Improved method for ex vivo expansion CD34+HSPCs into NK cells using an aryl hydrocarbon receptor antagonist
The present invention relates to the field of medicine, specifically the field of treatment of cancer. More specifically, the invention relates to a method for the ex vivo production of a population of highly functional NK cells from CD34-positive cells, to a population of highly functional NK cells obtained and to the use of such population of highly functional NK cells for adoptive cell therapy. Natural killer cells (NK cells) are CD3-negative CD56-positive lymphocytes, which are part of the innate immune system and play an important role in the defense against virus-infected and transformed cells. NK cell activation and subsequent killing of target cells is regulated by a balance in their expression levels of inhibitory receptors, including the killer-immunoglobulin like receptors (KIRs) and CD94/NKG2A heterodimer, versus activating receptors, such as DNAX accessory molecule-1 (DNAM-1), natural cytotoxicity receptors (NCRs) and NKG2D. In homeostasis, NK cells are inhibited by their inhibitory receptors recognizing self human leukocyte antigen (HLA) class I molecules and/or HLA-E molecules presenting conserved HLA class I leader sequences. However, an NK cell-mediated anti-tumor effect can be induced by up-regulation of activating ligands or down-regulation of HLA class I molecules on tumor cells. In addition, in the setting of haploidentical allogeneic stem cell transplantation (allo-SCT), donor NK cells may lack expression of inhibitory KIRs for recipient HLA class I molecules and hence be activated. This phenomenon is called missing-self recognition and can contribute to the curative graft-versus-tumor (GVT) effect. Because of their ability to kill tumor cells, NK cells are considered potent effectors for adoptive immunotherapy against cancer. So far, promising results have been obtained by infusion of haploidentical NK cells after immunosuppressive chemotherapy in adult and childhood acute myeloid leukemia (AML) [2-4]. However, a limitation in these studies is the relatively low NK cell numbers that can be enriched from aphaeresis products for multiple infusions. Furthermore, contaminating alloreactive T cells risk the induction of graft-versus-host disease (GVHD), especially when IL-2 of IL-15 are co-administrated to boost NK cell survival and expansion. In order to generate high numbers of allogeneic NK cells completely devoid of T cell contamination, a Good Manufacturing Practice (GMP)-compliant, cytokine-based ex vivo culture protocol has been developed [5,6]. Using this procedure, CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from umbilical cord blood (UCB) can be expanded over 2000-fold in large-scale bioreactors into a mixture of immature and mature NK cells with a purity >80%. Pre-clinical studies conducted in NOD/SCID-IL2Rγnull (NSG) mice demonstrated that these HSPC-NK cells have BM homing capacity, display IL-15-driven in vivo expansion, further mature in vivo by gaining CD16 and KIR expression, and effectively prolong survival of leukemia-bearing mice. Currently, administration of this HSPC-NK cell product following immunosuppressive chemotherapy is being investigated in a phase I clinical trial in older AML patients who are not eligible for allo-SCT (see www.trialregister.nl and search for 2818). In HLA-matched non-myeloablative and T cell-depleted allo-SCT, early NK cell repopulation has been associated with decreased relapse rates, without increasing GVHD incidence [8, 9]. Moreover, high NK cell numbers in stem cell grafts have been associated with a decreased incidence of GVHD. In addition, transplants from donors with KIR-B haplotypes, containing several activating KIRs, led to lower rates of relapse and improved survival [11-13]. For these reasons, it would be highly valuable to exploit HSPC-NK cell products for adoptive immunotherapy after allo-SCT. Since NK cells of donor origin will not be rejected, multiple NK cell infusions without the need for immunosuppressive chemotherapy to prevent rejection, could be administered after allo-SCT. Consequently, these cells may potentially induce long-term GVT effects. However, to obtain large numbers of NK cells from donor origin, peripheral blood (PB) or bone marrow (BM)-derived CD34-positive HSPCs, which have a lower expansion potential compared to UCB-derived CD34-positive HSPCs, should be expanded and differentiated into NK cells. As said, a protocol for obtaining high numbers of NK cells has been reported [5,6]. The protocol is however based on CD34-positive cells from umbilical cord blood, which cells have a higher expansion potential than peripheral blood (PB) or bone marrow (BM)-derived CD34-positive HSPCs. In addition, said protocol requires the use of heparin from animal origin which has the disadvantage that it may be contaminated with pathogens and its composition may not be constant over time. Moreover, the protocol requires a low dose cytokine cocktail throughout the procedure which renders the protocol complicated and expensive. Furthermore, GMP-compliant productions using this heparin-based protocol has shown some inconsistencies regarding NK purity of more than 80% (range 40-85%, n=12, Dolstra et al. manuscript submitted). Accordingly, there is a need for more robust and consistent ex vivo expansion and differentiation of CD34-positive HSPCs into NK cells. Such method should meet the requirements of resulting in high-fold expansion in large-scale bioreactors, high purity of the NK cells and high functionality of the NK cells. Effective HSPC-NK products can be exploited for adoptive cell therapy in allogeneic stem cell transplant as well as non-transplant setting against hematological and solid malignancies. In an aspect, the invention provides for a method, preferably a consistent method, for the ex vivo production of a population of highly functional NK cells from CD34-positive cells comprising culturing CD34-positive cells in an expansion medium and subsequently culturing the expanded CD34-positive cells in a differentiation medium, wherein the expansion medium comprises an aryl hydrocarbon receptor antagonist, wherein the CD34-positive cells are preferably peripheral blood, bone marrow or umbilical cord blood-derived CD34-positive cells. The invention further provides for a method according to the invention, with the proviso that the expansion medium and/or the differentiation medium do not comprise a glycosaminoglycan and/or do not comprise G-CSF, GM-CSF and/or IL-6. The invention further provides for a method according to the invention, wherein culturing CD34-positive cells in an expansion medium and subsequently culturing the expanded CD34-positive cells in a differentiation medium comprises:
The invention further provides for a method according to the invention, wherein the first differentiation medium and/or the second differentiation medium further comprises IL-2 or IL-12. The invention further provides for a method according to the invention, wherein the aryl hydrocarbon receptor antagonist is one selected from the group consisting of SR1 (StemRegenin 1), CH-223191, GNF351, 6,2′,4′ trimethoxyflavone and CB7993113, preferably the aryl hydrocarbon receptor antagonist is SR1. The invention further provides for a method according to the invention, wherein culturing in the expansion medium is performed for about 7 to 10 days, wherein culturing in the first differentiation medium is performed from about day 7 to 10 to about day 19 to 21, and wherein culturing in the second differentiation medium is performed from about day 19 to 21 for at least about 10 days, preferably for at least about 14 to 21 days or for at least 21 days. The invention further provides for a method according to the invention, wherein at least about 1E+9 highly functional NK cells are produced from a single donor and/or wherein the amount of CD3-positive cell is at most about 0.1%, preferably wherein at least about 1E+9 highly functional and highly pure CD56-positive, Perforin-positive and EOMES-positive NK cells are produced from a single donor and/or wherein the amount of CD3-positive cells is at most about 0.1%. The invention further provides for a method according to the invention, wherein the mean overall expansion is at least about two-fold higher than when no aryl hydrocarbon receptor antagonist is used. In an aspect, the invention provides for a population of highly functional NK cells obtainable by a method according to the invention, preferably wherein in the population, at least 80% of the NK cells are highly functional NK cells, more preferably wherein the highly functional NK cells are CD56-positive, Perforin-positive and EOMES-positive NK cells. The invention further provides for a population of highly functional NK cells according to the invention comprising at least 1E+9 highly functional NK cells from a single donor and/or wherein the amount of CD3-positive cell is at most about 0.1%, preferably wherein at least about 1E+9 highly functional and highly pure CD56-positive, Perforin-positive and EOMES-positive NK cells are produced from a single donor and/or wherein the amount of CD3-positive cells is at most about 0.1%. The invention further provides for a population of highly functional NK cells according to the invention, wherein at least 10% of the CD56-positive cells express CD16, at least 80% of the CD56-positive cells express NKG2A, at least 50% of the CD56-positive cells express NKG2D, at least 50% of the CD56-positive cells express DNAM1, at least 50% of the CD56-positive cells express NKp30, at least 60% of the CD56-positive cells express NKp44, at least 80% of the CD56-positive cells express NKp46, at least 50% of the CD56-positive cells express TRAIL, at least 20% of the CD56-positive cells express CD62L, at least 60% of the CD56-positive cells express CXCR3 and/or at least 10% of the CD56-positive cells is capable of secreting IFN-gamma upon stimulation with K562 target cells. The invention further provides for a population of highly functional NK cells according to the invention, wherein the CD56-positive cells further express at least the combination of NKG2D, DNAM1, NKp30, NKp44, NKp46, TRAIL, CD62L, CXCR3, perforin, granzyme B and are capable of secreting IFN-gamma upon stimulation with K562 target cells. The invention further provides for a population of NK cells, wherein in the population, at least 80% of the NK cells are highly functional NK cells, more preferably wherein the highly functional NK cells are CD56-positive, Perforin-positive and EOMES-positive NK cells. The invention further provides for a therapeutic product comprising a population of NK cells according to the invention. In an aspect, the invention provides for a population of highly functional NK cells according to the invention or a population of highly functional NK cells obtainable by a method according to the invention or a therapeutic product comprising a population of NK cells according to the invention, for use as a medicament. In an aspect, the invention provides for a population of highly functional NK cells according to the invention or a population of highly functional NK cells obtainable by a method according to the invention or a therapeutic product comprising a population of NK cells according to the invention, for use in the treatment of cancer. The invention further provides for a population of highly functional NK cells