Methods for generating populations of immune cells from pluripotent stem cells

Abstract

The present invention relates to methods of producing a population of immune cells, to a population of immune cells obtainable or obtained by the methods of the invention, to non-immunogenic T cells obtainable or obtained by the methods of the invention, to pharmaceutical compositions comprising the immune cells or non-immunogenic T cells of the invention, and also to the population of immune cells, the non-immunogenic T cells, or the pharmaceutical composition for use in methods of treatment or for use in cell therapy.

Description

[Technical field]

[0001] TECHNICAL FIELD OF THEINVENTION The present invention relates to methods of producing populations of immune cells, populations of immune cells obtainable or obtained by the methods of the invention, non-immunogenic T cells obtainable or obtained by the methods of the invention, pharmaceutical compositions comprising the immune cells or non-immunogenic T cells of the invention, and also to the populations of immune cells, non-immunogenic T cells, or pharmaceutical compositions for use in methods of treatment or for use in cell therapy. [Background technology]

[0002] 2. Background of the Invention The development of immune cell populations, specifically T cell populations, for use in diagnosis and therapy is a current challenge. Stem cells are an excellent source to enable the provision of such immune cell populations, for example differentiated T cell populations. An important characteristic of stem cells is their ability to self-renew indefinitely and to differentiate into multiple different types of cells or tissues. The self-renewal ability of stem cells is important for their characteristics as a pool of primitive undifferentiated cells. Furthermore, the high "plasticity" and "plasticity" characteristics of stem cells are based on their ability to trans-differentiate into tissues that may be different from their origin. Pluripotent stem cells (PSCs) have attracted attention as a source for the generation of T cells. In particular, induced pluripotent stem cells (iPS), which represent non-embryonic cells reprogrammed into pluripotent stem cells, are a useful source. Currently, the most utilized pluripotent cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Human ESC lines were first established by Thomson et al. (Thomson et al. (1998), Science 282:1145-1147 (Non-Patent Document 1)). Human ESC research has recently enabled the development of new technologies to reprogram somatic cells into ES-like cells. This technology was pioneered by Yamanaka et al. in 2006 (Takahashi & Yamanaka (2006), Cell, 126:663-676 (Non-Patent Document 2)). The resulting induced pluripotent cells (iPS cells) behave very similarly to ESCs and, importantly, can also differentiate into any cell type of the body. Furthermore, parthenogenetic stem cells have also been reported to be suitable for EHM generation in mouse models (Didie et al. (2013), J Clin Invest., 123:1285-1298 (Non-Patent Document 3)). The generation of low immunogenic T cells from genetically modified allogeneic human induced pluripotent stem cells is described, for example, in Wang et al., Nature Biomedical Engineering, Vol. 5, May 2021, 429-440 (Non-Patent Document 4). Individually modified T cells are a powerful tool for use in targeted treatment of diseases that require a directed immune response, such as cancer.It has been shown that T cells can be cultured and expanded in vitro for use in adoptive cellular immunotherapy or cancer treatment, and such T cells have been demonstrated to possess anti-tumor activity in tumor-bearing patients.

[0003] Methods for the generation of T cells derived from PSCs are known in the art (see Wang et al., 2021, supra). In addition, Montel-Hagen et al. (Montel-Hagen et al., 2019, Cell Stem Cell 24, 376-389, March 7, 2019 (Non-Patent Document 5)) disclose a method for the generation of T cells from pluripotent stem cells. According to this publication, immune cell differentiation is achieved by culturing cell aggregates in an air-liquid interface situation. Thus, the method described in Montel-Hagen involves complex and time-consuming cell culture techniques for the generation of T cells from PSCs. In addition, Iriguchi et al. (Iriguchi et al., 2021, Nature Communications, 12:430 Jan 18; 12(1):430. doi:10.1038 / s41467-020-20658-3 (Non-Patent Document 6)) disclose a method for the generation of T cells from iPSCs. Iriguchi et al. describe a method to predict T cell differentiation in DLL4-coated plates by adding additional factors. This requires a certain amount of cell culture volume, and therefore it is difficult to generate a larger number of differentiated T cells. These methods have the disadvantage that they are very laborious and are not suitable for generating immune cells in a cost-effective manner. Moreover, these prior art methods only allow the generation of T cells.

[0004] Therefore, there is a need to provide a method that allows the generation of populations of immune cells, such as T-cell-like cells, and ideally also NK-cell-like cells, which should also be suitable for implementation in large-scale approaches, such as bioreactors, to efficiently achieve larger yields of the desired immune cells.

[0005] Therefore, it is an object of the present invention to provide a method for producing a population of immune cells that meets this need. This object is solved by the subject matter of the independent claims, in particular the method for producing a population of immune cells, the population of immune cells obtainable or obtained by the method, the T cells obtainable or obtained by the method, pharmaceutical compositions, and the population of immune cells for use in methods of treatment. The provision of the population of immune cells described herein has the additional advantage that it is possible to selectively produce distinct subpopulations of the population of immune cells, in particular T cell-like cells and / or NK cell-like cells, depending on the desired application. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Thomson et al. (1998), Science 282:1145-1147 [Non-Patent Document 2] Takahashi & Yamanaka(2006),Cell,126:663-676 [Non-Patent Document 3] Didie et al. (2013), J Clin Invest., 123:1285-1298 [Non-Patent Document 4] Wang et al.,Nature Biomedical Engineering,Vol.5,May 2021,429-440 [Non-Patent Document 5] Montel-Hagen et al.,2019,Cell Stem Cell 24,376-389,March 7,2019 [Non-Patent Document 6] Iriguchi et al.,2021,Nature Communications,12:430 Jan 18;12(1):430.doi:10.1038 / s41467-020-20658-3 Summary of the Invention

[0007] A first aspect of the present invention relates to a method of generating a population of immune cells from pluripotent stem cells (PSCs), comprising: (i) (a) culturing PSCs that have undergone mesenchymal differentiation under suitable conditions in the presence of Notch ligand delta-like ligand 4 (DLL4) signaling activity in serum-free medium on a solid support suitable for the formation of 3D cell aggregates, thereby allowing the formation of 3D cell aggregates; (b) collecting the 3D cell aggregates and culturing them under suitable conditions in suspension culture in serum-free medium for about 3-6 days; and (c) inducing 3D cell aggregate formation and hematopoietic differentiation by culturing the 3D cell aggregates under suitable conditions in serum-free medium for a further period of about 4-7 days; (ii) inducing immune cell differentiation by culturing the 3D cell aggregates of (i) under suitable conditions in suspension culture in serum-free medium for a suitable time, thereby providing a population of immune cells.

[0008] A second aspect of the invention relates to a population of immune cells obtainable by the method of the invention.

[0009] A third aspect of the invention relates to a population of immune cells obtained by the method of the invention.

[0010] A fourth aspect of the invention relates to non-immunogenic T cells obtainable by the methods of the invention.

[0011] A fifth aspect of the invention relates to a non-immunogenic T cell obtained by the method of the invention.

[0012] A sixth aspect of the invention relates to a non-immunogenic T cell obtainable by the method of the invention, which lacks endogenous MHC class I molecules displayed on the cell surface of the T cell and which comprises an immunomodulatory protein on its surface.

[0013] A seventh aspect of the invention relates to a pharmaceutical composition comprising a population of immune cells of the invention or a non-immunogenic T cell of the invention.

[0014] An eighth aspect of the invention relates to a population of immune cells of the invention, a non-immunogenic T cell of the invention, or a pharmaceutical composition of the invention for use in a method of treatment.

