Media and methods for differentiating natural killer cells

Abstract

To provide media and methods to produce and expand NK cells from HSPCs in a stroma cell-free culture system while maintaining NK cell cytotoxic functions.SOLUTION: The present invention provides media and methods for differentiating NK progenitor cells to obtain differentiated NK cells. The NK differentiation media comprise a pyrimidoindole compound such as UM171 or UM729.SELECTED DRAWING: None

Description

[Technical field]

[0001] Field of the Disclosure The present disclosure relates to the culture of cells, and more particularly to the culture of immune-like cells or cells of the immune system. In particular, the present disclosure relates to the differentiation of immune-like cells or cells of the immune system. [Background technology]

[0002] background Natural killer (NK) cells are lymphocytes that have a key role in immunity against infections and malignancies due to their ability to secrete proinflammatory cytokines and lyse virus-infected and tumor cells. NK cell-based cancer immunotherapy is a growing field. Allogeneic haploidentical NK cells are reactive against leukemia cells without causing graft-versus-host disease (GVHD). "Off-the-shelf" NK cells engineered with chimeric antigen receptors (CARs) could be a desirable alternative to autologous CAR T cells.

[0003] Due to the tumor-killing properties of NK cells, there is an interest in developing methods to produce functional NK cells in sufficient quantities to address clinical needs, such as in immunotherapy applications. Although peripheral blood (PB) or umbilical cord blood (CB) NK cell expansion is a common method to generate sufficient numbers of NK cells required for these applications, these methods are challenging due to their dependency on feeder cells and may result in NK cell exhaustion. In vitro differentiation and expansion of NK cells from hematopoietic stem and progenitors (HSPCs) could provide a potentially much larger source for NK cells. In vitro production of NK cells from HSPCs faces similar challenges due to the need for co-culture with stromal cells and low expansion rates. Furthermore, expanded NK cells must retain their cytotoxic properties.

[0004] Therefore, there is a need for a robust method to generate and expand NK cells from HSPCs in a stromal cell-free culture system while maintaining the cytotoxic function of NK cells. Summary of the Invention

[0005] Summary of the Disclosure The present disclosure relates to media and methods for differentiating NK progenitor cells to obtain differentiated NK cells.

[0006] In one aspect of the present disclosure, a NK cell differentiation medium is provided. In one embodiment, the NK cell differentiation medium comprises a pyrimidoindole compound. In one embodiment, the pyrimidoindole compound is either UM171 or UM729. In another embodiment, the concentration of the pyrimidoindole compound is 10 nM to 3 μM. In one embodiment, the concentration of UM171 in the NK cell differentiation medium is about 100 nM. In one embodiment, the concentration of UM729 in the NK cell differentiation medium is about 1 μM.

[0007] In one embodiment, the NK cell differentiation medium further comprises a basal medium, which may include salts, buffers, lipids, amino acids, trace elements, certain proteins, and the like.

[0008] In one embodiment, the NK cell differentiation medium is supplemented with one or all of the following: one or more cytokines; one or more growth factors; or other proteins. The one or more cytokines can be SCF, FLT3L, IL-15, IL-2, IL-7, IL-3, IL-12, and IL-21. In one embodiment, the one or more cytokines can be either SCF or FLT3L and IL-15. In one embodiment, the NK cell differentiation medium is supplemented with each of SCF, FLT3L, IL-15, IL-2, IL-7, IL-3, IL-12, and IL-21.

[0009] In one embodiment, the NK cell differentiation medium is not supplemented with an aryl hydrocarbon receptor antagonist. In another embodiment, the NK cell differentiation medium is not conditioned by contact with stromal cells or stromal cell substitutes.

[0010] In another embodiment, the NK cell differentiation medium is serum-free.

[0011] In yet another embodiment, the NK cell differentiation medium differentiates NK progenitor cells, preferably isolated or derived from a primary sample, or derived from pluripotent stem cells, such as induced pluripotent stem cells.

[0012] In another aspect of the present disclosure, a method for differentiating into NK cells is provided. An exemplary method may include the following steps: Providing a population of NK progenitor cells in culture; and Contacting the population of NK precursor cells in culture with an NK cell differentiation medium as described above in an amount, concentration, and for a period of time sufficient to produce NK cells.

[0013] In one embodiment, the step of contacting the population of NK progenitor cells with the pyrimidoindole compound is about 1 week. In one embodiment, the step of contacting with the pyrimidoindole compound is at least 1 week. In one embodiment, the step of contacting with the pyrimidoindole compound is longer than 1 week. In one embodiment, the step of contacting with the pyrimidoindole compound is about 2 weeks. In one embodiment, the step of contacting with the pyrimidoindole compound is longer than 2 weeks.

[0014] In another embodiment, the population of NK progenitor cells is homogeneous with respect to phenotypic marker or heterogeneous with respect to phenotypic marker. In one embodiment, the phenotypic marker is CD7. In another embodiment, the phenotypic marker is CD5.

[0015] In another embodiment, during the contacting step, the frequency of NK cells is increased.

[0016] In another embodiment, the number of NK cells is increased during the contacting step.

[0017] In another embodiment, during the contacting step, the frequency or number of a population of NK precursor cells is increased or decreased.

[0018] In another embodiment, the providing and contacting steps are absent of stromal cells or stromal cell substitutes.

[0019] In another embodiment, the providing and contacting steps are absent an aryl hydrocarbon receptor antagonist.

[0020] In another embodiment, the NK cells express CD56.

[0021] In another embodiment, the NK cells are cytotoxic.

[0022] In another embodiment, the population of NK precursor cells is derived from a primary sample.

[0023] In another embodiment, the population of NK progenitor cells is isolated from a primary sample. In one embodiment, the primary sample is an umbilical cord blood sample, a bone marrow sample, or a peripheral blood sample.

[0024] In another embodiment, the population of NK progenitor cells is differentiated from pluripotent stem cells.

