Method of producing an immune cell devoid of endogenous effector functions from pluripotent stem cells

Abstract

The present invention relates to a method of producing an immune cell devoid of endogenous effector functions from pluripotent stem cells. Further, the present invention relates to a method of producing an immune cell devoid of endogenous effector functions, but comprising one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function; an immune cell or a population of immune cells obtainable or obtained by the methods of the invention, a pharmaceutical composition comprising the immune cells or the population of immune cells, as well as to the immune cell or the population of immune cells obtainable or obtained by the methods of the invention, or the pharmaceutical compositions for use in medicine.

Description

METHOD OF PRODUCING AN IMMUNE CELL DEVOID OF ENDOGENOUS EFFECTORFUNCTIONS FROM PLURIPOTENT STEM CELLSCROSS-REFERENCE TO RELATED APPLICATIONSThe present application claims the benefit of priority of EP Patent Application No. 23210624.5filed 17 November 2023, the content of which is hereby incorporated by reference in its entiretyfor all purposes. TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to a method of producing an immune cell devoid ofendogenous effector functions from pluripotent stem cells. Further, the present invention relates to a method of producing an immune cell devoid of endogenous effector functions, but comprising one or more exogenous modifications, wherein each of the one or more exogenousmodifications introduces a predetermined effector function, an immune cell or a population ofimmune cells obtainable or obtained by the methods of the invention, a pharmaceuticalcomposition comprising the immune cells or the population of immune cells, as well as to theimmune cell or the population of immune cells obtainable or obtained by the methods of theinvention or the pharmaceutical compositions for use in medicine.BACKGROUND OF THE INVENTION

[0002] The development of immune cell populations, in particular T cell populations, for use indiagnosis and therapy is a current task. To be able to provide such immune cell populations, such as differentiated T cell populations, stem cells are a good source. The crucial characteristic of stem cells is their capability to self-renew indefinitely and to differentiate in multiple different types of cells or tissue. The self-renewal capability of stem cells is crucial for their characteristic as pool of primitive undifferentiated cells. Further, the characteristic of high“flexibility” and “plasticity” of stem cells is founded on their capability to trans-differentiate intotissues which may be different from their origin. Pluripotent stem cells (PSCs) are in the focus as source for the generation of T cells. Currently, the most utilized pluripotent cells areembryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). In particular, iPSCswhich represent non-embryonic cells and have been isolated from an already developedorganism, are reprogrammed to pluripotent stem cells. ESCs being derived from embryonicblastocysts, thus being totipotent, have not been differentiated yet and never been programmed.

[0003] The generation of T cells from human iPSCs is known in the art. T cells, which areadditionally and individually engineered, are a powerful tool for the use in targeted therapiesof diseases which require a directed immune response, such as cancer. It has been shownthat T cells can be cultured and proliferated in vitro for use in adoptive cellular immunotherapyor cancer therapy in which such T cells have been proofed to possess anti-tumor activity in apatient having a tumor. However, such differentiated and engineered T cells when used intherapy can contribute to side effects during such treatments and therapies resulting in a notpredictable safety profile for the patient.

[0004] Therefore, there is a need for the provision of immune cells which can be applied intherapy and are safer for the patient, thus not providing any side effects to the patient.

[0005] Consequently, it is an object of the present invention to provide a method for producingan immune cell which fulfils said demand. This object is solved by the subject-matter of theindependent claims. SUMMARY OF THE INVENTION

[0006] The inventors discovered a method of producing an immune cell or a population ofimmune cells devoid of endogenous effector functions, meaning such immune cells as suchwhich can be characterized by the inventors e.g. as a NK like precursor cell via the expression of the phenotypic marker CD56 for NK cells as defined elsewhere herein do not have anynaturally occurring effector functions against any certain target and / or other effector cells, theimmune cell is interacting with (such as not having any cytolytic function when attacking acertain target cell, e.g. a target tumor cell, and / or not having any immunosuppressive orimmunomodulatory function against another effector cell, e.g. autoreactive T cell).

[0007] Being devoid of any endogenous effector functions may comprise not expressing anyendogenous (naturally occurring) surface molecules on the cell surface of the producedimmune cell, such surface molecules that would naturally be required for triggering any effectorfunction against said target cell, e.g. a tumor target cell. In this regard, such surface moleculerequired for triggering any effector function may refer to, but is not limited to, the T cell receptor(TCR); or the CD16 receptor, the CD94 / NKG2D receptor, or the NKp30 receptor (see Figure1A and B). Such term may also comprise that the produced immune cell is not able to exertany effector molecules such as cytolytic factors when interacting with a target cell or not ableto exert any modulatory molecules (e.g. cytokines) such as IL-10 and / or TGF-β cytokines.Such term “being devoid of any endogenous effector functions” may not comprise that theproduced immune cell can be deficient of endogenous MHC class I and / or MHC class IImolecules on their cell surface and additionally can express on their cell surface a recombinantimmunomodulatory protein as defined herein, preferably being a single chain fusion HLA classI protein, the resulting effect providing that the immune cell as such will not be attacked by therecipient’s T cells – in other words the risk of a rejection reaction of the immune system of therecipient is decreased when the immune cell is deficient of endogenous MHC class I and / orMHC class II molecules, additionally also expressing the recombinant immunomodulatory protein as defined herein.

[0008] Also the expansion potential is limited for such generated immune cells (see Figure 1Cand D) meaning that such immune cells die off more quickly for their own safety compared tonaturally occurring immune cells still comprising their natural effector functions.

[0009] In sum, such produced immune cells devoid of any endogenous effector functions maybe considered as “neutral cells” not providing any side effects (due to the lack of all effectorfunctions naturally inherent to such cell) if used in therapy of any kind and if converted to theparticular effector cell needed in such therapy as further described herein.

[0010] Providing such immune cells is achieved by the first method of the invention asdescribed herein, the method comprising the steps of: (i) inducing 3D cell aggregateformation, mesodermal differentiation and hematopoietic differentiation by (a) seeding PSCsunder suitable conditions on a solid support suitable for formation of 3D - cell aggregates, in afirst serum-free medium, thereby allowing the formation of 3D - cell aggregates; (b) collectingthe 3D - cell aggregates of step (a), resuspending the 3D – cell aggregates under suitableconditions in a second serum-free medium, and transferring the resuspended 3D – cellaggregates into and culturing in a third serum-free medium under suitable conditions insuspension culture for about 1 to about 3 days, thereby allowing the mesodermaldifferentiation; and (c) transferring the cultured 3D - cell aggregates of step (b) into and culturing in a fourth serum-free medium under suitable conditions in suspension culture forabout 2 to about 4 days and further culturing the 3D - cell aggregates under suitable conditionsin suspension culture for about 2 to about 6 days in a fifth serum-free medium, thereby allowingthe hematopoietic differentiation; (ii) inducing immune cell differentiation by culturing singlecells of the 3D - cell aggregates obtained from step (i)(c) under suitable conditions insuspension culture for a suitable time in a sixth serum-free medium, thereby providing animmune cell devoid of endogenous effector functions.

[0011] Additionally, the present inventors discovered that such immune cells obtained by theabovementioned method can be seen as cellular neutral vehicles for e.g. introducing targeted desired molecules in order to introduce a predetermined effector function which may bedesired in a particular therapy which then leads to a much more efficient therapeutic approach.In other words, such immune cells devoid of any endogenous effector functions can for example be the base to introduce exogenous gain-of-function genetic modifications so that the produced immune cell is devoid of any endogenous effector functions as described above, butcomprises one or more exogenous genetic modifications wherein each of the one or moreexogenous modifications introduces a predetermined effector function.

[0012] Providing such immune cells is achieved by the second method of the invention asdescribed herein, the method comprising the steps of: (i) inducing 3D cell aggregateformation, mesodermal differentiation and hematopoietic differentiation by (a) seeding PSCsunder suitable conditions on a solid support suitable for formation of 3D - cell aggregates, in afirst serum-free medium, thereby allowing the formation of 3D - cell aggregates; (b) collectingthe 3D - cell aggregates of step (a), resuspending the 3D – cell aggregates under suitableconditions in a second serum-free medium, and transferring the resuspended 3D – cellaggregates into and culturing in a third serum-free medium under suitable conditions insuspension culture for about 1 to about 3 days, thereby allowing the mesodermaldifferentiation; and (c) transferring the cultured 3D - cell aggregates of step (b) into and culturing in a fourth serum-free medium under suitable conditions in suspension culture forabout 2 to about 4 days and further culturing the 3D - cell aggregates under suitable conditionsin suspension culture for about 2 to about 6 days in a fifth serum-free medium, thereby allowing the hematopoietic differentiation; (ii) inducing immune cell differentiation by culturing singlecells of the 3D - cell aggregates obtained from step (i)(c) under suitable conditions insuspension culture for a suitable time in a sixth serum-free medium, wherein the method further comprises genetically introducing one or more exogenous modifications into the PSCs in anyone of before step (a), in step (a) to (c) of step (i) or in step (ii) , wherein each of the one ormore exogenous modifications introduces a predetermined effector function, thereby providinga genetically modified immune cell being devoid of endogenous effector functions, butcomprising one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function.

[0013] In sum, the provision of an immune cell or a population of immune cells devoid ofendogenous effector functions as described herein based on the particular steps and order of the first method of the invention has the advantage that the subsequent application of suchcell in therapy are safer for the patient. By then additionally genetically introducing one or moreexogenous modifications at the PSCs’ level (so into the PSC’s genome) either before step (a),or in any one of step (a), (b), or (c) of step (i), or in step (ii) of the second method of the invention, an immune cell or a population of immune cells devoid of endogenous effectorfunctions can be produced which - due to the artificially introduced modification into the PSCs’genome - then exhibits a predetermined effector function which is desired for a particularapplication of the immune cell.

[0014] The invention also relates to an immune cell or a population of immune cells devoid ofendogenous effector functions obtainable or obtained by the first method of the invention asdefined herein. Further, the invention also relates to an immune cell or a population of immune cells devoid of endogenous effector functions, but comprising one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces apredetermined effector function obtainable or obtained by the second method of the invention.

[0015] The invention also relates to a pharmaceutical composition comprising the immune cellor the population of immune cells obtainable or obtained by the methods of the invention asdefined elsewhere herein.

[0016] Additionally, the invention relates to the immune cell or the population of immune cellsobtainable or obtained by the methods of the invention as defined elsewhere herein or thepharmaceutical composition as defined elsewhere herein for use in medicine. Further, theinvention relates to the immune cell or the population of immune cells obtainable or obtainedby the methods of the invention as defined elsewhere herein or the pharmaceuticalcomposition as defined elsewhere herein for use in a method of preventing or treating cancer. BRIEF DESCRIPTION OF THE FIGURES

[0017] Figure 1 shows iPSC-derived cells having limited functional potential. (A) iPSCdifferentiated lymphocytes resemble cells from natural lymphocyte compartment. (B) iPSCdifferentiated lymphocytes such as NK like precursor cells do not express surface molecules(e.g. CD94, CD16, NKG2D, NKp30) required for triggering of the effector function. Figure 1(B)last panel contains CD3xCD19 staining, thus proving lack of TCR / CD3 receptor expressionthat is prerequisite for T cell identification and their endogenous cytolytic function. Lack of CD3signal also excludes identification as NKT cells. (C) and (D) iPSC differentiated lymphocyteshave limited expansion potential in in vitro culture.