[0015] Finally, a ninth aspect of the invention relates to a population of immune cells of the invention, a non-immunogenic T cell of the invention, or a pharmaceutical composition of the invention for use in cell therapy. [Brief description of the drawings]

[0016] [Figure 1] 1 shows a schematic timeline of an exemplary embodiment of the method of the present invention. [Figure 2A] Shown are microscopic images of cells seeded in AggreWell™ 800 6-well plates at day 0 (FIG. 2A) and day 3 (FIG. 2B) before being differentiated into immune cell populations by the methods described herein. [Figure 2B] See legend to Figure 2A. [Diagram 3] 1 shows flow cytometry analysis of immune cell populations including T-cell-like and NK-cell-like cells generated by the method of the present invention. Progenitor cells were pre-gated in a single live lymphoid cell population according to side and forward scatter. To separate progenitor cells from feeders, cells were gated according to CD29 / CD45 signature. All CD29-CD45+ cells were characterized for phenotype: CD3+CD56- (T-cell lineage marker), CD3-CD56+CD8+ / - (NK-cell lineage marker), and CD19+ (B-cell lineage marker). Furthermore, CD3+CD56- T-cell-like cells were further characterized for expression of CD4 and CD8 markers. These cells showed expression patterns similar to the classical thymocyte profile of CD4-CD8-, CD4+CD8+, CD4-CD8+, and CD4+CD8-. [Figure 4]Figure 1 shows flow cytometry analysis of cells of immune cell population obtained herein after stimulation with anti-human CD3 antibody. Progenitor cells were pre-gated in single live lymphoid cell population according to side scatter and forward scatter. To separate progenitor cells from feeders, cells were gated according to CD29 / CD45 signature. All CD29-CD45+ cells were characterized by phenotype: CD3+CD56- (T cell lineage marker) or CD3-CD56+ (NK cell lineage marker). [Diagram 5] Two figures are shown. The top figure shows the absolute cell numbers of CD45+CD56+ NK-like cells that were further expanded via the (differentiation) method of the invention at day 0 (start of expansion) and day 13 of expansion. The bottom figure shows that CD45+CD56+ NK-like cells expanded 19-fold from day 0 to day 13. [Figure 6] The results of lactate dehydrogenase (LDH) activity assay of differentiated immune cells are shown. The upper figure of Fig. 6 shows the mean value + / - SD of triplicate cultures of K562 target cells and differentiated immune cells of the present invention. The lower part of Fig. 6 shows the microscopic analysis of the control and co-culture groups of K562 cells and differentiated immune cells of the present invention used in LDH assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description of the Invention A first aspect of the present invention relates to a method of generating a population of immune cells from pluripotent stem cells (PSCs), comprising: (i) (a) culturing PSCs that have undergone mesenchymal differentiation under suitable conditions in the presence of Notch ligand delta-like ligand 4 (DLL4) signaling activity in serum-free medium on a solid support suitable for the formation of 3D cell aggregates, thereby allowing the formation of 3D cell aggregates; (b) collecting the 3D cell aggregates and culturing them under suitable conditions in suspension culture in serum-free medium for about 3-6 days; and (c) inducing 3D cell aggregate formation and hematopoietic differentiation by culturing the 3D cell aggregates under suitable conditions in serum-free medium for a further period of about 4-7 days; (ii) inducing immune cell differentiation by culturing the 3D cell aggregates of (i) under suitable conditions in suspension culture in serum-free medium for a suitable time, thereby providing a population of immune cells. In this regard, it is remarkable that it has surprisingly been found in the present invention that such a method not only provides T cells as described by Montel-Hagen et al., 2019 (supra) or Iriguchi et al., 2021 (supra), but also makes it possible to generate distinct subpopulations of a population of immune cells, in particular T cell-like cells and / or NK cell-like cells, simply depending on the intended use of the immune cells obtained.

[0018] As used herein, the term "about" when used in reference to an interval or period of time should be understood to include intervals that deviate from the explicit value of the given interval. For example, an interval of about 3-6 days, i.e., 72-144 hours, includes intervals that deviate from the given interval by 1, 2, 3, 4, 5, or 6 hours, and thus the given interval may be 1, 2, 3, 4, 5, or 6 hours shorter or longer with respect to the beginning and / or end of the interval.

[0019] One advantage of the present invention is that it allows the use of so-called feeder-free systems. That is, the 3D cell aggregates and the induction of hematopoietic differentiation can be performed without the use / assistance of stromal cells. Thus, in a preferred embodiment, the Notch ligand delta-like ligand 4 (DLL4) signaling activity is provided by alternative means / structures, instead of being provided by cells. Such alternative structures can be, for example, beads coated with DLL4, which will be described in more detail later. It may also be possible to use a soluble version of DLL4 to provide DLL4 signaling activity. Considering the subsequent steps of the method of the present invention, such a feeder-free approach offers the advantage that possible cellular contamination can be avoided, which does not have to be removed or sorted out when carrying out the method of the present invention. Thus, the feeder-free system of the present invention represents a pure system that is also efficient, since additional purification steps are not required.

[0020] In a further preferred embodiment of the invention, the method comprises a further step (0i) carried out prior to step (i) comprising: (a) seeding the PSCs under suitable conditions in serum-free medium; and (b) culturing the PSCs under suitable conditions in serum-free medium for about 2 to about 4 days; and (c) inducing mesodermal differentiation by resuspending the cells in serum-free medium. Preferably, the method of the invention does not require additional substances or treatments prior to mesodermal differentiation.

[0021] In one embodiment of the method of the present invention, the serum-free medium preferably contains, in step (0i), (a) activin A, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and an apoptosis inhibitor, preferably a ROCK inhibitor, each at a concentration of about 10 to about 50 ng / mL; (b) BMP4, VEGF, and FGF, each at a concentration of about 5 to 15 ng / mL; (c) an apoptosis inhibitor, preferably a ROCK inhibitor; and in step (i), (a) an inhibitor of the ALK receptor and a ROCK inhibitor, each at a concentration of about 5 to about 15 μM, (b) an inhibitor of the ALK receptor, about 5 to about 15 ng / mL, (c) an inhibitor of the ALK receptor, about 40 to about 60 ng / mL stem cell factor (SCF), about 5 to about 15 ng / mL. and in step (ii), about 1 to about 3% B27 supplement, about 5 to about 15 ng / mL SCF, about 5 to about 15 ng / mL Flt-3l, about 5 to about 15 ng / mL IL-7, and about 15 to about 45 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate.

[0022] As used herein, the term "about" with respect to a concentration or concentration range value should be understood to include concentrations or concentration ranges that fall outside the specific value of the given concentration or concentration range. For example, a concentration of about 50 ng / mL includes lower concentrations of 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 ng / mL, or higher concentrations of 51, 52, 53, 54, 55, 56, 57, 58, or 59 ng / mL.

[0023] In a preferred embodiment of the present invention, the ROCK inhibitor may be Y-27632 dihydrochloride, and the inhibitor of ALK receptor may be SB43152. The use of ROCK inhibitor provides the advantage that apoptosis and dedifferentiation are limited / avoided. Thus, the use of ROCK inhibitor and inhibitor of ALK receptor avoids the loss of cells due to apoptosis, i.e., a defined amount of cells is provided by the method of the present invention. Furthermore, the loss of differentiation is reduced, since the cells maintain the desired differentiation characteristics. That is, a defined quality of cells is provided using the method of the present invention.

[0024] Preferably, the suspension culture in steps (i) and (ii) is a dynamic suspension culture, i.e., a suspension culture that is agitated, for example, by culturing in a shaking device. Any suitable shaking device / shaker, for example, an orbital shaker, horizontal shaker, or linear shaker, may be used. The culture may be moved / agitated under any suitable conditions that may be experimentally determined by the skilled artisan. For example, a shaker, for example, an orbital shaker, may be operated at a speed of about 60 to about 80 revolutions per minute (rpm). In contrast to adherent cell culture, the suspension cell culture that may be used herein offers several advantages. Adherent cell culture requires more space and requires more delicate handling, for example, for passaging of cells. Suspension culture is a cell culture format that allows to provide a scalable cell culture process that is useful for the provision of higher cell numbers. Such higher cell numbers are specifically required for clinical use of cell products. The characteristics of dynamic cell culture are already very similar to those of culture in bioreactors, so that dynamic suspension cultures are much easier to transfer to bioreactor conditions compared to adherent cell cultures, e.g. 2D cell cultures or static cell cultures. Furthermore, in dynamic cell cultures, the growth factors present in the culture medium can circulate. This allows all cells to be in close contact with such growth factors. This can have a positive impact on the culture process, specifically the differentiation process of cells, and can help to achieve a more homogeneous and better defined cell population.

[0025] The induction of immune cell differentiation can be carried out for any suitable time. A suitable time can be determined experimentally, for example, by taking cell samples for a certain period of time and determining the nature / differentiation state of the cells, for example, by analysis of cell surface markers (see experimental section, where cells were first screened for CD29 / CD45 markers and all CD29-CD45+ cells were characterized for the phenotype of CD3+CD56- (T cell lineage marker), CD3-CD56+CD8+ / - (NK cell lineage), and CD19+ (B cell lineage). By doing so, a suitable time for differentiation can be determined, for example, depending on whether it is NK cell-like or T cell-like immune cells that are desired to be obtained as a product of the method of the present invention. In a preferred embodiment of the present invention, the step (ii) of inducing immune cell differentiation by culturing the 3D cell aggregates of (i) in suspension culture under suitable conditions is carried out in serum-free medium for about 21 to about 50 days.