according to the invention or a population of highly functional NK cells obtainable by a method according to the invention or a therapeutic product comprising a population of NK cells according to the invention for use as a medicament in the treatment of cancer, wherein treatment of the cancer further comprises allogeneic stem cell transplantation. Notably, it has now been demonstrated that peripheral blood (PB) or bone marrow (BM)-derived CD34-positive HSPCs, next to UCB-derived CD34-positive HSPCs, can be expanded and differentiated into a population of highly functional NK cells. Accordingly, in a first aspect the present invention provides for a method, preferably a consistent method, for the ex vivo production of a population of highly functional NK cells from CD34-positive cells comprising culturing CD34-positive cells in an expansion medium and subsequently culturing the expanded CD34-positive cells in a differentiation medium, wherein the expansion medium comprises an aryl hydrocarbon receptor antagonist, wherein the CD34-positive cells are preferably peripheral blood, bone marrow-derived or umbilical cord blood CD34-positive cells. Said method is herein referred to as a method according to the invention. The CD34-positive cells are preferably HSPCs and may be obtained or have been obtained from any source. Preferably, the CD34-positive cells are obtained or have been obtained from a human subject. A preferred source of the CD34-positive cells is blood, bone marrow or other post-embryonic tissue such as adult fat tissue (mesenchymal stem cells) and umbilical cord blood. The CD34-positive cells may also be obtained by reprogramming tissue specific cells such as skin stem cells or fibroblasts to produce HSPCs from induced pluripotent stem cells. The CD34-positive cells may or may not be obtained from embryonic stem cell lines. Particularly preferred are peripheral blood (PB), bone marrow (BM)-derived or umbilical cord blood (UCB) CD34-positive HSPCs. CD34-positive cells may be isolated or may have been isolated using any means known in the art. After collection of a sample (e.g. a bone marrow sample, peripheral blood sample or umbilical cord blood sample), mononuclear cells may be isolated using density gradient centrifugation. Peripheral blood cells may be obtained or may have been obtained by aphaeresis material from donors such as stem cell donors that may have been treated with G-SCF (granulocyte-colony stimulating factor). CD34-positive cells are preferably isolated from a population that may have been enriched for mononuclear cells using affinity purification such as using an anti-CD34 antibody, preferably using anti-CD34 immunomagnetic beads. A preferred method for the isolation of CD34-positive cells is a method as described in the examples herein. The isolated CD34-positive cells may be used fresh, i.e. immediately of shortly after isolation, or may be stored until further use. The expansion media and differentiation media described herein are collectively referred to as an expansion medium according to the invention and a differentiation medium according to the invention. An expansion medium according to the invention and a differentiation medium according to the invention comprise a basic medium supplemented with further components as defined herein. The basic medium may be any medium suitable for propagation and/or maintenance of CD34-positive HSPCs and/or for propagation and/or maintenance of CD56-positive cells. The basic medium is preferably a GMP (Good Manufacturing Practice) medium. The basic medium is preferably Cellgro GMP DC medium (Cellgenix, Freiburg, Germany) or NK MACS medium (Miltenyi, Bergisch Gladbach, Germany) supplemented with 2-10% human serum (such as human serum obtainable from Sanquin Bloodbank, Nijmegen, The Netherlands). A preferred basic medium is one described in the examples herein. Ex vivo production is to be construed that it does not involve any invasive manipulation of the human or animal body since it involves a method performed on a sample that has already been provided. A method according to the invention may be an in vitro method. A population of cells is to be construed as at least 1E+3 viable NK cells. Preferably, a population of cells comprises at least 1E+4, more preferably 1E+5, 1E+6, 1E+7, 1E+8, or most preferably 1E+9 viable NK cells. Preferably, in a method according to the invention the CD34-positive cells are cultured at an initial concentration of at least 1E+5 cells/ml, preferably between 1E+5 and 5E+5 cells/ml in a plate, flask or bag. Preferably, after about three days of culture, the cells are transferred to a new plate, flask or bag to deplete for stromal cells. Preferably, the cultures are refreshed with about at least 20% fresh medium every two to three days. Cell cultures are typically maintained under conditions conducive to the propagation and/or maintenance of the cells. Preferably, the cell cultures are maintained at around 37° C. in 75-95% humidity in 4.5-5.5% CO2. Preferably, the total culture time-frame for expansion and differentiation of CD34-positive cells into highly functional NK cells is about five weeks to about seven weeks, preferably about five to about six weeks and most preferably about five weeks. Preferably, in a method according to the invention, an expansion medium does not comprise a glycosaminoglycan and/or does not comprise G-CSF (granulocyte-colony-stimulating factor), GM-CSF (granulocyte-macrophage-colony-stimulating factor) and/or IL-6 (interleukin-6). Preferably, an expansion medium according to the invention does not comprise a glycosaminoglycan, G-CSF, GM-CSF or IL-6. Preferably, an expansion medium according to the invention does not comprise a glycosaminoglycan and G-CSF. Preferably, an expansion medium according to the invention does not comprise a glycosaminoglycan and GM-CSF. Preferably, an expansion medium according to the invention does not comprise a glycosaminoglycan and IL-6. Preferably, an expansion medium according to the invention does not comprise a glycosaminoglycan, G-CSF and GM-CSF. Preferably, an expansion medium according to the invention does not comprise a glycosaminoglycan, GM-CSF and IL-6. Preferably, an expansion medium according to the invention does not comprise a glycosaminoglycan, G-CSF, GM-CSF and IL-6. Preferably, in a method according to the invention, a differentiation medium does not comprise a glycosaminoglycan and/or does not comprise G-CSF, GM-CSF and/or IL-6. Preferably, a differentiation medium according to the invention does not comprise a glycosaminoglycan, G-CSF, GM-CSF or IL-6. Preferably, a differentiation medium according to the invention does not comprise a glycosaminoglycan and G-CSF. Preferably, a differentiation medium according to the invention does not comprise a glycosaminoglycan and GM-CSF. Preferably, a differentiation medium according to the invention does not comprise a glycosaminoglycan and IL-6. Preferably, a differentiation medium according to the invention does not comprise a glycosaminoglycan, G-CSF and GM-CSF. Preferably, a differentiation medium according to the invention does not comprise a glycosaminoglycan, GM-CSF and IL-6. Preferably, a differentiation medium according to the invention does not comprise a glycosaminoglycan, G-CSF, GM-CSF and IL-6. Preferably, such glycosaminoglycan is a heparin such as a low molecular weight heparin (LMWH). A LMWH typically is a heparin or heparin salt having an average molecular weight of between about 2000-10000 Dalton, preferably between 5000 and 8000 Dalton and more preferably about 8000 Dalton. Such heparin may be acetylated, desulphated and/or phosphorylated. Preferably, in a method according to the invention culturing CD34-positive cells in an expansion medium and subsequently culturing the expanded CD34-positive cells in a differentiation medium comprises:
More preferably, in a method according to the invention culturing CD34-positive cells in an expansion medium and subsequently culturing the expanded CD34-positive cells in a differentiation medium comprises:
In a method according to the invention, in the expansion medium, the concentration of IL-7 is preferably in the range of about 10 and 150 ng/ml, more preferably in the range of about 20 to 120 ng/ml, more preferably about 25 ng/ml. In a method according to the invention, in the expansion medium, the concentration of SCF is preferably in the range of about 10 and 150 ng/ml, more preferably in the range of about 20 to 120/ml, more preferably about 25 ng/ml. In a method according to the invention, in the expansion medium, the concentration of TPO is preferably in the range of about 10 and 150 ng/ml, more preferably in the range of about 20 to 120 ng/ml, more preferably about 25 ng/ml. In a method according to the invention, in the expansion medium, the concentration of Flt3L is preferably in the range of about 10 and 150 ng/ml, more preferably in the range of about 20 to 120 ng/ml, more preferably about 25 ng/ml. In a method according to the invention, in the expansion medium, the concentration of aryl hydrocarbon receptor antagonist is preferably in the range of about 0.5 and 5 μm, more preferably in the range of about 1 to 5 μm, more preferably about 2 μm. In a method according to the invention, in the first differentiation medium, the concentration of IL-7 is preferably in the range of about 10 and 40 ng/ml, more preferably in the range of about 20 to 30 ng/ml, more preferably about 25 ng/ml. In a method according to the invention, in the first differentiation medium, the concentration of SCF is preferably in the range of about 10 and 40 ng/ml, more preferably in the range of about 20 to 30 ng/ml, more preferably about 25 ng/ml. In a method according to the invention, in the first differentiation medium, the concentration of IL-15 is preferably in the range of about 10 and 100 ng/ml, more preferably in the range of about 25 to 75 ng/ml, more preferably about 50 ng/ml. Preferably, in a method according to the invention the first differentiation medium and/or the second differentiation medium further comprises IL-2 (interleukin-2) or IL-12 (interleukin-12). Preferably, the first differentiation medium further comprises IL-2 or IL-12, preferably IL-12. Preferably, the second differentiation medium further comprises IL-2 or IL-12, preferably IL-12. Preferably, the first differentiation medium and the second differentiation medium further comprises IL-2 or IL-12, preferably IL-12. Preferably, the concentration of IL-2 is in the range of about 500 to 2000 U/ml, more preferably in the range of about 750 to 1500 U/ml, more preferably about 1000 U/ml. Preferably, the concentration of IL-12 is in the range of about 0.05 to 0.4 ng/ml, more preferably in the range of about 0.1 to 0.3 ng/ml, more preferably about 0.2 ng/ml. Preferably, in a method according to the invention, the expansion medium comprises about 25 ng/ml IL-7, about 25 ng/ml CSF, about 25 ng/ml TPO, about 25 ng/ml Flt3L and about 2 μm aryl hydrocarbon receptor antagonist, preferably SR1. Preferably, in a method according to the invention, the first differentiation medium comprises about 25 ng/ml IL-7, about 25 ng/ml CSF, about 25 ng/ml Flt3L, about 50 ng/ml IL-15 and about 2 μm aryl hydrocarbon receptor antagonist, preferably SR1. Preferably, in a method according to the invention, the second differentiation medium comprises about 20 ng/ml IL-7, about 20 ng/ml CSF, about 250 ng/ml IL-15, about 1000 U/ml IL-2 or about 0.2 ng/ml IL-12 and about 2 μm aryl hydrocarbon receptor antagonist, preferably SR1. More preferably, the second differentiation medium comprises about 20 ng/ml IL-7, about 20 ng/ml CSF, about 250 ng/ml IL-15 and about 1000 U/ml IL-2 or about 0.2 ng/ml IL-12. In an embodiment:
In an embodiment:
In an embodiment:
In an embodiment:
In an embodiment, the recipes of the media are as depicted in the examples herein. In a method according to the invention, the aryl hydrocarbon receptor antagonist may be any the aryl hydrocarbon receptor antagonist that enhances expansion of CD34-positive HSPCs and induces expression of NK cell associated transcription factors promoting NK cell differentiation; it may be an antagonist that interferes with the aryl hydrocarbon receptor itself but is may also be an agent that reduces expression, transcription and/or translation of the aryl hydrocarbon receptor. A preferred aryl hydrocarbon receptor antagonist is one selected from the group consisting of SR1 (StemRegenin 1, WO2010/059401; Boitano et al. 2010), CH-223191 (Kim et al. 2006, Hughes et al. 2014), GNF351 (Smith et al. 2011), 6,2′,4′trimethoxyflavone (Zhao et al. 2010) and CB7993113 (Parks et al. 2014). A more preferred aryl hydrocarbon receptor in the embodiments of the invention is SR1. Preferably, in a method according to the invention, culturing in the expansion medium is performed for about 7 to 10 days. Preferably, culturing in the first differentiation medium is performed from about day 7 to 10 to about day 19 to 21. Preferably, culturing in the second differentiation medium is performed from about day 19 to 21 for at least about 10 days, more preferably for at least about 14 to about 21 days or for at least about 21 days. Preferably, in a method according to the invention, culturing in the expansion medium is performed for about 7 to 10 days, culturing in the first differentiation medium is performed from about day 7 to 10 to about day 19 to 21 and culturing in the second differentiation medium is performed from about day 19 to 21 for at least about 10 days, more preferably for at least about 14 days, more preferably for at least about 21 days. More preferably, in a method according to the invention, culturing in the expansion medium is performed for about 9 to 10 days, culturing in the first differentiation medium is performed from about day 9 to 10 to about day 19 to 21 and culturing in the second differentiation medium is performed from about day 19 to 21 for at least about 10 days, more preferably for at least about 14 days, more preferably for at least about 21. Preferably, in a method according to the invention at least about 1E+8, more preferably at least about 1E+9 highly functional NK cells are produced from a single donor or cord blood unit. Preferably, in a method according to the invention the amount of non-NK cells is 20% or less in the produced population of highly functional NK cells. Preferably, the amount of highly functional NK cells is at least about 70%, more preferably at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at most about 95%. Preferably, in a method according to the invention the amount of CD3-positive T cells is at most about 0.5%, more preferably at most about 0.4%, 0.3%, 0.2%, 0.1%, or at most about 0.05% in the produced population of highly functional NK cells. Preferably, in a method according to the invention the mean overall expansion is at least about two-fold higher, more preferably at least about three-fold higher than when no aryl hydrocarbon receptor antagonist is used. Preferably, the mean overall expansion is at least 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold or more preferably at least 2000-fold. Preferably, the mean overall expansion for peripheral blood (PB) or bone marrow (BM)-derived CD34-positive HSPCs is at least 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290-fold or more preferably at least 300-fold. Preferably, the mean overall expansion for umbilical cord blood (UCB) CD34-positive HSPCs is at least 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold or more preferably at least 2000-fold. In a second aspect, the invention provides for a population of highly functional NK cells obtainable by a method according to the invention, preferably a method according to the first aspect of the invention, preferably a method as depicted in the examples herein. Such population of highly functional NK cells is herein referred to as a population of highly functional NK cells according to the invention. A population of highly functional NK cells according to the invention is preferably an ex vivo or an in vitro population. Preferably, a population of highly functional NK cells according to the invention comprises at least about 1E+8, more preferably at least about 1E+9 highly functional NK cells from a single donor or cord blood unit. Preferably, in a population of highly functional NK cells according to the invention the amount of non-NK cells is 20% or less. Preferably, the amount of highly functional NK cells is at least about 70%, more preferably at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at most about 95%. Preferably, in a population of highly functional NK cells according to the invention the amount of CD3-positive cells is at most about 0.5%, more preferably at most about 0.4%, 0.3%, 0.2%, 0.1%, or at most about 0.05%. In the embodiments of the invention, a population of highly functional NK cells is defined as a population wherein at least 10% of the CD56-positive cells express CD16, at least 80% of the CD56-positive cells express NKG2A, at least 50% of the CD56-positive cells express NKG2D, at least 50% of the CD56-positive cells express DNAM1, at least 50% of the CD56-positive cells express NKp30, at least 60% of the CD56-positive cells express NKp44, at least 80% of the CD56-positive cells express NKp46, at least 50% of the CD56-positive cells express TRAIL, at least 50% of the CD56-positive cells express perforin, at least 50% of the CD56-positive cells express granzyme B, at least 20% of the CD56-positive cells express CD62L, at least 60% of the CD56-positive cells express CXCR3 and/or at least 10% of the CD56-positive cells is capable of secreting IFN-gamma upon stimulation with K562 target cells. Preferably, a population of highly functional NK cells according to the invention is defined as a population wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more preferable 100% of the CD56+ NK cells further express at least the combination of NKG2D, DNAM1, NKp46, TRAIL and CXCR3. Preferably, a population of highly functional NK cells according to the invention is defined as a population wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more preferable 100% of the CD56+ NK cells further express at least the combination of NKG2D, DNAM1, NKp30, NKp44, NKp46, TRAIL and CXCR3. Preferably, a population of highly functional NK cells according to the invention is defined as a population wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more preferable 100% of the CD56+ NK cells further express at least the combination of NKG2D, DNAM1, NKp46, TRAIL, CXCR3 and perforin, granzymeB. Preferably, a population of highly functional NK cells according to the invention is defined as a population wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more preferable 100% of the CD56+ NK cells further express at least the combination of NKG2D, DNAM1, NKp30, NKp44, NKp46, TRAIL, CXCR3, perforin, granzymeB and are capable of secreting IFN-gamma upon stimulation with K562 target cells. More preferably, a population of highly functional NK cells according to the invention is defined as a population wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more preferable 100% of the CD56+ NK cells further express at least the combination of NKG2A, NKG2D, DNAM1, NKp30, NKp44, NKp46, TRAIL, CD62L, CXCR3, perforin, granzyme B and are capable of secreting IFN-gamma upon stimulation with K562 target cells. The invention further provides for a population of NK cells, wherein in the population, at least 80% of the NK cells are highly functional NK cells, more preferably wherein the highly functional NK cells are CD56-positive, Perforin-positive and EOMES-positive NK cells. The invention further provides for a therapeutic product comprising a population of NK cells according to the invention In a third aspect, the invention provides for the medical use of a population of highly functional NK cells according to the invention, for the medical use of a population of highly functional NK cells obtainable by a method according to the invention and for the medical use of a therapeutic product comprising a population of NK cells according to the invention. In such medical use, a population of highly functional NK cells according to the invention or a population of highly functional NK cells obtainable by a method according to the invention, or a therapeutic product comprising a population of NK cells according to the invention can be transplanted into a patient by means known to the person skilled in the art. Accordingly, the invention provides for a population of highly functional NK cells according to the invention or a population of highly functional NK cells obtainable by a method according to the invention or a therapeutic product comprising a population of NK cells according to the invention for use as a medicament. Preferably, the use as a medicament is the treatment of a cancer, a viral disease, a fungal disease, or a solid transplant rejection, or an autoimmune disease and a loss of pregnancy. Preferably, the use as a medicament is the treatment of a cancer. Accordingly, there is provided a population of cells according to the invention or a population of highly functional NK cells obtainable by a method according to the invention or a therapeutic product comprising a population of NK cells according to the invention for use in the treatment of cancer. Said cancer may be any type of cancer suitable for treatment by NK cells, such as but not limited to a skin cancer, breast cancer, lung cancer, ovarian cancer, fallopian tube cancer, colorectal cancer, head and neck cancer, prostate cancer, bladder cancer, liver cancer, pancreatic cancer, stomach cancer, esophagus cancer, brain cancer and melanoma. Preferably, said cancer is a cancer of hematopoietic origin like leukemia such as acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), and lymphomas such as Hodgkin's and Non-Hodgkin's Lymphomas (HL and NHL) and their subtypes or multiple myeloma (MM). The medical use of a population of highly functional NK cells according to the invention or a therapeutic product comprising a population of NK cells according to the invention may further comprise another type of treatment such as treatment of a cancer with a therapeutic antibody specific for an antigen present on cells of said cancer in order to induce antibody-dependent cell-mediated cytotoxicity (ADDC) of the transferred NK cells. Preferably, such antibody is a therapeutic monoclonal antibody such as, but not limited to, Rituximab (anti-CD20), Ofatumumab (anti-CD20), Tositumomab (anti-CD20), Ibritumomab (anti-CD20), Brentuximab (anti-CD30), Dacetuzumab (anti-CD40), Trastuzumab (anti-Her2), Alemtuzumab (anti-CD52), Cetuximab (anti-EGFR), Panitumumab (anti-EGFR), Gemtuzumab (anti-CD33), Elotuzumab (anti-SLAM7), Catumaxomab (anti-EpCam), G250 (anti-CAIX). The medical use