived NK cells are provided, which do not require an isolation step.

[0160] Overview of culture methods The methods provided herein are capable of producing CD56+ / CD3- cells without any intervening isolation step. In some embodiments, the methods involve culturing pluripotent stem cells, such as iPSCs, in an HPC induction medium to obtain a population of cells comprising hematopoietic cells (HPCs) (HP cell bulk). The HP cell bulk population is then cultured in a CD4 / CD8 induction medium without an isolation step to obtain a cell population comprising CD4 / CD8 double-positive cells. In some embodiments, the cell population comprising CD4 / CD8 double-positive cells also comprises CD4+ / CD8- cells, CD4- / CD8- cells, and CD4- / CD8+ cells. Subsequently, during the culture period, the cells are cultured in an NK induction medium without an isolation step to obtain a population enriched for CD56+ / CD3- immune cells. Thus, in some embodiments, a method for producing CD56+ / CD3- cells comprises: 1) culturing pluripotent cells in HPC induction medium to obtain a population comprising hematopoietic cells (HP cell bulk); 2) culturing the HP cell bulk in CD4 / CD8 induction medium to obtain a population of cells comprising CD4+ / CD8- cells, CD4- / CD8- cells, and CD4- / CD8+ cells; and 3) culturing the population of cells comprising CD4+ / CD8- cells, CD4- / CD8- cells, and CD4- / CD8+ cells in NK induction for a period of time to obtain CD56+ / CD3- cells.

[0161] In some embodiments, a method for producing NK cells or iPS NK cells includes culturing cells (e.g., HP cell bulk) comprising hematopoietic progenitor cells to produce a cell population comprising CD4 / CD8 double-positive cells, followed by culturing the cell population in a medium for a subsequent culture period without an isolation step to obtain a population enriched in CD56+ / CD3- immune cells. In some embodiments, a method for producing pluripotent stem cell-derived CD56+ / CD3- immune cells includes culturing pluripotent stem cells in one or more media. In some embodiments, a method for producing CD56+ / CD3- immune cells includes culturing hematopoietic progenitor cells in one or more media. In some embodiments, a method for producing CD56+ / CD3- immune cells includes culturing a mixture or population of cells comprising CD4 / CD8 double-positive cells in one or more media. In some embodiments, the enriched population of CD56+ / CD3- immune cells can then be separated by any suitable method known in the art, such as, for example, FACS sorting or magnetic bead-based isolation techniques. In one embodiment, iPS cells can be differentiated into iPS NK cells in about 4 to 8 weeks, 5 to 7 weeks, or about 6 weeks. Thus, in some embodiments, iPS cells can be differentiated into NK cells in about 4 to 8 weeks. In some embodiments, iPS cells can be differentiated into NK cells in about 5 to 7 weeks. In some embodiments, iPS cells can be differentiated into NK cells in about 6 weeks.

[0162] The cell differentiation process can be assessed by various means known in the art. For example, in some embodiments, cell differentiation of cultured cells can be assessed by obtaining a sample of the cultured cells and subjecting the sample to one or more analytical methods to determine the cell phenotype of the cells. Known methods for determining cell phenotype include, for example, flow cytometry and immunofluorescence imaging. Any suitable sampling and phenotypic assay can be used with the cell culture methods described herein to determine the progress of the cell differentiation process. In some embodiments, the methods described herein include one or more sampling steps to determine the cell phenotype at a given time.

[0163] Overview from iPSC to HP cell bulk In some embodiments, pluripotent cells, such as iPSCs, are cultured in an HPC induction medium to produce a population comprising an HP cell bulk. In some embodiments, culturing iPSCs in an HPC induction medium to obtain an HPC cell bulk comprises culturing iPSCs in an HPC induction medium. The HPC induction medium can comprise various components that enable the production of an HP cell bulk. In some embodiments, the HPC induction medium comprises at least one compound selected from bone morphogenetic protein-4 (BMP4), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), ascorbic acid, a ROCK inhibitor, a GSK3 inhibitor, stem cell factor (SCF), thrombopoietin (TPO), Flt3L, and a TGFβ inhibitor. Thus, in some embodiments, the HPC induction medium for inducing HPCs from iPSCs comprises BMP4. In some embodiments, the culture medium for inducing HPCs from iPSCs comprises VEGF. In some embodiments, the culture medium for inducing HPCs from iPSCs comprises bFGF. In some embodiments, the culture medium for inducing HPCs from iPSCs comprises TGFβ.

[0164] The iPSCs are cultured in the HPC induction medium for a period sufficient to produce a HP cell bulk. In some embodiments, the iPSCs are cultured in the HPC induction medium for about 7-21 days, or 10-18 days, or about 14 days. In some embodiments, the iPSCs are cultured in the HPC induction medium for about 13 days.

[0165] In some embodiments, the cells are cultured in hypoxic conditions, such as, for example, about 3%, 4%, 5%, or 6% O. Thus, in some embodiments, the cells are cultured at about 3% O. Thus, in some embodiments, the cells are cultured at about 4% O. In some embodiments, the cells are cultured at about 5% O. In some embodiments, the cells are cultured at about 6% O.

[0166] Overview of HP cell bulk to CD4 / CD8 In some embodiments, HPC cell bulks, or HPCs obtained by other methods, such as primary HPCs isolated from a human donor or HPCs differentiated from other stem cell sources / species, such as from embryonic stem cells, are further cultured in medium without any intervening isolation step to obtain a population of cells comprising CD4 / CD8 double-positive (DP) cells. In some embodiments, HPC cell bulks, or HPCs obtained by other methods, such as primary HPCs isolated from a human donor or HPCs differentiated from other stem cell sources / species, such as from embryonic stem cells, are further cultured in medium without any intervening isolation step to obtain a population of cells comprising CD4+ / CD8 cells. In some embodiments, HPC cell bulks, or HPCs obtained by other methods, such as primary HPCs isolated from a human donor or HPCs differentiated from other stem cell sources / species, such as from embryonic stem cells, are further cultured in medium without any intervening isolation step to obtain a population of cells comprising CD4- / CD8- cells. In some embodiments, bulk HPC cells or HPCs obtained by other methods, such as primary HPCs isolated from a human donor or HPCs differentiated from other stem cell sources / types, such as from embryonic stem cells, are further cultured in culture medium without any intervening isolation step to obtain a cell population comprising CD4- / CD8+ cells. It is understood that cell populations obtained by this method include CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells. Cell populations include, for example, lymphocytes. In some embodiments, the HPC cell bulk is cultured in a CD4 / CD8 induction medium containing at least one compound selected from the group consisting of ascorbic acid, stem cell factor (SCF), IL-7, Flt3L, TPO, fibronectin or a variant thereof, a Notch ligand (e.g., Jag-1, Jag-2, DLL-1, DLL-3, DLL-4), a p38 inhibitor, and SDF-1 to obtain populations comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells.Thus, in some embodiments, bulk HPC cells are cultured in CD4 / CD8 induction medium containing ascorbic acid to obtain populations comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells. In some embodiments, bulk HPC cells are cultured in CD4 / CD8 induction medium containing SCF to obtain populations comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells. In some embodiments, bulk HPC cells are cultured in CD4 / CD8 induction medium containing IL-7 to obtain populations comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells. In some embodiments, bulk HPC cells are cultured in CD4 / CD8 induction medium containing Flt3L to obtain populations comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells. In some embodiments, bulk HPC cells are cultured in CD4 / CD8 induction medium containing a p38 inhibitor to obtain populations comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells. In some embodiments, bulk HPC cells are cultured in CD4 / CD8 induction medium containing SDF-1 to obtain populations comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells.

[0167] The culture period is a period suitable for obtaining a population comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells. In some embodiments, culturing the HP cell bulk to obtain a population of cells comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells is about 2 to 6 weeks, 3 to 5 weeks, or about 3 weeks. Thus, in some embodiments, the cell culture period is about 2 to 6 weeks. In some embodiments, the cell culture period is about 3 to 5 weeks. In some embodiments, the cell culture period is about 3 weeks.

[0168] In some embodiments, the cells are cultured in hypoxic conditions, such as, for example, about 3%, 4%, 5%, or 6% O. Thus, in some embodiments, the cells are cultured in hypoxic conditions, such as, for example, about 3% O. In some embodiments, the cells are cultured in hypoxic conditions, such as, for example, about 4% O. In some embodiments, the cells are cultured in hypoxic conditions, such as, for example, about 5% O. In some embodiments, the cells are cultured in hypoxic conditions, such as, for example, about 6% O.

[0169] Overview from CD4 / CD8 cells to CD56+ / CD3- cells To obtain a population of CD56+ / CD3− cells, the population of cells comprising CD4− / CD8− cells, CD4− / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8− cells can be further cultured in cell culture medium as described above. In some embodiments, the culture medium is an NK induction medium. In some embodiments, the NK induction medium comprises one or more of IL-7, IL-2, and an anti-CD3 antibody.

[0170] In some embodiments, populations of cells comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells are further cultured in culture medium without an intervening isolation step to obtain a population of cells enriched for CD56+ / CD3- cells. Such CD56+ cells include, for example, natural killer (NK) cells. In some embodiments, populations comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells are cultured in medium comprising a CD3 activator, IL-2, and / or IL-7. Thus, in some embodiments, populations comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells are cultured in medium comprising a CD3 activator. CD3 activators are known in the art and include, for example, antibody complexes that bind to CD3 and / or CD28 surface ligands. The CD3 activator may be, for example, an anti-CD3 antibody or a fragment thereof. In some embodiments, when an anti-CD3 antibody is used, the anti-CD3 antibody may be a polyclonal or monoclonal antibody. In some embodiments, the anti-CD3 antibody is a polyclonal antibody. In some embodiments, the anti-CD3 antibody is a monoclonal antibody. The antibody may belong to any immunoglobulin class, including IgG, IgA, IgM, IgD, IgE, or IgG. Various types of anti-CD3 antibodies may be used, including, for example, antibodies produced from the OKT3 clone or the UCHT1 clone. The concentration of the anti-CD3 antibody in the culture medium is, for example, 10 ng / ml to 1000 ng / ml.

[0171] In some embodiments, a population of cells comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells is cultured in a medium comprising a CD3 activator, IL-2. In some embodiments, a population of cells comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells is cultured in a medium comprising IL-7. In some embodiments, the cells are cultured in hypoxic conditions, for example, at about 3%, 4%, 5%, or 6% O2. Thus, in some embodiments, the cells are cultured at about 3% O2. Thus, in some embodiments, the cells are cultured at about 4% O2. Thus, in some embodiments, the cells are cultured at about 5% O2. In some embodiments, the cells are cultured at about 6% O2.