[0018] Figure 2 shows iPSC-derived cells showing no cytolytic function in vitro. (A)Conventional CAR construct (anti-CD19) was stably knocked-in into iPSC line to serve as acontrol (CAR iPSCs diff.). (B) Co-culture of CD19+GFP+ Raji (target) cells and CAR iPSC diff.(effectors) demonstrating interaction between CAR receptor and CD19 antigen by loss ofCD19 signal. (C) and (D) K652 and Raji co-cultures demonstrate lack of unspecific andantigen-specific cytolytic functions of CAR iPSC diff. conversely to controls: NK cells and CAR T cells, respectively.

[0019] Figure 3 shows iPSC-derived cells showing no cytolytic function in vivo. CAR iPSC diff.show no tumor control in in vivo NSG mouse model pre-injected with Raji cells when comparedto CAR T cell control.

[0020] Figure 4 shows that iPSC-derived cells can be used as a base to introduce gain-of-function genetic modifications. (A) Scheme depicting terminally differentiated lymphocyte fromunmodified iPSC (labeled with a star), effector such as T cell or CAR T cell (labeled with acircle) and target like tumor cell (not labeled). (B) Scheme depicting terminally differentiatedlymphocyte from genetically edited iPSC that can exert effector molecules (e.g., cytolytic factors) after an interaction with tumor target cell (under control of target-specific induciblesystem). (C) Scheme depicting boosting function of terminally differentiated lymphocyte fromgenetically modified iPSC, which carry tumor-specific antigen. Interaction with CAR T cell willenhance CAR T cell function to more effectively kill target tumor cell. (D) Scheme depictingimmuno-modulating (immuno-suppressing) function of terminally differentiated lymphocyte from genetically modified iPSC that can exert modulatory molecules (e.g., cytokines) after an interaction with effector cell (autoreactive T cell). In this instance, non-malignant target cell will be protected. DETAILED DESCRIPTION OF THE INVENTION

[0021] The method of the invention for producing an immune cell or a population of immunecells being devoid of any endogenous effector functions and which is derived from PSCs asdefined elsewhere herein comprises inter alia step (i) which refers to the step of a 3D cellaggregate formation which corresponds to step (i)(a), followed by the step of mesodermal differentiation which corresponds to step (i)(b), followed by the step of hematopoietic differentiation which corresponds to step (i)(c).

[0022] The 3D cell aggregate formation of step (i)(a) comprises the seeding of PSCs undersuitable conditions on a solid support suitable for formation of 3D - cell aggregates as definedelsewhere herein in a first serum-free medium, thereby allowing such formation of the 3D cellaggregates. In this context, the “first serum-free medium” refers to a medium not comprisingserum for culturing PSCs, such as an iPSC expansion medium, preferably being supplementedwith an inhibitor of GSK-3 and a ROCK inhibitor as defined elsewhere herein, more preferablyeach in a concentration of about 5 to about 15 µM (see Example 1). “Under suitable conditions”in this context may refer to conditions known to a person skilled in the art that allows theformation of 3D cell aggregates when PSCs are seeded on a solid support suitable forformation of 3D - cell aggregates in the first serum-free medium as described elsewhere herein.Preferably, about 1.5 to about 4.5x106 cells, preferably about 3 x106 cells, are seeded on thesolid support as defined herein. Adjusting the cell number of the PSCs within this distinct range may have impact on the growth factor ratio in the medium and may also influence the subsequent differentiation.

[0023] The mesodermal differentiation of step (i)(b) comprises the collection of the alreadyformed 3D cell aggregates in step (i)(a), followed by a resuspension step of the 3D cellaggregates under suitable conditions in a second serum-free medium (not the same as thefirst serum-free medium as used in step (i)(a)), and followed by transferring the resuspended3D – cell aggregates into a third serum-free medium and culturing the aggregates in said thirdserum-free medium (not the same as the first or the second serum-free medium) under suitableconditions in suspension culture as defined elsewhere herein for about 1 to about 3 days, suchas about 1 day, about 2 days, about 3 days, preferably for about 2 days. In this context, a“second serum-free medium” as used for the resuspension step refers to a medium notcomprising any serum which induces mesodermal differentiation, such as a mesoderm induction medium, preferably supplemented with growth factors such as bone morphogenicprotein 4 (BMP4), vascular endothelial growth factor (VEGF) and fibroblast growth factor(FGF), more preferably each in a concentration of about 10 to about 50 ng / ml (see Example1). The “third serum-free medium” as used for culturing the 3D cell aggregates within the mesodermal differentiation refers to a medium not comprising any serum which induces final mesodermal differentiation, such as an mesoderm induction medium, preferably supplemented with growth factors such as BMP4, VEGF, FGF and an inhibitor of ALKreceptors (see Example 1), more preferably each of BMP4, VEGF and FGF in a concentrationof about 10 to about 50 ng / ml and the inhibitor of ALK receptors in a concentration of about 5to about 15 µM. Such an inhibitor of ALK receptor (e.g. SB431542) may refer to a TGF-betainhibitor required for higher efficiency of mesoderm differentiation and may prevent TGF-beta enhanced differentiation of cell types other than mesodermal cells. “Under suitable conditions” in this context may refer to conditions known to a person skilled in the art that allowsmesodermal differentiation of the 3D cell aggregates collected after step (i)(a) in the secondand third serum-free medium as described elsewhere herein. The already formed 3D cellaggregates obtained from step (i)(a) when then cultured in suspension culture as defined herein do not attach to such culture plate, since such suspension culture plate is an ultra low attachment suspension culture plate. Such non-attachment of the aggregates to the plate dueto the ultra low attachment suspension culture the aggregates are comprised in, prevents thatsuch cells do not differentiate into undesired cell types.

[0024] The hematopoietic differentiation of step (i)(c) comprises transferring the cultured 3D –cell aggregates of step (b) into a fourth serum-free medium (not the same as the first, second or third serum-free medium) and culturing said aggregates in said fourth serum-free medium under suitable conditions in suspension culture for about 2 to about 4 days, such as about 2 days, about 3 days, or about 4 days, preferably about 3 days, followed by further culturing the 3D cell aggregates under suitable conditions in suspension culture for about 2 to about 6 days, such as about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days, preferablyfor about 4 days, in a fifth serum-free medium (not the same as the first, second, third or fourthserum-free medium). In this context, a “fourth serum-free medium” as used in the first culturing step within the hematopoietic differentiation refers to a medium not comprising any serum which induces hematopoietic differentiation, such as a hematopoietic induction medium,preferably supplemented with growth factors such as VEGF, FGF and stem cell factor (SCF),more preferably each in a concentration of about 10 to about 50 ng / ml (see Example 1). The“fifth serum-free medium” as used for the second culturing step within the hematopoieticdifferentiation refers to a medium not comprising any serum which induces final hematopoietic differentiation, such as an hematopoietic induction medium, preferably supplemented withgrowth factors such as VEGF, FGF, SCF and FMS-like tyrosine kinase 3 ligand (Flt-3l) as wellas Thrombopoietin (TPO), more preferably each of VEGF, FGF, SCF, FIt-3I and TPO in aconcentration of about 10 to about 50 ng / ml (see Example 1). Flt-3l and TPO may be requiredfor the final differentiation of the PSCs to allow hematopoietic differentiation. “Under suitable conditions” in this context may refer to conditions known to a person skilled in the art that allows hematopoietic differentiation of the 3D cell aggregates obtained after mesodermaldifferentiation in step (i)(b) in the fourth and fifth serum-free medium as described elsewhereherein. The suspension cultures as used in step (b) and (c) may be considered as different,even though the aggregates are in the same type of plate. However, the aggregates are atdifferent stages of development or differentiation as they are exposed to different growth factors within different media used for each step.

[0025] The method of the invention for producing an immune cell or a population of immunecells being devoid of any endogenous effector functions and which is derived from PSCs asdefined elsewhere herein additionally also comprises besides step (i) as defined above step (ii) which refers to the step of the immune cell differentiation. Such step (ii) comprises culturingsingle cells of the 3D cell aggregates obtained from step (i), in particular obtained from step(i)(c), under suitable conditions in suspension culture for a suitable time in a sixth serum-freemedium (not the same as the first, second, third, fourth or fifth serum-free medium). In thiscontext, a “sixth serum-free medium” as used in the immune cell differentiation refers to amedium not comprising any serum which induces immune cell differentiation, such as aimmune cell differentiation medium, preferably supplemented with IL-3, SCF, TPO, Flt3l, IL-7, IL-15, SDF and SB203580, more preferably each of IL-3, TPO, IL-15, SDF, SCF, FIt-3I, IL-7in a concentration of about 10 to about 50 ng / ml and SB203580 in a concentration of about 7to about 23 µM (see Example 1). “Under suitable conditions” in this context may refer toconditions known to a person skilled in the art that allows immune cell differentiation of the 3Dcell aggregates after hematopoietic differentiation in step (i)(c) in the sixth serum-free mediumas described elsewhere herein. Here, the suspension culture is considered as being different to the suspension cultures used in step (b) and (c) as the plates used in step (ii) are different compared to the plates used in step (b) and (c) as defined further herein (e.g. Notch ligandcoated or uncoated plates in step (ii)). Also in this step not the aggregates themselves arecultured in such suspension culture, but the single cells of the 3D cell aggregates afterdissociating such aggregates into single cells. “For a suitable time” in this context may refer toa particular time frame which is known to a person skilled in the art in which the PSCs as singlecells of the 3D cell aggregates in suspension culture as defined herein for step (ii) are converted to an immune cell of the invention, preferably such differentiation takes less than 50 days, more preferably less than 25 days, most preferably less than 21 days.

[0026] Additionally, the method of the invention as defined elsewhere herein can furthercomprise genetically introducing one or more exogenous modifications into the PSCs in any one of before step (a), or in any one of step (a), (b), or (c) of step (i), or in step (ii) of the methodof the invention to provide a genetically modified immune cell being devoid of endogenouseffector functions, but comprising one or more exogenous modifications, wherein each of theone or more exogenous modifications introduces a predetermined effector function. In otherwords, at the PSC level of the method of the invention as defined elsewhere herein, before step (a), or in any one of step (a), (b), or (c) of step (i), or in step (ii), the PSCs can be genetically modified, meaning that the PSCs’ genome can be genetically modified by introducing one ormore exogenous (genetic) modifications in order to then modify the later produced immunecell devoid of endogenous effector function which are derived from said genetically modifiedPSCs. In this context, the term “before step (a) of step (i)” may refer to any step which can beperformed on PSCs as defined herein, such as particularly culturing such PSCs, beforeseeding them as described in step (a).

[0027] The term “endogenous” within the context of “endogenous effector functions” as usedherein can be replaced by the term “naturally occurring”, meaning any effector functions asdefined herein that are inherent to the immune cell as such. The term “devoid of” as used inthe context of the invention means that such immune cell does not have or does not compriseany or lacks all endogenous effector functions as defined herein after being produced by themethods of the invention.