of a population of highly functional NK cells according to the invention may further comprise treatment with antibodies that influence migration or function of transferred NK cells such as, but not limited to, Bevacizumab (anti-VEGF), Tremelimumab (anti-CTLA-4), Ipilimumab (anti-CTLA4), Pidilizumab (anti-PD-1), Nivolumab (anti-PD-1), Pembrolizumab (anti-PD1), Lirilumab (anti-KIR). The medical use of a population of highly functional NK cells according to the invention may further comprise treatment with epigenetic modifiers such as, but not limited to, DNA methylation inhibitors such as, but not limited to, Decitabine and Azacitadine, and histon deacetylase inhibitors such as, but not limited to, Valproic acid, Vorinostat and Panobinostat. In an embodiment, there is provided a population of highly functional NK cells according to the invention or a population of highly functional NK cells obtainable by a method according to the invention or a therapeutic product comprising a population of NK cells according to the invention for use in the treatment in the treatment of cancer wherein treatment of the cancer further comprises allogenic stem cell transplantation. The medical use herein described is formulated as a product according to the invention for use as a medicament for treatment of the stated diseases but could equally be formulated as a method of treatment of the stated diseases using a product according to the invention, a product according to the invention for use in the preparation of a medicament to treat the stated diseases and use of a product according to the invention for the treatment of the stated diseases. Such medical uses are all envisaged by the present invention. In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”. The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1% of the value. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The present invention is further described by the following examples which should not be construed as limiting the scope of the invention. Unless stated otherwise, the practice of the invention will employ standard conventional methods of molecular biology, virology, microbiology or biochemistry. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Abstract Early natural killer (NK) cell repopulation after allogeneic stem cell transplantation (allo-SCT) has been associated with reduced relapse rates without an increased risk of graft-versus-host disease (GVHD), indicating that donor NK cells have specific anti-leukemic activity. Therefore, adoptive transfer of donor NK cells is an attractive strategy to reduce relapse rates after allo-SCT. Since NK cells of donor origin will not be rejected, multiple NK cell infusions could be administered in this setting. However, isolation of high numbers of functional NK cells from transplant donors is challenging. Hence, we developed a cytokine-based ex vivo culture protocol to generate high numbers of functional NK cells from G-CSF mobilized CD34+ stem and progenitor cells (HSPCs). In this study, we demonstrate that addition of aryl hydrocarbon receptor (AhR) antagonist SR1 to our culture protocol potently enhances expansion of CD34+ HSPCs, and induces expression of NK cell associated transcription factors promoting NK cell differentiation. As a result, high numbers of NK cells with an active phenotype can be generated using this culture protocol. These SR1-generated NK cells exert efficient cytolytic activity and IFN-γ production towards acute myeloid leukemia (AML) and multiple myeloma (MM) cells. Importantly, we observed that NK cell proliferation and function is not inhibited by cyclosporin A (CsA), an immunosuppressive drug often used after allo-SCT. These findings demonstrate that SR1 can be exploited to generate high numbers of functional NK cells from G-CSF mobilized CD34+ HSPCs, providing great promise for effective NK cell based immunotherapy after allo-SCT. Introduction Natural killer cells (NK cells) are CD3-CD56+ lymphocytes, which are part of the innate immune system and play an important role in the defense against virus-infected and transformed cells. NK cell activation and subsequent killing of target cells is regulated by a balance in their expression levels of inhibitory receptors, including the killer-immunoglobulin like receptors (KIRs) and CD94/NKG2A heterodimer, versus activating receptors, such as DNAX accessory molecule-1 (DNAM-1), natural cytotoxicity receptors (NCRs) and NKG2D. In homeostasis, NK cells are inhibited by their inhibitory receptors recognizing self human leukocyte antigen (HLA) class I molecules and/or HLA-E molecules presenting conserved HLA class I leader sequences. However, an NK cell-mediated anti-tumor effect can be induced by up-regulation of activating ligands or down-regulation of HLA class I molecules on tumor cells. In addition, in the setting of haploidentical allogeneic stem cell transplantation (allo-SCT), donor NK cells may lack expression of inhibitory KIRs for recipient HLA class I molecules and hence be activated. This phenomenon is called missing-self recognition and can contribute to the curative graft-versus-tumor (GVT) effect. Because of their ability to kill tumor cells, NK cells are considered potent effectors for adoptive immunotherapy against cancer. So far, promising results have been obtained by infusion of haploidentical NK cells after immunosuppressive chemotherapy in adult and childhood acute myeloid leukemia (AML) [2-4]. However, a limitation in these studies is the relatively low NK cell numbers that can be enriched from aphaeresis products for multiple infusions. Furthermore, contaminating alloreactive T cells risk the induction of graft-versus-host disease (GVHD), especially when IL-2 or IL-15 are co-administrated to boost NK cell survival and expansion. In order to generate high numbers of allogeneic NK cells completely devoid of T cell contamination, a Good Manufacturing Practice (GMP)-compliant, cytokine-based ex vivo culture protocol has been developed by our group [5,6]. Using this procedure, CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from umbilical cord blood (UCB) can be expanded over 2000-fold in large-scale bioreactors into a mixture of immature and mature NK cells with a purity >80%. Pre-clinical studies conducted in NOD/SCID-IL2Rγnull mice demonstrated that these HSPC-NK cells have BM homing capacity, display IL-15-driven in vivo expansion, and prolong survival of leukemia-bearing mice. Currently, administration of this HSPC-NK cell product following immunosuppressive chemotherapy is being investigated in a phase I clinical trial in older AML patients who are not eligible for allo-SCT (see www.trialregister.nl and search for 2818). In HLA-matched non-myeloablative and T cell-depleted allo-SCT, early NK cell repopulation has been associated with decreased relapse rates, without increasing GVHD incidence [8, 9]. Moreover, high NK cell numbers in stem cell grafts have been associated with a decreased incidence of GVHD. In addition, transplants from donors with KIR-B haplotypes, containing several activating KIRs, led to lower rates of relapse and improved survival [11-13]. For these reasons, it would be highly valuable to exploit HSPC-NK cell products for adoptive immunotherapy after allo-SCT. Since NK cells of donor origin will not be rejected, multiple NK cell infusions without the need for immunosuppressive chemotherapy to prevent rejection, could be administered after allo-SCT. Consequently, these cells may potentially induce long-term GVT effects. However, to obtain large numbers of NK cells from donor origin, peripheral blood (PB) or bone marrow (BM)-derived CD34+ HSPCs, which have a lower expansion potential compared to UCB-derived CD34+ HSPCs, should be expanded and differentiated into NK cells. Recently, it was described that expansion of CD34+ HSPCs can be enhanced by inhibition of the aryl hydrocarbon receptor (AhR) using the antagonist StemReginin1 (SR1). AhR is a ligand-inducible transcription factor, which plays an important role in biological responses towards xenobiotic agents such as digoxin [15, 16]. Yet, it has become clear that AhR also has multiple naturally occurring ligands, like tryptophan metabolites and dietary compounds. Furthermore, AhR was hypothesized to regulate differentiation of multiple immune cells including dendritic cells [17, 18], regulatory T cells [19, 20], γδ T cells and Th17 cells. Regarding NK cell differentiation, it has been observed that antagonism of AhR or silencing of AhR gene expression promotes differentiation of tonsillar IL-22-producing IL-1R1hi human ILC3s to CD56brightCD94+ interferon (IFN)-g-producing cytolytic mature NK cells expressing EOMES and TBET/TBX21. Based on these findings, we hypothesized that addition of SR1 to our culture system might improve expansion of CD34+ HSPCs, and differentiation of these cells into NK cells. We found that SR1 not only enhances expansion of CD34+ HSPCs, but also up-regulates the expression of early and late NK cell specific transcription factors such as TOX, ID2, EOMES, GATA-3 and EAT-2, thereby potentiating differentiation of SR1-expanded CD34+ cells into NK cells. These SR1-induced NK cells have a high purity, express high levels of activating receptors and efficiently target AML and multiple myeloma (MM) cells. Importantly, proliferation and cytolytic functions of SR1-induced HSPC-NK cells are not inhibited by cyclosporin A (CsA), in contrast to mycophenolic acid (MPA), which is used only shortly after allo-SCT, facilitating multiple infusions relatively shortly after allo-SCT. Therefore, our SR1 culture system holds great promise for future donor HSPC-NK cell adoptive immunotherapy after allo-SCT to boost anti-tumor and anti-viral immunity, leading to prolonged relapse-free survival. Materials and Methods Cell Lines Cell lines (K562, THP1, HL-60, U266, UM9, RPMI8226) were cultured in Iscove's modified Dulbecco's medium (IMDM; Invitrogen, Carlsbad, Calif., USA) containing 50 U/mL penicillin, 50 μg/mL streptomycin and 10% fetal calf serum (FCS; Integro, Zaandam, The Netherlands). Isolation of CD34+ Stem and Progenitor Cells Bone marrow samples were collected from healthy donors after written informed consent. Bone marrow-derived mononuclear cells (BM-MNCs) were isolated by Ficoll-hypaque (1.077 g/mL; GE Healthcare, Uppsala, Sweden) density gradient centrifugation. Peripheral blood-derived mononuclear cells (PB-MNCs) were obtained from aphaeresis material from stem cell donors who were treated with G-CSF (Neupogen®) 10 μg/kg/day subcutaneously for 5 to 6 days, after written informed consent. CD34+ HSPCs were isolated using anti-CD34 immunomagnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to manufacturer's instructions. CD34+ HSPCs were directly used for NK cell generation. Ex Vivo Expansion of CD34+HSPCs, and Differentiation Into NK Cells CD34+ HSPCs were plated into 24-well or 6-well tissue culture plates (Corning Incorporated, Corning, N.Y., USA). Cells were expanded during 9 or 10 days with a high-dose cytokine cocktail (Expansion cocktail I) consisting of 25 ng/mL IL-7 (Immunotools), 25 ng/mL SCF (Immunotools), 25 ng/mL TPO (Cellgenix) and 25 ng/mL Flt3L (Immunotools). From day 9 or 10 until day 14 or 15, TPO was replaced by 20 ng/mL IL-15 (Miltenyi). After day 14 or 15, cell differentiation was initiated by replacing Expansion cocktail I by a new high-dose cytokine cocktail (Differentiation cocktail) consisting of 