[0172] The cell population comprising CD56+ cells can be further cultured in NK induction medium to enrich for CD56+ / CD3- cells. In some embodiments, the cell population comprising CD56+ cells is cultured in NK induction medium containing IL-7 and / or IL-15 to further enrich for CD56+ cells. Thus, in some embodiments, the cell population comprising CD56+ cells is cultured in NK induction medium containing IL-7. In some embodiments, the cell population comprising CD56+ cells is cultured in NK induction medium containing IL-15. In some embodiments, the cells are cultured under hypoxic conditions, e.g., about 3%, 4%, 5%, or 6% O2. In some embodiments, the CD56+ / CD3- cells produced are NK cells. The percentage of CD56+ / CD3- NK cells produced is at least about 50%, 55%, 60%, 75%, 80%, 85%, 90%, 95%, or greater than 95%. Thus, in some embodiments, the method results in at least about 50% CD56+ / CD3- NK cells. In some embodiments, the method results in at least about 55% CD56+ / CD3- NK cells. In some embodiments, the method results in at least about 60% CD56+ / CD3- NK cells. In some embodiments, the method results in at least about 65% CD56+ / CD3- NK cells. In some embodiments, the method results in at least about 70% CD56+ / CD3- NK cells. In some embodiments, the method results in at least about 75% CD56+ / CD3- NK cells. In some embodiments, the method results in at least about 80% CD56+ / CD3- NK cells. In some embodiments, the method results in at least about 85% CD56+ / CD3- NK cells. In some embodiments, the method results in at least about 90% CD56+ / CD3- NK cells. In some embodiments, the method results in at least about 95% CD56+ / CD3- NK cells. In some embodiments, the method results in greater than about 95% CD56+ / CD3- NK cells.

[0173] In some embodiments, 5%, 10%, 15%, 20%, 25%, or about 30% of the cells produced by this culture method are CD3+ T cells. In some embodiments, less than 5% of the cells produced are CD3+ T cells. In some embodiments, about 5% of the cells produced are CD3+ T cells. In some embodiments, about 10% of the cells produced are CD3+ T cells. In some embodiments, about 15% of the cells produced are CD3+ T cells. In some embodiments, about 20% of the cells produced are CD3+ T cells. In some embodiments, about 25% of the cells produced are CD3+ T cells. In some embodiments, about 30% of the cells produced are CD3+ T cells.

[0174] In some embodiments, the enriched population of CD56+CD3− cells is isolated by methods known in the art, including, for example, flow cytometry (FACS)-based sorting methods and magnetic-based sorting (MACS).

[0175] In some embodiments, the culture period for obtaining CD56+ / CD3- NK cells from a population comprising CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8+ cells, and CD4+ / CD8- cells is about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days. In some embodiments, the culture period is about 7 days.

[0176] In some embodiments, the enriched CD56+ cell population comprises NK cells. In some embodiments, the enriched CD56+ cells comprise CD3- cells. In some embodiments, the enriched CD56+ cells comprise CD3+ cells.

[0177] Pluripotent cells suitable for differentiation into CD56+ / CD3- cells In some embodiments, any pluripotent, multipotent, or patient-derived HPCs can be used in the methods described herein. For example, in some embodiments, the cells are embryonic stem cells. In some embodiments, the cells are adult stem cells. Various adult stem cells are known in the art, including, for example, mesenchymal stem cells, hematopoietic stem cells, umbilical cord-derived cells, bone marrow stem cells, adipose stem cells, and the like. In some embodiments, the cells are induced pluripotent stem cells (iPSCs). Thus, NK cells produced according to the methods described herein can be generated from any pluripotent, multipotent, or patient-derived HPCs, such as primary HPCs derived directly from a donor.

[0178] In some embodiments, pluripotent cells include, for example, embryonic stem (ES) cells, embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), germline stem cells ("GS cells"), embryonic germ cells ("EG cells"), iPS cells, and pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (muse cells). In some embodiments, iPS cells can be derived from peripheral blood mononuclear cells of a healthy individual. Methods for producing iPS cells are known in the art. These cells can be produced by introducing reprogramming factors into any somatic cell. Examples of reprogramming factors herein include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Kif, and the like.These reprogramming factors include genes and their gene products such as ERas, ECAT15-2, Tel1, beta-catenin, Lin28b, Sal1l, Sal14, Esrrb, Nr5a2, Tbx3, and Glis1. These reprogramming factors can be used individually or in combination of two or more. Examples of combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, and WO2009 / 126251. , WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 068 955, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D,et al.(2008),Nat.Biotechnol.,26;795-797,Shi Y,et al.(2008),Cell Stem Cell,2;525-528,Eminli S,et al.(2008),Stem Cells.26;2467-2474,Eluangfu D,et al. al.(2008),Nat.Biotechnol.26;1269-1275,Shi Y,et al.(2008),Cell Stem Cell,3,568-574,Zhao Y,et al.(2008),Cell Stem Cell,3;475-479,Marson A,(2008),Cell Stem Cell,3,132-135,FengB,et al.(2009),Nat.Cell Biol.11;197-203,RLJudson et al.,(2009),Nat.Biotechnol.,27;459-461,Lyssiotis CA,et al.(2009),Proc Natl Acad Sci US A.106;8912-8917,Kim J,et al. (2009), Nature.461;649-643, Ichida JK, et al. (2009), Cell Stem Cell.5;491-503, Heng JC, et al. (2010), Cell Stem Cell.6;167-74, Han J, et al. (2010), Nature.463;1096-100, Mali P,et al.(2010),Stem Cells. 28; 713-720, and Maekawa M, et al. (2011), Nature. 474; 225-9.

[0179] iPSCs can be obtained from any suitable tissue, hi some embodiments, iPSCs are obtained from peripheral blood mononuclear cells.

[0180] Hematopoietic progenitor cells (HPCs) are cells that can differentiate into blood cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, and megakaryocytes. Hematopoietic progenitor cells or stem cells can be identified, for example, based on the presence of CD34 and / or CD43 surface antigens.

[0181] In some embodiments, the cells used to produce the CD56+ / CD3- NK cells described herein are genetically modified at any stage of cell differentiation. In some embodiments, the CD56+ / CD3- NK cells are genetically modified to contain a desired chimeric antigen receptor (CAR), T cell receptor (TCR), or other engineered protein.

[0182] In some embodiments, the cells used to produce the CD56+ / CD3- NK cells described herein are genetically modified at the pluripotent, multipotent, or unipotent stage. For example, in some embodiments, the cells used to produce the CD56+ / CD3- NK cells described herein are genetically modified at the pluripotent stage. For example, the cells may be genetically modified at the embryonic stem cell stage or the iPSC stem cell stage. In some embodiments, the cells used to produce the CD56+ / CD3- NK cells described herein are genetically modified at the multipotent stage. For example, the cells may be genetically modified at the hematopoietic stem cell (HSC) stage.

[0183] Culture conditions - iPSC to HPC cell bulk In some embodiments, a medium for producing hematopoietic progenitor cells from iPSCs (i.e., HPC induction medium) can be prepared by adding vitamin C to a basal medium used for culturing animal cells. Examples of basal media include Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fisher's Medium, and Neurobasal Medium (Life Technologies), as well as mixtures of two or more of these media. In some embodiments, the medium contains serum. In some embodiments, the medium is serum-free.

[0184] In some embodiments, the HPC induction medium may include StemPro™-34, which is a serum-free medium formulated to support the development of human hematopoietic cells in culture.

[0185] Optionally, in some embodiments, the HPC induction medium may also contain one or more substances such as albumin, human insulin, human transferrin, selenium or sodium selenate, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, α-monothioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and cytokines.

[0186] In some embodiments, HPC induction medium comprises IMDM medium containing serum, insulin, transferrin, selenium, thiolglycerol or α-monothioglycerol, L-glutamine, and ascorbic acid.

[0187] In some embodiments, the HPC induction medium comprises one or more substances that induce signaling in the bone morphogenetic protein 4 (BMP4) signaling pathway, including, but not limited to, BMP4.

[0188] In some embodiments, the HPC induction medium comprises one or more substances that induce signaling in the vascular endothelial growth factor (VEGF) signaling pathway, including, but not limited to, VEGF.

[0189] In some embodiments, the HPC induction medium comprises one or more substances that induce fibroblast growth factor (FGF) pathway signaling, including, but not limited to, bFGF and FGF2.

[0190] In some embodiments, the HPC induction medium comprises one or more substances that induce signaling in the stem cell factor / kit signaling pathway, including but not limited to SCF.

[0191] In some embodiments, the HPC induction medium comprises one or more substances that induce signaling in the Flt3-ligand signaling pathway, including but not limited to Flt3-ligand (Flt3L).

[0192] In some embodiments, the HPC induction medium comprises one or more substances that induce signaling in the thrombopoietin signaling pathway, including but not limited to TPO.

[0193] TGFβ inhibitors are small molecule inhibitors that interfere with signal transduction of the TGFβ family, and examples include SB431542, SB202190 (RK Lindemann et al., Mol. Cancer 2:20(2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, and LY580276 (Lilly Research Laboratories).

[0194] SB431542 is a potent and specific inhibitor of the transforming growth factor beta (TGFβ) superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7.

[0195] For example, when the TGFβ inhibitor is SB431542, its concentration in the medium is preferably 0.5 μM to 100 μM.

[0196] The HPC induction medium for producing HP cell bulk populations may be further supplemented with cytokine(s) selected from the group consisting of BMP4 (bone morphogenetic protein 4), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), SCF (stem cell factor), TPO (thrombopoietin), and Flt3L (Flt3 ligand).

[0197] In one embodiment, the HPC induction medium may include StemPro34 supplemented with human insulin (about 10 μg / ml), human transferrin (about 5.5 μg / ml), sodium selenite (about 6.7 ng / ml), L-glutamine (about 2 mM), α-monothioglycerol (about 0.4 mM), and SB431542 (about 6 μM).

[0198] In some embodiments, vitamin C can be added every 4 days, every 3 days, every 2 days, or every day during the culture period. Vitamin C can be added to the medium in an amount equivalent to about 5 μg / ml to about 500 μg / ml. In some embodiments, vitamin C is present in the medium at about 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, or 500 μg / ml.