[0026] The term "pluripotent stem cells" (PSCs), as used herein, refers to a stem cell type that can differentiate into a population of immune cells. In the context of the present invention, these pluripotent stem cells are preferably not generated using a process that involves modifying the genetic identity of human germline or using human embryos for industrial or commercial purposes. Preferably, the pluripotent stem cells are derived from a primate, more preferably, a human. In a further preferred embodiment of the present invention, the PSCs are human induced pluripotent stem (iPS) cells, preferably the human iPS cell line TC-1133. The cell line TC-1133 has been initialized under c-GMP conditions and can be purchased from Lonza. Further suitable PSCs, such as induced PSCs, can be obtained, for example, from the NIH human embryonic stem cell registry, the European Bank of Induced Pluripotent Stem Cells (EBiSC), Stem Cell Repository of the German Center for Cardiovascular Research (DZHK), or ATCC, to name just a few sources. Pluripotent stem cells are also available for commercial use, for example, from the NINDS Human Sequence and Cell Repository (https: / / stemcells.nindsgenetics.org), which is managed by the US National Institute of Neurological Disorders and Stroke (NINDS) and distributes human cell resources widely to academic and industrial researchers. Human iPS cells may be cultured in a matrigel-coated vessel, for example, a matrigel-coated T-flask. iPS cells are preferably cultured in iPS expansion medium, for example, Miltenyi StemMACS iPS-Brew XF.Further exemplary iPSC cell lines that may be used in the present invention include, but are not limited to, Gibco™ human episomal iPSC line (Order No. A18945, Thermo Fisher Scientific), or iPSC cell lines ATCC ACS-1004, ATCC ACS-1021, ATCC ACS-1025, ATCC ACS-1027, or ATCC ACS-1030 available from ATCC. Alternatively, a person skilled in the art of reprogramming can easily generate suitable iPSC lines by known protocols, for example those described by Okita et al., "A more efficient method to generate integration-free human iPS cells" Nature Methods, Vol. 8 No. 5, May 2011, pages 409-411 or Lu et al., "A defined xeno-free and feeder-free culture system for the derivation, expansion and direct differentiation of transgene-free patient-specific induced pluripotent stem cells", Biomaterials 35 (2014) 2816e2826. The (induced) pluripotent stem cells used in the present invention can be derived from any suitable cell type (e.g. stem cells, e.g. mesenchymal or epithelial stem cells, or differentiated cells, e.g. fibroblasts) and from any suitable source (body fluids or tissues). Examples of such sources (body fluids or tissues) include umbilical cord blood, skin, gums, urine, blood, bone marrow, any compartment of the umbilical cord (e.g., the amniotic membrane or Wharton's gel of the umbilical cord), the umbilical-placental junction, placenta, or adipose tissue, to name just a few. In one illustrative example, isolation of CD34 positive cells from umbilical cord blood is by magnetic cell sorting using an antibody specific for CD34 and subsequent reprogramming, for example, as described in Chou et al. (2011) Cell Research, 21:518-529.Baghbaderani et al. (2015), Stem Cell Reports, 5(4):647-659, show that the process of iPSC generation to generate cell line ND50039 can comply with good manufacturing practice standards. Thus, the pluripotent stem cells preferably meet the requirements of good manufacturing practice.

[0027] The PSCs used in the present invention may be pluripotent stem cells that lack endogenous MHC class I molecules presented on the cell surface of the pluripotent stem cells and contain immunomodulatory proteins on their surface.

[0028] A cell or pluripotent stem cell that is "lacking endogenous MHC class I molecules presented on its surface" does not present functional MHC class I molecules on its surface, i.e., on the surface of the cell or pluripotent stem cell. Such a deficient cell / pluripotent cell does not contain functional MHC class I molecules in its cell membrane either. In this regard, the term "endogenous" refers to MHC class protein I that is naturally contained in the cell or pluripotent stem cell and is not artificially introduced. Having endogenous MHC class I molecules increases the risk of rejection by the recipient's immune system, since the lack of MHC class I molecules on the cell surface can be interpreted by the immune system as a "missing self" signal. Thus, the feature that a cell or pluripotent stem cell lacks MHC class I molecules on its surface does not apply to immunomodulatory proteins and / or recombinant immunomodulatory proteins that may be introduced into the pluripotent stem cell. In one embodiment, the lack of MHC class I molecules on the cell surface can be achieved by disrupting all copies of the β2 microglobulin gene in the pluripotent stem cell. The MHC complex is a heterodimer of α-microglobulin and β2-microglobulin. Therefore, if β2-microglobulin is lost, functional MHC class I complexes cannot be assembled, resulting in the absence of MHC class I molecules on the cell membrane and / or cell surface. Many possible ways to modify the genome of pluripotent stem cells to lack MHC class I molecules and contain immunomodulatory proteins are known to those skilled in the art. It should be noted that pluripotent stem cells of the present invention that lack MHC class I molecules can express immunomodulatory proteins, even if they are MHC class I molecules as described herein, such as HLA-E. Thus, the term "lacking MHC class I molecules" can refer to endogenous MHC class I molecules and does not exclude the presence of (recombinant) immunomodulatory proteins.

[0029] In a further preferred embodiment of the invention, the immunomodulatory protein is a single chain fusion HLA class I protein.

[0030] Preferably, the single chain fusion HLA class I protein may comprise at least a portion of B2M covalently linked to at least a portion of an HLA class I α chain selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0031] In a preferred embodiment of the present invention, the pluripotent stem cells further express a target peptide antigen presented by a single-chain fusion HLA class I protein on the pluripotent cell surface.

[0032] Preferably, the target peptide antigen is covalently linked to the single chain fusion HLA class I protein.

[0033] In a preferred embodiment, the target peptide antigen comprises the sequence VMAPRTLFL (SEQ ID NO:1).

[0034] Preferably, essentially all copies of β microglobulin 2 gene are disrupted in pluripotent stem cells.In the pluripotent stem cells of the present invention, B2M gene may be disrupted so that functional endogenous B2M protein is not produced from the disrupted locus.In certain embodiments, disruption results in the expression of non-functional B2M protein, including but not limited to truncation, deletion, point mutation and insertion.In other embodiments, disruption does not result in protein expression from B2M gene.

[0035] In a preferred embodiment, the pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, and parthenogenetic stem cells.

[0036] Preferably, the pluripotent stem cells are primate-derived pluripotent stem cells, preferably human pluripotent stem cells.

[0037] In a preferred embodiment, the pluripotent stem cells generated from CD34 positive cells are isolated from umbilical cord blood.

[0038] Preferably, the pluripotent stem cell used in the present invention is ND-50039 of the NINDS Human Cell and Data Repository.

[0039] In a preferred embodiment, the PSC pluripotent stem cells lack endogenous MHC class II.

[0040] Preferably, the PSCs lack endogenous MHC class II by disruption of the C2TA gene-MHC class II transactivator.

[0041] In a preferred embodiment of the method of the present invention, in step (i), Notch ligand delta-like ligand 4 (DLL4) signaling activity is provided by co-culturing PSCs with stromal cells expressing DLL4 or by incubating PSCs with beads coated with DLL4.

[0042] Incubating PSCs with beads coated with DLL4 provides the advantage that DLL4 signaling activity is provided without the need for additional cells, such as stromal cells. Thus, a so-called feeder-free system can be provided by the method of the present invention. Thus, this avoids the step of sorting or selecting DLL4-expressing cells from the population of immune cells provided by the method of the present invention. If necessary, DLL4-coated beads can be separated by standard methods known to those skilled in the art, such as magnetic separation techniques or flow cytometry techniques. Various beads suitable for use for DLL4 coating, such as microbeads, or beads with larger diameters, such as Dynabeads (Thermo Fisher Scientific), are known to those skilled in the art.

[0043] When stromal cells are used, the ratio of PSCs to stromal cells can range from about 1:5 to about 1:40. Such a range is advantageous because it allows for the use of fewer stromal cells compared to prior art methods such as Montel-Hagen et al., 2019 (supra). Thus, when fewer stromal cells are used, this can have a beneficial effect on the composition of growth factors secreted by the stromal cells and the concentration of growth factors. For example, fewer stromal cells are likely to produce reduced amounts of growth factors, resulting in a reduced concentration of growth factors in the medium. Such a reduction in the concentration of growth factors is assumed to have a beneficial effect on the differentiation of pluripotent stem cells. Furthermore, fewer stromal cells require less effort to remove these stromal cells later in the procedure.

[0044] In a preferred embodiment of the present invention consisting of stromal cells, the stromal cells used in step (i) are the bone marrow-derived murine stromal cell line MS5. Murine stromal cells are advantageous because they can support immune cell differentiation.