[0028] The term “exogenous” on the other hand means “artificial” or “recombinant”.“Exogenous modification(s)” may thus refer to modification(s) as defined herein beinggenetically introduced into the PSCs (into the PSCs genome) by any suitable technique, including for example homologous recombination, gene editing methods or recombinase technology as known to a person skilled, so that such modification(s) within the PSCs genome is artificial / recombinant. The term "one or more (genetic) modification" as used hereingenerally refers to one or more alterations of a nucleic acid (DNA or RNA), e.g., the nucleicacid within a PSC's genome. For example, a genetic modification can refer to alterations, insertions (e., gene knock-ins), and / or deletion of genes (e.g., gene knock-outs). Preferably, this one or more genetic modification may result in incorporation and expression of one ormore recombinant genes encoding for a particular molecule which results in a desired effectorfunction of the genetically modified immune cell.

[0001] Within each of step of (a), (b) and / or (c) of step (i), or before step (a) of step (i) or instep (ii) of the method of the invention, when genetically introducing the exogenousmodifications, one modification can be introduced, but also more than one, such as two, three,four, five modifications. Preferably, said exogenous (genetic) modification is selected from thegroup consisting of a gene knock-in, a gene knock-out, a gene replacement, a point mutation,and a deletion, insertion or substitution of a gene, a gene fragment or a nucleotide, or acombination thereof. As used herein, the term "gene knock-out (deletion or inactivation)" refersto a genetic modification resulting from the deletion of the genetic information encoded in achromosomal locus within the PSC genome or resulting from a gene inactivation wherein thegenetic information encoded in a chromosomal locus is altered, thereby affecting transcription and / or translation of the gene. As used herein, the term "gene knock-in (insertion)" is a geneticmodification resulting in the insertion of a gene into a specific locus within the PSC genomethat was not previously present in said genome. As used herein, the term "gene replacement (substitution)" is a genetic modification resulting in the replacement of the genetic informationencoded in a chromosomal locus within the PSC genome (e.g. from human) with thecorresponding gene from another subject. The term "gene" is used herein broadly to refer to any segment of DNA containing a gene sequence associated with a biological function. Thus, genes include coding and / or non-coding sequences and / or the regulatory and control sequences required for their expression. A functional gene fragment may also include such sequences in order for the required expression.

[0029] In one embodiment, said exogenous modification being introduced into the PSCs is agene knock-in as defined herein. In another embodiment, said exogenous modification beingintroduced into the PSCs is a gene knock-out as defined herein. In another embodiment, saidexogenous modification being introduced into the PSCs is a gene replacement as definedherein. In another embodiment, said exogenous modification being introduced into the PSCsis a point mutation as defined herein. In another embodiment, said exogenous modificationbeing introduced into the PSCs is a deletion of a gene, a gene fragment or a nucleotide asdefined herein. In another embodiment, said exogenous modification being introduced into thePSCs is an insertion of a gene, a gene fragment or a nucleotide as defined herein. In anotherembodiment, said exogenous modification being introduced into the PSCs is a substitution ofa gene, a gene fragment or a nucleotide as defined herein. It may thus also be comprised herein, that said exogenous modification being introduced into the PSCs refers to a gene knock-out and another exogenous modification (in sum then two modifications) being introduced into the PSCs refers to a gene knock-in, or said exogenous modification beingintroduced into the PSCs refers to a gene knock-in and another exogenous modification (insum again then two modifications) being introduced into the PSCs refers to another geneknock-in and so on. If such modifications are modifications for e.g. a T cell, such modificationscould be introduced into the same PSCs used within the methods of the invention. If suchmodifications are modifications for e.g. a T cell and for e.g. a NK cell, such modifications wouldbe introduced into different PSCs used within the methods of the invention to provide a T celland a NK cell devoid of endogenous effector functions, but each exhibiting its desired effector function due to its modification being introduced.

[0030] Each exogenous modification on the gene level as defined herein results in introducinga predetermined effector function. Meaning, if you introduce e.g. two exogenous modificationsinto the PSCs as defined herein, the first modification may result in for example introducing apredetermined effector function and the second modification may result in introducing anotherpredetermined effector function. In this context, a “predetermined” effector function is a specificeffector function as it has been defined herein, e.g. against a certain target cell or againstanother effector cell, which the genetically modified immune cell produced by the methods of the invention should comprise so that the one or more exogenous modification being introduced into the PSCs’ genome is particularly chosen beforehand in order to achieve thespecific effector function / in order that the genetically modified immune cell which is devoid ofany endogenous effector function due to the method of the invention indeed will comprise the specific effector function as it has already been defined herein resulting from the exogenouslyintroduced modification (see Figure 4).

[0031] Preferably, the gene knock-in comprises the knock-in of at least one gene selectedfrom the group consisting of a gene encoding a chimeric antigen receptor (CAR); anexogenous T cell receptor (TCR); an exogenous IL-15 receptor; an exogenous CD16 receptor;an exogenous CD19 antigen; an exogenous IL-10 cytokine; an exogenous TGF-β cytokine; and an exogenous PD ligand 1 (PD-L1) antigen, or a combination thereof. In one embodiment, the gene knock-in comprises the knock-in of the gene encoding a CAR. In another embodiment, the gene knock-in comprises the knock-in of the gene encoding an exogenousTCR, such TCR providing cytolytic function against a target cell. In another embodiment, thegene knock-in comprises the knock-in of the gene encoding an exogenous IL-15 receptor,such receptor providing immunomodulatory function (e.g. persistency of the immune cell). Inanother embodiment, the gene knock-in comprises the knock-in of the gene encoding an exogenous CD16 receptor, such CD16 receptor providing cytolytic function against a target cell. In another embodiment, the gene knock-in comprises the knock-in of the gene encodingan exogenous CD19 antigen, such antigen providing immunomodulatory (immunostimulatory)function. In another embodiment, the gene knock-in comprises the knock-in of the geneencoding an exogenous IL-10 cytokine, such cytokine providing immunosuppressive function.In another embodiment, the gene knock-in comprises the knock-in of the gene encoding an exogenous TGF-β cytokine, such cytokine providing immunosuppressive function. In another embodiment, the gene knock-in comprises the knock-in of the gene encoding an exogenous PD ligand 1 (PD-L1) antigen, such ligand providing immunosuppressive function.

[0032] A CAR protein may comprise a single-chain variable fragment (scFv) of an antibodywith binding capacity for a specific tumor associated antigen, linked via a transmembrane peptide to intracellular co-stimulatory domains such as CD28, OX40 and CD137. Thesepeptides are subsequently fused to the signaling domains of the TCRζ chain that activates theCAR T cell, if it binds its epitope on a tumor cell. Subsequent release of granzymes andperforins leads to tumor cell lysis (June CH, O'Connor RS, Kawalekar OU, Ghassemi S, MiloneMC. CAR T cell immunotherapy for human cancer. Science (New York, N.Y.) 2018;359:1361– 65).

[0033] Each of the abovementioned gene knock-ins as defined above, preferably the CAR orthe exogenous TCR, may be introduced as defined herein into an endogenous TCR-α gene orinto an endogenous TCR-β gene for conditional expression. The introduction may beperformed with a constitutive promoter such as CAG so that every cell express the gene knock-in, or with a conditional promoter from an endogenous gene such TCR- α or β or any othergene with gene disruption or without gene disruption. Such gene knock-ins as defined abovemay alternatively be introduced into a safe harbor locus as known to a person skilled forcontinuous expression (to prevent the silencing of the gene knock-ins during differentiation),preferably with a conditional promoter.

[0034] Also preferably, the gene knock-out comprises the knock-out of at least one geneselected from the group consisting of a gene encoding an endogenous PD-1 cell surfaceprotein; an endogenous VEGF receptor; an endogenous VCAM-1 cell surface protein; and an endogenous ICAM-1 cell surface protein, or a combination thereof. In one embodiment, the gene knock-out comprises the knock-out of the gene encoding an endogenous PD-1 cell surface protein, such protein providing immunomodulatory function (e.g. persistency / efficiency of the immune cell). In another embodiment, the gene knock-out comprises the knock-out of the gene encoding an endogenous VEGF receptor, such receptor providing immunomodulatory function (e.g. persistency of the immune cell). In another embodiment, the gene knock-out comprises the knock-out of the gene encoding an endogenous VCAM-1 cell surface protein, such protein providing immunomodulatory function (e.g. persistency of the immune cell). In another embodiment, the gene knock-out comprises the knock-out of the gene encoding an endogenous ICAM-1 cell surface protein, such protein providing immunomodulatory function (e.g. persistency of the immune cell).

[0035] Each embodiment described herein concerning the methods of the invention ofproducing an immune cell as defined herein may also be applicable to the methods of the invention of producing a population of immune cells as defined herein.

[0036] As used herein, the term “about” when used with reference to intervals or periods oftime has to be understood such that also any interval which deviates from the distinct value ofthe given interval is also comprised. For example, an interval of about 2 to 4 days, which means48 to 96 hours, also comprises intervals which deviate for 1, 2, 3, or 4 hours from the giveninterval, such that the given interval may be 1, 2, 3, or 4 hours shorter or longer concerning thebeginning and / or the end to the interval.

[0037] In one embodiment of the method of the invention the first serum-free mediumpreferably comprises: in step (i)(a) an inhibitor of GSK-3 and a ROCK inhibitor, each in aconcentration of about 5 to about 15 µM. Additionally or alternatively, the second serum-freemedium used in step (i)(b) may comprise BMP4, VEGF, FGF, each in a concentration of about10 to about 50 ng / mL, and the third serum-free medium used in step (i)(b) may compriseBMP4, VEGF, and FGF, each in a concentration of about 10 to about 50 ng / mL and additionallyan inhibitor of ALK receptors in a concentration of about 5 to about 15 µM. Additionally oralternatively, the fourth serum-free medium used in step (i)(c) may comprise VEGF, FGF, andSCF, each in a concentration of about 10 to about 50 ng / mL, and the fifth serum-free medium used in step (i)(c) may comprise VEGF, FGF, and SCF, each in a concentration of about 10 to about 50 ng / mL, and additionally about 10 to about 50 ng / mL Flt-3l, and about 10 to about 50 ng / mL TPO. Additionally or alternatively, the sixth serum-free medium used in step (ii) may comprise about 7 to about 23 % BIT 9500 serum substitute, about 25 to about 75 mM B- mercaptoethanol, about 10 to about 50 ng / ml IL-3, about 10 to about 50 ng / ml TPO, about 10 to about 50 ng / ml IL-15, about 10 to about 50 ng / ml SDF, about 7 to about 23 µM SB203580, about 10 to about 50 ng / mL SCF, about 10 to about 50 ng / mL Flt-3l, about 10 to about 50 ng / mL IL-7, and about 15 to about 45 mM L-ascorbic acid 2-phophatate sesquimagnesium salt hydrate.

[0038] As used herein, the term “about” in relation to any value of a concentration or range ofa concentration has to be understood such that any concentration or range of concentration which deviates from the distinct value of the given concentration or range of concentration isalso comprised. For example, a concentration of about 50 ng / mL comprises also a lowerconcentration of 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 ng / mL, or a higher concentration of 51, 52, 53, 54, 55, 56, 57, 58, or 59 ng / mL.