[0199] As used herein, "vitamin C" refers to L-ascorbic acid and its derivatives, and "L-ascorbic acid derivatives" refer to derivatives that become vitamin C through an enzymatic reaction in vivo. Examples of L-ascorbic acid derivatives include vitamin C phosphate (e.g., ascorbic acid 2-phosphate), ascorbic acid glucoside, ascorbyl ethyl, vitamin C ester, ascorbyl tetrahexyldecanoate, ascorbyl stearate, and ascorbyl 2-phosphate 6-palmitate. Vitamin C phosphate is preferred. Examples of vitamin C phosphate (e.g., ascorbic acid 2-phosphate) include salts of L-ascorbic acid phosphate, such as L-ascorbic acid phosphate Na and L-ascorbic acid phosphate Mg.

[0200] In some embodiments, when the substance that causes signaling in the bone morphogenetic protein 4 (BMP4) signaling pathway is BMP4, the concentration of BMP4 in the HPC induction medium for producing hematopoietic progenitor cells is about 5 ng / ml to 500 ng / ml, for example, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, 300 ng / ml, 350 ng / ml, 400 ng / ml, 450 ng / ml or 500 ng / ml.

[0201] In some embodiments, when the substance that causes signal transduction of the vascular endothelial growth factor (VEGF) signaling pathway is VEGF, the concentration of VEGF in the HPC induction medium for producing hematopoietic progenitor cells is about 5 ng / ml to 500 ng / ml, for example, about 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, 300 ng / ml, 350 ng / ml, 400 ng / ml, 450 ng / ml, or 500 ng / ml.

[0202] In some embodiments, when the substance that induces signal transduction in the fibroblast growth factor (FGF) signaling pathway is bFGF, the concentration of bFGF in the HPC induction medium for producing hematopoietic progenitor cells is about 5 ng / ml to 500 ng / ml, for example, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, 300 ng / ml, 350 ng / ml, 400 ng / ml, 450 ng / ml, or 500 ng / ml.

[0203] In some embodiments, when the substance that induces signal transduction of the stem cell factor / kit signaling pathway is SCF, the concentration of SCF in the HPC induction medium for producing hematopoietic progenitor cells is about 5 ng / ml to 100 ng / ml, e.g., 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml.

[0204] In some embodiments, when the substance that causes signal transduction of the Flt3 ligand signaling pathway is Flt3L, the concentration of Flt3L in the HPC induction medium for producing hematopoietic progenitor cells is about 1 ng / ml to 100 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 20 ng / ml, 50 ng / ml, or 100 ng / ml.

[0205] In some embodiments, when the substance that induces signal transduction in the thrombopoietin signaling pathway is TPO, the concentration of TPO in the HPC induction medium for producing hematopoietic progenitor cells is about 1 ng / ml to 200 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100, 125 ng / ml, 150 ng / ml, 175 ng / ml, or 200 ng / ml.

[0206] In some embodiments, pluripotent stem cells can be cultured in adherent or suspension culture. In adherent culture, the culture may be performed in a culture vessel coated with a coating agent and / or co-cultured with other cells. Examples of other cells for co-culture include C3H10T1 / 2 (Takayama N., et al. J Exp Med. 2817-2830, 2010) and stromal cells derived from different species (Niwa A et al. J Cell Physiol. 2009 Nov;221(2):367-77). Examples of coating agents include Matrigel (Nivea A, et al. PLoS One. 6(7):e22261, 2011), iMatrix511 (Miyazaki T, et al. Nature Communication 2012;3:1236), gelatin, collagen, elastin, glycosaminoglycans and proteoglycans such as hyaluronic acid and chondroitin sulfate, and cell adhesion proteins such as fibronectin or its modifications, vitronectin, laminin, etc. Examples of suspension culture methods include those described in Chadwick et al. Blood 2003, 102:906-15, Vijayaragavan et al. Cell Stem Cell 2009, 4:248-62, and Saeki et al. Stem Cells 2009, 27:59-67.

[0207] In some embodiments, HP cell bulks can also be prepared from net-like structures (also called ES-sacs or iPS-sacs) obtained by culturing pluripotent stem cells. The term "net-like structure" as used herein refers to a three-dimensional sac-like structure (with internal space) derived from pluripotent stem cells. This structure is formed within an endothelial cell population and contains hematopoietic progenitor cells.

[0208] In some embodiments, the temperature conditions for culturing to produce HP cell bulks are about 37°C to about 42°C. In some embodiments, the temperature is, for example, about 37°C to about 42°C, preferably about 37°C to about 39°C. The culture period can be appropriately determined by one skilled in the art by obtaining samples from the cell culture for phenotypic analysis, such as monitoring the number of hematopoietic progenitor cells. Various methods for phenotypic analysis of cell samples are known in the art, including, for example, flow cytometry and immunofluorescence.

[0209] The culture period for obtaining HP cell bulk can vary, for example, from 6 to 14 days. Examples of culture periods include at least 6 days, 7 or more days, 8 or more days, 9 or more days, 10 or more days, 11 or more days, 12 or more days, 13 or more days, and 14 or more days. In some embodiments, the culture period is 6 days. In some embodiments, the culture period is 7 days. In some embodiments, the culture period is 8 days. In some embodiments, the culture period is 9 days. In some embodiments, the culture period is 10 days. In some embodiments, the culture period is 10 days. In some embodiments, the culture period is 11 days. In some embodiments, the culture period is 12 days. In some embodiments, the culture period is 13 days. In some embodiments, the culture period is 14 days. In some embodiments, the culture period is longer than 14 days. The culture can be performed under hypoxic conditions. Examples of hypoxic conditions include 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or less. The frequency of medium changes can be determined by one skilled in the art. In some embodiments, the medium can be changed every two days. In other embodiments, the medium can be changed every three days. In some embodiments, the medium is changed daily.

[0210] In some embodiments, culturing to produce HP cell bulks is carried out by combining one or more of the above conditions. For example, in some embodiments, a method for obtaining HP cell bulks from iPSCs includes (i) culturing pluripotent stem cells on C3H10T1 / 2 in a basal medium supplemented with vitamin C under hypoxic conditions; and (ii) further supplementing the culture medium of (1) with VEGF, SCF, and Flt3L and culturing the cells under normal oxygen conditions. The period during which step (i) is carried out is at least 6 days or more, preferably 7 days or more, and more preferably 7 days. The period during which step (ii) is carried out is at least 6 days or more, preferably 7 days or more, and more preferably 7 days.

[0211] In some embodiments, the hematopoietic progenitor cells obtained in the HP cell bulk can be isolated before further use. In some other embodiments, the obtained hematopoietic progenitor cells can be used as a cell population (HP cell bulk) that also contains other cell types. The HP cell bulk population is an unseparated cell preparation. In some embodiments, the method does not include an isolation step.

[0212] Culture conditions - HP cell bulk to CD4 / CD8 cells In some embodiments, the CD4 / CD8 induction medium results in a cell population comprising CD4+ / CD8+, CD4- / CD8- cells, CD4- / CD8+ cells, and CD4+ / CD8- cells. "CD4 / CD8 double positive cells" (DP cells) refer to cells that express both CD4 and CD8. CD4 / CD8 double positive cells can be identified as cells that are CD4, CD8, CD3, and CD45 positive. "CD4 / CD8 double negative cells" (DN cells) refer to cells that do not express both CD4- and CD8-. "CD4- / CD8+ cells" refer to cells that do not express CD4- but express CD8+. "CD4+ / CD8- cells" refer to cells that express CD4 but do not express CD8.

[0213] In some embodiments, cell populations comprising CD4+ / CD8+, CD4- / CD8- cells, CD4- / CD8+ cells, and CD4+ / CD8- cells can be induced to differentiate into CD56+ / CD3- cells.

[0214] In some embodiments, a cell population containing, inter alia, CD4 / CD8 double-positive cells can be produced by a method comprising culturing hematopoietic progenitor cells or HP cell bulk in a medium supplemented with vitamin C. The vitamin C added to the basal medium is the same as that used in the above-described induction of HP cell bulk.

[0215] In some embodiments, the medium used to produce cell populations containing CD4+ / CD8+, CD4- / CD8-, CD4- / CD8+, and CD4+ / CD8- cells from the HP cell bulk is a CD4 / CD8 induction medium. In some embodiments, the CD4 / CD8 induction medium can be prepared by adding vitamin C to a basal medium used to culture animal cells. Examples of basal media include Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM medium (Thermo Fisher Scientific (Gibco)), Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fisher's Medium, and Neurobasal Medium (Life Technologies), as well as mixtures of two or more of these media. The medium may contain serum or may be serum-free.

[0216] Optionally, the basal medium may also contain one or more of the following substances: albumin, human insulin, human transferrin, selenium or sodium selenate, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and cytokines.

[0217] The CD4 / CD8 induction medium for producing a cell population comprising, among other cells, CD4 / CD8 double positive cells may be further supplemented with cytokine(s) selected from the group consisting of ascorbic acid, SCF, IL-7, Flt3L, TPO, fibronectin or a variant thereof, Notch ligand, p38 inhibitor and SDF-1.

[0218] In some embodiments, vitamin C can be added every 4 days, every 3 days, every 2 days, or every day during the culture period. Vitamin C can be added to the medium in an amount equivalent to about 5 μg / ml to about 500 μg / ml. In some embodiments, vitamin C is present in the medium at about 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, or 500 μg / ml.

[0219] In some embodiments, the basal medium comprises one or more substances that induce signaling in the stem cell factor / kit signaling pathway, including but not limited to SCF.

[0220] In some embodiments, the basal medium comprises one or more substances that induce signal transduction of the Flt3-ligand signaling pathway, including but not limited to Flt3-ligand (Flt3L).

[0221] In some embodiments, the basal medium comprises one or more substances that induce signaling in the thrombopoietin signaling pathway, including but not limited to TPO.

[0222] In some embodiments, the basal medium includes one or more p38 inhibitors that are inhibitors of p38α and p38β, which suppress downstream activation of MAPKAP kinase-2 and heat shock protein 27. Examples of chemical inhibitors of p38 that may be used in the present invention include SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole) and derivatives thereof, SB202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole) and derivatives thereof, SB239063 (trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol) and derivatives thereof, SB220025 and derivatives thereof, PD169316, RPR200765A, AMG-548, BIRB-796, SCIO-469, SCIO-323, VX-702, and FR167653. These compounds are commercially available; for example, SB203580, SB202190, SC239063, SB220025, and PD169316 are available from Calbiochem, and SCIO-469 and SCIO-323 are available from Scios. Other examples of p38 inhibitors include dominant-negative mutants of p38, such as p38T180A, which is obtained by a point mutation of threonine to alanine at position 180 in the DNA-binding domain of p38, and p38Y182F, which is obtained by a point mutation of tyrosine to phenylalanine at position 182 of p38 in humans and mice. The p38 inhibitor is contained in the medium, for example, at about 0.5 μM to about 50 μM.

