Methods for inducing natural killer cell differentiation

Abstract

The present invention relates to a natural killer (NK) cell that has been differentiated in vitro, as well as corresponding methods, compositions, kits, and therapeutic uses.

Description

[0001] METHODS FOR INDUCING NATURAL KILLER CELL DIFFERENTIATION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a methodology for producing Natural Killer (NK) cells, the NK cells thereof and their use in therapy. Also disclosed herein are hematopoietic stem cells (HSCs) and / or hematopoietic stem and progenitor cells (HSPCs) produced using the present methodologies, and their use in therapy.

[0004] BACKGROUND OF THE INVENTION

[0005] Natural Killer (NK) cells are highly promising effector cell candidates for their use in adoptive cell therapies for immuno-oncology applications. Specifically, NK cell-based immunotherapies have gained significant traction to treat several cancer types due to their durable responses and absence of adverse toxicity events associated with CAR-T therapies. With no requirement for complete HLA-matching, the NK cells allow safe use of allogeneic cellular material obtained from such sources as donor peripheral or umbilical cord blood.

[0006] Current methodologies for obtaining HSC, HSPC and / or NK cells involve complex and resource-intensive processes. NK cell isolation and activation methods can be cumbersome, limiting their scalability and clinical applicability. Additionally, traditional approaches often rely on bone marrow (BM), cord blood (CB) or peripheral blood as sources of HSCs and / or HSPCs, with susbequent differentiation and expansion steps. There is a need for an improved and streamlined method that facilitates the production of high purity HSC, HSPC and / or NK cells in a more efficient and cost-effective manner. Also, the lack of consistency in cell performance due to donor variability and high manufacturing costs impede their use.

[0007] Human pluripotent stem cells such as human induced Pluripotent stem cells (hiPSC) and human Embryonic stem cells (hESC) represent unlimited sources of well characterized starting material for genetic modification and robust differentiation into committed progenitors and mature cells with defined characteristics, including hematopoietic stem and progenitor cells (HSPC) and immune cells of lymphoid origin, which alleviates many manufacturing and affordability constraints. iPSC-derived NK cells have shown similar effects as those from primary cell sources. Moreover, facile genetic modification of iPSC lines provides huge potential for ‘off-the-shelf’ therapies with increased functionality. As such, there remains a need for reliable methods of producing NK cells and / or HSCs, and / or HSPCs quickly and efficiently to increase their availability particularly for use in therapy.

[0008] SUMMARY OF THE INVENTION

[0009] The present inventors have developed several in vitro protocols for differentiating stem cells (such as induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs)) into NK cells. It was found that said protocols consistently yield stem cell-derived NK cells that exhibit: 1) a characteristic NK cell surface marker profile as shown in Figure 2, and 7; 2) potent cytotoxic activity against various cancer cell lines (e.g. breast, pancreatic, and lung) as shown in Figure 3; and 3) the capacity for activation upon encountering immune stimuli, such as cancer cells (e.g. the K562 cell line), as shown in Figures 7 and 8.

[0010] Surprisingly, the present inventors discovered that these protocols offer additional advantages, notably the ability to produce high yields of NK cells having a desired phenotype (CD45+ / CD56+) and / or produce NK cells exhibiting a distinct gene expression profile compared to blood-derived NK cells, and / or produce relatively high yields of NK cells having the characteristics mentioned above and / or are more cost-effective. The benefits of these inventive protocols will be further detailed below.

[0011] More specifically, the inventors have surprisingly developed feeder-free GMP-compliant protocols for the production of hematopoietic stem cells (HSCs) and / or hematopoietic stem and progenitor cells (HSPCs) and subsequently, natural killer (NK) cells from human induced pluripotent stem cells (iPSCs). The protocols herein disclosed use defined media compositions that effectively substitute the exogenous signals necessary for HSC, HSPC and NK cell poiesis. As such, the inventors have developed several protocols for producing highly pure NK cell populations (e.g. CD45+ / CD56+), capable of recognising cancer cells of hematopoietic and solid tissue origin and showing activation and potent cytotoxicity in in vitro assays.

[0012] The invention addresses the aforementioned challenges by providing novel protocols for the optimised production of HSC, HSPC, and / or NK cells. The protocols involve a series of carefully orchestrated steps, including but not limited to, cell selection, culture conditions, timing, and molecular modulation, to enhance the yield, purity and potency of HSC, HSPC and / or NK cells that are distinct and superior (e.g. more cytotoxic against cancer cells) to blood-derived NK cells. By combining these critical elements, the disclosed protocols enable the generation of therapeutic NK cell populations with enhanced efficacy (e.g. greater cancer killing activity), making it a valuable contribution to the field of cell therapy and regenerative medicine, in particular, cancer therapy.

[0013] It was found that the specific selection and combination of nutrients, media components and signals, as well as timing, reliably resulted in the differentiation of stem cells (e.g. iPSCs) into HSCs / HSPCs and then into NK cells. The protocols unexpectedly provide much-needed methods of producing such NK cells at a cost-effective and efficient yet consistently reliable pace that can be used in large scale manufacturing.

[0014] DETAILED DESCRIPTION OF THE INVENTION

[0015] An aspect of the present invention is a method for producing Natural Killer (NK) cells, the method comprising the steps of: i)providing a population of cell aggregates comprising induced Pluripotent Stem Cells (iPSCs) or Embryonic Stem Cells (ESCs); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises at least 4 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, Activin A, VEGF and bFGF; wherein the culturing media used in the culturing of step iii) comprises at least 4 components selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IL-6, thrombopoietin (TPO), Notch agonist, IGF-1 , estradiol, Nicotinamide and Tryptophan; wherein the culturing media used in the culturing of step iv) comprises at least 5 components selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF-p receptor kinase inhibitor, bFGF, IL-6, TPO, Nicotinamide, Tryptophan, Notch agonist, Anti-human IgG antibody, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha; wherein the culturing media used in the culturing of step v) comprises at least 3 components selected from FLT3L, SCF, IL-7, IL-15, Notch agonist, Hydrocortisone, IL-15R-alpha, Nicotinamide, Anti-human IgG antibody, IGF1 , TPO, IL-21 , and SMAD3 inhibitor. An aspect of the invention is a composition comprising NK cells, for use in therapy, wherein the NK cells have been produced according to the first aspect of the invention.

[0016] An aspect of the invention is a method of treatment comprising administering to a patient a composition comprising NK cells, wherein the NK cells have been produced according to the first aspect of the invention.

[0017] An aspect of the invention is use of a composition comprising NK cells in the manufacture of a medicament for use in therapy, wherein the NK cells have been produced according the first aspect of the invention.

[0018] An aspect of the invention is a kit for producing NK cells from the provided population of cell aggregates comprising iPSCs or ESCs according to the first aspect of the invention, wherein the kit comprises sterile elements for the differentiation of the cell aggregates, or pharmaceutically acceptable salts thereof.

[0019] In one aspect, the present invention relates to a NK cell that has been differentiated in vitro, wherein the NK cell comprises: increased expression of one or more of: LINC00511, LINC02315, TESC, TPM4, TEC, DELEC1, COTL1, DRAXIN, SLC1A5, GLUL, LPAR3, NFILZ, INPP4B, ITGB7, KIT, S100A4, MIR646HG, CNR2, TNFSF10, LEF1, PRF1, RASSF8, THEMIS, DPF3, IL32, CTSW, AFAP1L2, CD3E, STYK1, ZNF683, CCR6, CCR1, MIR181A1HG, ID2, SYTL3, ZFHX3, CCND2, ITGAX, TRPM2, BCL2, NDFIP2, LTB, PRKCA, NCAM1, DOCK5, and IL18RT, and / or decreased expression of one or more of: ADAM28, TMEM71, DIPK1A, MIR3667HG, PMEPA1, TIG IT, COLGALT2, BTN3A3, LINC00861, and MPP7, when compared to a blood-derived NK cell.

[0020] In one aspect, the present invention relates to a NK cell that has been differentiated in vitro, wherein the NK cell comprises: increased expression of one or more of: MAGI1, IGSF1, DLEU1, FBXL7, TIAM1, MAML3, PTPN14, DAPK2, MY01E, CSF1, T0X2, H0XB3, ITGA1, LINC00504, DPF3, ZFHX3, THEMIS, IL32, CTSW, TRPM2, NDFIP2, CCND2, ITGAX, IL18R1, PRKCA, LTB, CD3E, DOCK5, ZNF683, BCL2, MIR181A1HG, STYK1, RASSF8, CCR1, NCAM1, CCR6, ID2, AFAP1L2, and SYTL3: and / or decreased expression of one or more of: ZNF831, F2R, RAP1GAP2, TIGIT, BTN3A3, COLGALT2, MPP7, and LINC00861, when compared to a blood-derived NK cell. The NK cell may comprise: increased expression of one or more of: RASSF8, THEMIS, DPF3, IL32, CTSW, AFAP1L2, CD3E, SLC1A5, GLUL, STYK1 , LPAR3, ZNF683, NFILZ, INPP4B, ITGB7, KIT, S100A4, CCR6, CNR2, TNFSF10, CCR1, MIR646HG, MIR181A1HG, ID2, SYTL3, ZFHX3, LEF1 , CCND2, ITGAX, TRPM2, BCL2, NDFIP2, PRF1, LTB, PRKCA, NCAM1, DOCK5, and IL18R1 (preferably RASSF8, THEMIS, DPF3, IL32, AFAP1L2, and CTSI / V9; and / or decreased expression of one or more of: ADAM28, TMEM71, DIPK1A, MIR3667HG, PMEPA1, TIGIT, COLGALT2, BTN3A3, LINC00861, and MPP7 (preferably BTN3A3, LINC00861, MIR3667HG, MPP7, and PMEPAT), when compared to a blood- derived NK cell.

[0021] The NK cell may comprise: increased expression of one or more of: CSF1, DPF3, ZFHX3, THEMIS, IL32, TOX2, CTSW, HOXB3, TRPM2, NDFIP2, CCND2, ITGA1 , ITGAX, IL18R1, PRKCA, LINC00504, LTB, CD3E, DOCK5, ZNF683, BCL2, MIR181A1HG, STYK1, RASSF8, CCR1, NCAM1, CCR6, ID2, AFAP1L2, and SYTL3 (preferably CSF1, DPF3, ZFHX3, THEMIS, IL32, and TOX2); and / or decreased expression of one or more of: ZNF831, F2R, RAP1GAP2, TIGIT, BTN3A3, COLGALT2, MPP7, and LINC00861 (preferably RAP1GAP2, COLGALT2, MPP7, and LINC0086T), when compared to a blood-derived NK cell.

[0022] The NK cell may comprise: increased expression of one or more of: LINC00511, LINC02315, TESC, TPM4, TEC, DELEC1, COTL1, and DRAXIN (preferably LINC00511, LINC02315, TESC, and TPM4); and / or decreased expression of one or more of: ADAM28, TMEM71 , DIPK1A, MIR3667HG, PMEPA1 , TIGIT, COLGALT2, BTN3A3, LINC00861 , and MPP7 (preferably BTN3A3, LINC00861, MIR3667HG, MPP7, and PMEPAT), when compared to a blood-derived NK cell.

[0023] The NK cell may comprise: increased expression of one or more of: MAGI1, IGSF1, DLEU1, FBXL7, TIAM1, MAML3, PTPN14, DAPK2, and MYO 1E (preferably MAGI1, IGSF1, DLEU1 , FBXL7, and TIAM1): and / or decreased expression of one or more of: ZNF831 , F2R, RAP1 GAP2, TIGIT, BTN3A3, COLGALT2, MPP7, and LINC00861 (preferably RAP1GAP2, COLGALT2, MPP7, LINC0086T), when compared to a blood-derived NK cell.

[0024] The NK cell may comprise: increased expression of one or more of: SLC1A5, CTSW, ID2, IL18R1, IL32, NFILZ, PRF1, SYTL3, TNFSF10, ITGA1, ITGAX, and PRKCA', and / or decreased expression of one or more of: ADAM28, TMEM71 , DIPK1A, MIR3667HG, PMEPA1 , TIGIT, COLGALT2, BTN3A3, LINC00861 , and MPP7 (preferably BTN3A3, LINC00861, MIR3667HG, MPP7, and PMEPAT), when compared to a blood-derived NK cell. The NK cell may comprise: increased expression of one or more of: CTSW, ID2, IL18R1, IL32, NFILZ, PRF1, SYTL3, TNFSF10, ITGA1, ITGAX, and PRKCA; and / or decreased expression of one or more of: ZNF831 , F2R, RAP1GAP2, TIGIT, BTN3A3, COLGALT2, MPP7, and LINC00861 (preferably RAP1GAP2, COLGALT2, MPP7, LINC00861), when compared to a blood-derived NK cell.

[0025] An NK cell may comprise increased expression of one or more of the listed genes and decreased expression of one or more of the listed genes when compared to a blood-derived NK cell.

[0026] The one or more genes of the invention have the following National Center for Biotechnology Information (NCBI) identification (ID) numbers, with the gene version being the latest available as of April 24, 2025. The skilled person knows how to determine a NCBI ID number for a given gene by, for instance, searching the NCBI gene database (https: / / www.ncbi.nlm.nih.gov / gene).

[0027]

[0028] The term “increased expression” or “decreased expression” may mean increased and / or decreased expression of least 2 genes or more such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more genes, preferably at least 3 genes, more preferably at least 4 genes, yet more preferably at least 5 genes. Most preferably, an NK cell may comprise increased expression of all of the listed genes and / or (preferably and) decreased expression of all the listed genes when compared to a blood-derived NK cell.

[0029] Preferably, the increased expression and / or decreased expression is not the result of a genetic modification of the NK cell.

[0030] In one aspect, the present invention relates to a method for selecting an NK cell suitable for use in treating cancer, the method comprising: a. comparing a measured expression level of one or more genes selected from: SLC1A5, GLUL, LPAR3, NFILZ, INPP4B, ITGB7, KIT, S100A4, MIR646HG, CNR2, TNFSF10, LEF1 , PRF1, RASSF8, THEMIS, DPF3, IL32, CTSW, AFAP1L2, CD3E, STYK1, ZNF683, CCR6, CCR1, MIR181A1HG, ID2, SYTL3, ZFHX3, CCND2, ITGAX, TRPM2, BCL2, NDFIP2, LTB, PRKCA, NCAM1, DOCK5, IL18R1,CSF1, TOX2, HOXB3, ITGA1, LINC00504, LINC00511, LINC02315, TESC, TPM4, TEC, DELEC1, COTL1, DRAXIN, MAGI1, IGSF1, DLEU1, FBXL7, TIAM1, MAML3, PTPN14, DAPK2, MYO1E, ADAM28, TMEM71, DIPK1A, MIR3667HG, PMEPA1, TIGIT, COLGALT2, BTN3A3, LINC00861, MPP7, ZNF831, F2R, and RAP1GAP2; and b. selecting the NK cell when the measured expression level of: one or more of: LINC00511, LINC02315, TESC, TPM4, TEC, DELEC1, COTL1, DRAXIN, SLC1A5, GLUL, LPAR3, NFILZ, INPP4B, ITGB7, KIT, S100A4, MIR646HG, CNR2, TNFSF10, LEF1, PRF1, RASSF8, THEMIS, DPF3, IL32, CTSW, AFAP1L2, CD3E, STYK1, ZNF683, CCR6, CCR1, MIR181A1HG, ID2, SYTL3, ZFHX3, CCND2, ITGAX, TRPM2, BCL2, NDFIP2, LTB, PRKCA, NCAM1, DOCK5, and IL18R1 is increased and / or one or more of: ADAM28, TMEM71, DIPK1A, MIR3667HG, PMEPA1 , TIGIT, COLGALT2, BTN3A3, LINC00861, and MPP7 is decreased, when compared to a blood-derived NK cell.

[0031] In one aspect, the present invention relates to a method for selecting an NK cell suitable for use in treating cancer, the method comprising: a. comparing a measured expression level of one or more genes selected from: SLC1A5, GLUL, LPAR3, NFILZ, INPP4B, ITGB7, KIT, S100A4, MIR646HG, CNR2, TNFSF10, LEF1, PRF1, RASSF8, THEMIS, DPF3, IL32, CTSW, AFAP1L2, CD3E, STYK1, ZNF683, CCR6, CCR1, MIR181A1HG, ID2, SYTL3, ZFHX3, CCND2, ITGAX, TRPM2, BCL2, NDFIP2, LTB, PRKCA, NCAM1, DOCK5, IL18R1,CSF1, TOX2, HOXB3, ITGA1, LINC00504, LINC00511, LINC02315, TESC, TPM4, TEC, DELEC1, COTL1, DRAXIN, MAGI1, IGSF1, DLEU1, FBXL7, TIAM1, MAML3, PTPN14, DAPK2, MYO1E, ADAM28, TMEM71, DIPK1A, MIR3667HG, PMEPA1, TIGIT, COLGALT2, BTN3A3, LINC00861, MPP7, ZNF831, F2R, and RAP1GAP2; and b. selecting the NK cell when the measured expression level of: MAGI1, IGSF1, DLEU1, FBXL7, TIAM1, MAML3, PTPN14, DAPK2, MYO1E, CSF1, TOX2, HOXB3, ITGA1, LINC00504, DPF3, ZFHX3, THEMIS, IL32, CTSW, TRPM2, NDFIP2, CCND2, ITGAX, IL18R1, PRKCA, LTB, CD3E, DOCK5, ZNF683, BCL2, MIR181A1HG, STYK1, RASSF8, CCR1, NCAM1, CCR6, ID2, AFAP1L2, and SYTL3 is increased and / or one or more of: ZNF831, F2R, RAP1GAP2, TIGIT, BTN3A3, COLGALT2, MPP7, and LINC00861 is decreased, when compared to a blood-derived NK cell.

[0032] The selected NK cell may be an NK cell as described herein. Thus, the disclosure above relating to gene expression may be relevant to the method of selecting an NK cell suitable for use in treating cancer. In other words, the measured expression level may correspond to that of an NK cell described herein.

[0033] The term “comparing a measured expression level of one or more genes” means measuring the expression level of one or more of the genes disclosed herein in a NK cell that has been differentiated in vitro from a stem cell (e.g. iPSC) and comparing the expression level of one of more of said genes in the NK cell to the expression level of the said one or more genes in a blood-derived NK cell (e.g. peripheral blood NK cell or cord blood NK cell).

[0034] The term “measuring” as used in reference to expression of one or more genes of the invention encompasses measuring both negative (e.g. no expression) and positive expression (e.g. expression).

[0035] Measuring expression may be carried out by any means known to the person skilled in the art. In some embodiments expression may be measured using high-throughput techniques. For example, measuring expression may be at the level of transcription (e.g. transcriptomic techniques) or translation (e.g. proteomic techniques). Alternatively, or additionally, the invention may employ the use of genomics, e.g. to detect the presence or absence of single nucleotide polymorphisms (SNPs), promoter sequences, gene copy number (e.g. duplications), and / or enhancer or other relevant genetic features, preferably those that determine the expression level of one or more genes of the invention. High-throughput techniques can be used to analyse whole genomes, proteomes and transcriptomes rapidly, providing data, including the expression levels, of all of the genes, polypeptides and transcripts in a cell. Proteomics is a technique for analysing the proteome of a cell (e.g. at a particular point in time). The proteome is different in different cell types. Typically, proteomics is carried out by mass-spectrometry, including tandem mass-spectrometry, and gel-based techniques, including differential in-gel electrophoresis. Proteomics can be used to detect polypeptides expressed in a particular cell type and generate a proteomic profile to allow for the identification of specific cell types. mRNA of a target gene can be detected and quantified by e.g. Northern blotting or by quantitative reverse transcription PCR (RT-PCR). Single cell gene expression analysis or single cell RNA sequencing analysis may also be performed using commercially available systems. Alternatively, or in addition, gene expression levels can be determined by analysing polypeptide levels e.g. by using Western blotting techniques such as ELISA-based assays.

[0036] Gene expression levels may be determined by measuring the mRNA or cDNA levels of the genes of the invention, such as single cell RNA sequencing (scRNA-Seq). Preferably, the gene expression levels are determined by measuring mRNA levels using scRNA-sequencing techniques.

[0037] The term “increased” as used herein in reference to expression of the one or more genes of the invention may refer to an expression level that is statistically-significantly increased when compared to a blood-derived NK cell (peripheral blood NK cell or umbilical cord blood NK cell, preferably a peripheral blook NK cell). Such a gene may be considered to be upregulated.

[0038] An increased expression levels may mean greater than 1-fold, 1.25-fold to about 10-fold or more expression relative to a blood-derived NK cell. For instance, an increased expression level may mean greater than at least about 1.0-fold, 1.1-fold, 1.2-fold, 1.25-fold, 1.3-fold, 1.4- fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.75-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold,

[0039] 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1 -fold, 3.2-fold, 3.3-fold, 3.4- fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4.0-fold, 4.1 -fold, 4.2-fold, 4.3-fold, 4.4-fold,

[0040] 4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold, 5.0-fold, 5.1 -fold, 5.2-fold, 5.3-fold, 5.4-fold, 5.5- fold, 5.6-fold, 5.7-fold, 5.8-fold, 5.9-fold, 6.0-fold, 6.1 -fold, 6.2-fold, 6.3-fold, 6.4-fold, 6.5-fold,

[0041] 6.6-fold, 6.7-fold, 6.8-fold, 6.9-fold, 7.0-fold, 7.1-fold, 7.2-fold, 7.3-fold, 7.4-fold, 7.5-fold, 7.6- fold, 7.7-fold, 7.8-fold, 7.9-fold, 8.0-fold, 8.1 -fold, 8.2-fold, 8.3-fold, 8.4-fold, 8.5-fold, 8.6-fold,

[0042] 8.7-fold, 8.8-fold, 8.9-fold, 9.0-fold, 9.1 -fold, 9.2-fold, 9.3-fold, 9.4-fold, 9.5-fold, 9.6-fold, 9.7- fold, 9.8-fold, 9.9-fold, 10.0-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, 75- fold, 100-fold, 150-fold, 200-fold, or at least about 300-fold expression when compared to a blood-derived NK cell.