[0045] In a further preferred embodiment, in step (i), stromal cells are stromal cells differentiated from PSCs expressing DLL4.The use of such stromal cells provides the advantage that the method of the present invention does not require mouse-derived cells that may have to be removed or selected later in the procedure.Therefore, it is possible to provide a population of immune cells that does not contain non-human cells, such as mouse cells.

[0046] In a preferred embodiment, the PSCs are cultured in a cell culture medium / vessel containing at least one extracellular matrix protein during the induction of mesodermal differentiation in step (0i). 6Cells are seeded into each cell culture vessel, e.g., a well of a 6-well plate. Adjusting the cell number of PSCs to this defined range can affect the growth factor ratio in the medium and thus the subsequent differentiation. The above-mentioned range of cell numbers allows the beneficial differentiation of cells into the desired population of immune cells (see experimental section).

[0047] Preferably, the container is coated with at least one extracellular matrix protein.

[0048] In a preferred embodiment, at least one extracellular matrix protein can be selected from the group consisting of vitronectin, laminin, collagen, fibronectin, elastin, matrigel, peptides containing the amino acid sequence RGD, proteins containing the amino acid sequence RGD, and combinations thereof.Therefore, in order to provide the optimal structural and biochemical environment for PSCs and the subsequent differentiation procedure, those skilled in the art can empirically select the optimal extracellular matrix protein or the optimal combination of extracellular matrix proteins.Therefore, combinations of two, three, four or more extracellular matrix proteins can also be used in the method of the present invention.

[0049] Preferably, in step (i)(a) of the method of the invention, the solid support comprises one or more wells, wherein each well comprises a V-shaped or conical cavity.

[0050] In a preferred embodiment, the solid support is a microwell culture plate. Any suitable microwell culture plate can be used as long as it allows the generation of the desired immune cell population. Examples of suitable microwell culture plates include, but are not limited to, AggreWell™ plates (available from StemCell Technologies), BIOFLOAT™ 96-well plates (available from faCellitate), or Nunclon Sphera 3D culture (available from ThermoFisher). Such cell culture plates are advantageous because they allow the beneficial dynamic suspension cell culture described herein. For example, when AggreWell™ plates are used, the plates are preferably pretreated with an anti-adhesion solution. Such anti-adhesion solutions allow the surface tension to be reduced, preventing cell adhesion. This provides a low-adhesion surface that facilitates the formation of 3D cell aggregates. The formation of 3D cell aggregates can be carried out in a very easy and robust manner that also allows for scale-up. The 3D cell aggregate formation is different compared to the prior art methods such as those described in Montel-Hagen et al., 2019 (supra), which describe complex and time-consuming cell culture techniques that are not suitable for scale-up. Moreover, the 3D cell aggregates obtained using the method of Montel-Hagen et al., 2019 (supra) appear to be larger compared to the 3D cell aggregates of the method of the present invention. This provides an additional advantage of the present invention that growth factors can diffuse more easily into such smaller 3D cell aggregates compared to larger 3D cell aggregates. Thus, growth factors can reach cells in the innermost locations of the 3D cell aggregates more efficiently, which leads to efficient and more homogeneous differentiation of all cells of the 3D cell aggregates. In this regard, see Example 1 of the present application.

[0051] In a preferred embodiment, the 3D cell aggregates after step (ii) are dissociated to provide single cells.

[0052] As mentioned above, in the methods of the invention, it has surprisingly been found that the population of immune cells may comprise both T cell-like cells and NK cell-like cells, ie a mixture of different cell types.

[0053] Thus, the term "population of immune cells" as used herein includes a mixture of different immune cells contained in a population of immune cells. Thus, "population of immune cells" should not be understood as a single homogeneous group of immune cells, e.g., a pure or homogeneous population of T cells, where homogeneity can be assessed by the percentage of cells expressing a particular marker protein or cells lacking the expression of a particular marker protein. The term "population of immune cells" refers to different immune cells or different types of immune cells that can be distinguished from each other by methods known in the art, e.g., flow cytometry methods, and then isolated from each other by the skilled artisan. Thus, the isolation and subsequent expansion of specific and homogeneous cell populations characterized by the expression (pattern) of specific marker proteins is in fact part of the present invention (see Example 4 of the present application, where CD45+CD56+NK cell-like cells were expanded after differentiation). The term "population of immune cells" means that any type of immune cells can be included in the population of cells obtained by the method of the present invention. Immune cells include neutrophils, eosinophils (acidophilic leukocytes), basophils, lymphocytes, and monocytes, among which B cells, T cells, and natural killer (NK) cells. Furthermore, the population of immune cells according to the present invention may also be considered as a population of immune cells having a (desired) differentiation level or an intermediate differentiation level of immune cells. The population of immune cells may also include cells representing precursors of T cells or T cell-like cells, or precursors of NK cells or NK cell-like cells, and may also include cells representing mature T cells or T cell-like cells, or mature NK cells or NK cell-like cells. Specifically, the population of immune cells according to the present invention includes immune cells having the phenotype of T cell-like cells and NK cell-like cells.

[0054] Providing a population of immune cells has the advantage that it is possible to select between the obtained T cell-like cells and NK cell-like cells. Thus, in contrast to the prior art methods that can provide only T cells or T cell-like cells, the method of the present invention can provide T cell-like cells together with NK cell-like cells. Thus, depending on the desired application, one can choose between T cell-like cells and NK cell-like cells. The yield of these T cell-like cells and NK cell-like cells can be specifically shown in flow cytometry analysis (see Example 2 and Figure 3). The cytometry analysis shows that the population of immune cells provided by the method of the present invention contains distinct subpopulations that can be characterized as T cell-like cells and NK cell-like cells by the expression of distinct markers. The characterization of T cell-like cells and NK cell-like cells is described in detail below.

[0055] In a preferred embodiment of the present invention, step (ii) is followed by a further step (iii), which comprises inducing, selectively expanding and activating T-cell-like cells and NK-cell-like cells.

[0056] Preferably, in one embodiment of the method, in step (iii), T cell-like cells are induced, selectively expanded and activated under suitable conditions.Such suitable conditions for T cell-like cells are, for example, expanding cells with CD3 activating antibodies, for example, antibody OKT3 antibody, and / or interleukin 2 (IL-2).The antibody OKT3 and IL-2 activate the cells, and after activation, the cells can be expanded by propagating the cells in cell culture.The expansion of cells can be monitored, for example, by determining the level of one or more of intracellular or secreted IFN-g, TNF-a, and / or IL-2.

[0057] In one preferred embodiment of the present invention, in step (iii), T cell-like cells are induced, selectively expanded and activated in a medium comprising B27 supplement, SCF, Flt-31, IL-7, IL-2, IL-15 and a CD3 stimulatory binding molecule.

[0058] In this embodiment, the medium in step (iii) may contain about 1 to about 3% B27 supplement, about 5 to about 15 ng / mL SCF, about 2 to about 7 ng / mL Flt-13, about 5 to about 15 ng / mL IL-7, about 5 to about 15 ng / mL IL-2, about 5 to about 15 ng / mL IL-15. The CD3 stimulatory binding molecule may be selected, for example, from a multimerization reagent carrying an anti-CD3 antibody, such as the antibody OKT-3, or an antigen fragment of the antibody OKT-3, such as the Fab fragment of the antibody OKT-3, at a concentration of about 250 to about 750 ng / mL. Such a multimerization reagent may be defined as being a multimerization reagent as follows: The multimerization reagent has a first agent reversibly immobilized (bound) to it, which provides a primary activation signal to the cell; the multimerization reagent comprises at least one binding site Z1 for reversible binding of the first agent, the first agent comprises at least one binding partner C1, which can reversibly bind to the binding site Z1 of the multimerization reagent, the first agent being bound to the multimerization reagent via a reversible bond formed between the binding partner C1 and the binding site Z1, the first agent binding to a receptor molecule on the surface of the cell, thereby providing a primary activation signal to the cell, thereby activating the cell. Such multimerization reagents in general, as well as multimerization reagents loaded with a CD3-binding stimulatory antibody, for example a Fab fragment of the antibody OKT-3, are described in detail in the international patent application WO2015 / 158868. This multimerization reagent is also known by the term "Expamer". In one exemplary embodiment of the present invention, step (iii) is carried out for about 2 to about 5 days, preferably about 3 to about 4 days.

[0059] In a preferred embodiment, a further step (iv) is carried out after step (iii), which comprises further expanding the immune cells by culturing them under suitable conditions. In an illustrative example, this expansion can be carried out in serum-free medium containing B27 supplement and IL-2.

[0060] Preferably, the serum-free medium in step (iv) comprises about 1% to about 3% B27 supplement and about 30 to about 70 units / mL IL-2. In a preferred embodiment of the present invention, step (iv) is carried out for about 2 to 10 days, preferably about 7 days. Preferably, steps (iii) and (iv) do not require incubation on a plate coated with DLL4 (see Examples 2 and 3 of the present application).