[0025] [The present invention 1001] 1. An NK cell differentiation medium comprising a pyrimidoindole compound. [The present invention 1002] The NK cell differentiation medium of the present invention 1001, wherein the pyrimidoindole compound is UM171 or UM729. [The present invention 1003] The NK cell differentiation medium of the present invention 1001 or 1002, further comprising a basal medium. [The present invention 1004] The NK cell differentiation medium of the present invention 1003, further comprising one or more of SCF, FLT3L, IL-2, IL-3, IL-15, or IL-7. [The present invention 1005] The NK cell differentiation medium of any of the present inventions 1001 to 1004, which has not been conditioned by contact with stromal cells or stromal cell substitutes. [The present invention 1006] The NK cell differentiation medium according to any one of the present inventions 1001 to 1005, which does not contain an aromatic hydrocarbon receptor antagonist. [The present invention 1007] The NK cell differentiation medium according to any one of the present inventions 1001 to 1006, which is serum-free. [The present invention 1008] The NK cell differentiation medium according to any one of claims 1001 to 1007, which differentiates NK precursor cells. [The present invention 1009] The NK cell differentiation medium of the present invention, wherein the NK precursor cells are isolated or derived from a primary sample. [The present invention 1010] The NK cell differentiation medium of the present invention 1008 or 1009, wherein the NK precursor cells are derived from pluripotent stem cells. [The present invention 1011] The NK cell differentiation medium of the present invention 1010, wherein the pluripotent stem cells are induced pluripotent stem cells. [The present invention 1012] A method for differentiating into NK cells, comprising: Providing a population of NK progenitor cells; and contacting a population of NK precursor cells in culture with any one of the media of the present inventions 1001 to 1011 at a concentration and for a period of time sufficient to produce NK cells. A method comprising: [The present invention 1013] The method of claim 1012, wherein the population of NK progenitor cells is homogenous with respect to phenotypic markers or heterogeneous with respect to phenotypic markers. [The present invention 1014] The method of claim 1013, wherein the phenotypic marker is CD7. [The present invention 1015] The method of any one of claims 1013 to 1014, wherein the phenotypic marker is CD5. [The present invention 1016] The method of any of claims 1012 to 1015, wherein during the contacting step, the frequency of NK cells is increased. [The present invention 1017] Any of the methods of claims 1012 to 1016, wherein during the contacting step, the number of NK cells is increased. [The present invention 1018] The method of any of claims 1012 to 1017, wherein during the contacting step, the frequency or number of the population of NK precursor cells is increased or decreased. [The present invention 1019] The method of any of claims 1012 to 1018, wherein neither stromal cells nor stromal cell substitutes are present in the providing and contacting steps. [The present invention 1020] The method of any one of claims 1012 to 1019, wherein the providing and contacting steps are free of an aromatic hydrocarbon receptor antagonist. [The present invention 1021] The method according to any one of claims 1012 to 1020, wherein the concentration of the pyrimidoindole compound is 10 nM to 3 µM. [The present invention 1022] The method of any one of claims 1012 to 1021, wherein the period is at least one week. [The present invention 1023] The method of claim 1022, wherein said period is about two weeks. [The present invention 1024] The method of any of claims 1012 to 1023, wherein the NK cells express CD56. [The present invention 1025] The method of any of claims 1012 to 1024, wherein the NK cells are cytotoxic. [The present invention 1026] The method of any of claims 1012 to 1025, wherein the population of NK precursor cells is derived from a primary sample. [The present invention 1027] The method of any of claims 1012 to 1025, wherein the population of cells is isolated from a primary sample. [The present invention 1028] The method of claim 1027, wherein the primary sample is an umbilical cord blood sample, a bone marrow sample, or a peripheral blood sample. [The present invention 1029] The method of any of claims 1012 to 1025, wherein the population of cells is differentiated from a pluripotent stem cell. Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0026] For a better understanding of the various aspects described herein, and to show more clearly how these may be put into practice, reference is made, by way of example, to the accompanying drawings, which show at least one exemplary embodiment and which are described herein, and which are not intended to limit the scope of the teachings described herein. [Figure 1] FIG. 1 shows the effect of pyrimidoindole compounds on the differentiation of CD56+ NK cells from NK precursor cells. (A) The frequency of CD56+ NK cells after differentiation of NK precursor cells in a 14-day culture in the presence of a range of concentrations of UM171. At 100 nM, the frequency of NK cells is significantly increased compared to cultures lacking UM171 (paired t-test: p = 0.013). (B) The frequency of CD56+ NK cells after differentiation of NK precursor cells in a 14-day culture in the presence of a range of concentrations of UM729. At 1 μM, the frequency of CD56+ NK cells is significantly increased compared to cultures lacking UM729 (paired t-test: p = 0.017). Results for cultures in the presence of 100 nM UM171 are also shown. [Diagram 2] FIG. 2 shows the effect of pyrimidoindole compounds on the differentiation of CD56+ NK cells from NK precursor cells. (A) CD56+ NK cell yield per input cell (i.e., day 0 cells) after 14 days of culture of NK precursor cells in the presence of a range of concentrations of UM171. (B) CD56+ NK cell yield per input cell (i.e., day 0 cells) after 14 days of culture of NK precursor cells in the presence of a range of concentrations of UM729. At 1 μM, CD56+ NK cell yield is significantly increased compared to cultures lacking UM729 (paired t-test: p = 0.032). Results for cultures in the presence of 100 nM UM171 are also shown. [Diagram 3]FIG. 3 shows the effect of the presence or absence of pyrimidoindole compounds on the differentiation of CD56+ NK cells from NK precursor cells. (A) The results of the 100 nM data points in FIG. 1(A) and FIG. 2(A) were normalized to standard ("STD") cultures lacking the UM171 compound. n = 15, paired t-test on unnormalized data: p = 0.0001 for frequency of occurrence and p = 0.052 for amount of production (i.e., data points in FIG. 2(A) vs. STD). (B) The results of the 1 μM data points in FIG. 1(B) and FIG. 2(B) were normalized to STD cultures lacking the UM729 compound. n = 20, paired t-test on unnormalized data: p < 0.0001 for frequency of occurrence and p = 0.006 for amount of production. [Figure 4] Figure 4 shows the expression of natural cytotoxicity receptors on the surface of differentiated CD56+ NK cells. Cells were analyzed by flow cytometry for expression of (A) NKp46, (B) NKp44, and (C) NKp30. [Diagram 5] Figure 5 shows expression of phenotypic markers associated with more mature NK cells by differentiated CD56+ NK cells. Cells were analyzed by flow cytometry for expression of (A) NKG2D, (B) CD16, (C) CD94, (D) KIR, and (E) intracellular IFNγ. The histogram in (E) shows an overlay of gated PMA / ionomycin-stimulated CD56+ NK cells (black line only histogram) and unstimulated CD56+ NK cells (gray filled histogram). [Figure 6] Figure 6 shows that differentiated CD56+ NK cells do not express T cell markers. (A) NK precursor cells of a first donor cultured in the presence of UM171 do not express CD3. (B) NK precursor cells of a second donor cultured in the presence of UM171 do not express CD3. (C) NK precursor cells of a second donor cultured in the presence of UM729 do not express CD3. [Figure 7]Figure 7 shows that differentiated CD56+ NK cells are cytotoxic. Peripheral blood (PB) NK cells and monocytes were also co-cultured with calcein AM labeled K562 cells at an effector to target ratio of 5:1 as positive and negative controls, respectively. Results are expressed as % of specific lysis of K562 cells: [(test release - spontaneous release) x 100] / (maximum release - spontaneous release). Mean ± standard deviation is shown (CB CD34+ derived NK cells: n = 18, PB NK cells and monocytes: n = 7). [Figure 8] FIG. 8 shows that pyrimidoindole compounds have a specific effect on NK precursor cells but not on CD34+ HSPCs. Cord blood-derived CD34+ HSPCs were plated on day 0 and UM171 was added to the NK differentiation cultures for different periods: days 0 to 14 (+ / -); days 14 to 28 (- / +); or days 0 to 28 (+ / +). Cultures without UM171 were also performed: days 0 to 28 (- / -). The effect of UM171 addition on NK cell differentiation was evaluated (A) in terms of the frequency of CD56+ NK cells and (B) in terms of the yield of CD56+ NK cells per input cell (i.e., day 0 cells). [Figure 9] FIG. 9 shows that pyrimidoindole compounds inhibit the differentiation of CD34+ HSPCs into NK progenitors. CD7+CD5+ cells were cultured with or without UM171 and then analyzed on day 14 of the NK cell differentiation culture. The effect of UM171 addition on the differentiation of CD34+ cells into NK progenitors was assessed (A) in terms of the frequency of CD7+CD5+ NK progenitors and (B) in terms of the yield of CD7+CD5+ NK progenitors per input cell. [Figure 10]Figure 10 shows the phenotype of the intermediate population of cells differentiated from CD34+ HSPCs after 14 days of culture. (A) Cells were analyzed by flow cytometry for expression of CD56. (B) CD56- cells were analyzed by flow cytometry for expression or absence of CD7 and CD5, markers of NK precursor cells. [Figure 11] FIG. 11 shows that pyrimidoindole compounds promote the differentiation of CD7+CD5+ and CD7+CD5- NK progenitor cells into NK cells. In FIG. 10(B), each population of cells - CD7+CD5+, CD7+CD5-, CD7-CD5- - was sorted by flow cytometry and cultured in NK cell differentiation medium in the presence (square data points) or absence (circular data points) of UM729. The effect of UM729 addition on the differentiation into NK cells was evaluated (A) in terms of the frequency of CD56+ NK cells and (B) in terms of the yield of CD56+ NK cells per input progenitor cell (i.e., sorted day 14 cells). The horizontal lines are the average of three donors; each donor is represented by a symbol of different shading. [Figure 12] Figure 12 shows that the effect of pyrimidoindole compounds on NK cell differentiation is not reproduced when an aryl hydrocarbon receptor (AhR) antagonist is used. NK progenitor cells were cultured either in the presence or absence of 5 μM CH223191. The effect of CH223191 addition on NK cell differentiation was assessed (A) in terms of the frequency of CD56+ NK cells and (B) in terms of the yield of CD56+ NK cells per input cell (i.e., day 0 cells). [Figure 13]FIG. 13 shows that pyrimidoindole compounds selectively affect the CD34- subset of NK progenitors. Sorted populations of day 14 NK progenitors were plated in the presence (gray columns) or absence (white columns) of UM729 and cultured for 14 days. The effect of UM729 addition on NK cell differentiation was assessed (A) in terms of the frequency of CD56+ NK cells and (B) in terms of the yield of CD56+ NK cells per input progenitor cells. Each point represents an individual cord blood donor. [Figure 14] FIG. 14 shows that populations of NK precursor cells derived from four PSC lines, 1C, M001, H1, and F016, respond to pyrimidoindole compounds. PSC-derived CD34+ cells differentiated into populations of NK precursor cells were plated in the absence (-) or presence (+) of UM729 and cultured for 14 days. The effect of UM729 addition on differentiation into PSC-derived NK cells was assessed (A) in terms of the frequency of CD56+ PSC-derived NK cells and (B) in terms of the yield of CD56+ PSC-derived NK cells per input CD34+ cells. Each point represents an individual biological replicate. [Figure 15] FIG. 15 shows that pyrimidoindole compounds have a negative effect on the differentiation of a population of NK progenitor cells from PSC-derived CD34+ cells. CD34+ cells derived from four PSC lines, 1C, M001, H1, and F016, were plated in the absence (-) or presence (+) of UM729 and cultured for 14 days. The effect of UM729 addition on the differentiation of PSC-derived CD34+ cells into a population of PSC-derived NK progenitor cells was assessed (A) in terms of the frequency of CD5+CD7+ PSC-derived NK progenitor cells, and (B) in terms of the yield of CD5+CD7+ PSC-derived NK progenitor cells per input cell. Each point represents an individual biological replicate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description The present disclosure relates to media and methods for differentiating NK progenitor cells and to methods for obtaining differentiated NK cells.