[0043] The term “decreased” as used herein in reference to expression of the one or more genes of the invention may refer to an expression level that is statistically-significantly decreased when compared to a blood-derived NK cell (peripheral blood NK cell or umbilical cord blood NK cell, preferably a peripheral blook NK cell). Such a gene may be considered to be downregulated.

[0044] A decreased expression level may mean less than about -1.0-fold, -1.1-fold, -1.2-fold, -1.25- fold, -1.3-fold, -1.4-fold, -1.5-fold, -1.6-fold, -1.7-fold, -1.75-fold, -1.8-fold, -1.9-fold, -2.0-fold, - 2.1 -fold, -2.2-fold, -2.3-fold, -2.4-fold, -2.5-fold, -2.6-fold, -2.7-fold, -2.8-fold, -2.9-fold, -3.0- fold, -3.1 -fold, -3.2-fold, -3.3-fold, -3.4-fold, -3.5-fold, -3.6-fold, -3.7-fold, -3.8-fold, -3.9-fold, - 4.0-fold, -4.1 -fold, -4.2-fold, -4.3-fold, -4.4-fold, -4.5-fold, -4.6-fold, -4.7-fold, -4.8-fold, -4.9- fold, -5.0-fold, -5.1 -fold, -5.2-fold, -5.3-fold, -5.4-fold, -5.5-fold, -5.6-fold, -5.7-fold, -5.8-fold, - 5.9-fold, -6.0-fold, -6.1 -fold, -6.2-fold, -6.3-fold, -6.4-fold, -6.5-fold, -6.6-fold, -6.7-fold, -6.8- fold, -6.9-fold, -7.0-fold, -7.1 -fold, -7.2-fold, -7.3-fold, -7.4-fold, -7.5-fold, -7.6-fold, -7.7-fold, - 7.8-fold, -7.9-fold, -8.0-fold, -8.1 -fold, -8.2-fold, -8.3-fold, -8.4-fold, -8.5-fold, -8.6-fold, -8.7- fold, -8.8-fold, -8.9-fold, -9.0-fold, -9.1 -fold, -9.2-fold, -9.3-fold, -9.4-fold, -9.5-fold, -9.6-fold, - 9.7-fold, -9.8-fold, -9.9-fold, -10.0-fold, -15-fold, -20-fold, -25-fold, -30-fold, -35-fold, -40-fold, - 50-fold, -75-fold, -100-fold, -150-fold, -200-fold, or at least about -300-fold expression when compared to a blood-derived NK cell.

[0045] The fold change difference can be in absolute terms (e.g. CPM: counts per million) or Log2CPM (a standard measure in the field) of the expression level in a sample. Preferably the fold change is Log2 fold change. A Log2 change may be an increase of at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3,

[0046] 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4,

[0047] 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5,

[0048] 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6,

[0049] 8.7, 8.8, 8.9, 9.0, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0 or more. In one embodiment a Log2 change is a decrease of at least -0.1 , -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -1.0, -1.1 , -1.2, -1.3, -1.4, -1.5, -1.6, -1.7, -1.8, -1.9, -2.0, -2.1 , -2.2, -2.3, -2.4, -2.5, -2.6, -

[0050] 2.7, -2.8, -2.9, -3.0, -3.1 , -3.2, -3.3, -3.4, -3.5, -3.6, -3.7, -3.8, -3.9, -4.0, -4.1 , -4.2, -4.3, -4.4, -

[0051] 4.5, -4.6, -4.7, -4.8, -4.9, -5.0, -5.1 , -5.2, -5.3, -5.4, -5.5, -5.6, -5.7, -5.8, -5.9, -6.0, -6.1 , -6.2, -

[0052] 6.3, -6.4, -6.5, -6.6, -6.7, -6.8, -6.9, -7.0, -7.1 , -7.2, -7.3, -7.4, -7.5, -7.6, -7.7, -7.8, -7.9, -8.0,-

[0053] 8.1 , -8.2, -8.3, -8.4, -8.5, -8.6, -8.7, -8.8, -8.9, -9.0, -9.1 , -9.2, -9.3, -9.4, -9.5, -9.6, -9.7, -9.8, -

[0054] 9.9, -10.0 or more. A decrease may be indicated by the presence of a symbol prior to the value.

[0055] Preferably, the fold-change is measured and / or is determined by RNA sequencing (RNA- Seq), such as single cell RNA sequencing. The term “unchanged” or “the same” as used herein in reference to expression of the one or more genes of the invention may refer to an expression level that is not statistically- significantly different to a blood-derived NK cell. Preferably, an expression level that is the same as a blood-derived NK cell.

[0056] The expression level may be an average such as a mean expression level. The statistical significance may be determined by any standard means in the art. For example, the statistical significance may be determined using two-way ANOVA, e.g. where n is at least 3 and data are presented as mean + / - standard error of mean.

[0057] The term “one or more” when used in the context of a gene of the invention may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or more of the genes. The term “one of more” may mean all of the genes.

[0058] The term “blood-derived NK cell” as used herein refers to a NK cell that has been isolated (e.g. isolated directly) from the blood of a subject such as from peripheral blood or umbilical cord blood (also referred to as “cord blood”) of a subject. Preferably, a blood-derived NK cell has been isolated from peripheral blood of a subject.

[0059] As used herein the term “peripheral blood” has its conventional use in the art; that is generally blood which is circulating throughout the circulatory system. Human peripheral blood is within the scope of the present invention and is obtained with written informed preconsent and ethical approval.

[0060] Thus, the blood-derived NK cell may be a peripheral blood NK cell or an umbilical cord blood NK cell, preferably a peripheral blood NK cell.

[0061] The NK cell of the invention may be differentiated from a stem cell in vitro, such as an induced Pluripotent Stem Cell (iPSC) or an Embryonic Stem Cell (ESC). Preferably, the NK cell of the invention is differentiated from an iPSC in vitro.

[0062] The term “stem cell” as used herein refers to an undifferentiated or partially differentiated living cell characterised by its capacity for self-renewal through cell division and its potential to differentiate into one or more specialised cell types. This biological property typically enables the stem cell to regenerate tissues or cell populations lost due to injury, disease, or normal physiological turnover. The stem cell may be derived from various sources, including embryonic, fetal, or adult tissues. Non-limiting examples of a stem cell include an iPSC and an ESC.

[0063] As used herein the term induced Pluripotent Stem Cell(s) “iPSCs” (or iPSC) refers to specialised cells that have been reprogrammed from differentiated cells, such as skin or blood cells, through the introduction of specific genetic factors. iPSCs are pluripotent, possessing the capability to differentiate into various human cell types such as hematopoietic cells, epithelial cells, myocytes, adipocytes and hepatocytes. In one embodiment the iPSCs used in the present invention express OCT3 / 4, Nanog, TRA-1-60, TRA-1-81 , SSEA-4 or any combination thereof.

[0064] As used herein the term “Embryonic Stem Cell(s) (ESCs or ESC)” has its conventional meaning in the art and refers to stem cells derived from embryos that are 3 to 5 days old. ESCs are pluripotent cells which give rise to all somatic cell types in the embryo. In one embodiment the ESCs used in the present invention express OCT3 / 4, Nanog, TRA-1-60, TRA-1-81 , SSEA-4 or any combination thereof.

[0065] In a particular embodiment, the ESCs (or ESC) are human ESCs (or human ECS).

[0066] In some situation, the stem cell is not an ESC or is not a human ESC. Preferably, the term “stem cell” as used herein does not encompass a human embryonic stem cell.

[0067] Preferably, the stem cell is an iPSC.

[0068] Populations of the iPSCs (or iPCS) and ESCs (or ESC) are commercially available and there are many well-established cell lines. Suitably, the iPSCs and / or ESCs of the present invention have already been developed through reprogramming and / or clonal selection i.e., as already established iPSC and / or ESC cell lines.

[0069] The source of the iPSCs (or iPSC) includes, but is not restricted to, umbilical cord blood, bone marrow and peripheral blood. The cells may have been obtained from a source which is fresh or frozen or from a cell bank, wherein a fresh source has not been frozen prior to use If the sample is frozen, then the cells will be thawed before use in the method. The NK cell may further express cell surface markers (or biomarkers) such as CD45 and / or CD56. Preferably, NK cell expresses CD45 and CD56 (CD45+ / CD56+).

[0070] Within the biomarker terms (for example “CD45+”), the (+) designation indicates that the specified cluster of differentiation (CD) is expressed by the cell and is present on the cell surface. Within the biomarker terms (for example “CD45-"), the (-) designation indicates that the specified CD is not expressed or poorly expressed by the cell. Within the biomarker terms (for example CD45+ / CD56+), the ( / ) designation indicates that both specified CDs are expressed by the cell and are present on the cell surface.

[0071] The term “cell surface marker(s)” (or biomarkers) as used herein refers to proteins, glycoproteins, or carbohydrates or other molecules expressed on the outer membrane of a cell (e.g. immune cells). Cell surface markers are often used, alone or in combination, to identify, distinguish different type of immune, assess differentiation and maturation stage, and / or to assess phenotypically profile (e.g. phenotypic analysis using techniques such as flow cytometry and cell sorting assays) on NK cells or precursors thereof. Non-limiting examples of cell surface markers include “Clusters of Differentiation” (CD) markers. “CD” represents a standardised nomenclature system commonly used in the field of immunology. Non-limiting examples of cell surface markers that may be expressed on NK cells or precursors thereof, include CD7, CD16, CD45, CD56 (or NCAM1), CD57, CD69, NKp44, NKp46, NKG2A, NKG2D, DNAM, DNAM-1 (or CD226), TRAIL (or CD253), TIGIT, TIM3, and CXCR3, and others. For instance, a NK cell or a precursor thereof may express or lack one or more cell surface markers such as those described herein. A NK cell or a precursor thereof may express (or upregulate) or lack (or downregulate) one or more of the cell surface markers described herein in response to an immune stimulus such as exposure to a cancer cell (e.g. cancer cell line K562).

[0072] In one aspect the present invention provides a method for producing a NK cell, the method comprising the steps of: i) providing a population of cell aggregates comprising stem cells (preferably iPSCs); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises at least 4 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, Activin A, VEGF and bFGF; wherein the culturing media used in the culturing of step iii) comprises at least 4 components selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IL-6, thrombopoietin (TPO), Notch agonist, IGF-1 , estradiol, Nicotinamide and Tryptophan; wherein the culturing media used in the culturing of step iv) comprises at least 5 components selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF-p receptor kinase inhibitor, bFGF, IL-6, TPO, Nicotinamide, Tryptophan, Notch agonist, Anti-human IgG antibody, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha; wherein the culturing media used in the culturing of step v) comprises at least 3 components selected from FLT3L, SCF, IL-7, IL-15, Notch agonist, Hydrocortisone, IL-15R-alpha, Nicotinamide, Anti-human IgG antibody, IGF1 , TPO, IL-21 , and SMAD3 inhibitor.

[0073] The term “comprises” or “comprising” will take its usual meaning in the art, namely indicating that the component includes but is not limited to the relevant features (i.e. including, among other things). As such, the term “comprises” will include references to the component consisting essentially of (such as consisting of) the relevant features. The term “consists of’ or “consisting of” will take its usual meaning in the art, namely indicating that the component includes and is limited to the relevant features.

[0074] As used herein the term “hematopoietic stem and progenitor cells (HSPCs)” refer to cells found in the bone marrow, umbilical cord blood and peripheral blood, which can differentiate and / or proliferate to form blood cells. Examples of blood cells include, but are not restricted to, monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, megakaryocytes, platelets, T cells, B cells, and natural killer cells.

[0075] As used herein the term “hematopoietic stem cells (HSCs)” refer to multipotent or pluripotent cells which have the ability to differentiate into blood cells of all lineages and to regenerate themselves whilst maintaining their pluripotent characteristics. The term “one or more” as used herein may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20, etc. In one embodiment, wherein “one or more” precedes a list, “one or more” may mean all of the members of the list. Similarly, the term “at least one” as used herein may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20, etc. In one embodiment, wherein “at least one” precedes a list, “at least one” may mean all of the members of the list.

[0076] As used herein the term “Natural Killer (NK) cells” (and “NK cell”) refers to a type of immune cell that has granules (small particles) with enzymes that can kill tumour cells or cells infected with a virus. As used herein the term “induced NK (iNK) cells” (also referred to as “stem derived-NK cell” or iPSC-derived NK cell” or “ESC-derived NK cell”) refers to NK cells that have been genetically reprogrammed or induced (preferably induced and preferably not genetically reprogrammed) to become a specific type of cell such as an NK cell, which has been differentiated in vitro from a stem cell, such as an iPSC or an ESC, preferably an iPSC.

[0077] The term “NK cell precursor” or “precursor thereof’ as used herein refers to an immature, lineage-committed progenitor cell that typically has the inherent developmental potential and capacity to differentiate into a functionally competent NK cell. These precursors typically have undergone some level of restriction, losing the ability to develop into other lymphoid or myeloid lineages, but have not yet acquired the full phenotypic and functional characteristics of mature NK cells, such as the characteristic surface marker expression (e.g. CD56, CD16, etc.), cytotoxic machinery (e.g. perforin, granzymes, etc.), and the ability to mediate spontaneous cytotoxicity and cytokine production upon activation. Non-limiting examples of NK cells precursors include hematopoietic stem cells (HSCs) and hematopoietic stem and progenitor cells (HSPCs).

[0078] As used herein the term “live cells” (and “live cell”) will take its usual meaning in the art and refers to the viable cells within the whole cell culture. Live cells are determined by exclusion of the viability dye that enters only dead cells.

[0079] In one embodiment, the iPSCs (or iPSC) and / or ESCs (or ESC) have been obtained from a mammal, preferably a human.

[0080] In one embodiment, the iPSCs (or iPSC) have been obtained from any somatic human cell, preferentially blood cell. In a preferred embodiment, the iPSCs (or iPSC) have been obtained from umbilical cord blood.

[0081] In one embodiment, the ESCs (or ESC) have been obtained from embryos that are 3 to 5 days old. Preferably the embryos are human embryos.

[0082] Before the differentiation steps (ii-v) of the present invention, it may be important to prime the obtained commercially available iPSCs and / or ESCs (suitably in the form of single cells) into cell aggregates. Such priming (otherwise known herein as “cell aggregation”) is a well- known process in the field and there are many known methods to do so (for example, Cell aggregation is a feature of tissue formation that allows the binding of cells of the same type, in the context of the present invention, the pluripotent cells attach to each other during the priming.

[0083] As such and accordingly, step i) of the method of the present invention is providing a population of cell aggregates comprising induced Pluripotent Stem Cells (iPSCs) or Embryonic Stem Cells (ESCs). Preferably the cell aggregates comprise iPSCs.

[0084] Single cells that become partially differentiated do not form aggregates and remain as suspended cells. As such, the population of cell aggregates may further comprise single cells.

[0085] The cell aggregates are not particularly limited, and many forms may be used in the context of the present invention. Suitably, the cell aggregates comprising iPSCs and / or ESCs may comprise clumps of cells, cut monolayer fragments, spheroids, embryoid bodies, encapsulated cut monolayer fragments and / or hydrogel beads encapsulating cut monolayer fragments. In a preferred embodiment, the cell aggregates comprising iPSCs and / or ESCs comprise spheroids and / or embryoid bodies.

[0086] The term “cut monolayer fragments” refers to cut fragments of a monolayer culture, generally of one cell thickness, comprising 100-500 cells. The term “hydrogel beads encapsulating cut monolayer fragments” refers to the cut monolayer fragments encapsulated in hydrogel beads by electrospraying. Accordingly, cut monolayer fragments, encapsulated cut monolayer fragments and / or hydrogel encapsulating cut monolayer fragments of the present invention comprise iPSCs and / or ESCs. The term “spheroids” refers to three-dimensional aggregates comprising iPSCs and / or ESCs.

[0087] The term “Embryoid bodies (EBs)” refers to a type of spheroid comprising iPSC and / or ESCs.

[0088] As used herein the term “cultured population” refers to an isolated population of cells which has been propagated and / or differentiated in an artificial medium ex vivo. It will be obvious to a skilled person what type of artificial media to use in line with common general knowledge and the disclosures herein. Examples of suitable media are StemDiff APEL-2 medium, DMEM and F12. This is sometimes referred to herein as Base media or Basal media. The artificial media may also be supplemented with other factors or cytokines to signal the differentiation of the cells. Specific details and examples of such are detailed herein. The isolated population can be cultured or propagated over a number of days to form a cultured population. The term “up to X days” includes the X day (“X” being a whole number).

[0089] As used herein, the term “propagation” or “proliferation” refers to the process of cell division. Propagation is a fundamental aspect of cell growth and occurs during normal tissue growth, wound healing and in culture systems such as cell line or stem cell cultures.

[0090] As used herein, the term “differentiation” refers to the process by which the undifferentiated cells, such as iPSCs become specialised and acquire specific functions and characteristics. During differentiation, the cells undergo changes in gene expression and morphology to become more specialised cell types such as hematopoietic cells, epithelial cells, myocytes, adipocytes and hepatocytes.

[0091] Within the cells, wherein the cells may be part of the iPSCs and / or ESCs or the cultured population of cells, there are subtypes of cells. Examples of the cell subtypes are, but not restricted to; hematopoietic stem cells, hematopoietic progenitor cells and cells as defined by their phenotypic markers. Non-limiting examples of phenotypic markers are; CD34, CD43, CD45, or CD56, wherein the cells can also be defined by combinations of these phenotypic markers. As used herein the term “enriched” is used to refer to a set of cells which contains a high proportion of a specific subset / subtype of cell, wherein the set of cells may contain 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90% of the specific subset / subtype of cell. Within the present invention the term “enriched” can be used to refer to a population of cells wherein the cells have undergone differentiation and wherein a specific subtype of cells have increased in number proportionally more than other cells within the population. This enriched population of cells contains a significant proportion of a specific subtype of cells, wherein the significant proportion may be 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90% of the total population. Suitably the third cultured population of the method of the invention comprises an enriched proportion of HSCs and / or HSPCs. Suitably the fourth cultured population of the method of the invention comprises an enriched proportion of HSC, HSPC and / or NK cells.

[0092] As used herein, the term “serum-free media” refers to the culturing of cells in a media which has not been supplemented with serum derived from a human or an animal.

[0093] As used herein, the term “serum-containing media” refers to the culturing of cells in a media which has been supplemented with serum derived from a human or an animal.

[0094] As used herein the term “feeder free tissue culture system” refers to a method of culturing cells without utilising a layer of connective tissue cells to support and provide metabolites to the growing cells.

[0095] Preferably, the method for producing Natural Killer (NK) cells comprises the use of a feeder free tissue culture system.

[0096] As used herein the term “total culturing time” refers to the time in which steps ii), iii), iv) and v) are carried out. Suitably, the total culturing time is up to 45 days, such as up to 40 days. Suitably, the total culturing time is at least 15 days, preferably at least 20 days. Suitably, the culturing process of step ii) occurs for a period of up to 5 days to form the first cultured population, the culturing process of step iii) occurs for a period of up to 5 days to form the second cultured population, the culturing process of step iv) occurs for a period of up to 8 days to form the third cultured population and the culturing process of step v) occurs for a period of up to 5 or up to 25 days to form the fourth cultured population. During the total culturing time, the cells are allowed to grow on an appropriate media such as basal media, the end of the total culturing time is signified by the cells being harvested, pooled or analysed.

[0097] In some embodiments of the present invention, the culturing process of step ii) occurs for a period of up to 5 days, preferably from 2 to 4 days, such as 3 days. This is to allow the cells to begin the first stage of the differentiation process. In some embodiments of the present invention, the culturing process of step iii) occurs for a period of up to 5 days, preferably from 2 to 4 days, such as 3 days.

[0098] Each culturing step is important to provide the required nutrients and signals for the cells to differentiate into the desired HSCs, HSPCs and then NK cells. Naturally a certain amount of time is required for such processes to occur, however, the skilled person will appreciate that there can be considerable variability in the time frames for the invention to still function. In some embodiments of the present invention, the culturing process of step iv) occurs for a period of up to 8 days, preferably from 4 to 8 days, such as 6 days.

[0099] In some embodiments of the present invention, the culturing process of step v) occurs for a period of up to 5 days, preferably up to 4 days.

[0100] In some embodiments of the present invention, to produce the NK cells, the culturing process of step v) occurs for a period of at least 14 days, preferably from 20 to 30 days, preferably the culturing process of step v) occurs for a period of 24 to 30 days, such as 25 days.

[0101] In one embodiment, the steps ii), iii), iv) and v) are carried out over a total time sufficient for the iPSCs and / or ESCs to differentiate into HSCs and / or HSPCs. In some such embodiments, the percentage of cells expressingCD34+ / CD43+, CD34+ / CD45+ and / or CD34+ / CD43+ / CD45+ markers will increase. Suitably, the third cultured population of step iv) comprises HSCs and / or HSPCs. Suitably, the fourth cultured population of step v) further comprises HSCs and / or HSPCs and / or NK cells.

[0102] Suitably, the steps ii), iii), iv) and v) are carried out over a total time sufficient for the iPSCs and / or ESCs to differentiate into NK cells. In some such embodiments, the percentage of cells expressing CD45+ and CD56+ will increase.

[0103] In some embodiments of the present invention, the steps ii), iii), iv) and v) are carried out over a total time of about 26 to about 46 days.

[0104] In one embodiment the cells in the first cultured population are less pluripotent than the iPSCs and / or ESCs, and the number of cells in the first cultured population have increased.

[0105] In a preferred embodiment of the present invention, the total number of NK cells derived from the initial iPSCs and / or ESCs is between 20 and 200 NK cells per starting iPSC and / or ESC. In some embodiments of the present invention, the culturing media used in the culturing of steps ii), iii), iv) and / or v) further comprises at least 1 component, such as at least 2 components, selected from penicillin-streptomycin (P / S), B-mercaptoethanol, L-glutamine, L- ascorbic acid (L-AA), Sodium selenite and Ethanolamine, preferably P / S, B-mercaptoethanol, L-glutamine, L-AA, Sodium selenite and Ethanolamine.

[0106] In some embodiments of the present invention, the culturing media used in the culturing steps of ii), iii), iv) and / or v) comprises at least 3, such as at least 5 or at least 6 components, selected from penicillin-streptomycin (P / S), B-mercaptoethanol, L-glutamine, L-ascorbic acid (L-AA), Sodium selenite and Ethanolamine, preferably P / S, B-mercaptoethanol, L-glutamine, L-AA, Sodium selenite and Ethanolamine.