[0061] In a preferred embodiment of the method of the present invention, the population of immune cells is characterized by the expression of the surface proteins CD4, CD8, CD56, CD3, and CD45. Such characterization allows the population of immune cells to be differentiated into different subpopulations of T-cell-like cells and NK-cell-like cells. For example, to perform such characterization, a flow cytometric analysis of the population of immune cells can be performed. The skilled artisan can use the markers CD4, CD8, CD56, CD3, and CD45 to define the distinct subpopulations of T-cell-like cells and NK-cell-like cells. Thus, the T-cell-like cells obtainable by the method of the present invention are preferably characterized by the expression of either CD45, CD3, CD4 or CD8, and the lack of expression of CD56; thus, the T-cell-like cells are preferably characterized by the expression of either CD45, CD3, CD4 or CD8, and the lack of expression of CD56. + , CD3 + , CD56 - , and CD4 + or CD45 + , CD3 + , CD56 - , and CD8 +The NK cell-like cells obtainable by the method of the present invention are preferably characterized by the expression of CD45 and CD56, and the lack of expression of CD3, and thus the NK cell-like cells are preferably characterized by the expression of CD45 and CD56, and the lack of expression of CD3. + , CD56 + , and CD3 - Other suitable markers and / or methods that allow such discrimination may be used as well. Such cell selection based on marker expression profiles may be readily performed by one skilled in the art.

[0062] The method of the present invention is suitable for producing a population of immune cells with high cell numbers by using bioreactor conditions in one or all of steps (ii), (iii) and / or (iv). The use of a bioreactor provides the advantage that a higher yield of cells can be achieved by using a large-scale bioreactor format. The use of a bioreactor allows the optimization of the concentration of different factors and at the same time allows the production of a higher cell number. Thus, the number of cells of the immune cell population provided by the method of the present invention can be scaled up by using a bioreactor. Doing so makes it possible to provide the number of cells of the immune cell population required for clinical application. Furthermore, doing so makes it possible to provide a highly standardized and cost-effective method. Furthermore, the use of a bioreactor simplifies the procedure, since it provides a hassle-free method that can be easily implemented.

[0063] A second aspect of the invention relates to a population of immune cells obtainable by the method of the invention.

[0064] A third aspect of the invention relates to a population of immune cells obtained by the method of the invention.

[0065] A fourth aspect of the invention relates to non-immunogenic T cells obtainable by the methods of the invention.

[0066] A fifth aspect of the invention relates to non-immunogenic T cells obtained by the methods of the invention.

[0067] A sixth aspect of the invention relates to a non-immunogenic T cell obtainable by the method of the invention, which lacks endogenous MHC class I molecules displayed on the cell surface of the T cell and which comprises an immunomodulatory protein on its surface.

[0068] Preferably, non-immunogenic T cells express chimeric antigen receptors (CARs) or foreign T cell receptors (TCRs) on their surface. CAR proteins may contain single chain variable fragments (scFvs) of antibodies capable of binding to specific tumor-associated antigens, linked to intracellular costimulatory domains, such as CD28, OX40, and CD137, via transmembrane peptides. These peptides are then fused to the signaling domains of the TCR ζ chain, which activates the CAR T cells when they bind to epitopes on tumor cells. The subsequent release of granzymes and perforin leads to tumor cell lysis (June CH, O'Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science (New York, NY) 2018; 359: 1361-65). These synthetic receptor molecules enable MHC-independent T cell activation that differs from the typical response mediated by the TCR complex (Gideon Gross, Tova Waks, and Zelig Eshhar. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc. Natl. Acad. Sci. USA 1989:10024-28; Kuwana Yea. Expression of chimeric receptor composed of immunoglobulin-derived V regions and T-cell receptor-derived C regions. Biochemical and biophysical research communications 1987:960-68).This represents an important advantage in tumor cell recognition, since loss of MHC-associated antigen presentation is a major immune escape strategy by malignant cells (Garrido F, Aptsiauri N, Doorduijn EM, Garcia Lora AM, van Hall T. The urgent need to recover MHC class I in cancers for effective immunotherapy. Current opinion in immunology 2016;39:44-51).

[0069] In a further preferred embodiment of the present invention, essentially all copies of the β microglobulin 2 gene are disrupted in T cells, and thus the T cells lack endogenous MHC class I molecules on the cell surface.In the T cells of the present invention, the B2M gene may be disrupted so that functional endogenous B2M protein is not produced from the disrupted locus.In certain embodiments, the disruption results in the expression of non-functional B2M protein, including but not limited to truncation, deletion, point mutation, and insertion.In other embodiments, the disruption does not result in protein expression from the B2M gene.

[0070] Preferably, the immunomodulatory protein is a single chain fusion HLA class I protein.

[0071] In a further preferred embodiment, the single chain fusion HLA class I protein may comprise at least a portion of B2M covalently linked to at least a portion of an HLA class I α chain selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0072] Preferably, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A.

[0073] In a further preferred embodiment, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A0201.

[0074] Preferably, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-E.

[0075] In a preferred embodiment, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-G.

[0076] Preferably, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-B.

[0077] In a preferred embodiment, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-C.

[0078] Preferably, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-F.

[0079] In a preferred embodiment, the non-immunogenic T cells further express a target peptide antigen that is presented on the pluripotent cell surface by a single-chain fusion HLA class I protein.

[0080] Preferably, the target peptide antigen is covalently linked to the single chain fusion HLA class I protein.

[0081] In a preferred embodiment, the target peptide antigen comprises the sequence VMAPRTLFL (SEQ ID NO:1).

[0082] Preferably, the gene for the immunomodulatory protein is integrated into the (endogenous) B2M locus of the T cell genome.

[0083] In a preferred embodiment, the CAR or exogenous TCR is integrated into the endogenous TCR alpha gene, the endogenous TCR beta gene, or into both the endogenous TCR alpha gene and the endogenous TCR beta gene.

[0084] Preferably, the CAR or the foreign TCR binds to an antigen displayed on the surface of a target cell.

[0085] In a preferred embodiment, the antigen is a tumor-associated antigen, a viral antigen, or a bacterial antigen, preferably a tumor-associated antigen.

[0086] Preferably, the antigen is CD19 or a CMV derived antigen.

[0087] In a preferred embodiment, the T cells are primate-derived T cells, preferably human T cells.

[0088] A further aspect of the invention relates to a pharmaceutical composition comprising the population of immune cells of the method of the invention or the non-immunogenic T cells of the method of the invention.

[0089] A further aspect of the invention relates to a population of immune cells of the invention, a non-immunogenic T cell of the invention, or a pharmaceutical composition of the invention for use in a method of treatment.

[0090] As used herein, the term "treat", "treating" or "treatment" means to reduce (slow down (alleviate)), stabilize or inhibit, or at least partially alleviate or eliminate the progression of symptoms associated with the respective disease. It therefore includes the administration of cells of the immune system, preferably in the form of a medicament or pharmaceutical composition, to a subject as defined elsewhere herein. Those in need of treatment include those already suffering from a disease, here cancer. Preferably, treatment reduces (slow down (alleviate)), stabilizes or inhibits, or at least partially alleviates or eliminates the progression of symptoms associated with the presence of a disease or pathological condition and / or the progression of a disease or pathological condition. Thus, "treat", "treating" or "treatment" refers to therapeutic treatment. Specifically, in the context of the present invention, treating or treatment refers to the improvement of symptoms associated with cancer, as defined elsewhere herein. The term "subject" as used herein includes a mammalian subject. Preferably, the subject of the present invention is a mammal, e.g., a human. In some embodiments, the mammal is a mouse. Subjects include human and animal patients. If a subject is a living human who can receive treatment for a disease or condition as described herein, it is also referred to as a "patient." Those in need of treatment include those who already suffer from the disease.

[0091] A further aspect of the present invention relates to a population of immune cells of the present invention, such as a (non-immunogenic) T cell population or NK cell population of the present invention, or a pharmaceutical composition of the present invention, for use in cell (based) therapy. This medical use comprises administering the population of immune cells of the present invention or a cell population derived from the population of immune cells of the present invention to a subject in need thereof. The subject is typically a mammal, such as a human, monkey, cat, rodent (mouse, rat), dog, or other animal, such as a horse or cow.