[0039] In a preferred embodiment of the invention, the ROCK inhibitor is Y-27632dihydrochloride, the inhibitor of ALK receptors is SB43152 and the inhibitor of GSK-3 isCHIR99021. The use of the ROCK inhibitor provides the advantage that apoptosis and de- differentiation is limited / avoided. Accordingly, the use of ROCK inhibitor and of inhibitor of ALKreceptors avoids the loss of cells due to apoptosis which means that a distinct quantity of cellsis provided with the method of the invention. In addition, the loss of differentiation is reducedsince the cells maintain the desired differentiation characteristics. This means that a distinctquality of cells is provided using the method of the present invention. By adding an inhibitor of GSK-3, such as CHIR99021, to the first serum-free medium in step (i)(a) which activates the WNT pathway, more CD34-positive PSCs are produced after step (i)(c).

[0040] The PSCs used in the present invention may be pluripotent stem cells that are deficientof endogenous major histocompatibility complex (MHC) class I molecules presented on thecell surface of the PSCs and that comprise an immunomodulatory protein on their surface.

[0041] A cell or pluripotent stem cell, which is “deficient of endogenous MHC class I moleculespresented on the cell surface” does not present a functional MHC class I molecule on itssurface, i.e. the surface of the cell or the pluripotent stem cell. Nor does such a deficientcell / pluripotent cell comprise a functional MHC class I molecule in its cell membrane. In this context, the term “endogenous” relates to any MHC class protein I, which naturally is comprised in the cell or pluripotent stem cell and not artificially introduced. Having endogenous MHC class I molecules increases the risk of a rejection reaction of the immune system of the recipient because a lack of MHC class I molecules on the cell surface might be interpreted asa “missing self”-signal by the immune system. Accordingly, the feature that the cell orpluripotent stem cells is deficient of MHC class I molecules on their surface, does not apply to any immunomodulatory protein, which may be introduced into the pluripotent stem cell and / or a recombinant immunomodulatory protein. In one embodiment, the deficiency of MHC class I molecules on the cell surface can be achieved by disrupting all copies of the beta 2- microglobulin gene in the pluripotent stem cells. The MHC complex is a heterodimer of alpha- microglobulin and beta 2-microglobulin. Hence, if beta 2-microglobulin is missing, no functional MHC class I complex can be assembled and consequently, no MHC class I molecule is presenton the cell membrane and / or cell surface. Many possible ways are known to the person skilledin the art to modify the genome of the pluripotent stem cell in such a way that they are deficientof MHC class I molecules and comprise an immunomodulatory protein. It should be noted thata pluripotent stem cell of the invention, which is deficient of MHC class I molecules, may express an immunomodulatory protein, even if it is an MHC class I molecule such as HLA-E described herein. Accordingly, the term “deficient of MHC class I molecules” may relate to endogenous MHC class I molecules and does not exclude the presence of a (recombinant) immunomodulatory protein.

[0042] In a further preferred embodiment of the invention the immunomodulatory protein is asingle chain fusion HLA class I protein.

[0043] Preferably, the single chain fusion HLA class I protein may comprise at least a portionof B2M covalently linked to at least a portion of an HLA class Iα chain selected from the groupconsisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G, preferably HLA-E. Suchoptional introduction of the recombinant immunomodulatory protein, preferably being a singlechain fusion HLA class I protein, even more preferably being HLA-E, into the PSCs, results inthe PSCs being protected from NK-mediated host rejection, thus resulting in non-immunogenicPSCs as defined elsewhere herein.

[0044] In a preferred embodiment of the invention the PSCs express further a target peptideantigen that is presented by the single chain fusion HLA class I protein on the pluripotent cellsurface. The target peptide antigen is preferably covalently linked to the single chain fusionHLA class I protein. In an even more preferred embodiment the target peptide antigencomprises the sequence VMAPRTLFL (SEQ ID NO: 1).

[0045] Preferably, essentially all copies of the beta-microglobulin 2 gene are disrupted in thePSCs. In the PSCs of the invention the B2M gene may be disrupted so that no functionalendogenous B2M protein is produced from the disrupted genetic loci. In certain embodiments, the disruption results in expression of non-functional B2M proteins, including but not limited to truncations, deletions, point mutations and insertions. In other embodiments, the disruption results in no protein expression from the B2M gene.

[0046] In a further preferred embodiment the PSCs as defined herein are additionally oralternatively being deficient of endogenous MHC class II. Preferably, the PSCs are deficientof endogenous MHC class II by disrupting the C2TA gene-MHC class II transactivator.

[0047] By the abovementioned genetic modifications non-immunogenic immune cells may beproduced which are not attacked by the recipient’s immune cells, such as T and NK cells. Suchabovementioned genetic modifications may not be comprised by the one or more exogenous modifications introduced in any one of before step (a), in step (a), (b) or (c) of step (i), or in step (ii) of the method of the invention when genetically introducing one or more exogenousmodifications into the PSCs to introduce a predetermined effector function as defined herein.

[0048] In a preferred embodiment the PSCs are selected from the group consisting ofembryonic stem cells, induced pluripotent stem cells and parthenogenetic stem cells.

[0049] The term “pluripotent stem cell” (PSC) as used herein refers to any such stem cell typethat is able to differentiate into an immune cell. In the context of the present invention, thesePSCs are preferably not produced using a process which involves modifying the germ linegenetic identity of human beings or which involves use of a human embryo for industrial or commercial purposes. The PSCs used within the methods of the invention may refer to wild type PSCs (without having any non-immunogenic modifications as defined herein) or to non-immunogenic PSCs as defined herein. Thus, such PSCs-derived immune cells which aredevoid of any endogenous effector functions due to the method of the invention can additionallybe non-immunogenic immune cells as defined elsewhere herein. Preferably, the PSCs are ofprimate origin, more preferably human. In a further preferred embodiment of the invention, the PSC are human induced pluripotent stem (iPS) cells, preferably CD34-positive cells isolatedfrom umbilical cord blood, most preferably the human iPS cell line TC-1133 which also refersto the cell line ND-50039 of the NINDS Human Cell and Data Repository (see Example 1).The cell line TC-1133 has been reprogrammed under c-GMP conditions and can be purchasedfrom Lonza. Further suitable PSC’s, including induced PSCs, can for example, be obtained from the NIH human embryonic stem cell registry, the European Bank of Induced Pluripotent Stem Cells (EBiSC), the Stem Cell Repository of the German Center for CardiovascularResearch (DZHK), or ATCC, to name only a few sources. PSCs are also available forcommercial use, for example, from the NINDS Human Sequence and Cell Repository (https: / / stemcells.nindsgenetics.org) which is operated by the U.S. National Institute of Neurological Disorders and Stroke (NINDS) and distributes human cell resources broadly to academic and industry researchers.

[0050] Further exemplary iPSC cell lines that can be used in the present invention, include butare not limited to, the Human Episomal iPSC Line of Gibco™ (order number A18945, Thermo Fisher Scientific), or the iPSC cell lines ATCC ACS-1004, ATCC ACS-1021, ATCC ACS-1025, ATCC ACS-1027 or ATCC ACS-1030 available from ATTC. Alternatively, any person skilled in the art of reprogramming can easily generate suitable iPSC lines by known protocols such as the one 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 by 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”, Biomaterials35 (2014) 2816e2826. The (induced) pluripotent stem cell that is used in the present inventioncan be derived from any suitable cell type (for example, from a stem cell such as a mesenchymal stem cell, or an epithelial stem cell or a differentiated cells such as fibroblasts) and from any suitable source (bodily fluid or tissue). Examples of such sources (body fluids or tissue) include cord blood, skin, gingiva, urine, blood, bone marrow, any compartment of theumbilical cord (for example, the amniotic membrane of umbilical cord or Wharton’s jelly), thecord-placenta junction, placenta or adipose tissue, to name only a few. In one illustrative example, is the isolation of CD34-positive cells from umbilical cord blood for example bymagnetic cell sorting using antibodies specifically directed against CD34 followed byreprogramming 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 can be in compliance with the regulations of good manufacturing practice to generate the cell lineND-50039. Accordingly, the PSCs preferably fulfill the requirements of the good manufacturingpractice.

[0051] The PSCs as described herein may have been cultured on a solid support comprisingat least one extracellular matrix protein before seeding such cells on another solid support as described herein in step (i)(a). Such step (which may refer to the step (a’) as defined below) may refer to the culturing step of the PSCs before step (a) of the methods of the invention.Such at least one extracellular matrix protein may be selected from the group consisting ofvitronectin, laminin, collagen, fibronectin, elastin, Matrigel, a peptide containing the amino acidsequence RGD, a protein containing the amino acid sequence RGD and combinations thereof.The skilled person is able to empirically select the respective optimal extracellular matrix protein or the optimal combination of extracellular matrix proteins. Thus, also a combination of two, three, four or more extracellular matrix proteins can be used for culturing of the PSCs inthis step. Preferably, PSCs as described herein may be cultured on a Matrigel-coatedcontainer before step (i)(a), such as Matrigel-coated T-flasks. The PSCs are preferablycultured in iPSC expansion medium such as Miltenyi StemMACS iPS-Brew XF, on such aMatrigel-coated container so that they stay pluripotent and do not differentiate. Also comprised herein is the methods of the invention additionally comprising the step (a’) before step (a) comprising culturing PSCs under suitable conditions on a solid support comprising at least oneextracellular matrix protein in a serum-free medium. In this context, the “serum-free medium”refers to a medium not comprising serum for culturing PSCs so that they stay pluripotent and do not differentiate, such as an iPSC expansion medium, preferably being supplemented with Rock inhibitor Y-27632. “Under suitable conditions” in this context may refer to conditions known to a person skilled in the art that allows the culture of PSCs on a coated solid supportin the serum-free medium as described elsewhere herein so that the PSCs maintainpluripotency and do not differentiate.

[0052] Preferably, the PSCs are pluripotent stem cells of primate origin, more preferablyhuman pluripotent stem cells. In a preferred embodiment the PSCs are generated from CD34-positive cells that are isolated form umbilical cord blood. In an even more preferredembodiment, the PSCs used in the present invention are ND-50039 cells of the NINDS HumanCell and Data Repository.

[0053] Preferably, in step (i)(a) of the present methods the solid support comprises one ormore wells, wherein each well comprises a V-shaped or conical cavity. In a preferredembodiment 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 3D cell aggregates. Examples of suitablemicrowell culture plates include, but are not limited to, an AggreWellTM plate (available fromStemCell Technologies), a BIOFLOAT™ 96-well plate (available from faCellitate), or a NunclonSphera 3D culture (available from ThermoFisher). Such support as used in step (i)(a),preferably if an AggreWellTM plate is used, is preferably pre-treated with an anti-adherencesolution as mentioned above. Such an anti-adherence solution allows to reduce surfacetension and prevents cell adhesion to the support so that the cells only attach to each other.This provides a low adherence surface which facilitates the formation of 3D–cell aggregates. The formation of 3D-cell aggregates can be conducted in a very easy and robust way which allows also scale-up.