[0107] In some embodiments of the present invention, the culturing media used in the culturing steps of ii), iii), iv) and / or v) further comprises a basal medium, optionally selected from one or more of X-VIVO™ 15, StemSpan™ SFEM II, Stemline II Hematopoietic Stem Cell Expansion Medium, StemPro™-34 SFM (1X), StemDiff APEL-2 medium, DMEM, F12, IMDM and RPMI-1640, preferably the basal medium is selected from one or more of StemDiff APEL-2 medium, DMEM and F12.

[0108] In some embodiments of the present invention, the culturing media used in the culturing of step ii) comprises at least 5 components, such as at least 6 components, selected from VEGF, ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, Activin A, and bFGF.

[0109] Preferably, the culturing media used in the culturing of step ii) comprises VEGF.

[0110] In some embodiments of the present invention, the culturing media used in the culturing of step iii) comprises at least 5 components, such as at least 6 components, selected from IGF- 1 , Notch agonists, BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IL-6, TPO, estradiol, Nicotinamide and Tryptophan.

[0111] Preferably, the culturing media used in the culturing of step iii) comprises IGF-1 and / or Notch agonist (preferably DLL4 Fc).

[0112] Preferably, the culturing media used in the culturing of step iii) comprises IGF-1 and Notch agonist (preferably DLL4 Fc). Preferably, the culturing media used in the culturing of step ii) does not comprise VEGF.

[0113] In some embodiments of the present invention, the culturing media used in the culturing of step iv) comprises at least 8 components, such as at least 9 components, selected from Nicotinamide, Tryptophan, Notch agonists, Anti-human IgG antibody, BMP4, VEGF, SCF, IL- 1 , IL-3, IL-6, IL-7, IL-9, IL-15, TGF-|3 receptor kinase inhibitor, bFGF, TPO, aryl hydrocarbon receptor (AhR) antagonist and IL-15Ralpha.

[0114] Preferably, the culturing media used in the culturing of step iv) comprises at least one of: nicotinamide, tryptophan, Notch agonist (preferably DLL4 Fc), and anti-human IgG antibody.

[0115] Preferably, the culturing media used in the culturing of step iv) comprises nicotinamide, tryptophan, Notch agonist (preferably DLL4 Fc), and anti-human IgG antibody.

[0116] In some embodiment of the present invention, the culturing media used in the culturing of step v) comprises at least 6 components, such as at least 7 components, selected from Sodium selenite, Ethanolamine (MEA), FLT3L, SCF, IL-2, IL-7, IL-15, IL-18, IL-21 , Notch agonist, Hydrocortisone, IL-15Ralpha, IGF1 , TPO, IL-21 , SMAD3 inhibitor, Nicotinamide and Anti-human IgG antibody.

[0117] In some embodiments of the present invention, the culturing media used in the culturing of steps ii), iii) and / or iv) comprises BMP4 in combination with at least one component selected from VEGF, Activin A, SCF, LiCI and CHIR99021.

[0118] In some embodiments of the present invention, the culturing media used in the culturing of step v) further comprises a media additive. Suitably the media additive is serum-containing or serum-free.

[0119] Suitably, the serum-containing media additive is selected from human serum, human plasma replacement and human serum replacement, preferably the serum-containing media additive is HI human clotted AB serum.

[0120] Suitably, the serum-free media additive comprises Polyvinyl alcohol (PVA) and recombinant albumin (such as recombinant human serum albumin, cellastim-S). Suitably the serum-free media additive may further comprise one or more supplements and / or vitamins. Suitable supplements include Insulin-Transferrin-Selenium-Ethanolamine (ITS-X), non-essential amino acids, fatty acids, steroid hormones and cholesterol.

[0121] The term ROCK inhibitor as used herein refers to a compound which interferes or inhibits the activity of rho kinase (ROCK) and the ROCK pathway. The rho kinase belongs to the AGC family of serine-threonine specific protein kinases and is involved in regulating the shape and movement of cells by acting on the cytoskeleton. Suitably, the ROCK inhibitor is selected from Y-27632 dihydrochloride, fasudil, Y-39983 dihydrochloride, WF-536, SLx-2119, XD- 4000, rhostatin and VAS-012, preferably the ROCK inhibitor is Y-27632 dihydrochloride.

[0122] The term GSK-3 inhibitor as used herein refers to a compound which interferes or inhibits the activity of the glycogen synthase kinase-3 (GSK-3). GSK-3 is a serine or threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. Suitably, the GSK-3 inhibitor is selected from CHIR99021 , LiCI, BIO, TWS119 and Kenpaullone, preferably the GSK-3 inhibitor is CHIR99021 or LiCI and most preferably the GSK-3 inhibitor is CHIR99021 .

[0123] The term TGF-p receptor kinase inhibitor as used herein refers to a compound which interferes or inhibits the activity of TGF-p receptor kinase. TGF-p receptor kinases are single pass serine or threonine kinase receptors that belong to TGFp receptor family and are involved in paracrine signalling. Suitably, the TGF-p receptor kinase inhibitor is selected from SB431542, A 83-01 , RepSox, SB525334, Galunisertib and D4476, preferably the TGF- P receptor kinase inhibitor is SB431542.

[0124] The term Notch agonist as used herein refers to a compound which promotes the activity of Notch receptor proteins. The notch receptor is a hetero-oligomer composed of a large extracellular portion, which associates in a calcium-dependent, non-covalent interaction with a smaller piece of the notch protein composed of a short extracellular region, a single transmembrane-pass, and a small intracellular region. Notch signalling promotes proliferative signalling during neurogenesis and plays a major role in the role of embryonic development. Suitably, the Notch agonist is selected -from DLL4-FC, DLL1 , DLL3, DLL4, JAG1 and GAG2, preferably the Notch agonist is DLL4-Fc.

[0125] The term antagonist of the aryl hydrocarbon receptor (AhR antagonist) as used herein refers to a compound which interferes or inhibits the activity of the aryl hydrocarbon receptor. The AhR is a member of the family of basic helix-loop-helix transcription factors that regulates gene expression as a sensor of xenobiotic chemicals such as aryl hydrocarbons and as a regulator of enzymes such as cytochrome P450s.

[0126] Examples of AhR antagonists suitable for use in the invention include, but are not restricted to, SR1 , PD98059, GNF351 , BAY 2416964, CH-223191 , PDM-11 , BAY-218. Suitably, the AhR antagonist is selected from StemRegenin 1 (SR1), CH-223191 , BAY-218, BAY 2416964 (compound 192), GNF351 , PDM2 and PDM-11 , preferably the AhR antagonist is SR1 .

[0127] Interleukins (ILs) are a group of cytokines (secreted proteins and signal molecules) that are expressed and secreted by white blood cells (leukocytes) as well as some other body cells. There are many known ILs (the most commonly known being the 17 numbered from IL-1 to IL-17). In the claimed method, IL-1 , IL-2, IL-3, IL-6, IL-7, IL-9, IL-15, IL-18, and IL-21 are chosen due to their ability to promote differentiation in certain desired lineages. For example, IL-3 and IL-6 promote expansion and survival of HSCs and HSPCs.

[0128] The term IL-15Ralpha as used herein refers to a subunit of the interleukin 15 receptor that, in humans, is encoded by the IL15RA gene. IL-15Ralpha specifically binds IL15 with very high affinity, and is capable of binding IL-15 independently of other subunits. It is suggested that this property allows IL-15 to be produced by one cell, endocytosed by another cell, and then presented to a third party cell. This receptor is reported to enhance cell proliferation and expression of apoptosis inhibitor BCL2L1 / BCL2-XL and BCL2. IL-15Ralpha comprises a “Sushi domain”, which contains the shortest region (a 65-amino acid sequence) retaining IL- 15 binding activity.

[0129] The term mothers against decapentaplegic homolog 3 (SMAD3) inhibitor as used herein refers to a compound which interferes or inhibits the activity of the SMAD3 protein. SMAD3 is a member of the SMAD family of protein and acts as a mediator of the signals initiated by the transforming growth factor beta (TGF-P) superfamily of cytokines, which regulate cell proliferation, differentiation and death. Suitably, the SMAD3 inhibitor is selected from SIS3 and 16d, preferably the SMAD3 inhibitor is SIS3.

[0130] Suitably, wherein the culturing media used in the culturing of steps ii), iii), iv) and / or v) comprises Anti-human IgG antibody, the culturing media used in the culturing of steps ii), iii), iv) and v) further comprises DLL4-Fc. In some embodiments of the present invention, the BMP4 is used at a concentration of 1 ng / ml to 30 ng / ml, preferably 5 ng / ml to 25 ng / ml, such as about 10 ng / ml or about 20 ng / ml.

[0131] In some embodiments of the present invention, the VEGF is used at a concentration of 10 ng / ml to 30 ng / ml, preferably about 20 ng / ml.

[0132] In some embodiments of the present invention, the SCF is used at a concentration of 10 ng / ml to 100 ng / ml, 10 ng / ml to 50 ng / ml, preferably 15 ng / ml to 45 ng / ml, such as about 20 ng / ml or about 40 ng / ml.

[0133] In some embodiments of the present invention, the GSK-3 inhibitor is used at a concentration of 1 pM to 5 pM, preferably about 3 pM, or 1 mM to 5 mM, preferably about 2 mM.

[0134] In some embodiments of the present invention, the Activin A is used at a concentration of 5 ng / ml to 40 ng / ml, preferably 10 ng / ml to 35 ng / ml, such as about 15 ng / ml or about 25 ng / ml

[0135] In some embodiments of the present invention, the ROCK inhibitor is used at a concentration of 5 pM to 20 pM, preferably about 10 pM.

[0136] In some embodiments of the present invention, the bFGF is used at a concentration of 5 ng / ml to 20 ng / ml, preferably about 10 ng / ml.

[0137] In some embodiments of the present invention, the TGF-p receptor kinase inhibitor is used at a concentration of 5 pM to 20 pM, preferably about 10 pM.

[0138] In some embodiments of the present invention, each of the IL-3, IL-6, IL-7, IL-15 and IL-21 are independently used at concentrations of 1 ng / ml to 30 ng / ml, preferably 1 ng / ml to 25 ng / ml, such as about 5 ng / ml, 10 ng / ml, 15 ng / ml or 20 ng / ml.

[0139] In some embodiments of the present invention, the TPO is used at a concentration of 5 ng / ml to 20 ng / ml, preferably about 10 ng / ml.

[0140] In some embodiments of the present invention, the Nicotinamide is used at a concentration of 1 mM to 5 mM, preferably about 2.5 mM. In some embodiments of the present invention, the Notch agonist is used at a concentration of 250 ng / ml to 750 ng / ml, preferably about 500 ng / ml.

[0141] In some embodiments of the present invention, the IGF- 1 is used at a concentration of 50 ng / ml to 200 ng / ml, preferably about 100 ng / ml.

[0142] In some embodiments of the present invention, the tryptophan is used at a concentration of 0.001 pM to 1 pM or 0.1 pM to 1 pM, preferably about 0.1 pM.

[0143] In some embodiments of the present invention, the Anti-human IgG antibody is used at a concentration of 1 pg / ml to 5 pg / ml, preferably about 3.5 pg / ml.

[0144] In some embodiments of the present invention, the AhR antagonist is used at a concentration of 0.1 pM to 2 pM or 1 pM to 2 pM, preferably about 1 pM.

[0145] In some embodiments of the present invention, the FLT3L is used at a concentration of 5 ng / ml to 20 ng / ml, preferably about 10 ng / ml.

[0146] In some embodiments of the present invention, the hydrocortisone is used at a concentration of 0.1 pM to 2 pM or 1 pM to 2 pM, preferably about 1 pM.

[0147] In some embodiments of the present invention, the IL-15Ralpha is used at a concentration of 5 ng / ml to 20 ng / ml, preferably about 10 ng / ml.

[0148] In some embodiments of the present invention, the estradiol is used at a concentration of 5 nM to 15 nM, preferably about 10 nM.

[0149] In some embodiments of the present invention, the SIS3 is used at a concentration of 0.1 ng / ml to 0.2 ng / ml, preferably about 0.125 ng / ml.

[0150] Step ii) may comprise the use of one or more media components (preferably all) of Protocol 1 , Protocol 2, Protocol 3, Protocol 4, Protocol 5, Protocol 6, or Protocol 7 presented in Table 2A. Step iii) may comprise the use of one or more media components (preferably all) of Protocol 1 , Protocol 2, Protocol 3, Protocol 4, Protocol 5, Protocol 6, or Protocol 7 presented in Table 3A, respectively. Step iv) may comprise the use of one or more media components (preferably all) of Protocol 1 , Protocol 2, Protocol 3, Protocol 4, Protocol 5, Protocol 6, or Protocol 7 presented in Table 4A, respectively. Step v) may comprise the use of one or more media components (preferably all) of Protocol 1 , Protocol 2, Protocol 3, Protocol 4, Protocol 5, Protocol 6, or Protocol 7 presented in Table 5A, respectively. Where the one or more media components used in step ii) comprise one or more media components of a given protocol, the one or more media components used in steps iii) to v) are preferably also one or more media components of the same protocol. The amount of a given media component may be 1000 times less to 1000 times more, 100 times less to 100 times more, or 10 times less to 10 times more than the amount listed in said table. Preferably the amount of a given media component may be the same as the amount listed in said table.

[0151] As used herein, the term “feeding step” (also referred to as “media change step”) refers to a full or partial media exchange to a fresh media of the same kind. In case of a partial media exchange, half of the culture media is collected and span. The cells are transferred back to the growing culture, and the same volume of media containing double the amount of culture reagents is added to the culture. In case of a full media exchange, all cells and media are collected, the cells are span and transferred to a culture containing fresh media supplemented with single the amount of culture reagents.

[0152] As used herein, the term “full media change” refers to a change of culturing media, wherein all of the media volume is collected and replaced with an equal amount and typically an equal concentration of freshly prepared media.

[0153] As used herein, the term “half media change” refers to a change of culturing media, wherein about half of the media volume is collected and replaced with an equal amount of freshly prepared media.

[0154] In some embodiments of the present invention, the culturing of step ii) further optionally comprises a culturing media change step, wherein the culturing media is collected and replaced with an equal amount of step ii) culturing media at up to 4 days, preferably at 2 to 4 days and even more preferably at 3 days from the beginning of the culturing of step ii).

[0155] In some embodiments of the present invention, the culturing of step iii) further optionally comprises a culturing media change step, wherein the culturing media is collected and replaced with an equal amount of step iii) culturing media at up to 4 days, preferably at 2 to 4 days from the beginning of the culturing of step iii). In some embodiments of the present invention, the culturing of step iv) further optionally comprises a culturing media change step, wherein the culturing media is collected and replaced with an equal amount of step iv) culturing media at up to 6 days, preferably at 4 to 6 days from the beginning of the culturing of step iv).

[0156] In some embodiments of the present invention, the culturing of step v) further optionally comprises a culturing media change step, wherein the culturing media is collected and replaced with an equal amount of step v) culturing media every 2 to 5 days, preferably every 3 to 4 days from the beginning of the culturing of step v).

[0157] Suitably, the culturing media change step may be a full culturing media change, wherein all of the media volume is collected and replaced with freshly prepared media, or the culturing media change step may be a half culturing media change, wherein half of the media volume is collected and replaced with freshly prepared media.

[0158] Suitably, a half culturing media change step comprises replacing half of the media volume with a freshly prepared culturing media which is twice as concentrated as the previous media concentration.

[0159] Preferably, the method (e.g. encompassing protocol 7 in the examples) for producing a NK cell comprises the steps of: i) providing a population of cell aggregates comprising stem cells (such as induced Pluripotent Stem Cells (iPSCs), preferably iPSCs); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises VEGF and at least 3 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, and bFGF, and preferably wherein the culturing media used in the culturing of step ii does not comprise Activin A; wherein the culturing media used in the culturing of step iii) comprises at least 4 components selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IL-6, thrombopoietin (TPO), Notch agonist, IGF-1 , estradiol, Nicotinamide and Tryptophan; wherein the culturing media used in the culturing of step iv) comprises at least 5 components selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF-p receptor kinase inhibitor, bFGF, IL-6, TPO, Nicotinamide, Tryptophan, Notch agonist, Anti-human IgG antibody, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha; wherein the culturing media used in the culturing of step v) comprises at least 3 components selected from FLT3L, SCF, IL-7, IL-15, Notch agonist, Hydrocortisone, IL-15R- alpha, Nicotinamide, Anti-human IgG antibody, IGF1 , TPO, IL-21 , and SMAD3 inhibitor.

[0160] Advantageously, the above method (e.g. encompassing protocol 7 in the examples) may be particularly cost-effective (e.g. inexpensive) when compared to other methods for producing NK cells. The present inventors surprisingly found that, in the method for producing a NK cell of the invention (e.g. encompassing protocol 7 in the example section), it was particularly advantageous when the culturing media used in the culturing of step ii) comprised VEGF and at least 3 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, and bFGF, and did not comprise Activin A. This was found to reliably produce stem cell (iPSCs)- derived NK cells with the desired cell surface marker profile (e.g. see example 1 and Figure 7) and potent cytotoxicity against cancer cells (e.g. see example 1 and Figure 3C) in a more cost-effective manner compared to the other protocol tested. Notably, this was achieved without significantly compromising the yield of the mature stem (iPSC)-cell derived NK cells (CD45+ / CD56+ by day 38) compared to other protocols (e.g. example 1 and Figure 1 E). This makes said method (e.g. encompassing protocol 7 in the examples) particularly advantageous for industrial manufacturing where cost control and consistent quality of the product are paramount. Thus, this unique combination (“sweet spot”) of cost-efficiency and consistent quality of the stem (iPSC) cell-derived NK cells makes said method (e.g. encompassing protocol 7 in the examples) highly attractive for industrial manufacturing.

[0161] Additionally, it was surprisingly found that the stem cell (iPSC)-derived NK cells generated by said method (e.g. encompassing protocol 7 in the examples) not only exhibit the functional differences described above but also possessed a distinct gene expression profile (gene signature) compared to blood-derived NK cells (e.g. peripheral blood NK cells) (e.g. as shown in Figures 4, 5, and 6 of the examples). Without wishing to be bound to any theory, it is believed that the presence of VEGF and the absence of Actinin A in the culture medium at particular time during the in vitro differentiation phase (preferably during phase Day 0 to Day 2) and / or the presence of BMP4 at particular dosage (preferably in amount lower than 20 ng / ml preferably around 10 ng / ml) in the culture medium at particular time during the in vitro differentiation phase (preferably during phase Day 3 to Day 5 and / or D6 to D11) leads to the observed distinct functional characteristics and molecular profile. Such features are unique to said method (e.g. encompassing protocol 7 in the examples).

[0162] Preferably, the method (e.g. encompassing protocol 3 in the examples) for producing a NK cell comprises the steps of: i) providing a population of cell aggregates comprising stem cells (preferably induced Pluripotent Stem Cells (iPSCs)); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises Activin A and / or LiCI, and at least 2 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, and bFGF, and wherein the culturing media used in the culturing of step ii) does not comprise VEGF; wherein the culturing media used in the culturing of step iii) comprises IGF-1 and / or a Notch agonist (preferably DLL4 Fc) and at least 2 components selected from BMP4, VEGF, SCF, TGF- receptor kinase inhibitor, bFGF, IL-3, IL-6, TPO, estradiol, Nicotinamide and Tryptophan; wherein the culturing media used in the culturing of step iv) comprises one or more of Nicotinamide, Tryptophan, Notch agonist (preferably DLL4 Fc), and Antihuman IgG antibody and at least 1 component selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF-p receptor kinase inhibitor, bFGF, IL-6, TPO, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha; wherein the culturing media used in the culturing of step v) comprises at least 3 components selected from FLT3L, SCF, IL-7, IL-15, Notch agonist, Hydrocortisone, IL-15R-alpha, Nicotinamide, Anti-human IgG antibody, IGF1 , TPO, IL-21 , and SMAD3 inhibitor.

[0163] More preferably, the culturing media used in the culturing of step ii) comprises Activin A and LiCI, and at least 2 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, and bFGF, and does not comprise VEGF.

[0164] More preferably, the culturing media used in the culturing of step iii) comprises IGF- 1 and a Notch agonist (preferably DLL4 Fc) and at least 2 components selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IL-6, TPO, estradiol, Nicotinamide and Tryptophan.

[0165] More preferably, the culturing media used in the culturing of step iv) comprises Nicotinamide and Tryptophan and at least 1 component selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF-p receptor kinase inhibitor, bFGF, IL-6, TPO, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha. Yet more preferably, the culturing media used in the culturing of step iv) comprises Nicotinamide, Tryptophan, Notch agonist (preferably DLL4 Fc), and Anti-human IgG antibody and at least 1 component selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF-p receptor kinase inhibitor, bFGF, IL-6, TPO, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha.

[0166] Advantageously, the above method (e.g. encompassing protocol 3 in the examples), may be particularly suitable to produce higher yields (higher number of cells) of NK cells with a desired cell surface profile (e.g. CD45+ / CD56+). Specifically, the present inventors surprisingly found that, in the method for producing a NK cell of the invention (which is referred to as protocol 3 in the example section), it was particularly advantageous when the culturing media used in the culturing of step iii) comprises IGF-1 and / or a Notch agonist (preferably DLL4 Fc) and at least 2 components selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IL-6, TPO, estradiol, Nicotinamide and Tryptophan and when the culturing media used in the culturing of step iv) comprises one or more of Nicotinamide, Tryptophan, Notch agonist (preferably DLL4 Fc), and Anti-human IgG antibody and at least 1 component selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF-p receptor kinase inhibitor, bFGF, IL-6, TPO, aryl hydrocarbon receptor (AhR) antagonist and IL-15R- alpha. Said method (e.g. encompassing protocol 3 in the examples) was surprisingly found to yields a significantly higher number of CD45+ / CD56+ double-positive stem cell (iPSC)- derived NK cells by day 38 of differentiation (representative of mature NK cells) compared to the other tested protocols (e.g. example 1 and Figure 1 E). This is particularly advantageous for industrial manufacturing where a higher yield of NK cells with the desired cell surface expression profile is consistently achieved from batch to batch.