[0092] Typically, the present invention relates to a population of immune cells, or a non-immunogenic T cell population, or a NK cell population, for use according to the present invention, where the treatment or therapy is against cancer. That is, the population of immune cells is typically against a subject for cancer treatment or immunotherapy. The immune cell population of the present invention, e.g., T cells or NK cells, can be used in adoptive T cell transfer or similar therapeutic approaches. Among various immune cell therapies, e.g., T cell immunotherapy, adoptive cell therapy has attracted considerable attention and interest in recent years. Adoptive cell therapy is an individualized therapy in which the patient's own immune cells are removed from the patient, expanded in vitro in large quantities, and reinjected into the patient to eliminate the tumor. An overview of the development of adoptive cell therapy is given in Guedan et al., Rev Immunol. 2019 April 26; 37: 145-171. doi: 10.1146 / annurev-immunol-042718-041407.

[0093] Immune cell population, for example (non-immunogenic) T cell population, can be used for the treatment of essentially all cancers.The examples of cancers that can be treated with the cell population obtained by the present invention are lung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, skin cancer, melanoma, colon cancer, rectal cancer, stomach cancer, esophageal cancer, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, breast cancer, ovarian cancer, lymphatic system cancer, for example lymphoma and multiple myeloma, leukemia, sarcoma of bone or soft tissue, cervical cancer, or vulvar cancer.

[0094] Unless otherwise stated, the following terms used herein, e.g., in the description and claims, have the definitions given below.

[0095] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the present invention.

[0096] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "reagent" includes one or more of such various reagents, and reference to a "method" includes reference to equivalent steps and methods known to those of skill in the art that may be modified or substituted for the methods described herein.

[0097] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0098] The term "and / or" when used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by that term".

[0099] The term "about" or "approximately" means within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which will depend in part on the method by which the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more, as per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, "about" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. However, it also includes specific numbers, for example, about 20 includes 20. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. When a particular value is described in the present application and claims, unless otherwise indicated, it should be assumed that the term "about" means within an acceptable error range for that particular value.

[0100] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise," as well as variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps and not the exclusion of other integers or steps or groups of integers or steps. As used herein, the term "comprising" may be substituted with the terms "containing" or "including," or, sometimes, as used herein, with the term "having."

[0101] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0102] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms.

[0103] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc. described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0104] All publications cited throughout the text of this specification (e.g., all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) are incorporated herein by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. In the event that material incorporated by reference contradicts or is inconsistent with the present specification, the present specification takes precedence over such material. EXAMPLES

[0105] Example of the invention The following examples illustrate the present invention. These examples should not be construed as limiting the scope of the invention. The examples are included for illustrative purposes, and the present invention is limited only by the claims.

[0106] Example 1: Generation of populations of immune cells from pluripotent stem cells (PSCs) Human induced pluripotent stem (iPS) cell line TC-1133 initialized under c-GMP conditions was purchased from Lonza. Human iPS cells were maintained in iPS expansion medium (Miltenyi StemMACS iPS-Brew XF) in matrigel-coated T-flasks. Mouse stromal cells have been shown to support immune cell differentiation. For use in differentiation protocols, we generated mouse stromal cell lines expressing the Notch ligand delta-like ligand 4 (DLL4). Bone marrow-derived mouse stromal cell line (MS5) was purchased from the German Collection of Microorganisms and Cell Cultures GmbH. A plasmid carrying a CAG promoter, full-length human DLL4, and puromycin resistance gene, and bovine growth hormone polyadenylation signal sequence between left and right piggybac transposase-specific sequence arms was constructed. Cells were transfected with a plasmid carrying piggybac transposase and DLL4. Successful transfectants were selected and expanded in puromycin-containing medium. MS5-DLL4 cells were maintained in medium composed of DMEM (Gibco 42430025), 10% FBS, 1% penicillin / streptomycin, 1× Glutamax (Gibco 35050061), and 1 mM sodium pyruvate (Gibco 11360039).

[0107] Mesoderm induction (differentiation day -18 to day -14, see also FIG. 1). The following steps were carried out: 1. Wash human iPS cells with 5 mL of Dulbecco's phosphate buffered saline (DPBS; Gibco 14190136). 2. Add 3 mL of Accutase (Gibco A1110501) and incubate the cells for 4 minutes in a 37° C. incubator. 3. Detach the cells by tapping the flask, then wash the cells out with 5 mL of DPBS. 4. Transfer the cells to a Falcon and centrifuge at 300g for 4 minutes. 5. Discard the supernatant and resuspend the cells in 1 mL StemPro34-SFM medium (Gibco 10639011) and count. 6. Add 1.0 × 10 cells per well to a 6-well plate coated with Matrigel. 6 Seed the cells into mesoderm induction medium composed of StemPro34-SFM medium supplemented with Activin A (10 ng / mL), BMP4 (10 ng / mL), VEGF (10 ng / mL), FGF (10 ng / mL), and the ROCK inhibitor Y-27632 dihydrochloride (10 µM). 7. Change the medium daily on days 1, 2, and 3 with StemPro34-SFM medium supplemented with BMP4 (10 ng / mL), VEGF (10 ng / mL), and FGF (10 ng / mL). 8. On day 4, wash cells 3 times with DPBS. 9. Add 1 mL of Accutase to the cells and incubate in a 37° C. incubator for 10 minutes. 10. Gently detach the cells from the plate by pipetting up and down and transfer to a Falcon tube. 11. A centrifugation step is carried out at 300 g for 4 minutes. 12. Resuspend the cells in StemPro34-SFM medium supplemented with Y-27632 and count.

[0108] 3D aggregate formation and hematopoietic induction (differentiation-day 14 to day 0, again see FIG. 1). The following steps were carried out: 1. Pre-treat AggreWell™ 800 6-well plates (Stemcell Technologies, 34821) with 2 mL per well of Anti-Adhesion Rinse Solution (Stemcell Technologies, 07010). 2. Aspirate the anti-adhesion rinse solution from the wells. 3. Rinse each well with warm DPBS and keep at room temperature until use. 4. MS5-DLL4 cells are harvested by trypsinization (TrypL Express, Gibco 12604013), resuspended in StemPro34-SFM medium (Lonza CC-4176) and counted. 5. 5.0×10 6 MS5-DLL4 cells were cultured at 0.5 × 10 6 Resuspend with mesoderm cells (1:10 ratio per well) in 5 mL of hematopoietic induction medium composed of StemPro34-SFM medium supplemented with 10 μM SB431542 and 10 μM Y-27632. Gently pipette the cells up and down to achieve a homogenous distribution. 6. Aspirate DPBS from the wells of the AggreWell™ 800 6-well plate. 7. Seed the cells into the wells, carefully avoiding bubble formation. 8. Centrifuge the plate at 100g for 3 minutes to trap the cells in the microwells (see Figure 2A). 9. Incubate the plates in a 37°C, 5% CO2, 95% humidity incubator for 3 days. 10. On the third day, aggregate formation can be observed microscopically (Figure 2B). 11. To collect the 3D aggregates from the plate, gently tap the plate to dislodge the aggregates. Collect the aggregates in the center of the well by swirling. Use a serological pipette to collect the aggregates from the plate and transfer to a Falcon tube. 12. After all the aggregates have settled / settled to the bottom of the Falcon, carefully discard the medium without disturbing the aggregates. 13. Resuspend the aggregates in 2.5mL of hematopoietic induction medium composed of StemPro34-SFM medium supplemented with 10um of SB431542 and transfer to ultra-low attachment suspension culture plates. 14. Maintain the aggregates in dynamic suspension culture by placing the plates on an orbital shaker and rotating the shaker at 80 rpm. 15. From differentiation day-13 to differentiation day-7, replace the medium with fresh StemPro34-SFM medium supplemented with 10 μM SB431542 every 2-3 days. 16. Differentiation - Maintain aggregates in StemPro34-SFM medium supplemented with 10 μM SB431542, 50 ng / mL SCF, 5 ng / mL Flt-3l, 5 ng / mL TPO by refreshing the medium every 2-3 days from day 7 to day 0.

[0109] Immune cell differentiation (from day 0 to day 50 of differentiation). The following steps were performed: From differentiation day 0 to differentiation day 50, aggregates were maintained in dynamic suspension culture in immune cell differentiation medium composed of RPMI 1640 (Gibco 618736), 2% B27 (Gibco, 17504044), 10 ng / mL SCF, 5 ng / mL Flt-3l, 5 ng / mL IL-7, 30 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, and 1% penicillin / streptomycin.