[0028] As used herein, "hematopoietic stem and progenitor cells" or "HSPCs" refer to cells of the hematopoietic lineage that are capable of self-renewal and / or differentiation into more specialized cells of the hematopoietic lineage. HSPCs may be obtained from bone marrow (BM), umbilical cord blood (CB), peripheral blood (PB) from embryos to adults, thymus, peripheral lymph nodes, gastrointestinal tract, tonsils, pregnant uterus, liver, or any other tissue with a localized population of HSPCs. HSPCs may also be differentiated from pluripotent stem cells, such as induced pluripotent stem cells, embryonic stem cells, naïve stem cells, extended stem cells, etc. A distinguishing feature of human HSPCs is the expression of CD34, a transmembrane phosphorylated glycoprotein, and thus HSPCs are characterized by the expression of CD34. + Human HSPCs may be referred to as human HSPCs. Human HSPCs are defined by co-expression of CD45 and CD34, and may be further defined by a combination of markers, such as, for example, CD38, CD43, CD45RO, CD45RA, CD10, CD49f, CD59, CD90, CD109, CD117, CD133, CD166, HLA-DR, CD201, and integrin alpha 3, which may be used to distinguish HSPC subsets. HSPCs may lack expression or have only low expression of markers, such as, for example, glycophorin A, CD3, CD4, CD8, CD14, CD15, CD19, CD20, and CD56; such markers may be characteristic of mature blood cells.

[0029] As used herein, "natural killer cells" or "NK cells" refer to a type of lymphocyte of the hematopoietic lineage that can be derived from HSPCs. More specifically, NK cells can be derived from multipotent lymphoid progenitors (MLPs) or common lymphoid progenitors (CLPs). NK cells are typically characterized by the absence of T-cell and B-cell specific markers; expression of CD56 and CD16 (low affinity Fcγ receptor 3A, expressed on a subset of NK cells); and effector functions of the cells. More specifically, the effector functions of NK cells can include cytotoxicity and / or production of IFNγ. NK cells can be further characterized by the expression of activating and inhibitory receptors, termed killer immunoglobulin-like receptors (KIRs). Other activating receptors that NK cells may express include NKG2D, and natural cytotoxicity receptors (NCRs), including NKp30, NKp44, and NKp46. Differentiation of HSPCs into NK cells usually occurs via intermediate progenitors, such as NK precursors, but it is possible that HSPCs (or CD34 + It may also be possible to directly differentiate NK cells.

[0030] As used herein, "natural killer progenitor cell" or "NK progenitor cell" refers to a cell type that is more specialized than HSPC, but can further differentiate into NK cell. NK progenitor cell may be a direct descendant of HSPC or distant from HSPC. Furthermore, NK progenitor cell may directly differentiate into NK cell or may undergo one or more additional stages of differentiation before becoming NK cell. One example of NK progenitor cell is a cell that is positive for phenotypic markers CD7 and CD5. In another example, NK progenitor cell may be positive for CD7 but negative for CD5. Other phenotypic markers that NK progenitor cell may express include CD10, CD45RA, CD34, CD38, CD161, CD122, CD117, and / or integrin α4β7. CD34 may or may not be expressed in NK progenitor cell. As used herein, unless expressly specified, a population of NK precursor cells may refer to a homogenous population of cells or a heterogeneous population of cells capable of differentiating into NK cells.

[0031] As used herein, "pyrimidoindole compound" refers to a class of compounds that can be used to differentiate NK cells from a population of NK progenitor cells. In one embodiment, the pyrimidoindole compound can be pyrimido[4,5-b]indole. In a particular embodiment, the pyrimidoindole compound can be UM171, which has the formula C 25 H 27 N9(X ​​HBr [X H2O] or alternatively, (1r,4r)-N 1 -(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine. In another particular embodiment, the pyrimidoindole compound can be UM729, which has the formula C 20 H 25N5O2·XHCl [XH2O] or alternatively referred to as methyl 4-((3-(piperidin-1-yl)propyl)amino)-9H-pyrimido[4,5-b]indole-7-carboxylate. UM171 and UM729 are disclosed in U.S. Pat. No. 9,409,906, and UM729 is commercially available from STEMCELL Technologies.

[0032] Culture medium The medium of the present disclosure includes any medium that can be used to differentiate HSPCs into NK cells or can be used to differentiate a population of NK precursor cells into NK cells. Such differentiation into NK cells can refer to the differentiation of HSPCs into NK cells, which can include the induction of one or more populations of NK precursor cells between them. Alternatively, differentiation into NK cells can be the direct or indirect differentiation of a population of NK precursor cells into NK cells.

[0033] The medium of the present disclosure may contain serum or may be serum-free. In one embodiment, the medium of the present disclosure is serum-free. When the medium is serum-free, it may be necessary to include serum replacement supplements in such medium, such as BIT 9500 Serum Substitute (STEMCELL Technologies, Catalog No. 09500), or other commercially available serum replacement solutions. Alternatively, the components that are normally present in serum that are required to culture or differentiate any cell of the present disclosure can be added individually to the medium at acceptable concentrations.

[0034] The medium of the present disclosure may include a basal medium formulated to be suitable for culturing the cells of the present disclosure (e.g., HSPCs, NK progenitor cells, NK cells). The basal medium may be any basal medium that supports the culture of cells of the hematopoietic lineage. As a non-limiting example, the basal medium may be: StemSpan™ SFEM (STEMCELL Technologies, Catalog No. 09650), StemSpan™ SFEM II (STEMCELL Technologies, Catalog No. 09655), StemSpan™-ACF (STEMCELL Technologies, Catalog No. 09855), StemSpan™ H3000 (STEMCELL Technologies, Catalog No. 09850), or any other commercially available basal medium suitable for the purpose. Common ingredients used to formulate such basal media may include salts, buffers, lipids, amino acids, trace elements, certain proteins, etc.

[0035] The NK cell differentiation medium of the present disclosure may also include one or more pyrimidoindole compounds. In one embodiment, the NK cell differentiation medium includes only one pyrimidoindole compound. Examples of pyrimidoindole compounds include either UM171 or UM729. The concentration of the pyrimidoindole compound in the NK cell differentiation medium varies depending on the nature of the compound. For example, some pyrimidoindole compounds may be more potent than others. For example, UM171 has been reported to be approximately 10 times more potent than UM729. Thus, in order to observe the same effect in the differentiation of NK cells from a population of NK progenitor cells, it may be necessary to include approximately 10 times higher concentrations of UM729 relative to UM171. In one embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 1 nM to 10 μM. In a more specific embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 5 nM to 5 μM. In an even more specific embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 10 nM to 3 μM. In an even more specific embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 30 nM to 2 μM. In an even more specific embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 50 nM to 1 μM. In an even more specific embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 75 nM to 500 nM. In an even more specific embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 90 nM to 150 nM.

[0036] In an embodiment in which the pyrimidoindole compound is UM171, its concentration in the NK cell differentiation medium may be about 10 nM to 1000 nM, or about 20 nM to 500 nM, or about 50 nM to 200 nM. In a particular embodiment, the concentration of UM171 in the NK cell differentiation medium is about 100 nM.

[0037] In an embodiment in which the pyrimidoindole compound is UM729, its concentration in the NK cell differentiation medium may be about 50 nM to 10 μM, or about 100 nM to 5 μM, or about 250 nM to 2.50 μM. In a particular embodiment, the concentration of UM729 in the NK cell differentiation medium is about 1 μM.

[0038] To culture the cells of the present disclosure (e.g., HSPCs, NK progenitor cells, NK cells), it may be necessary to further add supplements to the NK cell differentiation medium of the present disclosure. The supplements added to the basal medium may vary depending on the particular type of cells to be cultured. In general, a non-exhaustive list of potential supplements includes one or more cytokines, one or more growth factors, or other proteins.

[0039] Specifically, SCF, FLT3L, IL-3, IL-2, IL-7, IL-15, IL-12, and IL-21 may be included in the NK cell differentiation medium. In one embodiment, the NK cell differentiation medium is supplemented with each of FLT3L, SCF, IL-3, IL-15, IL-2, IL7, IL-12, and IL-21. In one embodiment, the NK cell differentiation medium is supplemented with one or more of FLT3L, SCF, IL-3, IL-2, IL-15, IL-7, IL-12, and IL-21. In one embodiment, the NK cell differentiation medium is supplemented with one or more of FLT3L, SCF, IL-15, and IL-7. In one embodiment, the NK cell differentiation medium is supplemented with either FLT3L or SCF, and IL-15. In embodiments of NK cell differentiation media that include one or more of SCF, FLT3L, IL-3, IL-7, IL-2, IL-15, IL-12, and IL-21, such cytokines may be present at a concentration of about 1-1000 ng / mL, or about 1-100 ng / mL, or about 5-50 ng / mL, and IL-2 may be present at about 50-1500 IU / mL.

[0040] In some embodiments, any one or more of SCF, FLT3L, IL-3, IL-2, IL-7, IL-15, IL-12, and IL-21 may be omitted, but the efficacy of the NK cell differentiation medium may be compromised. In one embodiment, in order to differentiate NK cells from a population of NK precursor cells using the NK cell differentiation medium, the NK cell differentiation medium is required to contain IL-15.