20 ng/mL IL-7, 20 ng/mL SCF, 20 ng/mL IL-15 and 1000 U/mL IL-2 (Proleukin®; Chiron, München, Germany). Where mentioned, 2 μM StemRegenin1 (SR1; Cellagen Technology, San Diego, Calif., USA) was added to the culture medium (see RNA Isolation and Real-Time Quantitative RT-PCR (qRT-PCR) Total RNA from 0.5-2E+5 cells, collected weekly from HSPC-NK cell cultures, was isolated using the Quick-RNA™ MicroPrep Kit (Zymo Research, CA, USA). Next, cDNA was synthesized using M-MLV-reverse transcriptase (Invitrogen) in a standard reaction as described before, after which real-time PCR was performed using the following Taqman Gene expression assays (Applied Biosystems, Forster City, Calif., USA): AhRR (Hs01005075_m1); TOX (Hs01055573_m1); ID2 (Hs04187239_m1); EOMES (Hs00172872_m1); GATA3 (Hs00231122_m1); SH2D1B (Hs01592483_m1); IFNG (Hs00989291_m1); GZMB (Hs01554355_m1); PRF1 (Hs99999108_m1). For all genes, Ct values were normalized to GAPDH (Hs02758991_g1) by calculating ΔCt=Cttarget gene−CtGAPDH per sample. Finally, gene expression levels were quantified relative to GAPDH as follows: 2̂(−[ΔCt]). Flow Cytometry (FCM) Cell numbers and expression of cell-surface markers were determined by FCM. Anti-human CD45-ECD (J.33, Beckman Coulter, Woerden, The Netherlands) and anti-CD56-PC7 (HCD56, Biolegend, San Diego, Calif., USA) antibodies were used to follow cell number and NK cell differentiation during culture using the Coulter FC500 flow cytometer (Beckman Coulter). The population of viable CD45+ cells was determined by exclusion of 7-AAD (Sigma, St Louis, Mo., USA) positive cells. For phenotypical analysis, cells were incubated with antibodies in FCM buffer (PBS/0.5% bovine serum albumin (BSA; Sigma) for 30 minutes at 4° C. After washing, cells were resuspended in FCM buffer and analyzed. The following conjugated monoclonal antibodies were used for NK cell phenotyping: anti-NKG2A-PE (Z199; Beckman Coulter), anti-DNAM-1-FITC (DX11; BD Biosciences Pharmingen, Breda, The Netherlands), anti-CD16-FITC (3G8), anti-CD3-FITC (UCHT1), anti-NKG2D-PE (1D11), anti-NKp30-PE (P30-15), anti-NKp44-PE (P44-8), anti-NKp46-PD (9E5), anti-CD158b-PE (Dx27), anti-CD158e-PE (Dx9), anti-CD158a/h-PE (HP-MA4), anti-CD62L-PE (DREG56), anti-CD253-PE (RIK-2), anti-CXCR3-PE (G025H7), anti-CXCR4-PE (12G5), anti-IgG1-PE (MOPC-21), anti-IgG2a-PE (MOPC-173), anti-IgG2b-PE (MCP-11), anti-IgG1-FITC (MOPC-21; all from Biolegend). Fluorescence-Activated Cell Sorting (FACS) of HSCP-NK Cells After 5 weeks of culture, CD56+ cells were isolated from the total cultured cells. For this purpose cells were stained for 15 minutes at 4° C. using the appropriate concentration of anti-CD56-PEcy7 (CD56-PC7 (HCD56, Biolegend). Cells were washed and resuspended in FACS buffer at a concentration of 1.5E+6/mL, and subsequently sorted at the FACSAria Cell Sorter (BD Biosciences). FCM-Based Cytotoxicity Assays Cell lines were labeled with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes Invitrogen, Eugene, Oreg., USA) and primary AML blasts were labeled with 1.5 μM CFSE, both in a concentration of 1E+7/mL for 10 minutes at 37° C. The reaction was terminated by adding an equal volume of FCS. After washing, cells were resuspended in IMDM/10% FCS to a final concentration of 3E+5/mL. Target cells (3E+4) were co-cultured in triplicate with effector cells at different effector:target (E:T) ratios in a total volume of 200 μL IMDM/10% FCS in 96-wells round-bottom plates (Corning Incorporated). Effector cells and target cells alone were plated out in triplicate as controls. In experiments with primary AML blasts, AML blasts were derived from bone marrow samples from 5 patients at the time of diagnosis and were supplemented with IL-3 (50 ng/mL; Cellgenix), SCF (25 ng/mL), Flt3L (20 ng/mL), GM-CSF (100 ng/mL), G-CSF (100 ng/mL) and IL-15 (5 ng/mL). To measure degranulation of NK cells, anti-CD107a-PC7 (H4A3, Biolegend) was added to the co-culture. After overnight co-culture at 37° C., 50 μL supernatant was discarded and 50 μL Coulter® Isoton® II Dilutent containing 0.2 μL 7-AAD was added instead. Cells were harvested and the number of viable target cells was quantified by FCM by gating on forward scatter and side scatter and exclusion of 7-AAD positive cells. Target cell survival was calculated as follows: % survival=([absolute number of viable CFSE+ target cells co-cultured with NK cells]/[absolute number of viable CFSE+ target cells cultured in medium])×100%. The percentage specific lysis was calculated as follows: % lysis=(100−[% survival]), as described earlier by Jedema et al. [25, 26] Degranulation of NK cells during overnight co-culture was determined as the percentage of CD107a expressing cells measured by FCM. Enzyme-Linked Immunosorbent Assays The production capacity of IFN-γ and Granzyme B by NK cells were evaluated by ELISA according to manufacturer instructions (IFN-γ: Pierce Endogen, Rockfore, Ill., USA; Granzyme B; Mabtech, Sweden). To this end, NK cells (1E+5 ) were co-cultured in triplicate with target cells (1E+5 ) in a total volume of 200 μL IMDM/10% FCS in 96-wells round-bottom plates (Corning Incorporated). NK cells alone were plated out in triplicate as controls. After incubation overnight at 37° C., 150 μL supernatant was collected and stored at −20° C. until use. Proliferation Assays HSPC-NK cells were cultured according to our culture protocol for 35 days. After 35 days, NK cells were labeled with 1 μM CFSE as described previously. After staining, cells were resuspended in differentiation medium at a concentration of 2E+6/mL and plated in duplicate in a 96-well plate. CsA (Biovision Incorporated, Milpitas, Calif., USA) or MPA (Sigma-Aldrich, Zwijndrecht, The Netherlands) were added to final concentrations of 0.01 to 1 μg/mL (CsA) or 0.1 to 10 μg/mL (MPA). Half of the medium containing MPA or CsA in the final concentration was refreshed every 2-3 days. After 7 days, cells were harvested. The number of viable target cells was quantified by FCM by gating on forward scatter and side scatter and exclusion of 7-AAD positive cells. Proliferation was analyzed by determining the CFSE dilution within CD56+ cells. Statistical Analysis Results from different experiments are described as mean ±standard error of the mean (SEM). Statistical analysis was performed using a one-tailed paired student's t-test, a two-tailed unpaired student's t-test or one-way ANOVA if values had a normal distribution (normality was determined using the Kolmogorov-Smirnov test). For values without normal distribution we used the Wilcoxon matched-pairs signed rank test. Differences were considered to be significant for p values <0.05. Results SR1 Enhances Expansion of PB and BM-Derived CD34+ HSPCs and Improves NK Cell Differentiation In this study, we investigated whether NK cells could be generated in vitro from PB or BM-derived CD34+ HSPCs. We used the feeder-free culture protocol described in SR1 Influences Expression of Transcription Factors Important for NK Cell Differentiation and Maturation To gain insight into the molecular processes behind the SR1-enhanced differentiation of CD34+ HSPCs into NK cells, we analyzed the gene expression profile of several transcription factors that are described to be important for NK cell differentiation and maturation [27-31]. To analyze the culture composition, we concomitantly determined the percentage of CD56+ cells in our cultures at different time points ( To investigate whether the increased expression levels of these transcription factors reflected a difference between NK cells generated in the presence or absence of SR1, or resulted from the different composition of the cultures, CD56+ NK cells, generated in the presence or absence of SR1, were sorted by FACS from HSPC-NK cultures after 5 weeks. Subsequently, gene expression levels of the previously mentioned transcription factors were determined. We did not observe increased expression of the NK cell differentiation factors TOX, ID2 and EOMES suggesting that SR1 increases the number of NK cell progenitors resulting in more NK cells, but it does not intrinsically change HSPC-NK cells ( Collectively, these data demonstrate that addition of SR1 to our ex vivo culture system blocks function of AhR, resulting in the upregulation of several transcription factors that are required for NK cell differentiation and maturation. SR1-Generated HSPC-NK Cells Have an Activated and Mature Phenotype To further elucidate the effect of SR1 on NK cell activation status and function, we investigated the influence of SR1 on the phenotype of our ex vivo-generated CD56+ NK cells. After 35 days of culture in the presence of SR1, NK cell phenotype was analyzed using FCM ( SR1-Generated HSPC-NK Cells Have Efficient IFN-γ Production Capacity and Cytolytic Activity Against AML and MM Cells Next, we investigated the functional activity of SR1-generated NK cells, and compared them with NK cells generated in the absence of SR1. After 5 weeks of culture, NK cell products were harvested and co-cultured overnight with AML or MM tumor cell lines. Target cell induced IFN-γ production was determined. We found that our SR1-generated HSPC-NK cells have a good IFN-γ producing capacity, which is improved in cells generated in the presence of SR1 ( To further confirm the functional activity of SR1-generated NK cells, we investigated degranulation of these cells after overnight co-culture with AML or MM tumor cell lines. We found marked degranulation of our SR1-generated NK cells upon co-culture with the different cell lines ( Taken together, these data demonstrate that SR1-induced HSPC-NK cells, generated from G-CSF-mobilized CD34+ cells, mediate efficient IFN-γ production, degranulation and cytolytic activity against hematological tumor cells. Furthermore, as expected by increased expression of activating markers, SR1 augments IFN-γ production and cytotoxic activity of ex vivo-generated HSPC-NK cells. HSPC-NK Cell Proliferation, Viability and Function is Inhibited by Mycophenolic Acid (MPA) But Not by Cyclosporin A (CsA) Increased NK cell numbers after non-myeloablative allo-SCT have been associated with decreased relapse rates, without an increased risk for GVHD [8, 9]. Hence, adoptive transfer of ex vivo-generated NK cells shortly after allo-SCT is an attractive approach to improve patient outcome. Our therapeutic strategy is to apply HSPC-NK cell adoptive transfer for high-risk patients treated with non-myeloablative allo-SCT ( SR1 is a Key Ingredient Driving NK Cell Differentiation in a Cytokine-Based Culture Method In earlier studies, we exploited a phased based addition of cytokines in heparin-based basal media (5-7). Heparin is able to bind certain cytokines, reducing their degradation and perhaps presenting them in a more physiological manner. Furthermore, low dose cytokine cocktail containing G-CSF, GM-CSF and IL-6 was being used, which was based on studies using the fetal liver-derived stromal cell line AFT024. Since we observed that the AhR antagonist is a crucial component for the ex vivo expansion of NK cells from BM and mobilized blood CD34+ HSPCs, we investigated whether we could simplify the culture method by leaving out heparine and low dose cytokines G-CSF, GM-SCF and IL-6 that have mainly an effect on myeloid progenitor cells (see Discussion NK cells are the first lymphocyte population recovering after allo-SCT, and have several important functions shortly after allo-SCT. First of all, they are involved in defence against