[0110] Example 2: Activation of Differentiated Cells and Flow Cytometry Analysis The following steps were carried out: 1. On day 50 of differentiation, collect the aggregates from the culture plate and wash once with DPBS. 2. Dissociate the aggregates into single cells in a dissociation solution composed of 1 mg / mL collagenase type, 1 mg / mL collagenase / dispase, and 5000 units / mL at 37° C. for 1 hour. 3. Dissociated cells are centrifuged and resuspended in activation medium consisting of RPMI 1640 (Gibco 618736), 2% B27 (Gibco, 17504044), and 50 units / mL IL-2. 4. Seed the cells into cell culture flasks and culture them in an incubator at 37°C, 5% CO2, and 95% humidity for one week. 5. After one week, the cells are harvested from the cell culture flask and passed through a 100 μm sterile filter. 6. Singled cells are pelleted by centrifugation, washed once with PBS, and resuspended in staining buffer composed of PBS and 0.5% human serum albumin. 7. Stain the cells with anti-mouse anti-CD29, and anti-human anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-CD19, and anti-CD56 in the presence of Fc block for 20 minutes at 4°C. 8. Cells are washed with PBS and resuspended in staining buffer containing viability dye (PI). 9. Analyze cells on a Cytoflex LX flow cytometer according to the manufacturer's instructions.

[0111] The results of this experiment are shown in Figure 3. The following gating strategy was applied: Progenitor cells were pre-gated in a single live lymphoid cell population according to side and forward scatter. To separate progenitor cells from the feeder, cells were gated according to CD29 / CD45 signature. All CD29-CD45+ cells were characterized for CD3+CD56- (T cell lineage marker), CD3-CD56+CD8+ / - (NK cell lineage), and CD19+ (B cell lineage) phenotypes. In addition, CD3+CD56- T cell-like cells were further characterized for expression of CD4 and CD8 markers. These cells showed expression patterns similar to the classical thymocyte profile of CD4-CD8-, CD4+CD8+, CD4-CD8+, and CD4+CD8-.

[0112] Example 3: Early maturation of immune cell populations by anti-CD3 antibody stimulation To achieve early maturation of cells of the immune cell population, the following steps were performed: 1. At week 3 of differentiation (immune cell differentiation phase), collect the aggregates from the culture plate and wash once with DPBS. 2. Dissociate the aggregates into single cells with a dissociation solution consisting of 1 mg / ml collagenase type II. 3. Dissociated cells are centrifuged and resuspended in maturation medium consisting of RPMI 1640 (Gibco 618736), 2% B27 (Gibco, 17504044), 10 ng / mL SCF, 5 ng / mL Flt-3l, and 10 ng / mL IL-7, 10 ng / mL IL-2, 10 ng / mL IL-15, and 500 ng / ml monoclonal anti-human CD3 antibody (Biolegend 317347). 4. The cells are seeded into cell culture flasks and cultured at 37° C. in an incubator with 5% CO2 and 95% humidity for one week. 5. After one week, the cells are harvested from the cell culture flask and passed through a 100 μm sterile filter. 6. Singled cells are pelleted by centrifugation, washed once with PBS, and resuspended in staining buffer composed of PBS and 0.5% human serum albumin. 7. Stain the cells with anti-mouse anti-CD29, and anti-human anti-CD45, anti-CD3, and anti-CD56 in the presence of Fc block for 20 minutes at 4°C. 8. Cells are washed with PBS and resuspended in staining buffer containing viability dye (PI). 9. Analyze cells on a Cytoflex LX flow cytometer according to the manufacturer's instructions.

[0113] The results are shown in Figure 4. The gating strategy was as follows: progenitor cells were pre-gated in the single live lymphoid cell population according to side and forward scatter. To separate progenitor cells from the feeders, cells were gated according to CD29 / CD45 signatures. All CD29-CD45+ cells were phenotypically characterized as CD3+CD56- (T cell lineage markers), CD3-CD56+ (NK cell lineage).

[0114] Example 4: Differentiation into NK-like cells The following steps were carried out: 1. On day 50 of differentiation, collect the aggregates from the culture plate and wash once with DPBS. 2. Dissociate the aggregates into single cells with a dissociation solution consisting of 1 mg / mL collagenase type, 1 mg / mL collagenase / dispase, and 5000 units / mL at 37° C. for 1 hour. 3. Analyze the dissociated cells and select for CD45+CD56+ NK cell-like cells. 4. These CD45+CD56+ NK cell-like cells are centrifuged and resuspended in media composed of RPMI 1640 (Gibco 618736), 2% B27 (Gibco, 17504044), and 50 units / mL IL-2 and IL-15. 5. The cells are seeded into cell culture flasks and cultured in an incubator at 37° C., 5% CO2, and 95% humidity for 13 days.

[0115] The results are shown in Figure 5. CD45+CD56+ NK cell-like cells were found to be further expanded 19-fold after cytokine stimulation with IL-2 and IL-15 for 13 days.

[0116] Example 5: Measurement of cytotoxic activity of differentiated immune cells The cytotoxic activity of differentiated immune cells was measured in the following LDH assay using lactate dehydrogenase (LDH) activity assay. Lactate dehydrogenase (LDH) is an oxidoreductase that catalyzes the interconversion of pyruvate and lactate. Cells release LDH after tissue damage. LDH is a very stable enzyme, so it is useful for evaluating the presence of tissue and cell injury and toxicity. In the LDH assay described below, K562 cells, which are the target cells in the assay, were co-cultured with the differentiated immune cells of the present invention at target-effector ratios of 1:1, 1:2, 1:4, and 1:8.

[0117] The following steps were carried out: 1. Culture differentiated immune cells in immune cell differentiation medium containing IL-2 for 3 days prior to performing the LDH assay. The medium contains: Iscove's Modified Dulbecco's Medium (IMDM) (Thermo Fischer Sci Catalog No. 31980030), 15% BIT 9500 serum replacement (stem cell tech catalog number 09500), 1% penicillin-streptomycin, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (30 mM) (can range from 15 mM to 50 mM), SCF (10 ng / mL) (range can be 5 ng / mL to 50 ng / mL), TPO (10 ng / mL) (range can be 5 ng / mL to 50 ng / mL), Flt3l (10 ng / mL) (range can be 5 ng / mL to 50 ng / mL), IL-7 (10 ng / mL) (range can be 5 ng / ml to 50 ng / mL), IL-15 (10 ng / mL) (range can be 5 ng / ml to 50 ng / mL), IL-2 (10 ng / mL) (range can be 5 ng / ml to 20 ng / mL). 2. K562 cells are prepared by seeding K562 cells in cell culture well plate so that 3000 cells are added per well. As control, wells containing only K562 cells are similarly prepared. Therefore, the control wells are not for co-culture with differentiated immune cells. 3. Differentiated immune cells were added at ratios of 1:1, 1:2, 1:4, and 1:8, respectively. As an additional control, separate wells contained only differentiated immune cells, not co-cultured with K562 cells, and the number of differentiated immune cells was equal to the 1:8 ratio wells. 4. Co-cultures and controls were cultured in immune cell differentiation medium containing IL-2 for 24 hours at 37° C., 5% CO2. 5. After 24 hours, the medium was collected and LDH assay was performed and measured according to the manufacturer's protocol (Sigma Aldrich, Cat. No. MAK066-1KT). The percentage of specific release of LDH was calculated. The data is shown in Figure 6. The data shown therein represent the mean value + / - SD of triplicate cultures. The results of the LDH assay could show that the cytotoxicity measured by LDH activity was significantly higher in the 1:4 and 1:8 ratio groups of co-culture samples compared to the control group. In addition, microscopic analysis was also performed, which is shown in Figure 6. According to the microscopic analysis, K562 cells were observed to be extremely low or absent in the 1:4 and 1:8 mixed co-cultures.

[0118] In summary, cytotoxic activity could be observed for the differentiated immune cells of the present invention.

Claims

1. 1. A method for generating a population of immune cells from pluripotent stem cells (PSCs), comprising: (i) (a) culturing PSCs that have undergone mesenchymal differentiation in a serum-free medium on a solid support suitable for the formation of 3D cell aggregates in the presence of Notch ligand delta-like ligand 4 (DLL4) signaling activity under suitable conditions, thereby allowing the formation of 3D cell aggregates; (b) harvesting the 3D cell aggregates and culturing them under appropriate conditions in suspension culture in serum-free medium for about 3-6 days; and (c) culturing the 3D cell aggregates under appropriate conditions in serum-free medium for an additional period of about 4 to 7 days. inducing 3D cell aggregate formation and hematopoietic differentiation by (ii) inducing immune cell differentiation by culturing the 3D cell aggregates of (i) in serum-free medium in suspension culture for a suitable time under suitable conditions. thereby providing a population of immune cells.