[0054] Preferably, the suspension culture used in step (i)(b) and (c) of the methods of theinvention is a dynamic suspension culture, that means a suspension culture that is being agitated, for example by culturing on a shaking device. Any suitable shaking device / shakercan be used, for example an orbital shaker, a horizontal shaker or a linear shaker. The culturecan be moved / agitated under any suitable conditions that can be experimentally determinedby the skilled person. For example, a shaker such an orbital shaker may be operated at a rateof about 60 to about 80 rotations per minutes (rpm). In contrast to an adherent cell culture, the suspension cell culture as used herein provides several advantages. Adherent cell culturerequires more space and requires a more elaborate handling, for example for passaging ofcells. The suspension culture is a cell culture format which allows to provide a scalable cell culturing process which is useful for the provision of higher cell numbers. Such higher cellnumbers are in particular required for clinical use of cellular products. The dynamic suspensionculture can also be much easier transferred to bioreactor conditions compared to an adherent cell culture, such as 2D cell culture or static cell culture, since the features of the dynamic cell culture are already very similar to the features of a culture in a bioreactor. Moreover, growth factors which are present in the culture medium are able to circulate in the dynamic cell culture. This allows that every cell can be in close contact with such growth factors. This may positivelyinfluence the culture process, in particular the differentiation process of the cells and can helpto achieve a more homogenous and better-defined cell population.

[0055] Preferably, the methods of the invention further comprise dissociating the 3D cellaggregates into single cells after step (i)(c) and before step (ii) and optionally then sorting forCD34-positive PSCs. Such dissociation step may be performed by applying collagenase type II. Sorting for CD34-positive PSCs may be performed by MACS or FACs as known to a person skilled in the art.

[0056] The induction of the immune cell differentiation can be carried out over any suitabletime, preferably less than 50 days, more preferably less than 25 days, most preferably less than 21 days. A suitable time can be determined experimentally, for example, by taking cell samples over a certain period of time and determine the nature / differentiation state of the cells, for example by analysis of the cell surface markers (see the experimental section where cellswere first screened for the CD45 marker to scan for immune cells (leukocytes)) (see Figure1A). Such generated immune cells could then further be characterized for the phenotype of CD56high(which refers to NK-like precursor cells) (see Figure 1B). In a preferred embodiment of the invention, the step (ii) of inducing immune cell differentiation by culturing single cells ofthe 3D – cell aggregates obtained from (i)(c) under suitable conditions in a suspension cultureis conducted for about 7 to about 21 days, such as about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or about 21 days, preferably for about 14 days in serum-free medium. Thesuspension culture used in step (ii) of the method of the invention is not a dynamic suspensionculture as it may be the case for the suspension culture in step (i)(b) and (c) of the methods ofthe invention. Such suspension culture of step (ii) is preferably a Notch-ligand coated suspension culture plate. Immune cell differentiation may also be induced when not applyinga Notch-ligand coated suspension culture plate, namely a non-coated suspension cultureplate. The Notch-ligand may be the Notch-ligand Delta-like ligand 4 (DLL4) or the Notch-ligandDelta-like ligand 1 (DLL1). Notch signalling being reported to be important for immune celldifferentiation.

[0057] The immune cell generated by the method of the invention (not including anymodification steps at the PSC level) is preferably a NK like precursor cell devoid of endogenouseffector functions as described elsewhere herein. Also comprised herein is such method of theinvention as defined herein producing a population of immune cells which may comprise NK like precursor cells devoid of any endogenous effector functions. “A population of immune cells” with regard to such method may be understood as a single homogenous group of immune cells such as a pure or homogenous population of NK like precursor cells all devoidof endogenous effector functions as defined herein which can be characterized by a skilledperson with methods known in the art, such as flow cytometric methods. In case for the method where the PSCs are genetically modified as defined herein in order to achieve a genetically modified immune cell, the immune cell generated by such method is preferably a T cell like cell or a NK cell like cell devoid of endogenous effector functions, which comprises one or more exogenous modifications, wherein each of the one or more exogenous modificationsintroduces a predetermined effector function. In this context, a “T cell like cell” means that theproduced immune cell is not a naturally occurring T cell since based on the method includingthe introduction of exogenous modification a genetically modified T cell is generated being devoid of any endogenous effector function, but comprising e.g. just one predetermined effector function if one exogenous modification which results in said predetermined effector function has been introduced into the PSCs which then develop into the modified T cell. Thesame applies mutatis mutandis to “NK cell like cell”, “B cell like cell” etc. The term “populationof immune cells” as used herein with regard to the method comprising the modification stepincludes a mixture of (different) immune cells which is comprised in the population of immunecells. Thus, “a population of immune cells” in this regard can be understood as a singlehomogenous group of immune cells such as a pure or homogenous population of T cell likecells or NK cell like cells etc. (wherein homogeneity can be assessed by the percentage of cells that express or lack expression of specific markers proteins). The term “population of immune cells” can also be understood as a heterogenous group of immune cells comprisingany type of immune cells, but different immune cells or different types of immune cells (suchas for example T cell like cells and NK cell like cells) all devoid of endogenous effectorfunctions, but comprising a predetermined effector function due to the exogenously introducedgenetic modification dependent on the desired application, such cells which can bedistinguished from each other and subsequently also isolated from each other by a skilledperson with methods known in the art, such as flow cytometric methods. Meaning, if forexample an exogenous TCR is introduced into the PSCs’ genome and an exogenous CD16 receptor is introduced into the PSCs’ genome, the PSCs genetically modified with theexogenous TCR result in T cell like cells and the PSCs genetically modified with the exogenous CD16 receptor result in NK cell like cells, having a population of different immune cells as defined herein. Immune cells comprise neutrophils, eosinophils (acidophiles), basophils, lymphocytes, and monocytes, and among the lymphocytes B cells, T cells and natural killer (NK) cells. The population of immune cells may also comprise cells representing precursorcells of T cell like cells or precursor cells of NK cell like cells as well as cells representingmature T cell like cells or mature NK cell like cells.

[0058] The methods of the present invention may also be suitable of producing a populationof immune cells with high cell numbers by using bioreactor conditions in any one or all of thesteps (i)(a), (i)(b), (i)(c) and / or (ii) or even in the PSC culturing step before step (i)(a). The useof a bioreactor provides the advantage that higher yields of cells can be achieved due to theuse of a large-scale bioreactor format. The use of a bioreactor allows optimization of theconcentration of different factors and at the same time production of higher cell numbers. Therefore, the number of cells of the population of immune cells provided by the method of theinvention can be scaled up due to the use of a bioreactor. So doing allows providing the numberof cells of the population of immune cells need for clinical applications. In addition, so doingallows the provision of a highly standardized and cost-effective method. In addition, the use ofa bioreactor simplifies the procedure, as the use of a bioreactor provides a less laboriousmethod which can be easily conducted.

[0059] All definitions provided herein for the method of the invention can be applied to theimmune cell or the population of immune cells devoid of endogenous effector functionsobtainable or obtained by the method of the invention. The same applies mutatis mutandis tothe definitions provided herein for the method of the invention which can be applied to the immune cell or the population of immune cells devoid of endogenous effector functions, but comprising one or more exogenous modifications, wherein each of the one or more exogenousmodifications introduces a predetermined effector function obtainable or obtained by themethod of the invention.

[0060] In a preferred embodiment, an immune cell or a population of immune cells, preferablya NK like precursor cell, obtainable or obtained by the particular method of the invention isprovided herein, which expresses CD45, but does not express the endogenous CD94 receptor,the endogenous CD16 receptor, the endogenous NKG2D receptor, the endogenous NKp30receptor, the endogenous TCR and the endogenous CD19 antigen.

[0061] In a preferred embodiment, an immune cell or a population of immune cells obtainableor obtained by the particular method of the invention is provided herein, which is devoid ofendogenous effector functions as defined herein, but comprises one or more exogenousmodifications, wherein each of the one or more exogenous modifications introduces apredetermined effector function, wherein the one or more exogenous modification is a geneknock-in comprising the knock-in of at least one gene selected from the group consisting of agene encoding a CAR, an exogenous TCR, an exogenous IL-15 receptor, an exogenous CD16receptor, an exogenous CD19 antigen, an exogenous IL-10 cytokine, an exogenous TGF-βcytokine, and an exogenous PD ligand 1 (PD-L1) antigen. In other words, also comprisedherein, is an immune cell or a population of immune cells, preferably a T cell like cell or a population of T cell like cells, obtainable or obtained by the particular method as defined herein, which is devoid of endogenous effector functions, but expresses at least any one of a CAR, anexogenous TCR, an exogenous IL-10 cytokine, an exogenous TGF-β cytokine, or anexogenous PD ligand 1 (PD-L1) antigen. Also comprised herein, is an immune cell or apopulation of immune cells, preferably a NK cell like cell or a population of NK cell like cells,obtainable or obtained by the particular method as defined herein, which is devoid of endogenous effector functions, but expresses at least any one of an exogenous IL-15 receptor or an exogenous CD16 receptor. Also comprised herein, is an immune cell or a population ofimmune cells, preferably a B cell like cell or a population of B cell like cells, obtainable orobtained by the particular method as defined herein, which is devoid of endogenous effectorfunctions, but expresses an exogenous CD19 antigen and / or an exogenous PD ligand 1 (PD-L1) antigen.

[0062] The present invention also comprises a method of converting the immune cell devoidof endogenous effector functions obtainable or obtained by the method of the invention as defined herein into an immune cell devoid of endogenous effector functions, but comprising one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function, the method comprising the stepsof: of: (i) inducing 3D cell aggregate formation, mesodermal differentiation and hematopoieticdifferentiation by (a) seeding PSCs under suitable conditions on a solid support suitable forformation of 3D - cell aggregates in a first serum-free medium, thereby allowing the formationof 3D - cell aggregates; (b) collecting the 3D - cell aggregates of step (a), resuspending the 3D– cell aggregates under suitable conditions in a second serum-free medium, and transferringthe resuspended 3D – cell aggregates into and culturing in a third serum-free medium undersuitable conditions in suspension culture for about 1 to about 3 days, thereby allowing themesodermal differentiation; and (c) transferring the cultured 3D - cell aggregates of step (b)into and culturing in a fourth serum-free medium under suitable conditions in suspensionculture for about 2 to about 4 days and further culturing the 3D - cell aggregates under suitableconditions in suspension culture for about 2 to about 6 days in a fifth serum-free medium,thereby allowing the hematopoietic differentiation; followed by (ii) inducing immune celldifferentiation by culturing single cells of the 3D - cell aggregates obtained from step (i)(c)under suitable conditions in suspension culture for a suitable time in a sixth serum-freemedium; wherein the method further comprises genetically introducing one or more exogenousmodifications into the PSCs in any one of before step (a), in step (a) to (c) of step (i), or in step (ii), wherein each of the one or more exogenous modifications introduces a predeterminedeffector function, thereby providing a genetically modified immune cell being devoid ofendogenous effector functions, but comprising one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function. All definitions provided herein may also be applicable, if necessary, to the method of converting.

[0063] Additionally, the present invention comprises a pharmaceutical composition comprisingthe immune cell or the population of immune cells obtained / obtainable by the methods of theinvention as defined herein. Such pharmaceutical composition may further comprise at leastone pharmaceutically acceptable carrier. Said pharmaceutically acceptable carrier includesany carrier that does not itself elicit an adverse reaction harmful to the subject receiving thepharmaceutical composition. Said pharmaceutical composition is thus used herein for therapeutic purposes. In accordance with the present invention, the term "pharmaceutical composition" relates to a composition for administration to a subject as defined herein, preferably a human. Pharmaceutical compositions or formulations are usually in such a formas to allow the biological activity of the active ingredient to be effective and may therefore beadministered to a subject for therapeutic use as described herein. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and lipid aggregates such as e.g. oil droplets or liposomes. The carrier used in combination with the pharmaceuticalcomposition of the present invention may be water-based and forms an aqueous solution oran oil-based carrier solution.