[0223] In some embodiments, the basal medium comprises SDF-1.

[0224] In some embodiments, SDF-1 may be not only SDF-1α or its mature form, but also isoforms such as SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε, SDF-1φ, or their mature forms, or a mixture of these in any ratio. Preferably, SDF-1α is used. SDF-1 may also be called CXCL-12 or PBSF.

[0225] In some embodiments, one or more amino acids in the amino acid sequence of SDF-1 can be substituted, deleted, and / or added as long as the SDF-1 retains chemokine activity. Similarly, sugar chains can be substituted, deleted, and / or added. Amino acid mutations are permitted as long as at least four cysteine ​​residues (Cys30, Cys32, Cys55, and Cys71 in human SDF-1α) are maintained and the amino acid sequence exhibits 90% or greater identity with the naturally occurring substance. SDF-1 can be obtained from mammals, such as monkeys, sheep, cows, horses, pigs, dogs, cats, rabbits, rats, and mice, or from non-human mammals. For example, the protein registered under GenBank accession number NP_954637 can be used as human SDF-1α, and the protein registered under GenBank accession number NP_000600 can be used as SDF-1β. In some embodiments, when the substance that triggers signal transduction of the Flt3 ligand signaling pathway is Flt3L, the concentration of Flt3L in the culture medium for producing a cell population containing CD4 / CD8 double-positive cells is about 1 ng / ml to 100 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml.

[0226] In some embodiments, when the substance that induces signaling of the stem cell factor / kit signaling pathway is SCF, SCF is used to produce a cell population containing CD4 / CD8 double positive cells under the same conditions as above.

[0227] In some embodiments, when the substance that induces signaling of the stem cell factor / kit signaling pathway is TPO, TPO is used to produce a cell population containing CD4 / CD8 double positive cells under the same conditions as above.

[0228] The fibronectin or its variants used in the present invention are not particularly limited, as long as they are molecules capable of binding to CD3-positive cells. The fibronectin variants are not particularly limited, as long as they are molecules capable of binding to VLA-5 and VLA-4 on the surface of CD3-positive cells, and examples thereof include RetroNectin. Fibronectin and its variants can be present in any form in the culture medium. For example, they can be contained in the culture medium during culture or immobilized on the culture vessel, preferably immobilized on the culture vessel.

[0229] When fibronectin or a variant thereof is contained in the culture medium, the lower limit of the concentration of fibronectin or a variant thereof can be 10 ng / ml or more, preferably 100 ng / ml or more, and the upper limit can be 10,000 μg / ml or less, preferably 1,000 μg / ml or less.

[0230] In some embodiments, the concentration of IL-7 in the medium used to produce a cell population comprising CD4 / CD8 double positive cells is about 1 ng / ml to 100 ng / ml, e.g., 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml.

[0231] SDF-1 can be commercially available, purified from nature, or produced by peptide synthesis or genetic engineering. SDF-1 is contained in the medium at a concentration of, for example, about 10 ng / ml to about 100 ng / ml. Furthermore, SDF-1 substitutes with SDF-1-like activity can also be used instead of SDF-1. Examples of such SDF-1 substitutes include CXCR4 agonists, and low-molecular-weight compounds with CXCR4 agonist activity or the like can be added to the medium instead of SDF-1.

[0232] In some embodiments, when the SDF-1 is SDF1α, the concentration of SDF-1α in the culture medium for producing a cell population containing CD4 / CD8 double positive cells is about 1 nM to 100 nM, for example, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM.

[0233] In some embodiments, when the p38 inhibitor is SB203580, the concentration of SB203580 in the medium for producing a cell population of CD4 / CD8 double-positive cells is about 0.5 μM to 100 μM, for example, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, or 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, or 100 μM.

[0234] In some embodiments, a basal medium for producing a cell population comprising CD4 / CD8 double-positive cells comprises αMEM medium supplemented with about 15% FBS, about 4 mM L-glutamine, about 100 U / ml penicillin, about 100 μg / ml streptomycin, about 55 μM 2-mercaptoethanol, about 50 μg / ml ascorbic acid 2-phosphate, about 10 μg / ml human insulin, about 5.5 μg / ml human transferrin, about 6.7 ng / ml sodium selenate, about 50 ng / ml SCF, about 50 ng / ml IL-7, about 50 ng / ml Flt3L, about 100 ng / ml TPO, about 15 μM SB203580, and about 30 nM SDF-1α.

[0235] To produce a cell population containing CD4 / CD8 double-positive cells, hematopoietic progenitor cells can be cultured in adherent or suspension culture. In some embodiments, the culture vessel / dish is coated with DLL1 or DLL4, or a fusion protein of DLL4 or DLL1, and Fc, or the like. DLL1 or DLL4 can be recombinant human (rh)-DLL1 or rh-DLL4. In some embodiments, the culture vessel / dish is coated with rh-DLL4 / Fc chimera (Sino Biological) and RetroNectin (Takara Bio Inc.). For adherent culture, a coated culture vessel may be used, and / or the hematopoietic progenitor cells may be co-cultured with feeder cells, or the like. Examples of feeder cells for co-culture include the bone marrow stromal cell line, OP9 cells (available from Riken BioResource Center). OP9 cells are preferably OP-DL1 cells (Holmes RI and Zuniga-Pflucker JC. Cold Spring Harb Protoc. 2009(2)), which constantly express Dll1. In some embodiments, when OP9 cells are used as feeder cells, separately prepared Dll1, or a fusion protein of Dll1 with Fc or the like, they can be added to the medium to perform coculture. In some embodiments, Dll1 can include proteins encoded by genes having the nucleotide sequence of NCBI Accession No. NM#005618 for humans or NCBI Accession No. NM#007865 for mice, as well as naturally occurring mutants with high sequence identity (e.g., 90% or more sequence identity) to these proteins and equivalent functions. When feeder cells are used to produce a cell population containing CD4 / CD8 double-positive cells, the feeder cells can be appropriately replaced during culture. Feeder cell replacement can be performed by transferring the cultured target cells to pre-plated feeder cells. Exchanges may be performed every 5 days, every 4 days, every 3 days, or every 2 days.

[0236] In some embodiments, the culture temperature conditions for culturing the HP cell bulk to produce a cell population of CD4 / CD8 double-positive cells include the following: about 37°C to about 42°C, and about 37°C to about 39°C for CD4 / CD8 double-positive cells. In some embodiments, the culture can be performed under hypoxic conditions. Examples of hypoxic conditions include oxygen concentrations of 15%, 10%, 9%, 8%, 7%, 6%, 5%, and lower. The culture period can be appropriately determined by one skilled in the art by monitoring the number of different types of cells, including CD4 / CD8 double-positive cells. Examples of culture periods include 10 days or more, 12 days or more, 14 days or more, 16 days or more, 18 days or more, 20 days or more, 21 days or more, 23 days or more, 25 days or more, 28 days or more, 30 days or more, 35 days or more, or 42 days or more. In some other embodiments, the CD4 / CD8 double positive cells obtained from the culture are a cell population (DP cell bulk) that also contains other cell types.

[0237] When a population of a specific cell type (e.g., CD4- / CD8+ cells) is isolated from a cell population containing CD4 / CD8 double-positive cells, isolation may be performed using any one of indicators, including, but not limited to, CD4, CD8, CD3, and CD45, depending on the cell type to be isolated. The isolation method may be a method well known to those skilled in the art, such as a method in which cells are labeled with a specific antibody (e.g., a CD4, CD8, CD3, or CD45 antibody) and then isolated using a flow cytometer, or a method in which cells are purified using an affinity column that immobilizes a desired antigen.

[0238] Derivation of CD56+ / CD3- cells from a cell population containing CD4 / CD8 double-positive cells In some embodiments, the NK cells are CD56+ / CD3- immune cells.

[0239] In some embodiments, NK cells are produced by a method comprising culturing a cell population comprising CD4 / CD8 double positive cells or a bulk of DP cells in a medium supplemented with vitamin C. In some embodiments, an NK induction medium is used to produce CD56+ / CD3- cells.

[0240] In some embodiments, the medium is prepared by adding vitamin C to a basal medium used to culture animal cells. Examples of basal media include Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fisher's Medium, and Neurobasal Medium (Life Technologies), as well as mixtures of two or more of these media. In some embodiments, the medium contains serum. In some embodiments, the medium is serum-free. Optionally, the basal medium may also contain one or more of the following substances: albumin, human insulin, human transferrin, selenium or sodium selenate, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, lipids, amino acids, glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and cytokines.

[0241] In some embodiments, the medium used to produce CD56-positive NK cells further comprises an anti-CD3 antibody (UCHT1) and cytokines, examples of suitable cytokines include IL-2 and IL-7.

[0242] The CD3 antibody is not limited as long as it specifically recognizes CD3. In some embodiments, the concentration of the CD3 antibody in the NK induction medium is about 10 ng / ml to 1000 ng / ml, for example, 10 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, or 1000 ng / ml.

[0243] In some embodiments, vitamin C is used to generate CD56-positive K cells under the same conditions as above.

[0244] In some embodiments, the concentration of IL-2 in the NK induction medium for producing CD56-positive NK cells is about 1 U / ml to 1000 U / ml, e.g., about 1 U / ml, 5 U / ml, 10 U / ml, 20 U / ml, 30 U / ml, 40 U / ml, 50 U / ml, 60 U / ml, 70 U / ml, 80 U / ml, 90 U / ml, 100 U / ml, 500 U / ml, or 1000 U / ml. In some embodiments, the concentration of IL-2 in the NK induction medium for producing CD56-positive NK cells is about 1 ng / ml to 100 ng / ml, e.g., 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml.

[0245] In some embodiments, the concentration of IL-7 in the NK induction medium for producing CD56-positive NK cells is about 1 ng / ml to 100 ng / ml, e.g., 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml.

[0246] In some embodiments, the culture temperature conditions for culturing a population containing CD4 / CD8 double-positive cells to produce CD56-positive NK cells are about 37°C to about 42°C, or about 37°C to about 39°C. The culture period can be appropriately determined by one skilled in the art by monitoring the number of CD56-positive NK cells, etc. The number of days of culture is not limited as long as CD56-positive NK cells can be obtained. Examples of culture periods include at least 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more.

[0247] In some embodiments, the obtained CD56+ NK cells may be isolated before further use. In some other embodiments, the obtained CD56-positive NK cells may be used as a cell population that also includes other cell types, such as T cells (NK cell bulk).