viral infections such as cytomegalovirus (CMV) infections [37, 38], which can cause high morbidity shortly after allo-SCT. Furthermore, high NK cell numbers shortly after non-myeloablative and T cell-depleted allo-SCT have been associated with reduced relapse rates without an increased risk of GVHD [8, 9], indicating that allogeneic donor NK cells have specific antitumor activity. Nevertheless, it was shown that early engrafting NK cells have decreased cytokine producing capacity. So, in order to further exploit the beneficial effects of NK cells after allo-SCT, adoptive transfer of functional and rapidly maturating NK cells would be an attractive immunotherapeutic strategy. Since donor-derived NK cells will not be rejected post-transplant, multiple NK cells infusions without the need for immunosuppressive chemotherapy to prevent rejection, will be feasible in this setting. However, high numbers of functional NK cells of donor origin are needed to apply this strategy. Since isolation of sufficient numbers of NK cells from donors, without contaminating alloreactive T cells, is challenging; we investigated if high numbers of functional NK cells could be generated from G-CSF-mobilized CD34+ HSPCs, using an ex vivo cytokine-based culture protocol. This protocol was developed by our group earlier, and high numbers of pure and functional NK cells with in vivo maturation capacity, can be generated from UCB-derived CD34+ HSPCs using this GMP-compliant protocol [5-7]. However, we found that NK cell expansion and differentiation from G-CSF-mobilized and BM-derived CD34+ cells in the absence of SR1 was very limited, as compared to NK cell expansion and differentiation of UCB-derived CD34+ HSPCs observed in our previously developed culture protocol. Interestingly, we found that addition of the AhR antagonist SR1 to our cultures greatly enhanced NK cell expansion and differentiation. As a result, we can expand G-CSF-mobilized CD34+ cells on average 268-fold using our newly developed SR1-based protocol. In addition, SR1-generated NK cell products are 83%±9% pure. The remaining non-NK cells in the cultures represented mainly CD14+ and/or CD15+ mature monocytic and myelocytic cells. Probably due to SR1 addition still some remaining low amount CD34+ cells could be detected. However, contaminating CD3+ T cells were either not detectable or at very low frequency (0.1%±0.1%). Furthermore, we found that SR1-NK cells have a similar phenotype as our NK cells previously generated from UCB-derived CD34+ HSPCs, which is a highly active phenotype, characterized by expression of high levels of activating NK cell receptors [5-7]. Interestingly, expression levels of CD62L, CXCR3, DNAM-1 and TRAIL were even higher on NK cells generated in the presence of SR1. CD62L and CXCR3 are involved in homing to lymphoid organs and trafficking towards inflammation in vivo, so high expression of these markers can contribute to trafficking of NK cells toward hematological tumor cells in the lymphoid organs [7, 34, 35]. In addition, it was described that CD62L indicates an unique subset of polyfunctional NK cells combining the ability to produce IFN-γ with cytotoxic properties. DNAM-1 and TRAIL are activation markers, and increased expression levels of these markers suggests that SR1-generated NK cells are more active as compared to NK cells generated in the absence of SR1. This was indeed confirmed in functional studies showing that SR1-induced NK cells have increased INF-γ production capacity and an increased capability to kill AML cell lines, as compared to NK cells generated in the absence of SR1. Furthermore, SR1-generated NK cells showed efficient degranulation against AML and MM cell lines. Besides, even at low E:T ratios, these cells efficiently kill hematological tumor cell lines, and most importantly, patient-derived primary AML blasts. To further investigate the applicability of our SR1-NK cell product after allo-SCT, we investigated the effect of the immunosuppressive drugs MPA en CsA, which are commonly used after non-myeloablative allo-SCT, on our SR1-NK cell product. Importantly, we found that therapeutic CsA concentrations do not affect HSPC-NK cell viability, proliferation or function, while exposure to therapeutic levels of MPA did have a negative effect. Exposure to CsA did even enhance IFN-γ production by the HSPC-NK cells. This is in accordance with the effect of CsA and MPA on naturally occurring NK cells described in literature [41, 42]. These results provide strong rational to infuse SR1-generated HSPC-NK cells relatively short after allo-SCT, since MPA treatment is generally stopped at day 28 after allo-SCT. CsA, which is usually prescribed for at least six months, will most likely not affect NK cell viability, proliferation or function in vivo upon transfer. Several methods to generate NK cell products from donor origin for immunotherapy have been reported [43-48]. In most studies, NK cells are isolated from aphaeresis products using magnetic cell sorting systems. However, poor recovery and viability is a common problem in these procedures, therefore NK cells require cytokine stimulation and expansion before infusion [2, 46-50]. NK cell numbers up to 7.6E+8 have been reported using this method. Nevertheless, large-scale aphaeresis procedures are necessary to obtain this amount of NK cells, and contaminating T cells can potentially cause GVHD. To circumvent these problems, NK cell generation from CD34+ HSPCs is an attractive option. Yoon et al. recently reported safe infusion of NK cells generated using a feeder-free 6-week culture procedure after HLA-mismatched allo-SCT. Using this procedure, an average number of 9.28E+6 NK cells/kg was generated from 2.22E+6 donor CD34+ HSPCs/kg. In our system, the average NK cell expansion from mobilized CD34+ HSPCs was 235 fold (range 115-904 fold). Therefore, if we use 10% of the G-CSF-mobilized CD34+ HSPCs isolated from donors for allo-SCT, we will have on average 0.5E+6 CD34+ cells/kg. In case of a 70 kg donor and patient, we will get on average 8E+9 NK cells (>1E+8 NK cells/kg). So we will be able to infuse large numbers of NK cells from donor origin using our SR-1 based NK cell generation protocol. In the present study, we showed that the AhR antagonist SR1 greatly enhances NK cell expansion and differentiation. AhR is a ligand-inducible transcription factor which has extensively been studied in the context of its activation by environmental pollutants, such as dioxins and polycyclic aromatic hydrocarbons [51, 52]. Until recently, little was known about the physiological role of AhR, but a growing body of evidence shows that AhR has multiple endogenous activators, and is involved in several physiological processes [22, 53, 54]. Examples of endogenous ligands are metabolites of dietary substances [15, 55] and tryptophan metabolites like cinnabarinic acid, 6-formylindolo[3,2-b]carbazole (FICZ), which can be produces in the skin upon light exposure, and the indoleamine 2,3-dioxygenase 1 (IDO1)-metabolite kynurenin. AhR also regulates differentiation of various immune cells like T helper 17 (TH17) cells, dendritic cells [17, 18], regulatory T cells [19, 20] and γδ T cells. Recently, Hughes et al. reported that antagonism of AhR promotes differentiation of immature innate lymphoid cells into NK cells expressing EOMES and TBET. In our SR1-based HSPC-derived NK cell culture, we also observed up-regulation of EOMES, which is involved in NK cell maturation, and additionally we found upregulated expression of TOX, ID2, GATA-3 and EAT-2 after AhR inhibition. TOX is important for early NK cell development, ID2 is involved in NK cell maturation and GATA-3 and EAT-2 are important for NK cell effector functions [30, 31]. Interestingly, expression of TOX markedly decreased in the first week of culture in our culture system in the absence of SR1. In the presence of SR1, TOX expression is more preserved, resulting is significantly higher TOX expression after one week. This suggests that before induction of differentiation by addition of IL-15, early NK cell progenitors are expanded in the presence of SR1, explaining increased NK cell numbers generated in the presence of SR1, IL-15 and IL-2/IL-12. To our knowledge, this is the first report of an applying AhR blocking in an in vitro system to generate high numbers of functional NK cells for therapeutic application. In conclusion, we developed an SR1 and cytokine-based ex vivo culture system, which enables us to generate high numbers of very potent NK cells from G-CSF-mobilized CD34+ HSPCs. Addition of SR1 to the culture system induced up-regulation of multiple NK cell-related transcription factors, and resulted in generation of NK cell products with very high cell numbers and purity. These NK cells have an active phenotype and are highly functional in vitro, therefore they hold great promise for future adoptive HSPC-NK cell therapy after allo-SCT as well as for adoptive cell therapy in the non-transplant setting against hematological and solid malignancies. Abstract Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against haematological and solid cancers. Here, we report a highly efficient SR1-based culture method for the generation of CD56+ NKG2A+ Perforin+EOMES+LFA-1+ NK cell products with high cell number and purity. In this system, CD34+ HSPCs from umbilical cord blood are expanded and differentiated into NK cells under stroma-free conditions in the presence of SR1, IL15 and IL12. We demonstrate that this inventive method drives the generation of more pure, more mature and highly functional NK cells. Particularly, these SR1-expanded HSPC-NK cells mediate significant IFNγ production and cytotoxic activity towards tumor targets. Furthermore, HSPC-NK cells actively infiltrate, migrate and mediate killing of ovarian cancer spheroids using an in vivo-like model system and mouse xenograft model. These findings demonstrate that SR1/IL15/IL12-expanded HSPC-NK cells efficiently kill hematological tumor cells, destruct tumor spheroids in vitro and target intraperitoneal tumors in vivo, providing great promise for effective adoptive immunotherapy in cancer patients. Introduction Natural killer (NK) cells have gained significant attention for adoptive cell therapy (ACT) of cancer due to their well-documented antitumor function. Their natural ability to lyse tumor cells without prior sensitization, their pivotal role in antitumor responses and immunesurveillance against cancer, along with the observations that NK cells do not cause graft-versus-host disease (GVHD) while maintaining graft-versus-tumor (GVT) immunity after allogeneic stem cell transplantation (allo-SCT) have all been central to the overall promising application of NK cells for adoptive immunotherapy of cancer. However, effective ACT requires NK cells to be appropriately activated, available in sufficient numbers, have appreciable persistence in vivo, home to the tumor site and effectively kill tumor cells upon encounter. Generally, allogeneic NK cell products have been enriched from the peripheral blood (PB) of haplo-identical donors followed by overnight activation with IL-2 or IL-15. However, this cellular product is rather heterogeneous with 25-95% of infused cells being NK cells depending on the used enrichment method, and contains a variable number of monocytes, B cells and potentially alloreactive T cells capable of inducing GVHD [2, 3, 50, 60-62]. Furthermore, this approach yields relatively