[0041] Furthermore, the NK cell differentiation medium of the present disclosure may synergize with additional supplements. For example, on the one hand, stromal cells may be used with the cell culture medium of the present disclosure. Non-exhaustive examples of stromal cells include embryonic liver cell lines EL08.1D2, AFT024 cells, OP9 cells, or M2-10B4 cells. On the other hand, stromal cell-free culture approaches may be used with the cell culture medium of the present disclosure. Stromal cell-free culture systems may utilize media that has been preconditioned by stromal cells, or such systems may utilize stromal cell substitutes. Stromal cell substitutes may include one or more defined components that provide appropriate signals or binding sites to the cells under culture. Such components may be included in a coating applied to the inner culture surface of the culture vessel, or may be included on a solid surface suspended in the cell culture medium, for example, particles, beads, microcarriers, etc. A non-exhaustive list of such components may include: a coating of fibronectin, either full length or a fragment thereof, VCAM-1, immobilized Notch ligand, or a coating, such as, for example, StemSpan™ Lymphoid Differentiation Coating Supplement (STEMCELL Technologies, Cat. No. 09925). Alternatively, stromal cell substitutes may provide such components in soluble form in the cell culture medium, for example, by addition or as medium that has been pre-conditioned by stromal cells.

[0042] Although it may be necessary to include stromal cells or stromal cell substitutes to differentiate HSPCs into NK progenitor cells, stromal cells or stromal cell substitutes may not be essential to differentiate NK progenitor cells into NK cells. In a preferred embodiment, NK cell differentiation medium (i.e., medium for differentiating NK progenitor cells into NK cells) is not conditioned by contact with stromal cells or stromal cell substitutes. Thus, such NK cell differentiation medium may not have been previously or will not be contacted with stromal cells or stromal cell substitutes.

[0043] Compounds other than pyrimidoindole compounds have been reported to play a role in the culture of various types of hematopoietic cells. For example, compounds from the class of aryl hydrocarbon receptor antagonists may be included in the NK cell differentiation medium. Some examples of aryl hydrocarbon receptor antagonists include StemRegenin 1 (SR1) (STEMCELL Technologies, Catalog No. 72342) or CH223191 (STEMCELL Technologies, Catalog No. 72732). In an embodiment in which CH223191 is included in the NK cell differentiation medium, the compound may be present at a concentration ranging from about 100 nM to 10 μM, or ranging from about 500 nM to 8 μM, or ranging from about 1 μM to 6 μM. In a particular embodiment, the concentration of CH223191 is about 5 μM.

[0044] In one embodiment, the NK cell differentiation medium does not contain an aryl hydrocarbon receptor antagonist.

[0045] Thus, in one aspect of the present disclosure, an NK cell differentiation medium is provided. The NK cell differentiation medium can be used to differentiate NK cells from HSPCs or from a population of NK progenitor cells. The NK cell differentiation medium can include a basal medium (as described above) and a pyrimidoindole compound (as described above). In one embodiment, the basal medium is StemSpan™ SFEM II (STEMCELL Technologies, Cat. No. 09655) and the pyrimidoindole compound is either UM171 or UM729. Such NK cell differentiation medium is serum-free, while further including one or more of SCF, FLT3L, IL-15, IL-2, IL-3 IL-7, IL-12, and IL-21.

[0046] If it is important to obtain significant quantities of differentiated NK cells, it may be desirable to first culture and expand HSPCs before differentiating them into NK progenitors and NK cells.

[0047] HSPCs can be expanded by any known method known in the art. In one embodiment, HSPCs are expanded using StemSpan™ CD34 + Expansion Supplement (CD34 +HSPCs can be expanded to significant numbers using StemSpan™ SFEM II (STEMCELL Technologies, Catalog No. 09655) supplemented with StemSpan™ CC100 (STEMCELL Technologies, Catalog No. 02690) or StemSpan™ CC110 (STEMCELL Technologies, Catalog No. 02697) according to the manufacturer's protocol. Expansion of HSPCs using StemSpan™ SFEM II (STEMCELL Technologies, Catalog No. 09655) and supplemented with Expansion Supplement, or with any other suitable medium, can be further enhanced by adding a pyrimidoindole compound, such as UM171 or UM729, to the medium.

[0048] The concentration of the pyrimidoindole compound in the HSPC expansion protocol varies depending on the nature of the compound. For example, some pyrimidoindole compounds may be more potent than others. For example, UM171 has been reported to be approximately 10 times more potent than UM729. Thus, to observe the same effect with a different pyrimidoindole compound, it may be necessary to include a 10-fold higher concentration of UM729 relative to UM171. In one embodiment, the concentration of the pyrimidoindole compound in the HSPC expansion protocol may be 1 nM to 10 μM. In a more specific embodiment, the concentration of the pyrimidoindole compound in the HSPC expansion protocol may be 5 nM to 5 μM. In an even more specific embodiment, the concentration of the pyrimidoindole compound in the HSPC expansion protocol may be 10 nM to 3 μM. In an even more specific embodiment, the concentration of the pyrimidoindole compound in the HSPC expansion protocol may be 30 nM to 2 μM. In even more particular embodiments, the concentration of the pyrimidoindole compound in the HSPC expansion protocol can be 50 nM to 1 μM. In even more particular embodiments, the concentration of the pyrimidoindole compound in the HSPC expansion protocol can be 75 nM to 500 nM. In even more particular embodiments, the concentration of the pyrimidoindole compound in the HSPC expansion protocol can be 90 nM to 150 nM.

[0049] The medium for expanding HSPCs may further comprise an arylhydrocarbon receptor antagonist. Some examples of arylhydrocarbon receptor antagonists include stemregenin 1 (SR1) (STEMCELL Technologies, Catalog No. 72342) or CH223191 (STEMCELL Technologies, Catalog No. 72732). In the embodiment in which CH223191 is included in the HSPC expansion medium, the compound may be present at a concentration ranging from about 100 nM to 10 μM, or ranging from about 500 nM to 8 μM, or ranging from about 1 μM to 6 μM. In certain embodiments, the concentration of CH223191 is about 5 μM.

[0050] HSPCs, whether expanded as described above or not, can be cultured in a first medium to produce a temporary population of NK progenitor cells. The first medium (i.e., NK progenitor differentiation medium) can be essentially as described above for NK cell differentiation medium, and the addition of a pyrimidoindole compound is optional. In such an embodiment, the pyrimidoindole compound can be UM171 or UM729. In one embodiment, the NK progenitor medium is not supplemented with a pyrimidoindole compound.

[0051] The NK progenitor differentiation medium of the present disclosure may synergize with additional supplements. For example, on the one hand, stromal cells may be used with such cell culture medium. Non-exhaustive examples of stromal cells include embryonic liver cell lines EL08.1D2, AFT024 cells, OP9 cells, or M2-10B4 cells. On the other hand, stromal cell-free culture approaches may be used with such cell culture medium. Stromal cell-free culture systems may utilize media that have been preconditioned by stromal cells, or such systems may utilize stromal cell substitutes. Stromal cell substitutes may include one or more defined components that provide appropriate signals or binding sites to the cells under culture. Such components may be included in a coating applied to the inner culture surface of the culture vessel, or may be included on a solid surface suspended in the cell culture medium, for example, particles, beads, microcarriers, etc. A non-exhaustive list of such components may include: a coating of fibronectin, either full length or a fragment thereof, VCAM-1, immobilized Notch ligand, or a coating, such as, for example, StemSpan™ Lymphoid Cell Differentiation Coating Supplement (STEMCELL Technologies, Cat. No. 09925). Alternatively, stromal cell substitutes may provide such components in soluble form in the cell culture medium, for example, by addition or as medium that has been pre-conditioned by stromal cells.

[0052] In one embodiment, the NK progenitor differentiation medium is contacted with stromal cells (or the medium is conditioned by culturing stromal cells) or the medium includes a stromal cell substitute. For example, the stromal cell substitute can be: a coating of fibronectin, either full length or a fragment thereof, VCAM-1, immobilized Notch ligand, or a coating such as, for example, StemSpan™ Lymphoid Cell Differentiation Coating Supplement (STEMCELL Technologies, Catalog No. 09925).

[0053] Therefore, in another aspect of the present disclosure, there is provided a NK precursor cell differentiation medium as described above.Such medium can be used to differentiate HSPC into a population of NK precursor cells.Therefore, the induced NK precursor cells can continue to be cultured in the NK precursor cell differentiation medium to obtain differentiated NK cells, or the induced NK precursor cells can then be cultured in the above-mentioned NK cell differentiation medium.

[0054] method The method of the present disclosure includes such a step for differentiating NK cells from a population of NK precursor cells.The method of the present disclosure can also include such a step for differentiating NK cells from HSPCs, with or without passing through one or more intermediate populations of NK precursor cells.The method for differentiating NK cells disclosed herein is preferably an in vitro method.

[0055] The disclosed method can be started by providing a suitable starting population of cells. When starting from HSPCs, HSPCs can be purchased commercially, isolated from blood or tissue samples, or derived (i.e., differentiated from) pluripotent stem cells, such as induced pluripotent stem cells. Regardless of the source of HSPCs, it may be necessary to carry out one or more purification experiments to remove as many contaminating cells as possible. Contaminating cells can be removed from samples containing HSPCs by any known approach. For example, such samples can be subjected to density gradient centrifugation, or positive and / or negative cell selection. An exemplary approach involves conventional negative selection of mononuclear cells using RosetteSep™ reagents, e.g., RosetteSep™ Cord Blood CD34 Pre-Enrichment Cocktail (STEMCELL Technologies, Cat. No. 15896C), followed by depletion of CD34 cells using an appropriate EasySep™ reagent, e.g., EasySep™ Human CD34 Positive Selection Kit II (STEMCELL Technologies, Cat. No. 17896). + and positive selection of HSPCs.