2. (0i)(a) seeding PSCs in serum-free medium under appropriate conditions; (b) culturing the PSCs in serum-free medium under appropriate conditions for about 2-4 days; and (c) Resuspending the cells in serum-free medium Inducing mesoderm differentiation by 2. The method of claim 1, wherein a further step (0i) comprising:

3. Serum-free medium, In step (0i), (a) activin A, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and an apoptosis inhibitor, preferably a ROCK inhibitor, each at a concentration of about 10-50 ng / mL; (b) BMP4, VEGF, and FGF, each at a concentration of about 5-15 ng / mL; (c) an apoptosis inhibitor, preferably a ROCK inhibitor Including; In step (i), (a) an inhibitor of the ALK receptor and a ROCK inhibitor, each at a concentration of about 5-15 μM; (b) about 5 to 15 ng / mL of an inhibitor of the ALK receptor; (c) about 5-15 ng / mL inhibitor of the ALK receptor, about 40-60 ng / mL stem cell factor (SCF), about 5-15 ng / mL FMS-like tyrosine kinase 3 ligand (Flt-3l), and about 5-15 ng / mL thrombopoietin (TPO). Including; In step (ii), About 1-3% B27 supplement, about 5-15 ng / mL SCF, about 5-15 ng / mL Flt-3l, about 5-15 ng / mL IL-7, and about 15-45 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate Including, 3. The method of claim 2.

4. The method of claim 3, wherein the ROCK inhibitor is Y-27632 dihydrochloride and the inhibitor of the ALK receptor is SB43152.

5. 2. The method of claim 1, wherein the suspension culture in steps (i) and (ii) is a dynamic suspension culture by culturing in a shaker or a dynamic suspension culture by culturing in an orbital shaker with a rotation of about 60-80 rpm.

6. 2. The method of claim 1, wherein step (ii) is carried out for about 21 to 50 days.

7. 10. The method of claim 1, characterized by one or more of the following: - the PSCs are human induced pluripotent stem (iPS) cells, preferably the human iPS cell line TC-1133; ・PSC, Pluripotent stem cells that lack endogenous MHC class I molecules presented on the cell surface of the pluripotent stem cells and contain immunomodulatory proteins on the cell surface That is; - the immunomodulatory protein is a single chain fusion HLA class I protein; - Single chain fusion HLA class I protein, At least a portion of B2M covalently linked to at least a portion of an HLA class I α chain selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. Contains; - the pluripotent stem cells further express a target peptide antigen that is presented on the pluripotent cell surface by a single chain fusion HLA class I protein; - the target peptide antigen is covalently linked to a single chain fusion HLA class I protein; - the target peptide antigen comprises the sequence VMAPRTLFL (SEQ ID NO:1); - Disruption of essentially all copies of the β2 microglobulin gene in pluripotent stem cells; - the pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, and parthenogenetic stem cells; - the pluripotent stem cells are primate-derived pluripotent stem cells, preferably human pluripotent stem cells; - the pluripotent stem cells were generated from CD34 positive cells isolated from umbilical cord blood; and / or - The pluripotent stem cells are ND-50039 from the NINDS Human Cell and Data Repository.

8. 10. The method of claim 1, wherein the PSC is characterized by one or more of the following: - the PSCs are pluripotent stem cells that lack endogenous MHC class II; and / or -PSCs lack endogenous MHC class II due to disruption of the C2TA gene-MHC class II transactivator.

9. The method of claim 1 or 2, characterized by one or more of the following: - in step (i), Notch ligand delta-like ligand 4 (DLL4) signaling activity is provided by co-culturing the PSCs with stromal cells expressing DLL4 or by incubating the PSCs with beads coated with DLL4; The ratio of PSCs to stromal cells is in the range of approximately 1:5 to 1:40; In step (i), the stromal cells are the bone marrow-derived murine stromal cell line MS5; In step (i), the stromal cells are stromal cells differentiated from PSCs that express DLL4; - the PSCs are cultured in a cell culture vessel comprising at least one extracellular matrix protein during the induction of mesodermal differentiation in step (0i); - the container is coated with said at least one extracellular matrix protein; and / or - At least one extracellular matrix protein is selected from the group consisting of vitronectin, laminin, collagen, fibronectin, elastin, matrigel, peptides containing the amino acid sequence RGD, proteins containing the amino acid sequence RGD, and combinations thereof.

10. 10. The method of claim 1, characterized by one or more of the following: - in step (i)(a), the solid support comprises one or more wells, each well comprising a V-shaped or conical cavity; the solid support is a microwell culture plate; and / or - The microwell culture plate is an AggreWell™ plate (available from StemCell Technologies), a BIOFLOAT™ 96-well plate (available from faCellitate), or a Nunclon Sphera 3D culture (available from ThermoFisher).

11. 2. The method of claim 1, wherein the 3D cell aggregates are dissociated after step (ii) to provide single cells.

12. 10. The method of claim 1, characterized by one or more of the following: - the population of immune cells comprises T cell-like cells and NK cell-like cells; - a further step (iii) is carried out after step (ii) which comprises inducing, and preferably also selectively expanding and activating, T-cell-like cells and NK-cell-like cells; - in step (iii), inducing, selectively expanding and activating T-cell-like cells under appropriate conditions; - in step (iii), selectively expanding the T cell-like cells in a medium comprising B27 supplement, SCF, Flt-31, IL-7, IL-2, IL-15, and a CD3 stimulatory binding molecule; the medium in step (iii) comprises about 1-3% B27 supplement, about 5-15 ng / mL SCF, about 2-7 ng / mL Flt-13, about 5-15 ng / mL IL-7, about 5-15 ng / mL IL-2, about 5-15 ng / mL IL-15, and the CD3 stimulatory binding molecule is selected from an anti-CD3 antibody, e.g., an OKT-3 antibody, and a multimerization reagent carrying an OKT-3 Fab fragment, preferably at a concentration of about 250-750 ng / mL; - a further step (iv) is carried out after step (iii) which comprises further expanding the immune cells by culturing them under suitable conditions in serum-free medium containing B27 supplement and IL-2; the serum-free medium of step (iv) comprises 1-3% B27 supplement and 30-70 units / mL IL-2; and / or - Immune cell populations are characterized by expression of CD4, CD8, CD56, CD3, and CD45.

13. 13. The method of claim 1 or 12, which is suitable for generating a population of immune cells having high cell numbers by using bioreactor conditions in one or all of steps (ii), (iii) and / or (iv).

14. 2. A population of immune cells or non-immunogenic T cells accessible or obtained by the method of claim 1.

15. The non-immunogenic T cells obtainable by the method of claim 1, characterized by one or more of the following: - the T cell lacks an endogenous MHC class I molecule displayed on the cell surface of the T cell and contains an immunomodulatory protein on its surface; - Non-immunogenic T cells express a chimeric antigen receptor (CAR) or a foreign T cell receptor (TCR) on their surface; - essentially all copies of the β2 microglobulin 2 gene are disrupted in T cells, such that the T cells lack endogenous MHC class I molecules on the cell surface; - the immunomodulatory protein is a single chain fusion HLA class I protein; - Single chain fusion HLA class I protein, At least a portion of B2M covalently linked to at least a portion of an HLA class I α chain selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. Contains; - the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A; - the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A0201; - the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-E; - the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-G; - the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-B; - the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-C; - the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-F; - the non-immunogenic T cells further express target peptide antigens that are presented on the pluripotent cell surface by single-chain fusion HLA class I proteins; - the target peptide antigen is covalently linked to a single chain fusion HLA class I protein; - the target peptide antigen comprises the sequence VMAPRTLFL (SEQ ID NO:1); - the gene for the immunomodulatory protein is integrated into the (endogenous) B2M locus of the T cell genome; - the CAR or exogenous TCR is integrated into the endogenous TCR α gene, the endogenous TCR β gene, or into both the endogenous TCR α gene and the endogenous TCR β gene; - CAR or foreign TCR binds to an antigen displayed on the surface of a target cell; - the antigen is a tumor-associated antigen, a viral antigen or a bacterial antigen, preferably a tumor-associated antigen; - the antigen is CD19 or a CMV-derived antigen; - the T cells are T cells of primate origin; and / or - The T cells are human T cells.

16. 16. A pharmaceutical composition comprising the population of immune cells or the non-immunogenic T cells of claim 14 or the non-immunogenic T cells of claim 15.

17. 17. The pharmaceutical composition of claim 16 for use in a method of treatment or cell therapy.

18. 18. The pharmaceutical composition of claim 17, wherein the treatment or therapy is for cancer.

19. 19. The pharmaceutical composition of claim 18, wherein the cancer is selected from lung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, skin cancer, melanoma, colon cancer, rectal cancer, gastric cancer, esophageal cancer, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, breast cancer, ovarian cancer, lymphatic system cancer, e.g. lymphoma and multiple myeloma, leukemia, sarcoma of bone or soft tissue, cervical cancer, and vulvar cancer.

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