[0248] In some embodiments, the NK cell bulk may comprise 50%, 55%, 60%, 75%, 80%, 85%, 90%, or more than 90% NK cells. In some embodiments, the NK cell bulk comprises about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells. In some embodiments, the NK cell bulk comprises about 75% CD56+ / CD3- NK cells and about 25% CD56+ / CD3+ T cells. In some embodiments, the NK cell bulk may also comprise B cells and monocytes.

[0249] When CD56+ NK cells are isolated, the isolation method can be a method well known in the art, such as labeling cells with anti-CD56 and anti-CD3 antibodies and then isolating them using a flow cytometer (fluorescence-activated cell sorting), or purifying the cells using an affinity column that immobilizes the desired antigen, or the like.

[0250] In some embodiments, the CD56-positive NK cells are CD56+ / CD3+. In some embodiments, the CD56-positive NK cells are CD56+ / CD3-.

[0251] Preparation of CAR-NK cells In some embodiments, NK cells are engineered to contain one or more transgenes. For example, NK cells can be genetically engineered to express a tumor-targeting chimeric antigen receptor (CAR), thereby producing anti-tumor effector cells. In one example, NK cells or iPS NK cells can be engineered to express a CAR. In some embodiments, cell precursors to NK cells are engineered to express a CAR. For example, in some embodiments, iPS cells are engineered to express a CAR. In some embodiments, cells in HP cell bulk are engineered to express a CAR. In some embodiments, NK cells are engineered to express a CAR. Furthermore, in some embodiments, these transgenic receptors can be directed to tumor-associated antigens that are not derived from proteins. In certain embodiments, NK cells or iPS NK cells are modified to contain at least one CAR. In some embodiments, a single CAR targets two or more antigens.

[0252] In some embodiments, the iNK cells comprise chimeric, non-naturally occurring, engineered receptors. In some embodiments, the engineered chimeric antigen receptors (CARs) have one, two, three, four, or more components, and in some embodiments, one or more components facilitate targeting or binding of lymphocytes to one or more tumor antigen-containing cancer cells.

[0253] In some embodiments, a CAR generally comprises at least one transmembrane polypeptide comprising at least one extracellular ligand binding domain and one transmembrane polypeptide comprising at least one intracellular signaling domain, whereby the polypeptides assemble together to form a chimeric antigen receptor.

[0254] As used herein, the term "extracellular ligand-binding domain" is defined as an oligo- or polypeptide capable of binding to a ligand. Preferably, the domain will be capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.

[0255] In particular, the extracellular ligand-binding domain can comprise an antigen-binding domain derived from an antibody directed against a target antigen.

[0256] In some embodiments, NK cells or iPS NK cells can be genetically modified to express one or more chimeric antigen receptors (CARs). In some embodiments, the CAR comprises an extracellular ligand-binding domain that targets a tumor antigen selected from one or more of CD44, CD19, CD20, CD22, CD23, CD30, CD89, CD123, CS-1, ROR1, mesothelin, c-Met, PSMA, Her2, GD-2, CEA, MAGE A3 TCR, EGFR, HER2 / ERBB2 / neu, EPCAM, EphA2, CEA, and BCMA. In some embodiments, the tumor antigen comprises CD19.

[0257] In some embodiments, the extracellular ligand-binding domain is a single-chain antibody fragment (scFv) comprising a variable light (VL) fragment and a variable heavy (VH) fragment of a target antigen-specific monoclonal antibody joined by a flexible linker.

[0258] In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the transmembrane domain further comprises a stalk region between the extracellular ligand-binding domain and the transmembrane domain. As used herein, the term "stalk region" generally refers to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, the stalk region is used to confer more flexibility and accessibility to the extracellular ligand-binding domain. The stalk region can comprise up to 300 amino acids, 10-100 amino acids, and / or 25-50 amino acids. The stalk region can be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or all or part of an antibody constant region. Alternatively, the stalk region can be a synthetic sequence corresponding to a naturally occurring stalk sequence or can be a completely synthetic stalk sequence. In a preferred embodiment, the stalk region is a portion of the human CD8 alpha chain.

[0259] In some embodiments, the CAR comprises a signaling domain or an intracellular signaling domain that contributes to intracellular signaling after binding of the extracellular ligand-binding domain to a target, resulting in activation of iPS NK cells and an immune response. The term "signaling domain" refers to the portion of a protein that transmits an effector signal function signal and instructs the cell to perform a specialized function. In some embodiments, the iPS NK cells have a CAR that comprises a signaling domain.

[0260] In some embodiments, the iPS NK cells are genetically modified to express the IL-15Rα / IL-15 complex.

[0261] In some embodiments, the transmembrane polypeptides are expressed on the surface of immune cells, for example, on iPS NK cells, and have the ability to interact together to direct the cellular response of the immune cells against predefined target cells. Different transmembrane polypeptides of the CAR, including extracellular ligand-binding domains and / or signaling domains, interact together to participate in signal transduction and induce an immune response after binding to the target ligand. The transmembrane domains can be derived from either natural or synthetic sources. The transmembrane domains can be derived from any membrane-bound or transmembrane protein.

[0262] In some embodiments, genetic modification of NK cells to express a CAR may include the following steps: (1) synthesizing a gene corresponding to a particular CAR; (2) preparing a vector containing the gene corresponding to the CAR; and (3) transducing CD56+ NK cells with the vector containing the CAR gene.

[0263] The expression vector encoding the CAR can be introduced as one or more DNA molecules or constructs, and at least one marker can be present that will allow for selection of host cells containing the construct(s).

[0264] Constructs can be prepared by conventional methods, in which genes and regulatory regions can be isolated, ligated, cloned into a suitable cloning host, and analyzed by restriction or sequencing, or other convenient means. In particular, PCR can be used to isolate individual fragments containing all or part of the functional units, and one or more mutations can be introduced, if necessary, using "primer repair," ligation, in vitro mutagenesis, or the like. Once the construct(s) are completed and demonstrated to have the correct sequence, they can then be introduced into CTLs by any convenient means. Constructs can be packaged into non-replicating viral genomes, such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), or other vectors (such as retroviral or lentiviral vectors), for infection or cell transduction. Constructs can also include viral sequences for transfection, if desired. Alternatively, constructs can be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like. Host cells may be grown and expanded in culture prior to the introduction of the construct(s), followed by appropriate treatment for the introduction and integration of the construct(s). The cells are then expanded and screened for the marker present in the construct. Various markers that may be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.

[0265] In some cases, the construct may have a target site for homologous recombination, where it is desired that the construct be integrated into a specific locus. For example, this can be done by knocking out an endogenous gene and replacing it with a gene encoded by the construct (at the same locus or elsewhere) using materials and methods known in the art for homologous recombination. For homologous recombination, either the OMEGA or O-vector can be used.

[0266] The constructs can be introduced as a single DNA molecule encoding at least the CAR and optionally another gene, or as separate DNA molecules carrying one or more genes. The other genes can include, for example, genes encoding therapeutic molecules or suicide genes. The constructs can be introduced simultaneously or sequentially, with the same or different markers, respectively.

[0267] Vectors containing useful elements for expression in prokaryotes or eukaryotes, such as bacterial or yeast replication origins, selectable and / or amplifiable markers, promoter / enhancer elements, which can be used to prepare construct DNA stocks and to perform transfections, are well known in the art and many are commercially available.

[0268] How to use The cells according to the present invention can be used to treat cancer, viral infections or autoimmune disorders in a patient in need thereof. In another embodiment, the isolated cells according to the present invention can be used in the manufacture of a medicament for the treatment of cancer, autoimmune disorder viral infections in a patient in need thereof.

[0269] The present invention relates to a method for treating a patient in need thereof, the method comprising at least one of the steps of: (a) providing chimeric antigen receptor cells according to the present invention; and (b) administering the cells to the patient.

[0270] This treatment can be ameliorative, curative, or preventative. It can be either part of an autoimmune therapy or part of an allogeneic immunotherapy treatment. Autologous means that the cells, cell lines, or cell populations used to treat a patient are derived from the patient or a human leukocyte antigen (HLA)-matched donor. Allogeneic means that the cells or cell populations used to treat a patient are derived from a donor, rather than from the patient.

[0271] In some embodiments, the described cells are allogeneic. In some embodiments, the described cells are autologous.

[0272] The treatment can be used to treat patients diagnosed with cancer, viral infection, autoimmune disorders, or graft-versus-host disease (GvHD). Cancers that can be treated include non-vascularized or substantially non-vascularized tumors, as well as vascularized tumors. Cancers can include non-solid tumors (e.g., hematological tumors such as leukemia and lymphoma) or solid tumors. Cancer types that can be treated with the CARs of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0273] In some embodiments, the treatment is combined with one or more therapies for cancer selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiation therapy.

[0274] In some embodiments, the treatment can be administered to a patient undergoing immunosuppressive treatment.

[0275] In further embodiments, the cell composition is administered to a patient in combination with one or more additional therapies. For example, in some embodiments, the cell composition is administered to a patient in conjunction with (e.g., before, simultaneously with, or after) bone marrow transplantation, T-cell ablative therapy using chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell composition of the present invention is administered after B-cell ablative therapy using an agent reactive with CD20, such as Rituxan. For example, in one embodiment, the subject may receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of expanded immune cells of the present invention. In additional embodiments, the expanded cells are administered before or after surgery. The modified cells obtained by any one of the methods described herein can be used in certain aspects of the present invention to treat patients in need thereof for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD). Accordingly, within the scope of the present invention is a method of treating a patient in need thereof for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), the method comprising treating the patient by administering to the patient an effective amount of modified cells comprising an inactivated TCR alpha gene and / or TCR beta gene.

[0276] Cell administration In some embodiments, cells can be introduced into a host organism, e.g., a mammal, in a variety of ways. In certain embodiments, cells can be introduced at the site of a tumor, while in alternative embodiments, the cells are cancerous or modified to develop cancer. The number of cells used depends on several factors, including the purpose of introduction, the lifespan of the cells, and the protocol used, such as the number of doses, the ability of the cells to proliferate, and the stability of the recombinant construct. Cells can be applied as a dispersion and are generally injected at or near the site of interest. The cells can be in a physiologically acceptable medium. In one example, the NK cells or iPS NK cells of the present invention can express one or more CARs, TCRs, or any other engineered protein or polypeptide domain, such as high-affinity CD16. In some embodiments, the cells are encapsulated and placed at the site of the tumor to inhibit immune recognition.

[0277] The cells can be administered as needed. A variety of protocols can be used depending on the desired response, the method of administration, the longevity of the cells, and the number of cells present. The number of administrations will depend, at least in part, on the factors described above.