[0056] Depending on the number of NK cells required, it may be desirable to first expand the HSPCs (with or without passing through one or more NK progenitor populations) before differentiating the HSPCs into NK cells. The HSPCs may be expanded using any known approach. An exemplary approach for expanding HSPCs is to use an appropriate basal medium and StemSpan™ CD34 +The method may include culturing HSPCs in the presence of Expansion Supplement (STEMCELL Technologies, Catalog No. 02691), or StemSpan™ CC100 (STEMCELL Technologies, Catalog No. 02690), or StemSpan™ CC110 (STEMCELL Technologies, Catalog No. 02697). Using the above-mentioned approach, suitable basal media may include the following serum-free expansion media: StemSpan™ SFEM (STEMCELL Technologies, Catalog No. 09650), StemSpan™ SFEM II (STEMCELL Technologies, Catalog No. 09655), StemSpan™-ACF (STEMCELL Technologies, Catalog No. 09855), StemSpan™ H3000 (STEMCELL Technologies, Catalog No. 09850).

[0057] Whether or not the HSPCs have been subjected to prior expansion, the HSPCs may be differentiated into a population of NK progenitor cells. The population of NK progenitor cells may be a homogenous population of cells or may be a heterogenous population of cells. For example, a homogenous population of NK progenitor cells may be a homogenous population of cells that is CD7 + CD5 + or by phenotype of CD7 + CD5 - and the heterogeneous population of NK progenitors can be characterized by the phenotype of CD7 + CD5 + and phenotype, CD7 + CD5 - and phenotype, CD7 - CD5 - In one embodiment, the NK progenitor cells (e.g., CD7 + CD5 + cells, CD7 + CD5 - Cells, and / or CD7 - CD5 - Cells) are CD34 +, or CD34 - It may be either of the following.

[0058] In some embodiments, the induction of NK precursor cells from HSPCs is not essential. In one embodiment, it may be desirable to use the medium of the present disclosure to directly differentiate HSPCs into NK cells. In one embodiment, it may be desirable to use the medium of the present disclosure to directly differentiate pluripotent stem cells into NK cells, where the pluripotent stem cells include but are not limited to induced pluripotent stem cells. In one embodiment, it may be desirable to use the medium of the present disclosure to transdifferentiate cells corresponding to a specific germ layer into NK cells. In some of these embodiments, it may be necessary to further add additional factors, or cytokines and / or transcription factors, either in the form of proteins or nucleic acids, or in the form of activators of such transcription factors, to the medium.

[0059] When differentiating HSPCs into a population of NK progenitor cells, this can be performed by contacting the population of HSPCs with a suitable medium. The suitable medium can be any known medium capable of differentiating HSPCs into a population of NK progenitor cells. In one embodiment, HSPCs are differentiated into a population of NK progenitor cells by contacting them with NK progenitor differentiation medium as described above. In a different embodiment, the NK progenitor differentiation medium can include StemSpan™ SFEM II (STEMCELL Technologies, Catalog No. 09655) supplemented with Lymphoid Progenitor Expansion Supplement (STEMCELL Technologies, Catalog No. 09915). In one embodiment, HSPCs contacted with NK progenitor differentiation medium can be cultured with StemSpan™ Lymphoid Cell Differentiation Coating Supplement (STEMCELL Technologies, Catalog No. 09925) or with any other known coating useful for differentiating HSPCs into a population of NK progenitor cells.

[0060] In another aspect, the method of the present disclosure may begin by providing a population of NK progenitor cells, where, for example, the population may be differentiated from or derived from HSPCs or induced pluripotent stem cells, for example using an NK progenitor differentiation medium as described above, or, for example, the population may be directly isolated from a primary sample (such as umbilical cord blood, peripheral blood, bone marrow, thymus, uterus, liver, gastrointestinal tract, or secondary lymphoid tissues) based on distinguishing phenotypic markers (such as CD7 and / or CD5 ± CD34). In one embodiment, it may be possible to induce (i.e., differentiate) a population of NK progenitor cells, if such a population of NK progenitor cells is derived from a primary sample or derived from pluripotent stem cells, such as induced pluripotent stem cells or ES cells. In one embodiment, the population of NK progenitor cells may be isolated from a primary sample. Approaches for isolating cells are known in the art and may include either positive or negative cell selection, or both. As a non-limiting example, lineage positive cells may be depleted (e.g., using RosetteSep™ Human Hematopoietic Progenitor Cell Enrichment Cocktail, STEMCELL Technologies, Cat. No. 15066, etc.) and NK progenitor cells may be isolated by selecting cells expressing one or more of the following markers: CD34, CD38, CD45RA, CD10, CD7, and CD5. Thus, in one embodiment, the population of NK progenitor cells expresses CD7. In another embodiment, the population of NK progenitor cells may express both CD7 and CD5. In another embodiment, the population of NK progenitor cells may only express CD7 and not CD5. In another embodiment, the population of NK progenitor cells may or may not express CD34.

[0061] In another embodiment, the population of NK progenitor cells can be differentiated from pluripotent stem cells (PSCs). Differentiation of PSC-derived NK progenitor cells involves differentiation of one or more intermediate cell populations, such as PSC-derived CD34 + Differentiation of HSPCs etc. may be necessary.

[0062] The differentiation of a population of NK precursor cells into NK cells can be carried out by contacting a population of NK precursor cells in culture with a pyrimidoindole compound at a concentration and for a period of time sufficient to produce NK cells. The pyrimidoindole compound can be included in any medium that supports a population of NK precursor cells. In one embodiment, the pyrimidoindole compound is included in the NK cell differentiation medium according to the present disclosure.

[0063] As mentioned above, the concentration of the pyrimidoindole compound in the NK cell differentiation medium varies depending on the nature of the compound. For example, some pyrimidoindole compounds may be more potent than others. For example, UM171 has been reported to be approximately 10 times more potent than UM729. Thus, in order to observe the same effect in the differentiation of NK cells from a population of NK progenitor cells, it may be necessary to include a 10-fold higher concentration of UM729 relative to UM171. In one embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 1 nM to 10 μM. In a further specific embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 5 nM to 5 μM. In an even more specific embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 10 nM to 3 μM. In an even more specific embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 30 nM to 2 μM. In an even more specific embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 50 nM to 1 μM. In an even more specific embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 75 nM to 500 nM. In an even more specific embodiment, the concentration of the pyrimidoindole compound in the NK cell differentiation medium may be 90 nM to 150 nM.

[0064] As also described above, in an embodiment where the pyrimidoindole compound is UM171, its concentration in the NK cell differentiation medium may be about 10 nM to 1000 nM, or about 20 nM to 500 nM, or about 50 nM to 200 nM. In a particular embodiment, the concentration of UM171 in the NK cell differentiation medium is about 100 nM.

[0065] As also described above, in embodiments where the pyrimidoindole compound is UM729, its concentration in the NK cell differentiation medium may be about 50 nM to 10 μM, or about 100 nM to 5 μM, or about 250 nM to 2.50 μM. In certain embodiments, the concentration of UM729 in the NK cell differentiation medium is about 1 μM.

[0066] Other potential embodiments of the NK cell differentiation medium are as described above.

[0067] NK cell differentiation medium containing pyrimidoindole compounds can increase the frequency and / or production of NK cells differentiated from a population of NK precursor cells. During differentiation into NK cells, as the frequency and production of NK cells increases, the frequency and / or production of NK precursor cells can decrease. Therefore, it may be important to include pyrimidoindole compounds in NK cell differentiation medium to improve the efficacy of differentiating NK cells from a population of NK precursor cells in culture. The effect of pyrimidoindole compounds may be specific to NK precursor cells, because including pyrimidoindole compounds in a medium (such as NK precursor differentiation medium) for differentiating NK precursor cells from HSPCs may have a global inhibitory effect on the production and frequency of NK precursor cells induced thereby. However, inclusion of a pyrimidoindole compound in a medium for differentiating NK cells from a population of NK progenitor cells in culture (e.g., NK cell differentiation medium) can rescue or offset the inhibitory effect of the pyrimidoindole compound that may be included in a medium for differentiating NK progenitor cells from a population of HSPCs (e.g., NK progenitor differentiation medium), whether primary cells or cells differentiated therefrom, such as pluripotent stem cells, e.g., induced pluripotent stem cells or ES cells.

[0068] When differentiating NK cells to produce NK cells by providing a population of NK precursor cells and contacting such population of NK precursor cells in culture with a pyrimidoindole compound, the providing and contacting steps may be performed in the presence or absence of stromal cells or stromal cell substitutes. In one embodiment, the population of NK precursor cells is differentiated into NK cells in the absence of stromal cells and stromal cell substitutes.

[0069] Furthermore, when NK cells are differentiated by providing a population of NK progenitor cells and contacting such population of NK progenitor cells in culture with a pyrimidoindole compound to produce NK cells, the providing and contacting can be performed in the presence or absence of an arylhydrocarbon receptor agonist, such as stemregenin 1 (SR1) (STEMCELL Technologies, Catalog No. 72342) or CH223191 (STEMCELL Technologies, Catalog No. 72732).In one embodiment, the population of NK progenitor cells is differentiated into NK cells in the absence of an arylhydrocarbon receptor antagonist.