[0167] Additionally, the NK cells derived from said method (e.g. encompassing protocol 3 in the examples) exhibited superior cytotoxicity against various cancer cell lines compared to the NK-92 cell line, a standard positive control in research and known for its broad and potent anti-tumour activity exceeding that of blood-derived NK cells, as shown in the examples (e.g. Figure 3) (Suck et al (2016) Cancer Immunol Immunother, Vol 65, pages 485-492). Consequently, the stem cell (iPCS)-derived NK cells generated by said method (e.g. encompassing protocol 3 in the examples) possess cytotoxic activity surpassing both NK-92 cells and blood-derived NK cells. Surprisingly, the NK cells produced by said method (e.g. encompassing protocol 3 in the example) not only exhibited distinct functional differences compared to blood-derived NK cells (e.g. peripheral blood NK cells) but also possessed a distinct gene expression profile (gene signature) compared to blood-derived NK cells (e.g. as show in Figures 4, 5, and 6).

[0168] Without wishing to be bound to any theory, it is believed that the presence of Activin A and / or LiCI (preferably both) in the culture medium at particular time during the in vitro differentiation phase (preferably during phase Day 0 to Day 2), and / or the presence of DDL4 Fc and / or IGF-1 (preferably the presence of DDL4 Fc and IGF-1) in the culture medium at particular time during the in vitro differentiation phase (preferably during phase Day 3 to Day 5) and / or the presence of one or more of Nicotinamide, Tryptophan, Notch agonist (preferably DLL4 Fc), and Anti-human IgG antibody (preferably the presence of Nicotinamide, Tryptophan, Notch agonist (preferably DLL4 Fc), and Anti-human IgG antibody) in the culture medium at particular time during in vitro differentiation phase (preferably during phase Day 6 to Day 11) leads to the observed distinct functional characteristics and molecular profile. Such features are unique to said method (e.g. encompassing protocol 3 in the examples).

[0169] Other advantages associated with said method (e.g. encompassing protocol 3 in the examples) are shown in Figure 1C. Specifically, it was found that said method (e.g. encompassing protocol 3 in the examples) generated stem cell (iPSC)-derived NK cell populations with a higher percentage of CD45+ / CD56+ cells compared to other methods of the invention (e.g. protocol 5 in the examples), indicating greater stem cell (iPSC)-derived NK cell purity per batch. In other words, said method (e.g. encompassing protocol 3 in the examples) predominantly produced the desired stem cell (iPSC)-derived NK cells with minimal contamination from cells lacking the appropriate marker profile or cells from an undesirable cell lineage.

[0170] Furthermore, when examining the relationship between the cell surface marker profile and cell number, said method (e.g. encompassing protocol 3 in the examples) stands out. Figure 1 E shows that by day 38 of differentiation, said method (e.g. encompassing protocol 3 in the examples) resulted in the greatest number of CD45+ / CD56+ NK cells (indicative of mature NK cells) relative to the other protocols. Therefore, said method (e.g. encompassing protocol 3 in the examples) is associated with the highest yield of CD45+ / CD56+ NK cells, which is beneficial for industrial manufacturing process.

[0171] Thus, key considerations for selecting an in vitro stem cell (iPSCs or ESCs) differentiation protocol for NK cell production include its ability to generate NK cells with the desired cell surface marker profile, strong anti-cancer cytotoxicity (superior to NK-92 cells as well as blood-derived NK cells), and activation upon encountering immune stimuli (e.g. tumour cells). Importantly, the chosen protocol should preferably also deliver high yields of such stem cell (iPSC)-derived NK cells with the desired surface marker profile as well as functional characteristics. This dual focus on functionality and yield is important for reliable, industrialscale manufacturing of off-the-shelf stem cell (e.g. iPSC)-derived NK cells with consistent molecular and functional characteristics across batches, in a cost-effective manner.

[0172] The methods of the invention (particularly protocols 3 and 7 in the examples) meet the above criteria. Specifically, the methods of the invention (e.g. encompassing protocols 3 and 7 in the examples) are associated with several advantages including the production of stem cell (iPSC)-derived NK cells that differ significantly from blood-derived NK cells and NK-92 cells in both function, phenotype (cell surface markers), and genetic makeup. Specifically, these stem cell (iPSC)-derived NK cells show greater cytotoxic potency and stronger activation in response to stimuli both at baseline and against cancer cells (e.g. K562) together with a unique gene expression profile (gene signature). Additionally, both protocols deliver high- yield, cost-effective, and scalable production of CD45+ / CD56+ stem cell (iPSC)-derived NK cells.

[0173] Preferably, the method (e.g. encompassing protocol 6 in the examples) for producing a NK cell comprises the steps of: i) providing a population of cell aggregates comprising stem cells (preferably induced Pluripotent Stem Cells (iPSCs)); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises Activin A and / or LiCI, and at least 2 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, VEGF and bFGF; wherein the culturing media used in the culturing of step iii) comprises IL-6 and / or TPO, and at least 2 components selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IGF-1 , estradiol, Nicotinamide and Tryptophan, and wherein the culturing media used in the culturing of step iii) does not comprise Notch agonist (preferably DLL4 Fc); wherein the culturing media used in the culturing of step iv) comprises TGF-p receptor kinase inhibitor (preferably SB431542) and / or bFGF, and at least 3 components selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, bFGF, IL-6, TPO, Nicotinamide, Tryptophan, aryl hydrocarbon receptor (AhR) antagonist and IL-15R- alpha, and wherein the culturing media used in the culturing of step iii) does not comprise Notch agonist (preferably DLL4 Fc) and / or Anti-human IgG antibody; wherein the culturing media used in the culturing of step v) comprises at least 3 components selected from FLT3L, SCF, IL-7, IL-15, Notch agonist, Hydrocortisone, IL-15R-alpha, Nicotinamide, Anti-human IgG antibody, IGF1 , TPO, IL-21 , and SMAD3 inhibitor.

[0174] More preferably, the culturing media used in the culturing of step ii) comprises Activin A and LiCI, and at least 2 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, VEGF and bFGF.

[0175] More preferably, the culturing media used in the culturing of step iii) comprises IL-6 and TPO, and at least 2 components selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IGF-1 , estradiol, Nicotinamide and Tryptophan, and does not comprise Notch agonist (preferably DLL4 Fc).

[0176] More preferably, the culturing media used in the culturing of step iv) comprises TGF-p receptor kinase inhibitor (preferably SB431542) and bFGF, and at least 3 components selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, bFGF, IL-6, TPO, Nicotinamide, Tryptophan, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha, and wherein the culturing media used in the culturing of step iii) does not comprise Notch agonist (preferably DLL4 Fc) and Anti-human IgG antibody.

[0177] Advantageously, the method above (e.g. encompassing protocol 6 in the examples) was found to be particularly suitable for producing high yield of NK cells having a desired cell surface marker profile (CD45+ / CD56+) in a more cost-effective manner, is the following method. Specifically, the present inventors surprisingly found that, in the method for producing a NK cell of the invention (e.g. encompassing protocol 6 in the examples), it was particularly advantageous when the culturing media used in the culturing of step ii) comprised Activin A and / or LiCI (preferably both), and at least 2 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, VEGF and bFGF. This is surprising as compounds such as Activin A and LiCI are not typically used in methods for producing a NK cell.

[0178] The present inventors also found that it was particularly advantageous when the culturing media used in the culturing of step iii) comprised IL-6 and / or TPO (preferably both), and at least 2 components selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IGF-1 , estradiol, Nicotinamide and Tryptophan, and did not comprise Notch agonist DLL4 Fc. The absence of the Notch agonist DLL4 Fc is particularly advantageous as this compound is relatively expensive compared to other compounds used in the in vitro production of NK cells or other Notch agonists. Surprisingly, its absence did not impact or alter significantly the yield or quality of the stem cell (iPSC)-derived NK cells produced by said method (e.g. encompassing protocol 6 in the examples).

[0179] Similar advantageous were also observed when the culturing media used in the culturing of step iv) comprised TGF-p receptor kinase inhibitor (preferably SB431542) and bFGF, and at least 3 components selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, bFGF, IL-6, TPO, Nicotinamide, Tryptophan, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha, and did not comprise the Notch agonist DLL4 Fc and the Anti-human IgG antibody. This is surprising and advantageous for the same reasons as above. Additionally, the removal of the Anti-human IgG antibody simplifies the process and allows further cost savings as the Antihuman IgG antibody is often added the culture medium to promotes the effects of DLL4 Fc. Without wishing to be bound by theory, it is believed that the Anti-human IgG antibody helps or promotes the maintenance of the 3D structure of DLL4 Fc.

[0180] Said method (e.g. encompassing protocol 6 in the examples) was surprisingly found to yields a relatively high number of CD45+ / CD56+ double-positive stem cell (iPSC)-derived NK cells by day 38 of differentiation (representative of mature NK cells) compared to the other tested protocols (e.g. example 1 and Figure 1 E), however at a lower cost. This is particularly advantageous for industrial manufacturing where a high yield of NK cells with the desired cell surface expression profile is consistently achieved from batch to batch, and in a cost- effective manner.

[0181] Without wishing to be bound to any theory, it is believed that the presence of Activin A and / or LiCI (preferably both) in the culture medium at particular time during the in vitro differentiation phase (preferably during phase Day 0 - Day 2) and / or the presence of IL-6 and / or TPO and the absence of DLL4 Fc in the culture medium at particular time during in vitro differentiation phase (preferably during phase Day 3 to Day 5), and / or the presence of TGF-p receptor kinase inhibitor (preferably SB431542) and / or bFGF and absence of DLL4 Fc and Antihuman IgG antibody in the culture medium at particular time during in vitro differentiation phase (preferably during phase Day 6 to Day11) leads to the observed advantages. Such features are unique to said method (e.g. encompassing protocol 6 in the examples).

[0182] Preferably, the method (e.g. encompassing protocol 5 in the examples) for producing a NK cell comprises the steps of: i) providing a population of cell aggregates comprising stem cells (preferably induced Pluripotent Stem Cells (iPSCs)); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises at least 4 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, Activin A, VEGF and bFGF; wherein the culturing media used in the culturing of step iii) comprises bFGF and / or TGF-p receptor kinase inhibitor (preferably SB431542), and at least 2 components selected from BMP4, VEGF, SCF, bFGF, IL-3, IL-6, TPO, IGF-1 , estradiol, Nicotinamide and Tryptophan, and wherein the culturing media used in the culturing of step iii) does not comprise a Notch agonist (preferably DLL4 Fc); wherein the culturing media used in the culturing of step iv) comprises SR1 and / or Sushi, and at least 3 components selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF-p receptor kinase inhibitor, bFGF, IL-6, TPO, Nicotinamide, Tryptophan, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha, and wherein the culturing media used in the culturing of step iii) does not comprise a Notch agonist (preferably DLL4 Fc) and / or Anti-human IgG antibody; wherein the culturing media used in the culturing of step v) comprises at least 3 components selected from FLT3L, SCF, IL-7, IL-15, Notch agonist, Hydrocortisone, IL-15R-alpha, Nicotinamide, Anti-human IgG antibody, IGF1 , TPO, IL-21 , and SMAD3 inhibitor.

[0183] More preferably, the culturing media used in the culturing of step iii) comprises bFGF and TGF-p receptor kinase inhibitor (preferably SB431542), and at least 2 components selected from BMP4, VEGF, SCF, bFGF, IL-3, IL-6, TPO, IGF-1 , estradiol, Nicotinamide and Tryptophan, and does not comprise a Notch agonist (preferably DLL4 Fc).

[0184] More preferably, the culturing media used in the culturing of step iv) comprises SR1 and Sushi, and at least 3 components selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF-p receptor kinase inhibitor, bFGF, IL-6, TPO, Nicotinamide, Tryptophan, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha, and does not comprise a Notch agonist (preferably DLL4 Fc) and Anti-human IgG antibody.

[0185] Advantageously, the method above (e.g. encompassing protocol 5 in the examples) was found to be particularly suitable to produce NK cells having potent cytotoxic activity against cancer cells (having high or potent cancer killing activity), such as breast cancer cells. Specifically, the present inventors surprisingly found that the method above (e.g. encompassing protocol 5 in the examples) is associated with advantages such as the production of stem cell (iPSC) derived NK cells endowed with potent (high) (dose-dependent) cancer killing activity (cytotoxic activity against cancer cells), such as breast cancer cells (e.g. as shown in Figure 3C).

[0186] Further, the method above (e.g. encompassing protocol 5 in the examples) is associated with cost saving advantages since compounds like DLL4 Fc (which are expensive) as well as Anti-human IgG antibody are avoided.

[0187] Additionally, the method above (e.g. encompassing protocol 5 in the examples) is associated with the ability to produce moderate yield of (mature) stem cell (iPSC)-derived NK cells having a desired marker profile (CD45+ / CD56+) (e.g. as shown in Figure 1 D and E). Therefore, said method (e.g. encompassing protocol 5 in the examples) represents a good combination (“sweet spot”) of cost-efficiency and consistent quality of the stem (iPSC) cell- derived NK cells, which makes said method (e.g. encompassing protocol 5 in the examples) advantageous for industrial manufacturing.

[0188] Without wishing to be bound to any theory, it is believed that the presence of FGF and / or TGF-p receptor kinase inhibitor (preferably SB431542) (preferably both) and the absence of the Notch agonist (preferably DLL4 Fc) in the culture medium at particular time during the in vitro differentiation phase (preferably during phase Day 3 to Day 5) and / or the presence of SR1 and / or Sushi (preferably both) and the absence of DLL4 Fc and / or Anti-human IgG antibody (preferably both) in the culture medium at particular time during in vitro differentiation phase (preferably during phase Day 6 to Day 11) leads to the observed advantages. Such features are unique to said method (e.g. encompassing protocol 5 in the examples).

[0189] Most preferably, the method for producing a NK cell may comprise the steps of: i) providing a population of cell aggregates comprising stem cells (preferably induced Pluripotent Stem Cells (iPSCs)); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises VEGF, ROCK inhibitor (preferably Y-27632 dihydrochloride), BMP4, SCF, GSK-3 inhibitor (preferably CHIR99021), and bFGF, and wherein the culturing media used in the culturing of step ii does not comprise Activin A; wherein the culturing media used in the culturing of step iii) comprises BMP4, VEGF, SCF, IL-3, IL-6, and TPO; wherein the culturing media used in the culturing of step iv) comprises BMP4, VEGF, SCF, IL-3, IL-7, IL-15, IL-6, and TPO; wherein the culturing media used in the culturing of step v) comprises FLT3L, SCF, IL-7, and IL-15.

[0190] Most preferably, the method for producing a NK cell may comprise the steps of: i) providing a population of cell aggregates comprising stem cells (preferably induced Pluripotent Stem Cells (iPSCs)); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises ROCK inhibitor (preferably Y-27632 dihydrochloride), BMP4, SCF, GSK-3 inhibitor (preferably LiCI), Activin A, and bFGF, and wherein the culturing media used in the culturing of step ii) does not comprise VEGF; wherein the culturing media used in the culturing of step iii) comprises BMP4, VEGF, SCF, bFGF, Notch agonist (preferably DLL4 Fc), and IGF-1 ; wherein the culturing media used in the culturing of step iv) comprises BMP4, VEGF, SCF, IL-3, IL-7, IL-15, Nicotinamide, Tryptophan, Notch agonist (preferably DLL4 Fc), and Anti-human IgG antibody; wherein the culturing media used in the culturing of step v) comprises FLT3L, SCF, IL-7, IL-15, Notch agonist (preferably DLL4 Fc), and Hydrocortisone.

[0191] Preferably, the method for producing a NK cell may comprise the steps of: i) providing a population of cell aggregates comprising stem cells (preferably induced Pluripotent Stem Cells (iPSCs)); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor (preferably liCI), Activin A, and bFGF; wherein the culturing media used in the culturing of step iii) comprises BMP4, VEGF, SCF, IL-3, IL-6, and TPO, and wherein the culturing media used in the culturing of step iii) does not comprise Notch agonist (preferably DLL4 Fc); wherein the culturing media used in the culturing of step iv) comprises BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF- receptor kinase inhibitor (preferably SB431542), and bFGF, and wherein the culturing media used in the culturing of step iii) does not comprise Notch agonist (preferably DLL4 Fc) and / or Anti-human IgG antibody; wherein the culturing media used in the culturing of step v) comprises FLT3L, SCF, IL-7, IL-15, Notch agonist (preferably DLL4 Fc), and Hydrocortisone.

[0192] Preferably, the method for producing a NK cell may comprise the steps of: i) providing a population of cell aggregates comprising stem cells (preferably induced Pluripotent Stem Cells (iPSCs)); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor (preferably CHIR99021), and Activin A; wherein the culturing media used in the culturing of step iii) comprises BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor (preferably SB431542), and bFGF, and wherein the culturing media used in the culturing of step iii) does not comprise a Notch agonist (preferably DLL4 Fc); wherein the culturing media used in the culturing of step iv) comprises BMP4, VEGF, SCF, IL-3, IL-7, IL-15, IL-6, TPO, and aryl hydrocarbon receptor (AhR) antagonist (preferably SR1); wherein the culturing media used in the culturing of step v) comprises FLT3L, SCF, IL-7, IL-15, Notch agonist (preferably DLL4 Fc), Nicotinamide and Anti-human IgG antibody.

[0193] Preferably, the method for producing a NK cell may comprise the steps of: i) providing a population of cell aggregates comprising stem cells (preferably induced Pluripotent Stem Cells (iPSCs)); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor (preferably CHIR99021), and Activin A; wherein the culturing media used in the culturing of step iii) comprises BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor (preferably SB431542), and bFGF, and preferably wherein the culturing media used in the culturing of step iii) does not comprise a Notch agonist (preferably DLL4 Fc); wherein the culturing media used in the culturing of step iv) comprises BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF- receptor kinase inhibitor (preferably SB431542), and bFGF, and preferably wherein the culturing media used in the culturing of step iv) does not comprise a Notch agonist (preferably DLL4 Fc) and Anti-human IgG antibody; wherein the culturing media used in the culturing of step v) comprises FLT3L, SCF, IL-7, and IL-15. Preferably, the method for producing a NK cell may comprise the steps of: i) providing a population of cell aggregates comprising stem cells (preferably induced Pluripotent Stem Cells (iPSCs)); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises a ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor (preferably CHIR99021), and Activin A; wherein the culturing media used in the culturing of step iii) comprises BMP4, VEGF, SCF, bFGF, and TGF-|3 receptor kinase inhibitor (preferably SB431542), and preferably wherein the culturing media used in the culturing of step iii) does not comprises a Notch agonist (preferably DLL4 Fc); wherein the culturing media used in the culturing of step iv) comprises BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF- receptor kinase inhibitor (preferably SB431542), and bFGF, and preferably wherein the culturing media used in the culturing of step iv) does not comprise Notch agonist (preferably DLL4 Fc) and Anti-IgG antibody; wherein the culturing media used in the culturing of step v) comprises FLT3L, SCF, IL-7, and IL-15.

[0194] Preferably, the method for producing a NK cell may comprise the steps of: i) providing a population of cell aggregates comprising stem cells (preferably induced Pluripotent Stem Cells (iPSCs)); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor (preferably CHIR99021), and Activin A; wherein the culturing media used in the culturing of step iii) comprises BMP4, VEGF, SCF, Nicotinamide and Tryptophan, and preferably wherein the culturing media used in the culturing of step iii) does not comprise Notch agonist (preferably DLL4 Fc); wherein the culturing media used in the culturing of step iv) comprises BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF- receptor kinase inhibitor (preferably SB431542), and bFGF, and preferably wherein the culturing media used in the culturing of step iv) does not comprise a Notch agonist (preferably DLL4 Fc) and Anti-human IgG antibody; wherein the culturing media used in the culturing of step v) comprises FLT3L, SCF, IL-7, IL-15, Notch agonist (preferably DLL4 Fc), and Nicotinamide.

[0195] The above also apply for when the methods of the invention are used to produce intermediate cells (or NK cell precursors), such as HSCs and / or HSPCs.

[0196] Thus, during the method to produce the NK cells, intermediate cells (or NK cell precursors) may also be produced, suitably HSCs and / or HSPCs. As such, there is also provided a method for producing HSCs and / or HSPCs cells, the method comprising the steps of: i) providing a population of cell aggregates comprising stem cells (preferably induced Pluripotent Stem Cells (iPSCs)); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) optionally culturing the third cultured population from step iv) to form a fourth cultured population; wherein the culturing media used in the culturing of step ii) comprises at least 4 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, Activin A, VEGF and bFGF; wherein the culturing media used in the culturing of step iii) comprises at least 4 components selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IL-6, TPO, Notch agonist, IGF-1 , estradiol, Nicotinamide and Tryptophan; wherein the culturing media used in the culturing of step iv) comprises at least 5 components selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF-p receptor kinase inhibitor, bFGF, IL-6, TPO, Nicotinamide, Tryptophan, Notch agonist, Anti-human IgG antibody, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha; wherein the culturing media used in the culturing of step v) comprises at least 3 components selected from FLT3L, SCF, IL-7, IL-15, Notch agonist, Hydrocortisone, IL-15R- alpha, Nicotinamide, Anti-human IgG antibody, IGF1 , TPO, IL-21 , and SMAD3 inhibitor.

[0197] The method for producing HSCs and / or HSPCs may have any of the features described for the method of producing the NK cells.

[0198] Suitably, the third cultured population of step iv) comprises HSCs and / or HSPCs.

[0199] Suitably, the fourth cultured population of step v) further comprises HSCs and / or HSPCs.

[0200] Suitably in some embodiments of the present invention, the methods further comprise a step (vi) of isolating the produced HSC, HSPC and / or NK cells. Such isolating may be done by known methods in the art, such as density gradient centrifugation, magnetic-activated cell sorting (MACS), fluorescence-activated cell sorting (FACS). Such isolating may be used to enrich for desired populations.

[0201] In some embodiments of the present invention, the methods for producing HSC, HSPC and / or NK cells further comprise a step vi) of collecting the produced HSC, HSPC and / or NK cells from the fourth cultured population in step v). Such collecting may be done by known methods in the art, such as detaching adherent cells, neutralising enzymes, harvesting suspension cells, removing the supernatant and washing cells.