[0064] Also comprised herein is a kit comprising the pharmaceutical composition as definedherein. Such kit may additionally comprise a manual comprising instructions for applying said pharmaceutical composition to a subject in need thereof.

[0065] Further, the present invention is directed to the immune cell or the population of immunecells of the invention as defined herein, or the pharmaceutical composition of the invention asdefined herein for use in medicine. The present invention also comprises the immune cell orthe population of immune cells of the invention, or the pharmaceutical composition of the invention for use in a method of preventing or treating cancer.

[0066] As used herein, the term “treat”, “treating” or “treatment” means to reduce (slow down(lessen)), stabilize or inhibit or at least partially alleviate or abrogate the progression of thesymptoms associated with the respective disease. Thus, it includes the administration of said cell as defined herein, preferably in the form of a medicament or a pharmaceutical composition,to a subject, defined elsewhere herein. Those in need of treatment include those alreadysuffering from the disease, here cancer. Preferably, a treatment reduces (slows down(lessens)), stabilizes, or inhibits or at least partially alleviates or abrogates progression of asymptom that is associated with the presence and / or progression of a disease or pathological condition. “Treat”, “treating”, or “treatment” refers thus to a therapeutic treatment. In particular, in the context of the present invention, treating or treatment refers to an improvement of thesymptom that is associated with cancer, as defined elsewhere herein. The term “subject” whenused herein includes mammalian subjects. Preferably, the subject of the present invention isa mammal, including human. In some embodiment the mammal is a mouse. A subject also includes human and veterinary patients. Where the subject is a living human who may receive treatment for a disease or condition as described herein, it is also addressed as a “patient”.Those in need of treatment include those already suffering from the disease. In this case, thesubject may have a disease, here cancer or a symptom of the respective disease, here cancer.

[0067] The term “prevent”, “preventing”, or “prevention” may refer to prophylactic orpreventative measures so that a disease will not even occur or symptoms associated with the respective disease will not occur, meaning preventing the onset or recurrence of a disease,here cancer. Thus, it also includes the administration of said cell as defined herein, preferablyin the form of a medicament or a pharmaceutical composition, to a subject, defined elsewhere herein. The definitions referring to the “subject” provided above may also apply hereto. In thiscase, the subject may have a predisposition toward a particular disease, here cancer.

[0068] In this context, the present invention also comprises the immune cell or the populationof immune cells of the invention, or the pharmaceutical composition of the invention for use ina method of preventing or treating cancer as defined herein, wherein cancer is selected fromlung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, skin cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, tracheal cancer, head & neck cancer, pancreatic cancer, liver cancer, breast cancer, lymphoid cancers including lymphoma and multiple myeloma, leukemia, sarcomas of bone or soft tissue, cervical cancer, and vulvar cancer.

[0069] A further aspect of the invention is directed to the immune cell or the population ofimmune cells of the invention, or the pharmaceutical composition of the invention for use incell (based) therapy. This medical use includes administering the immune cell or the populationof immune cells of the invention or a cell population derived from the population of immunecells of the invention or the pharmaceutical composition to a subject in need thereof. Thesubject is typically a mammal, such as a human. The definitions above may also be appliedhereto, where necessary.

[0070] The immune cell or the population of immune cells of the invention or thepharmaceutical composition as defined elsewhere herein is thus typically administered to asubject for cancer treatment or immunotherapy. The immune cell or the population of immunecells of the invention or the pharmaceutical composition as defined elsewhere may be used in adoptive T cell transfer or similar therapeutic approaches. Among the different immune cell therapies such as T cell immunotherapies, adoptive cell therapy has attracted substantialattention and interest during the last years. The adoptive cell therapy is a personalized therapyin which a patient’s own immune cells are removed from, the methods of the inventions appliedto generate modified immune cells, expanded in vitro to large numbers, and reinfused backinto the patient to eliminate tumors. A summary of the developments of the adoptive celltherapy is given in Guedan et al., Rev Immunol. 2019 April 26; 37: 145–171.doi:10.1146 / annurev-immunol-042718-041407. ***

[0071] Unless otherwise stated, the following terms used in this document, including thedescription and claims, have the definitions given below.

[0072] Those skilled in the art will recognize, or be able to ascertain, using not more thanroutine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0073] It is to be noted that as used herein, the singular forms "a", "an", and "the", includeplural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0074] Unless otherwise indicated, the term "at least" preceding a series of elements is to beunderstood to refer to every element in the series. Those skilled in the art will recognize, or beable 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.

[0075] The term "and / or" wherever used herein includes the meaning of "and", "or" and "allor any other combination of the elements connected by said term".

[0076] The term "about" or "approximately" means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, morepreferably up to 5%, and more preferably still 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. It includes, however, also the concrete number, e.g., about 20 includes 20. Alternatively, particularly with respect to biological systems or processes, the term canmean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0077] Throughout this specification and the claims which follow, unless the context requiresotherwise, the word “comprise”, and 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 but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”.

[0078] When used herein “consisting of" excludes any element, step, or ingredient notspecified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0079] 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.

[0080] It should be understood that this invention is not limited to the particular methodology,protocols, material, reagents, and substances, etc., described herein and as such can 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.

[0081] All publications cited throughout the text of this specification (including all patents,patent applications, scientific publications, manufacturer’s specifications, instructions, etc.) are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.EXAMPLES OF THE INVENTION

[0082] The following examples illustrate the invention. These examples should not beconstrued as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims.

[0083] Example 1: Producing of a population of immune cells from pluripotent stemcells (PSC).

[0084] Human induced pluripotent stem (iPS) cell line TC-1133 that was reprogrammed underc-GMP conditions was purchased from Lonza. Such cell line also refers to ND-50039 cells of the NINDS Human Cell and Dara Repository. Human iPS cells were cultured and maintainedon matrigel-coated T-flasks in iPS expansion medium (Miltenyi StemMACS iPS-Brew XF) asit has been defined elsewhere herein.

[0085] 3D-aggregate formation and mesoderm induction (Differentiation day 1 to day 3):1. AggreWell™4006-well plate (Stemcell Technologies, 34425) are pre-treated with 2 mLAnti-Adherence Rinsing Solution (Stemcell Technologies, 07010) per well.2. Anti-Adherence Rinsing Solution is aspirated from the wells.3. Each well is rinsed with warm Dulbecco's phosphate-buffered saline (DPBS, Gibco14190136) and kept at room temperature until use.4. Human iPS cells are washed with 5 mL DPBS (Gibco 14190136) in T-flasks.5. 3 mL accutase (Gibco A1110501) is added and the cells are incubated in an incubatorat 37°C for 4 min.6. The cells are detached by tapping the flask and later the cells are washed down with 5mL DPBS.7. The cells are transferred into falcon and centrifuge at 300g for 4 min.8. The supernatant is discarded and the cells are resuspended in 1 mL iPSC expansionmedium (Miltenyi StemMACS iPS-Brew XF) and counted.9. 3.0x106 cells are seeded per one well of AggreWell™400 6-well plate in iPSCexpansion medium supplemented with CHIR99021 (10µM), BMP4 (50 ng / mL), VEGF(50 ng / mL), FGF (50 ng / mL), and ROCK inhibitor Y-27632 dihydrochloride (10 µM).10. Centrifuge the plate at 100g for 3 min to capture the cells in the microwells.11. Incubate the plate at 37°C with 5% CO2 and 95% humidity for 24 hours.12. On day 1 the aggregate formation can be observed on microscope.13. To collect the 3D-aggregates from the plate, the plate is gently tapped and theaggregates dislodged. By swirling the aggregates are collected in the center of the well.Using a serological pipette collect the aggregates from the plate and transfer into falcon.14. After all the aggregates sink down at the bottom of the falcon, the medium can becarefully discarded without disturbing the aggregates.15. The aggregates are resuspended in mesoderm induction medium composed ofStemPro34-SFM medium (Gibco 10639011) supplemented with BMP4 (50ng / ml), VEGF (50 ng / ml) and FGF (50 ng / ml) and transfered into ultra-low attachmentsuspension culture plate.16. The plate is placed onto an orbital shaker and the aggregates are maintained indynamic suspension culture by rotating the shaker at 80 rpm.17. The medium is changed on day 2 with mesoderm induction medium composed ofStemPro34-SFM medium supplemented with BMP4 (50 ng / mL), VEGF (50 ng / mL),FGF (50 ng / mL) and SB431542 (10µM).

[0086] Hematopoietic induction (Differentiation day 4 to day 11):1. On day 4 the medium is changed in the plate with hematopoietic induction mediumcomposed of StemPro34-SFM medium supplemented with VEGF (50 ng / ml), FGF (50 ng / ml) and SCF (50 ng / ml)2. On day 7 and day 9 the medium is changed again with hematopoietic induction mediumcomposed of StemPro34-SFM medium supplemented with VEGF (50 ng / ml), FGF (50 ng / ml), SCF (50 ng / ml), Flt-3l (10 ng / ml), and TPO (30 ng / ml).3. On day 11 the aggregates are collected from the wells and dissociated into single cellswith collagenase type II.4. The CD34 positive cells are then sorted using MACS or FACS.

[0087] Immune cell differentiation (Differentiation day 11 to day 25):From differentiation day 11 to day 25 the aggregates (iPSCs-derived CD34+ cells) are culturedin suspension culture in an immune cell differentiation medium composed of IMDM (ThermoFischer Sci, 31980030) and supplemented with 15% BIT 9500 serum substitute (StemcellTechnologies, 09500), B-mercaptoethanol (50mM), IL-3 (10ng / ml- only from day 11 to day 18),TPO (50ng / ml), IL15 (50ng / ml), SDF (10 ng / ml), SB203580 (15uM), 50ng / mL SCF, 50ng / mLFlt-3l, 50ng / mL IL-7, 30 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate and 1% penicillin / streptomycin on Notch-ligand coated (ex DLL4) or uncoated cell culture plates.

[0088] iPSC-derived immune cells as described by the differentiation protocol in Example 1are used in the following Examples. Explicitly, either wild-type (WT) PSCs (not non-immunogenic variants as defined herein) as well as CD19-CAR expressing cells were used.CD19-CAR construct was stably knocked into safe harbor locus at iPSC level as definedherein. Polyclonal population was used for downstream experiments.

[0089] Example 2: Phenotypic characterization of iPSC-derived immune cells – nofunctional potential of such immune cells in vitro.

[0090] For phenotypic characterization, iPSC-derived immune cells were analyzed using flowcytometry and the following antibodies: anti-CD45 (AF700), anti-CD94 (B525), anti-CD16 (V450), anti-NKG2D (V610), NKp30 (V763), anti-CD3 (FITC), anti-CD19 (PB) (all fromBioLegend) (see Figure 1). CAR expression has been monitored using an anti-idiotypeantibody specific for the CD19-directed CAR (Miltaneyi Biotec). Antibody staining was performed according to manufacturers’ instructions. Cell associated fluorescence was analyzed by flow cytometry using CytoFLEX LX flow cytometer (Beckman Coulter). For all displayed dot plots were pre-gated on live and single cells.