[0278] Administration of the cells or cell populations according to the present invention can be carried out by any convenient method, such as aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscular, intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

[0279] Nucleic Acid-Based Expression Systems In some embodiments, the NK cells or iPS NK cells of the present invention are engineered to express one or more CAR, TCR, or any other engineered protein or polypeptide domain, such as high-affinity CD16 or CD19. Recombinant techniques for generating expression vectors containing these polypeptides are well known in the art and are generally described below.

[0280] vector The term "vector" refers to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. The nucleic acid sequence can be "exogenous," meaning that the sequence is foreign to the cell into which the vector is introduced, or that the sequence is homologous to a sequence within the cell but is located anywhere within the nucleic acid of the host cell where the sequence is not normally found. Vectors can include plasmids, cosmids, and viruses (e.g., bacteriophages, animal viruses, and plant viruses), as well as artificial chromosomes (e.g., YACs). Those skilled in the art are well equipped to construct vectors via standard recombinant techniques (see, e.g., Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference).

[0281] The term "expression vector" refers to any type of genetic construct that contains a nucleic acid encoding a transcribable RNA. In some cases, the RNA molecule is translated into a protein, polypeptide, or peptide. In other cases, for example, in the production of antisense molecules or ribozymes, these sequences are not translated. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription, and possibly translation, of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences that serve other functions, as described below.

[0282] Promoters and Enhancers A "promoter" is a regulatory sequence, which is a region of a nucleic acid sequence at which the initiation and rate of transcription are controlled. This sequence can contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind to initiate specific transcription of the nucleic acid sequence. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation relative to a nucleic acid sequence to control transcription initiation and / or expression of that sequence.

[0283] Promoters generally contain sequences that function to position the start site for RNA synthesis. The most well-known example is the TATA box, but in some promoters that do not contain a TATA box, such as the mammalian terminal deoxynucleotide transferase gene promoter and the SV40 late gene promoter, individual elements that overlap the start site themselves help to fix the start location. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but several promoters have been shown to contain functional elements downstream of the start site as well. To bring a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of a transcriptional reading frame is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates DNA transcription and promotes expression of the encoded RNA.

[0284] Spacing between promoter elements is often flexible, allowing promoter function to be maintained when elements are inverted or moved relative to one another. In the tk promoter, promoter elements can be spaced as far apart as 50 bp before activity begins to decline. Depending on the promoter, individual elements may function cooperatively or independently to activate transcription. Promoters may or may not be used in combination with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0285] A promoter can be one naturally associated with a nucleic acid sequence, as can be obtained by isolating the 5' prime non-coding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer can be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, particular advantages can be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter. This refers to a promoter not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, promoters or enhancers isolated from other viruses, or prokaryotic or eukaryotic cells, and "non-naturally occurring" promoters or enhancers, i.e., promoters or enhancers containing different elements of different transcriptional regulatory regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA constructs include the lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences can be produced using nucleic acid amplification techniques such as recombinant cloning and / or PCR™ in conjunction with the compositions disclosed herein (see U.S. Patent Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference. Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, etc., can be used as well.

[0286] Naturally, it will be important to use a promoter and / or enhancer that effectively directs expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are generally aware of the use of promoter, enhancer, and cell type combinations for protein expression (see, e.g., Sambrook et al., 1989, incorporated herein by reference). The promoter used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, which would be advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0287] Furthermore, any promoter / enhancer combination can be used to drive expression. The use of T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided as part of the delivery complex or as an additional gene expression construct.

[0288] Assays for characterizing the identity of tissue-specific promoters or elements and their activity are well known to those of skill in the art.

[0289] Specific initiation signals may also be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, such as the ATG initiation codon, may need to be provided. One of ordinary skill in the art would be able to readily determine this and provide the necessary signals.

[0290] In certain embodiments of the present invention, the use of internal ribosome entry site (IRES) elements is used to create multigenic or polycistronic messages, which may be used in the present invention.

[0291] A vector can contain a multiple cloning site (MCS), a nucleic acid region containing multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant techniques to digest the vector. "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at specific locations within the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those skilled in the art. Often, vectors are linearized or fragmented using a restriction enzyme that cuts within the MCS, allowing exogenous sequences to be ligated into the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be adjacent to each other. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombinant technology.

[0292] Splice sites, termination signals, origins of replication, and selectable markers may also be used.

[0293] Plasmid vectors In certain embodiments, plasmid vectors are contemplated for use in transforming host cells. Generally, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with these hosts. The vector usually contains a replication site and marking sequences capable of providing phenotypic selection in transformed cells. In a non-limiting example, E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species. pBR322 contains ampicillin and tetracycline resistance genes, thus providing a means for easily identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage, must also contain, or be modified to contain, a promoter that can be used by the microorganism for expression of its own proteins, for example.

[0294] Additionally, phage vectors containing replicon and control sequences compatible with host microorganisms can be used as transforming vectors in connection with these hosts. For example, phage lambda GEM™-11 can be utilized to generate recombinant phage vectors that can be used to transform host cells such as E. coli LE392.

[0295] Further useful plasmid vectors include the pIN vectors (Inouye et al., 1985) and pGEX vectors for use in generating glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and isolation or cleavage. Other suitable fusion proteins are those with galactosidase, ubiquitin, etc.

[0296] Bacterial host cells, e.g., E. coli, containing the expression vector are grown in any of several suitable media, e.g., LB. Expression of the recombinant protein in a particular vector can be induced by contacting the host cells with an agent specific for the particular promoter, e.g., by adding IPTG to the medium or by switching the incubation to a higher temperature, as will be understood by those skilled in the art. After culturing the bacteria for an additional period (usually 2-24 hours), the cells are harvested by centrifugation and washed to remove residual medium.

[0297] viral vectors Certain viruses infect or enter cells through receptor-mediated endocytosis, and are integrated into the genome of host cells, allowing stable and efficient expression of viral genes, making them attractive candidates for transferring foreign nucleic acid into cells (e.g., mammalian cells).The components of the present invention can be viral vectors encoding one or more CARs, TCRs, or any other engineered protein or polypeptide domain, such as the high-affinity CD16 of the present invention.Non-limiting examples of viral vectors that can be used to deliver the nucleic acid of the present invention are described below.

[0298] Adenovirus vectors A particular method for delivering nucleic acids involves the use of adenoviral expression vectors. Adenoviral vectors are known to have a low ability to integrate into genomic DNA, but this characteristic is offset by the high efficiency of gene transfer afforded by these vectors. "Adenoviral expression vector" refers to a construct containing sufficient adenoviral sequences to (a) support packaging of the construct and (b) ultimately express the cloned tissue- or cell-specific construct. Knowledge of the genetic organization of adenovirus, a 36 kb, linear, double-stranded DNA virus, allows for the replacement of large pieces of adenoviral DNA with up to 7 kb of foreign sequence (Grunhaus and Horwitz, 1992).

[0299] AAV vectors Nucleic acids can be introduced into cells using adenovirus-assisted transfection. Improved transfection efficiency has been reported in cell lines using adenovirus-linked systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated virus (AAV) is an attractive vector system for use in the cells of the present invention because it has a high integration frequency and can infect non-dividing cells, and is therefore useful for delivering genes to mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a wide host range for infection (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details regarding the generation and use of rAAV vectors are described, for example, in U.S. Patent Nos. 5,139,941 and 4,797,368, each of which is incorporated herein by reference.

[0300] Retroviral vectors Retroviruses are useful as delivery vectors due to their ability to integrate genes into the host genome, transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specialized cell lines (Miller, 1992).

[0301] To construct a retroviral vector, a nucleic acid (e.g., encoding a desired sequence) is inserted into the viral genome in place of a specific viral sequence, resulting in the production of a replication-defective virus. To produce virions, packaging cell lines containing the gag, pol, and env genes but lacking the long-term repeat (LTR) and packaging components are constructed (Mann et al., 1983). When a recombinant plasmid containing a cDNA along with retroviral long-term repeat (LTR) and packaging sequences is introduced into a specialized cell line (e.g., by calcium phosphate precipitation), the packaging sequences allow the RNA transcripts of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect a wide variety of cell types; however, integration and stable expression require host cell division (Paskind et al., 1975).

[0302] Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (e.g., Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Patent Nos. 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency viruses (HIV-1, HIV-2), and simian immunodeficiency viruses (SIV). Lentiviral vectors are generated by multiple attenuation of HIV pathogenicity genes, such as deletion of genes env, vif, vpr, vpu, and nef, making the vector biologically safe.

[0303] Recombinant lentiviral vectors can infect non-dividing cells and can be used for gene transfer and expression of nucleic acid sequences both in vivo and ex vivo. For example, in recombinant lentiviruses capable of infecting non-dividing cells, suitable host cells are transfected with two or more vectors carrying packaging functions, i.e., gag, pol, and env, and rev and tat, as described in US Pat. No. 5,994,136 (incorporated herein by reference). To target receptors in specific cell types, recombinant viruses can be targeted by linking the envelope protein to an antibody or a specific ligand. Inserting a sequence of interest (including regulatory regions) into a viral vector along with another gene encoding a ligand for a receptor on a specific target cell, for example, makes the vector target-specific.

[0304] Combination therapy In certain embodiments of the present invention, the clinical aspects of the methods of the present invention are combined with other agents effective in treating hyperproliferative diseases, such as anti-cancer agents. "Anticancer" agents can adversely affect cancer in a subject by, for example, killing cancer cells, inducing apoptosis in cancer cells, slowing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to tumors or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting cancer progression, or extending the lifespan of a subject with cancer. More generally, these other compositions will be provided in a combined amount effective to kill or inhibit cell proliferation. This process can involve simultaneously contacting the cancer cells with the expression construct and the agent(s) or multiple factors. This can be accomplished by contacting the cells with a single composition or pharmacological formulation containing both agents, or by simultaneously contacting the cells with two separate compositions or formulations, where one composition contains the expression construct and the other composition contains the second agent(s).

[0305] Resistance of tumor cells to chemotherapy and radiotherapy agents is a major problem in clinical oncology. One of the goals of current cancer research is to find ways to improve the effectiveness of chemotherapy and radiotherapy by combining one treatment with another. In the context of the present invention, cell therapy can be used in combination with chemotherapy, radiotherapy, or immunotherapy intervention, as well as with proapoptotic agents or cell cycle regulators.

[0306] Alternatively, the treatment may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments in which the other agent and the present invention are administered separately to an individual, it is ensured that no significant period of time lapses between their respective delivery times, thereby allowing the agent and the present invention treatment to still exert their beneficially combined effect on the cell. In such cases, it is contemplated that both modalities may contact the cell within about 12-24 hours of each other, more preferably within about 6-12 hours of each other. In some circumstances, it may be desirable to significantly extend the treatment period, in which case a period of several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) lapses between each administration.