[0202] The collected HSC, HSPC and / or NK cells may be considered “obtained” by the methods of the invention or obtainable by the methods of the invention.

[0203] In other embodiments of the present invention, the methods for producing HSC, and / or HSPC further comprise a step vi) of collecting the produced HSCs and / or HSPCs from the fourth cultured population in step v) at up to 20 days, preferably at 12 to 16 days from the beginning of the culturing of step ii).

[0204] In other embodiments of the present invention, the methods for producing NK cells further comprise a step vi) of collecting the produced NK cells from the fourth cultured population in step v) at least 20 days, preferably at least 25 days, such as at least 30 days after the beginning of the culturing of step ii). Suitably the collecting occurs between 20 and 55 days after the beginning of the culturing of step ii), preferably between 25 and 50 days, such as between 30 and 45 days.

[0205] Suitably, the collected HSCs and / or HSPCs express CD34+, CD43+ and / or CD45+.

[0206] Suitably, the percentage of CD34+, CD43+ and / or CD45+ in the collected HSCs and / or HSPCs constitutes between about 30% to 70% of live cells of the total cell culture cells.

[0207] Preferably, the collected (or obtainable or obtained by the methods of the invention) NK cells express CD56+ and CD45+.

[0208] The term “obtainable” as used herein may mean obtained.

[0209] In some embodiments of the present invention, the culturing of step iii) and step iv) further comprises a “feeding step”, wherein a half culturing media change occurs.

[0210] In some embodiments of the present invention, the step i), providing a population of cell aggregates comprising iPSCs and / or ESCs, further comprises the steps of: ia) seeding the population of iPSCs and / or ESCs in base media to form cell aggregates comprising the iPSCs or ESCs; ib) removing non-aggregated cells; ic) collecting and resuspending the cell aggregates; iv) seeding the cell aggregates in full growth media. The cell aggregates are then ready to begin the NK differentiation process via steps ii)-v) of the methods of the present invention.

[0211] The starting conditions for priming the population of iPSCs and / or ESCs may also be varied in the present invention. Suitably the starting conditions for culturing the population of iPSCs and / or ESCs in step ia) may occur in static conditions, shaker flask conditions and / or bioreactor conditions, which are all well-known processes in the field.

[0212] Suitably the step ia) occurs for up to 5 days, preferably up to 3 days, more preferably up to 2 days, such as up to 1 day. Suitably the base media used in step ia) is IPS Brew Media (commercially available) optionally further comprising a ROCK inhibitor, preferably Y-27632 dihydrochloride.

[0213] During the step ia) the formation of the cell aggregates, the pluripotent cells attach to each other. The cells that are partially differentiated and are not pluripotent do not form cell aggregates and stay in suspension. These cells need to be removed. The removal of such non-aggregated cells in step ib) can be performed by any suitable means known to the skilled person, such as with a stripette.

[0214] The collecting and resuspending of step ic) can be performed by any suitable means known to the skilled person, such as using a cell culture filter in which single cells and smaller aggregates will pass through it and the larger cell aggregates left on the filter will be collected and / or resuspending in a basal medium commercially available (such as Ste m D iff AP EL-2).

[0215] Various cytokines may be incorporated into the media and / or incorporated during culture, examples of suitable cytokines include, but are not restricted to; interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL- 7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon-a (INF-a), interferon-p (INF-P), interferon-y (INF-y), granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), Wnt1 , bone morphogenetic protein 7 (BMP7), angiopoietin-like 5 (ANGPTL5), insulin growth factor binding protein 2 (IGFBP2), erythropoietin (EPO), thrombopoietin (TPO), Fms-like tyrosine kinase 3-ligand (FLT3LG).

[0216] Various growth factors may be incorporated into the media and / or incorporated during culture. Examples of suitable growth factors include but are not restricted to insulin-like growth factor (IGF), epidermal growth factor (EGF), human epidermal growth factor (hEGF), platelet- derived growth factor (PDGF), fibroblast growth factor 1 (FGF1), nerve growth factor (NGF), macrophage inflammatory protein 1-a (MIP-1a), leukaemia inhibitory factor (LIF). In an embodiment of the present invention, the population of iPSCs and / or ESCs is cultured at a temperature between 32°C to 39°C, preferably between 36°C to 38°C. In an embodiment of the invention the cells are cultured in a humidified incubator with between about 1% to about 50% CO2, preferably between about 1% to about 25% CO2, more preferably between about 1% to about 10% CO2. The present invention can be performed in a culture vessel suitable for animal cell culture. In one embodiment the present invention is performed in Nanex Hematopoietic Stem / Progenitor Cell (HSPC) Expansion Plates or TC treated Corning 24 well plates or suspension Greiner Bio 24 well plates. In a preferred embodiment, the present invention is performed in a conventional cell culture plate or a suitable closed system such as a cell culture bag (e.g VueLife®) or a stirred bioreactor.

[0217] Suitable methods for determining phenotype and purity of differentiating cells are known in the art and include, for example, multicolour flow cytometric analysis combined with total cell counting, use of absolute counting beads in combination with flow cytometric analysis, cell counts based on imaging analysis of a cell aliquot using a manual or automated hemocytometer (Viacell, Countess, Nucleocounter, Nexcelome).

[0218] In one aspect, the present invention relates to a composition comprising a (collected) HSC, HSPC and / or NK cell obtainable or obtained by the methods of the invention (preferable by protocols 3 or 7 in the examples).

[0219] In one aspect, the present invention relates to a composition comprising a NK cell that has been differentiated in vitro, wherein the NK cell comprises: increased expression of one or more of: LINC00511, LINC02315, TESC, TPM4, TEC, DELEC1, COTL1, DRAXIN, SLC1A5, GLUL, LPAR3, NFILZ, INPP4B, ITGB7, KIT, S100A4, MIR646HG, CNR2, TNFSF10, LEF1, PRF1, RASSF8, THEMIS, DPF3, IL32, CTSW, AFAP1L2, CD3E, STYK1, ZNF683, CCR6, CCR1, MIR181A1HG, ID2, SYTL3, ZFHX3, CCND2, ITGAX, TRPM2, BCL2, NDFIP2, LTB, PRKCA, NCAM1, DOCK5, and IL18RT, and / or decreased expression of one or more of: ADAM28, TMEM71, DIPK1A, MIR3667HG, PMEPA1, TIGIT, COLGALT2, BTN3A3, LINC00861, and MPP7, when compared to a blood-derived NK cell.

[0220] In one aspect, the present invention relates to a composition comprising a NK cell that has been differentiated in vitro, wherein the NK cell comprises: increased expression of one or more of: MAG 11, IGSF1, DLEU1, FBXL7, TIAM1, MAML3, PTPN14, DAPK2, MYO1E, CSF1, TOX2, HOXB3, ITGA1, LINC00504, DPF3, ZFHX3, THEMIS, IL32, CTSW, TRPM2, NDFIP2, CCND2, ITGAX, IL18R1, PRKCA, LTB, CD3E, DOCK5, ZNF683, BCL2, MIR181A1HG, STYK1, RASSF8, CCR1, NCAM1, CCR6, ID2, AFAP1L2, and SYTL3 and / or decreased expression of one or more of: ZNF831, F2R, RAP1GAP2, TIGIT, BTN3A3, COLGALT2, MPP7, and LINC00861, when compared to a blood-derived NK cell.

[0221] In one aspect, the present invention relates to a kit comprising the compositions or an NK cell of the invention and instructions for use in medicine.

[0222] The HSCs or HSPCs obtainable or obtained by the methods presented herein can be used as a cell transplant. The HSCs or HSPCs obtained by the method presented herein may be used to repopulate mammalian bone marrow. Therefore, an embodiment of the present invention is the HSCs or HSPCs obtainable or obtained by the methods presented herein for use in the treatment of a haematological disorder, immune disorder, metabolic disorder, or neurodegenerative disorder, wherein the subject is administered HSCs or HSPCs obtainable or obtained according to the methods described above. In a particular embodiment, the HSCs or HSPCs are for use in the treatment of a haematological disorder.

[0223] The HSCs and / or HSPCs obtainable or obtained from the methods presented herein can be used as a graft for hematopoietic stem cell therapy as a substitute for conventional bone marrow, cord blood or peripheral blood transplantation. The transplantation of the expanded population of cells may be carried out in the same manner as conventional bone marrow, cord blood or peripheral blood transplantation. The graft may comprise the expanded population of cells along with any of the following components: a buffer solution, an antibiotic, a pharmaceutical compound.

[0224] Examples of disorders that may be treated using the HSC, HSPC and / or NK cells obtainable or obtained from the methods of the present invention include hematopoietic malignancies, such as: Acute myeloid leukemia (AML), Acute lymphocytic leukemia (ALL), Chronic lymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML), Myelodysplastic syndromes (MDS), Multiple myeloma (MM), B Lymphoid Malignancies: B-cell non-Hodgkin lymphoma (B-NHL), Relapsed and Refractory B Cell Lymphoma, Relapsed / Refractory Diffuse Large B-Cell Lymphoma, Blastic Plasmacytoid Dendritic Cell Neoplasm, Plasma Cell Leukemia; solid tissue tumours, including metastatic cancers such as: Neuroblastoma, Melanoma, Renal Cell Carcinoma (including metastatic), Ovarian, fallopian tube, or primary peritoneal cancer, Breast cancer, Glioblastoma (including Recurrent HER2-positive Glioblastoma), Prostate cancer (including Castration-Resistant Prostate Cancer), Pancreatic Cancer (including locally advanced or metastatic pancreatic cancer), Triple negative breast, bladder, and lung cancers, Recurrent / Metastatic Gastric or Head and Neck Cancer, Head and neck squamous cell carcinoma (HNSCC), Non-small-cell lung cancer (NSCLC), Gastrointestinal cancer, Liver metastases of colorectal or pancreatic cancer, Hepatocellular cancer (including Advanced Hepatocellular Carcinoma), Mesothelioma, Osteosarcoma, Extensive Stage Small Cell Lung Cancer, Refractory Metastatic Colorectal Cancer, Recurrent / Metastatic Gastric or Head and Neck Cancer, Metastatic Castration-Resistant Prostate Cancer; bacterial, viral and fungal infections.

[0225] The HSC, HSPC and / or NK cells may be administered through the following administration routes; subcutaneous, intraparietal, intramuscular, intravenous, intratumor, intraocular, intraretinal, intravitreal, or intracranial.

[0226] The HSC, HSPC and / or NK cells may be combined with a pharmaceutically acceptable excipient, diluent, or carrier in order to improve and enhance administration, stability, uniformity, bioavailability, or any combination thereof. In certain embodiments, the extracellular vesicles or cells of the current disclosure are administered suspended in a sterile solution. In certain embodiments, the solution comprises 0.9% NaCI. In certain embodiments, the solution further comprises one or more of: buffers, for example, acetate, citrate, histidine, succinate, phosphate, bicarbonate, or hydroxymethylaminomethane (Tris); surfactants, for example, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), or poloxamer 188; polyol / disaccharide / polysaccharides, for example, glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, or dextran 40; amino acids, for example, glycine or arginine; antioxidants, for example, ascorbic acid or methionine; and chelating agents, for example, EGTA or EGTA.

[0227] In one aspect, the present invention relates to a NK cell of the invention or a composition of the invention for use in treating a disorder.

[0228] In one aspect, the present invention relates to a method of treating a disorder comprising administering to a subject in need thereof the NK cell of the invention or the composition of the invention.

[0229] Use of an NK cell of the invention or a composition of the invention in the manufacture of a medicament for treating a disorder.

[0230] The disorder may be one or more of: a viral, fungal, or bacterial infection, haematological disorder, immune disorder or cancer, optionally wherein the disorder is Acute myeloid leukemia (AML), Acute lymphocytic leukemia (ALL), Chronic lymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML), Myelodysplastic syndromes (MDS), Multiple myeloma (MM), B Lymphoid Malignancies: B-cell non-Hodgkin lymphoma (B-NHL), Relapsed and Refractory B Cell Lymphoma, Relapsed / Refractory Diffuse Large B-Cell Lymphoma, Blastic Plasmacytoid Dendritic Cell Neoplasm, Plasma Cell Leukemia or a solid tissue tumour, including metastatic cancers.

[0231] In one aspect, the present invention relates to an NK cell of the invention or a composition of the invention for use in treating cancer.

[0232] In one aspect, the present invention relates to a method for treating cancer comprising administering to a subject in need thereof the NK cell of the invention or the composition of the invention.

[0233] In one aspect, the present invention relates to the use of an NK cell of the invention or a composition of the invention in the manufacture of a medicament for treating cancer.

[0234] The term “treat” or “treating” as used herein encompasses prophylactic treatment (e.g. to prevent onset of a neurological disorder, e.g. CTE) as well as corrective treatment (e.g. treatment of a subject already suffering from a neurological disorder, e.g. PCS). Preferably “treat” or “treating” as used herein means corrective treatment of a neurological disorder, e.g. PCS.

[0235] As used herein, the term "subject" refers to any animal (for example, a mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a therapy in accordance with the use of the present invention. Human subjects are envisaged in particular. The term “Patient” may be used herein to refer to a human subject. A subject may be an adult or a child (under 18 years of age).

[0236] As used herein, an “effective amount” of a compound, compounds, cells or composition defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, but one that is sufficient to provide the desired effect, e.g., the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure.

[0237] The cancer may be one or more of: acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), multiple myeloma (MM), B lymphoid malignancies: B-cell non-Hodgkin lymphoma (B-NHL), relapsed and refractory B cell lymphoma, relapsed and refractory diffuse large B-cell lymphoma, blastic plasmacytoid dendritic cell neoplasm, plasma cell leukemia and solid tissue tumours and metastatic cancers.

[0238] The invention provides a provides a composition comprising HSC, HSPC and / or NK cells, for use in therapy, wherein the HSC, HSPC and / or NK cells have been produced (are obtainable or obtained) according to the described methods of the invention.

[0239] The invention provides a method of treatment comprising administering to a patient a composition comprising HSC, HSPC and / or NK cells, wherein the HSC, HSPC and / or NK cells have been produced (are obtainable or obtained) according to the described methods of the invention.

[0240] The invention provides a use of a composition comprising HSC, HSPC and / or NK cells in the manufacture of a medicament for use in therapy, wherein the HSC, HSPC and / or NK cells have been produced (are obtainable or obtained) according to the described methods of the invention.

[0241] The invention provides a kit for producing HSC, HSPC and / or NK cells from the provided population of cell aggregates comprising iPSCs and / or ESCs according to the described methods of the invention, wherein the kit comprises sterile elements for the differentiation of the cell aggregates, or pharmaceutically acceptable salts thereof.

[0242] Kits of the invention are described herein. In a preferred embodiment the kit also contains apparatus and / or materials for providing a population of cell aggregates comprising iPSCs or ESCs and obtaining the produced HSC, HSPC and / or NK cells. A person skilled in the art will know of suitable elements, apparatus and / or materials for performing the method described herein to provide a population of cell aggregates comprising iPSCs and / or ESCs and to obtain the produced HSC, HSPC and / or NK cells. Examples include magnetic bead isolation, MACS bead isolation columns, CliniMACS (Miltenyi) or FACS sorting.

[0243] In an embodiment of the present invention, the therapy or treatment is of a haematological disorder, immune disorder, viral, fungal and bacterial infections or cancer.

[0244] In a preferred embodiment, the therapy or treatment is for hematopoietic malignancies, such as: Acute myeloid leukemia (AML), Acute lymphocytic leukemia (ALL), Chronic lymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML), Myelodysplastic syndromes (MDS), Multiple myeloma (MM), B Lymphoid Malignancies: B-cell non-Hodgkin lymphoma (B-NHL), Relapsed and Refractory B Cell Lymphoma, Relapsed / Refractory Diffuse Large B-Cell Lymphoma, Blastic Plasmacytoid Dendritic Cell Neoplasm, Plasma Cell Leukemia; solid tissue tumours, including metastatic cancers such as: Neuroblastoma, Melanoma, Renal Cell Carcinoma (including metastatic), Ovarian, fallopian tube, or primary peritoneal cancer, Breast cancer, Glioblastoma (including Recurrent HER2-positive Glioblastoma), Prostate cancer (including Castration-Resistant Prostate Cancer), Pancreatic Cancer (including locally advanced or metastatic pancreatic cancer), Triple negative breast, bladder, and lung cancers, Recurrent / Metastatic Gastric or Head and Neck Cancer, Head and neck squamous cell carcinoma (HNSCC), Non-small-cell lung cancer (NSCLC), Gastrointestinal cancer, Liver metastases of colorectal or pancreatic cancer, Hepatocellular cancer (including Advanced Hepatocellular Carcinoma), Mesothelioma, Osteosarcoma, Extensive Stage Small Cell Lung Cancer, Refractory Metastatic Colorectal Cancer, Recurrent / Metastatic Gastric or Head and Neck Cancer, Metastatic Castration-Resistant Prostate Cancer.

[0245] Suitably, the therapy or treatment is cancer immunotherapy.

[0246] In one embodiment, the method of treatments described above and herein comprises a step of administering the HSCs, HSPCs and / or NK cells produced (or obtainable or obtained) by the methods of the invention to a subject. Suitably the method of treatment comprises administering a composition comprising an effective amount of the HSCs, HSPCs and / or NK cells.

[0247] Clauses 1 . A method for producing Natural Killer (NK) cells, the method comprising the steps of: i) providing a population of cell aggregates comprising induced Pluripotent Stem Cells (iPSCs) or Embryonic Stem Cells (ESCs); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises at least 4 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, Activin A, VEGF and bFGF; wherein the culturing media used in the culturing of step iii) comprises at least 4 components selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IL-6, TPO, Notch agonist, IGF-1 , estradiol, Nicotinamide and Tryptophan; wherein the culturing media used in the culturing of step iv) comprises at least 5 components selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF-p receptor kinase inhibitor, bFGF, IL-6, TPO, Nicotinamide, Tryptophan, Notch agonist, Anti-human IgG antibody, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha; wherein the culturing media used in the culturing of step v) comprises at least 3 components selected from FLT3L, SCF, IL-7, IL-15, Notch agonist, Hydrocortisone, IL-15R-alpha, Nicotinamide and Anti-human IgG antibody, IGF1 , TPO, IL-21 , and SMAD3 inhibitor.

[0248] 2. The method according to clause 1 , wherein the culturing process of step ii) occurs for a period of up to 5 days, preferably from 2 to 4 days.

[0249] 3. The method according to clause 1 or 2, wherein the culturing process of step iii) occurs for a period of up to 5 days, preferably from 2 to 4 days.

[0250] 4. The method according to any one of the preceding clauses, wherein the culturing process of step iv) occurs for a period of up to 8 days, preferably from 4 to 8 days.

[0251] 5. The method according to any one of the preceding clauses, wherein the HSCs and / or HSPCs express CD34+, CD43+, and / or CD45+. 6. The method according to any one of the preceding clauses, wherein the third cultured population of step iv) and the fourth cultured population of step v) producing hematopoietic stem cells (HSCs) and / or hematopoietic stem and progenitor cells (HSPCs)).

[0252] 7. The method according to any one of the preceding clauses, wherein the culturing process of step v) occurs for a period of at least 14 days, preferably from 20 to 30 days, preferably the culturing process of step v) occurs for a period of 24 to 30 days.

[0253] 8. The method according to any one of the preceding clauses, wherein the culturing media used in the culturing of steps ii), iii), iv) and / or v) further comprises at least 1 component, such as at least 2 components, selected from penicillin-streptomycin (P / S), B- mercaptoethanol, L-glutamine, L-ascorbic acid (L-AA), Sodium selenite and Ethanolamine, preferably P / S, B-mercaptoethanol, L-glutamine, L-AA, Sodium selenite and Ethanolamine.

[0254] 9. The method according to clause 8, wherein the culturing media used in the culturing of steps ii), iii), iv) and / or v) comprises at least 3 components, such as at least 4 components, selected from penicillin-streptomycin (P / S), B-mercaptoethanol, L-glutamine, L-ascorbic acid (L-AA), Sodium selenite and Ethanolamine, preferably P / S, B-mercaptoethanol, L-glutamine, L-AA, Sodium selenite and Ethanolamine.

[0255] 10. The method according to any one of the preceding clauses, wherein the culturing media used in the culturing of step ii) comprises at least 5 components, such as at least 6 components, selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, Activin A, VEGF and bFGF.

[0256] 11. The method according to any one of the preceding clauses, wherein the culturing media used in the culturing of step iii) comprises at least 5 components, such as at least 6 components, selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IL-6, TPO, Notch agonists, IGF-1 , estradiol, Nicotinamide and Tryptophan.

[0257] 12. The method according to any one of the preceding clauses, wherein the culturing media used in the culturing of step iv) comprises at least 8 components, such as at least 9 components, selected from BMP4, VEGF, SCF, IL-1 , IL-3, IL-6, IL-7, IL-9, IL-15, TGF-p receptor kinase inhibitor, bFGF, TPO, Nicotinamide, Tryptophan, Notch agonists, Anti-human IgG antibody, aryl hydrocarbon receptor (AhR) antagonist and IL-15Ralpha. 13. The method according to any one of the preceding clauses, wherein the culturing media used in the culturing of step v) comprises at least 6 components, such as at least 7 components, selected from Sodium selenite, Ethanolamine (MEA), FLT3L, SCF, IL-2, IL-7, IL-15, IL-18, IL-21 , Notch agonist, Hydrocortisone, IL-15Ralpha, IGF1 , TPO, IL-21 , SMAD3 inhibitor, Nicotinamide and Anti-human IgG antibody.

[0258] 14. The method according to any one of the preceding clauses, wherein the ROCK inhibitor is selected from Y-27632 dihydrochloride, fasudil, Y-39983 dihydrochloride, WF-536, SLx-2119, XD-4000, rhostatin and VAS-012, preferably the ROCK inhibitor is Y-27632 dihydrochloride.

[0259] 15. The method according to any one of the preceding clauses, wherein the GSK-3 inhibitor is selected from CHIR99021 , LiCI, BIO, TWS119 and Kenpaullone, preferably the GSK-3 inhibitor is CHIR99021 or LiCI and most preferably the GSK-3 inhibitor is CHIR99021 .