[0091] For cell growth and fold expansion, live nucleated cells were enumerated usingNuceloCounter NC-3000 (Chemotec) according to manufacturers’ instructions.

[0092] Example 3: Phenotypic characterization of iPSC-derived immune cells – nocytolytic function of such immune cells in vitro.

[0093] To test in vitro cytolytic function, iPSC-derived cells (effectors) were co-cultured withCD19+GFP+Raji cell line (targets) for 0, 24 and 48 hours at indicated cell E:T ratios. Changes CD19 antigen expression as well as changes in cell frequencies were assessed using flow cytometry as described above. Human primary T and NK cells isolated from PBMC fraction bypositive selection (Miltenyi Biotec kits) were used as positive controls (see Figure 2).

[0094] Human primary T cells controls were transfected for CAR expression as follows.Purified T cells were activated for 48 h and cultured with 300 IU ml−1recombinant human IL-2, 5 ng ml−1recombinant human IL-7 (Peprotech) and 5 ng ml−1IL-15. Afterward, 1 × 106cells were electroporated with Cas9 ribonucleoprotein and CD19-CAR DNA templates in 20 μl of Nucleofector Solution P3 (Lonza) with a 4D Nucleofector X unit (Lonza, (pulse code EH100). After electroporation, cells were cultured in serum free media with 180 IU ml−1IL-2 until co- culture experiments.

[0095] Example 4: Phenotypic characterization of iPSC-derived immune cells – nocytolytic function of such immune cells in vivo.

[0096] To evaluate CAR T cell function in vivo, 6–8-week-old male NSGS mice were selected.First, tumor cells were injected via tail vein with 5 × 105CD19+Raji / ffluc cells.7 days later, mice were additionally injected intravenously with PBS (control) or 0.75 × 106selected CAR+cells. For Bioluminescence imaging, mice received intraperitoneal injections of luciferin substrate (XenoLight d-Luciferin, Perkin Elmer) resuspended in PBS (15 μg / g body weight),anesthetized with isoflurane and imaged using an IVIS Lumina Imaging System (Perkin Elmer)10 min after luciferin injection (in small binning mode and with an acquisition time up to 1 min to obtain unsaturated images). Luciferase activity was analyzed using Living Image Software (Perkin Elmer) (see Figure 3).ITEMS1. A method of producing an immune cell devoid of endogenous effector functions frompluripotent stem cells (PSCs), the method comprising the steps of: (i) inducing 3D cell aggregate formation, mesodermal differentiation and hematopoieticdifferentiation by (a) seeding PSCs under suitable conditions on a solid support suitable for formationof 3D - cell aggregates, in a first serum-free medium, thereby allowing theformation of 3D - cell aggregates;(b) collecting the 3D - cell aggregates of step (a), resuspending the 3D – cellaggregates under suitable conditions in a second serum-free medium, and transferring the resuspended 3D – cell aggregates into and culturing in a thirdserum-free medium under suitable conditions in suspension culture for about 1to about 3 days, thereby allowing the mesodermal differentiation; and(c) transferring the cultured 3D - cell aggregates of step (b) into and culturing in afourth serum-free medium under suitable conditions in suspension culture for about 2 to about 4 days and further culturing the 3D - cell aggregates undersuitable conditions in suspension culture for about 2 to about 6 days in a fifth serum-free medium, thereby allowing the hematopoietic differentiation; and(ii) inducing immune cell differentiation by culturing single cells of the 3D - cell aggregatesobtained from step (i)(c) under suitable conditions in suspension culture for a suitable time in a sixth serum-free medium;thereby providing an immune cell devoid of endogenous effector functions.2. A method of producing a genetically modified immune cell from pluripotent stem cells(PSCs) being devoid of endogenous effector functions, but comprising one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function, the method comprising the steps of: (i) inducing 3D cell aggregate formation, mesodermal differentiation and hematopoieticdifferentiation by (a) seeding PSCs under suitable conditions on a solid support suitable for formationof 3D - cell aggregates, in a first serum-free medium, thereby allowing theformation of 3D - cell aggregates;(b) collecting the 3D - cell aggregates of step (a), resuspending the 3D – cellaggregates under suitable conditions in a second serum-free medium, and transferring the resuspended 3D – cell aggregates into and culturing in a thirdserum-free medium under suitable conditions in suspension culture for about 1to about 3 days, thereby allowing the mesodermal differentiation; and (c) transferring the cultured 3D - cell aggregates of step (b) into and culturing in afourth serum-free medium under suitable conditions in suspension culture for about 2 to about 4 days and further culturing the 3D - cell aggregates undersuitable conditions in suspension culture for about 2 to about 6 days in a fifth serum-free medium, thereby allowing the hematopoietic differentiation; and(ii) inducing immune cell differentiation by culturing single cells of the 3D - cell aggregatesobtained from step (i)(c) under suitable conditions in suspension culture for a suitabletime in a sixth serum-free medium; wherein the method further comprises genetically introducing one or more exogenous modifications into the PSCs in any one of before step (a), in step (a) to (c) of step (i), or in step (ii) wherein each of the one or more exogenous modifications introduces apredetermined effector function, thereby providing a genetically modified immune cell being devoid of endogenous effector functions, but comprising one or more exogenous modifications, wherein each of the one ormore exogenous modifications introduces a predetermined effector function.3. The method of item 2, wherein said exogenous modification is selected from the groupconsisting of a gene knock-in, a gene knock-out, a gene replacement, a point mutation, and a deletion, insertion or substitution of a gene, a gene fragment or a nucleotide, or acombination thereof.4. The method of item 3, wherein said gene knock-in comprises the knock-in of at least onegene selected from the group consisting of a gene encoding: i) a chimeric antigen receptor (CAR); ii) an exogenous T cell receptor (TCR); iii) an exogenous IL-15 receptor; iv) an exogenous CD16 receptor; v) an exogenous CD19 antigen; vi) an exogenous IL-10 cytokine; vii) an exogenous TGF-β cytokine; and viii) an exogenous PD-1 ligand 1 antigen, or a combination thereof, and / or wherein said gene knock-out comprises the knock-out of at least one gene selected from the group consisting of a gene encoding:ix) an endogenous PD-1 cell surface protein; x) an endogenous VEGF receptor; xi) an endogenous VCAM-1 cell surface protein; and xii) an endogenous ICAM-1 cell surface protein, or a combination thereof.5. The method of any one of the preceding items, wherein:in step (i) (a) the first serum-free medium comprises an inhibitor of GSK-3 and a ROCKinhibitor, each in a concentration of about 5 to about 15 µM; in step (i) (b) the second serum-free medium comprises bone morphogenic protein 4(BMP4), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), each in a concentration of about 10 to about 50 ng / mL, and the third serum-free medium comprises BMP4, VEGF, FGF, each in aconcentration of about 10 to about 50 ng / mL and additionally an inhibitor of ALKreceptors in a concentration of about 5 to about 15 µM; and / or in step (i) (c) the fourth serum-free medium comprises VEGF, FGF, and stem cell factor (SCF), each in a concentration of about 10 to about 50 ng / mL, and the fifth serum-free medium comprises VEGF, FGF, and SCF, each in a concentration of about 10 to about 50 ng / mL, and additionally about 10 to about50 ng / mL FMS-like tyrosine kinase 3 ligand (Flt-3l), and about 10 to about 50 ng / mL Thrombopoietin (TPO); and / or in step (ii) the sixth serum-free medium comprises about 7 to about 23 % BIT 9500 serumsubstitute, about 25 to about 75 mM B-mercaptoethanol, about 10 to about 50 ng / ml IL-3, about 10 to about 50 ng / ml TPO, about 10 to about 50 ng / ml IL-15, about 10 to about 50 ng / ml SDF, about 7 to about 23 µM SB203580, about 10 to about 50 ng / mL SCF, about 10 to about 50 ng / mL Flt-3l, about 10 to about 50 ng / mL IL-7, and about 15 to about 45 mM L-ascorbic acid 2-phophatate sesquimagnesium salt hydrate.6. The method of any one of the preceding items, wherein the ROCK inhibitor is Y-27632dihydrochloride, the inhibitor of ALK receptors is SB43152 and the inhibitor of GSK-3 is CHIR99021.7. The method of any one of the preceding items, wherein the PSCs are pluripotent stem cellsbeing deficient of endogenous MHC class I molecules presented on the cell surface of the PSCs and comprising an immunomodulatory protein on their surface.8. The method of item 7, wherein the immunomodulatory protein is a single chain fusion HLAclass I protein.9. The method of item 8, wherein the single chain fusion HLA class I protein comprises atleast a portion of beta-2 microglobulin (B2M) covalently linked to at least a portion of an HLA class Iα chain selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.10. The method of any one of items 8 to 9, wherein the PSCs further express a target peptideantigen that is presented by the single chain fusion HLA class I protein on the pluripotent cell surface.11. The method of item 10, wherein the target peptide antigen is covalently linked to the singlechain fusion HLA class I protein.12. The method of item 10 or 11, wherein the target peptide antigen comprises the sequenceVMAPRTLFL (SEQ ID NO: 1).13. The method of any one of items 7 to 12, wherein essentially all copies of a B2M gene aredisrupted in the PSCs.14. The method of any one of the preceding items, wherein the PSCs are pluripotent stem cellsbeing deficient of endogenous MHC class II.15. The method of item 14, wherein the PSCs are deficient of endogenous MHC class II bydisrupting the C2TA gene-MHC class II transactivator.16. The method of any one of the preceding items, wherein the PSCs are selected from thegroup consisting of embryonic stem cells, induced pluripotent stem cells and parthenogenetic stem cells.17. The method of any one of the preceding items, wherein the PSCs are pluripotent stem cellsof primate origin, preferably human pluripotent stem cells.18. The method of any one of the preceding items, wherein the PSCs are generated fromCD34-positive cells isolated from umbilical cord blood.19. The method of any one of the preceding items, wherein the PSCs are ND-50039 cells ofthe NINDS Human Cell and Data Repository. 20. The method of any one of the preceding items, wherein in step (i)(a) the solid support comprises one or more wells, wherein each well comprises a V-shaped or conical cavity.21. The method of item 20, wherein the solid support is a microwell culture plate.22. The method of item 21, wherein the microwell culture plate is any one of an AggreWellTMplate, a BIOFLOAT™ 96-well plate, or a Nunclon Sphera 3D culture.23. The method of any one of the preceding items, wherein the suspension culture in step (i)(b)and (c) is a dynamic suspension culture, preferably by culturing on a shaker at rotations of about 60 to about 80 rpm.24. The method of any one of the preceding items, further comprising dissociating the 3D –cell aggregates into single cells after step (i)(c) and sorting for CD34-positive PSCs.25. The method of any one of the preceding items, wherein step (ii) is conducted for about 7to about 21 days.26. The method of any one of the preceding items, wherein in step (ii) the suspension cultureis a Notch-ligand coated suspension culture plate.27. The method of any one of the preceding items, wherein the immune cell is a NK likeprecursor cell devoid of endogenous effector functions.28. An immune cell or a population of immune cells devoid of endogenous effector functionsobtainable by the method of any one of items 1, 3 to 27.29. An immune cell or a population of immune cells devoid of endogenous effector functionsobtained by the method of any one of items 1, 3 to 27.30. An immune cell or a population of immune cells devoid of endogenous effector functions,but comprising one or more exogenous modifications, wherein each of the one or moreexogenous modifications introduces a predetermined effector function obtainable by themethod of any one of items 2, 3 to 27.31. An immune cell or a population of immune cells devoid of endogenous effector functions,but comprising one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function obtained by themethod of any one of items 2, 3 to 27.32. A pharmaceutical composition comprising the immune cell or the population of immunecells of any one of items 28 to 31.33. The immune cell or the population of immune cells of any one of items 28 to 31, or thepharmaceutical composition of item 32 for use in medicine.34. The immune cell or the population of immune cells of any one of items 28 to 31, or thepharmaceutical composition of item 32 for use in a method of preventing or treating cancer.35. The immune cell or the population of immune cells, or the pharmaceutical composition forthe use of item 34, wherein the cancer is selected from lung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, skin cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, tracheal cancer, head & neck cancer, pancreatic cancer, liver cancer, breast cancer, lymphoid cancers including lymphoma and multiple myeloma, leukemia, sarcomas of bone or soft tissue, cervical cancer, and vulvar cancer.