[0070] The step of contacting the population of NK progenitor cells in culture with the pyrimidoindole compound can be any length of time sufficient to produce NK cells from the population of NK progenitor cells. Considering that the pyrimidoindole compound in the basal medium (or other components in the basal medium or in the supplemented basal medium) can be depleted over time, it may be necessary to perform medium change at regular intervals, such as every day, every other day, every 2nd day, every 3rd day, every 4th day, every 5th day, every 6th day, every 7th day, or less frequently, or at different frequencies, such as every 1-2 days, every 2-3 days, or every 3-4 days. The number of medium changes performed can be directly correlated with the duration of the method for differentiating into NK cells. In one embodiment, the duration of the method for differentiating into NK cells from NK progenitor cells (i.e., the duration of contacting the population of NK progenitor cells with the pyrimidoindole compound) is about one week. In another embodiment, the duration of the method for differentiating NK cells from NK precursor cells is at least 1 week. In another embodiment, the duration of the method for differentiating NK cells from NK precursor cells is longer than 1 week. In another embodiment, the duration of the method for differentiating NK cells from NK precursor cells is about 2 weeks. In another embodiment, the duration of the method for differentiating NK cells from NK precursor cells is longer than 2 weeks.

[0071] By contacting a population of NK precursor cells in culture with a pyrimidoindole compound, the frequency of NK cells can be increased. By increasing the frequency of NK cells, we mean an increase in the proportion of NK cells as a percentage of all cells in a culture at a time point compared to an earlier time point. For example, when expression of CD56 is used as a measure of differentiated NK cells, the frequency of NK cells at a given time point is determined by the following formula: CD56 + Number of cells / (CD56 + Number of cells + CD56 - number of cells).

[0072] Also, by contacting a population of NK precursor cells in culture with a pyrimidoindole compound, the number (i.e., production) of NK cells can be increased. By increasing the number of NK cells, we mean an increase in the number of NK cells at a time point compared to an earlier time point. For example, if expression of CD56 is used as a measure of differentiated NK cells, the number of NK cells at a given time point can be increased by increasing the expression of CD56. + The number of cells is determined by counting.

[0073] NK cells differentiated according to the methods disclosed herein may express the phenotypic marker CD56. For example, about 40% of the differentiated NK cells of the present disclosure express CD56 + Alternatively, about 50% of the differentiated NK cells of the present disclosure may be CD56 + Alternatively, about 60% of the differentiated NK cells of the present disclosure may be CD56 + Alternatively, about 70% of the differentiated NK cells of the present disclosure may be CD56 + Alternatively, more than 70% of the differentiated NK cells of the present disclosure may be CD56 + Alternatively, more than 80% of the differentiated NK cells of the present disclosure may be CD56 + In certain embodiments, >70% of the differentiated NK cells of the present disclosure may be CD56 + It could be.

[0074] NK cells differentiated according to the methods disclosed herein may also express natural cytotoxicity markers, such as NKp30, and / or NKp44, and / or NKp46. Thus, the differentiated NK cells of the present disclosure may be cytotoxic. In standard cytotoxicity assays, the differentiated NK cells of the present disclosure may exert cytotoxicity against target cells. In certain embodiments, the target cells may be K562 cell lines, and the differentiated NK cells of the present disclosure may acquire killing capacity comparable to NK cells isolated from peripheral blood of adult donors. For example, NK cells isolated from peripheral blood of adult donors may achieve a specific lysis percentage of about 90%, or about 80%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%. In certain embodiments, the differentiated NK cells of the present disclosure and the NK cells isolated from the peripheral blood of adult donors each acquire about 70% killing capacity. In some embodiments, it may be desirable to first activate the differentiated NK cells before performing a killing assay. Those skilled in the art are familiar with how to activate NK cells, which may include exposing the NK cells to IL-2 or IL-15 for a sufficient period of time.

[0075] NK cells differentiated according to the methods disclosed herein may also express other markers associated with mature NK cells, such as, for example, NKG2D, and / or CD16, and / or CD94, and / or KIR.

[0076] NK cells differentiated according to the methods disclosed herein may also produce IFNγ upon appropriate stimulation, e.g., with PMA / ionomycin. Intracellular IFNγ may be detected following treatment with Brefeldin A. In one embodiment, about 30% of differentiated NK cells of the present disclosure that are appropriately stimulated produce intracellular IFNγ. In another embodiment, about 40% of differentiated NK cells of the present disclosure that are appropriately stimulated produce intracellular IFNγ. In another embodiment, about 50% of differentiated NK cells of the present disclosure that are appropriately stimulated produce intracellular IFNγ. In another embodiment, about 60% of differentiated NK cells of the present disclosure that are appropriately stimulated produce intracellular IFNγ. In another embodiment, about 70% of differentiated NK cells of the present disclosure that are appropriately stimulated produce intracellular IFNγ. In another embodiment, about 80% of differentiated NK cells of the present disclosure that are appropriately stimulated produce intracellular IFNγ. In another embodiment, about 90% of the differentiated NK cells of the present disclosure that are appropriately stimulated produce intracellular IFNγ. In another embodiment, greater than 90% of the differentiated NK cells of the present disclosure that are appropriately stimulated produce intracellular IFNγ.

[0077] Importantly, the majority of NK cells differentiated according to the methods disclosed herein do not express some markers associated with T cell lineage. In one embodiment, less than 5% of the differentiated NK cells of the present disclosure express CD3. In another embodiment, less than 4% of the differentiated NK cells of the present disclosure express CD3. In another embodiment, less than 3% of the differentiated NK cells of the present disclosure express CD3. In another embodiment, less than 2% of the differentiated NK cells of the present disclosure express CD3. In another embodiment, less than 1% of the differentiated NK cells of the present disclosure express CD3. The absence or essentially absence of CD3 expression in a differentiated NK cell of the present disclosure indicates that the cell is not a T cell or an NKT cell.

[0078] By carrying out the above-mentioned method, it may be possible to obtain a large amount of in vitro differentiated NK cells. Such in vitro differentiated NK cells may be used for subsequent cell therapy applications. Before using such in vitro differentiated NK cells, it may be desirable to make such NK cells transgenic NK cells, for example, by gene editing techniques. Thus, when it is desired to produce transgenic NK cells, it may be desirable to clonally expand the transgenic HSPCs or transgenic NK progenitor cells before differentiating the population of NK progenitor cells into NK cells using the media and methods of the present disclosure.

[0079] The following non-limiting examples are illustrative of the present disclosure. EXAMPLES

[0080] Example 1: Cell preparation and culture Human CB samples were obtained from Bloodworks NW (Seattle, WA) and were cloned using CD34 + Cells were isolated using EasySep™ Human Cord Blood CD34 Positive Selection Kit II (STEMCELL Technologies, Cat. No. 17896). + The purity of the cells is typically greater than 90%.

[0081] Wells of a 24-well plate were coated with StemSpan™ Lymphoid Cell Differentiation Coating Supplement (STEMCELL Technologies, Cat. No. 09925) and 5000 CD34 cells / well were added. + Cells were plated. CD34 +Cells were cultured in StemSpan™ SFEM II (STEMCELL Technologies, Cat. No. 09605, 09655) supplemented with Lymphoid Progenitor Expansion Supplement (STEMCELL Technologies, Cat. No. 09915) for up to 14 days with medium changes every 3-4 days.

[0082] After 14 days of culture, CD34 + Cell-derived NK progenitors were harvested. 50,000 NK progenitors were plated per well in uncoated 24-well tissue culture plates. Plated NK progenitors were cultured in NK cell differentiation medium (i.e., StemSpan™ SFEM II (STEMCELL Technologies, Cat. No. 09605, 09655) supplemented with NK Cell Differentiation Supplement (STEMCELL Technologies, Cat. No. 09950) for up to 2 weeks and then cultured for an additional 2 weeks with medium changes every 3-4 days. UM729 or UM171 was added to the supplemented NK cell differentiation medium from days 14 to 28. Cells were harvested on day 28 for analysis or subsequent applications.

[0083] Harvested cells were stained and measured by flow cytometry for expression of CD56, a NK cell lineage marker. Dead cells were excluded by light scatter profile and 7-AAD or DRAQ7 staining. Results in Figures 1 and 2 under the condition "0" (plotted on the x-axis) represent the CD56 expression that results from experiments following the protocol described above but in the absence of UM171 or UM729. + NK cell frequency and yield are shown. Cell counts were obtained using a NucleoCounter NC250. Yields were calculated by dividing the resulting number of cells by the number of cells initially plated. Yields of specific cell types were obtained by multiplying the total yield by the frequency of the cells.

[0084] Example 2: Effect of pyrimidoindole compounds on differentiation into NK cells Cells were cultured as described in Example 1, except that culture of NK precursor cells in NK cell differentiation medium for up to 2 weeks (i.e., culture days 14 to 28) was performed in the presence or absence of UM171 or UM729.