[0260] 16. The method according to any one of the preceding clauses, wherein the TGF-p receptor kinase inhibitor is selected from SB431542, A 83-01 , RepSox, SB525334, Galunisertib and D4476, preferably the TGF-p receptor kinase inhibitor is SB431542.

[0261] 17. The method according to any one of the preceding clauses, wherein the Notch agonist is selected from DLL4-FC, DLL1 , DLL3, DLL4, JAG1 and GAG2, preferably the Notch agonist is DLL4-FC.

[0262] 18. The method according to any one of the preceding clauses, wherein the AhR antagonist is selected from StemRegenin 1 (SR1), CH-223191 , BAY-218, BAY 2416964 (compound 192), GNF351 , PDM2 and PDM-11 , preferably the AhR antagonist is SR1 .

[0263] 19. The method according to any one of the preceding clauses, wherein the culturing media used in the culturing of steps ii), iii), iv) and / or v) comprises a basal medium, optionally selected from one or more of X-VIVO 15, StemSpan SFEM II, Stemline II Hematopoietic Stem Cell Expansion Medium, StemPro-34 SFM (1X), StemDiff APEL-2 medium, DMEM, F12, IMDM and RPMI-1640, preferably the basal medium is one or more of StemDiff APEL-2 medium, DMEM and F12. 20. The method according to any one of the preceding clauses, wherein the culturing media used in the culturing of steps ii), iii) and / or iv) comprises BMP4 in combination with at least one component selected from VEGF, Activin A, SCF, LiCI and CHIR99021 .

[0264] 21. The method according to any one of the preceding clauses, wherein the culturing media used in the culturing of step v) further comprises a media additive, optionally wherein the media additive is serum-containing or serum-free.

[0265] 22. The method according to clause 21 , wherein the serum-containing media additive is selected from human serum, human plasma replacement and human serum replacement, preferably the serum-containing media additive is HI human clotted AB serum.

[0266] 23. The method according to clause 21 , wherein the serum-free media additive comprises Polyvinyl alcohol (PVA) and recombinant albumin, optionally the serum-free media additive further comprises one or more supplements and / or vitamins.

[0267] 24. The method according to any one of the preceding clauses, wherein the culturing of step ii) further optionally comprises a culturing media change step, wherein the culturing media is collected and replaced with an equal amount of step ii) culturing media at up to 4 days, preferably at 2 to 4 days and even more preferably at 3 days from the beginning of the culturing of step ii).

[0268] 25. The method according to any one of the preceding clauses, wherein the culturing of step iii) further optionally comprises a culturing media change step, wherein the culturing media is collected and replaced with an equal amount of step iii) culturing media at up to 4 days, preferably at 2 to 4 days from the beginning of the culturing of step iii).

[0269] 26. The method according to clause 20, wherein the culturing of step iv) further optionally comprises a culturing media change step, wherein the culturing media is collected and replaced with an equal amount of step iv) culturing media at up to 6 days, preferably at 4 to 6 days from the beginning of the culturing of step iv).

[0270] 27. The method according to any preceding clause, wherein the culturing of step v) further optionally comprises a culturing media change step, wherein the culturing media is collected and replaced with an equal amount of step v) culturing media every 2 to 5 days, preferably every 3 to 4 days from the beginning of the culturing of step v). 28. The method according to any one of the preceding clauses wherein the method further comprises a step vi) of collecting the HSC, HSPC and / or NK cells from the fourth cultured population in step v).

[0271] 29. The method according to any one of the preceding clauses wherein the cell aggregates comprise cut monolayer fragments, spheroids, embryoid bodies, encapsulated cut monolayer fragments and / or hydrogel beads encapsulating cut monolayer fragments.

[0272] 30. A composition comprising the collected HSC, HSPC and / or NK cells of step vi) for use in therapy, wherein the collected HSC, HSPC and / or NK cells have been produced according to the method of any one of the preceding clauses.

[0273] 31. A method of treatment comprising administering to a patient a composition comprising the collected HSC, HSPC and / or NK cells of step vi), wherein the collected HSC, HSPC and / or NK cells have been produced according to the method of any one of clauses 1- 28.

[0274] 32. Use of a composition comprising the collected HSC, HSPC and / or NK cells of step vi), in the manufacture of a medicament for use in therapy, wherein the collected HSC, HSPC and / or NK cells have been produced according to the method of any one of clauses 1-29.

[0275] 33. The composition according to clause 30, the method according to clause 31 , or the use according to clause 32, wherein the therapy or treatment is of a viral, fungal, or bacterial infection, haematological disorder, immune disorder or cancer.

[0276] 34. The composition according to clause 30, the method according to clause 31 , the use according to clause 32, or the composition, method, or use according to clause 33, wherein the therapy or treatment is of Acute myeloid leukemia (AML), Acute lymphocytic leukemia (ALL), Chronic lymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML), Myelodysplastic syndromes (MDS), Multiple myeloma (MM), B Lymphoid Malignancies: B- cell non-Hodgkin lymphoma (B-NHL), Relapsed and Refractory B Cell Lymphoma, Relapsed / Refractory Diffuse Large B-Cell Lymphoma, Blastic Plasmacytoid Dendritic Cell Neoplasm, Plasma Cell Leukemia and solid tissue tumours, including metastatic cancers. 35. The composition according to clause 30, the method according to clause 31 , or the use according to clause 32, wherein the therapy or treatment is cancer immunotherapy.

[0277] 36. The method, composition, or use according to any one of the preceding clauses, wherein the population of iPSCs is obtained from any somatic human cell, preferentially blood cell.

[0278] 37. The method, composition, or use according to any one of the preceding clauses, wherein the steps ii), iii), iv) and v) are carried out over a total time sufficient for the population of cell aggregates comprising iPSCs or ESCs to differentiate into NK cells.

[0279] 38. The method, composition, or use according to any one of the preceding clauses, wherein the steps ii), iii), iv) and v) are carried out over a total time of up to 46 days, preferably about 26 to about 46 days.

[0280] 39. The method, composition, or use according to any one of the preceding clauses, wherein the population of cell aggregates comprising iPSCs or ESCs is obtained from a mammal, preferably a human.

[0281] 40. The method, composition, or use according to any one of the preceding clauses, wherein the collected HSC, HSPC and / or NK cells express CD56+ and / or CD45+.

[0282] 41 . A kit for producing HSC, HSPC and / or NK cells from iPSCs and / or ESCs according to the method of clause 1 , wherein the kit comprises sterile elements for the differentiation of iPSCs and / or ESCs, or pharmaceutically acceptable salt thereof.

[0283] 42. The kit according to clause 41 further comprising any of the additional features of clauses 2 to 40.

[0284] 43. A method for producing Natural Killer (NK) cells, the method comprising the steps of: i) providing a population of cell aggregates comprising induced Pluripotent Stem Cells (iPSCs) or Embryonic Stem Cells (ESCs); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises at least 4 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, Activin A, VEGF and bFGF; wherein the culturing media used in the culturing of step iii) comprises at least 4 components selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IL-6, TPO, Notch agonist, IGF-1 , estradiol, Nicotinamide and Tryptophan; wherein the culturing media used in the culturing of step iv) comprises at least 5 components selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF-p receptor kinase inhibitor, bFGF, IL-6, TPO, Nicotinamide, Tryptophan, Notch agonist, Anti-human IgG antibody, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha; wherein the culturing media used in the culturing of step v) comprises at least 3 components selected from FLT3L, SCF, IL-7, IL-15, Notch agonist, Hydrocortisone, IL-15R-alpha, Nicotinamide and Anti-human IgG antibody, IGF1 , TPO, IL-21 , and SMAD3 inhibitor.

[0285] 44. The method according to clause 43, wherein the culturing process of step ii) occurs for a period of up to 5 days, preferably from 2 to 4 days.

[0286] 45. The method according to clause 43 or 44, wherein the culturing process of step iii) occurs for a period of up to 5 days, preferably from 2 to 4 days.

[0287] 46. The method according to any one of clauses 43-45, wherein the culturing process of step iv) occurs for a period of up to 8 days, preferably from 4 to 8 days.

[0288] 47. The method according to any one of clauses 43-46, wherein the HSCs and / or HSPCs express CD34+, CD43+, and / or CD45+.

[0289] 48. The method according to any one of clauses 43-47, wherein the third cultured population of step iv) and the fourth cultured population of step v) producing hematopoietic stem cells (HSCs) and / or hematopoietic stem and progenitor cells (HSPCs)). 49. The method according to any one of clauses 43-48, wherein the culturing process of step v) occurs for a period of at least 14 days, preferably from 20 to 30 days, preferably the culturing process of step v) occurs for a period of 24 to 30 days.

[0290] 50. The method according to any one of clauses 43-49, wherein the culturing media used in the culturing of steps ii), iii), iv) and / or v) further comprises at least 1 component, such as at least 2 components, selected from penicillin-streptomycin (P / S), B-mercaptoethanol, L- glutamine, L-ascorbic acid (L-AA), Sodium selenite and Ethanolamine, preferably P / S, B- mercaptoethanol, L-glutamine, L-AA, Sodium selenite and Ethanolamine.

[0291] 51. The method according to clause 50, wherein the culturing media used in the culturing of steps ii), iii), iv) and / or v) comprises at least 3 components, such as at least 4 components, selected from penicillin-streptomycin (P / S), B-mercaptoethanol, L-glutamine, L-ascorbic acid (L-AA), Sodium selenite and Ethanolamine, preferably P / S, B-mercaptoethanol, L-glutamine, L-AA, Sodium selenite and Ethanolamine.

[0292] 52. The method according to any one of clauses 43-51 , wherein the culturing media used in the culturing of step ii) comprises at least 5 components, such as at least 6 components, selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, Activin A, VEGF and bFGF.

[0293] 53. The method according to any one of clauses 43-52, wherein the culturing media used in the culturing of step iii) comprises at least 5 components, such as at least 6 components, selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IL-6, TPO, Notch agonists, IGF-1 , estradiol, Nicotinamide and Tryptophan.

[0294] 54. The method according to any one of clauses 43-53, wherein the culturing media used in the culturing of step iv) comprises at least 8 components, such as at least 9 components, selected from BMP4, VEGF, SCF, IL-1 , IL-3, IL-6, IL-7, IL-9, IL-15, TGF-p receptor kinase inhibitor, bFGF, TPO, Nicotinamide, Tryptophan, Notch agonists, Anti-human IgG antibody, aryl hydrocarbon receptor (AhR) antagonist and IL-15Ralpha.

[0295] 55. The method according to any one of clauses 43-54, wherein the culturing media used in the culturing of step v) comprises at least 6 components, such as at least 7 components, selected from Sodium selenite, Ethanolamine (MEA), FLT3L, SCF, IL-2, IL-7, IL-15, IL-18, IL- 21 , Notch agonist, Hydrocortisone, IL-15Ralpha, IGF1 , TPO, IL-21 , SMAD3 inhibitor, Nicotinamide and Anti-human IgG antibody. 56. The method according to any one of clauses 43-55, wherein the ROCK inhibitor is selected from Y-27632 dihydrochloride, fasudil, Y-39983 dihydrochloride, WF-536, SLx-2119, XD-4000, rhostatin and VAS-012, preferably the ROCK inhibitor is Y-27632 dihydrochloride.

[0296] 57. The method according to any one of clauses 43-56, wherein the GSK-3 inhibitor is selected from CHIR99021 , LiCI, BIO, TWS119 and Kenpaullone, preferably the GSK-3 inhibitor is CHIR99021 or LiCI and most preferably the GSK-3 inhibitor is CHIR99021 .

[0297] 58. The method according to any one of clauses 43-57, wherein the TGF-p receptor kinase inhibitor is selected from SB431542, A 83-01 , RepSox, SB525334, Galunisertib and D4476, preferably the TGF-p receptor kinase inhibitor is SB431542.

[0298] 59. The method according to any one of clauses 43-58, wherein the Notch agonist is selected from DLL4-FC, DLL1 , DLL3, DLL4, JAG1 and GAG2, preferably the Notch agonist is DLL4-FC.

[0299] 60. The method according to any one of clauses 43-59, wherein the AhR antagonist is selected from StemRegenin 1 (SR1), CH-223191 , BAY-218, BAY 2416964 (compound 192), GNF351 , PDM2 and PDM-11 , preferably the AhR antagonist is SR1 .

[0300] 61 . The method according to any one of clauses 43-60, wherein the culturing media used in the culturing of steps ii), iii), iv) and / or v) comprises a basal medium, optionally selected from one or more of X-VIVO™ 15, StemSpan™ SFEM II, Stemline II Hematopoietic Stem Cell Expansion Medium, StemPro™-34 SFM (1X), StemDiff APEL-2 medium, DMEM, F12, IMDM and RPMI-1640, preferably the basal medium is one or more of StemDiff APEL-2 medium, DMEM and F12.

[0301] 62. The method according to any one of clauses 43-61 , wherein the culturing media used in the culturing of steps ii), iii) and / or iv) comprises BMP4 in combination with at least one component selected from VEGF, Activin A, SCF, LiCI and CHIR99021.

[0302] 63. The method according to any one of clauses 43-62, wherein the culturing media used in the culturing of step v) further comprises a media additive, optionally wherein the media additive is serum-containing or serum-free. 64. A composition comprising the collected HSC, HSPC and / or NK cells of step vi) for use in therapy, wherein the collected HSC, HSPC and / or NK cells have been produced according to the method of any one of clauses 43-63.

[0303] 65. The composition according to clause 64, wherein the therapy or treatment is of a viral, fungal, or bacterial infection, haematological disorder, immune disorder or cancer, optionally wherein the therapy or treatment is of Acute myeloid leukemia (AML), Acute lymphocytic leukemia (ALL), Chronic lymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML), Myelodysplastic syndromes (MDS), Multiple myeloma (MM), B Lymphoid Malignancies: B- cell non-Hodgkin lymphoma (B-NHL), Relapsed and Refractory B Cell Lymphoma, Relapsed / Refractory Diffuse Large B-Cell Lymphoma, Blastic Plasmacytoid Dendritic Cell Neoplasm, Plasma Cell Leukemia and solid tissue tumours, including metastatic cancers, optionally wherein the therapy or treatment is cancer immunotherapy.

[0304] 66. The method, composition, or use according to any one of clauses 43-65, wherein the steps ii), iii), iv) and v) are carried out over a total time of up to 46 days, preferably about 26 to about 46 days.

[0305] 67. The method, composition, or use according to any one of clauses 43-66, wherein the collected HSC, HSPC and / or NK cells express CD56+ and / or CD45+.

[0306] BRIEF DESCRIPTION OF THE DRAWINGS

[0307] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples. Many of the Figures submitted herein are better understood in colour. The colour versions of the drawings are part of the application as filed and the right to present colour images of the drawings in later proceedings is hereby reserved.

[0308] Figure 1 shows the production of NK cells from iPSCs in feeder-free cultures. Panel A shows schematics of differentiation process. iPSC or ESC cell aggregates (iPCS cell aggregates are formed overnight during the priming (PR) step) are provided in step 1) and differentiated into HSCs and / or HSPCs during steps 2), 3), 4 and 5). Further commitment, differentiation and maturation into functional NK cells occurs during step 5) of the method of the present invention. Step 1 comprises a (full) change of culture medium for medium 1 according to

[0309] Table 2A. Step 2 comprises a (full) change of culture medium for medium 2 according to

[0310] Table 3A. Step 3 comprises a (full) change of culture medium for medium 3 according to Table 4A. Step 4 comprises a (full) change of culture medium for medium 4 according to Table 5A. Panel B shows the morphology of differentiating iPSCs at Day 3 and Day 35 of NK differentiation using Protocol 3 of the invention. Panels C and D show identification (%) of CD45+ / CD56+ induced NK cells (iNK) population, which are derived from Protocol 3, Protocol 5 or Protocol 6 of the invention, at Day 26 and Day 35 of differentiation using FACS. Panel E shows charts in which the number of NK cells produced by Protocol 1 , Protocol 2, Protocol 3, Protocol 5, Protocol 6 or Protocol 7 is plotted against the percentage of CD45+ / CD56+ cells of NK cells derived from said protocols. The number of NK cells represents the total NK cell yields for each cell culture started from the same number of iPSC cells, while the percentage of CD45+ / CD56+ indicate the purity of the NK cell population produced by these protocols and harvested at day 26, day 35, and day 38). Protocols leading to the highest yield and purity (highest number of CD45+ / CD56+ NK cells), such as protocols 3 and 6, are considered advantageous for industrial production or manufacturing purposes.

[0311] Figure 2 shows phenotypic analysis (expression of markers) of iPSC derived NK cells generated by the protocols of the invention (protocols 1 , 2, 3, and 4) compared to NK cells obtained from peripheral blood (PB-NK). Panel A shows the expression of inhibitory (NKG2A) and activating (NKp44) receptors. Panel B shows the expression of activating (NKp46 and NKG2D) receptors. Panel C shows the expression of perforin (intracellular molecule associated with cytotoxic activity) and chemotactic (CXCR3) receptors. The expression of NKp44, NKG2A, NKp46, NKG2D, perforin and CXCR3 was measured on the surface of iPSC-derived NK cells generated by the protocols of the invention, analysed by FACS at Day 35 and Day 41 of differentiation, and compared to the expression of the same receptors on the surface of BP-NK cells . High level of expression of these markers by iNK may be used as a predictor of anti-cancer properties of the iPSC derived NK cells of the invention.

[0312] Figure 3 shows the cytotoxic activity of iPSC-derived NK cells of the invention tested on multiple cancer cell lines and compared to the activity of human NK cell line NK92. Panel A shows the phase contrast and fluorescent images of T47D cancer cell line expressing GFP reporter before and after 24-hour coculture with iPSC derived NK cells generated by protocol 3 of the invention. Panels B and C shows an image analysis-based NK killing assay targeting T47D and demonstrated dose dependent killing of breast cancer cells. Panel D shows an image analysis-based NK killing assay targeting Pane 1 cells that express RFP reporter, and demonstrated dose dependent killing of pancreatic cancer cells. Panels E and F show a luciferase-based NK killing assay targeting A549 modified with luciferase reporter, and showed dose dependent killing of lung cancer cells. Panel B: NK cells generated by Protocol 2 were harvested at D69 and tested in the killing assay, NK cells generated by Protocol 1 were harvested at day 41 and Day 69 and tested in the killing assay, NK cells generated by Protocol 3 were harvested at day 41 and day 35 and tested in the killing assay. Panel C: NK cells generated by protocols 1 , 2, 3, 5, and 7 were harvested at day 39 and tested in the killing assay. Panels D, E and F: NK cells generated by protocols 1 , 2, 3 and 4 were harvested at day 40 and tested in the killing assay.

[0313] Figure 4. Panel A shows a Uniform Manifold Approximation and Projection (UMAP) embedding of the single cell RNA-seq data for the NK cells derived from protocols 3 and 7, the peripheral blood (PB)-derived CD34 positive cells (CD34_PB), the umbilical cord blood (UCB)-derived CD34 positive cells (CD34_CB), the induced pluripotent stem cells (iPSCs), the unsorted peripheral blood mononucleated cells (PBMCs) and the NK cells sorted from PBMCs (PB_NK). Each point represents a single cell, coloured by the sample of origin. The UMAP coordinates show that each sample / tissue type clusters separately. Panel B shows a magnification of the map shown at the bottom right corner of panel A. Panels C and D show a UMAP representation of log-transformed normalized expression for cluster markers for Protocol 3 and Protocol 7, respectively. Each point is coloured according to gene expression. Genes were selected by testing differential gene expression of Protocol 3 or Protocol 7 clusters against the rest of the data and prioritised according to the product of the difference in percentage of expression (% cells with at least one count for the gene in the cluster) and the Area Under the receiver-operator Curve (AUC) for classification.

[0314] Figure 5. Panels A-D are dot plots illustrating the mean log-transformed normalised expression (colour scale) and the proportion of cells expressing a given gene (size scale) in each cluster. Panel A shows transcription factors (TFs) important for NK lineage specification. Panel B shows NK maturation, activation, cytotoxicity, inhibition and general lineage specification markers. Panel C shows genes involved in the PRF1 / Granzyme network. Panel D shows genes involved in the TRAIL / FAS ligand network.

[0315] Figure 6 shows example of genes differentially expressed in NK cells derived from Protocols

[0316] 3 and 7 relative to the peripheral blood (PB)-derived CD34 positive cells (CD34_PB), the umbilical cord blood (UCB)-derived CD34 positive cells (CD34_CB), the induced pluripotent stem cells (iPSCs), the unsorted peripheral blood mononucleated cells (PBMCs) and the NK cells sorted from PBMCs (PB_NK), presented as violin plots to demonstrate levels of gene expression across all samples in the dataset. Figure 7 shows phenotypic changes in expression patterns of activating and inhibitory molecules expressed on the surface and internally (Perforin) by iPCS-derived NK cells generated by Protocols 2, 3, and 7 in response to exposure to K562 human leukemic cell line for 24 hours in vitro. The iPCS-derived NK cells were collected at Day 49 (Protocols 2 and 3) and Day 42 (Protocol 7) of differentiation. Panel A shows that the iPCS-derived NK cells express higher level of activating CD69 molecules compared to cord blood NK cells (CB NK), but lower levels of DNAM expression. The double-positive CD69+ / DNAM+ population is maintained at high level indicating the capability of iPCS-derived NK cells of the invention to maintain their cytotoxic potential. Panel B shows that, unlike the CB NK cells, the iPCS- derived NK cells of the invention do not express and do not upregulate TIGIT, a molecule correlated with exhaustion of the ability of NK cells to perform cytotoxic activity. The iPCS- derived NK cells of the invention express higher levels of TIM3, which is further upregulated upon exposure to K562 cells, indicating their activated status.

[0317] Figure 8 shows that upon stimulation with K562 or cytokines, the iPCS-derived NK cells of the invention retain high expression of activating and cytotoxic molecules (Nkp46, CD69, Perforin) and do not have significant up-regulation of exhaustion markers (CD57, TIGIT).

[0318] The following example[s] illustrate[s] the invention.

[0319] EXAMPLES

[0320] Example 1 : Production of NK cells differentiated in vitro from stem cells (IPSCs) according to Protocols 1, 2, 3, 4, 5, 6, or 7.