[0307] In some embodiments, the treatment cycles are repeated as necessary. It is also contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the cell therapy of the present invention.

[0308] chemotherapy Cancer treatment also includes a variety of combination therapies with both chemotherapy and radiation based treatments. Combination chemotherapy includes, for example, Abraxane, altretamine, docetaxel, Herceptin, methotrexate, novantrone, zoladex, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabine, navelbine, farnesyl protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, or any analogs or derived variants thereof, and combinations thereof.

[0309] In certain embodiments, chemotherapy for an individual is used in combination with the present invention, eg, before, during, and / or after administration of the present invention.

[0310] Radiation therapy Other commonly used agents that cause DNA damage are generally gamma rays, X-rays, and / or the direct delivery of radioisotopes to tumor cells. Other forms of DNA damage, such as microwave and ultraviolet radiation, are also considered. All of these agents most likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from 50 to 200 roentgens per day for prolonged periods (3-4 weeks) to 2000 to 6000 roentgens per single dose. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells.

[0311] The terms "contacting" and "exposing" are used herein to describe a process in which, when applied to a cell, a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to or directly juxtaposed with a target cell. To achieve cell death or stasis, for example, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent the cell from dividing.

[0312] immunotherapy Immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector can be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody alone can act as the therapeutic effector, or it can recruit other cells to actually kill the cells. Antibodies can also be conjugated to drugs or toxins (such as chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and simply act as targeting agents. Alternatively, the effector can be a lymphocyte bearing a surface molecule that interacts directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0313] Thus, immunotherapies other than the therapies of the present invention described herein can be used in conjunction with the present cell therapy as part of a combination therapy. General approaches to combination therapy are discussed below. In general, tumor cells must bear some marker that is compatible with targeting, i.e., that is not present on the majority of other cells. Many tumor markers exist, any of which may be suitable for targeting in the context of the present invention. Common tumor markers include PD-1, PD-L1, CTLA4, carcinoembryonic antigen, prostate-specific antigen, urinary tract tumor-associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erbB, and p155.

[0314] gene In yet another embodiment, the secondary treatment is gene therapy, in which a therapeutic polynucleotide is administered before, after, or simultaneously with the clinical embodiments of the present invention. A variety of expression products are encompassed by the present invention, including inducers of cell proliferation, inhibitors of cell proliferation, or regulators of programmed cell death.

[0315] surgery Approximately 60% of cancer patients will undergo some type of surgery, including preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in combination with other therapies, such as the treatment of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and / or alternative therapies.

[0316] Curative surgery includes the physical removal, excision, and / or destruction or resection of all or part of cancerous tissue. Tumor resection refers to the physical removal of at least part of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgical surgery (Mohs surgery). Furthermore, it is contemplated that the present invention may be used in conjunction with the removal of superficial cancers, pre-cancers, or incidental amounts of normal tissue.

[0317] When all cancerous cells, tissues, or parts of tumors are removed, a cavity may be formed in the body. Treatment can be achieved by perfusion, direct injection, or local application of the area with additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also vary in dosage. [Example]

[0318] Other features, objects, and advantages of the present invention will become apparent in the following examples. However, it should be understood that the examples, while illustrating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the examples.

[0319] Example 1. Differentiation of iPS cells into bulk HP cells The iPS cell line FfI-01s04 was derived from peripheral blood mononuclear cells of a healthy individual. FfI-01s04 cells dispersed in StemFit complete medium were cultured at a density of 6x10 5 Cells / well were seeded into ultra-low attachment-treated 6-well plates under hypoxic (5% O) conditions ("Day 0"). StemFit complete medium contained 10 μM CHIR99021 and 50 μM Y-27632. The following day (i.e., Day 1), FfI-01s04 cells were dispersed in hematopoietic progenitor cell (HPC) differentiation medium containing BMP4 (50 ng / ml), VEGF (50 ng / ml), bFGF (50 ng / ml), and ascorbic acid 2-phosphate (50 μg / ml). HPC induction culture medium contained StemPro34 supplemented with human insulin (10 μg / ml), human transferrin (5.5 μg / ml), sodium selenite (6.7 ng / ml), L-glutamine (2 mM), and α-monothioglycerol (0.4 mM). On day 2, SB431542 (6 μM in medium) was added to the culture medium (i.e., HPC differentiation medium containing the cells) and the cells were cultured for 2 days. On day 4, the cells were resuspended in a different medium containing VEGF (50 ng / ml), bFGF (50 ng / ml), SCF (50 ng / ml), and ascorbic acid 2-phosphate (50 μg / ml) and cultured for an additional 3 days. On day 7, the cells were exposed to a different medium containing VEGF (50 ng / ml), bFGF (50 ng / ml), SCF (50 ng / ml), ascorbic acid 2-phosphate (50 μg / ml), TPO (30 ng / ml), and Flt3L (10 ng / ml) and cultured for an additional 7 days using this medium. During this 7-day culture period, the medium was changed every 2–3 days.

[0320] Example 2. Differentiation of bulk HP cells into a population containing CD4 / CD8 cells On day 14, the cell population obtained from Example 1 ("HP cell bulk") was plated in a 15 cm dish at 3.12 x 10 6Cells were seeded per dish and cultured at 37°C and 5% O. Note that various seeding densities may be used, and the above seeding density is one embodiment. Each 15 cm dish was coated with rh-DLL4 / Fc chimera (Sino Biological) and RetroNectin (Takara Bio Inc.). During this culture period, the medium was changed every 2-3 days. These cells were cultured in MEMα (ThermoFisher Scientific (Gibco)) supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 55 μM 2-mercaptoethanol, 50 μg / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 6.7 ng / ml sodium selenite, 50 ng / ml SCF, 50 ng / ml IL-7, 50 ng / ml Flt3L, 100 ng / ml TPO, 15 μM SB203580, and 30 nM SDF-1α. On day 21, the cells were passaged onto new 15 cm dishes coated with hDLL4 / RetroNectin. On day 28, the cells were further passaged onto new 15 cm dishes freshly coated with hDLL4 / RetroNectin. On day 35, all cells, including CD4 / CD8 cells ("DP cell bulk"), were harvested.

[0321] Example 3. Differentiation of DP cell bulk into NK cell bulk On day 35, DP cell bulk, i.e., cells obtained in Example 2 without undergoing cell isolation, was cultured at 1x10 6Cells were seeded into 48-well plates at 1000 cells / well and cultured at 37°C in 5% CO for 3 days. MEMα medium supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 ng / ml streptomycin, 50 μg / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 6.7 ng / ml sodium selenite, 500 ng / ml anti-CD3 antibody (UCHT1), 10 ng / ml IL-2, and 10 ng / ml IL-7 was used as the culture medium. On day 38, the cells were dispersed in another MEMα medium supplemented with 15% FBS, 4 mL of glutamine, 100 U / ml penicillin, 100 ng / ml streptomycin, 50 μg / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 6.7 ng / ml sodium selenite, 10 ng / ml IL-2, and 10 ng / ml IL-7, and used as the culture medium. On day 42, all cells, including NK cells ("NK cell bulk"), were collected.

[0322] Example 4. Flow cytometry analysis Next, the NK cell bulk was stained with the set of antibodies listed in Table 1 and analyzed by flow cytometry. As shown in Figure 1, a portion of the CD3-negative NK cell bulk expressed CD56 ("CD56-positive immune cells"). The CD56-positive and CD3-negative immune cells in Figure 1 are natural killer cells (NK cells). Therefore, CD56-positive and CD3-negative immune cells were prepared from the HP cell bulk derived from iPS cells (FfI-01s04 line). The CD56-expressing cells obtained by the above process are sometimes referred to as iPS NK cells. The antibodies used for flow cytometry are listed in Table 1.

[0323] [Table 1]

[0324] Example 5. Single-cell RNA-seq (scRNAseq) analysis To identify different cell types in the NK cell bulk population (i.e., the cells obtained in Example 3), a nonlinear dimensionality reduction method (uniform manifold approximation and projection (UMAP) based on single-cell RNA-seq (scRNAseq)) was applied.

[0325] Single-cell RNA sequencing was performed on 10,000 cells from the NK cell bulk using Genewiz's IIuminaNextSeq500. Using the SingleR algorithm and manual cluster labeling, events were classified into four broad cell populations (monocytes, B cells, NK cells, and T cells in the PBMC sample). A freely available PBMC dataset from 10XGenomics was used as a control for data analysis.

[0326] As shown in Figure 2, approximately 75% of the cells in the NK cell bulk were identified as NK cells, and approximately 25% of the cells in the NK cell bulk were identified as T cells. Therefore, the NK cell bulk contained both NK cells and T cells based on mRNA profiling by single-cell RNA sequencing.

[0327] Example 6. Preparation of CAR-NK cells The NK cells were further modified to express one or more CARs. The modification of the NK cells included the steps of (1) synthesizing an anti-CD19 CAR gene and an IL-15Rα / IL-15 gene; (2) preparing a retroviral vector containing the anti-CD19 CAR gene and the IL-15Rα / IL-15 gene; and (3) transducing the NK cells with the retroviral vector containing the anti-CD19 CAR gene and the IL-15Rα / IL-15 gene.

[0328] Preparation of CAR / IL15-NK cells The anti-CD19 CAR gene was prepared by synthesizing oligopeptides designed to be positioned from the N-terminus, as shown in Table 2.

[0329] [Table 2]

[0330] The anti-CD19 CAR was constructed according to WO2014 / 153270 (hereby incorporated by reference in its entirety).

[0331] Preparation of IL-15Rα / IL-15 gene The IL-15Rα / IL-15 gene was prepared by synthesizing an oligopeptide designed to be positioned from the N-terminus. IL-15Rα / IL-15 was constructed according to Mortier et al., 2006, The Journal of Biological Chemistry, Vol. 281, No. 3, pages 1612-1619, January 20, 2006; Chertova et al., The Journal of Biological Chemistry, Vol. 288, No. 25, pages 18093-18103, June 21, 2013; and Rowley et al., Eur J Immunol, 2009 February;39(2):491-506 (each of which is incorporated by reference in its entirety).

[0332] [Table 3]

[0333] Preparation of retroviral vector containing anti-CD19 CAR gene The anti-CD19 CAR gene was integrated into the multiple cloning site of the pMY retroviral vector, which was generated using FRY-RD18 cells to produce the retroviral vector.