low NK cell numbers enough for a single dose, but higher and multiple dosing requires further expansion before adoptive transfer [63-65]. For these reasons, development of more homogeneous, scalable and “off-the-shelf” allogeneic NK cell products is preferable for adoptive immunotherapy. Alternatively, NK cells can be efficiently generated ex vivo from hematopoietic stem and progenitor cells (HSPC) or induced pluripotent stem cells (iPSC) [5-7, 45, 66, 67]. Previously, we have reported good manufacturing practice (GMP)-compliant, cytokine-based culture protocols for the ex vivo generation of highly active NK cells from CD34+ HSPCs isolated from various stem cell sources [5, 7, 66]. By applying the aryl hydrocarbon (AhR) antagonist StemRegenin1 (SR1 ), and the combination of IL-15 and IL-12 we have shown that highly active CD56+ NK cells can be generated with potent cytolytic activity towards hematological tumor cells in vitro as well as anti-leukemic effects in vivo following IV administration. In the present study, we investigated the preclinical efficacy of ex vivo generated, highly functional CD56+Perforin+ HSPC-NK cells generated by an optimized SR1/IL15/IL12 based culture protocol in clinically relevant tumor models. Materials and Methods Cell Lines Tumor cell lines SKRC52, SKRC17, SKOV-3 and IGROV1 were cultured in Roswell Park Memorial Institute medium (RPMI 1640; Invitrogen, Carlsbad, Calif.) medium with 10% Fetal Calf Serum (FCS; Integro, Zaandam, The Netherlands). The OVCAR-3 cell line was cultured in RPMI 1640 medium with 20% FCS and 1 μg/ml insulin. K562 cells were cultured in Iscove's Dulbecco's medium (IMDM; Invitrogen, Carlsbad, Calif.) containing 10% FCS. SKOV-3-GFP-luc cells were generated by stable transduction of parental cells with lentiviral particles LVP20 encoding the reporter genes green fluorescent protein (GFP) and luciferase (luc) under control of the CMV promoter (GenTarget, San Diego). Transduced cells were cloned and an optimal SKOV-3-GFP-luc clone for in vitro and in vivo experiments was selected based on GFP expression, luciferase activity, and comparable susceptibility to HSPC-NK killing as the parental cells. Patient-Derived Ovarian Cancer (OC) Cells Patient material from stage III and IV OC patients was obtained before primary treatment in the Radboud University Medical Center (RUMC) after written informed consent. Fresh ascites was filtered using a 100 μm filter, centrifuged and resuspended in PBS/0.5% BSA. Subsequently, mononuclear cells were isolated using a Ficoll-Hypaque (1.077 g/ml; GE Healthcare, Uppsala, Sweden) density gradient centrifugation. Isolated cells were washed and cultured in RPMI 1640 medium with 10% FCS. After 24 hours, non-adherent cells were removed by washing with PBS. After 3 days, medium was refreshed. At day 6-8, cells were trypsinized and analyzed for percentage tumor cells by flow cytometry (FCM). After reaching 90% confluency, OC cell layers were used for either tumor spheroid culture or directly for co-culture experiments. Multicellular Tumor Spheroids OC tumor spheroids were generated by seeding 3×104 cells/well in a volume of 200 μl/well of culture medium in 96-well plates coated with 1% agarose in DMEM F12 medium as described previously by Giannattasio et al. with minor adaptations. 3D tumor spheroids were used for functional assays upon reaching a solid state after 72 h after initial seeding. HSPC-NK Cell Generation Umbilical cord blood (UCB) units were collected at Caesarean sections after full term pregnancy and obtained informed consent of the mother. CD34+ HSPCs were isolated from mononuclear cells after Ficoll-Hypaque density gradient centrifugation and CD34-positive immunomagnetic bead selection (Miltenyl Biotec, Bergisch Gladbach, Germany). After isolation, CD34+ HSPCs were either cryopreserved or directly used for NK cell generation. Cultures were performed over 5-6 weeks in 6-well tissue culture plates (Corning Inc., NY) or Vuelife™ AC-bags (Cellgenix, Freiburg, Germany), using CellGro DC medium (Cellgenix) or NK MACS medium (Miltenyi) supplemented with 10% and 2% human serum (Sanquin Bloodbank, Nijmegen, The Netherlands) during the expansion and the differentiation phase, respectively. CD34+ cells were first cultured for 9 or 10 days with 2 μM SR1 (Cellagen Technology, San Diego, Calif.), 25 ng/mL IL-7 (Immunotools), 25 ng/mL SCF (Immunotools), 25 ng/mL Flt3L (Immunotools) and 25 ng/mL TPO (CellGenix). From day 9 or 10 until day 14 or 15, TPO was replaced by 50 ng/mL IL-15 (Miltenyi). After day 14 or 15, cells were cultured in differentiation medium consisting of 20 ng/mL IL-7, 20 ng/mL SCF, 50 ng/mL IL-15, and 0.2 ng/ml IL-12 (Miltenyi). SR1 was added till day 21. Total cell number and CD56 acquisition were analyzed twice a week by flow cytometry, and medium was refreshed every 2 to 4 days to keep cell density between 1.5E+6 and 2.5E+6 cells/ml. Cultures were maintained at 37° C., in 95% humidity, and 5% CO2. HSPC-NK cell products were used in experiments after 5 to 6 weeks of culture with >80% CD56+ cells. Flow Cytometry (FCM) Cell numbers and expression of cell-surface markers were determined by FCM. Anti-human CD45-ECD (J.33; Beckman Coulter, Woerden, The Netherlands) and anti-CD56-PC7 (HCD56; BioLegend, San Diego, Calif.) antibodies were used to follow cell number and NK cell differentiation during culture using the Coulter FC500 flow cytometer (Beckman Coulter). The population of viable cells was determined by exclusion of 7-AAD-positive cells (Sigma, St. Louis, Mo.). For phenotypical analysis, cells were incubated with panels of antibodies in the FCM buffer (PBS/0.5% BSA) for 30 min at 4° C. After washing, cells were resuspended in the FCM buffer and analyzed. FCM-Based Degranulation and IFNγProduction Assay HSPC-NK cell degranulation and IFNγ production were determined upon stimulation with K562 cells at an E:T ratio of 1:1. Anti-CD107a (H4A3; BD Biosciences) was added at a 1:100 dilution at the start of the co-culture, and brefeldin A (1 ng/mL, BD Biosciences) was added 1 h later. Cells were collected the following day, stained for cell surface markers, and then treated with fixation/permeabilization buffer (eBioscience) and stained for intracellular Perforin and IFNγ. Flow cytometric analysis was performed with exclusion of dead cells with Fixable Viability Dye (eBioSciences) and using non-stimulated cells as a control. FCM-Based Cytotoxicity and Infiltration Assays In monolayer cytotoxicity assays, tumor cells were first seeded at a concentration of 3E+4 in flat bottom 96-well plates in triplicate for each test condition. The following day, HSPC-NK cells were labeled with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes; Invitrogen, Eugene, Oreg.) and were added at 3 different effector-target (E:T) ratio's (1:1, 3:1 and 10:1). For cytotoxicity assays with SKOV-3-GFPluc cells, HSPC-NK cells were added without CFSE staining. After 24 hours, co-culture supernatants were harvested and stored at −20° C. until use for ELISA. Subsequently, cells were detached using trypsine (for solid tumor cell lines), resuspended and collected in micronic tubes. The life/dead marker 7AAD (1/1000 final dilution) was added to the cells and absolute numbers of viable targets present in each well were determined by FCM (FC500, Beckman Coulter). Specific lysis of tumor cells by NK cells was calculated with the following formula: 100%−[(absolute number of viable target cells after co-culture with NK cells/absolute number of viable target cells cultured in medium)×100%]. In cytotoxicity assays with OC spheroids, day three multicellular spheres were used. HSPC-NK cells were added to the tumor spheroids at 3 different E:T ratio's. After 24 hours of co-culture, supernatant was collected for ELISA. Subsequently, spheroids were washed, trypsinized and viable 7AAD-negative tumor cells were counted using the FC500 flow cytometer. The production capacity of Granzyme-B and Interferon-γ by HSPC-NK cells were evaluated by ELISA according to the manufacturer's instructions (IFN-γ; Pierce Endogen, Rockford, USA and Granzyme-B; Mabtech, Sweden). For the FCM-based infiltration assay, we performed the OC spheroid-NK cell co-culture assay as described above. After different incubation time points, tumor spheroids were harvested with a cut 1000 μl-tip, pooled and separated from the supernatant containing the surrounding NK cells and residual target cells by mild centrifugation. Following two washing steps in PBS/2% FCS, tumor spheroids were dissociated using trypsin. The resulting single cell suspension was washed again and stained with CD56 and NKG2A antibodies, and the life/dead marker 7AAD. The percentage of viable CD56+NKG2A+ NK cells was determined by FCM (FC500 flow cytometer). Confocal Microscopic Imaging of HSPC-NK Cell Invasion in Tumor Spheroids Co-culture of OC spheroids and HSPC-NK cells was performed as described above. For confocal microscopic experiments, SKOV-3-GFPluc spheroids were co-cultured with CD56APC-labeled NK cells. At 4, 18 and 24 hours of co-culture, spheres were collected, washed mildly with PBS, and placed in an ibidi 1 μ-slide 8-wells plate (ibidi GmbH) in RPMI without phenol red. Imaging was performed with the Leica TCS SP5 microscope. Images were processed with Fiji imageJ software. HSPC-NK Cell Adoptive Transfer, Intraperitoneal SKOV-3 Model, and Bioluminescence Imaging NOD/SCID/IL2Rgnull (NSG) mice originally purchased from Jackson laboratories were housed and bred in the RUMC Central Animal Laboratory. All animal experiments were approved by the Animal Experimental Committee of the RUMC and conducted in accordance with institutional and national guidelines under the university permit number 10300. A preclinical OC xenograft model was established using SKOV-3 cells previously engineered to express the GFP and luciferase reporter genes (SKOV-3-GFPLuc). Efficient OC engraftment in 6-12 weeks old NSG mice was achieved using 2E+5 cells SKOV-3-GFPLuc cells injected intraperitoneally (IP). Tumor load was monitored by bioluminescence imaging (BLI) as previously described (7). In brief, mice were injected i.p. with D-luciferin (150 mg/kg) and after 10 min BLI images were collected using the IVIS imager with the LivingImage processing software. Regions of interest (ROIs) were drawn around the abdominal area of the mice, and measurements were automatically generated as integrated flux of photons (photons/s). In HSPC-NK cell adoptive transfer studies, SKOV-3 injected mice were equally divided into two groups (no treatment versus IP NK treatment groups) after the first BLI at day 3. After 4 and 11 days, mice received two IP NK cell infusions (12×106 cells/mouse/infusion). Recombinant human IL-15 (Miltenyi Biotech) was administrated subcutaneously at a dose of 1 μg (5,000 units) per mouse every 2-3 d, starting the day of HSPC-NK cell infusion till day 21. BLI images were collected weekly till day 63. Statistical Analysis Data analysis was conducted by Prism software (GraphPad, version 5.03 for Windows). The survival probability was estimated by Kaplan-Meier methods. Two-way ANOVA, or Student t-test was used to calculate statistically significant differences between groups. A P-value of <0.05 was considered statistically significant. Results SR1-Generated HSPC-NK Cell Products From UCB Units Recently, we reported that inhibition of AhR using SR1 improved NK cell generation from CD34+ HSPCs, by enhancing the expression of several