[0321] Abbreviations

[0322] Abbreviations as used herein will be known to those skilled in the art. In particular, the following abbreviations may be used herein:

[0323] Abbreviation Definition

[0324] DMEM Dulbecco’s Modified Eagle Medium

[0325] FACS Fluorescence-activated cell sorting iNK Induced Natural Killer cell (differentiated from iPSC and / or ESC) iPSC Induced Pluripotent Stem Cell

[0326] L-AA L-ascorbic acid

[0327] MEA Ethanolamine

[0328] PB Peripheral blood

[0329] Pen / Strep or P / S Penicillin / Streptomycin

[0330] TC (plate) Tissue culture (plate) NCR Natural cytotoxicity receptor

[0331] CB Cord blood

[0332] Materials and Methods

[0333] Cells Established iPSC and / or ESC cell lines are used to form cell aggregates comprising iPSCs and / or ESCs. All experiments were performed starting from these cell aggregates.

[0334] Table 1. Materials and Reagents

[0335] Cell Culture Media iPSC Brew Media (cell culture media defined by iPSCs line requirement)

[0336] STEMdiff™ APEL™2 Medium

[0337] Gibco™ DMEM, high glucose, GlutaMAX Gibco™ Ham's F-12 Nutrient Mix, GlutaMAX

[0338] Media Supplements

[0339] L-ascorbic acid (L-aa or L-AA)

[0340] Human AB serum

[0341] Gibco™ L-Glutamine

[0342] Pen / Strep

[0343] Cytokines / Growth Factors*

[0344] Recombinant Human BMP4

[0345] Recombinant Human SCF

[0346] Recombinant Human FGF basic

[0347] Recombinant Human VEGF

[0348] Recombinant Human IL-3

[0349] Recombinant Human IL-7

[0350] Recombinant Human IL-15

[0351] Recombinant Human FLT-3 Ligand

[0352] Chemical Compounds

[0353] ROCK inhibitor (Y-27632 dihydrochloride)

[0354] GSK3 inhibitor (CHIR 99021)

[0355] Beta-mercaptoethanol

[0356] Sodium selenite

[0357] Ethanolamine (MEA)

[0358] Equipment for Cell Culture

[0359] 60 mm Petri dishes

[0360] 6-well suspension plates

[0361] 12-well TC plates

[0362] StemPro EZPassage Disposable Stem Cell Passaging Tool, (Cutting tool) Plastic Pasteur pipettes.

[0363] *AII cytokines / growth factors are reconstituted in buffers and at concentrations as per manufacturer’s instructions on the product information sheets. All stock solutions are aliquoted and kept at -20°C. All components and exemplary sources used in the protocols of the invention are listed in Table 1A below:

[0364] Table 1A: List of components and exemplary sources used in protocols 1 to 7

[0365] Protocols 1 to 7 for production of HSC, HSPC and / or NK cells (starting from iPSCs):

[0366] Day -1 (or Day -2) (iPSC cell line differentiation priming step): iPSCs were mechanically cut or collected as single cells and seeded in iPSC cell culture media with 5 pM Y-27632 dihydrochloride. The cell aggregates were allowed to form over 1 day (or 2 days) before the start of differentiation.

[0367] The aggregate culture was initiated from cell at 80% confluency. Cell aggregates were seeded in full iPSC cell culture media in 6-well suspension plates at the density 10-20 aggregates per cm2.

[0368] Day 0 (Start of the differentiation process) using protocols 1 , 2, 3, 4, 5, 6, or 7: Cell aggregates were collected using a 40 pm cell strainer, allowing the smaller fragments and suspension cells to pass the strainer. The collected cell aggregates were distributed into 12- well TC plates at the density of 38-76 cell aggregates / well (10-20 cell aggregates / cm2) and the media was changed for the Step 1 (as shown in Figure 1A) media composition (medium 1) at 1 ml / well using the media formulations described in Table 2A.

[0369] Table 2. Media Composition 1 Plasticell Control

[0370] Specifically, the following media composition (“Medium 1”) were used at DAY 0 for Protocols 1 , 2, 3, 4, 5, 6 and 7 (as shown in Table 2A): Table 2A: Composition of medium 1 for Protocols 1-7.

[0371] Day 2 (Half media change to Media Composition 1 (medium 1): Half of the media were collected from each well and replaced with equal amount of freshly prepared Media Composition 1 (Table 2A). Cell aggregates at this stage started to adhere to the plate.

[0372] Day 3 (Full media change to Media Composition 2 (medium 2)): All media were collected from the well and replaced by the Media Compositions 2 (medium 2) of Table 3A in the same manner as described for the half media change above on Day 2. Table 3. Media Composition 2

[0373] Specifically, the following media composition (“Medium 2”) were used at DAY 3 for Protocols 1 , 2, 3, 4, 5, 6 and 7 (as shown in Table 3A): Table 3A: Composition of medium 2 for Protocols 1-7.

[0374] Day 6 (Full media change to Media Composition 3 (medium 3)): All media were collected from the well and replaced with the Media Compositions 3 (medium 3) of Table 4A in the same manner as described for the half media change.

[0375] Specifically, the following media composition (“Medium 3”) were used at DAY 6 for Protocols 1 , 2, 3, 4, 5, 6 and 7 (as shown in Table 4A): Table 4A: Composition of medium 3 for protocols 1-7.

[0376] Days 8, 10 (Half media change): Half of the media were collected from each well and replaced with an equal amount of freshly prepared media from any one of the Media Compositions 3 (medium 3) of Table 4A.

[0377] Days 12-35 (Full media change to the Media Composition 4 (medium 4)): As previously, the media were collected from the wells / flasks and replaced by either freshly prepared Media Composition 4 (medium 4) (Table 5A). Starting from Day 12, the suspension cells can be collected and analysed for the markers of HSC / HSPC. At Day 15 more than 30-90% cells are CD34-positive. There is a progressive increase in expression of CD43 and CD45 markers, which is accompanied by decline in CD34 expression (Table 6).

[0378] The cells were left to mature into NK cells until Day 35 or longer. Half of the media were replaced every 3-4 days with an equal amount of freshly prepared media from the Media Compositions 4 (medium 4) of Table 5A.

[0379] As the higher cell density is important for NK differentiation, it is recommended that the cell numbers are kept above 100,000 cells / ml.

[0380] Table 5. Media Composition 4

[0381] *serum-free media additive comprises Polyvinyl alcohol (PVA) and recombinant albumin (cellastim-S) and supplements and vitamins (Insulin-Transferrin-Selenium-Ethanolamine (ITS -X), non-essential amino acids, fatty acids, steroid hormones and cholesterol). Specifically, the following media composition (“Medium 4”) were used at DAY 12 for Protocols 1 , 2, 3, 4, 5, 6 and 7 (as shown in Table 5A):

[0382] Table 5A: Composition of medium 4 for protocols 1-7.

[0383]

[0384]

[0385]

[0386]

[0387] Collection and analysis of suspension cells during culturing

[0388] Cell cultures were checked under the microscope. The appearance of suspension cells may indicate the presence of hematopoietic differentiation at Day 6. If there were suspension cells visible under the microscope during media changes, the removed media containing suspension cells were spun at 300g for 5 min, the supernatant removed and the sedimented cells re-suspended in fresh media before adding back to the well.

[0389] Flow cytometry analysis in aggregate cell cultures To assess the efficiency of the differentiation procedure, the phenotype and purity of differentiating cells were analyzed by flow cytometry.

[0390] The suspension cells were collected from cultures starting from day 12 onwards. The cellular aggregates can be dissociated using Accutase. Fluorescence-Activated Cell Sorting (FACS) was performed at Days 12, 18, 24, 28, 35 or at later time points.

[0391] For the early and late cell phenotype markers, antibody panels for stage-specific surface markers to identify developing haemangioblasts / HSCs and differentiating NK cells were used as described in Tables 6 and 7. The markers used in the FACS assays are shown in Panels 1 , 2, and 3 below

[0392] Table 6. Early markers

[0393] Table 7. NK specific (CD56) and exclusion (CD3) markers

[0394] Panel 1 : NK maturation and purity markers Panel 2: Functionality markers

[0395] Panel 3: Functionality markers

[0396] Cytotoxicity assay

[0397] If not specifically indicated, the iPSC-derived NK (iNK) cells produced by protocols 1 , 2, 3, 4, 5, 6 or 7 were collected at Day 40 of differentiation.

[0398] Imaging-based cytotoxicity assay was performed using T47D breast cancer cells with nuclear green fluorescent protein expression and PANC-1 pancreatic cancer cells with nuclear red fluorescent protein expression. Luciferase-based cytotoxicity assay was done using A549 lung cancer cells with luciferase expression.

[0399] Target cancer cells were seeded to 96 well plates one day prior to the addition of effector cells (iNK cells) for cytotoxicity assay. Effector cells were added to target cells at various E:T (effector to target) ratios, ranging from 0.25:1 to 2:1. After 24hours and / or 48hours of coculture of effector and target cells, relative percentage of live target cells remained in each well was measured and quantified.

[0400] For imaging-based assay, the 96 well plates were scanned using HSC Celllnsight CX5 (ThermoFisher). Number of live target cells in each well was quantified based on the count of cells positive for the nucleus fluorescent protein. The number of live target cells in each condition was normalised to the number of live target cells in the condition without effector cells (i.e. cancer cells only), and the percentages of live cells were used to plot the cytotoxicity graphs.

[0401] For luciferase-based assay, ONE-Glo Reagent (Promega) was used according to manufacturer’s protocol. Luminescence was measured using CLARIOstar (BMG LABTECH). Total luminescence is proportional to the number of live target cells in each well. The total luminescence in each condition was normalised to the total luminescence in the condition without effector cells (i.e. cancer cells only), and the percentages of live cells were used to plot the cytotoxicity graphs.

[0402] NK-92 cells were used as a control. NK-92 cells are an immortalised human NK cell line derived from the peripheral blood mononuclear cells of a male patient with rapidly progressive non-Hodgkin's lymphoma. These cells exhibit many of the functional characteristics of activated NK cells found in human blood, including the ability to recognise and kill cancer cells as well as virally infected cells by secreting cytotoxic molecules such as perforin and granzymes. NK-92 cells are often used as positive control in research, particularly in assays measuring NK cell cytotoxicity, and have been shown to more potent than blood-derived NK cells (Suck et al (2016) Cancer Immunol Immunother, Vol 65, pages 485-492).

[0403] Figure 3 shows that iPSC-derived NK cells generated via Protocols 1 , 2, 3, 4, 5, and 7 exhibit superior, dose-dependent cytotoxic activity against breast, pancreatic, and lung cancer cell lines (Figure 3A-F) compared to the already potent NK-92 cells. Given the established greater potency of NK-92 cells over blood-derived NK cells, these findings also show that the iPSC-derived NK cells generated via the Protocols of the invention are more potent than blood-derived NK cells.

[0404] Puritv and yield of CD45+ / CD56+ iPSC-derived NK cells

[0405] The results indicate that Protocols 1 through 7 showed variations in their capacity to generate populations (batches) of mature iPSC-derived NK cells (day 35 and older) that comprise a high proportion of CD45+ / CD56+ NK cells. The presence of both CD45+ and CD56+ markers indicates mature and functional iPSC-derived NK cells capable of cytotoxicity and cytokine production, especially when considered alongside with the expression of other NK-specific receptors such as NKp44, NKp46, and NKG2D (as shown in Figure 2).

[0406] Therefore, a higher percentage of CD45+ / CD56+ iPSC-derived NK cells produced within a batch signifies indicates a “purer” batch, with less contamination from undesirable cells like immature NK cells or cells of other lineages. Consequently, protocols yielding a greater proportion of CD45+ / CD56+ iPSC-derived NK cells per batch offer significant manufacturing advantages. Specifically, they lead to: 1) increased efficiency by maximising the conversion of starting material (iPSCs) into the desired NK cell product; 2) enhanced cost-effectiveness through higher yields, which lower the cost per unit and minimise waste and the need for extensive downstream purification; 3) improved scalability; 4) reduced batch-to-batch variability, ensuring more consistent product quality; and 5) enhanced potency and efficacy, as a higher proportion of the desired NK cells in the final product contributes to greater therapeutic impact.

[0407] In sum, a protocol that efficiently directs iPSC differentiation towards the CD45+ / CD56+ NK cell phenotype simplifies manufacturing, lowers costs, and improves the therapeutic potential of the resulting cell therapy. The results show that Protocol 3 effectively meets these criteria.

[0408] Specifically, Figure 1 (Panels C, D, and E) reveals that Protocol 3 produced a high percentage (> 90%) of mature (> day 35) CD45+ / CD56+ NK cells per batch and furthermore generated a higher number of iPSC-derived CD45+ / CD56+ NK cells compared to the other protocols tested. This indicates that Protocol 3 is particularly advantageous for producing superior yield of the desired NK cells such as iPSC-derived NK cells with the desired phenotype (e.g. CD45+ / CD56+) and function (e.g. cytotoxicity).

[0409] NK cell-like phenotypic profile

[0410] The results show that the iPSC-derived NK cells generated by the protocols of the invention all express cell surface marker profile characteristic of NK cells, regardless of the Protocol used. Specifically, Figure 2 shows that mature NK cells (day 35 or Day 41) generated by Protocols 1 , 2, 3 or 4, which are double positive for CD45 and CD56 (CD45+ / CD56+) also express NK cell-specific markers such as NKp44, NKp46, perforin, NKG2A, NKG2D, and CXCR3. These results indicate that the protocols of the invention reliably generate iPSC- derived NK cells expressing a set of markers characteristic of NK cells or displaying an NK cell-like phenotypic profile. Activation of iNK cells by K562 cells with multiparametric flow cytometry detection iNK cells were produced by Protocols 1 , 2, 3, 4, 5, 6, or 7 (as explained above) and collected at 26, 35, 38, 30 or 40 days of differentiation. iNK cells were contacted for 24 hrs with K562 suspension cells, which are capable of activating iNK cells, at a ratio iNK : K562 = 1 : 2. CB- derived NK cells were used as control for the assay. After co-incubation, the iNK cells were collected and stained using the Panel 4 antibodies for the multiparametric detection of changes in expression of activating and inhibitory molecules characteristic of NK cells cytotoxicity response. The expression of these molecules on unstimulated iNK cells was used to identify the baseline expression.

[0411] Panel 4: Multiparametric flow cytometry

[0412] CD69 / DNAM The results show that the iPSC-derived NK cells produced by the protocols of the invention such as Protocols 2, 3, and 7, expressed higher levels of CD69 (a marker of early activation of NK cells) at their cell surface both at baseline and in response to activation by cancer cells (K562 cells) compared to CB NK cells (as shown in Figures 7A and 8A). This is a surprising finding as such high levels of CD69 are not expected or typical for NK cells. Further, the iPSC-derived NK cells produced by Protocols 2, 3, and 7, expressed lower levels of DNAM (a marker of ) both at baseline and in response to activation by cancer cells (K562 cells) compared to CB NK cells (as shown in Figures 7A and 8B). Figure 7A shows that the doublepositive CD69+ / DNAM+ population is maintained at high level indicating the capability of iPCS-derived NK cells produced by the protocols of the invention (Protocols 2, 3, and 4) to maintain their cytotoxic potential. Overall, the NK cells of the invention are distinct in terms of their CD69+DNMA expression profile compared to blood-derived NK cells (peripheral blood NK cells).

[0413] Overall, these results indicate that both CB NK cells and iPSC-derived NK cells (from Protocols 2, 3, and 4) can be triggered by immune stimuli such as K562 cells. However, the iPSC-derived NK cells of the invention displayed a more pronounced activation profile, showing higher activity even without stimulation (e.g. CD69 expression profile) and a stronger response when challenged.

[0414] TIGIT / TIM3

[0415] The results show+ that the iPCS-derived NK cells produced by Protocols 2, 3, and 4 express lower levels of TIGIT (a marker of exhaustion of the ability of NK cells to perform cytotoxic activity) at baseline and in response to activation by cancer cells (K562 cells) as compared to CB NK cells (as shown in Figure 7B and 8A). Conversely, the iPCS-derived NK cells produced by Protocols 2, 3, and 4 expressed higher levels of TIM3 both at baseline and in response to activation by cancer cells (K562 cells) compared to CB NK cells. Overall, these results show that the iPCS-derived NK cells produced by the Protocols 2, 3, and 4 have a higher activated status and cytotoxic potential compared to CB NK cells.

[0416] Other markers

[0417] Figure 8 shows that upon stimulation with K562, the iPCS-derived NK cells of the invention retain high expression of activating and cytotoxic molecules (Nkp46, CD69, Perforin) and do not have significant up-regulation of exhaustion markers (CD57, TIGIT).

[0418] In summary, the results show that the iPCS-derived NK cells produced by Protocols 2, 3, and 4 exhibit a distinct activation profile, including elevated basal and stimulated activation. These characteristics, combined with their demonstrated superior cytotoxic capacity (as shown in Figure 3), show their potential as efficacious new therapeutic drugs. Example 2: Sinqle Cell RNA-sequencina analysis iPSCs were thawed and recovered for one passage before fixation. PB-CD34+ cells were thawed and recovered overnight before fixation. UCB-CD34+ cells were thawed and recovered overnight before fixation. Donor PBMCs were thawed and recovered overnight before fixation. PBMC-NK were isolated from thawed and recovered donor PBMCs using NK cell Isolation Kit, human (Miltenyi Biotec), according to manufacturer’s protocol. iPSC-derived NK cells produced by protocols 3 and 7 were collected from differentiation experiment (example 1) for fixation. All cells were fixed using Evercode™ Cell Fixation v2 (Parse Biosciences), according to manufacturer’s protocol V2.1.2.

[0419] Libraries for single cell RNA sequencing (scRNA-seq) were prepared from fixed cells using Evercode™ WT v2 (Parse Biosciences), according to manufacturer’s protocol V2.2.2. Number of fixed cells used for sample preparation was calculated using Parse Biosciences Evercode™ WT Sample Loading Table v2 (Parse Biosciences), with targeted 2750 barcoded cells for each control sample (i.e. iPSC, PB-CD34+, UCB-CD34+, PBMC and PB-NK samples) and targeted 4125 barcoded cells for each iPSC-derived NK sample. Libraries were sequenced using service from Novogene (UK). The results of the scRNA-seq are shown in Figure 4.

[0420] Figure 4 (A-D) shows that iPSC-derived NK cells produced via Protocols 3 and 7 exhibit unique gene expression profiles ("gene signatures") that clearly distinguish them from peripheral blood NK cells. Tables A-D below show the differentially expressed genes (relative to PB-NK cells), which are categorised by increased or decreased (or no expression) expression in the iPSC-derived NK cells of the invention relative to peripheral blood NK cells.

[0421] The “LogFc” value represents the Iog2 fold change. A positive value indicates that a given gene is expressed at a higher level (upregulated) in the iPSC-derived NK cells of the invention compared to the peripheral blood NK cells. For instance, the gene RASSF8 (Table A) has a LogFc value of 2.73. A Iog2 fold change of approximately 2.73 translates to a fold change of 2273, which is about 6.6. This means that the gene RASSF8 is about 6.6 times more highly expressed in the iPSC-derived cells on average compared to peripheral blood NK cells. Thus, the expression of RASSF8 is considered significantly upregulated in the iPSC-derived NK cells of the invention compared to the peripheral blood NK cells.

[0422] Conversely, a logFc value, which is negative, indicates that a given gene is downregulated in the iPSC-derived NK cells of the invention compared to the peripheral blood NK cells. For instance, ADAM28 (Table B) has a LogFc value of -0.93. A Iog2 fold change of approximately -0.93 translates to a fold change of 2093, which is about 1.914. This means that the gene ADAM28 is about 1.914 times more highly expressed in the peripheral blood NK cells compared to the iPSC-derived cells. Said otherwise, the expression of ADAM28 in the iPSC-derived NK cells of the invention can be calculated as the Iog2 fold change: 2-093, which is about 0.522. This means that the expression of ADAM28 in the iPSC-derived NK cells of the invention is about 0.522 times the expression in peripheral blood NK cells (or about 52.2% of the level in blood-derived NK cells), which is about two-fold higher expression of ADAM28 in peripheral blood NK cells relative to the iPSC-derived NK cells of the invention.

[0423] The “score” value is a numerical value assigned to each gene that aims to rank them based on the strength of their differential expression between groups. A higher positive score typically indicates a more robust and biologically meaningful difference (upregulation or increased expression) in gene expression in one group compared to the other group. For instance, the gene RASSF8 (Table A) has a score of 89.35 while the gene BCL2 (Table A) has a score of 50.16. This means that RASSF8 ranks higher than BCL2 in terms of the strength of the differential expression values (e.g. fold changes - upregulation) compared to BCL2. However, despite the difference in score, both genes can be considered as being significantly more expressed (upregulated or increased expression) in the iPSC-derived NK cells of the invention compared to the peripheral blood NK cells. As shown in Tables A and C, all genes are associated with a score above 35 (e.g. scores range from 35 up to 90), which indicates that these genes are significantly more expressed (upregulated or have an increased expression) in the iPSC-derived NK cells of the invention compared to the blood- derived NK cells. A score of zero typically indicates that a given gene is not considered differentially expressed (e.g. no meaningful fold change or not upregulated or downregulated or no increased or decreased expression) between the groups compared.

[0424] Conversely, a higher negative score typically indicates a more robust and biologically meaningful difference (downregulation or decreased expression) in gene expression in one group compared to the other group. For instance, the gene ADAM28 (Table B) has a score of -12.23 while the gene TIGIT (Table B) has a score of -11.89494682. This means that ADAM28 ranks higher than TIGIT in terms of the strength of the differential expression values (e.g. fold changes - downregulation or decreased expression) compared to TIGIT. However, despite the difference in score, both genes can be considered as being significantly less expressed (downregulated or decreased expression) in the iPSC-derived NK cells of the invention compared to the peripheral blood NK cells. As shown in Tables B and D, all genes are associated with a score above -3.0 (e.g. scores range from -3.0 up to - 13.0), which indicates that these genes are significantly less expressed (downregulated or have a decreased expression) in the iPSC-derived NK cells of the invention compared to the peripheral blood NK cells.