Claims

CLAIMS1. A method of producing an immune cell devoid of endogenous effector functions frompluripotent stem cells (PSCs), the method comprising the steps of: (i) inducing 3D cell aggregate formation, mesodermal differentiation and hematopoieticdifferentiation by (a) seeding PSCs under suitable conditions on a solid support suitable for formationof 3D - cell aggregates, in a first serum-free medium, thereby allowing theformation of 3D - cell aggregates;(b) collecting the 3D - cell aggregates of step (a), resuspending the 3D – cellaggregates under suitable conditions in a second serum-free medium, and transferring the resuspended 3D – cell aggregates into and culturing in a thirdserum-free medium under suitable conditions in suspension culture for about 1to about 3 days, thereby allowing the mesodermal differentiation; and (c) transferring the cultured 3D - cell aggregates of step (b) into and culturing in afourth serum-free medium under suitable conditions in suspension culture for about 2 to about 4 days and further culturing the 3D - cell aggregates undersuitable conditions in suspension culture for about 2 to about 6 days in a fifth serum-free medium, thereby allowing the hematopoietic differentiation; and(ii) inducing immune cell differentiation by culturing single cells of the 3D - cell aggregatesobtained from step (i)(c) under suitable conditions in suspension culture for a suitable time in a sixth serum-free medium; thereby providing an immune cell devoid of endogenous effector functions.

2. A method of producing a genetically modified immune cell from pluripotent stem cells(PSCs) being devoid of endogenous effector functions, but comprising one or moreexogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function, the method comprising the steps of: (i) inducing 3D cell aggregate formation, mesodermal differentiation and hematopoieticdifferentiation by (a) seeding PSCs under suitable conditions on a solid support suitable for formationof 3D - cell aggregates, in a first serum-free medium, thereby allowing theformation of 3D - cell aggregates;(b) collecting the 3D - cell aggregates of step (a), resuspending the 3D – cellaggregates under suitable conditions in a second serum-free medium, andtransferring the resuspended 3D – cell aggregates into and culturing in a thirdserum-free medium under suitable conditions in suspension culture for about 1to about 3 days, thereby allowing the mesodermal differentiation; and (c) transferring the cultured 3D - cell aggregates of step (b) into and culturing in afourth serum-free medium under suitable conditions in suspension culture for about 2 to about 4 days and further culturing the 3D - cell aggregates undersuitable conditions in suspension culture for about 2 to about 6 days in a fifth serum-free medium, thereby allowing the hematopoietic differentiation; and(ii) inducing immune cell differentiation by culturing single cells of the 3D - cell aggregatesobtained from step (i)(c) under suitable conditions in suspension culture for a suitabletime in a sixth serum-free medium; wherein the method further comprises genetically introducing one or more exogenous modifications into the PSCs in any one of before step (a), in step (a) to (c) of step (i), or in step (ii) wherein each of the one or more exogenous modifications introduces a predetermined effector function, thereby providing a genetically modified immune cell being devoid of endogenous effector functions, but comprising one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function.

3. The method of claim 2, wherein said exogenous modification is selected from the groupconsisting of a gene knock-in, a gene knock-out, a gene replacement, a point mutation, and a deletion, insertion or substitution of a gene, a gene fragment or a nucleotide, or acombination thereof.

4. The method of claim 3, wherein said gene knock-in comprises the knock-in of at least onegene selected from the group consisting of a gene encoding: i) a chimeric antigen receptor (CAR); ii) an exogenous T cell receptor (TCR); iii) an exogenous IL-15 receptor; iv) an exogenous CD16 receptor; v) an exogenous CD19 antigen; vi) an exogenous IL-10 cytokine; vii) an exogenous TGF-β cytokine; and viii) an exogenous PD-1 ligand 1 antigen, or a combination thereof, and / orwherein said gene knock-out comprises the knock-out of at least one gene selected from the group consisting of a gene encoding: ix) an endogenous PD-1 cell surface protein; x) an endogenous VEGF receptor; xi) an endogenous VCAM-1 cell surface protein; and xii) an endogenous ICAM-1 cell surface protein, or a combination thereof.

5. The method of any one of the preceding claims, wherein:in step (i) (a) the first serum-free medium comprises an inhibitor of GSK-3 and a ROCKinhibitor, each in a concentration of about 5 to about 15 µM;in step (i) (b) the second serum-free medium comprises bone morphogenic protein 4(BMP4), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), each in a concentration of about 10 to about 50 ng / mL, and the third serum-free medium comprises BMP4, VEGF, FGF, each in aconcentration of about 10 to about 50 ng / mL and additionally an inhibitor of ALKreceptors in a concentration of about 5 to about 15 µM; and / or in step (i) (c) the fourth serum-free medium comprises VEGF, FGF, and stem cell factor (SCF), each in a concentration of about 10 to about 50 ng / mL, and the fifth serum-free medium comprises VEGF, FGF, and SCF, each in a concentration of about 10 to about 50 ng / mL, and additionally about 10 to about50 ng / mL FMS-like tyrosine kinase 3 ligand (Flt-3l), and about 10 to about 50 ng / mL Thrombopoietin (TPO); and / or in step (ii) the sixth serum-free medium comprises about 7 to about 23 % BIT 9500 serumsubstitute, about 25 to about 75 mM B-mercaptoethanol, about 10 to about 50 ng / ml IL-3, about 10 to about 50 ng / ml TPO, about 10 to about 50 ng / ml IL-15, about 10 to about 50 ng / ml SDF, about 7 to about 23 µM SB203580, about 10 to about 50 ng / mL SCF, about 10 to about 50 ng / mL Flt-3l, about 10 to about 50 ng / mL IL-7, and about 15 to about 45 mM L-ascorbic acid 2-phophatate sesquimagnesium salt hydrate.

6. The method of any one of the preceding claims, wherein the ROCK inhibitor is Y-27632dihydrochloride, the inhibitor of ALK receptors is SB43152 and the inhibitor of GSK-3 is CHIR99021.

7. The method of any one of the preceding claims, wherein the PSCs are pluripotent stemcells being deficient of endogenous MHC class I molecules presented on the cell surface of the PSCs and comprising an immunomodulatory protein on their surface.

8. The method of claim 7, wherein the immunomodulatory protein is a single chain fusion HLAclass I protein.

9. The method of claim 8, wherein the single chain fusion HLA class I protein comprises atleast a portion of beta-2 microglobulin (B2M) covalently linked to at least a portion of an HLA class Iα chain selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.

10. The method of any one of claims 8 to 9, wherein the PSCs further express a target peptideantigen that is presented by the single chain fusion HLA class I protein on the pluripotent cell surface.

11. The method of claim 10, wherein the target peptide antigen is covalently linked to the singlechain fusion HLA class I protein.

12. The method of claim 10 or 11, wherein the target peptide antigen comprises the sequenceVMAPRTLFL (SEQ ID NO: 1).

13. The method of any one of claims 7 to 12, wherein essentially all copies of a B2M gene aredisrupted in the PSCs.

14. The method of any one of the preceding claims, wherein the PSCs are pluripotent stemcells being deficient of endogenous MHC class II.

15. The method of claim 14, wherein the PSCs are deficient of endogenous MHC class II bydisrupting the C2TA gene-MHC class II transactivator.

16. The method of any one of the preceding claims, wherein the PSCs are selected from thegroup consisting of embryonic stem cells, induced pluripotent stem cells and parthenogenetic stem cells.

17. The method of any one of the preceding claims, wherein the PSCs are pluripotent stemcells of primate origin, preferably human pluripotent stem cells.

18. The method of any one of the preceding claims, wherein the PSCs are generated fromCD34-positive cells isolated from umbilical cord blood.

19. The method of any one of the preceding claims, wherein the PSCs are ND-50039 cells ofthe NINDS Human Cell and Data Repository.

20. The method of any one of the preceding claims, wherein in step (i)(a) the solid support comprises one or more wells, wherein each well comprises a V-shaped or conical cavity.

21. The method of claim 20, wherein the solid support is a microwell culture plate.

22. The method of claim 21, wherein the microwell culture plate is any one of an AggreWellTMplate, a BIOFLOAT™ 96-well plate, or a Nunclon Sphera 3D culture.

23. The method of any one of the preceding claims, wherein the suspension culture in step(i)(b) and (c) is a dynamic suspension culture, preferably by culturing on a shaker at rotations of about 60 to about 80 rpm.

24. The method of any one of the preceding claims, further comprising dissociating the 3D –cell aggregates into single cells after step (i)(c) and sorting for CD34-positive PSCs.

25. The method of any one of the preceding claims, wherein step (ii) is conducted for about 7to about 21 days.

26. The method of any one of the preceding claims, wherein in step (ii) the suspension cultureis a Notch-ligand coated suspension culture plate.

27. The method of any one of the preceding claims, wherein the immune cell is a NK likeprecursor cell devoid of endogenous effector functions.

28. An immune cell or a population of immune cells devoid of endogenous effector functionsobtainable or obtained by the method of any one of claims 1, 3 to 27.

29. An immune cell or a population of immune cells devoid of endogenous effector functions,but comprising one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function obtainable orobtained by the method of any one of claims 2, 3 to 27.

30. A pharmaceutical composition comprising the immune cell or the population of immunecells of any one of claims 28 to 29.

31. The immune cell or the population of immune cells of any one of claims 28 to 29, or thepharmaceutical composition of claim 30 for use in medicine.

32. The immune cell or the population of immune cells of any one of claims 28 to 29, or thepharmaceutical composition of claim 30 for use in a method of preventing or treatingcancer.

33. The immune cell or the population of immune cells, or the pharmaceutical composition forthe use of claim 32, wherein the cancer is selected from lung cancer, prostate cancer,ovarian cancer, testicular cancer, brain cancer, skin cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, tracheal cancer, head & neck cancer, pancreatic cancer, liver cancer, breast cancer, lymphoid cancers including lymphoma and multiple myeloma, leukemia, sarcomas of bone or soft tissue, cervical cancer, and vulvar cancer.

Images