[0334] Preparation of retroviral vector containing IL-15Rα / IL-15 gene The IL-15Rα / IL-15 gene was integrated into the multiple cloning site of another pMY retroviral vector, which was generated using FRY-RD18 cells for retroviral vector production.

[0335] Transduction of anti-CD19 CAR gene and IL-15Rα / IL-15 gene into iPS NK cells iPS NK cells were transduced with a retroviral vector containing the anti-CD19CAR gene and a retroviral vector containing the IL-15Rα / IL-15 gene to generate anti-CD19CAR-expressing iPSNK cells ("iNK-CAR19").

[0336] Example 7. In vivo antitumor activity of iNK-CAR19 Luciferase-expressing Nalm6 cells (ATCC; cancer cells) (5 x 10 5 Nalm6 cells) were transplanted into NOD / Shi-scidIL-2R gamma null mice ("NSG mice") via the tail vein. NSG mice (male, 4-5 weeks old) were obtained from the Jackson Laboratory. Four days after Nalm6 cell transplantation, iNK-CAR19 (1x10) cells dispersed in 0.2 ml of PBS or just 0.2 ml of PBS without cells were transplanted into NOD / Shi-scidIL-2R gamma null mice ("NSG mice"). 7 iNK-CAR19 cells were administered via the tail vein to Nalm6-implanted NSG mice. After administration of iNK-CAR19 cells or PBS, the mice were administered luciferin via the tail vein. Luciferase activity was measured over 70 days using an IVIS imaging system (PerkinElmer).

[0337] Figure 3 shows the antitumor effects of Nalm6-implanted NSG mice untreated (treated with buffer only) and treated with iNK-CAR19 cells. After 3 days, luminescence was detected in all mice treated with D-PBS buffer. In contrast, no luminescence was detected in other mice (treated with either primary CAR T cells or iNK-CAR cells) until 28 days after administration, and no luminescence was detected in one mouse treated with iNK-CAR19 cells even at 70 days after treatment. Therefore, iNK-CAR19 cells clearly demonstrated enhanced toxicity against Nalm6 cancer cells.

[0338] Additional equivalents and claims Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many additional equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above description, but rather is as set forth in the following claims.

Claims

1. 1. A method for producing pluripotent stem cell-derived NK cells, comprising: (A) providing a bulk cell population (HP cell bulk) comprising hematopoietic progenitor cells (HPCs) derived from pluripotent stem cells; (B) culturing the HP cell bulk in a CD4 / CD8 induction medium containing a p38 inhibitor and SDF-1 to obtain an intermediate heterogeneous cell population (DP cell bulk) containing CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8- cells, and CD4- / CD8+ cells; (C) culturing the DP cell bulk in an NK induction medium containing at least one compound selected from the group consisting of a CD3 activator, IL-2, and IL-7 to produce a cell population containing at least 50% CD56+ / CD3- NK cells; The above method does not include a cell isolation step.

2. The method of claim 1, wherein the HP cell bulk of (A) comprises CD34+ cells.

3. The method of claim 2, wherein 20% or more of the cells in the HP cell bulk are CD34+ cells.

4. The method of claim 3, wherein 20% to 90% of the HP cell bulk are CD34+ cells.

5. The method of claim 1, wherein the method does not include a step of isolating CD4+ / CD8+ cells.

6. 2. The method of claim 1, wherein step (A) comprises culturing pluripotent stem cells in an HPC induction medium to produce an HP cell bulk.

7. The method of claim 6, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

8. 8. The method of claim 6 or 7, wherein the HPC induction medium comprises at least one compound selected from bone morphogenetic protein-4 (BMP4), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), ascorbic acid, Flt3 ligand (Flt3L), thrombopoietin (TPO), and a TGFβ inhibitor.

9. The method of claim 8, wherein the HPC induction medium contains BMP4 at a concentration of 5 ng / mL to 500 ng / ml.

10. The method of claim 9, wherein the BMP4 is at a concentration of 50 ng / ml.

11. 9. The method of claim 8, wherein the bulk cell culture medium comprises VEGF at a concentration of 5 ng / mL to 500 ng / ml.

12. 12. The method of claim 11, wherein the VEGF is at a concentration of 50 ng / ml.

13. The method of claim 8, wherein the HPC induction medium contains bFGF at a concentration of 5 ng / mL to 500 ng / ml.

14. 14. The method of claim 13, wherein the bFGF is at a concentration of 50 ng / ml.

15. 15. The method of claim 14, wherein the bulk cell culture medium comprises ascorbic acid at a concentration of 5 μg / mL to 500 μg / ml.

16. 16. The method of claim 15, wherein the ascorbic acid is at a concentration of 50 μg / ml.

17. The method of claim 8, wherein the HPC induction medium comprises Flt3L at a concentration of 1 ng / mL to 100 ng / ml.

18. 18. The method of claim 17, wherein the Flt3L is at a concentration of 50 ng / ml.

19. The method of claim 8, wherein the HPC induction medium contains TPO at a concentration of 1 ng / mL to 200 ng / mL.

20. 20. The method of claim 19, wherein the TPO is at a concentration of 100 ng / ml.

21. 2. The method of claim 1, wherein the CD4 / CD8 induction medium further comprises at least one compound selected from the group consisting of ascorbic acid, stem cell factor (SCF), IL-7, Flt3L, and thrombopoietin (TPO).

22. 22. The method of claim 21, wherein the CD4 / CD8 induction medium comprises ascorbic acid at a concentration of 5 μg / ml to 500 μg / ml.

23. 23. The method of claim 22, wherein the ascorbic acid is at a concentration of 50 μg / ml.

24. 22. The method of claim 21, wherein the CD4 / CD8 induction medium comprises SCF at a concentration of 5 ng / mL to 100 ng / ml.

25. 25. The method of claim 24, wherein the SCF is at a concentration of 50 ng / ml.

26. 22. The method of claim 21, wherein the CD4 / CD8 induction medium comprises IL-7 at a concentration of 1 ng / mL to 100 ng / ml.

27. 27. The method of claim 26, wherein the IL-7 is at a concentration of 50 ng / ml.

28. 22. The method of claim 21, wherein the CD4 / CD8 induction medium comprises Flt3L at a concentration of 1 ng / mL to 100 ng / ml.

29. 29. The method of claim 28, wherein the Flt3L is Flt3L at a concentration of 50 ng / ml.

30. 22. The method of claim 21, wherein the CD4 / CD8 induction medium comprises TPO at a concentration of 1 ng / mL to 200 ng / ml.

31. 31. The method of claim 30, wherein the TPO is at a concentration of 100 ng / ml.

32. 22. The method of claim 21, wherein the CD4 / CD8 induction medium comprises a p38 inhibitor at a concentration of 0.5 μM to 100 μM.

33. 33. The method of claim 32, wherein the p38 inhibitor is SB203580.

34. 34. The method of claim 33, wherein the SB203580 is at a concentration of 15 μM.

35. 22. The method of claim 21, wherein the CD4 / CD8 induction medium comprises an SDF-1 inhibitor at a concentration of 10 ng / mL to 100 ng / ml.

36. 36. The method of claim 35, wherein the SDF-I inhibitor is at a concentration of 30 nM.

37. The method of claim 1, wherein the NK induction medium comprises a CD3 activator, IL-2, and IL-7.

38. The method of claim 1, wherein the NK induction medium contains IL-2 at a concentration of 1 ng / mL to 100 ng / ml.

39. 39. The method of claim 38, wherein the IL-2 is at a concentration of 10 ng / ml.

40. 2. The method of claim 1, wherein the second induction medium comprises IL-7 at a concentration of 1 ng / mL to 100 ng / ml.

41. 41. The method of claim 40, wherein the IL-7 is at a concentration of 10 ng / ml.

42. 42. The method of any one of claims 1 to 41, wherein each of the culturing steps is carried out at greater than 14% oxygen.

43. 42. The method of any one of claims 1 to 41, wherein each of the culturing steps is carried out in atmospheric oxygen.

44. 42. The method of any one of claims 1 to 41, wherein each of the culturing steps is carried out at 3-6% oxygen.

45. 45. The method of claim 44, wherein each of the culturing steps is carried out at 5% oxygen.

46. 7. The method of claim 6, wherein pluripotent stem cells are cultured in the HPC induction medium for more than 10 days to obtain a HP cell bulk.

47. 7. The method of claim 6, wherein pluripotent stem cells are cultured in the HPC induction medium for 11 to 15 days to obtain a HP cell bulk.

48. The method of claim 7, wherein pluripotent stem cells are cultured in the HPC induction medium for 14 days to obtain a HP cell bulk.

49. 8. The method of claim 7, wherein the iPSCs are obtained from peripheral blood mononuclear cells.

50. 2. The method of claim 1, wherein, without an enrichment step, at least 60% of the cells produced in step (C) are CD56+ / CD3- cells.

51. 51. The method of claim 50, wherein less than 25% of the cells produced are CD3+ cells.

52. 52. The method of claim 50 or 51, wherein the percentage of cells produced is determined by flow cytometry.

53. 52. The method of claim 50 or 51, wherein the percentage of cells produced is determined by single-cell RNA sequencing (scRNAseq).

54. 2. The method of claim 1, wherein the CD56+ / CD3- NK cells are genetically modified to express one or more chimeric antigen receptors (CARs).

55. 55. The method of claim 54, wherein the CAR is CD19.

56. 55. The method of claim 54, wherein the cells are further genetically modified to express the IL-15Rα / IL-15 complex.

57. (1) culturing iPSCs in an HPC induction medium containing at least one compound selected from vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and ascorbic acid to obtain a heterogeneous cell population (HP cell bulk) containing hematopoietic progenitor cells (HPCs); (2) culturing the HP cell bulk obtained in (1) in a CD4 / CD8 induction medium containing ascorbic acid, a p38 inhibitor, and SDF-1, to obtain an intermediate heterogeneous cell population (DP cell bulk) containing CD4- / CD8- cells, CD4- / CD8+ cells, CD4+ / CD8- cells, and CD4- / CD8+ cells, without a cell isolation step; (3) culturing the DP cell bulk in an NK induction medium containing at least one compound selected from the group consisting of a CD3 activating factor, IL-2, and IL-7, to obtain a cell population containing at least 50% CD56+ / CD3- NK cells, without a cell isolation step.

58. 60. A population of NK cells produced using the method of claim 1 or 57.

59. 60. Use of NK cells obtained by the method of claim 1 or 57 for the manufacture of a medicament for cell therapy.

60. 60. Use of NK cells obtained by the method of claim 1 or 57 for the manufacture of a medicament for treating cancer.

61. 61. The use of claim 60, wherein the cancer is leukemia or lymphoma.

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