[0425] Table A. Differentially expressed genes with an increased expression in iPSC-derived NK cells generated by Protocol 3 relative to PB-NK cells

[0426]

[0427] Table B. Differentially expressed genes with a decreased expression (or no expression) in iPSC-derived NK cells generated by Protocol 3 relative to PB-NK cells Table C. Differentially expressed genes with an increased expression in iPSC-derived NK cells generated by Protocol 7 relative to PB-NK cells

[0428] Table D. Differentially expressed genes with a decreased expression (or no expression) in iPSC-derived NK cells generated by Protocol 7 relative to PB-NK cells

[0429] Figure 5 (panels A-D) show examples of dot plot graphs illustrating the mean log- transformed normalised expression (colour scale) and the proportion of cells expressing a given gene (size scale) in a cluster (each group tested). For instance, Figure 5B shows that the expression of CD56 is significantly increased (upregulated) in the iPSC-derived NK cells generated by Protocols 3 and 7 compared to peripheral blood NK cells, as evidenced by the higher (darker) intensity in colour as well as by the larger size of the dot (Figure 5B). Thus, the iPSC-derived NK cells generated by Protocols 3 and 7 expressed high levels of CD56 and are referred to as CD56+ brightNK cells. Compared to blood-derived NK cells (e.g. peripheral blood NK cells), CD56+ brightNK cells are more cytotoxic against cancer cells. Further examples of differentially expressed genes include PRF1 (Figure 5C) and TNFRS10A and TNFRS10B (Figure 5D).

[0430] Figure 6 (panels A-D) shows violin plots, which represents another way to illustrate examples of genes (from Tables A and C) differentially expressed in the iPSC-derived NK cells generated by Protocols 3 and 7 compared to the peripheral blood NK cells. For instance, Figure 6A shows that the expression of CTSI / I / , ID2, and IL18R1 is increased (upregulated) in the iPSC-derived NK cells generated by Protocols 3 and 7 compared to the peripheral blood NK cells. Figure 6B shows that NFILZ is uniquely increased (upregulated) in the iPSC- derived NK cells generated by Protocol 3 compared to the peripheral blood NK cells while Figure 6D shows that ITGA1 is uniquely increased (upregulated) in the iPSC-derived NK cells generated by Protocol 7 compared to the peripheral blood NK cells.

[0431] Results and Discussion

[0432] As shown by the Examples, the applicant has developed novel serum-free feeder free and GMP-ready protocols (protocols 1 through 7) for the differentiation of human iPSCs and / or ESCs into fully functioning mature NK cells. The differentiation was a multistep process that started from priming of iPSC through the formation of cell aggregates using iPSC culturing media (Figure 1A). At the end of this step, the largest aggregates were collected and transferred to the first differentiation media (medium 1 - Table 2A) to induce mesoderm specification (Step ii)). Some of the aggregates attached and formed colonies of proliferating and differentiating cells (Figure 1 B). Some of the aggregates remained non-attached and cells differentiated inside the aggregates. It was followed by Step iii) media (medium 2 - Table 3A) to induce the formation of haemogenic endothelium, and Step iv) media (medium 3 - Table 4A) to promote haemotopoietic differentiation and induction of lymphoid cell lineages. During these 2 stages the budding of suspension cells became visible under the microscope. The last Step v) (medium 4 - Table 5A) was necessary to continue specification and full maturation of NK cells. By Day 35 of differentiation or earlier, such cell culture was predominantly formed by suspension cells (Figure 1 B). These suspension cells were collected and stained for the expression of the surface markers. The expression of NK cell markers (as defined by CD56+ / CD45+) gradually increased within the cell population, reaching at least 90% of suspension cells by Day 35 of differentiation, depending on the differentiation protocol used (Figure 1C-E).

[0433] The functional activity of NK cells was determined by the balanced expression of the activating and inhibitory receptors on the cell surface, which coordinate recognition of infected or cancer cells by NK cells. In contrast to T-cells, this recognition does not require antigen presentation by MHC molecules, which advocates for the use of NK cells as a more universal cell therapy product. However, as induced NK cells (iNK) are created in vitro from the iPSC source, their characteristics should be compared to the healthy donor peripheral blood (PB) NK cells as a golden standard. iPSC-derived NK cells differentiated using the claimed methods were close (or even better) to the donor PB NK cells in the expression of activating (NKp44, NKp46, NKG2D) and maturation / inhibitory (NKG2A) molecules (Figure 2A and B). They also showed very high expression of perforin (Figure 2C), a component of cytotoxic granules, necessary to lyse target cancer or infected cells when the granules are released upon NK cells activation via the cell-to-cell contact. Moreover, it was shown that high expression of the CXCR3 chemokine receptor on induced NK cells can potentially guide these cells to the sites of tumour growth to facilitate its destruction (Figure 2C).

[0434] To understand whether induced NK cells are indeed capable of recognizing and killing cancer cells, several types of assays were developed where iPSC-derived NK cells of the invention (iNK) cells were mixed and co-cultured with various lines of cancer cells for a different time period up to 48 hrs. The cancer cells expressed reporter genes such as luciferase or a fluorescent protein, which facilitated quantification of relative numbers of cancer cells following their exposure to the iNK cells. The effect of the presence of iNK cells is shown in Figure 3A-C using breast cancer cell line expressing the GFP reporter: the decrease in numbers of fluorescent cells and a deformed morphology of fluorescent cells as evidenced by bright field images indicated cytotoxic activity of iNK cells. Using high throughput screening technology, the cytotoxic activity on iNK cells produced by different differentiation protocols against several cancer lines was quantified. Human NK-92 cell line was used, which was previously proposed as a therapeutic product for immuno-oncology, as the assay control. The cytotoxic effect was observed at a ratio of 0.25:1 of iNK cell : target (cancer) cell, which was rapidly increasing with the higher ratios of 0.5:1 or 1 :1. The cytotoxicity of iNK cells was equal or higher to this of NK-92 cells (Figures 3B-F). Interestingly, different cancer lines showed variable sensitivity to the exposure to iNK cells, which could be attributed to the ability of cancer cells to escape the immune response by the expression of inhibitory surface molecules or release of inhibitory cytokines that shut down the immune response (such as TGF or prostaglandins).

[0435] In conclusion, serum-free feeder-free GMP-compliant differentiation protocols (protocols 1- through 7) have been successfully developed for the production of iNK cells from human iPSCs, which can be produced as highly pure populations in cultures suitable for the scale- up bioreactor conditions. Phenotypically, these induced NK cells were comparable to PB NK cells and express a variety of surface and internal molecules predicting their cytotoxic activity, which has been proved by in vitro co-culture with cancer cell lines.

[0436] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A natural killer (NK) cell that has been differentiated in vitro, wherein the NK cell comprises: a. increased expression of one or more of: LINC00511, LINC02315, TESC, TPM4, TEC, DELEC1, COTL1, DRAXIN, SLC1A5, GLUL, LPAR3, NFILZ, INPP4B, ITGB7, KIT, S100A4, MIR646HG, CNR2, TNFSF10, LEF1 , PRF1, RASSF8, THEMIS, DPF3, IL32, CTSW, AFAP1L2, CD3E, STYK1, ZNF683, CCR6, CCR1, MIR181A1HG, ID2, SYTL3, ZFHX3, CCND2, ITGAX, TRPM2, BCL2, NDFIP2, LTB, PRKCA, NCAM1, DOCK5, and IL18R1; and / or decreased expression of one or more of: ADAM28, TMEM71, DIPK1A, MIR3667HG, PMEPA1, TIGIT, COLGALT2, BTN3A3, LINC00861, and MPP7, when compared to a blood-derived NK cell; or b. increased expression of one or more of: MAGI1, IGSF1, DLEU1, FBXL7, TIAM1, MAML3, PTPN14, DAPK2, MY01E, CSF1, TOX2, HOXB3, ITGA1, LINC00504, DPF3, ZFHX3, THEMIS, IL32, CTSW, TRPM2, NDFIP2, CCND2, ITGAX, IL18R1, PRKCA, LTB, CD3E, DOCK5, ZNF683, BCL2, MIR181A1HG, STYK1, RASSF8, CCR1, NCAM1, CCR6, ID2, AFAP1L2, and SYTL3 and / or decreased expression of one or more of: ZNF831, F2R, RAP1GAP2, TIGIT, BTN3A3, COLGALT2, MPP7, and LINC00861, when compared to a blood- derived NK cell.

2. A method for selecting an NK cell suitable for use in treating cancer, the method comprising: a. comparing a measured expression level of one or more genes selected from: SLC1A5, GLUL, LPAR3, NFILZ, INPP4B, ITGB7, KIT, S100A4, MIR646HG, CNR2, TNFSF10, LEF1, PRF1, RASSF8, THEMIS, DPF3, IL32, CTSW, AFAP1L2, CD3E, STYK1, ZNF683, CCR6, CCR1, MIR181A1HG, ID2, SYTL3, ZFHX3, CCND2, ITGAX, TRPM2, BCL2, NDFIP2, LTB, PRKCA, NCAM1, DOCK5, IL18R1,CSF1, TOX2, HOXB3, ITGA1, LINC00504, LINC00511, LINC02315, TESC, TPM4, TEC, DELEC1, COTL1, DRAXIN, MAGI1, IGSF1, DLEU1, FBXL7, TIAM1, MAML3, PTPN14, DAPK2, MY01E, ADAM28, TMEM71, DIPK1A, MIR3667HG, PMEPA1, TIGIT, COLGALT2, BTN3A3, LINC00861, MPP7, ZNF831, F2R, RAP1GAP2; and b. selecting the NK cell when the measured expression level of:i. one or more of: LINC00511, LINC02315, TESC, TPM4, TEC, DELEC1, COTL1, DRAXIN, SLC1A5, GLUL, LPAR3, NFILZ, INPP4B, ITGB7, KIT, S100A4, MIR646HG, CNR2, TNFSF10, LEF1 , PRF1, RASSF8, THEMIS, DPF3, IL32, CTSW, AFAP1L2, CD3E, STYK1, ZNF683, CCR6, CCR1 , MIR181A1HG, ID2, SYTL3, ZFHX3, CCND2, ITGAX, TRPM2, BCL2, NDFIP2, LTB, PRKCA, NCAM1, DOCK5, and IL18R1 is increased and / or one or more of: ADAM28, TMEM71, DIPK1A, MIR3667HG, PMEPA1 , TIGIT, COLGALT2, BTN3A3, LINC00861, and MPP7 is decreased, when compared to a blood- derived NK cell; or ii. one or more of: MAGI1, IGSF1, DLEU1, FBXL7, TIAM1, MAML3, PTPN14, DAPK2, MYO1E, CSF1, TOX2, HOXB3, ITGA1, LINC00504, DPF3, ZFHX3, THEMIS, IL32, CTSW, TRPM2, NDFIP2, CCND2, ITGAX, IL18R1, PRKCA, LTB, CD3E, DOCK5, ZNF683, BCL2, MIR181A1HG, STYK1, RASSF8, CCR1, NCAM1, CCR6, ID2, AFAP1L2, and SYTL3 is increased and / or one or more of: ZNF831, F2R, RAP1GAP2, TIGIT, BTN3A3, COLGALT2, MPP7, and LINC00861 is decreased, when compared to a blood-derived NK cell.

3. The NK cell according to claim 1 or the method according to claim 2, wherein the blood-derived NK cell is a peripheral blood NK cell or an umbilical cord blood NK cell.

4. The NK cell or the method according to any one of the preceding claims, wherein the increased expression and / or decreased expression is not the result of a genetic modification of the NK cell.

5. The NK cell or the method according to any one of the preceding claims, wherein the NK cell has been differentiated from a stem cell in vitro.

6. The NK cell or the method according to any one of the preceding claims, wherein the NK cell has been differentiated from a stem cell in vitro and wherein the stem cell is an induced pluripotent stem cell (iPSC).

7. The NK cell according to any one of the preceding claims, wherein the NK cell expresses CD45 and / or CD56, preferably wherein the NK cell expresses CD45 and CD56.

8. A method for producing Natural Killer (NK) cells, the method comprising the steps of: i) providing a population of cell aggregates comprising stem cells (preferably induced Pluripotent Stem Cells (iPSCs)); ii) culturing the population of cell aggregates from step i) to form a first cultured population; iii) culturing the first cultured population from step ii) to form a second cultured population; iv) culturing the second cultured population from step iii) to form a third cultured population; v) culturing the third cultured population from step iv) to form a fourth cultured population comprising NK cells; wherein the culturing media used in the culturing of step ii) comprises at least 4 components selected from ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, Activin A, VEGF and bFGF; wherein the culturing media used in the culturing of step iii) comprises at least4 components selected from BMP4, VEGF, SCF, TGF-p receptor kinase inhibitor, bFGF, IL-3, IL-6, TPO, Notch agonist, IGF-1 , estradiol, Nicotinamide and Tryptophan; wherein the culturing media used in the culturing of step iv) comprises at least5 components selected from BMP4, VEGF, SCF, IL-3, IL-7, IL-15, TGF-p receptor kinase inhibitor, bFGF, IL-6, TPO, Nicotinamide, Tryptophan, Notch agonist, Antihuman IgG antibody, aryl hydrocarbon receptor (AhR) antagonist and IL-15R-alpha; wherein the culturing media used in the culturing of step v) comprises at least 3 components selected from FLT3L, SCF, IL-7, IL-15, Notch agonist, Hydrocortisone, IL-15R-alpha, Nicotinamide, Anti-human IgG antibody, IGF1 , TPO, IL-21 , and SMAD3 inhibitor.

9. The method according to claim 8, wherein the culturing process of step ii) occurs for a period of up to 5 days, preferably from 2 to 4 days.

10. The method according to claim 8 or 9, wherein the culturing process of step iii) occurs for a period of up to 5 days, preferably from 2 to 4 days.

11. The method according to any one of claims 8-10, wherein the culturing process of step iv) occurs for a period of up to 8 days, preferably from 4 to 8 days.

12. The method according to any one of claims 8-11 , wherein the third cultured population of step iv) and the fourth cultured population of step v) produce hematopoietic stem cells (HSCs) and / or hematopoietic stem and progenitor cells (HSPCs)).

13. The method according to claim 12, wherein the HSCs and / or HSPCs express CD34+, CD43+, and / or CD45+.

14. The method according to any one of claims 8-13, wherein the culturing process of step v) occurs for a period of at least 14 days, preferably from 20 to 30 days, preferably the culturing process of step v) occurs for a period of 24 to 30 days.

15. The method according to any one of the claims 8-14, wherein the culturing media used in the culturing of steps ii), iii), iv) and / or v) further comprises at least 1 component, such as at least 2 components, selected from penicillin-streptomycin (P / S), B-mercaptoethanol, L-glutamine, L-ascorbic acid (L-AA), Sodium selenite and Ethanolamine, preferably P / S, B-mercaptoethanol, L-glutamine, L-AA, Sodium selenite and Ethanolamine.

16. The method according to claim 15, wherein the culturing media used in the culturing of steps ii), iii), iv) and / or v) comprises at least 3 components, such as at least 4 components, selected from penicillin-streptomycin (P / S), B-mercaptoethanol, L- glutamine, L-ascorbic acid (L-AA), Sodium selenite and Ethanolamine, preferably P / S, B-mercaptoethanol, L-glutamine, L-AA, Sodium selenite and Ethanolamine.

17. The method according to any one of claims 8-16, wherein the culturing media used in the culturing of step ii) comprises at least 5 components, such as at least 6 components, selected from VEGF, ROCK inhibitor, BMP4, SCF, GSK-3 inhibitor, Activin A, and bFGF.

18. The method according to claim 17, wherein the culturing media used in the culturing of step ii) comprises VEGF.

19. The method according to any one of the claims 8-18, wherein the culturing media used in the culturing of step iii) comprises at least 5 components, such as at least 6 components, selected from IGF-1 , Notch agonist, BMP4, VEGF, SCF, TGF-preceptor kinase inhibitor, bFGF, IL-3, IL-6, TPO, estradiol, Nicotinamide and Tryptophan.

20. The method according to claim 19, wherein the culturing media used in the culturing of step iii) comprises IGF-1 and / or Notch agonist (preferably DLL4 Fc).

21. The method according to claim 20, wherein the culturing media used in the culturing of step ii) does not comprise VEGF.

22. The method according to any one of claims 8-21 , wherein the culturing media used in the culturing of step iv) comprises at least 8 components, such as at least 9 components, selected from nicotinamide, tryptophan, Notch agonist, anti-human IgG antibody, BMP4, VEGF, SCF, IL-1 , IL-3, IL-6, IL-7, IL-9, IL-15, TGF-p receptor kinase inhibitor, bFGF, TPO, aryl hydrocarbon receptor (AhR) antagonist and IL-15Ralpha.

23. The method according to claim 22, wherein the culturing media used in the culturing of step iv) comprises at least one of: nicotinamide, tryptophan, Notch agonist (preferably DLL4 Fc), and anti-human IgG antibody.

24. The method according to claim 23, wherein the culturing media used in the culturing of step iv) comprises nicotinamide, tryptophan, Notch agonist (preferably DLL4 Fc), and anti-human IgG antibody.

25. The method according to any one of claims 8-24, wherein the culturing media used in the culturing of step v) comprises at least 6 components, such as at least 7 components, selected from Sodium selenite, Ethanolamine (MEA), FLT3L, SCF, IL-2, IL-7, IL-15, IL-18, IL-21 , Notch agonist, Hydrocortisone, IL-15Ralpha, IGF1 , TPO, IL- 21 , SMAD3 inhibitor, Nicotinamide and Anti-human IgG antibody.26 The method according to any one of claims 8-25 wherein the ROCK inhibitor is selected from Y-27632 dihydrochloride, fasudil, Y-39983 dihydrochloride, WF-536, SLx-2119, XD-4000, rhostatin and VAS-012, preferably the ROCK inhibitor is Y- 27632 dihydrochloride.

27. The method according to any one of the claims 8-26, wherein the GSK-3 inhibitor is selected from CHIR99021 , LiCI, BIO, TWS119 and Kenpaullone, preferably the GSK-3 inhibitor is CHIR99021 or LiCI and most preferably the GSK-3 inhibitor is CHIR99021.

28. The method according to any one of claims 8-27, wherein the TGF-p receptor kinase inhibitor is selected from SB431542, A 83-01 , RepSox, SB525334, Galunisertib and D4476, preferably the TGF-p receptor kinase inhibitor is SB431542.

29. The method according to any one of claims 8-28, wherein the Notch agonist is selected from DLL4-FC, DLL1 , DLL3, DLL4, JAG1 and GAG2, preferably the Notch agonist is DLL4-FC.

30. The method according to any one of claims 8-29, wherein the AhR antagonist is selected from StemRegenin 1 (SR1), CH-223191 , BAY-218, BAY 2416964 (compound 192), GNF351 , PDM2 and PDM-11 , preferably the AhR antagonist is SR1.

31. The method according to any one of claims 8-30, wherein the culturing media used in the culturing of steps ii), iii), iv) and / or v) comprises a basal medium, optionally selected from one or more of X-VIVO™ 15, StemSpan™ SFEM II, Stemline II Hematopoietic Stem Cell Expansion Medium, StemPro™-34 SFM (1X), StemDiff APEL-2 medium, DMEM, F12, IMDM and RPMI-1640, preferably the basal medium is one or more of StemDiff APEL-2 medium, DMEM and F12.32 The method according to any one of claims 8-31 , wherein the culturing media used in the culturing of steps ii), iii) and / or iv) comprises BMP4 in combination with at least one component selected from VEGF, Activin A, SCF, LiCI and CHIR99021 .

33. The method according to any one of claims 8-32, wherein the culturing media used in the culturing of step v) further comprises a media additive, optionally wherein the media additive is serum-containing or serum-free.

34. The method according to any one of the claims 8-33, wherein the steps ii), iii), iv) and v) are carried out over a total time of up to 46 days, preferably about 26 to about 46 days.

35. A composition comprising HSC, HSPC and / or NK cells obtainable by the method according to any one of claims 8-34.no36 The composition according to claim 35, wherein the HSC, HSPC and / or NK cells express CD56+ and / or CD45+.37 A composition comprising the NK cell according to any one of claims 1 or 3-7.38 A kit comprising: a. the NK cell according to any one of claims 1 or 3-7; or b. the composition according to any one of claims 35-37; and c. instruction for use in medicine.

39. An NK cell according to any one of claims 1 or 3-7 or a composition according to any one of claims 35-37 for use in treating a disorder.

40. A method of treating a disorder comprising administering to a subject in need thereof the NK cell according to any one of claims 1 or 3-7 or the composition according to any one of claims 35-37.41 . Use of an NK cell according to any one of claims 1 or 3-7 or a composition according to any one of claims 35-37 in the manufacture of a medicament for treating a disorder42. The NK cell or the composition for use according to claim 39, the method according to claim 40, or the use according to claim 41 , wherein the disorder is one or more of: a viral, fungal, or bacterial infection, haematological disorder, immune disorder or cancer, optionally wherein the disorder is Acute myeloid leukemia (AML), Acute lymphocytic leukemia (ALL), Chronic lymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML), Myelodysplastic syndromes (MDS), Multiple myeloma (MM), B Lymphoid Malignancies: B-cell non-Hodgkin lymphoma (B-NHL), Relapsed and Refractory B Cell Lymphoma, Relapsed / Refractory Diffuse Large B-Cell Lymphoma, Blastic Plasmacytoid Dendritic Cell Neoplasm, Plasma Cell Leukemia or a solid tissue tumour, including metastatic cancers.

43. An NK cell according to any one of claims 1 or 3-7 or a composition according to any one of claims 35-37 for use in treating cancer.I l l44. A method for treating cancer comprising administering to a subject in need thereof the NK cell according to any one of claims 1 or 3-7 or the composition according to any one of claims 35-37.

45. Use of an NK cell according to any one of claims 1 or 3-7 or a composition according to any one of claims 35-37 in the manufacture of a medicament for treating cancer.

46. The NK cell or composition for use according to claim 43, the method according to claim 44, or the use according to claim 45, wherein the cancer is one or more of: acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), multiple myeloma (MM), B lymphoid malignancies: B-cell nonHodgkin lymphoma (B-NHL), relapsed and refractory B cell lymphoma, relapsed and refractory diffuse large B-cell lymphoma, blastic plasmacytoid dendritic cell neoplasm, plasma cell leukemia and solid tissue tumours and metastatic cancers.

Images