[0085] The effects of UM171 and UM729 were tested over the following range of concentrations: 0 nM, 50 nM, 100 nM, 150 nM, and 200 nM for UM171; and 0 μM, 0.25 μM, 0.5 μM, 1 μM, and 2 μM for UM729. The results of these experiments are summarized in Figures 1 and 2.

[0086] CD56 + The frequency of NK cells appeared to be greatest at a concentration of 100 nM for UM171 (Fig. 1A) and at a concentration of 1 μM for UM729 (Fig. 1B). + NK cell production also appeared to be maximal at a concentration of 100 nM for UM171 (Figure 2A) and at a concentration of 1 μM for UM729 (Figure 2A). Based on these results, a concentration of 100 nM for UM171 and a concentration of 1 μM for UM729 were selected for further experiments.

[0087] Thus, the addition of UM171 or UM729 to differentiation cultures of NK precursor cells increased the uptake of CD56 as the concentrations of these molecules increased. + The frequency of NK cells was increased: up to 100 nM for UM171 and up to 1 μM for UM729. Overall, the mean frequency and mean yield of NK cells was increased by an average of about 30% and 2-fold in culture conditions containing 100 nM UM171 (FIG. 3A) or 1 μM UM729 (FIG. 3B) compared to culture conditions without pyrimidoindole compounds.

[0088] Example 3: Expression of natural cytotoxicity receptors in differentiated NK cells In the presence of 1 μM UM729, CD56 + NK cells were differentiated and analyzed by flow cytometry for expression of known NK cell markers on day 28. Cells were stained with fluorescently conjugated antibodies against the indicated NK cell markers for 15 minutes at 4°C. Non-specific binding was blocked with FcR blockers and 5% human or rat serum. Dead cells were excluded by light scatter profile and 7-AAD or DRAQ7 staining. Prepared samples were analyzed by flow cytometry.

[0089] The results show that: approximately 70% of the analyzed cells co-express CD56 and NKp46 (Figure 4A); approximately 80% of the analyzed cells co-express CD56 and NKp44 (Figure 4B); and approximately 80% of the analyzed cells co-express CD56 and NKp30 (Figure 4C).

[0090] Example 4: Expression of markers associated with more mature NK cells in differentiated NK cells In the presence of 1 μM UM729, CD56 + NK cells were differentiated and analyzed by flow cytometry on day 28 for the expression of known NK cell markers. Cells were stained essentially as described in Example 3 for the markers described in this example. Staining for KIR molecules was performed using a combination of two clones of antibodies, 180704 and HP-MA4, each recognizing a different subset of KIR molecules. Dead cells were excluded by light scattering profile and 7-AAD or DRAQ7 staining.

[0091] The results show that: approximately 50% of the analyzed cells co-express CD56 and NKG2D (Figure 5A); approximately 17% of the analyzed cells co-express CD56 and CD16 (Figure 5b); approximately 18% of the analyzed cells co-express CD56 and CD94 (Figure 5c); and approximately 10% of the analyzed cells co-express CD56 and KIR (Figure 5d).

[0092] Differentiated CD56 + IFNγ production by NK cells was also measured. For intracellular staining of IFNγ, differentiated CD56 + NK cells were stimulated with PMA / ionomycin for 2 hours. Brefeldin A was added and the cells were incubated for an additional 2 hours. Cells were harvested and stained for CD56 and Zombie NIR viability dye, then fixed, permeabilized, and stained for intracellular IFNγ. The results in Figure 5E show that differentiated CD56 + Approximately 58% of NK cells express intracellular IFNγ upon stimulation.

[0093] Example 5: Lack of expression of T cell markers in differentiated cells In the presence of pyrimidoindole compounds, CD56 was expressed as described in Examples 1 and 2. + NK cells were differentiated and analyzed by flow cytometry for expression of T cell markers on day 28. Analyzed cells were stained for CD3 essentially as described in Example 3. Dead cells were excluded by light scatter profile and 7-AAD or DRAQ7 staining.

[0094] CD3 expression was virtually absent in cells differentiated from the first donor's cord blood sample and cultured in the presence of 100 nM UM171 (FIG. 6A). Similarly, CD3 expression was virtually absent in cells differentiated from the second donor's cord blood sample and cultured in the presence of 100 nM UM171 (FIG. 6B) or 1 μM UM729 (FIG. 6C).

[0095] These results suggest that differentiated CD56 + This suggests that the cells are neither T cells nor NKT cells.

[0096] Example 6: Differentiated CD56 + NK cells are cytotoxic In the presence of 1 μM UM729, CD56 + NK cells were differentiated and analyzed on day 28 for their cytotoxicity against K562 target cells.

[0097] In vitro generated CD56 for cytotoxicity assay + NK cells were co-cultured with calcein AM-labeled K562 cells at a 5:1 effector (NK cells) to target (K562 cells) ratio for 4 h, and supernatants were analyzed for release of fluorescent calcein from killed target cells.

[0098] Peripheral blood (PB) NK cells and monocytes isolated using EasySep™ (STEMCELL Technologies, catalog numbers 17955 and 19359, respectively) were also co-cultured with the labeled K562 cells at the same ratios as above as positive and negative controls, respectively. PB NK cells were cultured overnight in the medium of Example 2, except that it did not contain the pyrimidoindole compound, while PB monocytes were cultured overnight in StemSpan™ SFEM II alone.

[0099] To detect spontaneous release of calcein, control wells containing only calcein AM labeled K562 targets were set up. To measure maximum release, labeled K562 cells were treated with 1% Triton™ X-100. After incubation, the plate was centrifuged at 500 xg for 5 min, and 100 μL of the supernatant was transferred to a black plate and analyzed using a SpectraMax® microplate reader (excitation 485 nm / emission 530 nm). Results are expressed as % of specific lysis: [(test release - spontaneous release) x 100] / (maximum release - spontaneous release).

[0100] FIG. 7 shows that NK cells differentiated as described herein are capable of killing K562 target cells, and their killing capacity is similar to that of NK cells isolated from peripheral blood of adult donors.

[0101] Example 7: The effect of pyrimidoindole compounds is specific to NK precursor cells CD34 + CB cells were isolated and cultured as described in Example 1. CD34 + To examine the effect of UM171 on the differentiation of HSPCs into NK cells, different media were used for cultures from days 0 to 14 and days 14 to 28 as in Examples 1 and 2, except that the medium from days 0 to 14 was further supplemented with 100 nM UM171.

[0102] In these experiments, UM171 was added to the cultures at different time points: days 0 to 14 (+ / -); days 14 to 28 (- / +); or days 0 to 28 (+ / +). Cultures without UM171 were also set up (- / -). Cells were analyzed for either CD56 expression at day 28 (Figure 8) or CD7 and CD5 expression at day 14 (Figure 9).

[0103] Cultures exposed to UM171 from days 14 to 28 (- / +) had increased frequency and production of NK cells when compared to cultures lacking UM171 (- / -) during this period, and cultures exposed to UM171 from days 0 to 14 (+ / -) had increased CD56 + Cell frequency and production were reduced (FIGS. 8A and 8B). The (+ / +) condition indicates that inclusion of UM171 between days 14 and 28 can compensate for the effect of this compound on cultures from days 0 to 14.

[0104] 9A and 9B show the CD34 + Results of HSPC cultures are shown. At day 14, CD7 + CD5 + A clear decrease in the frequency and production of NK precursor cells was observed (FIGS. 9A and 9B).

[0105] Taken together, these data suggest that UM171 inhibits CD34 + Promoting the generation of NK progenitor cells from HSPCs do not The results demonstrate that UM171 promotes (and can actually inhibit) differentiation of NK precursor cells into NK cells (Figure 8) whereas UM171 inhibits (and can actually inhibit) differentiation of NK precursor cells (Figure 8) (Figure 8 and 9).

[0106] Example 8: Phenotypic characterization of NK progenitor cells As described in Example 1, CD34 + CB cells were isolated and cultured up to day 14. After 14 days of culture, CB CD34 + NK progenitor cells derived from HSPCs were stained with antibodies against the markers CD5, CD7, and CD56 essentially as described in Example 3. CD56 was stained using a BD FACSAria Fusion fluorescence-activated cell sorter. - Cells were gated and CD7 + CD5 -cells, CD7 + CD5 + cells, CD5 - CD7 - The cells were sorted.

[0107] After 2 weeks of culture, CB CD34 + The majority of cells differentiated from cells expressing CD56 - (FIG. 10A), and these cells were CD7 + CD5 - cells, CD7 + CD5 + cells, and CD7 - CD5 - It comprises a heterogeneous population consisting essentially of cells.

[0108] To determine which subset of the day 14 NK cell progenitor population is targeted by pyrimidoindole compounds, we used CD7 + CD5 - , CD7 + CD5 + , and CD7 - CD5 - The sorted populations were cultured for an additional 14 days in NK cell differentiation medium without or with UM729, respectively.

[0109] The results are CD7 + CD5 + Cells and CD7 + CD5 - These results show that CD7 cells respond to UM729 and differentiate into NK cells at higher frequency (FIG. 11A) and higher yield (FIG. 11B) compared to culture conditions lacking UM729. - CD5 - The cells did not expand and differentiate into NK cells either in the presence or absence of UM729. + CD5 + The cells are CD7 + CD5 - NK cells at a higher frequency and abundance compared to UM729 cells, and thus this population responds to UM729 with CD56 +This appears to be the major population of NK precursor cells that differentiate into NK cells (Figure 11).