transcription factors involved in early NK cell development. In addition, we reported that combining IL-15 with IL-12 drives the differentiation of more mature and highly functional HPC-NK cells, which display potent alloreactivity towards haematological cancer cells. In the present study, we combined SR1, IL-15 and IL-12 to generate HPC-NK cells and test their alloreactivity potential against solid tumor cells. As illustrated in Upscaling of SR1-Expanded HSPC-NK Cell Products Next, we translated our SR1/IL15/IL12-based method into a GMP-compliant, closed system bioprocess in Vuelife™ AC culture bags, and compared this with research scale culturing in 6-well plates. For this, we expanded CD34+ UCB cells for 5 weeks in the presence of SR1 using our inventive ex vivo culture protocol depicted in SR1-Expanded HSPC-NK Cells Efficiently Kill Malignant Cells To explore the cytotoxic potential of SR1-expanded HSPC-NK cells against tumor cells, we first performed flow cytoemtry-based cytotoxicity assays with panels of tumor cell lines including frequently used renal cell carcinoma (RCC) and ovarian carcinoma (OC) cell lines (SKRC17, SKRC52, SKOV-3, IGROV1, OVCAR-3) as well as primary OC cells cultured from patient's ascites. The NK-sensitive K562 cells were included as control. Functional analysis demonstrated that SR1-expanded HSPC-NK cells mediate potent cytolytic activity against all solid tumor cell lines and patient-derived OC mono-layers tested ( SR1-Expanded HSPC-NK Cells Effectively Infiltrate and Destroy Tumor Spheroids Next, we tested the potency of SR1-expanded HSPC-NK cells to infiltrate and kill malignant cells in clinically relevant three-dimensional tumor spheroids. Microscopic evaluation and flow cytometry-based tumor cell quantification demonstrated that HSPC-NK cells target and destroy SKOV-3 spheroids as well as patient-derived OC spheroids after 24 hour co-culture ( SR1-Expanded HSPC-NK Cells Efficiently Target SKOV-3 OC Tumors In Vivo To study the in vivo anti-tumor potential of SR1-expanded HSPC-NK cells, adoptive transfer studies were designed in adult NSG mice injected IP with SKOV-3 cells, followed by two HSPC-NK cell infusions, supportive rhIL-15 injections, and weekly bioluminescence measurements ( Conclusion and Discussion In the past decade, adoptive transfer of allogeneic NK cells gained much attention as a promising adjuvant treatment approach against cancer. Thereby, a great number of reports described new methodologies to achieve higher NK cell numbers and activation. Particularly, expansion and differentiation of HSPCs allows generation of clinically applicable NK cell products with high purity and functionality. Nevertheless, we and others have observed that NK cells generated under stroma-free culture conditions using heparin, IL-15 and IL-2 have low surface expression of CD16 and only a subset (5-10%) expresses KIRs (5-7, 69), raising the question about NK cell potency and antitumor reactivity following adoptive transfer. Recently, we demonstrated that more mature and highly functional HSPC-NK cells can be generated under stroma-free conditions by replacement of IL-2 for IL-12 (68). Interestingly, we showed that IL-12 particularly favours the generation of CD16 and KIR-expressing NK cells, resulting in the rapid development of hyper-responsive CD16+KIR+NK cells following adoptive transfer. In addition, we also reported recently that inhibition of AhR using SR1 improved NK cell generation from CD34+ HSPCs, by enhancing the expression of several transcription factors involved in early NK cell development. In the present study, we combined SR1, IL-15 and IL-12 to generate HSPC-NK cells and tested their cytotoxic potential against solid tumor cells. We demonstrate that our inventive SR1/IL15/IL12-based method results in consistent, clinically applicable ex vivo production of highly active HSPC-NK cells. In addition, we demonstrate that the SR1, IL15 and IL12 combination not only improves NK production yields and purity, but also functionality is superior as SR1/IL15/IL2-expanded HSPC-NK cells mediate higher IFNγ production and cytotoxic activity towards tumor targets. Furthermore, these next generation HSPC-NK cells actively infiltrate into and destruct tumor spheroids in vitro and in vivo, providing great promise for effective adoptive immunotherapy in cancer patients. Previous methods to generate NK cell products from CD34+ HSPCs have been reported (5, 6, 69, 70). Spanholtz et al. reported a heparin-based culture method for CD34+ UCB cells in a medium comprising heparin, SCF, Flt3L, IL-7, TPO, IL-15, IL-2 and a cocktail of low-dose cytokines (5, 6, 69). Using this method 3-log expansion of HSPC-NK cells with a purity of >80% of CD56+ cells can be obtained in a bioreactor (6). However, next to relative low CD16 and KIR positivity (6), CD107a-based degranulation assays using K562 as target cells showed a mean frequency of CD107a+ degranulating NK cells of 28%±5% and IFNγ secreting NK cells of 2.5%±1% (n=10), for these NK cell products generated with this heparin/IL15/IL2 based protocol. In contrast, NK cells generated with our new inventive SR1/IL15/IL12-based protocol contained not only higher purity (i.e. >90%) of total CD56+ cells and >80% conventional CD56+Perforin+ NK cells, but also more CD56+Perforin+ NK cells (>10%) capable of mediating an IFNγ response towards K562 target cells. Previously, we showed that the AhR antagonist SR1 greatly enhances NK cell expansion and differentiation (66). AhR is a ligand-inducible transcription factor which has extensively been studied in the context of its activation by environmental pollutants, such as dioxins and polycyclic aromatic hydrocarbons [51, 52]. Until recently, little was known about the physiological role of AhR, but a growing body of evidence shows that AhR has multiple endogenous activators, and is involved in several physiological processes including NK cell development [22, 53, 54]. Recently, Hughes et al. reported that antagonism of AhR promotes differentiation of immature innate lymphoid cells into NK cells expressing EOMES and TBET. In our SR1-based HSPC-derived NK cell culture, we also observed up-regulation of EOMES, which is involved in NK cell maturation, and additionally we found upregulated expression of TOX, ID2, GATA-3 and EAT-2 after AhR inhibition. TOX is important for early NK cell development, ID2 is involved in NK cell maturation and GATA-3 and EAT-2 are important for NK cell effector functions [30, 31]. Interestingly, expression of TOX markedly decreased in the first week of culture in our culture system in the absence of SR1 (66). In the presence of SR1, TOX expression is more preserved, resulting is significantly higher TOX expression after one week. This suggests that before induction of differentiation by addition of IL-15 and IL-12, early NK cell progenitors are expanded in the presence of SR1, explaining increased numbers of conventional CD56+Perforin+NKG2A+LFA-1+ NK cells, and less CD56+ ILC3 cells, generated in the presence of SR1, IL-15 and IL-12. In this regard, Ambrosini et al. very recently reported a culture protocol for CD34+ UCB cells in a medium comprising SCF, Flt3L, IL-7, IL-15, IL-21 in the presence or absence of IL-1β (70). They showed that the addition of IL-1β at the beginning of the culture, i.e. at early stages of differentiation, also inhibits the proliferation of LFA-1-CD161+CD56+ ILC3 cells, while favoring the generation of mature NK cells expressing LFA-1 (CD11a), NKG2A/CD94, KIR, NKp46 and perforin. Although they showed that after 25 days of culture with IL-1β more CD56+ NK cells express CD161, LFA1, NKG2A, NKp46, EOMES and perforin (i.e. conventional NK cells or ILC1 cells) the overall percentage is only <20% in their culture protocol. Furthermore, Ambrosini et al. does not report total CD56+Perforin+ NK cell yields achievable with their IL-1β-based protocol (70). In sharp contrast, our inventive SR1/IL15/IL12 based protocol yields purities of mature CD56+NKG2A+NKp46+Perforin+EOMES+LFA1+ NK cells of >80%. Furthermore, we obtain with our SR1/IL15/IL12-based protocol more than 3-log NK cell expansion resulting in producing at least 1E+9 conventional CD56+Perforin+ NK cells with a high maturation and activation status from one single UCB unit containing >2E+6 CD34+ cells. In conclusion, we demonstrate that our inventive and scalable SR1/IL15/IL12-based method consistently drives the generation of more pure, more mature and highly functional HSPC-NK cell products with enhanced cytolytic activity and IFNγ production towards tumor cells. Furthermore, we demonstrated that these SR1-expanded HSPC-NK cells actively infiltrate, migrate and mediate killing of solid tumor spheroids in an in vivo-like 3D model system and mouse xenograft model. Collectively, our findings demonstrate that SR1/IL15/IL12-expanded HSPC-NK cells efficiently kill hematological tumor cells, destruct tumor spheroids in vitro and target intraperitoneal tumors in vivo, providing great promise for effective adoptive immunotherapy in cancer patients. The present invention relates to the field of medicine, specifically the field of treatment of cancer. More specifically, the invention relates to a method for the ex vivo production of a population of highly functional NK cells from CD34-positive cells, to a population of highly functional NK cells obtained and to the use of such population of highly functional NK cells for adoptive cell therapy. 1.-15. (canceled) 16. A method for the ex vivo production of a population of highly functional NK cells from CD34-positive cells comprising culturing CD34-positive cells in an expansion medium and subsequently culturing the expanded CD34-positive cells in a differentiation medium, wherein the expansion medium comprises an aryl hydrocarbon receptor antagonist. 17. The method of 18. The method of 19. The method of culturing in an expansion medium comprising IL-7, SCF, TPO, Flt3L and an aryl hydrocarbon receptor antagonist, further culturing in a first differentiation medium comprising Il-7, SCF, Flt3L, IL-15 and an aryl hydrocarbon receptor antagonist, culturing in a second differentiation medium comprising IL-7, SCF and IL-15. 20. The method of 21. The method of 22. The method of 23. The method of 24. The method of 25. The method of 26. The method of 27. A population of highly functional NK cells obtainable by the method of 28. The population of highly functional NK cells of 29. The population of highly functional NK cells of 30. The population of highly functional NK cells of 31. The population of highly functional NK cells of 32. The population of highly functional NK cells of 33. The population of highly functional NK cells of 34. A method of treatment of cancer in a subject in need thereof comprising administering to the subject the population of highly functional NK cells obtainable by the method of 35. The method of treatment of FIELD OF THE INVENTION
BACKGROUND OF THE INVENTION
SUMMARY OF THE INVENTION
DETAILED DESCRIPTION OF THE INVENTION
FIGURE LEGENDS
EXAMPLES
Example 1
The Aryl Hydrocarbon Receptor Antagonist StemRegenin 1 (SR1) Improves In Vitro Generation of Highly Functional NK Cells From CD34+ Hematopoietic Stem and Progenitor Cells
Example 2
The Aryl Hydrocarbon Receptor Antagonist StemRegenin1 Combined With IL-15 and IL-12 Drives the Generation of CD34+ Umbilical Cord Blood HSPC-Derived NK Cell Products With Superior Purity and Functional Characteristics
REFERENCE LIST