[0110] Example 9: Arylhydrocarbon receptor antagonists negatively affect NK cell differentiation Compounds other than pyrimidoindole compounds, such as aryl hydrocarbon receptor (AhR) antagonists, have been reported to expand HSPCs in culture, so the effect of AhR antagonists on the differentiation of NK progenitors was also tested.

[0111] As described in Example 1, CD34 + CB cells were isolated and cultured until day 14. On day 14, NK progenitor cells were further cultured in NK cell differentiation medium essentially as described in Example 1, except that no pyrimidoindole compound was added, but in the presence or absence of 5 μM CH223191.

[0112] Data show that the addition of CH223191 to differentiation cultures of NK progenitor cells inhibits CD56 + 12A) and production (FIG. 12B) of NK cells, suggesting that the observed effects on differentiation of NK precursor cells into NK cells are specific to pyrimidoindole compounds, but not AhR antagonists.

[0113] Example 10: CD34 + NK cell precursors do not respond to pyrimidoindole compounds As described in Example 1, CD34 + CB cells were isolated and cultured up to day 14. After 14 days of culture, CB CD34 + NK progenitor cells derived from HSPCs were stained with antibodies against the markers CD5, CD7, CD34, and CD56 essentially as described in Example 3. CD56 was stained using a BD FACSAria Fusion fluorescence-activated cell sorter. - Cells were gated and CD34 + CD7 + CD5- and CD34 + CD7 + CD5 + , and CD34 - CD7 + CD5 - and CD34 - CD7 + CD5 + was sorted. CD7 - Cells were sorted as a negative control.

[0114] To determine which subset of the day 14 NK cell progenitor population is targeted by pyrimidoindole compounds, we used CD34 + CD7 + CD5 - and CD34 + CD7 + CD5 + , and CD34 - CD7 + CD5 - and CD34 - CD7 + CD5 + The sorted populations were cultured for an additional 14 days in NK cell differentiation medium with or without UM729, respectively.

[0115] The results are as follows: - CD7 + CD5 - Cells and CD34 - CD7 + CD5 + We show that only CD34+ cells respond to UM729 and differentiate into NK cells at higher frequency (FIG. 13A) and yield (FIG. 13B) compared to culture conditions lacking UM729. Thus, the more immature CD34+ cells + The NK progenitor subset does not appear to be responsive to the effects of UM729.

[0116] Example 11: Effect of pyrimidoindole compounds on differentiation of NK cells from PSCs Three induced pluripotent stem cell (iPSC) lines - WLS-1C, STiPSC M001, and STiPS F016 - and one embryonic stem cell (ESC) line - H1 - were maintained in mTeSR™ 1. PSCs were harvested and dissociated into a single cell suspension using Accutase and filtered using a 37 μm reversible strainer (STEMCELL Technologies).

[0117] Single cell suspensions of PSCs were seeded into AggreWell plates (STEMCELL Technologies) to form aggregates of 500 cells. The seeded cells were cultured in mesoderm differentiation medium and 10 μM Y027632 (STEMCELL Technologies). After 3 days of culture (i.e., day 3), the medium was changed to induce differentiation into hematopoietic lineages. After 7 days of culture (i.e., day 10), the aggregates were harvested and dissociated using a solution containing collagenase II and trypsin.

[0118] Dissociated cells were purified by CD34 cloning using the EasySep™ Human CD34 Positive Selection Kit II (STEMCELL Technologies, Cat. No. 17856). + Enriched for cells. CD34 + Enriched CD34 cells to differentiate into NK progenitor cells + Cells were seeded at 5 x 10 cells onto plates coated with StemSpan™ Lymphoid Differentiation Coating Material (STEMCELL Technologies) in StemSpan™ SFEM II medium (STEMCELL Technologies) supplemented with StemSpan™ Lymphoid Progenitor Expansion Supplement (STEMCELL Technologies). 4 Cells / mL were plated and cultured for 14 days, with half-medium changes performed every 3-4 days.

[0119] After 14 days of culture, the NK progenitor population was transferred to uncoated plates at 1 x 10 cells in NK cell differentiation medium in either the presence or absence of UM729. 5 The cells were replated at 1000 cells / mL and cultured for an additional 14 days.

[0120] For CD56, cells were stained essentially as described in Example 3, and CD56 + Regarding the frequency of cell occurrence, and the input 5 x 10 4 PSC-derived CD34 + CD56 per cell + The cells were analyzed for their production. + Both the frequency (FIG. 14A) and yield (FIG. 14B) of cells were increased after culturing PSC-derived NK progenitor populations in the presence of UM729 compared to culturing such cells in the absence of UM729. Thus, the presence of UM729 between days 24 and 38 in PSC-derived NK progenitor cell cultures enhanced the expression of CD56 + The frequency and production of cells increases.

[0121] Example 12: Pyrimidoindole compounds inhibit PSC-derived CD34 + Inhibits differentiation of cells into NK cell precursors PSC-derived CD34 + The cells were generated as described in Example 11. PSC-derived CD34 cells were cultured using a 5% HO-immunoglobulin (HO)-immunoglobulin (IPC)-immunoglobulin (IMG ... + Cells were cultured as described in Example 11.

[0122] Cells were stained for CD5 and CD7, and CD5 was stained for CD7 essentially as described in Example 3. + CD7 + Regarding the frequency of cell occurrence, and the input 5 x 10 4 PSC-derived CD34 + CD5 per cell + CD7 +The cells were analyzed for their production. + CD7 + Both the frequency (Figure 15A) and yield (Figure 15B) of PSC-derived CD34 + After culturing the cells, the PSC-derived CD34 + The presence of UM729 between days 10 and 24 of cell cultures increased the CD5 + CD7 + The frequency and production of NK precursor cells is decreased.

[0123] While the invention has been described with reference to what are presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but rather, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0124] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

1. 1. An NK cell differentiation medium comprising a pyrimidoindole compound.

2. 2. The NK cell differentiation medium of claim 1, wherein the pyrimidoindole compound is UM171 or UM729.

3. 3. The NK cell differentiation medium of claim 1 or 2, further comprising a basal medium.

4. 4. The NK cell differentiation medium of claim 3, further comprising one or more of SCF, FLT3L, IL-2, IL-3, IL-15, or IL-7.

5. The NK cell differentiation medium of any one of claims 1 to 4, which has not been conditioned by contact with stromal cells or stromal cell substitutes.

6. The NK cell differentiation medium of any one of claims 1 to 5, which does not contain an aryl hydrocarbon receptor antagonist.

7. The NK cell differentiation medium of any one of claims 1 to 6, which is serum-free.

8. The NK cell differentiation medium according to any one of claims 1 to 7, which differentiates NK precursor cells.

9. 9. The NK cell differentiation medium of claim 8, wherein the NK progenitor cells are isolated or derived from a primary sample.

10. 10. The NK cell differentiation medium of claim 8 or 9, wherein the NK progenitor cells are derived from pluripotent stem cells.

11. The NK cell differentiation medium of claim 10, wherein the pluripotent stem cells are induced pluripotent stem cells.

12. A method for differentiating into NK cells, comprising: Providing a population of NK progenitor cells; and contacting a population of NK progenitor cells in culture with the medium of any one of claims 1 to 11 at a concentration and for a period of time sufficient to generate NK cells. A method comprising:

13. The method of claim 12, wherein the population of NK progenitor cells is homogenous with respect to phenotypic markers or heterogeneous with respect to phenotypic markers.

14. The method of claim 13, wherein the phenotypic marker is CD7.

15. The method of claim 13 or 14, wherein the phenotypic marker is CD5.

16. The method of any one of claims 12 to 15, wherein during the contacting step, the frequency of NK cells is increased.

17. The method of any one of claims 12 to 16, wherein during the contacting step, the number of NK cells is increased.

18. The method of any one of claims 12 to 17, wherein during the contacting step, the frequency or number of a population of NK progenitor cells is increased or decreased.

19. The method of any one of claims 12 to 18, wherein the providing and contacting steps are performed in the absence of stromal cells or stromal cell substitutes.

20. The method of any one of claims 12 to 19, wherein the providing and contacting steps are in the absence of an aryl hydrocarbon receptor antagonist.

21. The method of any one of claims 12 to 20, wherein the concentration of the pyrimidoindole compound is from 10 nM to 3 μM.

22. The method of any one of claims 12 to 21, wherein the period is at least one week.

23. 23. The method of claim 22, wherein the period is about two weeks.

24. The method of any one of claims 12 to 23, wherein the NK cells express CD56.

25. The method of any one of claims 12 to 24, wherein the NK cells are cytotoxic.

26. The method of any one of claims 12 to 25, wherein the population of NK progenitor cells is derived from a primary sample.

27. The method of any one of claims 12 to 25, wherein the population of cells is isolated from a primary sample.

28. 28. The method of claim 27, wherein the primary sample is an umbilical cord blood sample, a bone marrow sample, or a peripheral blood sample.

29. The method of any one of claims 12 to 25, wherein the population of cells is differentiated from a pluripotent stem cell.

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